Patent application title: INFLUENZA H5 VACCINES
Inventors:
Mauricio Realpe-Quintero (Zapopan, MX)
Paulino Carlos Gonzalez-Hernandez (Hamburg, DE)
Eric Vaughn (Ames, IA, US)
Eric Vaughn (Ames, IA, US)
Assignees:
BOEHRINGER INGELHEIM VETMEDICA, S.A. DE C.V.
BOEHRINGER INGELHEIM VETMEDICA GMBH
IPC8 Class: AA61K39145FI
USPC Class:
4241861
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same disclosed amino acid sequence derived from virus
Publication date: 2014-07-17
Patent application number: 20140199337
Abstract:
The present invention is based on the surprising finding that H5 protein
of clade 1 H5N1 induces, in particular by a single-shot vaccination, a
cross-clade protective immune response to influenza viruses with H5N1 HA.
In one aspect, the invention is thus directed to H5 protein of clade 1
H5N1 virus for use in a method of treating or preventing infections with
H5N1 virus of a different clade, namely of a clade different from clade 1
or from any clade with the exception of clade 1, respectively.Claims:
1. An H5 protein of a clade 1 H5N1 virus for use in a method of treating
or preventing infections with a H5N1 virus of a different clade, wherein
said H5 protein comprises a polypeptide sequence having at least 98%
sequence identity with the polypeptide sequence of SEQ ID NO: 1.
2. The H5 protein according to claim 1, wherein said H5 protein comprises a polypeptide sequence having at least 98.1%, preferably at least 98.2%, more preferably at least 98.3%, and most preferably at least 98.4% sequence identity with the polypeptide sequence of SEQ ID NO: 1.
3. The H5 protein according to claim 1, wherein said H5 protein has the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2, and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted.
4. The H5 protein according to claim 1, wherein such H5 protein has the amino acid 94N.
5. The H5 protein according to claim 1, wherein such H5 protein has the amino acid 120N.
6. The H5 protein according to claim 1, wherein such H5 protein has the amino acid 155N.
7. The H5 protein according to claim 1, wherein such H5 protein has one or more of the following amino acid clusters selected from the group consisting of: a. aa 93-95: GNF b. aa 123-125: SDH c. aa 128-130: SSG d. aa 138-140: GSS e. aa 226-228: MDF f. aa 270-272: EVE g. aa 309-311: NKL.
8. The H5 protein according to claim 1, wherein such H5 protein comprises a peptide comprising: i. the amino acid sequences of SEQ ID NO:5; SEQ ID NO:6 or SEQ ID NO:7; or ii. any peptide that has at least 85% sequence homology to the polypeptide of i) and that comprises hemagglutinin inhibition in a standard hemagglutinin inhibition assay; or iii. any part of the polypeptides of i) or ii) comprising at least 8 contiguous amino acids of any of such peptides of i) or ii) and wherein any of such peptide comprises hemagglutinin inhibition in a standard hemagglutinin inhibition assay; or iv. any peptide of i), ii) or iii) having one of the amino acids 36T, 36K, 83A, 83T, 83D, 86A, 86V, 120S, 155S, 156A, 156T, 189R, 189K, 212K, 212R, 212E, 263A or 263T; or v. any peptide of i), ii), iii) or iv) having one or more of the following amino acid clusters selected from the group consisting of: a. aa 93-95: GNF b. aa 123-125: SDH c. aa 128-130: SSG d. aa 138-140: GSS e. aa 226-228: MDF f. aa 270-272: EVE g. aa309-311: NKL.
9. The H5 protein according to claim 1, wherein such H5 protein comprises the amino acid sequence of SEQ ID NO:5.
10. The H5 protein according to claim 1, wherein such H5 protein is recombinantly expressed and/or produced by a baculovirus expression system, preferably in cultured insect cells.
11. The H5 protein according to claim 1, wherein said H5N1 virus of a different clade is selected from the group consisting of clade 0 H5N1 virus, clade 2 H5N1 virus, clade 3 H5N1 virus, clade 4 H5N1 virus, clade 5 H5N1 virus, clade 6 H5N1 virus, clade 7 H5N1 virus, clade 8 H5N1 virus and clade 9 H5N1 virus.
12. The H5 protein according to claim 1, wherein said H5N1 virus of a different clade is clade 2.2 H5N1 virus or a clade 2.3 H5N1 virus.
13. The H5 protein according to claim 1, wherein said H5N1 virus of a different clade is a clade 2.2.1 H5N1 virus or a clade 2.3.2 H5N1 virus.
14. The H5 protein according to claim 1, wherein said H5N1 virus of a different clade is a H5N1 virus of North African or of Vietnamese origin.
15. The H5 protein according to claim 14, wherein said H5N1 virus of North African origin is a H5N1 virus comprising a second H5 protein of influenza virus, wherein said second H5 protein encodes an amino acid sequence including at least one member of a group consisting of: (a) the amino acids 113D, 126H, 145(-), 156R, 160F, 167T, and 181N, wherein the modification 145(-) means that amino acid position 145 of H5 is deleted, or (b) the amino acids 87P, 145L, 172T, 201E, 206I, 208K, 254T, 341G and 421K, or (c) the amino acids 145L, 172T, and 254V, and wherein the numbering of the amino acid positions of said second H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:8; or wherein said second H5 protein consists of an amino acid sequence which is at least 95%, preferably at least 96%, more preferably at least 97%, still more preferably at least 98%, yet more preferably at least 99%, or in particular preferred 100% homolog with any one of the sequences as set forth in SEQ ID NOs: 9 to 46.
16. The H5 protein according to claim 1, wherein said H5N1 virus of a different clade comprising a second H5 protein encodes an amino acid sequence including at least one member of the group consisting of: (a) the amino acids 87L, 113D, 126H, 145(-), 156R, 160F, 167T, and 181N, or (b) the amino acids 87P, 113N, 126R, 145L, 160Y, 172T, 181H, 201E, 206I, 208K, 254T, 341G and 421K, or (c) the amino acids 87L, 113N, 126R, 145L, 156G, 160Y, 172T, 181H, and 254V, and/or wherein such second H5 protein comprises a peptide comprising: i. any one of the amino acid sequences of SEQ ID NOs: 9 to 46; ii. any peptide that has at least 85%, preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence homology to the polypeptide of i) and that comprises hemagglutinin inhibition in a standard hemagglutinin inhibition assay; or iii. any part of the polypeptides of i) or ii) comprising at least 334 contiguous amino acids of any of such peptides of i) or ii) and wherein any of such peptide comprises hemagglutinin inhibition in a standard hemagglutinin inhibition assay, and/or wherein such second H5 protein comprises a contiguous amino acid sequence which has at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with any one of the sequences as set forth in SEQ ID NOs: 9 to 46.
17. The H5 protein according to claim 1, wherein said H5N1 virus of a different clade comprising a second H5 protein consists of an amino acid sequence which is at least 95%, preferably at least 96%, more preferably at least 97%, still more preferably at least 98%, yet more preferably at least 99%, or in particular preferred 100% homolog with any one of the sequences as set forth in SEQ ID NOs: 15 or 20, and wherein such second H5 protein consisting of the amino acid sequence set forth in SEQ ID NO:20 is more preferred.
18. The H5 protein according to claim 1, for use in a method of treating or preventing viral infection, wherein said viral infection includes a virus from at least one member of a group selected from: (A) Subclade A H5N1 virus of North African origin, namely an infection with a H5N1 virus comprising a second H5 protein encoded by the amino acid sequence according to at least one of the sequences as set forth in SEQ ID NOs: 9 to 19, or 42 or 43, or (B) with Subclade B H5N1 virus of North African origin, namely an infection with a H5N1 virus comprising a H5 protein encoded by the amino acid sequence according to at least one of the sequences as set forth in SEQ ID NOs: 20 to 41, or 44 to 46.
19. A combination of an H5 protein of a clade 1 H5N1 virus for use in a method of treating or preventing infections with a H5N1 virus of a different clade, wherein said H5 protein comprises a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO: 1 and an inactivated Newcastle disease virus.
20. The combination of claim 19, wherein the inactivated Newcastle disease virus is an inactivated whole Newcastle disease virion.
21. The combination of claim 19, wherein the inactivated Newcastle disease virus is an inactivated Newcastle disease virus obtained by inactivation of a Newcastle disease virus comprising a RNA polynucleotide having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity with a RNA copy of the polynucleotide set forth in SEQ ID NO: 51, which has been inactivated.
22. The combination of claim 19, wherein the Newcastle disease virus is a Newcastle disease LaSota strain virus.
23. The combination of claim 19, wherein the Newcastle Disease Virus is inactivated with a reagent selected from the group consisting of Formaldehyde, BEI, Beta-Propio-Lactone (BPL), and combinations thereof.
24. A vaccine for use in a method of treating or preventing infections with a H5N1 virus, comprising: a. the H5 protein of a different clade, wherein said H5 protein comprises a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO:1 of, and b. a pharmaceutical acceptable carrier and/or excipient.
25. The vaccine according to claim 24, wherein the excipient is one or more adjuvants.
26. The vaccine according to claim 25, wherein the adjuvant is an Emulsigen-based adjuvant.
27. The vaccine according to claim 24, wherein the vaccine comprises one or more further antigens.
28. The vaccine according to claim 27, wherein the one or more further antigen is an antigen of a poultry pathogen.
29. The vaccine according to claim 28, wherein the one or more further antigen is H5, H7, or H9 of influenza virus.
30. The vaccine according to claim 29, wherein the H5 of influenza virus is H5 protein of a H5N1 virus of a clade different than clade 1.
31. Use of the H5 protein wherein said H5 protein comprises a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO:1, for the preparation of a pharmaceutical composition, preferably of a single-shot vaccine or a one dose vaccine, for the prophylaxis or treatment of infections caused by H5N1 virus of a clade other than clade 1.
32. A method for the treatment or prophylaxis of influenza virus infections caused by H5N1 virus of a clade other than clade 1, wherein the method comprises administration of a therapeutically effective amount of the H5 protein, wherein said H5 protein comprises a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO: 1, to a subject in need of such a treatment.
33. A method for the treatment or prophylaxis of influenza virus infections caused by H5N1 virus of a clade other than clade 1, wherein the method comprises administration of a therapeutically effective amount of a vaccine comprising: a. the H5 protein of a different clade, wherein said H5 protein comprises a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO: 1 of, and b. a pharmaceutical acceptable carrier and/or excipient to a subject in need of such a treatment.
34. The method of claim 32 wherein said administration is a single-shot administration or a one dose administration.
35. A kit of parts, comprising: a. the H5 protein according to claim 1; and b. a package leaflet indicating the use of such H5 protein, combination or vaccine of a) for the treatment or prophylaxis of infections caused by H5N1 virus of a clade other than clade 1.
36. The kit according to claims 35, wherein such kit comprises at least one or more further antigens of poultry or mammalian pathogen.
37. (canceled)
38. A method of reducing the incidence of or severity of influenza infection comprising the step of administering a composition selected from the group consisting of: a. an H5 protein of a clade 1 H5N1 virus, wherein said H5 protein comprises a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO: 1, and b. a combination of the H5 protein of group a) and an inactivated Newcastle disease virus.
39. (canceled)
40. Use of the H5 protein wherein said H5 protein comprises a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO:1, for the preparation of a medicament for reducing viral shedding in a subject infected with or at risk of a viral infection with H5N1 virus of a clade other than clade 1.
41. A vaccine for use in a method of treating or preventing infections with a H5N1 virus, comprising: a. the combination of the H5 protein of a different clade and an inactivated Newcastle disease virus, and b. a pharmaceutical acceptable carrier and/or excipient.
42. The vaccine according to claim 41, wherein the excipient is one or more adjuvants.
43. The vaccine according to claim 41, wherein the adjuvant is an Emulsigen-based adjuvant.
44. The vaccine according to claim 41, wherein the vaccine comprises one or more further antigens.
45. The vaccine according to claim 44, wherein the one or more further antigen is an antigen of a poultry pathogen.
46. The vaccine according to claim 45, wherein the one or more further antigen is H5, H7, or H9 of influenza virus.
47. The vaccine according to claim 46, wherein the H5 of influenza virus is H5 protein of a H5N1 virus of a clade different than clade 1.
48. Use of the combination of the H5 protein wherein said H5 protein comprises a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO:1 and an inactivated Newcastle disease virus, for the preparation of a pharmaceutical composition, preferably of a single-shot vaccine or a one dose vaccine, for the prophylaxis or treatment of infections caused by H5N1 virus of a clade other than clade 1.
49. A method for the treatment or prophylaxis of influenza virus infections caused by H5N1 virus of a clade other than clade 1, wherein the method comprises administration of a therapeutically effective amount of the combination of the H5 protein wherein said H5 protein comprises a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO:1 and an inactivated Newcastle disease virus combination to a subject in need of such a treatment.
50. The method of claim 41, wherein said administration is a single-shot administration or a one dose administration.
51. A method of reducing the incidence of or severity of influenza infection comprising the step of administering a composition selected from the group consisting of: a. an H5 protein of clade 1 H5N1 virus, wherein said H5 protein comprises or consists of a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO: 1, and b. a combination of the H5 protein of group a) and an inactivated Newcastle disease virus.
52. The H5 protein of claim 1, for use in a method for reducing viral shedding in a subject, wherein said H5 protein is to be administered to a subject infected with or at risk of a viral infection with H5N1 virus of a clade other than clade 1.
53. The method of claim 52, further comprising use of an inactivated Newcastle disease virus in combination with the H5 protein.
54. Use of a H5 protein wherein said H5 protein comprises a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO:1, for the preparation of a medicament for reducing viral shedding in a subject infected with or at risk of a viral infection with H5N1 virus of a clade other than clade 1.
55. The medicament of claim 54, further comprising use and an inactivated Newcastle disease virus in combination with the H5 protein, wherein the combination reduces viral shedding in a subject infected with or at risk of a viral infection with H5N1 virus of a clade other than clade 1.
Description:
FIELD OF THE INVENTION
[0001] The present invention relates to the field of medicine, preferably to the field of infectious diseases. In particular the present invention relates to influenza proteins and vaccines. Most particularly, the present invention relates to the use of any of such proteins or vaccines for the treatment and prevention of influenza infections, furthermore for the prevention of intra- and inter-species transmission of influenza virus.
BACKGROUND OF THE INVENTION
[0002] Influenza infection remains an important infection in animals and humans. Influenza is caused by viruses that undergo continuous antigenic changes/modifications and that possess an animal reservoir. Thus new epidemics and pandemics may occur in the future, and eradication of the disease will be difficult to achieve. Influenza viruses are well known in the art and described more in detail for example by P. Palese, Nature Medicine, vol. 10, no. 12, pp. S 82 to S 86 of December 2004, with further references. Briefly, the genome of the influenza A virus consists of eight single-stranded segments, and the viral particles has two major glycoproteins on its surface: hemagglutinin (H) and neuraminidase (N). With at least 16 different hemagglutinin (H1 to H16) and 9 different neuraminidase (N1 to N9) subtypes, there is a considerable antigenic variation among influenza viruses.
[0003] Influenza virus of type H5N1 Fowl Plague virus has been demonstrated to infect poultry, pigs and man. The viruses can also be transmitted directly from avian species to humans (Claas et al., Lancet 1998, 351: 472; Suarez et al., J. Virol. 1998, 72: 6678; Subbarao et al., Science 1998, 279: 393; Shortridge, Vaccine 1999, 17 (Suppl. 1): S26-S29). Mortality in known human clinical cases approaches about 50%.
[0004] Over the last century pigs have been an important vector for influenza pandemics. Pigs, camels, and seals, preferably pigs, can serve as a `mixing chamber` for avian influenza viruses, and therefore represent a potential risk factor for overcoming the species hurdles from poultry, the naturally reservoir of influenza viruses, to mammals. This normally occurs by double infections of the susceptible animals, e.g. pig, with both, an established mammalian (porcine), as well as an avian influenza virus. This double infection may create new recombinant viruses that may be the cause of human or porcine pandemics. Recent evidence would, however, indicate that a recombination of current avian H5 strains with mammalian influenza viruses will not result in highly virulent recombinants. On the other hand, avian influenza virus can infect pigs and by spontaneous mutations can become adapted to pigs. The critical hurdle will be overcome as soon as the virus can cause horizontal infections within a pig (or other mammalian) population.
[0005] Yet, a major part of Southeast Asian pigs have been infected with avian (H5) influenza virus strains originating from neighbouring poultry husbandry. As those infections have so far been sub-clinical, they can only be diagnosed by laboratory methods and thus are frequently overlooked. There is a high risk that those sub-clinically-infected pigs will serve as an opportunity for the virus to adapt to the mammalian system, spread within the porcine population, and also infect human beings.
[0006] Current influenza vaccines include a subunit vaccine (Babai et al., Vaccine 1999, 17(9-10):1223-1238; Crawford at al., Vaccine 1999, 17(18):2265-2274; Johansson et al., Vaccine 1999, 17(15-16):2073-2080) attenuated vaccine (Horimoto et al., Vaccine 2004, 22(17-18):2244-2247), DNA vaccine (Watabe et al., Vaccine 2001, 19(31):4434-4444) and inactivated influenza vaccine (Cao at al., Vaccine 1992, 10(4):238-242), with the latter being the most widely used on a commercial scale (Lipatov at al., J Virol 2004, 78(17):8951-8959).
[0007] Subunit vaccines, recombinant hemagglutinin and neuraminidase (Babai et al., Vaccine 1999, 17(9-10):1223-1238; Crawford et al., Vaccine 1999, 17(18):2265-2274; Johansson et al., Vaccine 1999, 17(15-16):2073-2080) may be an attractive alternative to the inactivated vaccine, although none are currently in use as commercial vaccines. The preparation of such vaccines is obviously safer than for an inactivated vaccine. Moreover, subunit vaccines do not generate antibody responses to internal influenza viral proteins and thus allow distinction between vaccinated and infected animals (Crawford at al., Vaccine 1999, 17(18):2265-2274).
[0008] Hemagglutinin protein is the receptor-binding and membrane fusion glycoprotein of influenza virus and the target for infectivity-neutralizing antibodies. The entire hemagglutinin protein (HA) from the H5N1 is composed of 568 amino acids, with a molecular weight of 56 kDa. The HA molecule consists of HA1 and HA2 subunits, with the HA1 subunit mediating initial contact with the cell membrane and HA2 being responsible for membrane fusion (Chizmadzhev, Bioelectrochemistry 2004, 63(1-2):129-136).
[0009] Baculovirus/insect cell systems have been used to express hemagglutinin genes isolated from avian influenza subtypes (Babai at al., Vaccine 1999, 17(9-10):1223-1238; Crawford et al., Vaccine 1999, 17(18):2265-2274; Johansson et al., Vaccine 1999, 17(15-16):2073-2080); Nwe et al., BMC Mircobiology 2006, 6(16):doi:10.1186/1471-2180-6-16). However, those recombinant proteins seem not to be protective in any case, or only less effective at least for some species (Treanor et al., Vaccine 2001, 19: 1732-1737).
[0010] The document Lin et al. (J Vet Med Sci. 2008 70(11):1147-52) discloses the use of a baculovirus/insect cell system for the production of H5 protein of clade 2 H5N1 virus A/duck/China/E319-2/03, which is usable for a prime-booster vaccination for preventing an infection with the clade 2 virus A/duck/China/E319-2/03.
[0011] Bright at al. (PLoS One. 2008 30; 3(1):e1501) describes the use of a baculovirus/insect cell system for generating virus-like particles (VLPs) which include neuraminidase, hemagglutinin and matrix 1 protein from clade 2 H5N1 virus for inducing a cross-clade protective immune response against a challenge with clade 1 H5N1 virus A/VN/1203/2004 in mice. However, the production of VLPs is not without problems, since in order to generate a functional VLP that effectively mimic a real virus, multiple virus structural proteins are needed which must then be correctly assembled into a particle that reproduces the confirmation of the outer shell (capsid) of the infectious virus. Further, study also reals that in vitro assembly of VLPs competes with aggregation (Ding at al. Biotechnology and Bioengineering 107 (3): 550-560).
[0012] Thus, there is a need to increase availability of improved vaccines and new vaccination approaches to provide better approaches to control influenza infections and to have a positive impact on disease load. In particular, there is a strong need for a simple, effective and easy-to-handle system inducing, preferably by a single-shot vaccination, a cross-clade protective immune response to influenza viruses with H5N1 HA.
DESCRIPTION OF THE INVENTION
[0013] Before the embodiments of the present invention it shall be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a preparation" includes a plurality of such preparations; reference to the "carrier" is a reference to one or more carriers and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All given ranges and values may vary by 1 to 5% unless indicated otherwise or known otherwise by the person skilled in the art, therefore, the term "about" was omitted from the description. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the substances, excipients, carriers, and methodologies as reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
[0014] The solution to the above technical problem is achieved by the description and the embodiments characterized in the claims.
Influenza Proteins and Nucleic Acid Molecules Coding for Those
[0015] The present invention is based on the surprising finding that H5 protein of clade 1 H5N1 induces, in particular by a single-shot vaccination, a cross-clade protective immune response to influenza viruses with H5N1 HA. As one feature, the H5 protein of clade 1 H5N1 virus, which is for reasons of clarity also termed "H5 protein (1)" herein, comprises or consists of a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence set forth in SEQ ID NO:1.
[0016] A "single-shot vaccination" refers to an immunogenic composition that is effective at reducing the incidence of or severity of infection after a single dose thereof, without the need for a booster.
[0017] In one aspect, the invention is thus directed to H5 protein (1) of clade 1 H5N1 virus for use in a method of treating or preventing infections with H5N1 virus of a different clade, namely of a clade different from clade 1 or from any clade with the exception of clade 1, respectively, wherein said H5 protein (1) comprises or consists of a polypeptide sequence having at least 98% sequence identity with the polypeptide sequence of SEQ ID NO:1.
[0018] The term "clade" or "clades" as used herein relates to the clade(s) of the WHO Nomenclature System for the highly Pathogenic Avian Influenza Virus (H5N1), which is summarized at the WHO website URL: who.int/csr/disease/avian_influenza/guidelines/nomencature/en/(12 Aug. 2011), which is incorporated herein by reference.
[0019] 10 distinct initial clades of viruses (numbered 0-9) are defined (WHO/OIE/FAO H5N1 Evolution Working Group, 2008), which are called first order clades. Clades are strictly defined on the nucleotide level as meeting the following three specific clade definition criteria developed by the WHO/OIE/FAO H5N1 Evolution Working Group:
[0020] sharing of a common (clade-defining) node;
[0021] monophyletic grouping with a bootstrap value of ≧60 at the clade-defining node (after 1000 neighbor-joining bootstrap replicates); and
[0022] average percentage pairwise nucleotide distances between and within clades of >1.5% and <1.5%, respectively.
[0023] As the viruses within these 10 clades continue to evolve, new sublineages (potential H5N1 clades) periodically emerge. Once these sublineages meet the same three specific clade definition criteria as the initial 10 clades (numbered 0-9), they are designated as separate clades (WHO/OIE/FAO H5N1 Evolution Working Group Emerg. Inf. Dis. 14, 7 (2008). These new clades are defined as second (or third, etc) order clades and assigned a numerical `address` which links them to their original clade using a hierarchical decimal numbering system. For example, within the antigenically distinct clade 2.3, third order clades meeting the clade definition are designated as clades 2.3.1 and 2.3.2 and so on. This logical hierarchal numbering system is objectively related to HA phylogeny.
[0024] The criteria used for the clade designation according to the WHO Nomenclature System for H5Nlare:
[0025] 1 Maintain previously designated clade numbers where possible (i.e., clade 2.2 remains 2.2 and clade 1 remains 1)
[0026] 2 New clade designations based on phylogenetic tree topology derived from all available sequences (the large tree)
[0027] H5N1 progenitors (closest to Gs/Guangdong/1/96) re-designated as clade 0
[0028] Subsequent clades numbered starting from clade 3 (i.e., clades 3-9)
[0029] Clades designated by presence of a distinct common node shared by at least 4 isolates (in a monophyletic group)
[0030] Additional branches designated as a single clade evolves into more than one distinct lineage (i.e., clade 2.2 or clade 2.3.1; based on sharing of a common node and monophyletic grouping)
[0031] 3 Average percentage pairwise distances between and within clades (using Kimura 2-parameter)
[0032] Distinct clades should have >1.5% average distances between other clades
[0033] Distinct clades should have <1.5% average distances within the clade (may be slightly higher in clades with highly evolved outliers; i.e., Ck/Shanxi/2/2006 in clade 7)
[0034] 4 Bootstrap (based on 1,000 neighbor-joining bootstrap replicates) ≧60% bootstrap value at clade-defining node
[0035] (taken from Table 1 of: WHO/OIE/FAO H5N1 Evolution Working Group Emerg. Inf. Dis. 14, 7 (2008)).
[0036] The prototype strain for each clade is listed in the following Table:
TABLE-US-00001 Clade Prototype strain 0 Gs/Guangdong/1/96 3 Ck/Hong Kong/YU562/2001 4 Gs/Guiyang/337/2006 5 Gs/Guangxi/914/2004 6 Ck/Hunan/01/2004 7 Ck/Shanxi/2/2006 8 Ck/Hong Kong/YU777/2002 9 Dk/Guangxi/2775/2005 1 Vietnam/1203/2004 2.1.1 Ck/Indonesia/BL/2003 2.1.2 Indonesia/538H/2006 2.1.3 Indonesia/5/2005 2.2 BHGs/Qinghai/1A/2005 2.3.1 Dk/Hunan/303/2004 2.3.2 Ck/Guangxi/2461/2004 2.3.3 Ck/Guiyang/3055/2005 2.3.4 Dk/Fujian/1734/2005 2.4 Ck/Yunnan/115/2004 2.5 Ck/Korea/ES/2003 2.5 Ck/Korea/ES/2003
(taken from Table 2 of: WHO/OIE/FAO H5N1 Evolution Working Group Emerg. Inf. Dis. 14, 7 (2008)).
[0037] The publication WHO/OIE/FAO H5N1 Evolution Working Group Emerg. Inf. Dis. 14, 7 (2008), which is incorporated herein by reference, is found at the CDC website URL: cdc.gov/EID/content/14/7/e1.htm (12 Aug. 2011).
[0038] An overview of the clade classification of known H5N1 viruses is provided by the phylogenetic tree at the WHO website URL: who.int/csr/disease/avian_influenza/H5CompleteTree.pdf (15 Aug. 2011), which is hereby incorporated by reference.
[0039] For determining the clade of a H5 protein of H5N1, for example, the web based tool "Highly Pathogenic Avian Influenza (HPAI) H5N1 HA clade prediction" can be used, which is described by Lu, Davis, Rowley, and Donis: "A Web-based tool for the clade designation of highly pathogenic avian influenza H5N1 viruses" in Options for the Control of Influenza VI. J. M. Katz, N. Cox & A. W. Hampson (Eds.) London: Blackwell, 2007, herein incorporated by reference, and which is found at the website URL: h5n1.flugenome.org/grouping.php (12 Aug. 2011).
[0040] For example, a H5 protein of clade 1 H5N1 virus (H5 protein (1)) is thus a HA with an amino acid sequence encoded by a nucleotide sequence of a clade 1 according to the above-mentioned WHO Nomenclature System for H5N1.
[0041] A clade 2.3.1 H5N1 virus, for instance, is hence a H5N1 falling under the criteria of a clade 2.3.1 according to the above-mentioned WHO Nomenclature System for H5N1.
[0042] In a preferred embodiment, the H5 protein (1) according to the invention, namely the H5 protein of clade 1 H5N1 virus as described herein, comprises or consists of a polypeptide sequence having at least 98.1%, preferably at least 98.2%, more preferably at least 98.3%, and most preferably at least 98.4% sequence identity with the polypeptide sequence of SEQ ID NO:1.
[0043] Sequence identity in the context of the invention is understood as being based on determined pairwise similarity between protein sequences. The determination of percent similarity between two sequences is preferably accomplished using a computational algorithm, in particular the well-known Basic Local Alignment Search Tool (Altschul S F, Gish W, Miller W, Myers E W, Lipman D J: Basic local alignment search tool. J Mol Biol 1990, 215(3):403-410). For purposes of the present invention, percent sequence identity of an amino acid sequence is determined using the BLAST blastp homology search algorithm using the following parameters: an expected threshold of 10, word size 3, BLOSUM62 matrix, gap opening penalty of 11, a gap extension penalty of 1, and conditional compositional score matrix adjustment. The database to search against is the set of non-redundant protein sequences (nr). The BLAST homology search algorithm is described in Altschul SF (1990), J Mol Biol 1990, 215(3):403-410, which is herein incorporated by reference.
[0044] A variant may, for example, differ from the reference accession number BAE07201 molecule without signal peptide (N-terminal 16 amino acid residues are not shown in SEQ ID NO:1) by as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
[0045] In one exemplary embodiment, the H5 protein (1) according to the invention, i.e. the H5 protein (1) of clade 1 H5N1 virus for use in a method of treating or preventing infections with H5N1 virus of a different clade, is preferably a H5 protein of influenza virus, wherein the H5 protein having the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted. Said preferred H5 protein (1) is also termed Mut k+ or mutK+ in the following. Preferably, such H5 protein and any further H5 protein according to the invention is an isolated H5 protein.
[0046] The term "H5 protein (1) of clade 1 H5N1", as used herein, preferably means "H5 protein (1) as single antigen of clade 1 H5N1 virus" or in particular "H5 protein (1) as single antigen".
[0047] The terms "hemagglutinin 5 (H5)" or "H5 of avian influenza virus" or "H5 protein" as used herein are equivalent and mean, but are not limited to any naturally occurring H5 protein and any modified forms of H5 protein, including any deletion, substitution and/or insertion mutant of H5 protein.
[0048] The numbering of the amino acid positions of the H5 protein (1) Mut k+ as used herein refers to the amino acid position as exemplarily given in SEQ ID NO:2. SEQ ID NO:2 represents the amino sequence of the hemagglutinin of strain duck/China/E319-2/03 but lacking the amino terminal signal peptide. In other words, if reference is made to the amino acid at position 223 (amino acid 223), the amino acid residue is meant which corresponds to amino acid 223 of SEQ ID NO:2. However, this does not mean that the H5 protein Mut k+ according to the invention has the identical amino acid sequence with SEQ ID NO:2. It only says, that the corresponding amino acids of the H5 proteins according to the inventions code for the amino acid residue, as explicitly mentioned. In the current case, amino acid 223 would be Serine (S). The terms "223N", or "155N" exemplarily mean, that the amino acid at positions 223 and 155, respectively--numbering according to the amino acid positions of SEQ ID NO:2--,that shall code for the amino acid Asparagine (N). In other words, if reference is made to "H5 protein (1) having the amino acid 223N", a H5 amino acid molecule that normally codes for Serine at amino acid position 223--numbering according to the amino acid positions of SEQ ID NO:2--that amino acid shall be substituted by an Asparagine (N). The term "328K+" or "modification 328K+" means, that at amino acid position 328 of H5 protein--numbering according to the amino acid positions of SEQ ID NO:2--, a second Lysine (K+) is inserted. In cases were amino acids sequences at positions 328 and 329 naturally codes for Lysine-Lysine, no further Lysine (K) shall be inserted. However, most of the known H5 sequences code at amino acid positions 328 and 329 for Lysine-Arginine. In any such cases, the term 328K+ modification means, that a second Lysine (K) shall be inserted between Lysine at position 328 and Arginine at position 329. The modified sequence would read then Lysine-Lysine-Arginine (KKR).
[0049] Regarding the present example, the hemagglutinin of strain duck/China/E319-2/03 is shifted to a H5 protein (1) of clade 1 H5N1, since it resembles the H5 sequence of the clade 1 H5N1 virus A/HongKong/213/2003, the year/location/host of this HK isolate, and shows reactivity with clade-1-specific antibodies. Hence the Mut K+ sequence is classified as a H5 sequence of a clade 1 H5N1. Within the context of the invention, the designed Mut K+ sequence is thus understood and defined to be a H5 protein of clade 1 H5N1 virus.
[0050] Thus, in particular also any designed H5 protein is understood and defined as a H5 protein of clade 1 H5N1 virus according to the invention, if it is encoded by a nucleotide sequence which fulfils the criteria of a nucleotide sequence of a clade 1 according to the above-mentioned WHO Nomenclature System for H5N1.
[0051] Thus, in one embodiment, the present invention is implemented with a H5 protein and any modified forms of H5 protein, including any deletion, substitution and/or insertion mutant of H5 protein, wherein those H5 proteins having the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted. It is self-explanatory, that any of the H5 proteins as provided herewith are antigenic, which mean they show antigenic properties in an standard hemagglutinin inhibition assay for influenza viruses.
[0052] According to a further embodiment, the present invention also relates to any part of the H5 protein (1), which means any peptide-fragment which shows antigenic properties in an standard hemagglutinin inhibition assay, having in one embodiment at least the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted.
[0053] A H5 protein (1) shows antigenic properties if it inhibits hemagglutination in a standard hemagglutinin inhibition assay, for example, as described in Example 2. Normally said antigenic part of H5 protein (1) comprises 200, 180, 160, 150, 140, 130, 120, 110 or most preferably 105 contiguous amino acids of the amino acid sequence that codes for the H5 protein as mentioned above, modified or non-modified, which shows antigenic properties in an standard hemagglutinin inhibition assay as described in Example 2. A standard hemagglutinin inhibition assay for example is also described in Stephenson et al., Virus Research vol. 103, pp. 91-95 (2004) with further references. However, the HI assay as described in Example 2 shall be understood to be the relevant reference assay in connection with all aspects of the invention as described herein:
[0054] Briefly, HI assay was performed to detect the presence of HA-specific antibodies. A heterologous H5N2 virus, A/chicken/Mexico/232/94, was used at a concentration of four hemagglutinating units [4 HA units] in the HI assay. In U-bottomed microtiter plates serial two-fold serum dilutions in PBS were subsequently mixed with equal volumes (25 μL) containing 4 HA units of virus, and incubated at room temperature (about 25° C.) for 30 min. Chicken red blood cells, at a concentration of 0.5% in PBS, were added to the serum-virus containing wells and incubated for 40 min at room temperature. The HI titers were determined as reciprocals of the highest serum dilutions in which inhibition of hemagglutination was observed.
