Patent application title: Influenza vaccines
Inventors:
Anders Fomsgaard (Frederiksberg, DK)
Assignees:
STATENS SERUM INSTITUT
IPC8 Class: AA61K39145FI
USPC Class:
4242061
Class name: Virus or component thereof reassortant or deletion mutant virus influenza virus
Publication date: 2008-12-04
Patent application number: 20080299151
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Patent application title: Influenza vaccines
Inventors:
Anders Fomsgaard
Agents:
HOWSON AND HOWSON
Assignees:
Statens Serum Institut
Origin: FT WASHINGTON, PA US
IPC8 Class: AA61K39145FI
USPC Class:
4242061
Abstract:
Described herein are vaccines and the use of naked DNA and/or RNA encoding
hemagglutinin (HA) from pandemic influenza, e.g., the 1918 H1N1 and/or
the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, as a vaccine
component against present day and coming H1, H2, H3, H5, N1, N2
containing influenza A infections in humans and swine optionally with the
naked DNA and/or RNA encoding Neuraminidase (NA) and/or matrix protein
(M) and/or the nucleoprotein (NP) from pandemic influenza virus included.
If the vaccine components are used as DNA or RNA vaccines with or without
the corresponding protein, the codons can optionally be "humanized" using
preferred codons from highly expressed mammalian genes and the
administration of this DNA vaccine can be by saline or buffered saline
injection of naked DNA or RNA, or injection of DNA plasmid or linear gene
expressing DNA fragments coupled to particles. Addition of the matrix
protein (M) and/or the nucleoprotein (NP) from the 1918 influenza strain
is also disclosed.Claims:
1. A method of treating or preventing infection with a H1, H2, H3
containing influenza A in a subject, said method comprising delivering to
the subject a naked DNA and/or RNA molecule encoding hemagglutinin (HA)
from pandemic influenza selected from one or more of the 1918 H1N1, the
1957 H2N2, and the 1968 H3N2 influenza A virus.
2. The method according to claim 1, wherein the method comprises administering a vaccine for N1, N2 containing influenza A in humans or swine, the method further comprising delivering to the subject a naked DNA and/or RNA molecule encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from a pandemic influenza virus therapeutically or prophylactically.
3. The method according to claim 1, wherein the codons of the DNA or RNA are humanized using codons of highly expressed human proteins.
4. The method according to claim 1, further comprising delivering an adjuvant to the subject.
5. The method according to claim 1, where a DNA vaccine is administered by saline injection of naked DNA and/or RNA, inoculated by gene gun or is delivered coupled to particles.
6. A vaccine for human use comprising a naked DNA and/or RNA molecule encoding hemagglutinin (HA) from a pandemic influenza selected from the group consisting of the 1918 H1N1, the 1957 H2N2, and the 1968 H3N2 influenza A virus.
7. The vaccine according to claim 6, further comprising a naked DNA and/or RNA molecule encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from a pandemic influenza virus.
8. The vaccine according to claim 6, wherein the vaccine consists of as its antigenic component the HA and one or more of a neuraminidase (NA), a matrix (M) protein, and/or nucleoprotein (NP).
9. The vaccine according to claim 6, where the vaccine consists of as its antigenic component naked DNA or RNA molecule coding for the HA and one or more of a neuraminidase (NA) protein, matrix protein, and/or nucleoprotein (NP).
10. The vaccine according to claim 9, where the DNA or RNA codons are humanized using codons of highly expressed human proteins.
11. The vaccine according to claim 6, wherein the vaccine further comprises an adjuvant.
12. The vaccine according to claim 6, where the vaccines are administered therapeutically to already infected humans or swine.
13. A method of treating or preventing infection with H5, H7 or H2 containing influenza A in a subject, said method comprising delivering to the subject a naked DNA and/or RNA molecule encoding hemagglutinin (HA) and/or neuraminidase (NA) from an influenza strain selected from the group consisting of a 2001 H5N7 low pathogenic Avian influenza virus (AIV) strain (A/Mallard/Denmark/64650/03(H5N7)) and a March 2006 Denmark H5N1 high pathogenic AIV strain (A/buzzard/Denmark/6370/06(H5N1)).