[0055] Of note, Haesebrouck and Pensaert (1986) found "that there may exist a correlation between the HI titers against the challenge virus and protection from challenge". Haesebrouck and Pensaert (1986) also determined that pigs with HI titers of ≧40 were "completely resistant to challenge and no replication of the virus occurred in the respiratory tract at challenge". Thus, the development of HI titers ≧40 in the vaccinated swine would correlate to protection. (F. Haesebrouck and M. B. Pensaert, 1986). Effect of intratracheal challenge of fattening pigs previously immunized with an inactivated influenza H1N1 vaccine (Veterinary Microbiology, 11 (1986) 239-249. It has to assume that equivalent or at least nearly equivalent H5 HI titers will also result in a complete immune protection of swine against avian influenza virus. Lower titers, at least result in a seroconversion of the vaccinated animals and result in partial immune protection of those animals, which also can dramatically reduce the risk of a pandemics.
[0056] Moreover, an antigenic part of the H5 protein (1) according to the invention includes, but is not limited to deletion mutants of H5 protein, which comprises:
[0057] i. at least 35, 30, 25, 20, 18, 15, 13, 10, 9, or most preferably 8 contiguous amino acids of the amino acid sequence that surrounds and includes the amino acid 223N; and
[0058] ii. at least 35, 30, 25, 20, 18, 15, 13, 10, 9, or most preferably 8 contiguous amino acids of the amino acid sequence that surrounds and includes the amino acid modification 328K+, and
[0059] iii. wherein any of such antigenic part of H5 protein shows hemagglutinin inhibition in a standard hemagglutinin inhibition assay as described in Example 2.
[0060] Preferably, those surrounding amino acids of amino acid 223N and/or 328K+ are encoded by SEQ ID NO:2 or SEQ ID NO:5.
[0061] Furthermore preferred H5 proteins (1) according to the invention are:
[0062] i. any of those mentioned above having the amino acid 223N and the modification 328K+;
[0063] ii. any of those mentioned above having the amino acid 94N/223N and the modification 328K+;
[0064] iii. any H5 protein of avian origin having the amino acid 223N, and the modification 328K+, wherein avian origin means that the H5 sequence derived form a virus isolate that was originally isolated from a poultry infected with avian influenza virus type 5; or
[0065] iv. any H5 protein of avian origin having the amino acids 94N/223N and the modification 328K+, wherein avian origin means that the H5 sequence derived from a virus isolate that was originally isolated from poultry infected with avian influenza virus type 5; or
[0066] v. any H5 protein of avian origin having the amino acids 155N/223N and the modification 328K+, wherein avian origin means that the H5 sequence derived from a virus isolate that was originally isolated from poultry infected with avian influenza virus type 5; or
[0067] vi. any H5 protein of avian origin having the amino acid 120N/155N/223N and the modification 328K+, wherein avian origin means that the H5 sequence derived from a virus isolate that was originally isolated from poultry infected with avian influenza virus type 5; or
[0068] vii. any H5 protein having the modifications 94N/223N and the modification 328K+; or
[0069] viii. any H5 protein having the modifications 94N/155N/223N and the modification 328K+; or;
[0070] ix. any H5 protein having the modifications 94N/120N/155N/223N and the modification 328K+; or
[0071] x. any H5 protein having the modifications 223N, the modification 328K+, and one or more of the following amino acid clusters selected from the group consisting of:
[0072] a. aa 93-95: GNF
[0073] b. aa 123-125: SDH
[0074] c. aa 128-130: SSG
[0075] d. aa 138-140: GSS
[0076] e. aa 226-228: MDF
[0077] f. aa 270-272: EVE
[0078] g. aa 309-311: NKL; or
[0079] xi. any H5 protein having the amino acid 223N, and the modification 328K+, and one or more of the following amino acid clusters selected from the group consisting of:
[0080] a. aa 93-95: GNF
[0081] b. aa 128-130: SSG
[0082] c. aa 138-140: GSS; or
[0083] xii. any H5 protein having the amino acid sequence of SEQ ID NO:5.
[0084] Furthermore preferred H5 proteins (1) as provided herewith include the H5 proteins as described by Hoffmann et al, PNAS, vol. 106, no. 36, pp. 12915-12920 of Sep. 6, 2005, wherein that H5 proteins includes one or more of the modifications as described above, at least the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted. The disclosure of this reference shall be entirely included herein by reference.
[0085] Furthermore preferred H5 proteins (1) as provided herewith include H5 proteins which comprise a peptide that comprises the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted, and:
[0086] i. the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 or SEQ ID NO:7 or,
[0087] ii. any peptide that has at least 85% sequence homology, more preferably at least about 90% sequence homology, still more preferably at least about 95% sequence homology, even more preferably at least about 97% sequence homology, still even more preferably at least about 98% sequence homology, and even more preferably at least about 99% sequence homology to the polypeptide of i) that comprises hemagglutinin inhibition in a standard hemagglutinin inhibition as described above; or
[0088] iii. any antigenic part of the polypeptides of i) or ii) comprising at least 35, 30, 25, 20, 18, 15, 13, 10, 9, or most preferably 8 contiguous amino acids of any of peptides of i) or ii).
[0089] iv. any peptides of i), ii) or iii) having the amino acids 36T, 36K, 83A, 83T, 83D, 86A, 86V, 120N, 120S, 155N, 155S, 156A, 156T, 189R, 189K, 212K, 212R, 212E, 223N, 223N, or 120N/155N.
[0090] v. any peptide of i), ii), iii) or iv) having one or more of the following amino acid clusters selected from the group consisting of:
[0091] a. aa 93-95: GNF
[0092] b. aa 123-125: SDH
[0093] c. aa 128-130: SSG
[0094] d. aa 138-140: GSS
[0095] e. aa 226-228: MDF
[0096] f. aa 270-272: EVE
[0097] g. aa 309-311: NKL; or
[0098] vi. any peptide of i), ii) iii) or iv) having one or more of the following amino acid clusters selected from the group consisting of:
[0099] a. aa 93-95: GNF
[0100] b. aa 128-130: SSG
[0101] c. aa 138-140: GSS.
[0102] "Sequence homology", as used herein, refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced if necessary. In contrast to sequence identity, conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence. Preferably the homolog sequence comprises at least a stretch of 50, even more preferred of 100, even more preferred of 250, even more preferred of 500 nucleotides. Upon such alignment, sequence homology is ascertained on a position-by-position basis, e.g., the sequences are "homolog" at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or amino acid residues in the reference sequence to give % sequence homology. Sequence homology can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence homology are designed to give the largest match between the sequences tested. Methods to determine sequence homology are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence homology between the given and reference sequences.
[0103] Furthermore preferred H5 proteins (1) include H5 proteins which comprise the 328K+ modification as mentioned above, and the amino acid sequence provided in TABLE 1, or any immunogenic part thereof:
TABLE-US-00002 TABLE 1 H5 antigens Basic- Amino acid positions# Sequence name sequence 36 83 86 120 155 156 189 212 223 263 223N/328K+ any HA H5 -- -- -- -- -- -- -- -- N -- 36T/223N/328K+ any HA H5 T -- -- -- -- -- -- -- N -- 36K/223N/328k+ any HA H5 K -- -- -- -- -- -- -- N -- 83A/223N/328k+ any HA H5 -- A -- -- -- -- -- -- N -- 83T/223N/328k+ any HA H5 -- T -- -- -- -- -- -- N -- 83D/223N/328k+ any HA H5 -- D -- -- -- -- -- -- N -- 86A/223N/328k+ any HA H5 -- -- A -- -- -- -- -- N -- 86V/223N/328k+ any HA H5 -- -- V -- -- -- -- -- N -- 120N/223N/328k+ any HA H5 -- -- -- N -- -- -- -- N -- 120S/223N/328k+ any HA H5 -- -- -- S -- -- -- -- N -- 155N/223N/328k+ any HA H5 -- -- -- -- N -- -- -- N -- 155S/223N/328k+ any HA H5 -- -- -- -- S -- -- -- N -- 156A/223N/328k+ any HA H5 -- -- -- -- -- A -- -- N -- 156T/223N/328k+ any HA H5 -- -- -- -- -- T -- -- N -- 189R/223N/328k+ any HA H5 -- -- -- -- -- -- R -- N -- 189K/223N/328k+ any HA H5 -- -- -- -- -- -- K -- N -- 212K/223N/328k+ any HA H5 -- -- -- -- -- -- -- K N -- 212R/223N/328k+ any HA H5 -- -- -- -- -- -- -- R N -- 212E/223N/328k+ any HA H5 -- -- -- -- -- -- -- E N -- 223N/263A/328k+ any HA H5 -- -- -- -- -- -- -- -- N A 223N/263T/328k+ any HA H5 -- -- -- -- -- -- -- -- N T 120N/155N/223N/ any HA H5 -- -- -- N N -- -- -- N -- 328k+ A/duck/China/E319- AAR99628 T A A S D A R K N A 2/03/328k+ A/duck/China/E319- AAR99628 T A A S D A R K N A 2/03_223N/328k+ A/duck/China/E319- AAR99628 T A A N D A R K N A 2/03_120N/223N/ 328k+ A/duck/China/E319- AAR99628 T A A S N A R K N A 2/03_155N/223N/ 328k+ A/duck/China/E319- AAR99628 T A A S N N R K N A 2/03_120N/155N/ 223N/328k+ HA/HK/213/03/328k+ AY518362 T A A N N A R K N A HA/Vietnam/1203/04 K T V S S T K R N T HA/Vietnam/1203/ K T V S S T K R N T 04_223N/328k+ HA/Vietnam/3046/ T A V S S T K R N T 04_223N/328k+ HA/Vietnam/3062/ T A V S S T K R N T 04_223N/328k+ HA/chicken/Vietnam/ T A V S S T K R N T 39/04_223N/328k+ HA/falcon/HK- T A A S S A K E N A D0028/04_223N/328k+ HA/duck/Singapore/ T D V S N A K E N A 3/97_223N/328k+ HA/HK/156/97/328k+ T A A S S A K E N T #the amino acid positions given in TABLE 1 refers to the positions as exemplarily defined in SEQ ID NO:2. In other words amino acid 223 of TABLE 1 refers to the amino acid 223 of the sequence of SEQ ID NO:2. -- means that the amino acids at this positions are variable as compared to the reference sequence.
[0104] Furthermore, the present invention also relates to H5 proteins (1) having at least the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted, and comprises:
[0105] i. a peptide having the sequences of NCBI Accession No. AAT65209, CAJ32556, ABC47656, CAF21874, CAF21870, AAC58998, AAC58997, AAC58996, AAC58994, AAC58993, AAC58992, AAC58991, AAC58990, AAC58995, AAS45134, AAN17270, AAN17269, AAN17268, AAN17267, AAN17266, AAN17265, AAN17264, AAN17263, AAN17262, AAN17261, AAN17260, AAN17259, AAN17257, AAN17256, AAN17255, AAN17254, AAA43083, AAA43082, AAB19079, BAE48696, BAE48693, BAE48696, BAE48695, BAE48694, BAE48692, BAE48691, BAE48690, BAE48689, BAE48688, BAE48687, BAE48686, BAE48685, BAE48684, BAE48683, AAC58999, ABC72082, AAV91149, AAP71993, AAP71992, AAP71991, AAP71990, AAP71989, AAP72011, AAP72010, AAP72009, AAP72008, AAP72007, AAP72006, AAP72005, AAP72004, AAP72003, AAP72002, AAP72001, AAP72000, AAP71999, AAP71998, AAP71997, AAP71996, AAP71995, AAP71994, AAF99718, ABF58847, AAG38534, AAC32102, AAC32099, AAL75847, AAC32101, AAC32098, AAC32088, AAC32078, AAR99628, AAC32100, AAM49555, AAL75843, AAL75839, AAD13573, AAD13568, AAF04720, AAF04719, AAC34263, AAR16155, AAD13574, AAD13570, AAD13575, AAD13572, AAD13569, AAD13567, AAD13566, AAK57506, AAG01225, AAG01215, AAG01205, AAG01195, or ABD83813 modified in a manner described above, which means that those sequences include the above-mentioned modifications 223N and 328 K+ which are not part of the wild-type sequences; or
[0106] ii. any peptide that has at least 85% sequence homology, more preferably at least about 90% sequence homology, still more preferably at least about 95% sequence homology, even more preferably at least about 97% sequence homology, still even more preferably at least about 98% sequence homology, and even more preferably at least about 99% sequence homology to the polypeptide of i) and that show hemagglutinin inhibition in a standard hemagglutinin inhibition as described above;
[0107] iii. any of the peptides of i) or ii) having the amino acids 36T, 36K, 83A, 83T, 83D, 86A, 86V, 120N, 120S, 155N, 155S, 156A, 156T, 189R, 189K, 212K, 212R, 212E, 263A, 263T, or 120N/155N; or
[0108] iv. any of such peptides of i), ii), or iii) having one or more of the following amino acid clusters selected from the group consisting of:
[0109] a. aa 93-95:GNF
[0110] b. aa 123-125 SDH
[0111] c. aa 128-130: SSG
[0112] d. aa 138-140:GSS
[0113] e. aa 226-228: MDF
[0114] f. as 270-272: EVE
[0115] g. aa 309-311: NKL; or
[0116] v. any peptide of i), ii) iii) or iv) having one or more of the following amino acid clusters selected from the group consisting of:
[0117] a. as 93-95:GNF
[0118] b. aa 128-130: SSG
[0119] c. aa 138-140:GSS
[0120] Preferably, the H5 protein (1) for use in a method of treating or preventing infections with H5N1 virus of a different clade is recombinantly expressed and/or produced by a baculovirus expression system, preferably in cultured insect cells.
[0121] The term "H5 protein (1)" as mentioned herein is thus, in particular, equivalent to the term "recombinant H5 protein" used herein.
[0122] Regarding the H5N1 virus of a different clade, as mentioned herein, said H5N1 virus of a different clade is preferably selected from the group consisting of clade 0 H5N1 virus, clade 2 H5N1 virus, clade 3 H5N1 virus, clade 4 H5N1 virus, clade 5 H5N1 virus, clade 6 H5N1 virus, clade 7 H5N1 virus, clade 8 H5N1 virus and clade 9 H5N1 virus.
[0123] In a further preferred embodiment of the invention, the H5N1 virus of a different clade is clade 2.2 H5N1 virus or a clade 2.3 H5N1 virus.
[0124] In a particular preferred embodiment of the invention, the H5N1 virus of a different clade is a clade 2.2.1 H5N1 virus or a clade 2.3.2 H5N1 virus.
[0125] For reasons of clarity, H5 protein of the H5N1 virus of a different clade is termed "H5 protein (2)" hereinafter. Hence, H5 protein (2) as mentioned herein is in particular a H5 protein coded by the genome of a H5N1 of any clade with the exception of clade 1.
[0126] In still a further preferred embodiment, the H5N1 virus of a different clade is a H5N1 virus of North African or of Vietnamese origin, wherein said H5N1 virus of North African origin is preferably a H5N1 virus comprising a H5 protein (2) of influenza virus,
wherein said H5 protein (2) has
[0127] (a) the amino acids 113D, 126H, 145(-), 156R, 160F, 167T, and 181N, wherein the modification 145(-) means that amino acid position 145 of H5 is deleted, or
[0128] (b) the amino acids 87P, 145L, 172T, 201E, 206I, 208K, 254T, 341G and 421K, or
[0129] (c) the amino acids 145L, 172T, and 254V,
[0130] and wherein the numbering of the amino acid positions of the H5 protein (2) refers to the amino acid position as exemplarily given in SEQ ID NO:8;
[0131] or wherein said H5 protein (2) consists of or comprises an amino acid sequence which is at least 95%, preferably at least 96%, more preferably at least 97%, still more preferably at least 98%, yet more preferably at least 99%, or in particular preferred 100% homolog with any one of the sequences as set forth in SEQ ID NOs: 9 to 46.
[0132] In the context of the invention, said H5 protein (2) according to (a) is a Subclade A protein, and said H5 protein according to (b) or (c) is a Subclade B protein.
[0133] Within the context of the invention, it is understood that the term "amino acid" in particular refers to an amino acid residue or, respectively, to an amino acid which has been covalently linked via peptide bonds to two further amino acids or, if the amino acid is N- or C-terminally located in the peptide sequence, to one further amino acid.
[0134] In a still more preferred embodiment of the invention, the H5N1 virus of a different clade comprises H5 protein (2) having
[0135] (a) the amino acids 87L, 113D, 126H, 145(-), 156R, 160F, 167T, and 181N, or
[0136] (b) the amino acids 87P, 113N, 126R, 145L, 160Y, 172T, 181H, 201E, 206I, 208K, 254T, 341G and 421K, or
[0137] (c) the amino acids 87L, 113N, 126R, 145L, 156G, 160Y, 172T, 181H, and 254V,
[0138] and/or wherein such H5 protein (2) comprises a peptide that comprises:
[0139] i. any one of the amino acid sequences of SEQ ID NOs: 9 to 46;
[0140] ii. any peptide that has at least 85%, preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence homology to the polypeptide of i) and that comprises hemagglutinin inhibition in a standard hemagglutinin inhibition assay; or
[0141] iii. any part of the polypeptides of i) or ii) comprising at least 334 contiguous amino acids of any of such peptides of i) or ii) and wherein any of such peptide comprises hemagglutinin inhibition in a standard hemagglutinin inhibition assay,
[0142] and/or
[0143] wherein such H5 protein (2) consists of or comprises a contiguos amino acid sequence which has at least 95% even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with any one of the sequences as set forth in SEQ ID NOs: 9 to 46.
[0144] More particular, the H5N1 virus of a different clade preferably comprises H5 protein (2) which consists of or comprises an amino acid sequence which is at least 95%, preferably at least 96%, more preferably at least 97%, still more preferably at least 98%, yet more preferably at least 99%, or in particular preferred 100% homolog with any one of the sequences as set forth in SEQ ID NOs: 15 or 20, and wherein such H5 protein (2) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:20 are in particular more preferred.
[0145] In particular, the present invention is directed to the H5 protein (1) described herein for use in a method of treating or preventing infections
[0146] (A) with Subclade A H5N1 virus of North African origin, namely an infection with a H5N1 virus comprising a H5 protein (2) having the amino acids according to (a) of claim 13 or 14 or comprising a H5 protein according to claim 16 or 17 relating to any one of the sequences as set forth in SEQ ID NOs: 9 to 19, or 42 or 43,
[0147] or
[0148] (B) with Subclade B H5N1 virus of North African origin, namely an infection with a H5N1 virus comprising a H5 protein having the amino acids according to (b) or (c) of claim 13 or 14 or comprising a H5 protein according to claim 16 or 17 relating to any one of the sequences as set forth in SEQ ID NOs: 20 to 41, or 44 to 46.
[0149] According to a further embodiment, the present invention also relates to nucleic acid molecules, which code for any of the H5 proteins (1), as described supra, for use in a method of treating or preventing infections with H5N1 virus of a different clade. Preferably, those nucleic acid molecules are RNA, DNA or copy (c)DNA molecules. Thus, the present invention relates to a nucleic acid molecule, preferably a cDNA molecule coding for a H5 protein or any modified forms of H5 protein, including any deletion, substitution and/or insertion mutant of H5 protein, wherein those H5 proteins having the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted.
[0150] According to a further embodiment, the present invention also relates to a nucleic acid molecule, preferably a cDNA molecule coding for any part of the H5 protein (1), which means encoding for any peptide-fragment which shows antigenic properties in an standard hemagglutinin inhibition assay as described supra, and having at least the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted. Normally such nucleic acid molecules, which code for an antigenic part of H5 protein, comprise 600, 540, 480, 450, 420, 390, 360, 330 or most preferably 315 contiguous nucleotides of the nucleotide sequence that codes for the H5 protein as mentioned above, modified or non-modified, and which shows antigenic properties in an standard hemagglutinin inhibition assay as described herein.
[0151] Further embodiments of antigenic parts of the H5 protein (1) are described supra. It is in the common knowledge of a person skilled in the art to construct any such nucleic acid molecules, preferably cDNA molecules which codes for the antigenic part of the H5 protein as described supra. This also include but is not limited to the construction of nucleic acid molecules, preferably of cDNA molecules, which codes for antigenic parts of the H5 protein as mentioned above including deletion mutants of H5 protein, which comprises:
[0152] i. at least 105, 90, 75, 60, 48, 45, 39, 30, 27, or most preferably 24 contiguous amino nucleotides of the nucleotide sequence that surrounds and includes the coding sequence that codes for amino acid 223N; and
[0153] ii. at least 105, 90, 75, 60, 48, 45, 39, 30, 27, or most preferably 24 contiguous amino nucleotides of the nucleotide sequence that surrounds and includes the coding sequence that codes for modification 328K+, and
[0154] iii. wherein any of such antigenic part of H5 protein show hemagglutinin inhibition in a standard hemagglutinin inhibition assay as described in Example 2.
[0155] Preferably, those surrounding nucleotides of the nucleotides, which code for amino acids 223N and/or 328K+, coding for SEQ ID NO:2 or SEQ ID NO:5.
[0156] Furthermore preferred nucleic acid molecules encoding for the H5 protein (1) according to the invention are:
[0157] i. any of those mentioned supra encoding for the amino acid 223N and the modification 328K+;
[0158] ii. any of those mentioned supra encoding for the amino acid 94N/223N and the modification 328K+;
[0159] iii. any nucleic acid molecules of avian origin encoding for the amino acid 223N, and the modification 328K+, wherein avian origin means that the H5 sequence derived from a virus isolate that was originally isolated from poultry infected with avian influenza virus type 5; or
[0160] iv. any nucleic acid molecules of avian origin encoding for the amino acids 94N/223N and the modification 328K+, wherein avian origin means that the H5 sequence derived from a virus isolate that was originally isolated from poultry infected with avian influenza virus type 5; or.
[0161] v. any nucleic acid molecules of avian origin encoding for the amino acids 155N/223N and the modification 328K+, wherein avian origin means that the H5 sequence derived from a virus isolate that was originally isolated from poultry infected with avian influenza virus type 5; or
[0162] vi. any nucleic acid molecule encoding for H5 protein of avian origin having the amino acid 120N/155N/223N and the modification 328K+, wherein avian origin means that the H5 sequence derived from a virus isolate that was originally isolated from poultry infected with avian influenza virus type 5; or
[0163] vii. any nucleic acid molecule encoding for H5 protein having the modifications 94N/223N and the modification 328K+; or
[0164] viii. any nucleic acid molecule encoding for H5 protein having the modifications 94N/155N/223N and the modification 328K+; or;
[0165] ix. any nucleic acid molecule encoding for H5 protein having the modifications 94N/120N/155N/223N and the modification 328K+; or
[0166] x. any nucleic acid molecule encoding for H5 protein having the modifications 223N, the modification 328K+, and one or more of the following amino acid clusters selected from the group consisting of:
[0167] a. aa 93-95: GNF
[0168] b. aa 123-125: SDH
[0169] c. aa 128-130: SSG
[0170] d. aa 138-140: GSS
[0171] e. as 226-228: MDF
[0172] f. aa 270-272: EVE
[0173] g. aa 309-311: NKL; or
[0174] xi. any nucleic acid molecule encoding for H5 protein having the amino acid 223N, the modification 328K+, and one or more of the following amino acid clusters selected from the group consisting of:
[0175] a. as 93-95: GNF
[0176] b. aa 128-130: SSG
[0177] c. aa 138-140: GSS; or
[0178] xii. any nucleic acid molecule encoding for H5 protein having the amino acid sequence of SEQ ID NO:5.
[0179] Furthermore preferred H5 proteins (1) as provided herewith include the H5 proteins as described by Hoffmann et al, PNAS, vol. 106, no. 36, pp. 12915-12920 of Sep. 6, 2005, wherein that H5 proteins includes one or more of the modifications as described above, at least the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted. The disclosure of this reference shall be entirely included herein by reference. Thus according to a further embodiments, the present invention also relates to any nucleic acid molecule, preferably a cDNA molecule coding for any of such proteins described by Hoffmann et al, PNAS, vol. 106, no. 36, pp. 12915-12920 of Sep. 6, 2005, wherein that H5 proteins includes one or more of the modifications as described above, at least the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted.
[0180] Methods, of how to introduce any of the above-mentioned modifications within the nucleotide sequence, including the encoding sequence of the H5 protein of an influenza virus, are well known in the art. The genomic sequence of the entire influenza virus can be modified according to the invention, for example according to the methods described in U.S. Pat. No. 6,951,754, with further references.
[0181] Furthermore, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art to modify a nucleic acid sequence coding for an antigen as described herein. Such techniques are explained fully in the literature. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1994).
[0182] According to a further embodiment, the present invention also relates to a vector that comprises any of such nucleic acid molecules as described supra. In other words, the present invention relates to a vector, that includes the coding sequence of any such H5 protein (1), or part thereof as described supra. Preferably, said vector is an expression vector, which allows the expression of any such H5 protein (1) or part thereof as described supra. Vectors according to the invention are those which are suitable for the transfection or infection of bacterial, yeast or animal cells, in vitro or in vivo.
[0183] Vectors and methods for making and/or using vectors (or recombinants) for expression can be by or analogous to the methods disclosed in: U.S. Pat. Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, 382,425, PCT publications WO 94/16716, WO 96/39491, WO 95/30018, Paoletti, "Applications of pox virus vectors to vaccination: An update, "PNAS USA 93: 11349-11353, October 1996, Moss, "Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety," PNAS USA 93: 11341-11348, October 1996, Smith et al., U.S. Pat. No. 4,745,051, (recombinant baculovirus), Richardson, C. D. (Editor), Methods in Molecular Biology 39, "Baculovirus Expression Protocols" (1995 Humana Press Inc.), Smith et al., "Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector", Molecular and Cellular Biology, December, 1983, Vol. 3, No. 12, p. 2156-2165; Pennock et al., "Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector, "Molecular and Cellular Biology March 1984, Vol. 4, No. 3, p. 399-406; EPA0 370 573, U.S. application Ser. No. 920,197, filed Oct. 16, 1986, EP Patent publication No. 265785, U.S. Pat. No. 4,769,331 (recombinant herpesvirus), Roizman, "The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors," PNAS USA 93:11307-11312, October 1996, Andreansky et al., "The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors," PNAS USA 93: 11313-11318, October 1996, Robertson et al. "Epstein-Barr virus vectors for gene delivery to B lymphocytes", PNAS USA 93: 11334-11340, October 1996, Frolov et al., "Alphavirus-based expression vectors: Strategies and applications," PNAS USA 93: 11371-11377, October 1996, Kitson et al., J. Virol. 65, 3068-3075, 1991; U.S. Pat. Nos. 5,591,439, 5,552,143, WO 98/00166, allowed U.S. application Ser. Nos. 08/675,556, and 08/675,566 both filed Jul. 3, 1996 (recombinant adenovirus), Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol. 3) p. 237-52, 1993, Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham, Tibtech 8, 85-87, April, 1990, Prevec et al., J. Gen Virol. 70,42434, PCT WO 91/11525, Feigner et al. (1994), J. Biol. Chem. 269, 2550-2561, Science, 259: 1745-49, 1993 and McClements et al., "Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease", PNAS USA 93: 11414-11420, October 1996, and U.S. Pat. Nos. 5,591,639, 5,589,466, and 5,580,859, as well as WO 90/11092, WO93/19183, WO94/21797, WO95/11307, WO95/20660, Tang et al., Nature and Furth et al. Analytical Biochemistry, relating to DNA expression vectors, inter alia. See also WO 98/33510; Ju et al., Diabetologia, 41: 736-739, 1998 (lentiviral expression system); Sanford et al., U.S. Pat. No. 4,945,050; Fischbach et al. (Intracel), WO 90/01543; Robinson et al., seminars in Immunology vol. 9, pp. 271-283 (1997), (DNA vector systems); Szoka et al., U.S. Pat. No. ______ (method of inserting DNA into living cells); McCormick et al., U.S. Pat. No. 5,677,178 (use of cytopathic viruses); and U.S. Pat. No. 5,928,913 (vectors for gene delivery), as well as other documents cited herein, each of which is incorporated by reference herein.
[0184] A viral vector, for instance, selected from pig herpes viruses, such as Aujeszky's disease virus, porcine adenovirus, poxviruses, especially vaccinia virus, avipox virus, canarypox virus, and swinepox virus, as well as DNA vectors (DNA plasmids) are advantageously employed in the practice of the invention.
Methods of Producing the H5 Proteins (1) According to the Present Invention
[0185] According to another aspect, the present invention provides methods of producing and/or recovering high amounts of recombinant H5 protein: i) by permitting infection of susceptible cells in culture with a recombinant viral vector containing H5 DNA coding sequences, wherein H5 protein is expressed by the recombinant viral vector, and ii) thereafter recovering the H5 protein from cell culture. High amounts of H5 protein means, but are not limited to, more than about 20 μg/mL cell culture, preferably more than about μg/mL, even more preferred more than about 30 μg/mL, even more preferred more than about 40 μg/mL, even more preferred more than about 50 μg/mL, even more preferred more than about 60 μg/mL, even more preferred more than about 80 μg/mL, even more preferred more than about 100 μg/mL, even more preferred than about 150 μg/mL, most preferred more than about 190 μg/mL.
[0186] According to a preferred embodiment, the H5 protein (1) is recovered by harvesting the whole (i.e. intact) SF+ cells expressing the H5 protein.
[0187] Preferred cells are those susceptible for infection with an appropriate recombinant viral vector, containing a H5 DNA and expressing the H5 protein (1). Preferably the cells are insect cells, and more preferably, they include the insect cells sold under the trademark SF+ insect cells (Protein Sciences Corporation, Meriden, Conn.). Preferred cell cultures have a cell count between about 0.3-2.0×106 cells/mL, more preferably from about 0.35-1.9×106 cells/mL, still more preferably from about 0.4-1.8×106 cells/mL, even more preferably from about 0.45-1.7×106 cells/mL, and most preferably from about 0.5-1.5×106 cells/mL.
[0188] Preferred viral vectors include baculovirus such as BaculoGold (BD Biosciences Pharmingen, San Diego, Calif.), in particular provided that the production cells are insect cells. Although the baculovirus expression system is preferred, it is understood by those of skill in the art that other expression systems will work for purposes of the present invention, namely the expression of H5 into the supernatant of a cell culture. Such other expression systems may require the use of a signal sequence in order to cause H5 expression into the media.
[0189] Appropriate growth media will also be determinable by those of skill in the art with a preferred growth media being serum-free insect cell media such as Excell 420 (JRH Biosciences, Inc., Lenexa, Kans.) and the like.
[0190] The recombinant viral vector containing the H5 DNA sequences has a preferred multiplicity of infection (MOI) of between about 0.03-1.5, more preferably from about 0.05-1.3, still more preferably from about 0.09-1.1, and most preferably from about 0.1-1.0, when used for the infection of the susceptible cells. Preferably the MOls mentioned above relates to one mL of cell culture fluid. Preferably, the method described herein comprises the infection of 0.35-1.9×106 cells/mL, still more preferably of about 0.4-1.8×106 cells/mL, even more preferably of about 0.45-1.7×106 cells/mL, and most preferably of about 0.5-1.5×106 cells/mL with a recombinant viral vector containing a H5 DNA and expressing the H5 protein having a MOI (multiplicity of infection) of between about 0.03-1.5, more preferably from about 0.05-1.3, still more preferably from about 0.09-1.1, and most preferably from about 0.1-1.0.
[0191] The infected cells are then incubated over a period of up to ten days, more preferably from about two days to about ten days, still more preferably from about four days to about nine days, and most preferably from about five days to about eight days. Preferred incubation conditions include a temperature between about 22-32° C., more preferably from about 24-30° C., still more preferably from about 25-29° C., even more preferably from about 26-28° C., and most preferably about 27° C. Preferably, the SF+ cells are observed following inoculation for characteristic baculovirus-induced changes. Such observation may include monitoring cell density trends and the decrease in viability during the post-infection period. It was found that peak viral titer is observed 3-5 days after infection and peak H5 protein expression in the cells is obtained between days 5 and 8, and/or when cell viability decreases to less than 10%.
[0192] Thus, one aspect of the present invention provides a method of producing and/or recovering recombinant H5 protein, preferably in amounts described above, by i) permitting infection of a number of susceptible cells (see above) in culture with a recombinant viral vector with a MOI as defined above, ii) expressing H5 protein by the recombinant viral vector, and iii) thereafter recovering the H5 protein from the cells obtained between days 5 and 8 after infection and/or cell viability decreases to less then 10%. Preferably, the recombinant viral vector is a recombinant baculovirus containing H5 DNA coding sequences and the cells are SF+ cells. Additionally, it is preferred that the culture be periodically examined for macroscopic and microscopic evidence of contamination or for atypical changes in cell morphology during the post-infection period. Any culture exhibiting any contamination should be discarded.
[0193] For recovery of H5 protein (1) that will be used in an immunogenic or immunological composition such as a vaccine, the inclusion of an inactivation step is preferred in order to inactivate the viral vector.
[0194] An "immunogenic or immunological composition" refers to a composition of matter that comprises at least one antigen which elicits an immunological response in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or gamma-delta T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.
[0195] As used herein, "vaccine" refers to that term as it is used by those of skill in the art. More particularly, "vaccine" refers to an immunogenic composition that, when administered to an animal in need thereof, results in a reduction in the incidence of or severity of clinical signs of influenza infection up to an including the complete prevention of such clinical signs. Preferably, the reduction in incidence or severity is at least 10%, more preferably at least 20%, still more preferably at least 30%, even more preferably at least 40%, more preferably at least 50%, still more preferably at least 60%, even more preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, and most preferably 100% in comparison to an animal or group of animals that did not receive the compositions of the present invention but that were exposed to infectious levels of influenza virus that would normally result in influenza infection resulting in exhibiting clinical signs.
[0196] Thus, the present invention also relates to a method of producing and/or recovering recombinant H5 protein, preferably in amounts described above, by i) permitting infection of a number of susceptible cells (see above) in culture with a recombinant viral vector with a MOI as defined above, ii) expressing H5 protein by the recombinant viral vector, iii) recovering the H5 expressed in cells obtained between days 5 and 8 after infection and/or cell viability decreases to less then 10%, and iv) inactivating the recombinant viral vector.
[0197] Preferably, this inactivation is done either just before or just after the filtration step, with after the filtration step being the preferred time for inactivation. Any conventional inactivation method can be used for purposes of the present invention. Thus, inactivation can be performed by chemical and/or physical treatments. In preferred forms, the volume of harvest fluids is determined and the temperature is brought to between about 32-42° C., more preferably between about 34-40° C., and most preferably between about 35-39° C. Preferred inactivation methods include the addition of cyclized binary ethylenimine (BEI), preferably in a concentration of about 1 to about 20 mM, preferably of about 2 to about 10 mM, still more preferably of about 2 to about 8 mM, still more preferably of about 3 to about 7 mM, most preferably of about 5 mM. For example the inactivation includes the addition of a solution of 2-bromoethyleneamine hydrobromide, preferably of about 0.4M, which has been cyclized to 0.2M binary ethylenimine (BEI) in 0.3N NaOH, to the fluids to give a final concentration of about 5 mM BEI. Preferably, the fluids are then stirred continuously for 72-96 hours and the inactivated harvest fluids can be stored frozen at -40° C. or below or between about 1-7° C. After inactivation is completed a sodium thiosulfate solution, preferably at 1.0M is added to neutralize any residual BEI. Preferably, the sodium thiosulfate is added in equivalent amount as compared to the BEI added prior to for inactivation. For example, in the event BEI is added to a final concentration of 5 mM, a 1.0M sodium thiosulfate solution is added to give a final minimum concentration of 5 mM to neutralize any residual BEI.
[0198] Thus, one further aspect of the present invention relates to a method of producing recombinant H5 protein, preferably in amounts described above, by i) permitting infection of a number of susceptible cells (see above) in culture with a recombinant viral vector with a MOI as defined above, ii) expressing H5 protein by the recombinant viral vector, iii) recovering the H5 expressed in the cells obtained between days 5 and 8 after infection and/or cell viability decreases to less then 10%, and iv) inactivating the recombinant viral vector. Preferably, the recombinant viral vector is a baculovirus containing H5 DNA coding sequences and the cells are SF+ cells. Preferred inactivation steps are those described above. Preferably, inactivation is performed between about 35-39° C. and in the presence of 2 to 8 mM BEI, still more preferred in the presence of about 5 mM BEI.
[0199] According to one further aspect of the present invention, the method described above also includes a neutralization step after step iv). This step v) comprises adding of an equivalent amount of an agent that neutralizes the inactivation agent within the solution. Preferably, if the inactivation agent is BEI, addition of sodium thiosulfate to an equivalent amount is preferred. Thus, according to a further aspect, step v) comprises adding of a sodium thiosulfate solution to a final concentration of about 1 to about 20 mM, preferably of about 2 to about 10 mM, still more preferably of about 2 to about 8 mM, still more preferably of about 3 to about 7 mM most preferably of about 5 mM, when the inactivation agent is BEI.
[0200] In preferred forms and especially in forms that will use the recombinant H5 protein in an immunogenic composition such as a vaccine, each lot of harvested H5 protein will be tested for inactivation by passage in the anchorage dependent, baculovirus susceptible insect cells, such as Sf9 cells. In a preferred form of this testing, 150 cm2 of appropriate cell culture monolayer is inoculated with 1.0 mL of inactivated H5 fluids and maintained at 25-29° C. for 14 days with at least two passages. At the end of the maintenance period, the cell monolayers are examined for cytopathogenic effect (CPE) typical of H5 baculovirus. Preferably, positive virus controls are also used. Such controls can consist of one culture of Sf9 cells inoculated with a non-inactivated reference H5 baculovirus and one flask of Sf9 cells that remain non-inoculated. After incubation and passage, the absence of virus-infected cells in the BEI treated viral fluids would constitute a satisfactory inactivation test. The control cells inoculated with the reference virus should exhibit CPE typical of H5 baculovirus and the non-inoculated flask should not exhibit any evidence of H5 baculovirus CPE. Alternatively, at the end of the maintenance period, the supernatant samples could be collected and inoculated onto a Sf9 96 well plate, which has been loaded with Sf9 cells, and then maintained at 25-29° C. for 5-6 days. The plate is then fixed and stained with anti-H5 antibody conjugated to FITC or any labeled antibody directed to baculovirus specific proteins (i.e. gp64). The absence of CPE, H5 expression, or expression of baculovirus specific proteins (i.e. gp64) in the BEI treated viral fluids constitutes a satisfactory inactivation test. The control cells inoculated with the reference virus should exhibit CPE and IFA activity and the non-inoculated flask should not exhibit any evidence of H5 baculovirus CPE and contain no IFA activity.
[0201] Thus a further aspect described herein, relates to an inactivation test for determining the effectiveness of the inactivation of the recombination viral vector expressing H5 protein (1), comprises the steps: i) contacting at least a portion of the culture fluid containing the recombinant viral vector with an inactivating agent, preferably as described above, ii) adding a neutralization agent to neutralize the inactivation agent, preferably as described above, and iii) determining the residual infectivity by the assays as described above.
[0202] After inactivation, the relative amount of recombinant H5 protein in a sample can be determined in a number of ways. Preferred methods of quantitation include SDS-PAGE densitometry, ELISA, and animal vaccination studies that correlate known quantities of vaccine with clinical outcomes (serology, etc.). When SDS-PAGE is utilized for quantitation, the sample material containing an unknown amount of recombinant H5 protein is run on a gel, together with samples that contain different known amounts of recombinant H5 protein. A standard curve can then be produced based on the known samples and the amount of recombinant H5 in the unknown sample can be determined by comparison with this standard curve. Because ELISAs are generally recognized as the industry standard for antigen quantitation, they are preferred for quantitation.
Vaccines Comprising H5 Proteins (1 or Nucleic Acid Molecules or Vectors Coding for Those
[0203] The invention further provides a combination of
[0204] (a) the H5 protein (1) described herein and
[0205] (b) an inactivated Newcastle disease virus for use in a method of treating or preventing infections with H5N1 virus of a different clade, in particular for use in any method of treating or preventing infections with H5N1 virus of a different clade as described herein.
[0206] Said combination is also termed "the combination described herein" hereinafter.
[0207] According to the invention it is understood that the combination described herein is preferably included in a multivalent combination vaccine or the combination described herein is in particular directed to a combined vaccination, more particular to an administration of the H5 protein (1) described herein and of the inactivted Newcastle disease virus within a maximum of 24 hours to an animal, in particular poultry, or human being in need thereof.
[0208] Preferably, the inactivated Newcastle disease virus is an inactivated whole Newcastle disease virion.
[0209] In another preferred embodiment, the inactivated Newcastle disease virus is an inactivated Newcastle disease virus obtained by inactivation of a Newcastle disease virus comprising a RNA polynucleotide having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity with a RNA copy of the polynucleotide set forth in SEQ ID NO: 51 (cDNA sequence of LaSota strain virus), which has been inactivated.
[0210] In particular, the inactivated Newcastle disease virus is an inactivated Newcastle disease LaSota strain virus.
[0211] In one preferred embodiment the inactivated Newcastle Disease Virus is a Newcastle Disease Virus which has been inactivated with a reagent selected from the group consisting of Formaldehyde, binary ethyleneimine (BEI), Beta-Propio-Lactone (BPL), and combinations thereof.
[0212] The amount of inactivated Newcastle disease virus in the combination described herein is preferably between 102 and 1010 equivalents of egg infectious doses (EID50), preferably between 106 and 109 EID50, in particular preferably between 107 and 109 EID50. The amount of the H5 protein (1) in the combination described herein is preferably the same as mentioned hereinafter.
[0213] The amount of the H5 protein (1) according to the invention is preferably between 10 and 1000 Hemagglutination units (HAU's) per dose, more preferably between 50 and 950 HAU's per dose, even more preferably between 100 and 900 HAU's per dose, even more preferably between 200 and 800 HAU's per dose, even more preferably between 300 and 700 HAU's per dose, still more preferably between 300 and 500 HAU's per dose.
[0214] According to a further aspect, the present invention relates to vaccines or pharmaceutical compositions in general, that comprises,
[0215] i. one or more of the H5 proteins (1) as described herein or the combination described herein;
[0216] ii. one or more of the nucleic acid molecules as described herein, coding for any such H5 proteins (1); and/or
[0217] iii. one or more of the vectors as described herein, including any such nucleic acid molecules and coding for any such H5 proteins (1) as described herein; and
[0218] iv. a pharmaceutical acceptable carrier and/or excipient.
[0219] The term "pharmaceutical composition" "Pharmaceutical/vaccine composition" as described herein, includes but is not limited to, vaccines for the reduction or prevention of an infection or to a composition of matter for the treatment and lessening of an infection.
[0220] The preparation of nucleic acid based vaccines, preferably cDNA vaccines, coding for influenza hemagglutinin are described for example in Deck et al, Vaccine 1997; 15(1):71-78; Ulmer et al., Science 1993; 259:1745-1749; Ulmer et al., Vaccine 1994; 12(16):1541-1544. Any of those methods can be used for the production of nucleic acid based vaccines, preferably cDNA vaccines, coding for an influenza H5 protein as described herein.
[0221] Moreover, a vaccine, which comprises H5 protein (1) or parts thereof as described herein, can be produced by conventional approaches, e.g. by recombinant expression techniques or by biochemical purification and separation techniques. Recombinant expression techniques, including the expression in insect cells are well known in the art, and described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1994). Further examples of well established recombinant expression systems are bacterial expression systems such as E. coli or B. subtilis, yeast-based expression systems such as S. cerevisiae or S. pombe, or mammalian cell expression systems such as the BHK-, CHO- and/or NSO-based expression systems. Such systems are well known in the art and generally available, e.g. commercially through Clontech Laboratories, Inc. 4030 Fabian Way, Palo Alto, Calif. 94303-4607, USA. Further expression strategies are for example described in Luschow et al., Vaccine no. 19 (2001), pp. 4249-4259, or Veit et al., PNAS vol. 103 (2006), pp. 8197-8202. Furthermore, recombinant adeno-associated virus systems are well established and for example described in U.S. Pat. No. 5,436,146 or WO200203872 with further references. Moreover, vaccinia (pox) virus based expression systems, for example as described in U.S. Pat. No. 6,265,183 with further references, are also well established and suitable to produce recombinant antigen(s), antigenic composition(s) as used according to the invention. Further suitable expression systems make use of recombinant popova viruses, such as SV40, fowl pox virus, pseudorabies viruses and retroviruses.
[0222] The relevant pharmaceutical/vaccine compositions as described herein, can also comprise inactivated virus which comprises H5 protein (1) as described herein, an apathogenic version of a live virus comprising H5 protein (1) as described herein, preparation and/or fragments of a virus, wherein said preparation and/or fragment comprise the H5 protein (1) as described herein.
[0223] The skilled person knows additional components which may be comprised in said compositions/vaccines together with antigen (see for example, Remington's Pharmaceutical Sciences. (1990). 18th ed. Mack Publ., Easton). The expert may use known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution, aqueous isotonic solutions, such as e.g. saline or corresponding plasma protein solutions, are readily available. The pharmaceutical composition/vaccine may be present as lyophylisates or dry preparations, which can be reconstituted with a known injectable solution directly before use under sterile conditions, e.g. as a kit of parts.
[0224] In addition the pharmaceutical/vaccine compositions of the present invention can include one or more veterinary-acceptable carriers. As used herein, "a veterinary-acceptable carrier" includes but is not limited to any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like.
[0225] Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others.
[0226] A preservative as used herein, refers to an anti-microbiological active agent, such as for example Gentamycin, Merthiolate, and the like. In particular adding of a preservative is most preferred for the preparation of a multi-dose composition. Those anti-microbiological active agents are added in concentrations effective to prevent the composition of interest for any microbiological contamination or for inhibition of any microbiological growth within the composition of interest.
[0227] "Adjuvants" as used herein, can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
[0228] The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopoeia type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.). John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997). Examples for suitable oil-in water emulsions are Emulsigen-based adjuvants, such as EMULSIGEN®, EMULSIGEN-D®, EMULSIGEN-P®, EMULSIGEN-75® (MVP Laboratories, Inc. Omaha, Nebr., USA). It has been surprisingly found, that pharmaceutical/vaccine compositions that comprise H5 protein, preferably recombinant H5 protein as described herein, have been effectively adjuvanted with oil-in water emulsions, preferably with such Emulsigen-based adjuvants, more preferably with EMULSIGEN® and EMULSIGEN-D®.
[0229] Moreover, it is possible to use the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.
[0230] A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars orpolyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol; (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol 974P, 934P and 971P. Most preferred is the use of Carbopol 971P. Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA (Monsanto) which are copolymers of maleic anhydride and ethylene. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.
[0231] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, or muramyl dipeptide among many others.
[0232] Preferably, the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. Even more preferred the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. Even more preferred the adjuvant is added in an amount of about 500 μg to about 5 mg per dose. Even more preferred the adjuvant is added in an amount of about 750 μg to about 2.5 mg per dose. Most preferred the adjuvant is added in an amount of about 1 mg per dose.
[0233] The pharmaceutical/vaccine compositions, can further include one or more other immunomodulatory agents such as, e.g., interleukins, interferons, or other cytokines. The pharmaceutical/vaccine compositions can also include Gentamicin and Merthiolate. While the amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan, the present invention contemplates compositions comprising from about 50 μg to about 2000 μg of adjuvant and preferably about 250 ug/1 ml dose of the vaccine composition. In another preferred embodiment, the present invention contemplates vaccine compositions comprising from about 1 ug/ml to about 60 μg/ml of antibiotics, and more preferably less than about 30 μg/ml of antibiotics.
[0234] Thus, according to a further embodiment, the present invention also relates to a pharmaceutical/vaccine composition comprising
[0235] i. a therapeutically effective amount of any one of the H5 proteins of influenza virus as described herein, wherein the H5 protein having the amino acid 223N and the modification 328K+, wherein numbering of the amino acid positions of the H5 protein refers to the amino acid position as exemplarily given in SEQ ID NO:2 and wherein the modification 328K+ means that at amino acid position 328 of H5 protein a second Lysine (K+) is inserted; and
[0236] ii. a pharmaceutically acceptable adjuvants as described above.
[0237] Preferably, the adjuvant is selected from the group consisting of:
[0238] a) EMULSIGEN®, a oil-in-water emulsion (o/w);
[0239] b) EMULSIGEN-D®, a oil-in-water (o/w) with dimethyldioctadecylammonum bromide (DDA);
[0240] c) a Polygen, a copolymer
[0241] d) EMULSIGEN-P®, a oil-in-water (o/w) with a proprietary immunostimulant
[0242] e) Carbigen is a cross-linked polymer
[0243] f) EMULSIGEN-75®, a double adjuvants comprise of a oil-in-water (o/w) with a cross-linked polymer
[0244] g) ISA 70 is a water-in-oil (w/o)
[0245] Most preferably, the adjuvants is a oil-in-water emulsion such as an emulsigen-based adjuvant selected from the group consisting of EMULSIGEN®, EMULSIGEN-D®, EMULSIGEN-P®, EMULSIGEN-75®, EMULSIGEN® and EMULSIGEN-P®. Most preferably EMULSIGEN® and EMULSIGEN-P® are used in the formulation of the current invention.
[0246] According to a further aspect, the pharmaceutical/vaccine compositions as provided herewith, comprise one or more antigen. Preferably, that further antigen is an antigen of a poultry or mammalian pathogen. According to a further embodiments, that additional antigen is an further influenza antigen such as hemagglutinin H5, H7, H9, or any other hemagglutinin of influenza virus, wherein the H5 is preferably a H5 protein of a H5N1 virus of a clade different than clade 1, in particular of a H5N1 virus of North African origin, such as the H5 protein (2) described herein. The additional antigen(s) can be added in a purified form, as part of an antigenic preparation, in the form of a killed microorganism or in the form of a modified live microorganism.
[0247] The term "antigen", as used herein means, but is not limited to, peptides, polypeptides, glycopeptides, or polysaccharides which are capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor in order to elicit, activate or stimulate an immune response directed to said antigen in a host to which said antigen is administered. The term "antigen" also refers to nucleic acid molecules, preferably DNA- or RNA-molecules, each of which codes for and express a peptide, polypeptide, or glycopeptide that is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor in order to elicit, activate or stimulate an immune response against the antigen that is coded by the nucleic acid molecule. The antigen used for the preparation of the pharmaceutical composition which is used according to the invention is a microorganism or an antigenic part and/or preparation of said microorganism. In this connection, the term "immunization", as used herein, means but is not limited to, any cause or enhancement of an immune response. The term "immune response" is already described supra.
[0248] Administration strategies for influenza vaccines are well known in the art. Mucosal vaccination strategies for inactivated and attenuated virus vaccines are contemplated. While the mucosa can be targeted by local delivery of a vaccine, various strategies have been employed to deliver immunogenic proteins to the mucosa.
[0249] In a specific embodiment, the vaccine can be administered in an admixture with, or as a conjugate or chimeric fusion protein with, cholera toxin, such as cholera toxin B or a cholera toxin A/B chimera (Hajishengallis, J Immunol., 154:4322-32, 1995; Jobling and Holmes, Infect Immun., 60:4915-24, 1992). Mucosal vaccines based on use of the cholera toxin B subunit have been described (Lebens and Holmgren, Dev Biol Stand 82:215-27, 1994). In another embodiment, an admixture with heat labile enterotoxin (LT) can be prepared for mucosal vaccination.
[0250] Other mucosal immunization strategies include encapsulating the virus in microcapsules (U.S. Pat. No. 5,075,109, U.S. Pat. No. 5,820,883, and U.S. Pat. No. 5,853,763) and using an immunopotentiating membranous carrier (WO 98/0558). Immunogenicity of orally administered immunogens can be enhanced by using red blood cells (rbc) or rbc ghosts (U.S. Pat. No. 5,643,577), or by using blue tongue antigen (U.S. Pat. No. 5,690,938).
[0251] According to another aspect, the present invention relates to a method for preparing a pharmaceutical/vaccine composition as described above, preferably a method for producing a vaccine which comprises a recombinant, baculovirus expressed H5 protein as described supra. Generally, this method includes the steps of transfecting a construct into a virus, wherein the construct comprises i) recombinant H5 cDNA as described herein, ii) infecting cells in growth media with the transfected virus, iii) causing the virus to express the recombinant H5 protein as described herein iv) recovering the expressed H5 protein from the culture v) and preparing the composition by blending the expressed H5 protein with a suitable adjuvant and/or other pharmaceutically acceptable carrier.
[0252] Preferred adjuvants are those described above. Thus according to a further aspect, the method for preparing an antigenic composition, such as for example a vaccine, for invoking an immune response against influenza infections comprises i) preparing and recovering H5 protein, and ii) admixing this with a suitable adjuvants.
[0253] In addition, the vaccine composition of the present invention can also include diluents, isotonic agents, stabilizers, an/or preservatives. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include anorganic or organic salts, e.g. sodium chloride, dextrose, mannitol, sorbitol, and lactose, saccharides, trehalose, mannitol, saccharose among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others. Suitable adjuvants, are those described above.
Medicinal Use of any of Such H5 Proteins (1), Nucleic Acid Molecules, Vectors, Vaccines, and Combinations Described Herein
[0254] The H5 proteins (1) as provided herewith, the nucleic acid molecules coding for any such H5 proteins (1), the vectors comprising any such nucleic acid molecules coding for any such H5 proteins (1) as described herein, and any pharmaceutical/vaccine composition comprising any of such H5 protein (1), nucleic acid molecule or vector or the combination described herein can be used as a medicine, preferably for the treatment and prophylaxis of infections, caused by influenza virus, most preferably by influenza A virus. The H5 proteins (1) as provided herewith, the nucleic acid molecules encoding for any such H5 proteins, the vectors comprising any such nucleic acid molecules encoding for any such H5 proteins (1) as described herein, and any pharmaceutical/vaccine composition comprising any of such H5 protein (1), nucleic acid molecule or vector, as described herein, or the combination described herein can be used for the treatment or prophylaxis of human beings as well as in veterinary medicine. When used in veterinary medicine, the treatment of poultry, preferably bird, chicken, duck, turkey and the like as well as mammals, preferably pigs, cattle, horses, seals, camels, dogs, cats, hamsters, mice and the like, is preferred.
[0255] In terms of the present invention, "prophylaxis" refers to the reduction in the incidence of or severity of clinical signs of influenza infection up to an including the complete prevention of such clinical signs. Preferably, the reduction in incidence or severity is at least 10%, more preferably at least 20%, still more preferably at least 30%, even more preferably at least 40%, more preferably at least 50%, still more preferably at least 60%, even more preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, and most preferably 100% in comparison to an animal or group of animals that did not receive the compositions of the present invention but that were exposed to infectious levels of influenza virus that would normally result in influenza infection resulting in exhibiting clinical signs.
[0256] Thus, according to another aspect the present invention relates to the use of H5 proteins (1) as provided herewith, the nucleic acid molecules encoding for any such H5 proteins (1), the vectors comprising any such nucleic acid molecules encoding for any such H5 proteins (1) as described herein and any pharmaceutical/vaccine compositions comprising any of such H5 protein (1), nucleic acid molecule or vector as described herein or the combination described herein, can be used as a medicine, preferably as a medicine for human beings and/or as veterinary medicine, preferably for poultry, in particular for chicken.
[0257] Moreover, H5 proteins (1) as provided herewith, the nucleic acid molecules coding for any such H5 proteins (1), the vectors comprising any such nucleic acid molecules coding for any such H5 protein (1), as described herein, or the combination described herein can be used for the preparation of a pharmaceutical composition, as described herein, preferably of a single-shot vaccine or a one dose vaccine, for the prophylaxis or treatment of infections caused by H5N1 virus of a clade other than clade 1, wherein said H5N1 virus of a clade other than clade 1 is preferably the H5N1 virus of a different clade as described herein. As mentioned above, those pharmaceutical compositions/vaccine compositions can be used for the treatment and/or prophylaxis of human beings as well as for the treatment and/or prophylaxis of animals, such as poultry, preferably bird, chicken, duck, turkey and the like as well as mammals, preferably pigs, cattle, horses, seals, camels, dogs, cats, hamsters, mice and the like.
[0258] According to a further aspect, the present invention also relates to a method for the treatment or prophylaxis of influenza virus infections caused by H5N1 virus of a clade other than clade 1, wherein said H5N1 virus of a clade other than clade 1 is preferably the H5N1 virus of a different clade as described herein, wherein the method comprising administration of a therapeutically effective amount of the H5 protein (1) as described herein or of the combination described herein, to a subject in need of such a treatment. Moreover, the present invention also relates to a method for the treatment or prophylaxis of influenza virus infections caused by H5N1 virus of a clade other than clade 1, wherein said H5N1 virus of a clade other than clade 1 is preferably the H5N1 virus of a different clade as described herein, wherein the method comprising administration of a therapeutically effective amount of any H5 nucleic acid molecule or vector as described herein, that codes for any H5 protein (1) as described herein, to a subject in need of such a treatment. Furthermore, the present invention also relates to a method for the treatment or prophylaxis of influenza virus infections caused by H5N1 virus of a clade other than clade 1, wherein said H5N1 virus of a clade other than clade 1 is preferably the H5N1 virus of a different clade described herein, wherein the method comprising administration of a therapeutically effective amount of the vaccine comprising any such H5 protein (1), nucleic acid molecule or vector, as described herein, to a subject in need of such a treatment. The subject in need thereof can be a human being as well as an animal, preferably poultry, even more preferably bird, chicken, duck, turkey or a mammal, preferably pig, cattle, horse, seal, camel, dog, cat, hamster, mouse and the like.
[0259] Preferably, the administration, as described herein, is a single-shot administration or a one dose administration.
[0260] Preferably, when chicken are vaccinated, the H5 protein as described herein can be used for vaccination at day 1 of age or later, e.g. at day 10, or at day 1 to 10, or at day 10 or later.
[0261] Preferably the influenza infection that can be treated by the administration of any H5 protein (1), the nucleic acid molecule or vector encoding for any such H5 protein, or any pharmaceutical/vaccine compositions as described herein, is caused by H5N1 virus of a clade other than clade 1, wherein said H5N1 virus of a clade other than clade 1 is preferably the H5N1 virus of a different clade as described herein and, as the case may be, also in combination with another avian, swine or human influenza virus or any combination or hybrid thereof.
[0262] A further advantage of the present invention is that it benefits a `DIVA` (Differentiation of Infected and Vaccinated Animals) concept with specific Elisa Kits for differentiating between vaccinated human beings or animals and human beings or animals infected with H5N1 virus.
[0263] According to another aspect, the present invention relates to a kit of parts, that comprises i) any of such H5 protein (1) as described herein, the nucleic acid molecule or vector encoding for any such H5 protein, or any pharmaceutical/vaccine composition comprising any of such H5 protein, nucleic acid molecule or vector as described herein, and ii) a package leaflet indicating the use of such H5 protein, nucleic acid molecule, vector or vaccine for the treatment or prophylaxis of infections caused by H5N1 virus of a clade other than clade 1, wherein said H5N1 virus of a clade other than clade 1 is preferably the H5N1 virus of a different clade as described herein. When chicken are vaccinated, the H5 protein (1) as described herein can be used for vaccination at day I on age or later.
[0264] It is thus understood that the kit of parts as mentioned herein is for the use, or is used, respectively, for the treatment or prophylaxis of infections caused by H5N1 virus of a clade other than clade 1, wherein said H5N1 virus of a clade other than clade 1 is preferably the H5N1 virus of a different clade as described herein.
[0265] According to a further embodiment, that kit in parts comprises at least one further antigen of a poultry or mammalian pathogen and the information indication the medicinal, human or veterinary use of that additional antigen, in particular the further antigen as mentioned above.
[0266] The invention further provides a method for reducing viral shedding in a subject, comprising administering the H5 protein (1) described herein or the combination as described herein to a subject infected with or at risk of a viral infection with H5N1 virus of a clade other than clade 1, wherein said H5N1 virus of a clade other than clade 1 is preferably the H5N1 virus of a different clade as described herein.
[0267] The invention also relates to the H5 protein (1) described herein or the combination as described herein for use in a method for reducing viral shedding in a subject, wherein said H5 protein (1) or said combination is to be administered to a subject infected with or at risk of a viral infection with H5N1 virus of a clade other than clade 1, and wherein said H5N1 virus of a clade other than clade 1 is preferably the H5N1 virus of a different clade as described herein.
[0268] Also, the invention provides the use of the H5 protein (1) described herein or of the combination as described herein for the preparation of a medicament for reducing viral shedding in a subject infected with or at risk of a viral infection with H5N1 virus of a clade other than clade 1, wherein said H5N1 virus of a clade other than clade 1 is preferably the H5N1 virus of a different clade as described herein.
[0269] Preferably, the H5 protein (1) according to the invention, the combination described herein, the vaccine as described herein or the kit mentioned herein is for use as a single-shot vaccine or in a one-dose vaccination.
EXAMPLES
[0270] The following examples set forth preferred materials and procedures in accordance with the present invention. It is to be understood, however, that these examples are provided by way of illustration only, and nothing therein should be deemed a limitation upon the overall scope of the invention.
Example 1
Construction of a Recombinant Baculoviruses Coding for and Expressing HA H5 Antigens
[0271] The recombinant baculovirus containing the H5 HA antigen was generated as follows: the coding sequences of the H5 HA (SEQ ID NO:3) was chemically synthesized and subcloned into the transfer vector pVL1392 (BD Biosciences Pharmingen, San Diego, Calif.). The H5 HA MutK+ (SEQ ID NO:5) was generated by using oligonucleotide primers and the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) and subcloned into the transfer vector pVL1392 (BD Biosciences Pharmingen, San Diego, Calif.). The pVL1392 plasmids containing the genes coding for H5 HA antigen (SEQ ID NO:3) and H5 HA MutK+ (SEQ ID NO:5) were then co-transfected with DiamondBac® (Sigma) baculovirus DNA into Sf9 insect cells (BD Biosciences Pharmingen) to generate the recombinant baculovirus containing the genes H5 HA coding for SEQ ID NO:3 and H5 HA mutK+ coding for SEQ ID NO:5. The recombinant baculoviruses containing the genes coding for H5 HA (SEQ ID NO:3) and H5 HA MutK+ (SEQ ID NO:5) were plaque-purified and Master Seed Viruses (MSVs) were propagated on the SF+ cell line, aliquoted, and stored at -70° C. Insect cells infected with H5 HA baculoviruses As described above to generate MSV or Working Seed Viruses express H5 HA antigen (SEQ ID NO:3) and H5 HA MutK+ (SEQ ID NO:5) antigen as detected by polyclonal serum or monoclonal antibodies in an indirect fluorescent antibody assay or Western blot.
[0272] After being seeded with the appropriate amounts of recombinant baculoviruses (H5 HA and H5 HA MutK+, respectively), spinner flasks containing SF+ cells (Protein Sciences, Inc., Meriden, Conn.) were then incubated at 27±2° C. for 7 days and with stirring 100 rpm during that time. The flasks used ventilated caps to allow for air flow. The crude whole cell culture containing baculovirus infected SF+ cells and the cell culture supernatents of each culture were harvested.
Example 2
Preparation of Pharmaceutical Compositions (Vaccines) Comprising HA H5 Antigens
[0273] The crude whole cell H5 HA protein and H5 HA Mutk+ protein expressed in insect cells by baculovirus-based expression system were harvested. Baculoviruses were inactivated in the presence of 5 mM cyclized binary ethylenimine (BEI) (final concentration) between about 32 and 39° C. for 72 to 96 hours. After inactivation is completed, a 0.3 M sodium thiosulfate solution was added to a final concentration of 5 mM to neutralize any residual BEI. After neutralization, various adjuvants were added and the following vaccine/pharmaceutical compositions were generated.
TABLE-US-00003 VACCINES Generic product 501 name Antigen Crude whole-cell H5 HA protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Emulsigen. Generic product 502 name Antigen Crude whole-cell H5 HA protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Emulsigen-D. Generic product 503 name Antigen Crude whole-cell H5 HA protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Polygen. Generic product 504 name Antigen Crude whole-cell H5 HA protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Emulsigen-P. Generic product 505 name Antigen Crude whole-cell H5 HA protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Carbigen. Generic product 506 name Antigen Crude whole-cell H5 HA protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Emulsigen-75. Generic product 507 name Antigen Crude whole-cell H5 HA protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with ISA 70. Generic product 508 name Antigen Crude whole-cell H5 HA mutK+ protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Emulsigen. Generic product 509 name Antigen Crude whole-cell H5 HA mutK+ protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Emulsigen-D. Generic product 510 name Antigen Crude whole-cell H5 HA mutK+ protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Polygen. Generic product 511 name Antigen Crude whole-cell H5 HA mutK+ protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Emulsigen-P. Generic product 512 name Antigen Crude whole-cell H5 HA mutK+ protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Carbigen. Generic product 513 name Antigen Crude whole-cell H5 HA mutK+ protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with Emulsigen-75. Generic product 514 name Antigen Crude whole-cell H5 HA K+ protein expressed in insect cells by a baculovirus-based expression system. Formulation An experimental vaccine comprised of cultured insect cells and supernatant expressing recombinant H5 HA. The vaccine was adjuvanted with ISA 70.
Example 3
Vaccination of Chicken Against Avian Influenza
[0274] A combination vaccine comprising H5 HA Mutk+ (Fraction 1) and inactivated Newcastle disease virus (Fraction 2), named "BACULO AI+ND KV" has been evaluated in animal trials. The vaccine was formulated with the haemmagglutinin H5 produced in the Baculovirus expression system based on the MutK+ construct (Examples 1 and 2). The origin of the Newcastle Disease (ND) virus fraction is the whole virus.
Fraction 1:
[0275] Recombinant, baculovirus-expressed, H5 hemagglutinin (H5 HA) from Avian Influenza H5N1 virus. Avian Influenza (AI) fraction.
[0276] AI fraction is inactivated with binary ethyleneimine (BEI). No residual infectivity coming from Baculovirus vector is allowed.
Fraction 2:
[0277] Whole virion, Newcastle Disease Virus (ND), LaSota Strain. Newcastle Disease fraction.
[0278] ND fraction is inactivated with Formaldehyde, BEI or Beta-Propio-Lactone (BPL). No residual infectivity coming from ND virus is allowed.
Formula Composition:
[0279] Inactivated harvest material from H5 HA protein and ND are blended into a water/oil emulsion. The mixture includes mineral oil as an adjuvant.
[0280] For evaluation of vaccine efficacy, three clinical parameters were considered: 1) Morbility/mortality. 2) Antibodies levels. 3) Viral shedding.
[0281] In all studies SPF chickens were vaccinated, administration of the vaccine was by subcutaneous route, in the back of the neck. A dose of 0.5 ml was administered unless otherwise stated.
[0282] Chickens were maintained inside isolator units during the whole duration of the studies. Studies were compliant with OIE international guidelines for evaluation of Avian Influenza vaccines.
[0283] Challenge was conducted to evaluate the Avian Influenza (AI) antigenic fraction. Chickens were inoculated 3 weeks after vaccination by the intra-nasal (50 μl) and oral (50 μl) route administering a total of 100 μl of allantoic fluid containing 106 EID50 of the challenge virus.
[0284] To evaluate protection from challenge against HPAI H5N1 two studies were conducted:
1) Protectotypes study, using a single or double vaccination (evaluating boosting effect), ages of 1 day old or 10 days old chickens (evaluating age effect), and doses of 0.5 or 0.2 ml (evaluating dose effect).
[0285] Two different challenge strains were used for this study: a) A subclade 2.3.2 Vietnamese strain (isolated in 2006) which has been recently causing disease in South-East-Asia (China, Vietnam) Poultry production. b) A subclade 2.2.1 group B1 Egyptian strain (isolated in 2010), which has been recently causing disease in Egyptian Poultry production. Challenge strains are not genetically close to the vaccine baculovirus construct (MutK+). Results are interpreted in the context of protectotypes as broadening up the protection conferred for two immunizations with similar or different vaccines.