14. The method according to claim 13, wherein the naked DNA and/or RNA molecule encoding HA or NA are delivered in a composition containing the HA and/or NA proteins.
15. The method according to claim 13, wherein the HA or NA are delivered in a composition containing the naked DNA and/or RNA molecule encoding the HA and/or NA proteins.
16. The method according to claim 15, where the DNA or RNA codons are humanized.
17. A vaccine for preventing infection with H5, H7 or H2 containing influenza A infections in humans or swine comprising a naked DNA and/or RNA molecule encoding hemagglutinin (HA) and/or neuraminidase (NA) from an influenza strain selected from the group consisting of 2001 H5N7 low pathogenic Avian influenza virus (AIV) strain (A/Mallard/Denmark/64650/03(H5N7)), March 2006 Denmark H5N1 high pathogenic AIV strain (A/buzzard/Denmark/6370/06(H5N 1)), (A/duck/Denmark/53-147-8/08 (H7N1)) and (A/widegeon/Denmark/66174/G18/04 (H2N3)).
18. The vaccine according to claim 17, wherein the vaccine comprises naked DNA and/or RNA molecules encoding the HA or NA proteins.
19. The vaccine according to claim 17, where the DNA or RNA codons are humanized.
20. The vaccine according to claim 17, wherein the vaccine further comprises an adjuvant.
Description:
CROSS-REFERENCE TO RELATED APPLICATION
[0001]This application claims the benefit under 35 USC 119(e) of U.S. Patent Application No. 60/934,117, filed Jun. 11, 2007.
BACKGROUND OF THE INVENTION
[0002]The invention concerns therapeutic and prophylactic vaccines for humans and swine, for influenza A infections in humans and swine.
[0003]Influenza is one of the oldest and most common diseases known to man, causing between three and five million cases of severe illness and between 250,000 and 500,000 deaths every year around the world. Also, swine are susceptible to human and avian influenza virus, since they possess both receptors in their respiratory tract. Because swine get infection and pneumonia from human influenza strains, they may serve as a dangerous mixing vessel for the generation of new recombinant influenza strains with pandemic potential.
[0004]Influenza rapidly spreads in seasonal epidemics affecting 5-15% of the population and the burden on health care costs and lost productivity are extensive (World Health Organization (WHO)). Influenza like illness was first described by Hippocrates in the year 412 BC. Up to the 19th century, influenza was thought to be a bacterial infection. Virus as the causative agent was first determined in 1931 by Richard Shope. The first known influenza A pandemic was in 1580. Since then there have been 31 pandemics of which three appeared in the 20th century, namely the `Spanish flu` in 1918, the `Asian flu` in 1957 and the `Hong Kong flu` in 1968, respectively. The pandemic of 1918 influenza A H1N1 was the worst pandemic in recent times causing 20 to 50 million deaths worldwide. Influenza most commonly presents as seasonal outbreaks and epidemics of variable severity.
[0005]Zoonosis of avian influenza virus (AIV) able to infect humans and swine and its spread in Asia, parts of Europe and the Middle East has recently evoked the concern about a pandemic occurring also in the 21st century. The causative strain of such a pandemic will probably be unknown until the pandemic emerges, at which time there will be an urgent need for a vaccine. Therefore, fast diagnosis and characterisation of circulating strains as well as emerging strains, new alternative vaccines approaches and production methods will be required in order to minimise the severity of the pandemic.
[0006]Since seasonal influenza A vaccines are produced on eggs, an epidemic of highly pathogenic AIV among poultry will also influence the production of seasonal vaccines. Moreover, the traditional influenza protein vaccines only have a limited protective effect. Also, seasonal vaccines have to be changed every season because of the genetic drift of influenza A virus and the narrow type specific antibody induction by traditional influenza A protein vaccines. Therefore, there is a need for new alternative influenza A vaccines with different properties.