Conclusions:
[0286] 1) Protection between 80 and 100% was observed depending on the age or dose. 100% protection was observed when administered as 0.5 ml dose at 10 days old of the bivalent formulation.
[0287] 2) When administered as a single 0.5 ml immunization of BACULO AI+ND KV at 10 days of age, the same protection is observed than administering two shots of the inactivated traditionally-produced comercial Volvac AI KV vaccine.
[0288] 3) When administered as a single 0.5 ml immunization of BACULO AI+ND KV at 10 days of age, similar level of H5-specific antibodies were detected in comparison with administering two shots of the inactivated traditionally-produced comercial Volvac AI KV vaccine.
[0289] 4) Low levels of viral shedding were observed until 3 days post-challenge, when the vaccine was administered as a single 0.5 ml immunization of BACULO AI+ND KV at 10 days of age. 2) BACULO efficacy study, using a single, unique vaccination at 10 days of age.
[0290] Three different challenge strains were used for this study: a) A subclade 2.2.1 Egyptian strain (isolated in 2008). b) A subclade 2.2.1 group A1 Egyptian strain (isolated in 2010). c) A subclade 2.2.1 group B1 Egyptian strain (isolated in 2010). The last two have been recently causing disease in Egyptian Poultry production.
Conclusions:
[0291] 1) Protection between 90 and 100% was observed.
[0292] 2) Vaccine BACULO AI+ND KV showed performance compliant with European Medicine Agency (EMA) guidelines for vaccines against HPAI virus in birds.
[0293] 3) This is the first report available demonstrating efficacy with a single shot administration for a baculovirus-based vaccine including a hemagglutinin genetically distant from those of the viruses used for challenge.
Example 4
1. Experimental Design
[0294] This experiment was designed and conducted similar to the above described Example 3:
[0295] For evaluation of vaccine efficacy, three clinical parameters were considered: 1) Morbility/mortality. 2) Antibodies levels. 3) Viral shedding.
[0296] In all studies SPF chickens were vaccinated, the administration of the vaccine was by subcutaneous route, in the back of the neck. A vaccine prototype containing a clade 1 H5 protein was used (called Mut K+) formulated as a bivalent product with a second, ND (Newcastle disease virus) antigenic fraction.
[0297] A dose of 0.5 ml was administered unless otherwise stated. Animals were vaccinate at 10 days of age.
[0298] Chickens were maintained inside isolator units during the whole duration of the studies. Studies were compliant with OIE international guidelines for evaluation of Avian Influenza vaccines.
[0299] Challenge was conducted to evaluate the Avian Influenza (AI) antigenic fraction. Chickens were inoculated 3 weeks after vaccination by the intra-nasal (50 μl) and oral (50 μl) route administering a total of 100 μl of allantoic fluid containing 106 EID50 of the challenge virus.
[0300] This is also summarized in the table (Table A) below (Vaccination was performed at 10 days of age, column 1 (ID of experimental groups according to the vaccine applied), column 2 (Vaccine dose), and column 3 (Challenge age)).
[0301] Challenge virus was A/Chicken/Egypt/1063/2010, which is classified as subclade 2.2.1.1 HP AIV H5N1 subtype. This is the official challenge strain used in Egypt for evaluation of vaccine batches. The challenge dose was 106 EID50.
2. Results & Data Analysis
[0302] Results & Data analysis are summarized in the table below (Table A): Column 4 (HI GMT (Geometric Mean Titre) 3 weeks post-vaccination, pre-challenge), column 5 (Percentage of survival, 2 weeks post-challenge), and column 6 (Detection of viral shedding, RT-PCR positive samples).
TABLE-US-00004 TABLE A Summary of the experimental design and of the results and data analysis of Example 4. Experimental group Percentage (10 Challenge GMT measured at of Viral Sheddi chickens dose 31 days of age survival Detection each) Vaccine (age) Homologous Heterologous post- viral RNA us -Vaccine Dose -Strain (vaccine (challenge challenge RT-PCR- ID- (age) 1063- strain) virus) (%) (#positives/to Mut K+ 0.5 ml 106 9.1 0.9 100 2/10 No vaccine (10 days EID50 -- -- 0 10/10 of age) (31 days of age) indicates data missing or illegible when filed
3. Conclusions
[0303] The vaccinated group survived the challenge. The vaccine prototype triggered an efficient immune response, as measured as HI titration using the homologous antigen.
[0304] The Mut K+ vaccine prototype provided good virological protection, as measured as ability to reduce viral shedding. RT-PCR Ct values were far low to represent infectious virus but only residual genetical material instead.
In the Sequence Listing (SEQ ID NOs: 1 to 51):
[0305] SEQ ID NO: 1 corresponds to H5 of A/Hong Kong/213/2003(H5N1) without signal peptide,
[0306] SEQ ID NOs: 2-7 correspond to SEQ ID NOs: 1-6 of the international (PCT) application number PCT/US2007/082699,
[0307] SEQ ID NO: 8 corresponds to H5 sequence of H5N1 "1709-6",
[0308] SEQ ID NO: 9 corresponds to H5 sequence of H5N1 "1553-1/A1",
[0309] SEQ ID NO: 10 corresponds to H5 sequence of H5N1 "1553-15/A1",
[0310] SEQ ID NO: 11 corresponds to H5 sequence of H5N1 "2095-50/A1",
[0311] SEQ ID NO: 12 corresponds to H5 sequence of H5N1 "3982-2/A1",
[0312] SEQ ID NO: 13 corresponds to H5 sequence of H5N1 "3982-5/A1",
[0313] SEQ ID NO: 14 corresponds to H5 sequence of H5N1 "3982-7/A1",
[0314] SEQ ID NO: 15 corresponds to H5 sequence of H5N1 "3982-8/A1",
[0315] SEQ ID NO: 16 corresponds to H5 sequence of H5N1 "3982-9/A1",
[0316] SEQ ID NO: 17 corresponds to H5 sequence of H5N1 "3982-12/A1",
[0317] SEQ ID NO: 18 corresponds to H5 sequence of H5N1 "3982-20/A1",
[0318] SEQ ID NO: 19 corresponds to H5 sequence of H5N1 "3982-44/A1",
[0319] SEQ ID NO: 20 corresponds to H5 sequence of H5N1 "1553-2/B1",
[0320] SEQ ID NO: 21 corresponds to H5 sequence of H5N1 "1553-6/B1",
[0321] SEQ ID NO: 22 corresponds to H5 sequence of H5N1 "1553-13/B2",
[0322] SEQ ID NO: 23 corresponds to H5 sequence of H5N1 "1553-26/B2",
[0323] SEQ ID NO: 24 corresponds to H5 sequence of H5N1 "1553-28/B1",
[0324] SEQ ID NO: 25 corresponds to H5 sequence of H5N1 "2095-39/B2",
[0325] SEQ ID NO: 26 corresponds to H5 sequence of H5N1 "2095-46/B1",
[0326] SEQ ID NO: 27 corresponds to H5 sequence of H5N1 "2095-49/B1",
[0327] SEQ ID NO: 28 corresponds to H5 sequence of H5N1 "2095-65/B1",
[0328] SEQ ID NO: 29 corresponds to H5 sequence of H5N1 "2095-68/B2",
[0329] SEQ ID NO: 30 corresponds to H5 sequence of H5N1 "2095-70/B2",
[0330] SEQ ID NO: 31 corresponds to H5 sequence of H5N1 "2095-73/B2",
[0331] SEQ ID NO: 32 corresponds to H5 sequence of H5N1 "2095-75/B2",
[0332] SEQ ID NO: 33 corresponds to H5 sequence of H5N1 "3982-3/B1",
[0333] SEQ ID NO: 34 corresponds to H5 sequence of H5N1 "3982-4/B1",
[0334] SEQ ID NO: 35 corresponds to H5 sequence of H5N1 "3982-13/B1",
[0335] SEQ ID NO: 36 corresponds to H5 sequence of H5N1 "3982-14/B2",
[0336] SEQ ID NO: 37 corresponds to H5 sequence of H5N1 "3982-19/B3",
[0337] SEQ ID NO: 38 corresponds to H5 sequence of H5N1 "3982-21/B2",
[0338] SEQ ID NO: 39 corresponds to H5 sequence of H5N1 "3982-43/B1",
[0339] SEQ ID NO: 40 corresponds to H5 sequence of H5N1 "3982-50/B1",
[0340] SEQ ID NO: 41 corresponds to H5 sequence of H5N1 "3982-52/B1",
[0341] SEQ ID NO: 42 corresponds to H5 sequence of H5N1 "3982-55/A1",
[0342] SEQ ID NO: 43 corresponds to H5 sequence of H5N1 "3982-56/A1",
[0343] SEQ ID NO: 44 corresponds to H5 sequence of H5N1 "3982-78/B2",
[0344] SEQ ID NO: 45 corresponds to H5 sequence of H5N1 "4794-17/B",
[0345] SEQ ID NO: 46 corresponds to H5 sequence of H5N1 "4794-18/B",
[0346] SEQ ID NO: 47 corresponds to H5 sequence translated from SEQ ID NO: 50,
[0347] SEQ ID NO: 48 codes for a H5 sequence of H5N1 "3982-8/A1" (SEQ ID NO: 15),
[0348] SEQ ID NO: 49 codes for a H5 sequence of H5N1 "1553-2/B1" (SEQ ID NO: 20),
[0349] SEQ ID NO: 50 corresponds to the consensus sequence obtained after analysis of the 38 H5 HA gene sequences coding for SEQ ID NOs: 9 to 46,
[0350] SEQ ID NO: 51 corresponds to the cDNA of Newcastle Disease Virus LaSota strain.
Sequence CWU
1
1
511552PRTAvian influenza virus 1Asp Gln Ile Cys Ile Gly Tyr His Ala Asn
Asn Ser Thr Glu Gln Val 1 5 10
15 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp
Ile 20 25 30 Leu
Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 35
40 45 Pro Leu Ile Leu Arg Asp
Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 50 55
60 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu
Trp Ser Tyr Ile Val 65 70 75
80 Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn
85 90 95 Asp Tyr
Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 100
105 110 Lys Ile Gln Ile Ile Pro Lys
Asn Ser Trp Ser Ser His Glu Ala Ser 115 120
125 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys
Ser Ser Phe Phe 130 135 140
Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Ala Tyr Pro Thr Ile 145
150 155 160 Lys Arg Ser
Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 165
170 175 Gly Ile His His Pro Asn Asp Ala
Ala Glu Gln Thr Arg Leu Tyr Gln 180 185
190 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu
Asn Gln Arg 195 200 205
Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Asn Gly 210
215 220 Arg Met Glu Phe
Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 225 230
235 240 Phe Glu Ser Asn Gly Asn Phe Ile Ala
Pro Glu Tyr Ala Tyr Lys Ile 245 250
255 Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Leu Glu
Tyr Gly 260 265 270
Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser
275 280 285 Met Pro Phe His
Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 290
295 300 Tyr Val Lys Ser Asn Arg Leu Val
Leu Ala Thr Gly Leu Arg Asn Ser 305 310
315 320 Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu
Phe Gly Ala Ile 325 330
335 Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
340 345 350 Gly Tyr His
His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys 355
360 365 Glu Ser Thr Gln Lys Ala Ile Asp
Gly Val Thr Asn Lys Val Asn Ser 370 375
380 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly
Arg Glu Phe 385 390 395
400 Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp
405 410 415 Gly Phe Leu Asp
Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met 420
425 430 Glu Asn Glu Arg Thr Leu Asp Phe His
Asp Ser Asn Val Lys Asn Leu 435 440
445 Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu
Leu Gly 450 455 460
Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 465
470 475 480 Ser Val Arg Asn Gly
Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala 485
490 495 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val
Lys Leu Glu Ser Ile Gly 500 505
510 Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu
Ala 515 520 525 Leu
Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 530
535 540 Ser Leu Gln Cys Arg Ile
Cys Ile 545 550 2551PRTAvian influenza virus 2Asp
Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 1
5 10 15 Asp Thr Ile Met Glu Lys
Asn Val Thr Val Thr His Ala Gln Asp Ile 20
25 30 Leu Glu Lys Thr His Asn Gly Lys Leu Cys
Asp Leu Asp Gly Val Lys 35 40
45 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu
Gly Asn 50 55 60
Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 65
70 75 80 Glu Lys Ala Asn Pro
Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 85
90 95 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser
Arg Ile Asn His Phe Glu 100 105
110 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala
Ser 115 120 125 Ser
Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Ser Ser Ser Phe Phe 130
135 140 Arg Asn Val Val Trp Leu
Ile Lys Lys Asn Asp Ala Tyr Pro Thr Ile 145 150
155 160 Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp
Leu Leu Val Leu Trp 165 170
175 Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln
180 185 190 Asn Pro
Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 195
200 205 Leu Val Pro Lys Ile Ala Thr
Arg Ser Lys Val Asn Gly Gln Ser Gly 210 215
220 Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn
Asp Ala Ile Asn 225 230 235
240 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile
245 250 255 Val Lys Lys
Gly Asp Ser Ala Ile Met Lys Ser Glu Val Glu Tyr Gly 260
265 270 Asn Cys Asn Thr Lys Cys Gln Thr
Pro Met Gly Ala Ile Asn Ser Ser 275 280
285 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu
Cys Pro Lys 290 295 300
Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 305
310 315 320 Pro Gln Arg Glu
Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala Ile Ala 325
330 335 Gly Phe Ile Glu Gly Gly Trp Gln Gly
Met Val Asp Gly Trp Tyr Gly 340 345
350 Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
Lys Glu 355 360 365
Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile 370
375 380 Ile Asp Lys Met Asn
Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn 385 390
395 400 Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn
Lys Lys Met Glu Asp Gly 405 410
415 Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met
Glu 420 425 430 Asn
Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr 435
440 445 Asp Lys Val Arg Leu Gln
Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn 450 455
460 Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn
Glu Cys Met Glu Ser 465 470 475
480 Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg
485 490 495 Leu Lys
Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr 500
505 510 Tyr Gln Ile Leu Ser Ile Tyr
Ser Thr Val Ala Ser Ser Leu Ala Leu 515 520
525 Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys
Ser Asn Gly Ser 530 535 540
Leu Gln Cys Arg Ile Cys Ile 545 550
3567PRTAvian influenza virus 3Met Glu Lys Thr Val Leu Leu Leu Ala Ile Val
Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30 Asp Thr
Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35
40 45 Leu Glu Lys Thr His Asn Gly
Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55
60 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp
Leu Leu Gly Asn 65 70 75
80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val
85 90 95 Glu Lys Ala
Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 100
105 110 Asp Tyr Glu Glu Leu Lys His Leu
Leu Ser Arg Ile Asn His Phe Glu 115 120
125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His
Glu Ala Ser 130 135 140
Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Ser Ser Ser Phe Phe 145
150 155 160 Arg Asn Val Val
Trp Leu Ile Lys Lys Asn Asp Ala Tyr Pro Thr Ile 165
170 175 Lys Arg Ser Tyr Asn Asn Thr Asn Gln
Glu Asp Leu Leu Val Leu Trp 180 185
190 Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu
Tyr Gln 195 200 205
Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 210
215 220 Leu Val Pro Lys Ile
Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225 230
235 240 Arg Met Asp Phe Phe Trp Thr Ile Leu Lys
Pro Asn Asp Ala Ile Asn 245 250
255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys
Ile 260 265 270 Val
Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Val Glu Tyr Gly 275
280 285 Asn Cys Asn Thr Lys Cys
Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295
300 Met Pro Phe His Asn Ile His Pro Leu Thr Ile
Gly Glu Cys Pro Lys 305 310 315
320 Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335 Pro Gln
Arg Glu Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala Ile Ala 340
345 350 Gly Phe Ile Glu Gly Gly Trp
Gln Gly Met Val Asp Gly Trp Tyr Gly 355 360
365 Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala
Ala Asp Lys Glu 370 375 380
Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile 385
390 395 400 Ile Asp Lys
Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn 405
410 415 Asn Leu Glu Arg Arg Ile Glu Asn
Leu Asn Lys Lys Met Glu Asp Gly 420 425
430 Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val
Leu Met Glu 435 440 445
Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr 450
455 460 Asp Lys Val Arg
Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn 465 470
475 480 Gly Cys Phe Glu Phe Tyr His Lys Cys
Asp Asn Glu Cys Met Glu Ser 485 490
495 Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu
Ala Arg 500 505 510
Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr
515 520 525 Tyr Gln Ile Leu
Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu 530
535 540 Ala Ile Met Val Ala Gly Leu Ser
Leu Trp Met Cys Ser Asn Gly Ser 545 550
555 560 Leu Gln Cys Arg Ile Cys Ile 565
4568PRTAvian influenza virus 4Met Glu Lys Ile Val Leu Leu Phe Ala
Ile Val Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu
Gln Val 20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45 Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp
Ser Tyr Ile Val 85 90
95 Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn
100 105 110 Asp Tyr Glu
Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115
120 125 Lys Ile Gln Ile Ile Pro Lys Asn
Ser Trp Ser Ser His Glu Ala Ser 130 135
140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser
Ser Phe Phe 145 150 155
160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Ala Tyr Pro Thr Ile
165 170 175 Lys Arg Ser Tyr
Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180
185 190 Gly Ile His His Pro Asn Asp Ala Ala
Glu Gln Thr Arg Leu Tyr Gln 195 200
205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn
Gln Arg 210 215 220
Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Asn Gly 225
230 235 240 Arg Met Glu Phe Phe
Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 245
250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro
Glu Tyr Ala Tyr Lys Ile 260 265
270 Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Leu Glu Tyr
Gly 275 280 285 Asn
Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290
295 300 Met Pro Phe His Asn Ile
His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310
315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser 325 330
335 Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350 Ala Gly
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355
360 365 Gly Tyr His His Ser Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375
380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser 385 390 395
400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415 Asn Asn Leu
Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420
425 430 Gly Phe Leu Asp Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu 450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465
470 475 480 Asn Gly Cys Phe
Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485
490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala 500 505
510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
Ile Gly 515 520 525
Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530
535 540 Leu Ala Ile Met Val
Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550
555 560 Ser Leu Gln Cys Arg Ile Cys Ile
565 5568PRTAvian influenza virus 5Met Glu Lys Thr Val
Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser 1 5
10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn
Asn Ser Thr Glu Gln Val 20 25
30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp
Ile 35 40 45 Leu
Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp
Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu
Trp Ser Tyr Ile Val 85 90
95 Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asp Tyr
Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115
120 125 Lys Ile Gln Ile Ile Pro Lys
Asn Ser Trp Ser Asp His Glu Ala Ser 130 135
140 Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Ser
Ser Ser Phe Phe 145 150 155
160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Ala Tyr Pro Thr Ile
165 170 175 Lys Arg Ser
Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180
185 190 Gly Ile His His Pro Asn Asp Ala
Ala Glu Gln Thr Arg Leu Tyr Gln 195 200
205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu
Asn Gln Arg 210 215 220
Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Asn Gly 225
230 235 240 Arg Met Asp Phe
Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 245
250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala
Pro Glu Tyr Ala Tyr Lys Ile 260 265
270 Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Val Glu
Tyr Gly 275 280 285
Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290
295 300 Met Pro Phe His Asn
Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310
315 320 Tyr Val Lys Ser Asn Lys Leu Val Leu Ala
Thr Gly Leu Arg Asn Ser 325 330
335 Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala
Ile 340 345 350 Ala
Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355
360 365 Gly Tyr His His Ser Asn
Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375
380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr
Asn Lys Val Asn Ser 385 390 395
400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415 Asn Asn
Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420
425 430 Gly Phe Leu Asp Val Trp Thr
Tyr Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn
Val Lys Asn Leu 450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465
470 475 480 Asn Gly Cys
Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485
490 495 Ser Val Arg Asn Gly Thr Tyr Asp
Tyr Pro Gln Tyr Ser Glu Glu Ala 500 505
510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu
Ser Ile Gly 515 520 525
Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530
535 540 Leu Ala Ile Met
Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550
555 560 Ser Leu Gln Cys Arg Ile Cys Ile
565 6263PRTAvian influenza virus 6His Ala Asn Asn
Trp Thr Glu Gln Val Asp Thr Ile Met Glu Lys Asn 1 5
10 15 Val Thr Val Thr His Ala Gln Asp Ile
Leu Glu Lys Thr His Asn Gly 20 25
30 Lys Leu Cys Asp Leu Asp Gly Val Lys Pro Leu Ile Leu Arg
Asp Cys 35 40 45
Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Ile 50
55 60 Asn Val Pro Glu Trp
Ser Tyr Ile Val Glu Lys Ala Asn Pro Ala Asn 65 70
75 80 Asp Leu Cys Tyr Pro Gly Asp Phe Asn Asp
Tyr Glu Glu Leu Lys His 85 90
95 Leu Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gln Ile Ile Pro
Lys 100 105 110 Asn
Ser Trp Ser Ser His Glu Ala Ser Leu Gly Val Ser Ser Ala Cys 115
120 125 Pro Tyr Gln Gly Lys Ser
Ser Phe Phe Arg Asn Val Val Trp Leu Ile 130 135
140 Lys Lys Asn Asn Ala Tyr Pro Thr Ile Lys Arg
Ser Tyr Asn Asn Thr 145 150 155
160 Asn Gln Glu Asp Leu Leu Val Leu Trp Gly Ile His His Pro Asn Asp
165 170 175 Ala Ala
Glu Gln Thr Arg Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser 180
185 190 Val Gly Thr Ser Thr Leu Asn
Gln Arg Leu Val Pro Lys Ile Ala Thr 195 200
205 Arg Ser Lys Val Asn Gly Gln Asn Gly Arg Met Glu
Phe Phe Trp Thr 210 215 220
Ile Leu Lys Pro Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe 225
230 235 240 Ile Ala Pro
Glu Tyr Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Ala 245
250 255 Ile Met Lys Ser Glu Leu Glu
260 7290PRTAvian influenza virus 7Gly Ser Ala Thr Met
Glu Lys Thr Val Leu Leu Leu Ala Ile Val Ser 1 5
10 15 Leu Val Lys Ser Asp Gln Ile Cys Ile Gly
Tyr His Ala Asn Asn Ser 20 25
30 Thr Glu Gln Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr
His 35 40 45 Ala
Gln Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu 50
55 60 Asp Gly Val Lys Pro Leu
Ile Leu Arg Asp Cys Ser Val Ala Gly Trp 65 70
75 80 Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Ile
Asn Val Pro Glu Trp 85 90
95 Ser Tyr Ile Val Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro
100 105 110 Gly Asn
Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile 115
120 125 Asn His Phe Glu Lys Ile Gln
Ile Ile Pro Lys Ser Ser Trp Ser Asp 130 135
140 His Glu Ala Ser Ser Gly Val Ser Ser Ala Cys Pro
Tyr Gln Gly Ser 145 150 155
160 Ser Ser Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asp Ala
165 170 175 Tyr Pro Thr
Ile Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu 180
185 190 Leu Val Leu Trp Gly Ile His His
Pro Asn Asp Ala Ala Glu Gln Thr 195 200
205 Arg Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly
Thr Ser Thr 210 215 220
Leu Asn Gln Arg Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn 225
230 235 240 Gly Gln Ser Gly
Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn 245
250 255 Asp Ala Ile Asn Phe Glu Ser Asn Gly
Asn Phe Ile Ala Pro Glu Tyr 260 265
270 Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Ala Ile Met Lys
Ser Glu 275 280 285
Val Glu 290 8562PRTH5N1 8Met Glu Lys Ile Val Leu Leu Leu Ala Ile Val
Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30 Asp Thr
Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35
40 45 Leu Glu Lys Thr His Asn Gly
Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55
60 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp
Leu Leu Gly Asn 65 70 75
80 Pro Met Cys Asp Glu Phe Leu Asn Val Ser Glu Trp Ser Tyr Ile Val
85 90 95 Glu Lys Ile
Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 100
105 110 Asn Tyr Glu Glu Leu Lys His Leu
Leu Ser Arg Ile Asn Arg Phe Glu 115 120
125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Pro Asp His
Glu Ala Ser 130 135 140
Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Gly Pro Ser Phe Tyr 145
150 155 160 Arg Asn Val Val
Trp Leu Ile Lys Lys Asp Asn Ala Tyr Pro Thr Ile 165
170 175 Lys Lys Ser Tyr His Asn Thr Asn Gln
Glu Asp Leu Leu Val Leu Trp 180 185
190 Gly Ile His His Pro Asn Asp Glu Ala Glu Gln Thr Arg Leu
Tyr Gln 195 200 205
Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 210
215 220 Leu Val Pro Lys Ile
Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225 230
235 240 Arg Val Glu Phe Phe Trp Thr Ile Leu Lys
Ser Asn Asp Ala Ile Asn 245 250
255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Asn Ala Tyr Lys
Ile 260 265 270 Val
Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly 275
280 285 Asn Cys Asn Thr Lys Cys
Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser 290 295
300 Met Pro Phe His Asn Ile His Pro Leu Thr Ile
Gly Glu Cys Pro Lys 305 310 315
320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335 Pro Gln
Gly Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile 340
345 350 Ala Gly Phe Ile Glu Gly Gly
Trp Gln Gly Met Val Asp Gly Trp Tyr 355 360
365 Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr
Ala Ala Asp Lys 370 375 380
Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser 385
390 395 400 Ile Ile Asp
Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe 405
410 415 Asn Asn Leu Glu Arg Arg Ile Glu
Asn Leu Asn Lys Lys Met Glu Asp 420 425
430 Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu
Val Leu Met 435 440 445
Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu 450
455 460 Tyr Asp Lys Val
Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465 470
475 480 Asn Gly Cys Phe Glu Phe Tyr His Arg
Cys Asp Asn Glu Cys Met Glu 485 490
495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu
Glu Ala 500 505 510
Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly
515 520 525 Thr Tyr Gln Ile
Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530
535 540 Leu Ala Ile Met Val Ala Gly Leu
Phe Leu Trp Met Cys Ser Asn Gly 545 550
555 560 Ser Leu 9567PRTH5N1 9Met Glu Lys Ile Met Leu Leu
Leu Ala Ile Val Ser Leu Val Lys Ser 1 5
10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn
Ser Thr Glu Gln Val 20 25
30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp
Ile 35 40 45 Leu
Glu Lys Thr His Asn Gly Lys Leu Cys Asn Leu Asp Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp
Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Leu Asn Val Pro Glu
Trp Ser Tyr Ile Val 85 90
95 Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Lys Phe Asn
100 105 110 Asp Tyr
Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115
120 125 Lys Ile Gln Ile Ile Pro Arg
Asn Ser Trp Ser Asp His Glu Thr Ser 130 135
140 Gly Val Ser Ser Ala Cys Gln Tyr Gln Gly Arg Ser
Ser Phe Phe Arg 145 150 155
160 Asn Val Val Trp Leu Thr Lys Lys Asp Asn Ala Tyr Ser Thr Ile Lys
165 170 175 Arg Ser Tyr
Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp Gly 180
185 190 Ile His His Pro Asn Asp Ala Ala
Glu Gln Thr Arg Leu Tyr Gln Asn 195 200
205 Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn
Gln Arg Leu 210 215 220
Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly Arg 225
230 235 240 Met Glu Phe Phe
Trp Thr Ile Leu Lys Ser Asn Asp Ala Ile Asn Phe 245
250 255 Glu Ser Asn Gly Asn Phe Ile Ala Pro
Glu Asn Ala Tyr Lys Ile Val 260 265
270 Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr
Gly Asn 275 280 285
Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser Met 290
295 300 Pro Phe His Asn Ile
His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr 305 310
315 320 Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser Pro 325 330
335 Gln Glu Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
Ala 340 345 350 Gly
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly 355
360 365 Tyr His His Ser Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu 370 375
380 Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser Ile 385 390 395
400 Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn
405 410 415 Asn Leu
Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly 420
425 430 Phe Leu Asp Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met Glu 435 440
445 Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr 450 455 460
Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn 465
470 475 480 Gly Cys Phe
Glu Phe Tyr His Arg Cys Asp Asn Glu Cys Met Glu Ser 485
490 495 Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala Arg 500 505
510 Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
Ile Gly Thr 515 520 525
Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu 530
535 540 Ala Ile Met Val
Ala Gly Leu Phe Leu Trp Met Cys Ser Asn Gly Ser 545 550
555 560 Leu Gln Cys Arg Ile Cys Ile
565 10567PRTH5N1 10Met Glu Lys Ile Val Leu Leu Leu Ala
Ile Val Ser Ile Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu
Gln Val 20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45 Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asn Leu Asp Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Leu Asn Val Pro Glu Trp
Ser Tyr Ile Val 85 90
95 Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asp Tyr Glu
Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115
120 125 Lys Ile Gln Ile Ile Pro Lys Gly
Ser Trp Ser Asp His Glu Ala Ser 130 135
140 Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Arg Ser Ser
Phe Phe Arg 145 150 155
160 Asn Val Val Trp Leu Thr Lys Lys Asn Asn Ala Tyr Pro Thr Ile Lys
165 170 175 Lys Ser Tyr Asn
Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp Gly 180
185 190 Ile His His Pro Asn Asp Ala Ala Glu
Gln Thr Arg Leu Tyr Gln Asn 195 200
205 Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln
Arg Leu 210 215 220
Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly Arg 225
230 235 240 Met Glu Phe Phe Trp
Thr Ile Leu Lys Ser Asn Asp Ala Ile Asn Phe 245
250 255 Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu
Asn Ala Tyr Lys Ile Val 260 265
270 Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly
Asn 275 280 285 Cys
Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser Met 290
295 300 Pro Phe His Asn Ile His
Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr 305 310
315 320 Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly
Leu Arg Asn Ser Pro 325 330
335 Gln Gly Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala
340 345 350 Gly Phe
Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly 355
360 365 Tyr His His Ser Asn Glu Gln
Gly Ser Gly Tyr Ala Ala Asp Lys Glu 370 375
380 Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys
Val Asn Ser Ile 385 390 395
400 Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn
405 410 415 Asn Leu Glu
Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly 420
425 430 Phe Leu Asp Val Trp Thr Tyr Asn
Ala Glu Leu Leu Val Leu Met Glu 435 440
445 Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys
Asn Leu Tyr 450 455 460
Asp Arg Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn 465
470 475 480 Gly Cys Phe Glu
Phe Tyr His Arg Cys Asp Asn Glu Cys Met Glu Ser 485
490 495 Val Arg Asn Gly Thr Tyr Asp Tyr Pro
Gln Tyr Ser Glu Glu Ala Arg 500 505
510 Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile
Gly Thr 515 520 525
Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu 530
535 540 Ala Ile Met Val Ala
Gly Leu Phe Leu Trp Met Cys Ser Asn Gly Ser 545 550
555 560 Leu Gln Cys Arg Ile Cys Ile
565 11560PRTH5N1misc_feature(560)..(560)Xaa can be any
naturally occurring amino acid 11Met Glu Lys Ile Val Leu Leu Leu Ala Ile
Val Ser Leu Val Lys Gly 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln
Val 20 25 30 Asp
Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35
40 45 Leu Glu Lys Thr His Asn
Gly Lys Leu Cys Asn Leu Asp Gly Val Lys 50 55
60 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly
Trp Leu Leu Gly Asn 65 70 75
80 Pro Met Cys Asp Glu Phe Leu Asn Val Pro Glu Trp Ser Tyr Ile Val
85 90 95 Glu Lys
Ile Tyr Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 100
105 110 Asp Tyr Glu Glu Leu Lys His
Leu Leu Ser Arg Ile Asn His Phe Glu 115 120
125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp
His Glu Ala Ser 130 135 140
Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Arg Ser Ser Phe Phe Arg 145
150 155 160 Asn Val Val
Trp Leu Thr Lys Lys Asp Asn Ala Tyr Pro Thr Ile Lys 165
170 175 Lys Ser Tyr Asn Asn Thr Asn Gln
Glu Asp Leu Leu Ile Leu Trp Gly 180 185
190 Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu
Tyr Gln Asn 195 200 205
Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg Leu 210
215 220 Val Pro Lys Ile
Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly Arg 225 230
235 240 Met Glu Phe Phe Trp Thr Ile Leu Lys
Ser Asn Asp Ala Ile Asn Phe 245 250
255 Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Asn Ala Tyr Lys
Ile Val 260 265 270
Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn
275 280 285 Cys Asn Thr Lys
Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser Met 290
295 300 Pro Phe His Asn Ile His Pro Leu
Thr Ile Gly Glu Cys Pro Lys Tyr 305 310
315 320 Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu
Arg Asn Ser Pro 325 330
335 Gln Gly Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala
340 345 350 Gly Phe Ile
Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly 355
360 365 Tyr His His Ser Asn Glu Gln Gly
Ser Gly Tyr Ala Ala Asp Lys Glu 370 375
380 Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val
Asn Ser Ile 385 390 395
400 Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn
405 410 415 Asn Leu Glu Arg
Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly 420
425 430 Phe Leu Asp Val Trp Thr Tyr Asn Ala
Glu Leu Leu Val Leu Met Glu 435 440
445 Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
Leu Tyr 450 455 460
Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn 465
470 475 480 Gly Cys Phe Glu Phe
Tyr His Arg Cys Asp Asn Glu Cys Met Glu Ser 485
490 495 Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln
Tyr Ser Glu Glu Ala Arg 500 505
510 Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly
Thr 515 520 525 Tyr
Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu 530
535 540 Ala Ile Met Val Ala Gly
Leu Phe Leu Trp Met Cys Ser Asn Gly Xaa 545 550
555 560 12544PRTH5N1misc_feature(1)..(1)Xaa can be
any naturally occurring amino acid 12Xaa Leu Leu Ala Ile Val Ser Ile Val
Lys Ser Asp Gln Ile Cys Ile 1 5 10
15 Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile
Met Glu 20 25 30
Lys Asn Val Thr Val Thr His Ala Gln Asp Ile Leu Glu Lys Thr His
35 40 45 Asn Gly Lys Leu
Cys Asn Leu Asp Gly Val Lys Pro Leu Ile Leu Arg 50
55 60 Asp Cys Ser Val Ala Gly Trp Leu
Leu Gly Asn Pro Met Cys Asp Glu 65 70
75 80 Phe Leu Asp Val Pro Glu Trp Ser Tyr Ile Val Glu
Lys Ile Asn Pro 85 90
95 Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn Asp Tyr Glu Glu Leu
100 105 110 Lys His Leu
Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gln Ile Ile 115
120 125 Pro Lys Asn Ser Trp Ser Asp His
Glu Thr Ser Gly Val Ser Ser Ala 130 135
140 Cys Pro Tyr Gln Gly Arg Ser Ser Phe Phe Arg Asn Val
Val Trp Leu 145 150 155
160 Thr Lys Lys Asn Thr Ala Tyr Pro Thr Ile Lys Lys Ser Tyr Asn Asn
165 170 175 Thr Asn Gln Glu
Asp Leu Leu Val Leu Trp Gly Ile His His Pro Asn 180
185 190 Asp Ala Ala Glu Gln Thr Arg Leu Tyr
Gln Asn Pro Thr Thr Tyr Ile 195 200
205 Ser Val Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro Lys
Ile Ala 210 215 220
Thr Arg Ser Lys Val Asn Gly Gln Ser Gly Arg Met Glu Phe Phe Trp 225
230 235 240 Thr Ile Leu Lys Ser
Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn 245
250 255 Phe Ile Ala Pro Glu Asn Ala Tyr Lys Ile
Val Lys Lys Gly Asp Ser 260 265
270 Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys
Cys 275 280 285 Gln
Thr Pro Ile Gly Ala Ile Asn Ser Ser Met Pro Phe His Asn Ile 290
295 300 His Pro Leu Thr Ile Gly
Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg 305 310
315 320 Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro
His Gly Glu Arg Arg 325 330
335 Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
340 345 350 Gly Trp
Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn 355
360 365 Glu Gln Gly Ser Gly Tyr Ala
Ala Asp Lys Glu Ser Thr Gln Lys Ala 370 375
380 Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile
Asp Lys Met Asn 385 390 395
400 Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Arg Arg
405 410 415 Ile Glu Asn
Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp 420
425 430 Thr Tyr Asn Ala Glu Leu Leu Val
Leu Met Glu Asn Glu Arg Thr Leu 435 440
445 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys
Val Arg Leu 450 455 460
Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe 465
470 475 480 Tyr His Arg Cys
Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr 485
490 495 Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu
Ala Arg Leu Lys Arg Glu Glu 500 505
510 Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln Ile
Leu Ser 515 520 525
Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met Val Xaa 530
535 540
13534PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 13Xaa Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser 1
5 10 15 Thr Glu Gln
Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His 20
25 30 Ala Gln Asp Ile Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asn Leu 35 40
45 Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val
Ala Gly Trp 50 55 60
Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Leu Asn Val Glu Trp Ser 65
70 75 80 Tyr Ile Val Glu
Lys Ile Asn Pro Thr Asn Asp Leu Cys Tyr Pro Gly 85
90 95 Asn Phe Asn Asp Tyr Glu Glu Leu Lys
His Leu Leu Ser Arg Ile Asn 100 105
110 His Phe Glu Lys Ile Gln Ile Ile Pro Lys Asn Tyr Trp Ser
Asp His 115 120 125
Glu Thr Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Arg Pro Ser 130
135 140 Phe Phe Arg Asn Val
Val Trp Leu Thr Lys Lys Asn Asn Ala Tyr Pro 145 150
155 160 Thr Ile Lys Lys Ser Tyr Asn Asn Thr Asn
Gln Glu Asp Leu Leu Val 165 170
175 Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg
Leu 180 185 190 Tyr
Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn 195
200 205 Gln Arg Leu Val Pro Lys
Ile Ala Thr Arg Ser Lys Val Asn Gly Gln 210 215
220 Ser Gly Arg Met Glu Phe Phe Trp Thr Ile Leu
Lys Ser Asn Asp Ala 225 230 235
240 Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Asn Ala Tyr
245 250 255 Lys Ile
Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu 260
265 270 Tyr Gly Asn Cys Asn Thr Lys
Cys Gln Thr Pro Ile Gly Ala Ile Asn 275 280
285 Ser Ser Met Pro Phe His Asn Ile His Pro Leu Thr
Ile Gly Glu Cys 290 295 300
Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg 305
310 315 320 Asn Ser Pro
Gln Gly Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly 325
330 335 Ala Ile Ala Gly Phe Ile Glu Gly
Gly Trp Gln Gly Met Val Asp Gly 340 345
350 Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly
Tyr Ala Ala 355 360 365
Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val 370
375 380 Asn Ser Ile Ile
Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg 385 390
395 400 Glu Phe Asn Asn Leu Glu Arg Arg Ile
Glu Asn Leu Asn Lys Lys Met 405 410
415 Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu
Leu Val 420 425 430
Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys
435 440 445 Asn Leu Tyr Asp
Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu 450
455 460 Leu Gly Asn Gly Cys Phe Glu Phe
Tyr His Arg Cys Asp Asn Glu Cys 465 470
475 480 Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro
Gln Tyr Ser Glu 485 490
495 Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
500 505 510 Ile Gly Thr
Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser 515
520 525 Leu Ala Leu Ala Ile Met 530
14548PRTH5N1misc_feature(1)..(1)Xaa can be any naturally
occurring amino acid 14Xaa Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser 1 5 10
15 Thr Glu Gln Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His
20 25 30 Ala Gln Asp
Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asn Leu 35
40 45 Asp Gly Val Lys Pro Leu Ile Leu
Arg Asp Cys Ser Val Ala Gly Trp 50 55
60 Leu Leu Gly Asn Pro Met Cys Asp Lys Phe Leu Asn Val
Pro Glu Trp 65 70 75
80 Ser Tyr Ile Val Glu Lys Ile Asn Pro Thr Asn Asp Leu Cys Tyr Pro
85 90 95 Gly Asn Phe Asn
Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile 100
105 110 Asn His Phe Glu Lys Ile Gln Ile Ile
Pro Lys Asn Ser Trp Ser Asp 115 120
125 His Glu Ala Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly
Arg Ser 130 135 140
Ser Phe Phe Arg Asn Val Val Trp Leu Thr Lys Lys Asn Asn Ala Tyr 145
150 155 160 Pro Thr Ile Lys Lys
Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu 165
170 175 Val Leu Trp Gly Ile His His Pro Asn Asp
Ala Ala Glu Gln Thr Arg 180 185
190 Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr
Leu 195 200 205 Asn
Gln Arg Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly 210
215 220 Gln Ser Gly Arg Met Glu
Phe Phe Trp Thr Ile Leu Lys Ser Asn Asp 225 230
235 240 Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile
Ala Pro Glu Asn Ala 245 250
255 Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu
260 265 270 Glu Tyr
Gly Asp Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile 275
280 285 Asn Ser Ser Met Pro Phe His
Asn Ile His Pro Leu Thr Ile Gly Glu 290 295
300 Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu
Ala Thr Gly Leu 305 310 315
320 Arg Asn Ser Pro Gln Gly Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe
325 330 335 Gly Ala Ile
Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp 340
345 350 Gly Trp Tyr Gly Tyr His His Ser
Asn Glu Gln Gly Ser Gly Tyr Ala 355 360
365 Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val
Thr Asn Lys 370 375 380
Val Asn Ser Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly 385
390 395 400 Arg Glu Phe Asn
Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys 405
410 415 Met Glu Asp Gly Phe Leu Asp Val Trp
Thr Tyr Asn Ala Glu Leu Leu 420 425
430 Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser
Asn Val 435 440 445
Lys Asn Leu Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys 450
455 460 Glu Leu Gly Asn Gly
Cys Phe Glu Phe Tyr His Arg Cys Asp Asn Glu 465 470
475 480 Cys Met Glu Ser Val Arg Asn Gly Thr Tyr
Asp Tyr Pro Gln Tyr Ser 485 490
495 Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu
Glu 500 505 510 Ser
Ile Gly Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser 515
520 525 Ser Leu Ala Leu Ala Ile
Met Val Ala Gly Leu Phe Leu Trp Met Cys 530 535
540 Ser Asn Gly Xaa 545
15541PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 15Xaa Ile Val Ser Ile Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His 1
5 10 15 Ala Asn Asn
Ser Thr Glu Gln Val Asp Thr Ile Met Glu Lys Asn Val 20
25 30 Thr Val Thr His Ala Gln Asp Ile
Leu Glu Lys Thr His Asn Gly Lys 35 40
45 Leu Cys Asn Leu Asp Gly Val Lys Pro Leu Ile Leu Arg
Asp Cys Ser 50 55 60
Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Leu Asn 65
70 75 80 Val Pro Glu Trp
Ser Tyr Ile Val Glu Lys Ile Asn Pro Thr Asn Asp 85
90 95 Leu Cys Tyr Pro Gly Asn Phe Asn Asp
Tyr Glu Glu Leu Lys His Leu 100 105
110 Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gln Ile Ile Pro
Lys Asn 115 120 125
Ser Trp Ser Asp His Glu Ala Ser Gly Val Ser Ser Ala Cys Pro Tyr 130
135 140 Gln Gly Arg Ser Ser
Phe Phe Arg Asn Val Val Trp Leu Thr Lys Lys 145 150
155 160 Asn Asn Ala Tyr Pro Thr Ile Lys Lys Ser
Tyr Asn Asn Thr Asn Gln 165 170
175 Glu Asp Leu Leu Val Leu Trp Gly Ile His His Pro Asn Asp Ala
Ala 180 185 190 Glu
Gln Thr Arg Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly 195
200 205 Thr Ser Thr Leu Asn Gln
Arg Leu Val Pro Lys Ile Ala Thr Arg Ser 210 215
220 Lys Val Asn Gly Gln Ser Gly Arg Met Glu Phe
Phe Trp Thr Ile Leu 225 230 235
240 Lys Ser Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala
245 250 255 Pro Glu
Asn Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met 260
265 270 Lys Ser Glu Leu Glu Tyr Gly
Asp Cys Asn Thr Lys Cys Gln Thr Pro 275 280
285 Ile Gly Ala Ile Asn Ser Ser Met Pro Phe His Asn
Ile His Pro Leu 290 295 300
Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu 305
310 315 320 Ala Thr Gly
Leu Arg Asn Ser Pro Gln Gly Glu Arg Arg Arg Lys Lys 325
330 335 Arg Gly Leu Phe Gly Ala Ile Ala
Gly Phe Ile Glu Gly Gly Trp Gln 340 345
350 Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn
Glu Gln Gly 355 360 365
Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly 370
375 380 Val Thr Asn Lys
Val Asn Ser Ile Ile Asp Lys Met Asn Thr Gln Phe 385 390
395 400 Glu Ala Val Gly Arg Glu Phe Asn Asn
Leu Glu Arg Arg Ile Glu Asn 405 410
415 Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr
Tyr Asn 420 425 430
Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His
435 440 445 Asp Ser Asn Val
Lys Asn Leu Tyr Asp Lys Val Arg Leu Gln Leu Arg 450
455 460 Asp Asn Ala Lys Glu Leu Gly Asn
Gly Cys Phe Glu Phe Tyr His Arg 465 470
475 480 Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly
Thr Tyr Asp Tyr 485 490
495 Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly
500 505 510 Val Lys Leu
Glu Ser Ile Gly Thr Tyr Gln Ile Leu Ser Ile Tyr Ser 515
520 525 Thr Val Ala Ser Ser Leu Ala Leu
Ala Ile Met Val Xaa 530 535 540
16520PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 16Xaa Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser 1
5 10 15 Thr Glu Gln
Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His 20
25 30 Ala Gln Asp Ile Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asn Leu 35 40
45 Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val
Ala Gly Trp 50 55 60
Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Leu Asn Val Pro Glu Trp 65
70 75 80 Ser Tyr Ile Val
Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro 85
90 95 Gly Asn Phe Asn Asp Tyr Glu Glu Leu
Lys His Leu Leu Ser Arg Ile 100 105
110 Asn His Phe Glu Lys Ile Gln Ile Ile Pro Lys Asn Ser Trp
Ser Asp 115 120 125
His Glu Ala Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Arg Ser 130
135 140 Ser Phe Phe Arg Asn
Val Val Trp Leu Thr Lys Lys Asn Asn Xaa Tyr 145 150
155 160 Pro Thr Ile Lys Lys Ser Tyr Asn Asn Thr
Asn Gln Glu Asp Leu Leu 165 170
175 Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr
Lys 180 185 190 Leu
Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu 195
200 205 Asn Gln Arg Leu Val Pro
Lys Ile Ala Thr Arg Ser Lys Val Asn Gly 210 215
220 Gln Ser Gly Arg Met Glu Phe Phe Trp Thr Ile
Leu Lys Ser Asn Asp 225 230 235
240 Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Asn Ala
245 250 255 Tyr Lys
Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu 260
265 270 Glu Tyr Gly Asn Cys Asn Thr
Lys Cys Gln Thr Pro Ile Gly Ala Ile 275 280
285 Asn Ser Ser Met Pro Phe His Asn Ile His Pro Leu
Thr Ile Gly Glu 290 295 300
Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu 305
310 315 320 Arg Asn Ser
Pro Gln Gly Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe 325
330 335 Gly Ala Ile Ala Gly Phe Ile Glu
Gly Gly Trp Gln Gly Met Val Asp 340 345
350 Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly Ser
Gly Tyr Ala 355 360 365
Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys 370
375 380 Val Asn Ser Ile
Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly 385 390
395 400 Arg Glu Phe Asn Asn Leu Glu Arg Arg
Ile Glu Asn Leu Asn Lys Lys 405 410
415 Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu
Leu Leu 420 425 430
Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
435 440 445 Lys Asn Leu Tyr
Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys 450
455 460 Glu Leu Gly Asn Gly Cys Phe Glu
Phe Tyr His Arg Cys Asp Asn Glu 465 470
475 480 Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser 485 490
495 Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu
500 505 510 Ser Ile Gly
Thr Tyr Gln Ile Leu 515 520
17537PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 17Xaa Ser Ile Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His Ala Asn 1
5 10 15 Asn Ser Thr
Glu Gln Val Asp Thr Ile Met Glu Lys Asn Val Thr Val 20
25 30 Thr His Ala Gln Asp Ile Leu Glu
Lys Thr His Asn Gly Lys Leu Cys 35 40
45 Asn Leu Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys
Ser Val Ala 50 55 60
Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Leu Asn Val Pro 65
70 75 80 Glu Trp Ser Tyr
Ile Val Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys 85
90 95 Tyr Pro Gly Asn Phe Asn Asp Tyr Glu
Glu Leu Lys His Leu Leu Ser 100 105
110 Arg Ile Asn His Phe Glu Lys Ile Gln Ile Ile Pro Lys Asn
Ser Trp 115 120 125
Ser Asp His Glu Ala Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly 130
135 140 Arg Ser Ser Phe Phe
Arg Asn Val Val Trp Leu Thr Lys Lys Asn Asn 145 150
155 160 Ala Tyr Pro Thr Ile Lys Lys Ser Tyr Asn
Asn Thr Asn Gln Glu Asp 165 170
175 Leu Leu Val Leu Trp Gly Ile His His Pro Asn Asp Glu Ala Glu
Gln 180 185 190 Thr
Arg Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser 195
200 205 Thr Leu Asn Gln Arg Leu
Val Pro Lys Ile Ala Thr Arg Ser Lys Val 210 215
220 Asn Gly Gln Ser Gly Arg Met Glu Phe Phe Trp
Thr Ile Leu Lys Ser 225 230 235
240 Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu
245 250 255 Asn Ala
Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser 260
265 270 Glu Leu Glu Tyr Gly Asn Cys
Asn Thr Lys Cys Gln Thr Pro Ile Gly 275 280
285 Ala Ile Asn Ser Ser Met Pro Phe His Asn Ile His
Pro Leu Thr Ile 290 295 300
Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr 305
310 315 320 Gly Leu Arg
Asn Ser Pro Gln Gly Glu Arg Arg Arg Lys Lys Arg Gly 325
330 335 Leu Phe Gly Ala Ile Ala Gly Phe
Ile Glu Gly Gly Trp Gln Gly Met 340 345
350 Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gln
Gly Ser Gly 355 360 365
Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr 370
375 380 Asn Lys Val Asn
Ser Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala 385 390
395 400 Val Gly Arg Glu Phe Asn Asn Leu Glu
Arg Arg Ile Glu Asn Leu Asn 405 410
415 Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn
Ala Glu 420 425 430
Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser
435 440 445 Asn Val Lys Asn
Leu Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn 450
455 460 Ala Lys Glu Leu Gly Asn Gly Cys
Phe Glu Phe Tyr His Arg Cys Asp 465 470
475 480 Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr
Asp Tyr Pro Gln 485 490
495 Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys
500 505 510 Leu Glu Ser
Ile Gly Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val 515
520 525 Ala Ser Ser Leu Ala Leu Ala Ile
Xaa 530 535
18528PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 18Xaa Ile Val Ser Ile Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His 1
5 10 15 Ala Asn Asn
Ser Thr Glu Gln Val Asp Thr Ile Met Glu Lys Asn Val 20
25 30 Thr Val Thr His Ala Gln Asp Ile
Leu Glu Lys Thr His Asn Gly Lys 35 40
45 Leu Cys Asn Leu Asp Gly Val Lys Pro Leu Ile Leu Arg
Asp Cys Ser 50 55 60
Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Leu Asn 65
70 75 80 Val Pro Glu Trp
Ser Tyr Ile Val Glu Lys Ile Asn Pro Ala Asn Asp 85
90 95 Leu Cys Tyr Pro Gly Asn Phe Asn Asp
Tyr Glu Glu Leu Lys His Leu 100 105
110 Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gln Ile Ile Pro
Lys Asn 115 120 125
Ser Trp Ser Asp His Glu Ala Ser Gly Val Ser Ser Ala Cys Pro Tyr 130
135 140 Gln Gly Arg Ser Ser
Phe Phe Arg Asn Val Val Trp Leu Thr Lys Lys 145 150
155 160 Asn Asn Ala Tyr Pro Thr Ile Lys Lys Ser
Tyr Asn Asn Thr Asn Gln 165 170
175 Glu Asp Leu Leu Val Leu Trp Gly Ile His His Pro Asn Asp Ala
Ala 180 185 190 Glu
Gln Thr Arg Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly 195
200 205 Thr Ser Thr Leu Asn Gln
Arg Leu Val Pro Lys Ile Ala Asn Arg Ser 210 215
220 Lys Val Asn Gly Gln Ser Gly Arg Met Glu Phe
Phe Trp Thr Ile Leu 225 230 235
240 Lys Ser Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala
245 250 255 Pro Glu
Asn Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met 260
265 270 Lys Ser Glu Leu Glu Tyr Gly
Asn Cys Asn Thr Lys Cys Gln Thr Pro 275 280
285 Ile Gly Ala Ile Asn Ser Ser Met Pro Phe His Asn
Ile His Pro Leu 290 295 300
Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu 305
310 315 320 Ala Thr Gly
Leu Arg Asn Ser Pro Gln Gly Glu Arg Arg Arg Lys Lys 325
330 335 Arg Gly Leu Phe Gly Ala Ile Ala
Gly Phe Ile Glu Gly Gly Trp Gln 340 345
350 Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn
Glu Gln Gly 355 360 365
Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly 370
375 380 Val Thr Asn Lys
Val Asn Ser Ile Ile Asp Lys Met Asn Thr Gln Phe 385 390
395 400 Glu Ala Val Gly Arg Glu Phe Asn Asn
Leu Glu Arg Arg Ile Glu Asn 405 410
415 Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr
Tyr Asn 420 425 430
Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His
435 440 445 Asp Ser Asn Val
Lys Asn Leu Tyr Asp Lys Val Arg Leu Gln Leu Arg 450
455 460 Asp Asn Ala Lys Glu Leu Gly Asn
Gly Cys Phe Glu Phe Tyr His Arg 465 470
475 480 Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly
Thr Tyr Asp Tyr 485 490
495 Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly
500 505 510 Val Lys Leu
Glu Ser Ile Gly Thr Tyr Gln Ile Leu Ser Ile Tyr Xaa 515
520 525
19535PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 19Xaa Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser 1
5 10 15 Thr Glu Gln
Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His 20
25 30 Ala Gln Asp Ile Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asn Leu 35 40
45 Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val
Ala Gly Trp 50 55 60
Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Leu Asn Val Pro Glu Trp 65
70 75 80 Ser Tyr Ile Val
Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro 85
90 95 Gly Asn Phe Asn Asp Tyr Glu Glu Leu
Lys His Leu Leu Ser Arg Ile 100 105
110 Asn His Phe Glu Lys Ile Gln Ile Ile Pro Lys Asn Ser Trp
Ser Asp 115 120 125
His Glu Ala Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Arg Ser 130
135 140 Ser Phe Phe Arg Asn
Val Val Trp Leu Thr Lys Lys Asp Asn Ala Tyr 145 150
155 160 Pro Thr Ile Lys Arg Ser Tyr Asn Asn Thr
Asn Gln Glu Asp Leu Leu 165 170
175 Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr
Arg 180 185 190 Leu
Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu 195
200 205 Asn Gln Arg Leu Val Pro
Lys Ile Ala Thr Arg Ser Lys Val Asn Gly 210 215
220 Gln Ser Gly Arg Met Glu Phe Phe Trp Thr Ile
Leu Lys Ser Asn Asp 225 230 235
240 Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Asn Ala
245 250 255 Tyr Lys
Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu 260
265 270 Glu Tyr Gly Asn Cys Asn Thr
Lys Cys Gln Thr Pro Ile Gly Ala Ile 275 280
285 Asn Ser Ser Met Pro Phe His Asn Ile His Pro Leu
Thr Ile Gly Glu 290 295 300
Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu 305
310 315 320 Arg Asn Ser
Pro Gln Gly Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe 325
330 335 Gly Ala Ile Ala Gly Phe Ile Glu
Gly Gly Trp Gln Gly Met Val Asp 340 345
350 Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly Ser
Gly Tyr Ala 355 360 365
Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys 370
375 380 Val Asn Ser Ile
Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly 385 390
395 400 Arg Glu Phe Asn Asn Leu Glu Arg Arg
Ile Glu Asn Leu Asn Lys Lys 405 410
415 Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu
Leu Leu 420 425 430
Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
435 440 445 Lys Asn Leu Tyr
Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys 450
455 460 Glu Leu Gly Asn Gly Cys Phe Glu
Phe Tyr His Arg Cys Asp Asn Glu 465 470
475 480 Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser 485 490
495 Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu
500 505 510 Ser Ile Gly
Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser 515
520 525 Ser Leu Ala Leu Ala Ile Xaa
530 535 20568PRTH5N1 20Met Glu Lys Ile Val Leu Leu Leu
Ala Ile Val Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr
Glu Gln Val 20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45 Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asp Leu Gly Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Pro Asn Val Ser Glu Trp
Ser Tyr Ile Val 85 90
95 Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asn Tyr Glu
Glu Leu Lys His Leu Leu Ser Arg Ile Asn Arg Phe Glu 115
120 125 Lys Ile Gln Ile Ile Pro Lys Ser
Ser Trp Pro Asp His Glu Ala Ser 130 135
140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Gly Pro
Ser Phe Tyr 145 150 155
160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asp Thr Tyr Pro Thr Ile
165 170 175 Lys Glu Ser Tyr
His Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180
185 190 Gly Ile His His Pro Asn Asn Glu Glu
Glu Gln Lys Arg Ile Tyr Lys 195 200
205 Asn Pro Thr Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn
Gln Arg 210 215 220
Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225
230 235 240 Arg Val Glu Phe Phe
Trp Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn 245
250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro
Glu Asn Ala Tyr Lys Ile 260 265
270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr
Gly 275 280 285 Asn
Cys Ser Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Thr Ser 290
295 300 Met Pro Phe His Asn Ile
His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310
315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser 325 330
335 Pro Gln Gly Glu Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350 Ala Gly
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355
360 365 Gly Tyr His His Ser Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375
380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser 385 390 395
400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415 Asn Asn Leu
Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420
425 430 Gly Phe Leu Asp Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Arg Asn Leu 450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465
470 475 480 Asn Gly Cys Phe
Glu Phe Tyr His Arg Cys Asp Asn Glu Cys Met Glu 485
490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala 500 505
510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
Ile Gly 515 520 525
Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530
535 540 Leu Ala Ile Met Val
Ala Gly Leu Phe Leu Trp Met Cys Ser Asn Gly 545 550
555 560 Ser Leu Gln Cys Arg Ile Cys Ile
565 21568PRTH5N1misc_feature(375)..(375)Xaa can be
any naturally occurring amino acid 21Met Glu Lys Ile Val Leu Leu Leu Ala
Ile Val Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu
Gln Val 20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45 Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Pro Asn Val Ser Glu Trp
Ser Tyr Ile Val 85 90
95 Glu Lys Thr Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asn Tyr Glu
Glu Leu Lys His Leu Leu Ser Arg Ile Asn Arg Phe Glu 115
120 125 Lys Ile Lys Ile Ile Pro Lys Ser
Ser Trp Pro Asp His Glu Ala Ser 130 135
140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Glu Pro
Ser Phe Tyr 145 150 155
160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile
165 170 175 Lys Glu Ser Tyr
His Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180
185 190 Gly Ile His His Pro Asn Asp Glu Glu
Glu Gln Thr Arg Ile Tyr Lys 195 200
205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn
Gln Arg 210 215 220
Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225
230 235 240 Arg Val Glu Phe Phe
Trp Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn 245
250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro
Glu Asn Ala Tyr Lys Ile 260 265
270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr
Gly 275 280 285 Asn
Cys Ser Thr Lys Cys Gln Thr Pro Val Gly Ala Ile Asn Ser Ser 290
295 300 Met Pro Phe His Asn Ile
His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310
315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser 325 330
335 Pro Gln Gly Glu Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350 Ala Gly
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355
360 365 Gly Tyr His His Ser Asn Xaa
Gln Gly Ser Gly Tyr Ala Ala Asp Arg 370 375
380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser 385 390 395
400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415 Asn Asn Leu
Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420
425 430 Gly Phe Leu Asp Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu 450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465
470 475 480 Asn Gly Cys Phe
Glu Phe Tyr His Arg Cys Asp Asn Glu Cys Ile Glu 485
490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala 500 505
510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
Ile Gly 515 520 525
Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530
535 540 Leu Ala Ile Ile Val
Ala Gly Leu Phe Leu Trp Met Cys Ser Asn Gly 545 550
555 560 Ser Leu Gln Cys Arg Ile Cys Ile
565 22568PRTH5N1 22Met Glu Lys Ile Val Leu Leu Leu
Ala Ile Val Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr
Glu Gln Val 20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45 Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Pro Asn Val Ser Glu Trp
Ser Tyr Ile Val 85 90
95 Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asn Tyr Glu
Glu Leu Lys His Leu Leu Ser Arg Ile Asn Arg Phe Glu 115
120 125 Lys Ile Lys Ile Ile Pro Lys Ser