[0007]The influenza virus belongs to the Orthomyxoviridae family. The family includes three genera; influenza A, B and C viruses, identified by antigenic differences in their nucleoprotein (NP) and matrix protein (M). The influenza A genus is further divided into subtype combinations based on the antigenic differences of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). The A strain has evolved to be able to infect several other mammalian species (e.g. horses and swine). Influenza A viruses of all recognised 16 HAs and 9 NAs antigenic subtypes have been recovered from aquatic birds, but few infect other animal species which indicates that aquatic birds are the natural reservoirs of influenza A.
[0008]The influenza A viruses have been the causative agents for the major pandemics and most of the annual outbreaks of epidemic influenza. The current nomenclature system for human influenza viruses includes the geographical location of first isolation, strain number, and year of isolation. The antigenic description of HA and NA is given in brackets, e.g., A/Moscow/10/99 (H3N2). Nonhuman strains also include the host of origin in the nomenclature, e.g., A/mallard/Denmark/64650/03(HSN7).
[0009]The influenza A virus genome consist of eight negative sense single stranded (ss) ribonucleic acid (RNA) segments packed in the viral core comprised of host cell membrane and a matrix 1 (M1) protein layer. The eight segments are associated with nucleoprotein (NP) and three large proteins; polymerase basic 1 (PB1) and 2 (PB2) protein, and polymerase acidic (PA) protein, which are responsible for RNA replication and transcription. NP encapsulates the RNA and forms ribonucleoprotein (RNP) complexes that protect and stabilise the RNA. Each segment include a sequence of 11-13 nucleotides at the 5' ends and 9-12 nucleotides at the 3' ends which are highly conserved and similar for A, B and C viruses. The major glycoproteins HA and NA, and the ion channel M2 protein, are embedded in a host derived lipid bilayer. Influenza viruses are somewhat pleomorphic in shape, but mostly spherical (80-120 nm in diameter).
[0010]All subtypes of influenza A are perpetuated in the wild aquatic bird population, believed to be the natural reservoir of influenza. Under normal circumstances an influenza infection in wild ducks is asymptomatic. The virus replicates in the intestinal tract and is excreted in high concentrations with the feces for a period up to 30 days. An avian influenza virus can persist in water and retain infectivity for about 100 days at 17° C. and can be stored indefinitely at -50° C. The continuous circulation of influenza A viruses might be due to bird overwintering sites in the subtropics. The 2004 H5N1 strains have become very stable and can survive for 6 days at 37° C. The virus is killed by heat at 56° C. for 3 hours or 60° C. for 30 minutes. Disinfectants like formalin and iodine compounds can also efficiently kill the influenza virus. Avian influenza viruses have been believed to be in evolutionary stasis in its natural host, the virus and the host tolerate each other. Generally no severe clinical symptoms are seen when poultry are infected with avian influenza, and the virus is described as a low pathogenic avian influenza virus (LP AIV). The subtypes H5 and H7 have the potential to become highly pathogenic (HP) to chickens through accumulation of mutations after transmission to poultry. Contrary to previous belief, wild migratory birds might play some role in the transmission of HP AIV. Thousands of wild aquatic birds in Hong Kong 2002 and China 2005 became infected with HP AIV H5N1 and this contributed to the spread of HP H5N1 to Europe and Africa in 2005.
[0011]Seasonal influenza strains have been isolated from humans and swine all year round. However, in temperate climates it is a winter disease, probably because people come together and stay in less ventilated rooms due to the cold weather.