Ser Trp Pro Asp His Glu Ala Ser 130 135
140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Arg Gly Pro
Ser Phe Tyr 145 150 155
160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile
165 170 175 Lys Lys Ser Tyr
His Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180
185 190 Gly Ile His His Pro Asn Asp Glu Glu
Glu Gln Thr Arg Ile Tyr Lys 195 200
205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn
Gln Arg 210 215 220
Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225
230 235 240 Arg Val Glu Phe Phe
Trp Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn 245
250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro
Glu Asn Ala Tyr Lys Ile 260 265
270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr
Gly 275 280 285 Asn
Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser 290
295 300 Met Pro Phe His Asn Ile
His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310
315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser 325 330
335 Pro Gln Gly Glu Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350 Ala Gly
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355
360 365 Gly Tyr His His Ser Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375
380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser 385 390 395
400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415 Asn Asn Leu
Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420
425 430 Gly Phe Leu Asp Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu 450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465
470 475 480 Asn Gly Cys Phe
Glu Phe Tyr His Arg Cys Asp Asn Glu Cys Met Glu 485
490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala 500 505
510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
Ile Gly 515 520 525
Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530
535 540 Leu Ala Ile Met Met
Ala Gly Leu Phe Leu Trp Met Cys Ser Asn Gly 545 550
555 560 Ser Leu Gln Cys Arg Ile Cys Ile
565 23568PRTH5N1 23Met Glu Lys Ile Val Leu Leu Leu
Ala Ile Val Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr
Glu Gln Val 20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45 Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Pro Asn Val Ser Glu Trp
Ser Tyr Ile Val 85 90
95 Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asn Tyr Glu
Glu Leu Lys His Leu Leu Ser Arg Ile Asn Arg Phe Glu 115
120 125 Lys Ile Lys Ile Ile Pro Lys Ser
Ser Trp Pro Asp His Glu Ala Ser 130 135
140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Gly Pro
Ser Phe Tyr 145 150 155
160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile
165 170 175 Lys Glu Ser Tyr
His Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180
185 190 Gly Ile His His Pro Asn Asp Glu Glu
Glu Gln Thr Arg Ile Tyr Lys 195 200
205 Asn Pro Thr Thr Tyr Ile Ser Ile Gly Thr Ser Thr Leu Asn
Gln Arg 210 215 220
Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Arg Gly 225
230 235 240 Arg Val Glu Phe Phe
Trp Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn 245
250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro
Glu Asn Ala Tyr Lys Ile 260 265
270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr
Gly 275 280 285 Asn
Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser 290
295 300 Met Pro Phe His Asn Ile
His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310
315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser 325 330
335 Pro Gln Gly Glu Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350 Ala Gly
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355
360 365 Gly Tyr His His Ser Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375
380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser 385 390 395
400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415 Asn Asn Leu
Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420
425 430 Gly Phe Leu Asp Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu 450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465
470 475 480 Asn Gly Cys Phe
Glu Phe Tyr His Arg Cys Asp Asn Glu Cys Met Glu 485
490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala 500 505
510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
Ile Gly 515 520 525
Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530
535 540 Leu Ala Ile Met Met
Ala Gly Leu Phe Leu Trp Met Cys Ser Asn Gly 545 550
555 560 Ser Leu Gln Cys Arg Ile Cys Ile
565 24568PRTH5N1 24Met Glu Lys Ile Val Leu Leu Leu
Ala Ile Val Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr
Glu Gln Val 20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45 Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Pro Asn Val Ser Glu Trp
Ser Tyr Ile Val 85 90
95 Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asn Tyr Glu
Glu Leu Lys His Leu Leu Ser Arg Ile Asn Arg Phe Glu 115
120 125 Lys Ile Gln Ile Ile Pro Lys Ser
Ser Trp Pro Asp His Glu Ala Ser 130 135
140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Glu Pro
Ser Phe Tyr 145 150 155
160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile
165 170 175 Lys Glu Ser Tyr
His Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180
185 190 Gly Ile His His Pro Asn Asp Glu Glu
Glu Gln Lys Arg Ile Tyr Lys 195 200
205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn
Gln Arg 210 215 220
Leu Val Pro Lys Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Ser Gly 225
230 235 240 Arg Val Glu Phe Phe
Trp Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn 245
250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro
Lys Asn Ala Tyr Lys Ile 260 265
270 Val Lys Lys Gly Ser Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr
Gly 275 280 285 Asn
Cys Ser Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser 290
295 300 Met Pro Phe His Asn Ile
His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310
315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser 325 330
335 Pro Gln Gly Glu Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350 Ala Gly
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355
360 365 Gly Tyr His His Ser Asn Glu
Gln Gly Thr Gly Tyr Ala Ala Asp Lys 370 375
380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser 385 390 395
400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415 Asn Asn Leu
Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420
425 430 Gly Phe Leu Asp Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Arg Asn Leu 450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465
470 475 480 Asn Gly Cys Phe
Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485
490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Lys Glu Ala 500 505
510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
Ile Gly 515 520 525
Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Thr Ser Ser Leu Ala 530
535 540 Leu Ala Ile Met Val
Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550
555 560 Ser Leu Gln Cys Arg Ile Cys Ile
565 25556PRTH5N1misc_feature(556)..(556)Xaa can be
any naturally occurring amino acid 25Met Glu Lys Ile Val Leu Leu Leu Ala
Ile Val Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu
Gln Val 20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45 Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Pro Asn Val Ser Glu Trp
Ser Tyr Ile Val 85 90
95 Glu Lys Thr Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asn Tyr Glu
Glu Leu Lys His Leu Leu Ser Arg Ile Asn Arg Phe Glu 115
120 125 Lys Ile Lys Ile Ile Pro Lys Ser
Ser Trp Pro Asp His Glu Ala Ser 130 135
140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Arg Pro
Ser Phe Tyr 145 150 155
160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile
165 170 175 Lys Glu Ser Tyr
His Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180
185 190 Gly Ile His His Pro Asn Asp Glu Glu
Glu Gln Thr Arg Ile Tyr Lys 195 200
205 Asn Pro Thr Thr Tyr Ile Ser Ile Gly Thr Ser Thr Leu Asn
Gln Arg 210 215 220
Leu Ile Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225
230 235 240 Arg Val Glu Phe Phe
Trp Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn 245
250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro
Glu Asn Ala Tyr Lys Ile 260 265
270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr
Gly 275 280 285 Asn
Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser 290
295 300 Met Pro Phe His Asn Ile
His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310
315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser 325 330
335 Pro Gln Gly Glu Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350 Ala Gly
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355
360 365 Gly Tyr His His Ser Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375
380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser 385 390 395
400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415 Asn Asn Leu
Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420
425 430 Gly Phe Leu Asp Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu 450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465
470 475 480 Asn Gly Cys Phe
Glu Phe Tyr His Arg Cys Asp Asn Glu Cys Met Glu 485
490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala 500 505
510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
Ile Gly 515 520 525
Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530
535 540 Leu Ala Ile Met Met
Ala Gly Leu Phe Leu Trp Xaa 545 550 555
26554PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 26Xaa Leu Leu Ala Ile Val Ser Leu Val Lys Ser Asp Gln Ile Cys
Ile 1 5 10 15 Gly
Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile Met Glu
20 25 30 Lys Asn Val Thr Val
Thr His Ala Gln Asp Ile Leu Glu Lys Thr His 35
40 45 Asn Gly Lys Leu Cys Asp Leu Asp Gly
Val Lys Pro Leu Ile Leu Arg 50 55
60 Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met
Cys Asp Glu 65 70 75
80 Phe Pro Asn Val Ser Glu Trp Ser Tyr Ile Val Glu Lys Ile Asn Pro
85 90 95 Ala Asn Asp Leu
Cys Tyr Pro Gly Asn Phe Asn Asn Tyr Glu Glu Leu 100
105 110 Lys His Leu Leu Ser Arg Ile Asn Arg
Phe Glu Lys Ile Gln Ile Ile 115 120
125 Pro Lys Ser Ser Trp Pro Asp His Glu Ala Ser Leu Gly Val
Ser Ser 130 135 140
Ala Cys Pro Tyr Gln Gly Glu Pro Ser Phe Tyr Arg Asn Val Val Trp 145
150 155 160 Leu Ile Lys Lys Asn
Asn Thr Tyr Pro Thr Ile Lys Glu Asn Tyr His 165
170 175 Asn Thr Asn Gln Glu Asp Leu Leu Val Leu
Trp Gly Ile His His Pro 180 185
190 Asn Asp Glu Glu Glu Gln Lys Arg Ile Tyr Lys Asn Pro Thr Thr
Tyr 195 200 205 Ile
Ser Val Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro Lys Ile 210
215 220 Ala Thr Arg Pro Lys Val
Asn Gly Gln Ser Gly Arg Val Glu Phe Phe 225 230
235 240 Trp Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn
Phe Glu Ser Asn Gly 245 250
255 Asn Phe Ile Ala Pro Lys Asn Ala Tyr Lys Ile Val Lys Lys Gly Ser
260 265 270 Ser Thr
Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Ser Thr Lys 275
280 285 Cys Gln Thr Pro Ile Gly Ala
Ile Asn Ser Ser Met Pro Phe His Asn 290 295
300 Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr
Val Lys Ser Asn 305 310 315
320 Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Gly Glu Gly
325 330 335 Arg Arg Lys
Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 340
345 350 Gly Gly Trp Gln Gly Met Val Asp
Gly Trp Tyr Gly Tyr His His Ser 355 360
365 Asn Glu Gln Gly Thr Gly Tyr Ala Ala Asp Lys Glu Ser
Thr Gln Lys 370 375 380
Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met 385
390 395 400 Asn Thr Gln Phe
Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Lys 405
410 415 Arg Ile Glu Asn Leu Asn Lys Lys Met
Glu Asp Gly Phe Leu Asp Val 420 425
430 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu
Arg Thr 435 440 445
Leu Asp Phe His Asp Ser Asn Val Arg Asn Leu Tyr Asp Lys Val Arg 450
455 460 Leu Gln Leu Arg Asp
Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu 465 470
475 480 Phe Tyr His Lys Cys Asp Asn Glu Cys Met
Glu Ser Val Arg Asn Gly 485 490
495 Thr Tyr Asp Tyr Pro Gln Tyr Ser Lys Glu Ala Arg Leu Lys Arg
Glu 500 505 510 Glu
Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln Ile Leu 515
520 525 Ser Ile Tyr Ser Thr Val
Ala Ser Ser Leu Ala Leu Ala Ile Met Val 530 535
540 Ala Gly Leu Ser Leu Trp Met Cys Ser Xaa 545
550 27564PRTH5N1 27Val Leu Leu Leu Ala
Ile Val Ser Leu Val Lys Ser Asp Gln Ile Cys 1 5
10 15 Ile Gly Tyr His Ala Asn Asn Ser Thr Glu
Gln Val Asp Thr Ile Met 20 25
30 Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile Leu Glu Lys
Thr 35 40 45 His
Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys Pro Leu Ile Leu 50
55 60 Arg Asp Cys Ser Val Ala
Gly Trp Leu Leu Gly Asn Pro Met Cys Asp 65 70
75 80 Glu Phe Pro Asn Val Ser Glu Trp Ser Tyr Ile
Val Glu Lys Ile Asn 85 90
95 Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn Asn Tyr Glu Glu
100 105 110 Leu Lys
His Leu Leu Ser Arg Ile Asn Arg Phe Glu Lys Ile Gln Ile 115
120 125 Ile Pro Lys Ser Ser Trp Pro
Asp His Glu Ala Ser Leu Gly Val Ser 130 135
140 Ser Ala Cys Pro Tyr Gln Gly Glu Pro Ser Phe Tyr
Arg Asn Val Val 145 150 155
160 Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile Lys Glu Asn Tyr
165 170 175 His Asn Thr
Asn Gln Glu Asp Leu Leu Val Leu Trp Gly Ile His His 180
185 190 Pro Asn Asp Glu Glu Glu Gln Lys
Arg Ile Tyr Lys Asn Pro Thr Thr 195 200
205 Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg Leu
Val Pro Lys 210 215 220
Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Ser Gly Arg Val Glu Phe 225
230 235 240 Phe Trp Thr Ile
Leu Lys Ser Asn Asp Thr Ile Asn Phe Glu Ser Asn 245
250 255 Gly Asn Phe Ile Ala Pro Lys Asn Ala
Tyr Lys Ile Val Lys Lys Gly 260 265
270 Ser Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys
Ser Thr 275 280 285
Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser Met Pro Phe His 290
295 300 Asn Ile His Pro Leu
Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser 305 310
315 320 Asn Arg Leu Val Leu Ala Thr Gly Leu Arg
Asn Ser Pro Gln Gly Glu 325 330
335 Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe
Ile 340 345 350 Glu
Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His 355
360 365 Ser Asn Glu Gln Gly Thr
Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln 370 375
380 Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn
Ser Ile Ile Asp Lys 385 390 395
400 Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu
405 410 415 Lys Arg
Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp 420
425 430 Val Trp Thr Tyr Asn Ala Glu
Leu Leu Val Leu Met Glu Asn Glu Arg 435 440
445 Thr Leu Asp Phe His Asp Ser Asn Val Arg Asn Leu
Tyr Asp Lys Val 450 455 460
Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe 465
470 475 480 Glu Phe Tyr
His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn 485
490 495 Gly Thr Tyr Asp Tyr Pro Gln Tyr
Ser Lys Glu Ala Arg Leu Lys Arg 500 505
510 Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr
Tyr Gln Ile 515 520 525
Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met 530
535 540 Val Ala Gly Leu
Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gln Cys 545 550
555 560 Arg Ile Cys Ile
28553PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 28Xaa Val Leu Leu Leu Ala Ile Ile Ser Leu Val Lys Ser Asp Gln Ile 1
5 10 15 Cys Ile Gly
Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile 20
25 30 Met Glu Lys Asn Val Thr Val Thr
His Ala Gln Asp Ile Leu Glu Lys 35 40
45 Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys
Pro Leu Ile 50 55 60
Leu Arg Gly Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys 65
70 75 80 Asp Glu Phe Pro
Asn Val Ser Glu Trp Ser Tyr Ile Val Glu Lys Ile 85
90 95 Asn Pro Ala Asn Asp Leu Cys Tyr Pro
Gly Asn Phe Asn Asn Tyr Glu 100 105
110 Glu Leu Lys His Leu Leu Ser Arg Ile Asn Arg Phe Glu Lys
Ile Gln 115 120 125
Ile Ile Pro Lys Ser Ser Trp Pro Asp His Glu Ala Ser Leu Gly Val 130
135 140 Ser Ser Ala Cys Pro
Tyr Gln Gly Glu Pro Ser Phe Tyr Arg Asn Val 145 150
155 160 Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr
Pro Thr Ile Lys Glu Ser 165 170
175 Tyr His Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp Gly Ile
His 180 185 190 His
Pro Asn Asp Glu Glu Glu Gln Lys Arg Ile Tyr Lys Asn Pro Thr 195
200 205 Thr Tyr Ile Ser Val Gly
Thr Ser Thr Leu Asn Gln Arg Leu Val Pro 210 215
220 Lys Ile Ala Thr Arg Pro Lys Val Asn Gly Gln
Ser Gly Arg Val Glu 225 230 235
240 Phe Phe Trp Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn Phe Glu Ser
245 250 255 Asn Gly
Asn Phe Ile Ala Pro Lys Asn Ala Tyr Lys Ile Val Lys Lys 260
265 270 Gly Ser Ser Thr Ile Met Lys
Ser Glu Leu Glu Tyr Gly Asn Cys Ser 275 280
285 Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser
Ser Met Pro Phe 290 295 300
His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys 305
310 315 320 Ser Asn Arg
Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Gly 325
330 335 Glu Gly Arg Arg Lys Lys Arg Gly
Leu Phe Gly Ala Ile Ala Gly Phe 340 345
350 Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
Gly Tyr His 355 360 365
His Ser Asn Glu Gln Gly Thr Gly Tyr Ala Ala Asp Lys Glu Ser Thr 370
375 380 Gln Lys Ala Ile
Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp 385 390
395 400 Lys Met Asn Thr Gln Phe Glu Ala Val
Gly Arg Glu Phe Asn Asn Leu 405 410
415 Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly
Phe Leu 420 425 430
Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu
435 440 445 Arg Thr Leu Asp
Phe His Asp Ser Asn Val Arg Asn Leu Tyr Asp Lys 450
455 460 Val Arg Leu Gln Leu Arg Asp Asn
Ala Lys Glu Leu Gly Asn Gly Cys 465 470
475 480 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met
Glu Ser Val Arg 485 490
495 Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Lys Glu Ala Arg Leu Lys
500 505 510 Arg Glu Glu
Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln 515
520 525 Ile Leu Ser Ile Tyr Ser Thr Val
Ala Ser Ser Leu Ala Leu Ala Ile 530 535
540 Met Val Ala Gly Leu Ser Leu Trp Met 545
550 29558PRTH5N1misc_feature(558)..(558)Xaa can be any
naturally occurring amino acid 29Ile Val Leu Leu Leu Ala Ile Val Ser Leu
Val Lys Ser Asp Gln Ile 1 5 10
15 Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr
Ile 20 25 30 Met
Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile Leu Glu Lys 35
40 45 Thr His Asn Gly Lys Leu
Cys Asp Leu Asp Gly Val Lys Pro Leu Ile 50 55
60 Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu
Gly Asn Pro Met Cys 65 70 75
80 Asp Glu Phe Pro Asn Val Ser Glu Trp Ser Tyr Ile Val Glu Lys Ile
85 90 95 Asn Pro
Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn Asn Tyr Glu 100
105 110 Glu Leu Lys His Leu Leu Ser
Arg Ile Asn Arg Phe Glu Lys Ile Lys 115 120
125 Ile Ile Pro Lys Ser Ser Trp Pro Asp His Glu Ala
Ser Leu Gly Val 130 135 140
Ser Ser Ala Cys Pro Tyr Gln Gly Gly Pro Ser Phe Tyr Arg Asn Val 145
150 155 160 Val Trp Leu
Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile Lys Lys Ser 165
170 175 Tyr His Asn Thr Asn Gln Glu Asp
Leu Leu Val Leu Trp Gly Ile His 180 185
190 His Pro Asn Asp Glu Glu Glu Gln Thr Arg Ile Tyr Lys
Asn Pro Thr 195 200 205
Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro 210
215 220 Lys Ile Ala Thr
Arg Ser Lys Val Asn Gly Gln Ser Gly Arg Val Glu 225 230
235 240 Phe Phe Trp Thr Ile Leu Lys Ser Asn
Asp Thr Ile Asn Phe Glu Ser 245 250
255 Asn Gly Asn Phe Ile Ala Pro Glu Asn Ala Tyr Lys Ile Val
Lys Lys 260 265 270
Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu His Gly Asn Cys Asn
275 280 285 Thr Lys Cys Gln
Thr Pro Ile Gly Ala Ile Asn Ser Ser Met Pro Phe 290
295 300 His Asn Ile His Pro Leu Thr Ile
Gly Glu Cys Pro Lys Tyr Val Lys 305 310
315 320 Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn
Ser Pro Gln Gly 325 330
335 Glu Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe
340 345 350 Ile Glu Gly
Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His 355
360 365 His Ser Asn Glu Gln Gly Ser Gly
Tyr Ala Ala Asp Lys Glu Ser Thr 370 375
380 Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser
Ile Ile Asp 385 390 395
400 Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu
405 410 415 Glu Lys Arg Ile
Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu 420
425 430 Asp Val Trp Thr Tyr Asn Ala Glu Leu
Leu Val Leu Met Glu Asn Glu 435 440
445 Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr
Asp Lys 450 455 460
Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys 465
470 475 480 Phe Glu Phe Tyr His
Arg Cys Asp Asn Glu Cys Met Glu Ser Val Arg 485
490 495 Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser
Glu Glu Ala Arg Leu Asn 500 505
510 Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr
Gln 515 520 525 Ile
Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile 530
535 540 Met Met Ala Gly Leu Phe
Leu Trp Met Cys Ser Asn Gly Xaa 545 550
555 30557PRTH5N1misc_feature(130)..(130)Xaa can be any
naturally occurring amino acid 30Leu Leu Ala Ile Val Ser Leu Val Lys Ser
Asp Gln Ile Cys Ile Gly 1 5 10
15 Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile Met Glu
Lys 20 25 30 Asn
Val Thr Val Thr His Ala Gln Asp Ile Leu Glu Lys Thr His Asn 35
40 45 Gly Lys Leu Cys Asp Leu
Asp Gly Val Lys Pro Leu Ile Leu Arg Asp 50 55
60 Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro
Met Cys Asp Glu Phe 65 70 75
80 Pro Asn Val Ser Glu Trp Ser Tyr Ile Val Glu Lys Ile Asn Pro Ala
85 90 95 Asn Asp
Leu Cys Tyr Pro Gly Asn Phe Asn Asn Tyr Glu Glu Leu Lys 100
105 110 His Leu Leu Ser Arg Ile Asn
Arg Phe Glu Lys Ile Lys Ile Ile Pro 115 120
125 Lys Xaa Ser Trp Pro Asp His Glu Ala Ser Leu Gly
Val Ser Ser Ala 130 135 140
Cys Pro Tyr Gln Gly Gly Pro Ser Phe Tyr Arg Asn Val Val Trp Leu 145
150 155 160 Ile Lys Lys
Asn Asn Thr Tyr Pro Thr Ile Lys Glu Ser Tyr His Asn 165
170 175 Thr Asn Gln Glu Asp Leu Leu Val
Leu Trp Gly Ile His His Pro Asn 180 185
190 Asp Glu Glu Glu Gln Thr Arg Ile Tyr Lys Asn Pro Thr
Thr Tyr Ile 195 200 205
Ser Ile Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro Lys Ile Ala 210
215 220 Thr Arg Ser Lys
Val Asn Gly Gln Ser Gly Arg Val Glu Phe Phe Trp 225 230
235 240 Thr Ile Leu Lys Ser Asn Asp Thr Ile
Asn Phe Glu Ser Asn Gly Asn 245 250
255 Phe Ile Ala Pro Glu Asn Ala Tyr Lys Ile Val Lys Lys Gly
Asp Ser 260 265 270
Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys Cys
275 280 285 Gln Thr Pro Ile
Gly Ala Ile Asn Ser Ser Met Pro Phe His Asn Ile 290
295 300 His Pro Leu Thr Ile Gly Glu Cys
Pro Lys Tyr Val Lys Ser Asn Arg 305 310
315 320 Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln
Gly Glu Gly Arg 325 330
335 Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
340 345 350 Gly Trp Gln
Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn 355
360 365 Glu Gln Gly Ser Gly Tyr Ala Ala
Asp Lys Glu Ser Thr Gln Lys Ala 370 375
380 Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp
Lys Met Asn 385 390 395
400 Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Lys Arg
405 410 415 Ile Glu Asn Leu
Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp 420
425 430 Thr Tyr Asn Ala Glu Leu Leu Val Leu
Met Glu Asn Glu Arg Thr Leu 435 440
445 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val
Arg Leu 450 455 460
Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe 465
470 475 480 Tyr His Arg Cys Asp
Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr 485
490 495 Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala
Arg Leu Lys Arg Glu Glu 500 505
510 Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln Ile Leu
Ser 515 520 525 Ile
Tyr Ser Thr Val Ala Gly Ser Leu Ala Leu Ala Ile Met Met Ala 530
535 540 Gly Leu Phe Leu Trp Met
Cys Ser Asn Gly Ser Leu Gln 545 550 555
31547PRTH5N1misc_feature(547)..(547)Xaa can be any naturally
occurring amino acid 31Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys
Ser Asp Gln Ile 1 5 10
15 Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile
20 25 30 Met Glu Lys
Asn Val Thr Val Thr His Ala Gln Asp Ile Leu Glu Lys 35
40 45 Thr His Asn Gly Lys Leu Cys Asp
Leu Asp Gly Val Lys Pro Leu Ile 50 55
60 Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
Pro Met Cys 65 70 75
80 Asp Glu Phe Pro Asn Val Ser Glu Trp Ser Tyr Ile Val Glu Lys Ile
85 90 95 Asn Pro Ala Asn
Asp Leu Cys Tyr Pro Gly Asn Phe Asn Asn Tyr Glu 100
105 110 Glu Leu Lys His Leu Leu Ser Arg Ile
Asn Arg Phe Glu Lys Ile Lys 115 120
125 Ile Ile Pro Lys Ser Ser Trp Pro Asp His Glu Ala Ser Leu
Gly Val 130 135 140
Ser Ser Ala Cys Pro Tyr Gln Glu Gly Pro Ser Phe Tyr Arg Asn Val 145
150 155 160 Val Trp Leu Ile Lys
Lys Asn Asn Thr Tyr Pro Thr Ile Lys Lys Ser 165
170 175 Tyr His Asn Thr Asn Gln Glu Asp Leu Leu
Val Leu Trp Gly Ile His 180 185
190 His Pro Asn Asp Glu Glu Glu Gln Thr Arg Ile Tyr Lys Asn Pro
Thr 195 200 205 Thr
Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro 210
215 220 Lys Ile Ala Thr Arg Ser
Lys Val Asn Gly Gln Ser Gly Arg Val Glu 225 230
235 240 Phe Phe Trp Thr Ile Leu Lys Ser Asn Asp Thr
Ile Asn Phe Glu Ser 245 250
255 Asn Gly Asn Phe Ile Ala Pro Glu Asn Ala Tyr Lys Ile Val Lys Lys
260 265 270 Gly Asp
Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn 275
280 285 Thr Lys Cys Gln Thr Pro Ile
Gly Ala Ile Asn Ser Ser Met Pro Phe 290 295
300 His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro
Lys Tyr Val Lys 305 310 315
320 Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Gly
325 330 335 Glu Gly Arg
Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340
345 350 Ile Glu Gly Gly Trp Gln Gly Met
Val Asp Gly Trp Tyr Gly Tyr His 355 360
365 His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys
Glu Ser Thr 370 375 380
Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp 385
390 395 400 Lys Met Asn Thr
Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu 405
410 415 Glu Lys Arg Ile Glu Asn Leu Asn Lys
Lys Met Glu Asp Gly Phe Leu 420 425
430 Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu
Asn Glu 435 440 445
Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys 450
455 460 Val Arg Leu Gln Leu
Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys 465 470
475 480 Phe Glu Phe Tyr His Arg Cys Asp Asn Glu
Cys Met Glu Ser Val Arg 485 490
495 Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu
Lys 500 505 510 Arg
Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln 515
520 525 Ile Leu Ser Ile Tyr Ser
Thr Val Ala Ser Ser Leu Ala Leu Ala Ile 530 535
540 Met Val Xaa 545
32547PRTH5N1misc_feature(154)..(154)Xaa can be any naturally occurring
amino acid 32Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser Asp Gln
Ile 1 5 10 15 Cys
Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile
20 25 30 Met Glu Lys Asn Val
Thr Val Thr His Ala Gln Asp Ile Leu Glu Lys 35
40 45 Thr His Asn Gly Lys Leu Cys Asp Leu
Asp Gly Val Lys Pro Leu Ile 50 55
60 Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
Pro Met Cys 65 70 75
80 Asp Glu Phe Pro Asn Val Ser Glu Trp Ser Tyr Ile Val Glu Lys Ile
85 90 95 Asn Pro Ala Asn
Asp Leu Cys Tyr Pro Gly Asn Phe Asn Asn Tyr Glu 100
105 110 Glu Leu Lys His Leu Leu Ser Arg Ile
Asn Arg Phe Glu Lys Ile Lys 115 120
125 Ile Ile Pro Lys Ser Ser Trp Pro Asp His Glu Ala Ser Leu
Gly Val 130 135 140
Ser Ser Ala Cys Pro Tyr Gln Gly Gly Xaa Ser Phe Tyr Arg Asn Val 145
150 155 160 Val Trp Leu Ile Lys
Lys Asn Asn Thr Tyr Pro Thr Ile Lys Glu Ser 165
170 175 Tyr His Asn Thr Asn Gln Glu Asp Leu Leu
Val Leu Trp Gly Ile His 180 185
190 His Pro Asn Asp Glu Glu Glu Gln Thr Arg Ile Tyr Lys Asn Pro
Thr 195 200 205 Thr
Tyr Ile Ser Ile Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro 210
215 220 Lys Ile Ala Thr Arg Ser
Lys Val Asn Gly Gln Ser Gly Arg Val Glu 225 230
235 240 Phe Phe Trp Thr Ile Leu Lys Ser Asn Asp Thr
Ile Asn Phe Glu Ser 245 250
255 Asn Gly Asn Phe Ile Ala Pro Glu Asn Ala Tyr Lys Ile Val Lys Lys
260 265 270 Gly Asp
Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn 275
280 285 Thr Lys Cys Gln Thr Pro Ile
Gly Ala Ile Asn Ser Ser Met Pro Phe 290 295
300 His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro
Lys Tyr Val Lys 305 310 315
320 Ser Asn Arg Leu Val Leu Ala Thr Xaa Leu Arg Asn Ser Pro Gln Gly
325 330 335 Glu Gly Arg
Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340
345 350 Ile Glu Gly Gly Trp Gln Gly Met
Val Asp Gly Trp Tyr Gly Tyr His 355 360
365 His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys
Glu Ser Thr 370 375 380
Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp 385
390 395 400 Lys Met Asn Thr
Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu 405
410 415 Glu Lys Arg Ile Glu Asn Leu Asn Lys
Lys Met Glu Asp Gly Phe Leu 420 425
430 Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu
Asn Glu 435 440 445
Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys 450
455 460 Val Arg Leu Gln Leu
Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys 465 470
475 480 Phe Glu Phe Tyr His Arg Cys Asp Asn Glu
Cys Met Glu Ser Val Arg 485 490
495 Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu
Lys 500 505 510 Arg
Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln 515
520 525 Ile Leu Ser Ile Tyr Ser
Thr Val Ala Gly Ser Leu Ala Leu Ala Ile 530 535
540 Met Val Xaa 545
33559PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 33Xaa Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser Asp Gln Ile Cys 1
5 10 15 Ile Gly Tyr
His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile Met 20
25 30 Glu Lys Asn Val Thr Val Thr His
Ala Gln Asp Ile Leu Glu Lys Thr 35 40
45 His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys Pro
Leu Ile Leu 50 55 60
Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp 65
70 75 80 Glu Phe Pro Asn
Val Ser Glu Trp Ser Tyr Ile Val Glu Lys Ile Asn 85
90 95 Pro Ala Asn Asp Leu Cys Tyr Pro Gly
Asn Phe Asn Asn Tyr Glu Glu 100 105
110 Leu Lys His Leu Leu Ser Arg Ile Asn Arg Phe Glu Lys Ile
Gln Ile 115 120 125
Ile Pro Lys Ser Ser Trp Pro Asp His Glu Ala Ser Leu Gly Val Ser 130
135 140 Ser Ala Cys Pro Tyr
Gln Gly Glu Pro Ser Phe Tyr Arg Asn Val Val 145 150
155 160 Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro
Thr Ile Lys Glu Asn Tyr 165 170
175 His Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp Gly Ile His
His 180 185 190 Pro
Asn Asp Glu Glu Glu Gln Lys Arg Ile Tyr Lys Asn Pro Thr Thr 195
200 205 Tyr Ile Ser Val Gly Thr
Ser Thr Leu Thr Gln Arg Leu Val Pro Lys 210 215
220 Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Ser
Gly Arg Val Glu Phe 225 230 235
240 Phe Trp Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn Phe Glu Ser Asn
245 250 255 Gly Asn
Phe Ile Ala Pro Lys Asn Ala Tyr Lys Ile Val Lys Lys Gly 260
265 270 Ser Ser Thr Ile Met Lys Ser
Glu Leu Glu Tyr Gly Asn Cys Ser Thr 275 280
285 Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser
Met Pro Phe His 290 295 300
Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser 305
310 315 320 Asn Arg Leu
Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Gly Glu 325
330 335 Gly Arg Arg Lys Lys Arg Gly Leu
Phe Gly Ala Ile Ala Gly Phe Ile 340 345
350 Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly
Tyr His His 355 360 365
Ser Asn Glu Gln Gly Thr Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln 370
375 380 Lys Ala Ile Asp
Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys 385 390
395 400 Met Asn Thr Gln Phe Glu Ala Val Gly
Arg Glu Phe Asn Asn Leu Glu 405 410
415 Lys Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe
Leu Asp 420 425 430
Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg
435 440 445 Thr Leu Asp Phe
His Asp Ser Asn Val Arg Asn Leu Tyr Asp Lys Val 450
455 460 Arg Leu Gln Leu Arg Asp Asn Ala
Lys Glu Leu Gly Asn Gly Cys Phe 465 470
475 480 Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu
Ser Val Arg Asn 485 490
495 Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Lys Glu Ala Arg Leu Lys Arg
500 505 510 Glu Glu Ile
Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln Ile 515
520 525 Leu Ser Ile Tyr Ser Thr Val Ala
Ser Ser Leu Ala Leu Ala Ile Met 530 535
540 Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser
Leu Gln 545 550 555
34554PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 34Xaa Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser 1
5 10 15 Thr Glu Gln
Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His 20
25 30 Ala Gln Asp Ile Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asp Leu 35 40
45 Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val
Ala Gly Trp 50 55 60
Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Pro Asn Val Ser Glu Trp 65
70 75 80 Ser Tyr Ile Val
Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro 85
90 95 Gly Asn Phe Asn Asn Tyr Glu Glu Leu
Lys His Leu Leu Ser Arg Ile 100 105
110 Asn Arg Phe Glu Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp
Pro Asp 115 120 125
His Glu Ala Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Glu 130
135 140 Pro Ser Phe Tyr Arg
Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr 145 150
155 160 Tyr Pro Thr Ile Lys Glu Ser Tyr His Asn
Thr Asn Gln Glu Asp Leu 165 170
175 Leu Val Leu Trp Gly Ile His His Pro Asn Asp Glu Glu Glu Gln
Lys 180 185 190 Arg
Ile Tyr Lys Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr 195
200 205 Leu Asn Gln Arg Leu Val
Pro Lys Ile Ala Thr Arg Pro Lys Val Asn 210 215
220 Gly Gln Ser Gly Arg Val Glu Phe Phe Trp Thr
Ile Leu Lys Ser Asn 225 230 235
240 Asp Thr Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Lys Asn
245 250 255 Ala Tyr
Lys Ile Val Lys Lys Gly Ser Ser Thr Ile Met Lys Ser Glu 260
265 270 Leu Glu Tyr Gly Asn Cys Ser
Thr Lys Cys Gln Thr Pro Ile Gly Ala 275 280
285 Ile Asn Ser Ser Met Pro Phe His Asn Ile His Pro
Leu Thr Ile Gly 290 295 300
Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly 305
310 315 320 Leu Arg Asn
Ser Pro Gln Gly Glu Gly Arg Arg Lys Lys Arg Gly Leu 325
330 335 Phe Gly Ala Ile Ala Gly Phe Ile
Glu Gly Gly Trp Gln Gly Met Val 340 345
350 Asp Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly
Thr Gly Tyr 355 360 365
Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn 370
375 380 Lys Val Asn Ser
Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val 385 390
395 400 Gly Arg Glu Phe Asn Asn Leu Glu Lys
Arg Ile Glu Asn Leu Asn Lys 405 410
415 Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala
Glu Leu 420 425 430
Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn
435 440 445 Val Arg Asn Leu
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala 450
455 460 Lys Glu Leu Gly Asn Gly Cys Phe
Glu Phe Tyr His Lys Cys Asp Asn 465 470
475 480 Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp
Tyr Pro Gln Tyr 485 490
495 Ser Lys Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu
500 505 510 Glu Ser Ile
Gly Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala 515
520 525 Ser Ser Leu Ala Leu Ala Ile Met
Val Ala Gly Leu Ser Leu Trp Met 530 535
540 Cys Ser Asn Gly Ser Leu Gln Cys Arg Xaa 545
550 35537PRTH5N1misc_feature(1)..(1)Xaa can be
any naturally occurring amino acid 35Xaa Ser Asp Gln Ile Cys Ile Gly Tyr
His Ala Asn Asn Ser Thr Glu 1 5 10
15 Gln Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His
Ala Gln 20 25 30
Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly
35 40 45 Val Lys Pro Leu
Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu 50
55 60 Gly Asn Pro Met Cys Asp Glu Phe
Pro Asn Val Ser Glu Trp Ser Tyr 65 70
75 80 Ile Val Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys
Tyr Pro Gly Asn 85 90
95 Phe Asn Asn Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn Arg
100 105 110 Phe Glu Lys
Ile Gln Ile Ile Pro Lys Ser Ser Trp Pro Asp His Glu 115
120 125 Ala Ser Leu Gly Val Ser Ser Ala
Cys Pro Tyr Gln Gly Glu Pro Ser 130 135
140 Phe Tyr Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn
Thr Tyr Pro 145 150 155
160 Thr Ile Lys Glu Asn Tyr His Asn Thr Asn Gln Glu Asp Leu Leu Val
165 170 175 Leu Trp Gly Ile
His His Pro Asn Asp Glu Glu Glu Gln Lys Arg Ile 180
185 190 Tyr Lys Asn Pro Thr Thr Tyr Ile Ser
Val Gly Thr Ser Thr Leu Asn 195 200
205 Gln Arg Leu Val Pro Lys Ile Ala Thr Arg Pro Lys Val Asn
Gly Gln 210 215 220
Ser Gly Arg Val Glu Phe Phe Trp Thr Ile Leu Lys Ser Asn Asp Thr 225
230 235 240 Ile Asn Phe Glu Ser
Asn Gly Asn Phe Ile Ala Pro Lys Asn Ala Tyr 245
250 255 Lys Ile Val Lys Lys Gly Ser Ser Thr Ile
Met Lys Ser Glu Leu Glu 260 265
270 Tyr Gly Asn Cys Ser Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile
Asn 275 280 285 Ser
Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys 290
295 300 Pro Lys Tyr Val Lys Ser
Asn Arg Leu Val Leu Ala Thr Gly Leu Arg 305 310
315 320 Asn Ser Pro Gln Gly Glu Gly Arg Arg Lys Lys
Arg Gly Leu Phe Gly 325 330
335 Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly
340 345 350 Trp Tyr
Gly Tyr His His Ser Asn Glu Gln Gly Thr Gly Tyr Ala Ala 355
360 365 Asp Lys Glu Ser Thr Gln Lys
Ala Ile Asp Gly Val Thr Asn Lys Val 370 375
380 Asn Ser Ile Ile Asp Lys Met Asn Thr Gln Phe Glu
Ala Val Gly Arg 385 390 395
400 Glu Phe Asn Asn Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Met
405 410 415 Glu Asp Gly
Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val 420
425 430 Leu Met Glu Asn Glu Arg Thr Leu
Asp Phe His Asp Ser Asn Val Arg 435 440
445 Asn Leu Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn
Ala Lys Glu 450 455 460
Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys 465
470 475 480 Met Glu Ser Val
Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Lys 485
490 495 Glu Ala Arg Leu Lys Arg Glu Glu Ile
Ser Gly Val Lys Leu Glu Ser 500 505
510 Ile Gly Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala
Ser Ser 515 520 525
Leu Ala Leu Ala Ile Met Val Ala Xaa 530 535
36554PRTH5N1 36Lys Gly Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser
Thr Glu 1 5 10 15
Gln Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln
20 25 30 Asp Ile Leu Glu Lys
Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly 35
40 45 Val Lys Pro Leu Ile Leu Arg Asp Cys
Ser Val Ala Gly Trp Leu Leu 50 55
60 Gly Asn Pro Met Cys Asp Glu Phe Pro Asn Val Ser Glu
Trp Ser Tyr 65 70 75
80 Ile Val Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn
85 90 95 Phe Asn Asn Tyr
Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn Arg 100
105 110 Phe Glu Lys Ile Lys Ile Ile Pro Lys
Ser Ser Trp Pro Asp His Glu 115 120
125 Ala Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Gly
Pro Ser 130 135 140
Phe Tyr Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro 145
150 155 160 Thr Ile Lys Glu Ser
Tyr His Asn Thr Asn Gln Glu Asp Leu Leu Val 165
170 175 Leu Trp Gly Ile His His Pro Asn Asp Glu
Glu Glu Gln Thr Arg Ile 180 185
190 Tyr Lys Asn Pro Asn Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu
Asn 195 200 205 Gln
Arg Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln 210
215 220 Ser Gly Arg Val Glu Phe
Phe Trp Thr Ile Leu Lys Ser Asn Asp Thr 225 230
235 240 Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala
Pro Glu Asn Ala Tyr 245 250
255 Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu
260 265 270 Tyr Gly
Asn Cys Ser Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn 275
280 285 Ser Ser Met Pro Phe His Asn
Ile His Pro Leu Thr Ile Gly Glu Cys 290 295
300 Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala
Thr Gly Leu Arg 305 310 315
320 Asn Ser Pro Gln Glu Glu Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly
325 330 335 Ala Ile Ala
Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly 340
345 350 Trp Tyr Gly Tyr His His Ser Asn
Glu Gln Gly Ser Gly Tyr Ala Ala 355 360
365 Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr
Asn Lys Val 370 375 380
Asn Ser Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg 385
390 395 400 Glu Phe Asn Asn
Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Met 405
410 415 Glu Asp Gly Phe Leu Asp Val Trp Thr
Tyr Asn Ala Glu Leu Leu Val 420 425
430 Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn
Val Lys 435 440 445
Asn Leu Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu 450
455 460 Leu Gly Asn Gly Cys
Phe Glu Phe Tyr His Arg Cys Asp Asn Glu Cys 465 470
475 480 Met Glu Ser Val Arg Asn Gly Thr Tyr Asp
Tyr Pro Gln Tyr Ser Glu 485 490
495 Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu
Ser 500 505 510 Ile
Gly Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser 515
520 525 Leu Ala Leu Ala Ile Met
Val Ala Gly Leu Phe Leu Trp Met Cys Ser 530 535
540 Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 545
550 37511PRTH5N1misc_feature(1)..(1)Xaa
can be any naturally occurring amino acid 37Xaa His Ala Asn Asn Ser Thr
Glu Gln Val Asp Thr Ile Met Glu Lys 1 5
10 15 Asn Ile Thr Val Thr His Ala Gln Asp Ile Leu
Glu Lys Thr His Asn 20 25
30 Gly Lys Leu Cys Asp Leu Asp Gly Val Lys Pro Leu Ile Leu Arg
Asp 35 40 45 Cys
Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe 50
55 60 Leu Asn Val Ser Glu Trp
Ser Tyr Ile Val Glu Lys Ile Asn Pro Ala 65 70
75 80 Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn Asn
Tyr Glu Glu Leu Lys 85 90
95 His Leu Leu Ser Arg Ile Asn Arg Phe Glu Lys Ile Gln Ile Ile Ser
100 105 110 Lys Asn
Ser Trp Pro Asp His Glu Ala Ser Leu Gly Val Ser Ala Ala 115
120 125 Cys Pro Tyr Gln Gly Gly Leu
Ser Phe Tyr Arg Asn Val Val Trp Leu 130 135
140 Ile Glu Lys Asn Asn Thr Tyr Pro Leu Ile Lys Lys
Asn Tyr His Asn 145 150 155
160 Thr Asn Gln Glu Asp Leu Leu Val Leu Trp Gly Ile His His Pro Asn
165 170 175 Asp Glu Ala
Glu Gln Xaa Arg Leu Tyr Lys Asn Ser Thr Thr Tyr Ile 180
185 190 Ser Val Gly Thr Ser Thr Leu Xaa
Gln Arg Leu Val Pro Lys Ile Ala 195 200
205 Thr Arg Pro Lys Val Asn Gly Gln Ser Gly Arg Val Glu
Phe Phe Trp 210 215 220
Thr Ile Leu Lys Ser Asn Asp Val Ile Asn Phe Glu Ser Asn Gly Asn 225
230 235 240 Phe Ile Ala Pro
Glu Asn Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser 245
250 255 Thr Ile Met Lys Ser Asp Leu Glu Tyr
Gly Asn Cys Ser Thr Lys Cys 260 265
270 Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser Met Pro Phe His
Asn Ile 275 280 285
His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg 290
295 300 Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser Pro Gln Gly Glu Arg Arg 305 310
315 320 Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
Ala Gly Phe Ile Glu Gly 325 330
335 Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser
Asn 340 345 350 Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala 355
360 365 Ile Asp Gly Val Thr Asn
Lys Val Asn Ser Ile Ile Asp Lys Met Asn 370 375
380 Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn
Asn Leu Glu Arg Arg 385 390 395
400 Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp
405 410 415 Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu 420
425 430 Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr Asp Lys Val Arg Leu 435 440
445 Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly
Cys Phe Glu Phe 450 455 460
Tyr His Arg Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr 465
470 475 480 Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu Glu 485
490 495 Ile Ser Gly Ala Lys Leu Glu Ser
Ile Gly Thr Tyr Gln Ile Xaa 500 505
510 38537PRTH5N1misc_feature(1)..(1)Xaa can be any naturally
occurring amino acid 38Xaa Leu Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr
His Ala Asn Asn 1 5 10
15 Ser Thr Glu Gln Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr
20 25 30 His Ala Gln
Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp 35
40 45 Leu Asp Gly Val Lys Pro Leu Ile
Leu Arg Asp Cys Ser Val Ala Gly 50 55
60 Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Pro Asn
Val Ser Glu 65 70 75
80 Trp Ser Tyr Ile Val Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr
85 90 95 Pro Gly Asn Phe
Asn Asn Tyr Glu Glu Leu Lys His Leu Leu Ser Arg 100
105 110 Ile Asn Arg Phe Glu Lys Ile Lys Ile
Ile Pro Lys Ser Ser Trp Pro 115 120
125 Asp His Glu Ala Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr
Gln Arg 130 135 140
Gly Pro Ser Phe Tyr Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn 145
150 155 160 Thr Tyr Pro Thr Ile
Lys Lys Ser Tyr His Asn Thr Asn Gln Glu Asp 165
170 175 Leu Leu Val Leu Trp Gly Ile His His Pro
Asn Asp Glu Glu Glu Gln 180 185
190 Thr Arg Ile Tyr Lys Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr
Ser 195 200 205 Thr
Leu Asn Gln Arg Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val 210
215 220 Asn Gly Gln Ser Gly Arg
Val Glu Phe Phe Trp Thr Ile Leu Lys Ser 225 230
235 240 Asn Asp Thr Ile Asn Phe Glu Ser Asn Gly Asn
Phe Ile Ala Pro Glu 245 250
255 Asn Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser
260 265 270 Glu Leu
Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly 275
280 285 Ala Ile Asn Ser Ser Met Pro
Phe His Asn Ile His Pro Leu Thr Ile 290 295
300 Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu
Val Leu Ala Thr 305 310 315
320 Gly Leu Arg Asn Ser Pro Gln Gly Glu Gly Arg Arg Lys Lys Arg Gly
325 330 335 Leu Phe Gly
Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met 340
345 350 Val Asp Gly Trp Tyr Gly Tyr His
His Ser Asn Glu Gln Gly Ser Gly 355 360
365 Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp
Gly Val Thr 370 375 380
Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala 385
390 395 400 Val Gly Arg Glu
Phe Asn Asn Leu Glu Lys Arg Ile Glu Asn Leu Asn 405
410 415 Lys Lys Met Glu Asp Gly Phe Leu Asp
Val Trp Thr Tyr Asn Ala Glu 420 425
430 Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His
Asp Ser 435 440 445
Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn 450
455 460 Ala Lys Glu Leu Gly
Asn Gly Cys Phe Glu Phe Tyr His Arg Cys Asp 465 470
475 480 Asn Glu Cys Met Glu Ser Val Lys Asn Gly
Thr Tyr Asp Tyr Pro Gln 485 490
495 Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val
Lys 500 505 510 Leu
Glu Ser Ile Gly Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val 515
520 525 Ala Ser Ser Leu Ala Leu
Ala Ile Met 530 535 39534PRTH5N1 39Lys Ser
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu 1 5
10 15 Gln Val Asp Thr Ile Met Glu
Lys Asn Val Thr Val Thr His Ala Gln 20 25
30 Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys
Asp Leu Asp Gly 35 40 45
Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu
50 55 60 Gly Asn Pro
Met Cys Asp Glu Phe Pro Asn Val Ser Glu Trp Ser Tyr 65
70 75 80 Ile Val Glu Lys Ile Asn Pro
Ala Asn Asp Leu Cys Tyr Pro Gly Asn 85
90 95 Phe Asn Asn Tyr Glu Glu Leu Lys His Leu Leu
Ser Arg Ile Asn Arg 100 105
110 Phe Glu Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Pro Asp His
Glu 115 120 125 Ala
Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Gly Pro Ser 130
135 140 Phe Tyr Arg Asn Val Val
Trp Leu Ile Lys Lys Asn Asp Thr Tyr Pro 145 150
155 160 Thr Ile Lys Glu Ser Tyr His Asn Thr Asn Gln
Glu Asp Leu Leu Val 165 170
175 Leu Trp Gly Ile His His Pro Asn Asp Glu Glu Glu Gln Lys Arg Ile
180 185 190 Tyr Lys
Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn 195
200 205 Gln Arg Leu Val Pro Lys Ile
Ala Thr Arg Ser Lys Val Asn Gly Gln 210 215
220 Ser Gly Arg Val Glu Phe Phe Trp Thr Ile Leu Lys
Ser Asn Asp Thr 225 230 235
240 Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Asn Ala Tyr
245 250 255 Lys Ile Val
Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu 260
265 270 Tyr Gly Asn Cys Ser Thr Lys Cys
Gln Thr Pro Ile Gly Ala Ile Asn 275 280
285 Thr Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile
Gly Glu Cys 290 295 300
Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg 305
310 315 320 Asn Ser Pro Gln
Gly Glu Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly 325
330 335 Ala Ile Ala Gly Phe Ile Glu Gly Gly
Trp Gln Gly Met Val Asp Gly 340 345
350 Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr
Ala Ala 355 360 365
Asp Lys Glu Ser Thr Gln Lys Ala Ile Asn Gly Val Thr Asn Lys Val 370
375 380 Asn Ser Ile Ile Asp
Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg 385 390
395 400 Glu Phe Asn Asn Leu Glu Lys Arg Ile Glu
Asn Leu Asn Lys Lys Met 405 410
415 Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu
Val 420 425 430 Leu
Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Arg 435
440 445 Asn Leu Tyr Asp Lys Val
Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu 450 455
460 Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Arg
Cys Asp Asn Glu Cys 465 470 475
480 Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Lys
485 490 495 Glu Ala
Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser 500
505 510 Ile Gly Thr Tyr Gln Ile Leu
Ser Ile Tyr Ser Thr Val Ala Ser Ser 515 520
525 Leu Ala Leu Ala Ile Met 530
40536PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 40Xaa Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser 1
5 10 15 Thr Glu Gln
Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His 20
25 30 Ala Gln Asp Ile Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asp Leu 35 40
45 Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val
Ala Gly Trp 50 55 60
Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Pro Asn Val Ser Glu Trp 65
70 75 80 Ser Tyr Ile Val
Glu Lys Thr Asn Pro Ala Asn Asp Leu Cys Tyr Pro 85
90 95 Gly Asn Phe Asn Asn Tyr Glu Glu Leu
Lys His Leu Leu Ser Arg Ile 100 105
110 Asn Arg Phe Glu Lys Ile Lys Ile Ile Pro Lys Ser Ser Trp
Pro Asp 115 120 125
His Glu Ala Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Glu 130
135 140 Pro Ser Phe Tyr Arg
Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr 145 150
155 160 Tyr Pro Thr Ile Lys Glu Ser Tyr His Asn
Thr Asn Gln Glu Asp Leu 165 170
175 Leu Val Leu Trp Gly Ile His His Pro Asn Asp Glu Glu Glu Gln
Thr 180 185 190 Arg
Ile Tyr Lys Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr 195
200 205 Leu Asn Gln Arg Leu Val
Pro Lys Ile Ala Thr Arg Ser Lys Val Asn 210 215
220 Gly Gln Ser Gly Arg Val Glu Phe Phe Trp Thr
Ile Leu Lys Ser Asn 225 230 235
240 Asp Thr Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Asn
245 250 255 Ala Tyr
Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu 260
265 270 Leu Glu Tyr Gly Asn Cys Ser
Thr Lys Cys Gln Thr Pro Val Gly Ala 275 280
285 Ile Asn Ser Ser Met Pro Phe His Asn Ile His Pro
Leu Thr Ile Gly 290 295 300
Glu Cys Pro Lys Tyr Val Xaa Ser Asn Arg Leu Val Leu Ala Thr Gly 305
310 315 320 Leu Arg Asn
Ser Pro Gln Gly Glu Gly Arg Arg Lys Lys Arg Gly Leu 325
330 335 Phe Gly Ala Ile Ala Gly Phe Ile
Glu Gly Gly Trp Gln Gly Met Val 340 345
350 Asp Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly
Ser Gly Tyr 355 360 365
Ala Ala Asp Arg Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn 370
375 380 Lys Val Asn Ser
Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val 385 390
395 400 Gly Arg Glu Phe Asn Asn Leu Glu Lys
Arg Ile Glu Asn Leu Asn Lys 405 410
415 Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala
Glu Leu 420 425 430
Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn
435 440 445 Val Lys Asn Leu
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala 450
455 460 Lys Glu Leu Gly Asn Gly Cys Phe
Glu Phe Tyr His Arg Cys Asp Asn 465 470
475 480 Glu Cys Ile Glu Ser Val Arg Asn Gly Thr Tyr Asp
Tyr Pro Gln Tyr 485 490
495 Ser Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu
500 505 510 Glu Ser Ile
Gly Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala 515
520 525 Ser Ser Leu Ala Leu Ala Ile Met
530 535 41515PRTH5N1misc_feature(1)..(1)Xaa can
be any naturally occurring amino acid 41Xaa Leu Val Lys Ser Asp Gln Ile
Cys Ile Gly Tyr His Ala Asn Asn 1 5 10
15 Ser Thr Glu Gln Val Asp Thr Ile Met Glu Lys Asn Val
Thr Val Thr 20 25 30
His Ala Gln Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp
35 40 45 Leu Asp Gly Val
Lys Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly 50
55 60 Trp Leu Leu Gly Asn Pro Met Cys
Asp Glu Phe Pro Asn Val Ser Glu 65 70
75 80 Trp Ser Tyr Ile Val Glu Lys Ile Asn Pro Ala Asn
Asp Leu Cys Tyr 85 90
95 Pro Gly Asn Phe Asn Asn Tyr Glu Glu Leu Lys His Leu Leu Ser Arg
100 105 110 Ile Asn Arg
Phe Glu Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Pro 115
120 125 Asp His Glu Ala Ser Leu Gly Val
Ser Ser Ala Cys Pro Tyr Gln Gly 130 135
140 Gly Pro Ser Phe Tyr Arg Asn Val Val Trp Leu Ile Lys
Lys Asn Asp 145 150 155
160 Thr Tyr Pro Thr Ile Lys Glu Ser Tyr His Asn Thr Asn Gln Glu Asp
165 170 175 Leu Leu Val Leu
Trp Gly Ile His His Pro Asn Asp Glu Glu Glu Gln 180
185 190 Lys Arg Ile Tyr Lys Asn Pro Thr Thr
Tyr Val Ser Val Gly Thr Ser 195 200
205 Thr Leu Asn Gln Arg Leu Val Pro Lys Ile Ala Thr Arg Ser
Lys Val 210 215 220
Asn Gly Gln Ser Gly Arg Val Glu Phe Phe Trp Thr Ile Leu Lys Ser 225
230 235 240 Asn Asp Thr Ile Asn
Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu 245
250 255 Asn Ala Tyr Lys Ile Val Lys Lys Gly Asp
Ser Thr Ile Met Lys Ser 260 265
270 Glu Leu Glu Tyr Gly Asn Cys Ser Thr Lys Cys Gln Thr Pro Ile
Gly 275 280 285 Ala
Ile Asn Thr Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile 290
295 300 Gly Glu Cys Pro Lys Tyr
Val Lys Ser Asn Arg Leu Val Leu Ala Thr 305 310
315 320 Gly Leu Arg Asn Ser Pro Gln Gly Glu Gly Arg
Arg Lys Lys Arg Gly 325 330
335 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met
340 345 350 Val Asp
Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly 355
360 365 Tyr Ala Ala Asp Lys Glu Ser
Thr Gln Lys Ala Ile Asp Gly Val Thr 370 375
380 Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn Thr
Gln Phe Glu Ala 385 390 395
400 Val Gly Arg Glu Phe Asn Asn Leu Glu Lys Arg Ile Glu Asn Leu Asn
405 410 415 Lys Lys Met
Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu 420
425 430 Leu Leu Val Leu Met Glu Asn Glu
Arg Thr Leu Asp Phe His Asp Ser 435 440
445 Asn Val Arg Asn Leu Tyr Asp Lys Val Arg Leu Gln Leu
Arg Asp Asn 450 455 460
Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Arg Cys Asp 465
470 475 480 Asn Glu Cys Met
Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln 485
490 495 Tyr Ser Glu Glu Ala Arg Leu Lys Arg
Glu Glu Ile Ser Gly Val Lys 500 505
510 Leu Glu Xaa 515
42519PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 42Xaa Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser 1
5 10 15 Thr Glu Gln
Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His 20
25 30 Ala Gln Asp Ile Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asn Leu 35 40
45 Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val
Ala Gly Trp 50 55 60
Leu Leu Gly Asn Pro Met Cys Asp Lys Phe Leu Asn Val Pro Glu Trp 65
70 75 80 Ser Tyr Ile Val
Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro 85
90 95 Gly Asp Phe Asn Asp Tyr Glu Glu Leu
Lys His Leu Leu Ser Arg Ile 100 105
110 Asn His Phe Glu Lys Ile Gln Ile Ile Pro Lys Asn Ser Trp
Ser Asp 115 120 125
His Glu Ala Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Arg Ser 130
135 140 Ser Phe Phe Arg Asn
Val Val Trp Leu Thr Lys Lys Asn Asn Ala Tyr 145 150
155 160 Pro Thr Ile Lys Lys Ser Tyr Asn Asn Thr
Asn Gln Glu Asp Leu Leu 165 170
175 Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr
Met 180 185 190 Leu
Tyr Gln Asn Pro Thr Thr Tyr Val Ser Val Gly Thr Ser Thr Leu 195
200 205 Asn Gln Arg Leu Val Pro
Lys Ile Ala Thr Arg Ser Lys Val Asn Gly 210 215
220 Gln Ser Gly Arg Met Glu Phe Phe Trp Thr Ile
Leu Lys Ser Asn Asp 225 230 235
240 Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Asn Ala
245 250 255 Tyr Lys
Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu 260
265 270 Glu Tyr Gly Asn Cys Asn Thr
Lys Cys Gln Thr Pro Ile Gly Ala Ile 275 280
285 Asn Ser Ser Met Pro Phe His Asn Ile His Pro Leu
Thr Ile Gly Glu 290 295 300
Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu 305
310 315 320 Arg Asn Ser
Pro Gln Gly Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe 325
330 335 Gly Ala Ile Ala Gly Phe Ile Glu
Gly Gly Trp Gln Gly Met Val Asp 340 345
350 Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly Ser
Gly Tyr Ala 355 360 365
Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys 370
375 380 Val Asn Ser Ile
Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly 385 390
395 400 Arg Glu Phe Asn Asn Leu Glu Arg Arg
Ile Glu Asn Leu Asn Lys Lys 405 410
415 Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu
Leu Leu 420 425 430
Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
435 440 445 Lys Asn Leu Tyr
Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys 450
455 460 Glu Leu Gly Asn Gly Cys Phe Glu
Phe Tyr His Arg Cys Asp Asn Glu 465 470
475 480 Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser 485 490
495 Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu
500 505 510 Ser Ile Gly
Thr Xaa Gln Ile 515
43510PRTH5N1misc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 43Xaa His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile Met Glu Lys 1
5 10 15 Asn Val Thr
Val Thr His Ala Gln Asp Ile Leu Glu Lys Thr His Asn 20
25 30 Gly Lys Leu Cys Asn Leu Asp Gly
Val Lys Pro Leu Ile Leu Arg Asp 35 40
45 Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys
Asp Glu Phe 50 55 60
Pro Asn Val Leu Glu Trp Ser Tyr Ile Val Glu Lys Ile Asn Pro Ala 65
70 75 80 Asn Asp Leu Cys
Tyr Pro Gly Asn Phe Asn Asp Tyr Glu Glu Leu Lys 85
90 95 His Leu Leu Ser Arg Ile Asn His Phe
Glu Lys Ile Gln Ile Ile Pro 100 105
110 Lys Asn Ser Trp Ser Asp His Glu Ala Ser Gly Val Ser Ser
Ala Cys 115 120 125
Pro Tyr Gln Arg Arg Ser Ser Phe Phe Arg Asn Val Val Trp Leu Thr 130
135 140 Lys Lys Asn Asn Ala
Tyr Pro Thr Ile Lys Lys Ser Tyr Asn Asn Thr 145 150
155 160 Asn Gln Glu Asp Leu Leu Val Leu Trp Gly
Ile His His Pro Asn Asp 165 170
175 Ala Ala Glu Gln Thr Arg Leu Tyr Gln Asn Pro Thr Thr Tyr Ile
Ser 180 185 190 Val
Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro Lys Ile Ala Thr 195
200 205 Arg Ser Lys Val Asn Gly
Gln Ser Gly Arg Met Glu Phe Phe Trp Thr 210 215
220 Ile Leu Lys Ser Asn Asp Ala Ile Asn Phe Glu
Ser Asn Gly Asn Phe 225 230 235
240 Ile Ala Pro Glu Asn Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr
245 250 255 Ile Met
Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gln 260
265 270 Thr Pro Ile Gly Ala Ile Asn
Ser Ser Met Pro Phe His Asn Ile His 275 280
285 Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys
Ser Asn Arg Leu 290 295 300
Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Gly Glu Arg Arg Arg 305
310 315 320 Lys Lys Arg
Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly 325
330 335 Trp Gln Gly Met Val Asp Gly Trp
Tyr Gly Tyr His His Ser Asn Glu 340 345
350 Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln
Lys Ala Ile 355 360 365
Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn Thr 370
375 380 Gln Phe Glu Ala
Val Gly Arg Glu Phe Asn Asn Leu Glu Arg Arg Ile 385 390
395 400 Glu Asn Leu Asn Lys Lys Met Glu Asp
Gly Phe Leu Asp Val Trp Thr 405 410
415 Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr
Leu Asp 420 425 430
Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Leu Gln
435 440 445 Leu Arg Asp Asn
Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr 450
455 460 His Arg Cys Asp Asn Glu Cys Met
Glu Ser Val Arg Asn Gly Thr Tyr 465 470
475 480 Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys
Arg Glu Glu Ile 485 490
495 Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Xaa Gln Ile Xaa
500 505 510
44523PRTH5N1misc_feature(523)..(523)Xaa can be any naturally occurring
amino acid 44His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile Met Glu Lys
Asn 1 5 10 15 Val
Thr Val Thr His Ala Gln Asp Ile Leu Glu Lys Thr His Asn Gly
20 25 30 Lys Leu Cys Asp Leu
Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys 35
40 45 Ser Val Ala Gly Trp Leu Leu Gly Asn
Pro Met Cys Asp Glu Phe Pro 50 55
60 Asn Val Ser Glu Trp Ser Tyr Ile Val Glu Lys Ile Asn
Pro Ala Asn 65 70 75
80 Asp Leu Cys Tyr Pro Gly Asn Phe Asn Asn Tyr Glu Glu Leu Lys His
85 90 95 Leu Leu Ser Arg
Ile Asn Arg Phe Glu Lys Ile Lys Ile Ile Pro Lys 100
105 110 Ser Ser Trp Pro Asp His Glu Ala Ser
Leu Gly Val Ser Ser Ala Cys 115 120
125 Pro Tyr Gln Gly Gly Pro Ser Phe Tyr Arg Asn Val Val Trp
Leu Ile 130 135 140
Lys Lys Asn Asn Thr Tyr Pro Thr Ile Lys Lys Ser Tyr His Asn Thr 145
150 155 160 Asn Gln Glu Asp Leu
Leu Val Leu Trp Gly Ile His His Pro Asn Asp 165
170 175 Glu Glu Glu Gln Thr Arg Ile Tyr Lys Asn
Pro Thr Thr Tyr Ile Ser 180 185
190 Val Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro Lys Ile Ala
Thr 195 200 205 Arg
Ser Lys Val Asn Gly Gln Ser Gly Arg Val Glu Phe Phe Trp Thr 210
215 220 Ile Leu Lys Ser Asn Asp
Thr Ile Asn Phe Glu Ser Asn Gly Asn Phe 225 230
235 240 Ile Ala Pro Glu Asn Ala Tyr Lys Ile Val Lys
Lys Gly Asp Ser Thr 245 250
255 Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gln
260 265 270 Thr Pro
Ile Gly Ala Ile Asn Ser Ser Met Pro Phe His Asn Ile His 275
280 285 Pro Leu Thr Ile Gly Glu Cys
Pro Lys Tyr Val Lys Ser Asn Arg Leu 290 295
300 Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Gly
Glu Gly Arg Arg 305 310 315
320 Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly
325 330 335 Trp Gln Gly
Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn Glu 340
345 350 Gln Gly Ser Gly Tyr Ala Ala Asp
Lys Glu Ser Thr Gln Lys Ala Ile 355 360
365 Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys
Met Asn Thr 370 375 380
Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Lys Arg Ile 385
390 395 400 Glu Asn Leu Asn
Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr 405
410 415 Tyr Asn Ala Glu Leu Leu Val Leu Met
Glu Asn Glu Arg Thr Leu Asp 420 425
430 Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg
Leu Gln 435 440 445
Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr 450
455 460 His Arg Cys Asp Asn
Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr 465 470
475 480 Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg
Leu Lys Arg Glu Glu Ile 485 490
495 Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln Ile Leu Ile
Tyr 500 505 510 Ser
Thr Val Ala Ser Ser Leu Ala Leu Ala Xaa 515 520
45541PRTH5N1 45Leu Leu Ala Ile Val Ser Leu Val Lys Ser Asp Gln
Ile Cys Ile Gly 1 5 10
15 Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile Met Glu Lys
20 25 30 Asn Val Thr
Val Thr His Ala Gln Asp Ile Leu Glu Lys Thr His Asn 35
40 45 Gly Lys Leu Cys Asp Leu Asp Gly
Val Lys Pro Leu Ile Leu Arg Asp 50 55
60 Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys
Asp Glu Phe 65 70 75
80 Pro Asn Val Ser Glu Trp Ser Tyr Ile Val Glu Lys Ile Asn Pro Ala
85 90 95 Asn Asp Leu Cys
Tyr Pro Gly Asn Phe Asn Asn Tyr Glu Glu Leu Lys 100
105 110 His Leu Leu Ser Arg Ile Asn Arg Phe
Glu Lys Ile Lys Ile Ile Pro 115 120
125 Lys Ser Ser Trp Pro Asp His Glu Ala Ser Leu Gly Val Ser
Ser Ala 130 135 140
Cys Pro Tyr Gln Gly Gly Pro Ser Phe Tyr Arg Asn Val Val Trp Leu 145
150 155 160 Ile Lys Lys Asn Asn
Thr Tyr Pro Thr Ile Lys Glu Ser Tyr His Asn 165
170 175 Ile Asn Lys Glu Asp Leu Leu Val Leu Trp
Gly Ile His His Pro Asn 180 185
190 Asp Glu Glu Glu Gln Ile Arg Ile Tyr Lys Asn Pro Thr Thr Tyr
Ile 195 200 205 Ser
Val Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro Lys Ile Ala 210
215 220 Thr Arg Ser Lys Val Asn
Gly Gln Ser Gly Arg Val Glu Phe Phe Trp 225 230
235 240 Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn Phe
Glu Ser Asn Gly Asn 245 250
255 Phe Ile Ala Pro Glu Asn Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser
260 265 270 Thr Ile
Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys Cys 275
280 285 Gln Thr Pro Ile Gly Ala Ile
Asn Ser Ser Met Pro Phe His Asn Ile 290 295
300 His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val
Lys Ser Asn Arg 305 310 315
320 Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Gly Glu Gly Arg
325 330 335 Arg Lys Lys
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 340
345 350 Gly Trp Gln Gly Met Val Asp Gly
Trp Tyr Gly Tyr His His Ser Asn 355 360
365 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr
Gln Lys Ala 370 375 380
Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn 385
390 395 400 Thr Gln Phe Glu
Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Lys Arg 405
410 415 Ile Glu Asn Leu Asn Lys Lys Met Glu
Asp Gly Phe Leu Asp Val Trp 420 425
430 Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg
Thr Leu 435 440 445
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Leu 450
455 460 Gln Leu Arg Asp Asn
Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe 465 470
475 480 Tyr His Arg Cys Asp Asn Glu Cys Met Glu
Ser Val Arg Asn Gly Thr 485 490
495 Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu
Glu 500 505 510 Ile
Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln Ile Leu Ser 515
520 525 Ile Tyr Ser Thr Val Ala
Ser Ser Leu Ala Leu Ala Ile 530 535
540 46555PRTH5N1misc_feature(1)..(1)Xaa can be any naturally
occurring amino acid 46Xaa Leu Ala Ile Val Ser Leu Val Lys Ser Asp Gln
Ile Cys Ile Gly 1 5 10
15 Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile Met Glu Lys
20 25 30 Asn Val Thr
Val Thr His Ala Gln Asp Ile Leu Glu Lys Thr His Asn 35
40 45 Gly Lys Leu Cys Asp Leu Asp Gly
Val Lys Pro Leu Ile Leu Arg Asp 50 55
60 Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys
Asp Glu Phe 65 70 75
80 Pro Asn Val Ser Glu Trp Ser Tyr Ile Val Glu Lys Ile Asn Pro Ala
85 90 95 Asn Asp Leu Cys
Tyr Pro Gly Asn Phe Asn Asn Tyr Glu Glu Leu Lys 100
105 110 His Leu Leu Ser Arg Ile Asn Arg Phe
Glu Lys Ile Lys Ile Ile Pro 115 120
125 Lys Ser Ser Trp Pro Asp His Glu Ala Ser Leu Gly Val Ser
Ser Ala 130 135 140
Cys Pro Tyr Gln Gly Gly Pro Ser Phe Tyr Arg Asn Val Val Trp Leu 145
150 155 160 Thr Lys Lys Asn Asn
Thr Tyr Pro Thr Ile Lys Lys Ser Tyr His Asn 165
170 175 Ile Asn Lys Glu Asp Leu Leu Val Leu Trp
Gly Ile His His Pro Asn 180 185
190 Asp Glu Glu Glu Gln Ile Arg Ile Tyr Lys Asn Pro Thr Thr Tyr
Ile 195 200 205 Ser
Val Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro Lys Ile Ala 210
215 220 Thr Arg Ser Lys Val Asn
Gly Gln Ser Gly Arg Val Glu Phe Phe Trp 225 230
235 240 Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn Phe
Glu Ser Asn Gly Asn 245 250
255 Phe Ile Ala Pro Glu Asn Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser
260 265 270 Thr Ile
Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys Cys 275
280 285 Gln Thr Pro Ile Gly Ala Ile
Asn Ser Ser Met Pro Phe His Asn Ile 290 295
300 His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val
Lys Ser Asn Arg 305 310 315
320 Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Gly Glu Gly Arg
325 330 335 Arg Lys Lys
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 340
345 350 Gly Trp Gln Gly Met Val Asp Gly
Trp Tyr Gly Tyr His His Ser Asn 355 360
365 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr
Gln Lys Ala 370 375 380
Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn 385
390 395 400 Thr Gln Phe Glu
Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Lys Arg 405
410 415 Ile Glu Asn Leu Asn Lys Lys Met Glu
Asp Gly Phe Leu Asp Val Trp 420 425
430 Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg
Thr Leu 435 440 445
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Leu 450
455 460 Gln Leu Arg Asp Asn
Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe 465 470
475 480 Tyr His Arg Cys Asp Asn Glu Cys Met Glu
Ser Val Arg Asn Gly Thr 485 490
495 Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu
Glu 500 505 510 Ile
Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln Ile Leu Ser 515
520 525 Ile Tyr Ser Thr Val Ala
Ser Ser Leu Ala Leu Ala Ile Met Met Ala 530 535
540 Gly Leu Phe Leu Trp Met Cys Ser Asn Gly Xaa
545 550 555 47568PRTArtificialconsensus
sequence translated into protein 47Met Glu Lys Ile Val Leu Leu Leu Ala
Ile Val Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu
Gln Val 20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45 Leu Glu Lys Thr
His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50
55 60 Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Pro Asn Val Ser Glu Trp
Ser Tyr Ile Val 85 90
95 Glu Lys Ile Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asn Tyr Glu
Glu Leu Lys His Leu Leu Ser Arg Ile Asn Arg Phe Glu 115
120 125 Lys Ile Gln Ile Ile Pro Lys Ser
Ser Trp Pro Asp His Glu Ala Ser 130 135
140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Gly Pro
Ser Phe Tyr 145 150 155
160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile
165 170 175 Lys Glu Ser Tyr
His Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180
185 190 Gly Ile His His Pro Asn Asp Glu Glu
Glu Gln Thr Arg Ile Tyr Lys 195 200
205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn
Gln Arg 210 215 220
Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225
230 235 240 Arg Val Glu Phe Phe
Trp Thr Ile Leu Lys Ser Asn Asp Thr Ile Asn 245
250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro
Glu Asn Ala Tyr Lys Ile 260 265
270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr
Gly 275 280 285 Asn
Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser 290
295 300 Met Pro Phe His Asn Ile
His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310
315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser 325 330
335 Pro Gln Gly Glu Gly Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340 345 350 Ala Gly
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355
360 365 Gly Tyr His His Ser Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375
380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser 385 390 395
400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415 Asn Asn Leu
Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420
425 430 Gly Phe Leu Asp Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu 450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465
470 475 480 Asn Gly Cys Phe
Glu Phe Tyr His Arg Cys Asp Asn Glu Cys Met Glu 485
490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala 500 505
510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
Ile Gly 515 520 525
Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530
535 540 Leu Ala Ile Met Val
Ala Gly Leu Phe Leu Trp Met Cys Ser Asn Gly 545 550
555 560 Ser Leu Gln Cys Arg Ile Cys Ile
565 481620DNAH5N1 48caatagtcag tattgttaaa agtgatcaga
tttgcattgg ttaccatgca aacaactcga 60cagagcaggt tgacacaata atggaaaaga
acgtcactgt tacacacgcc caagacatac 120tggaaaagac acacaacggg aaactctgca
atctagatgg agtgaagcct ctaattttaa 180gagattgtag tgtagctgga tggctcctag
ggaacccaat gtgcgacgaa ttcctcaatg 240tgccggaatg gtcttacata gtggagaaga
tcaatccaac caatgacctc tgttatccag 300ggaatttcaa cgactatgaa gaactgaaac
acctattgag cagaataaac cattttgaga 360aaattcagat cattcccaaa aattcttggt
cagatcatga agcctcagga gtgagctcag 420catgtccata ccagggaaga tcctcctttt
ttagaaatgt ggtatggctt accaaaaaga 480acaatgcata cccaacaata aagaaaagtt
acaataatac caaccaagaa gatcttttgg 540tattatgggg gattcaccat ccaaatgatg
cggcagagca gacaaggctt tatcaaaacc 600caactaccta tatttccgtt gggacatcaa
cactaaacca gagattggta cccaaaatag 660ctactagatc taaggtaaac gggcaaagtg
gaaggatgga gttcttttgg acaattttaa 720aatcgaatga tgcaataaac tttgagagta
atggaaattt cattgctcca gaaaatgcat 780acaaaattgt caagaaaggg gactcaacaa
ttatgaaaag tgagttggaa tatggtgact 840gcaacaccaa gtgtcagact ccaatagggg
cgataaactc cagtatgcca ttccacaaca 900tccaccctct caccatcggg gaatgcccca
aatatgtgaa atcaaacaga ttagtccttg 960ctactgggct cagaaatagc cctcaaggag
agagaagaag aaaaaagaga ggactatttg 1020gagctatagc aggttttata gagggaggat
ggcagggaat ggtagatggt tggtatgggt 1080accaccatag caacgagcag gggagtgggt
acgctgcaga caaagaatcc actcaaaagg 1140caatagatgg agtcaccaat aaggtcaact
cgatcattga caaaatgaac actcagtttg 1200aggctgttgg gagggaattt aataacttag
aaaggagaat agaaaattta aacaagaaga 1260tggaagacgg attcctagat gtctggactt
ataatgctga acttctggtt ctcatggaaa 1320atgagagaac tctagacttt catgactcaa
atgtcaagaa cctttatgac aaggtccgac 1380tacagcttag ggataatgca aaggagcttg
gtaacggttg tttcgagttc tatcacagat 1440gtgataatga atgtatggaa agtgtaagaa
acggtacgta tgactacccg cagtattcag 1500aagaagcaag attaaaaaga gaggaaataa
gtggagtaaa attggagtca ataggaactt 1560accaaatact gtcaatttat tcaacagtgg
cgagctccct agcactggca atcatggtgg 1620491707DNAH5N1 49atggagaaaa
tagtgcttct tcttgcaata gtcagtcttg ttaaaagtga tcagatttgc 60attggttacc
atgcaaacaa ctcaacagag caggttgaca caataatgga aaagaacgtc 120actgttacac
acgctcaaga catactggaa aagacacaca atgggaaact ctgcgatcta 180ggtggagtga
agcctctaat tttaagagat tgtagtgtag ctggatggct cctcgggaac 240ccaatgtgtg
acgaattccc caatgtgtcg gaatggtcct acatagtgga gaagatcaat 300ccagccaatg
acctctgtta cccagggaat ttcaacaact atgaagaact gaaacatcta 360ttgagcagaa
taaaccggtt tgagaaaatt cagatcatcc ccaaaagttc ttggccagat 420catgaagcct
cattaggagt gagctcagca tgtccatacc agggaggacc ctccttttat 480agaaatgtgg
tatggcttat caaaaagaac gatacatacc caacaataaa ggaaagttac 540cataatacca
atcaagaaga tcttttggtg ctgtggggga tccaccatcc aaataatgag 600gaagaacaga
aaaggatcta taaaaaccca actacctatg tttccgttgg gacatcaaca 660ctaaaccaga
gattggtacc gaagatagcc actagatcta aggtaaacgg gcaaagtgga 720agagtggagt
tcttttggac aattttaaaa tcaaatgata caataaactt tgagagtaat 780ggaaatttca
ttgctccaga aaatgcatac aaaattgtca agaaagggga ctcaacaatt 840atgaaaagtg
agttggaata tggtaactgc agcaccaagt gtcaaactcc aataggggcg 900ataaacacca
gtatgccatt ccacaacatc caccctctca ccatcgggga atgccccaaa 960tatgtgaaat
caaacagatt agtccttgct actgggctta gaaatagccc tcaaggagag 1020ggaagaagaa
aaaagagagg actatttgga gctatagcag gttttataga gggaggatgg 1080cagggaatgg
tagatggttg gtatgggtac caccatagta acgagcaggg gagtgggtac 1140gctgcagaca
aagaatccac tcaaaaggca atagatggag tcaccaataa ggtcaactcg 1200atcattgaca
aaatgaatac tcagtttgag gctgttggga gggaatttaa taacttggaa 1260aagagaatag
aaaatttaaa caagaagatg gaagacgggt tcctagatgt ctggacttat 1320aatgctgaac
ttctggttct catggaaaat gagagaactc tagactttca tgactcaaat 1380gtcaggaacc
tttacgacaa ggtgcgacta cagcttaggg acaatgcaaa ggagcttggt 1440aacggttgtt
tcgagttcta tcacagatgc gataatgaat gtatggaaag tgtaagaaac 1500ggaacgtatg
actacccgca gtattcagaa gaagcaagat taaaaagaga ggaaataagt 1560ggagtaaaat
tggaatcaat aggaacttac caaatactat caatttattc aacagtggca 1620agttccctag
cactggcaat catggtggct ggtctatttt tatggatgtg ctccaatgga 1680tcgttacaat
gcagaatttg catttaa
1707501707DNAArtificialconsensus of 38 H5 gene sequences 50atggagaaaa
tagtgcttct tcttgcaata gtcagtcttg ttaaaagtga tcagatttgc 60attggttacc
atgcaaacaa ctcaacagag caggttgaca caataatgga aaagaacgtc 120actgttacac
acgctcaaga catactggaa aagacacaca acgggaaact ctgcgatcta 180gatggagtga
agcctctaat tttaagagat tgtagtgtag ctggatggct cctcgggaac 240ccaatgtgtg
acgaattccc caatgtgtcg gaatggtcct acatagtgga gaagatcaat 300ccagccaatg
acctctgtta cccagggaat ttcaacaact atgaagaact gaaacaccta 360ttgagcagaa
taaaccggtt tgagaaaatt cagatcatcc ccaaaagttc ttggccagat 420catgaagcct
cattaggagt gagctcagca tgtccatacc agggaggacc ctccttttat 480agaaatgtgg
tatggcttat caaaaagaac aatacatacc caacaataaa ggaaagttac 540cataatacca
atcaagaaga tcttttggtg ctgtggggga ttcaccatcc aaatgatgag 600gaagagcaga
caaggatcta taaaaaccca actacctata tttccgttgg gacatcaaca 660ctaaaccaga
gattggtacc aaagatagcc actagatcta aggtaaacgg gcaaagtgga 720agagtggagt
tcttttggac aattttaaaa tcaaatgata caataaactt tgagagtaat 780ggaaatttca
ttgctccaga aaatgcatac aaaattgtca agaaagggga ctcaacaatt 840atgaaaagtg
agttggaata tggtaactgc aacaccaagt gtcaaactcc aataggggcg 900ataaactcca
gtatgccatt ccacaacatc caccctctca ccatcgggga atgccccaaa 960tatgtgaaat
caaacagatt agtccttgct actgggctca gaaatagccc tcaaggagag 1020ggaagaagaa
aaaagagagg actatttgga gctatagcag gttttataga gggaggatgg 1080cagggaatgg
tagatggttg gtatgggtac caccatagca acgagcaggg gagtgggtac 1140gctgcagaca
aagaatccac tcaaaaggca atagatggag tcaccaataa ggtcaactcg 1200atcattgaca
aaatgaatac tcagtttgag gctgttggga gggaatttaa taacttggaa 1260aagagaatag
aaaatttaaa caagaagatg gaagacgggt tcctagatgt ctggacttat 1320aatgctgaac
ttctggttct catggaaaat gagagaactc tagactttca tgactcaaat 1380gtcaagaacc
tttacgacaa ggtgcgacta cagcttaggg acaatgcaaa ggagcttggt 1440aacggttgtt
tcgagttcta tcacagatgc gataatgaat gtatggaaag tgtaagaaac 1500ggaacgtatg
actacccgca gtattcagaa gaagcaagat taaaaagaga ggaaataagt 1560ggagtaaaat
tggaatcaat aggaacttac caaatactat caatttattc aacagtggca 1620agttccctag
cactggcaat catggtggct ggtctatttt tatggatgtg ctccaatgga 1680tcgttacaat
gcagaatttg catttaa
17075115186DNANewcastle disease virus 51accaaacaga gaatccgtga gttacgataa
aaggcgaagg agcaattgaa gtcgcacggg 60tagaaggtgt gaatctcgag tgcgagcccg
aagcacaaac tcgagaaagc cttctgccaa 120catgtcttcc gtatttgatg agtacgaaca
gctcctcgcg gctcagactc gccccaatgg 180agctcatgga gggggagaaa aagggagtac
cttaaaagta gacgtcccgg tattcactct 240taacagtgat gacccagaag atagatggag
ctttgtggta ttctgcctcc ggattgctgt 300tagcgaagat gccaacaaac cactcaggca
aggtgctctc atatctcttt tatgctccca 360ctcacaggta atgaggaacc atgttgccct
tgcagggaaa cagaatgaag ccacattggc 420cgtgcttgag attgatggct ttgccaacgg
cacgccccag ttcaacaata ggagtggagt 480gtctgaagag agagcacaga gatttgcgat
gatagcagga tctctccctc gggcatgcag 540caacggaacc ccgttcgtca cagccggggc
cgaagatgat gcaccagaag acatcaccga 600taccctggag aggatcctct ctatccaggc
tcaagtatgg gtcacagtag caaaagccat 660gactgcgtat gagactgcag atgagtcgga
aacaaggcga atcaataagt atatgcagca 720aggcagggtc caaaagaaat acatcctcta
ccccgtatgc aggagcacaa tccaactcac 780gatcagacag tctcttgcag tccgcatctt
tttggttagc gagctcaaga gaggccgcaa 840cacggcaggt ggtacctcta cttattataa
cctggtaggg gacgtagact catacatcag 900gaataccggg cttactgcat tcttcttgac
actcaagtac ggaatcaaca ccaagacatc 960agcccttgca cttagtagcc tctcaggcga
catccagaag atgaagcagc tcatgcgttt 1020gtatcggatg aaaggagata atgcgccgta
catgacatta cttggtgata gtgaccagat 1080gagctttgcg cctgccgagt atgcacaact
ttactccttt gccatgggta tggcatcagt 1140cctagataaa ggtactggga aataccaatt
tgccagggac tttatgagca catcattctg 1200gagacttgga gtagagtacg ctcaggctca
gggaagtagc attaacgagg atatggctgc 1260cgagctaaag ctaaccccag cagcaaggag
gggcctggca gctgctgccc aacgggtctc 1320cgaggagacc agcagcatag acatgcctac
tcaacaagtc ggagtcctca ctgggcttag 1380cgaggggggg tcccaagctc tacaaggcgg
atcgaataga tcgcaagggc aaccagaagc 1440cggggatggg gagacccaat tcctggatct
gatgagagcg gtagcaaata gcatgaggga 1500ggcgccaaac tctgcacagg gcactcccca
atcggggcct cccccaactc ctgggccatc 1560ccaagataac gacaccgact gggggtattg
ataaacaaaa cccagcctgc ttccacaaaa 1620acatcccaat gccctcaccc gtagtcgacc
cctcgatttg cggctctata tgaccacacc 1680ctcaaacaaa catccccctc tttcctccct
ccccctgctg tacaactccg cacgccctag 1740ataccacagg cacaatgcgg ctcactaaca
atcaaaacag agccgaggga attagaaaaa 1800agtacgggta gaagagggat attcagagat
cagggcaagt ctcccgagtc tctgctctct 1860cctctacctg atagaccagg acaaacatgg
ccacctttac ggatgcagag atcgacgagc 1920tatttgagac aagtggaact gtcattgaca
acataattac agcccagggt aaaccagcag 1980agactgttgg aaggagtgca atcccacaag
gcaagaccaa ggtgctgagc gcagcatggg 2040agaagcatgg gagcatccag ccaccggcca
gtcaagacaa ccccgatcga caggacagat 2100ctgacaaaca accatccaca cccgagcaaa
cgaccccgca tgacagcccg ccggccacat 2160ccgccgacca gccccccacc caggccacag
acgaagccgt cgacacacag ctcaggaccg 2220gagcaagcaa ctctctgctg ttgatgcttg
acaagctcag caataaatcg tccaatgcta 2280aaaagggccc atggtcgagc ccccaagagg
ggaatcacca acgtccgact caacagcagg 2340ggagtcaacc cagtcgcgga aacagtcagg
aaagaccgca gaaccaagtc aaggccgccc 2400ctggaaacca gggcacagac gtgaacacag
catatcatgg acaatgggag gagtcacaac 2460tatcagctgg tgcaacccct catgctctcc
gatcaaggca gagccaagac aatacccttg 2520tatctgcgga tcatgtccag ccacctgtag
actttgtgca agcgatgatg tctatgatgg 2580aggcgatatc acagagagta agtaaggttg
actatcagct agatcttgtc ttgaaacaga 2640catcctccat ccctatgatg cggtccgaaa
tccaacagct gaaaacatct gttgcagtca 2700tggaagccaa cttgggaatg atgaagattc
tggatcccgg ttgtgccaac atttcatctc 2760tgagtgatct acgggcagtt gcccgatctc
acccggtttt agtttcaggc cctggagacc 2820cctctcccta tgtgacacaa ggaggcgaaa
tggcacttaa taaactttcg caaccagtgc 2880cacatccatc tgaattgatt aaacccgcca
ctgcatgcgg gcctgatata ggagtggaaa 2940aggacactgt ccgtgcattg atcatgtcac
gcccaatgca cccgagttct tcagccaagc 3000tcctaagcaa gttagatgca gccgggtcga
tcgaggaaat caggaaaatc aagcgccttg 3060ctctaaatgg ctaattacta ctgccacacg
cagcgggtcc ctgtccactc ggcatcacac 3120ggaatctgca ccgagttccc ccccgcagac
ccaaggtcca actctccaag cggcaatcct 3180ctctcgcttc ctcagcccca ctgaatggac
gcgtaaccgt aattaatcta gctacattta 3240agattaagaa aaaatacggg tagaattgga
gtgccccaat tgtgccaaga tggactcatc 3300taggacaatt gggctgtact ttgattctgc
ccattcttct agcaacctgt tagcatttcc 3360gatcgtccta caaggcacag gagatgggaa
gaagcaaatc gccccgcaat ataggatcca 3420gcgccttgac ttgtggactg atagtaagga
ggactcagta ttcatcacca cctatggatt 3480catctttcaa gttgggaatg aagaagccac
tgtcggcatg atcgatgata aacccaagcg 3540cgagttactt tccgctgcga tgctctgcct
aggaagcgtc ccaaataccg gagaccttat 3600tgagctggca agggcctgtc tcactatgat
agtcacatgc aagaagagtg caactaatac 3660tgagagaatg gttttctcag tagtgcaggc
accccaagtg ctgcaaagct gtagggttgt 3720ggcaaacaaa tactcatcag tgaatgcagt
caagcacgtg aaagcgccag agaagattcc 3780cgggagtgga accctagaat ataaggtgaa
ctttgtctcc ttgactgtgg taccgaagaa 3840ggatgtctac aagatcccag ctgcagtatt
gaaggtttct ggctcgagtc tgtacaatct 3900tgcgctcaat gtcactatta atgtggaggt
tgacccgagg agtcctttgg ttaaatctct 3960gtctaagtct gacagcggat actatgctaa
cctcttcttg catattggac ttatgaccac 4020cgtagatagg aaggggaaga aagtgacatt
tgacaagctg gaaaagaaaa taaggagcct 4080tgatctatct gtcgggctca gtgatgtgct
cgggccttcc gtgttggtaa aagcaagagg 4140tgcacggact aagcttttgg cacctttctt
ctctagcagt gggacagcct gctatcccat 4200agcaaatgct tctcctcagg tggccaagat
actctggagt caaaccgcgt gcctgcggag 4260cgttaaaatc attatccaag caggtaccca
acgcgctgtc gcagtgaccg ccgaccacga 4320ggttacctct actaagctgg agaaggggca
cacccttgcc aaatacaatc cttttaagaa 4380ataagctgcg tctctgagat tgcgctccgc
ccactcaccc agatcatcat gacacaaaaa 4440actaatctgt cttgattatt tacagttagt
ttacctgcct atcaagttag aaaaaacacg 4500ggtagaagat tctggatccc ggttggcgcc
ctccaggtgc aagatgggct ccagaccttc 4560taccaagaac ccagcaccta tgatgctgac
tatccgggtt gcgctggtac tgagttgcat 4620ctgtccggca aactccattg atggcaggcc
tcttgcagct gcaggaattg tggttacagg 4680agacaaagcc gtcaacatat acacctcatc
ccagacagga tcaatcatag ttaagctcct 4740cccgaatctg cccaaggata aggaggcatg
tgcgaaagcc cccttggatg catacaacag 4800gacattgacc actttgctca ccccccttgg
tgactctatc cgtaggatac aagagtctgt 4860gactacatct ggagggggga gacaggggcg
ccttataggc gccattattg gcggtgtggc 4920tcttggggtt gcaactgccg cacaaataac
agcggccgca gctctgatac aagccaaaca 4980aaatgctgcc aacatcctcc gacttaaaga
gagcattgcc gcaaccaatg aggctgtgca 5040tgaggtcact gacggattat cgcaactagc
agtggcagtt gggaagatgc agcagtttgt 5100taatgaccaa cttaataaaa cagctcagga
attagactgc atcaaaattg cacagcaagt 5160tggtgtagag ctcaacctgt acctaaccga
attgactaca gtattcggac cacaaatcac 5220ttcacctgct ttaaacaagc tgactattca
ggcactttac aatctagctg gtggaaatat 5280ggattactta ttgactaagt taggtgtagg
gaacaatcaa ctcagctcat taatcggtag 5340cggcttaatc accggtaacc ctattctata
cgactcacag actcaactct tgggtatacg 5400ggtaactcta ccttcagtcg ggaacctaaa
taatatgcgt gccacctact tggaaacctt 5460atccgtaagc acaaccaggg gatttgcctc
ggcacttgtc cccaaagtgg tgacacaggt 5520cggttctgtg atagaagaac ttgacacctc
atactgtata gaaactgact tagatttata 5580ttgtacaaga atagtaacgt tccctatgtc
ccctggtatt tattcctgct tgagcggcaa 5640tacgtcggcc tgtatgtact caaagaccga
aggcgcactt actacaccat acatgactat 5700caaaggttca gtcatcgcca actgcaagat
gacaacatgt agatgtgtaa accccccggg 5760tatcatatcg caaaactatg gagaagccgt
gtctctaata gataaacaat catgcaatgt 5820tttatcctta ggcgggataa ctttaaggct
cagtggggaa ttcgatgtaa cttatcagaa 5880gaatatctca atacaagatt ctcaagtaat
aataacaggc aatcttgata tctcaactga 5940gcttgggaat gtcaacaact cgatcagtaa
tgctttgaat aagttagagg aaagcaacag 6000aaaactagac aaagtcaatg tcaaactgac
tagcacatct gctctcatta cctatatcgt 6060tttgactatc atatctcttg tttttggtat
acttagcctg attctagcat gctacctaat 6120gtacaagcaa aaggcgcaac aaaagacctt
attatggctt gggaataata ctctagatca 6180gatgagagcc actacaaaaa tgtgaacaca
gatgaggaac gaaggtttcc ctaatagtaa 6240tttgtgtgaa agttctggta gtctgtcagt
tcagagggtt aagaaaaaac taccggttgt 6300agatgaccaa aggacgatat acgggtagaa
cggtaagaga ggccgcccct caattgcgag 6360ccaggcttca caacctccgt tctaccgctt
caccgacaac ggtcctcaat catggaccgc 6420gccgttagcc aagttgcgtt agagaatgat
gaaagagagg caaaaaatac atggcgcttg 6480atattccgga ttgcaatctt attcttaaca
gtagtgacct tggctatatc tgtagcctcc 6540cttttatata gcatgggggc tagcacacct
agcgatcttg taggcatacc gactaggaat 6600tccagggcag aagaaaagat tacatctaca
cttggttcca atcaagatgt agtagatagg 6660atatataagc aagtggccct tgagtctccg
ttggcattgt taaaaactga gaccacaatt 6720atgaacgcaa taacatctct ctcttatcag
attaatggag ctgcaaacaa cagtgggtgg 6780ggggcactta tccatgaccc agattatata
ggggggatag gcaaagaact cattgtagat 6840gatgctagtg atgtcacatc attctatccc
tctgcatttc aagaacatct gaattttatc 6900ccggcgccta ctacaggatc aggttgcact
cgaataccct catttgacat gagtgctacc 6960cattactgct acacccataa tgtaatattg
tctggatgca gagatcactc acattcatat 7020cagtatttag cacttggtgt gctccggaca
tctgcaacag ggagggtatt cttttctact 7080ctgcgttcca tcaacctgga cgacacccaa
aatcggaagt cttgcagtgt gagtgcaact 7140cccctgggtt gtgatatgct gtgctcgaaa
gtcacggaga cagaggaaga agattataac 7200tcagctgtcc ctacgcggat ggtacatggg
aggttagggt tcgacggcca gtaccacgaa 7260aaggacctag atgtcacaac attattcggg
gactgggtgg ccaactaccc aggagtaggg 7320ggtggatctt ttattgacag ccgcgtatgg
ttctcagtct acggagggtt aaaacccaat 7380tcacccagtg acactgtaca ggaagggaaa
tatgtgatat acaagcgata caatgacaca 7440tgcccagatg agcaagacta ccagattcga
atggccaggt cttcgtataa gcctggacgg 7500tttggtggga aacgcataca gcaggctatc
ttatctatca aggtgtcaac atccttaggc 7560gaagacccgg tactgactgt accgcccaac
acagtcacac tcatgggggc cgaaggcaga 7620attctcacag tagggacatc tcatttcttg
tatcaacgag ggtcatcata cttctctccc 7680gcgttattat atcctatgac agtcagcaac
aaaacagcca ctcttcatag tccttataca 7740ttcaatgcct tcactcggcc aggtagtatc
ccttgccagg cttcagcaag atgccccaac 7800ccgtgtgtta ctggagtcta tacagatcca
catcccctaa tcttctatag aaaccacacc 7860ttgcgagggg tattcgggac aatgcttgat
ggtgtacaag caagacttaa ccctgcgtct 7920gcagtattcg atagcacatc ccgcagtcgc
attactcgag tgagttcaag cagtaccaaa 7980gcagcataca caacatcaac ttgttttaaa
gtggtcaaga ctaataagac ctattgtctc 8040agcattgctg aaatatctaa tactctcttc
ggagaattca gaatcgtccc gttactagtt 8100gagatcctca aagatgacgg ggttagagaa
gccaggtctg gctagttgag tcaattataa 8160aggagttgga aagatggcat tgtatcacct
atcttctgcg acatcaagaa tcaaaccgaa 8220tgccggcgcg tgctcgaatt ccatgttgcc
agttgaccac aatcagccag tgctcatgcg 8280atcagattaa gccttgtcaa tagtctcttg
attaagaaaa aatgtaagtg gcaatgagat 8340acaaggcaaa acagctcatg gtaaataata
cgggtagaac atggcgagct ccggtcctga 8400aagggcagag catcagatta tcctaccaga
gtcacacctg tcttcaccat tggtcaagca 8460caaactactc tattactgga aattaactgg
gctaccgctt cctgatgaat gtgacttcga 8520ccacctcatt ctcagccgac aatggaaaaa
aatacttgaa tcggcctctc ctgatactga 8580gagaatgata aaactcggaa gggcagtaca
ccaaactctt aaccacaatt ccagaataac 8640cggagtgctc caccccaggt gtttagaaga
actggctaat attgaggtcc cagattcaac 8700caacaaattt cggaagattg agaagaagat
ccaaattcac aacacgagat atggagaact 8760gttcacaagg ctgtgtacgc atatagagaa
gaaactgctg gggtcatctt ggtctaacaa 8820tgtcccccgg tcagaggagt tcagcagcat
ccgtacggac ccggcattct ggtttcactc 8880aaaatggtcc acagccaagt ttgcatggct
ccatataaaa cagatccaga ggcatctgat 8940ggtggcagct aggacaaggt ctgcggccaa
caaattggtg atgctaaccc ataaggtagg 9000ccaagtcttt gtcactcctg aacttgtcgt
tgtgacgcat acgaatgaga acaagttcac 9060atgtcttacc caggaacttg tattgatgta
tgcagatatg atggagggca gagatatggt 9120caacataata tcaaccacgg cggtgcatct
cagaagctta tcagagaaaa ttgatgacat 9180tttgcggtta atagacgctc tggcaaaaga
cttgggtaat caagtctacg atgttgtatc 9240actaatggag ggatttgcat acggagctgt
ccagctactc gagccgtcag gtacatttgc 9300aggagatttc ttcgcattca acctgcagga
gcttaaagac attctaattg gcctcctccc 9360caatgatata gcagaatccg tgactcatgc
aatcgctact gtattctctg gtttagaaca 9420gaatcaagca gctgagatgt tgtgtctgtt
gcgtctgtgg ggtcacccac tgcttgagtc 9480ccgtattgcc gcaaaggcag tcaggagcca
aatgtgcgca ccgaaaatgg tagactttga 9540tatgatcctt caggtactgt ctttcttcaa
gggaacaatc atcaacgggt acagaaagaa 9600gaatgcaggt gtgtggccgc gagtcaaagt
ggatacaata tatgggaagg tcattgggca 9660actacatgca gattcagcag agatttcaca
cgatatcatg ttgagagagt ataagagttt 9720atctgcactt gaatttgagc catgtataga
atatgaccct gtcaccaacc tgagcatgtt 9780cctaaaagac aaggcaatcg cacaccccaa
cgataattgg cttgcctcgt ttaggcggaa 9840ccttctctcc gaagaccaga agaaacatgt
aaaagaagca acttcgacta atcgcctctt 9900gatagagttt ttagagtcaa atgattttga
tccatataaa gagatggaat atctgacgac 9960ccttgagtac cttagagatg acaatgtggc
agtatcatac tcgctcaagg agaaggaagt 10020gaaagttaat ggacggatct tcgctaagct
gacaaagaag ttaaggaact gtcaggtgat 10080ggcggaaggg atcctagccg atcagattgc
acctttcttt cagggaaatg gagtcattca 10140ggatagcata tccttgacca agagtatgct
agcgatgagt caactgtctt ttaacagcaa 10200taagaaacgt atcactgact gtaaagaaag
agtatcttca aaccgcaatc atgatccgaa 10260aagcaagaac cgtcggagag ttgcaacctt
cataacaact gacctgcaaa agtactgtct 10320taattggaga tatcagacaa tcaaattgtt
cgctcatgcc atcaatcagt tgatgggcct 10380acctcacttc ttcgaatgga ttcacctaag
actgatggac actacgatgt tcgtaggaga 10440ccctttcaat cctccaagtg accctactga
ctgtgacctc tcaagagtcc ctaatgatga 10500catatatatt gtcagtgcca gagggggtat
cgaaggatta tgccagaagc tatggacaat 10560gatctcaatt gctgcaatcc aacttgctgc
agctagatcg cattgtcgtg ttgcctgtat 10620ggtacagggt gataatcaag taatagcagt
aacgagagag gtaagatcag acgactctcc 10680ggagatggtg ttgacacagt tgcatcaagc
cagtgataat ttcttcaagg aattaattca 10740tgtcaatcat ttgattggcc ataatttgaa
ggatcgtgaa accatcaggt cagacacatt 10800cttcatatac agcaaacgaa tcttcaaaga
tggagcaatc ctcagtcaag tcctcaaaaa 10860ttcatctaaa ttagtgctag tgtcaggtga
tctcagtgaa aacaccgtaa tgtcctgtgc 10920caacattgcc tctactgtag cacggctatg
cgagaacggg cttcccaaag acttctgtta 10980ctatttaaac tatataatga gttgtgtgca
gacatacttt gactctgagt tctccatcac 11040caacaattcg caccccgatc ttaatcagtc
gtggattgag gacatctctt ttgtgcactc 11100atatgttctg actcctgccc aattaggggg
actgagtaac cttcaatact caaggctcta 11160cactagaaat atcggtgacc cggggactac
tgcttttgca gagatccagc gactagaagc 11220agtgggatta ctgagtccta acattatgac
taatatctta actaggccgc ctgggaatgg 11280agattgggcc agtctgtgca acgacccata
ctctttcaat tttgagactg ttgcaagccc 11340aaatattgtt cttaagaaac atacgcgaag
agtcctattt gaaacttgtt caaatccctt 11400attgtctgga gtgcacacag aggataatga
ggcagaagag aaggcattgg ctgaattctt 11460gcttaatcaa gaggtgattc atccccgcgt
tgcgcatgcc atcatggagg caagctctgt 11520aggtaggaga aagcaaattc aagggcttgt
tgacacaaca aacaccgtaa ttaagattgc 11580gcttactagg aggccattag gcataaagag
gctgatgcgg atagtcaatt attctagcat 11640gcatgcaatg ctgtttagag acgatgtttt
ttcctccagt agatccaacc accccttagt 11700ctcttctaat atgtgttctc tgacactggc
agactatgca cggaatagaa gctggtcacc 11760tttgacggga ggcaggaaaa tactgggtgt
atctaatcct gatacgatag aactcgtaga 11820gggtgagatt cttagtgtaa gcggagggtg
tacaagatgt gacagcggag atgaacaatt 11880tacttggttc catcttccaa gcaatataga
attgaccgat gacaccagca agaatcctcc 11940gatgagggta ccatatctcg ggtcgaagac
acaggagagg agagctgcct cacttgcaaa 12000aatagctcat atgtcgccac atgtaaaggc
tgccctaagg gcatcatccg tgttgatctg 12060ggcttatggg gataatgacg taaattggac
tgctgctctt acgattgcaa aatctcggtg 12120taatgtaaac ttagagtatc ttcggttact
gtccccttta cccacggctg ggaatcttca 12180acatagacta gatgatggta taactcagat
gacattcacc cctgcatctc tctacaggtg 12240tcaccttaca ttcacatatc caatgattct
caaaggctgt tcactgaaga aggagtcaaa 12300gaggggaatg tggtttacca acagagtcat
gctcttgggt ttatctctaa tcgaatcgat 12360atttccaatg acagcaacca ggacatatga
tgagatcaca ctgcacctac atagtaaatt 12420tagttgctgt atcagagaag cacctgttgc
ggttcctttc gagctacttg gggtggtacc 12480ggaactgagg acagtgacct caaataagtt
tatgtatgat cctagccctg gatcggaggg 12540agactttgcg agacttgact tagctatctt
caagagttat gagcttaatc tggagtcata 12600tcccacgata gagctaatga acattctttc
aatatccagc gggaagttga ttggccagtc 12660tgtggtttct tatgatgaag atacctccat
aaagaatgac gccataatag tgtacgacaa 12720tacccgaaat tggatcagtg aagctcagaa
ttcagatgtg gtccgcctat ttgaatatgc 12780agcacttgaa gtgctcctca actgttctta
ccaactctat tacctgagag taagaggcct 12840agacaatatt gtcttatata tgggtgattt
atacaagaat atgccaggaa ttctactttc 12900caacattgca gctacaatat ctcatcccgt
cattcattca aggttacatg cagtgggcct 12960ggtcaaccat gacggatcac accaacttgc
agatacggat tttatcgaaa tgtctgcaaa 13020actattagta tcttgcaccc gacgtgtgat
ctccggctta tattcaggaa ataagtatga 13080tctgctgttc ccatctgtct tagatgataa
cctgaatgag aagatgcttc agctgatatc 13140ccggttatgc tgtctgtaca cggtactctt
tgctacaaca agagaaatcc cgaaaataag 13200aggcttaact acaaaagaga aatgttcaat
acccactgag tatttactgt cggatgctgt 13260gaaaccatta cttagccccg atcaagtgag
ctctatcatg tctcctaaca taattacatt 13320cccagctaat ccgtactaca tgtctcggaa
gagcctcaat ttgatcaggg aaagggagga 13380cagggatact atcctggtgt tgttgttccc
ccaagagcca ttattagagt tcccttctgt 13440gcaagatatt ggtgctcgag tgaaagatcc
attcacccga caacctgcgg catttttgca 13500agagttagat ttgagtgctc cagcaaggta
tgacgcattc acacttagtc agattcatcc 13560tgaactcaca tctccaaatc cggaggaaga
ctacttagta cgatacttgt tcagagggat 13620agggactgca tcttcctctt ggtataaggc
atctcatctc ctttctgtac ccgaggtaag 13680atgtgcaaga cacgggaact ccttatactt
agctgaaggg agcggagcca tcatgagtct 13740tctcgaactg catgtaccac atgaaactat
ctattacaat acgctctttt caaatgagat 13800gaaccccccg caacgacatt tcgggccgac
cccaactcag tttttgaatt cggttgttta 13860taggaatcta caggcggagg taacatgcaa
agatggattt gtccaagagt tccgtccatt 13920atggagagaa aatacagagg aaagtgacct
gacctcagat aaagcagtgg ggtatattac 13980atctgcagtg ccctacagat ctgtatcatt
gctgcattgt gacattgaaa ctcctccagg 14040gtccaatcaa agcttactag atcaactagc
tatcaattta tctctgattg ccatgcattc 14100tgtaagggag ggcggggtag taatcatcaa
agtgttgtat gcaatgggat actactttca 14160tctactcatg aacttgtttg ctccgtgttc
cacaaaagga tatattctct ctaatggtta 14220tgcatgtcga ggagatatgg agtgttacct
ggtatttgtc atgggttacc tgggcgggcc 14280tacatttgta catgaggtgg tgaggatggc
aaaaactctg gtgcagcggc acggtacgct 14340cttgtctaaa tcagatgaga tcacactgac
caggttattc acctcacagc ggcagcgtgt 14400gacagacatc ctatccagtc ctttaccaag
attaataaag tacttgagga agaatattga 14460cactgcgctg attgaagccg ggggacagcc
cgtccgtcca ttctgtgcgg agagtctggt 14520gagcacgcta gcgaacataa ctcagataac
ccagattatc gctagtcaca ttgacacagt 14580tatccggtct gtgatatata tggaagctga
gggtgatctc gctgacacag tatttctatt 14640taccccttac aatctctcta ctgacgggaa
aaagaggaca tcacttaaac agtgcacgag 14700acagatccta gaggttacaa tactaggtct
tagagtcgaa aatctcaata aaataggcga 14760tataatcagt ctagagctta aaggcatgat
ctccatggag gaccttatcc cactaaggac 14820atacttgaag catagtacct gccctaaata
tttgaaggct gtcctaggta ttaccaaact 14880caaagaaatg tttacagaca cttctgtact
gtacttgact cgtgctcaac aaaaattcta 14940catgaaaact ataggcaatg cagtcaacgg
atattacagt aactgtgact cttaacgaaa 15000atcacatatt aataggctcc ttttttggcc
aattgtattc ttgttgattt aatcatatta 15060tgttagaaaa aagttgaacc ctgactcctt
aggactcgaa ttcgaactca aataaatgtc 15120ttaaaaaaag gttgcgcaca attattcttg
agtgtagtct cgtcattcac caaatctttg 15180tttggt
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