[0012]Of the 16 recognised subtypes of HA and 9 NAs, only H1, H2, H3, N1 and N2 have circulated in humans and swine in the last century. The pandemic introduction in humans of these types were 1918 H1N1, 1957 H2N2 ("Asiatic flu"), 1968 H3N2 ("HongKong Flu") and non-pandemic introduction of the reasserted new type H1N2 in 2001, respectively. The antigenicity of human influenza viruses are constantly changing by accumulation of mutations in the HA and NA antigenic sites, thereby making the virus capable of evading the host immune system causing epidemics. Viral mutagenesis is enhanced by the lack of "proof reading" in the replication of RNA. The mutation frequency is approximately one in 100,000 nucleotides. In the northern hemisphere seasonal influenza outbreaks usually occur between October and April. In the southern hemisphere, these outbreaks usually occur between April and October. The antigenic drift of human influenza viruses is closely monitored by WHO's global influenza surveillance program. The components of the next seasons' influenza vaccine for the northern hemisphere is determined in February based on the knowledge about the current circulating strains, and re-evaluated in September for the southern hemisphere.
[0013]Antigenic shift can occur in three ways. Either by direct transmission of an avian strain adapted to humans, genetic reassortment or reintroduction of an "old" strain. The possibility of an avian influenza virus crossing the species barrier and infecting humans directly was not recognised before 1997 when 18 people in Hong Kong became ill with HP AIV H5N1.
[0014]The origin of the 1918 pandemic is controversial. Taubenberger et al., (Characterization of the 1918 influenza virus polymerase genes. Nature, 2005, 437:889-893) suggested based on phylogenetics of the polymerase genes that the virus was entirely of avian origin. If the virus was of avian origin it might imply that the HP avian viruses circulating currently could cause a new pandemic by direct transmission to humans. However, there is consideration disagreement about the actual origin of the virus and many still believe that also this 1918 pandemic strain is a reassortant between a mammalian and avian virus most likely occurring from swine. Antigenic reassortment occurs when viral segments from two antigenic different viruses infect the same cell. The reasserted virus contains segments of both strains and if the newly introduced segment is HA (and NA) the complete antigenicity of the virus might change and the virus escapes the host immunity. The reassortant might be catastrophic if the virus is capable of efficient replication in the new host. In the worst case, such a reasserted strain might lead to a pandemic, world-spanning infection to which there is no pre-existing immunity in the human population. The pandemics of 1957 and 1968 were reassortants that acquired the HA, NA and PB1 and HA and PB1 genes from an aquatic source, respectively. In 1977, a strain identical to the H1N1 strains that circulated before 1957 re-emerged. Pigs are possible "mixing vessels" for reassorted viruses due to their receptor tropism for both α-(2,3) and α-(2,6) linkage to galactose. Other species like chicken and man might also serve as mixing vessels in the light of direct crossover to humans from an avian source after the discovery of α-(2,3) avian like receptor on cells also in humans and chickens.
[0015]The interpandemic evolution of influenza viruses has been thought to be caused by progressive antigenic drift due to the mutability of the RNA genome. H3N2 has been the predominant subtype circulating in humans since 1968 and has been in rapid drift as a single lineage while there has been slow replacement of antigenic variants of the H1N1 viruses. It has been shown that the rate of accumulating mutations is approximately 4-5×10-3 substitutions per nucleotide per year for HA1; others predict a rate of 5.7×10-3 substitutions per nucleotide per year. The HA and NA might evolve independently from each other and reassortments of the internal genes are also known. Positive selection has been inferred on codons involved at antibody antigenic sites, T-cell epitopes and sites important for virus egg growth properties. Recent research on viruses has suggested that the evolution of influenza does not always follow a constant rate, but is characterised by stochastic processes, short intervals of rapid evolution, and long intervals of neutral sequence evolution and slow extinction of coexisting virus lineages. The evolution seems also more influenced by reassortment events between co-circulating lineage and viral migration than previously believed.
[0016]Vaccination is the preferred choice for influenza prophylaxis. Inactivated influenza vaccines are licensed worldwide, while cold-adapted live vaccines are licensed only in Russia and the USA. The preferred prophylaxis of annual influenza infections is vaccination with inactivated protein vaccines from virus propagated in hens' eggs. Thus, the common vaccines are the inactivated vaccine viruses which are propagated in hens' eggs and inactivated by formaldehyde or β-propiolactone. There are three classes of inactivated vaccines; whole, split (chemically disrupted with ether or tributyl phosphate) and subunit (purified surface glycoproteins) administrated intramuscularly or subcutaneously. Whole inactivated influenza vaccine is not currently used due to high levels of side effects. The seasonal influenza vaccine (split and subunit) is trivalent, comprising H3N2 and H1N1 influenza A virus strains and an influenza B virus. The normal human vaccine dose is standardised to 15 μg HA protein of each virus component administered once in normal healthy adults and twice in children and other persons with no pre-existing influenza A immunity. The conventional vaccines induce merely a humoral immune response. The protective effect of the traditional protein split vaccine is very limited and because of the continuous evolution of influenza A virus strains and the type specific antibodies induced by the conventional vaccines, a new vaccine has to be produced every year based on the most recent circulating influenza A strain. Several vaccine improvements are necessary in case of a new emerging human strain. Egg production is too slow (6-12 months) in the case of emerging strains. If this strain is also an AIV virus highly pathogenic (HP) for poultry, egg production might be impossible because the virus kills the egg embryo. Also the availability of eggs might be limited and slow down vaccine production. In the case of no pre-existing immunity in the population, two vaccinations would be necessary, thereby further delaying the vaccine production. Even if there are no new pandemic influenza A among humans but only spread of a HPV AIV among poultry, a shortage of eggs will limit production of traditional seasonal influenza vaccines on eggs. In addition, traditional influenza protein vaccines do not have optimal protection as prophylaxis and no therapeutic effect. Thus, there is a need for new alternative influenza vaccines.
[0017]Although DNA vaccines were developed more than 16 years ago, clinical trials proceeding to stage I and II in humans are rare. Two veterinary DNA vaccines however, have been licensed; one for West Nile Virus (in horse) and a second for Infectious Hematopoietic Necrosis virus in Salmon. This demonstrates that DNA vaccines can have good protective effects and that new DNA vaccines are not limited by the size of the animal or species. The great success with DNA vaccines observed for the murine model for first generation DNA vaccines did not translate well to humans; nonetheless, researchers have recently demonstrated protective antibodies levels by a single dose of gene gun administrated HA DNA vaccine to humans.
[0018]Nucleic acid immunization" or the commonly preferred name "DNA vaccines" are the inoculation of antigen encoding DNA or RNA as expression cassettes or expression vectors or which may be incorporated into viral vectors with the purpose of inducing immunity to the gene product. Thus, as used herein, DNA vaccines refer to all kinds of delivery systems for the antigen encoding naked DNA or RNA but exclude viral vector-based delivery. The vaccine gene can be in form of circular plasmid or a linear expression cassette with just the key features necessary for expression (promoter, the vaccine gene and polyadenylation signal). Delivery systems may most often be naked DNA in buffer with or without adjuvant, DNA coupled to nanoparticles and/or formulated into adjuvant containing compounds or inserted into live viral or bacterial vectors, such as adenovirus, adeno-associated virus, alphavirus, poxviruses, herpes virus etc. DNA vaccines hold great promise since they evoke both humoral and cell-mediated immunity, without the same dangers associated with live virus vaccines. In contrast to live attenuated virus vaccines DNA vaccines may be delivered to same or different tissue or cells than the live virus that has to bind to specific receptors. The production of antigens in their native forms improves the presentation of the antigens to the host immune system. Unlike live attenuated vaccines, DNA vaccines are not infectious and can not revert to virulence.
[0019]WO2006063101 describes a pandemic avian influenza vaccine based on an adenovirus vehicle with HA DNA isolated from the avian H5N1 influenza virus isolated during the outbreak in 2003-2005. The vaccine was tested in animals challenged with the same H5N1 influenza virus strain.
[0020]DNA vaccines induce an immune response which may be comparable to the response acquired by natural virus infection by activating both humoral and cell-mediated immunity (6,30). The broad response to DNA vaccines is a result of the encoded genes being expressed by the transfected host cell, inducing both a Th1 and Th2 immune responses. The production of antigens in their native form improves the presentation of the antigens to the host immune system. In contrast, the conventional inactivated influenza protein based vaccines only induce a humoral response (Th2), directed against the influenza surface glycoproteins. This type of response is ineffective against drifted virus variants and therefore the virus composition of the seasonal influenza vaccine has to be assessed every season. Antigenic cross-reactive responses are mainly induced by the more conserved influenza proteins like the nucleoprotein (NP) and the matrix (M) protein. By including these genes in a DNA vaccine higher cross reactivity between drifted and heterologous strains have been shown (4,7;8,13). However, cellular immunity alone cannot protect against infection since this requires antibodies.
[0021]Influenza infection and symptoms in ferrets are highly comparable to what is observed in humans. Therefore ferrets are one of the best models for influenza vaccination trials (22). Influenza HA DNA vaccines in ferrets has also previously proved effective (18,32).
[0022]It has previously been shown that H1N1 whole inactivated virus vaccine induced partly protection against infection with 1918 in mice (28). Also, recently, a DNA vaccine encoding the HA from 1918 showed complete protection of mice against a 1918 H1N1 challenge (16).
[0023]Influenza vaccines that have the ability to induce immune responses able to cross-react with drifted virus variants and even heterologous strains would be of great advantage for both annual vaccine development and in cases of emerging new strains.
SUMMARY OF THE INVENTION
[0024]The present invention provides regimens and compositions containing the hemagglutinin (HA) from pandemic influenza A, e.g. the 1918 H1N1 and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, useful as a therapeutic or prophylactic vaccine component against present day and future influenza A strains. The invention may optionally provide naked DNA and/or RNA molecules encoding the Neuraminidase (NA), matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus. The invention also concerns vaccines comprising naked DNA and/or RNA coding HA and/or NA from the new circulating 2001 H5N7 low pathogenic (LP) Avian influenza virus (AIV) strain (A/Mallard/Denmark/64650/03(H5N7)), the newly introduced and circulating March 2006 Denmark H5N I high pathogenic Avian influenza A virus (AIV) strains A/buzzard/Denmark/6370/06 (H5N 1), A/duck/Denmark/53-147-8/08 (H7N1) and A/widegeon/Denmark/66174/G 18/04 (H2N3).
[0025]The data herein demonstrates that gene gun administrated codon optimised DNA vaccine in plasmid encoding HA and NA with or without M and NP based on the H1N1 pandemic virus from 1918 induced protection in ferrets against infection with a H1N1 (A/New Caledonia/20/99(H1N1)) present day virus. The circulating H1N1 strain in Europe in the 2006-2007 seasons is New Caledonia-like. The viruses are separated by a time interval of 89 years and differ by 21.2% in the HA1 protein. By comparison, a similar DNA vaccine encoding the HA and NA of (A/New Caledonia/20/99 (H1N1)) induced less protection. These results suggest not only a unique ability of the DNA vaccines but also a unique and unexpected feature of the 1918 HA and/or NA in inducing especially broad and efficient protective immunity against even extremely drifted strain variants.
[0026]The present invention discloses that an induced immune response with a DNA vaccine encoding HA and/or NA of the 1918 H1N1 influenza A gives a high level of cross protection against present day influenza infection. Tests were carried out in ferrets vaccinated with this DNA vaccine synthesised using human preferred codons of the 1918 H1N1 influenza and challenged with a contemporary H1N1 virus.
[0027]The results surprisingly show that the 1918 H1N1 DNA vaccines are as good as or better candidates for influenza prophylaxis than annual conventional protein based vaccines which frequently need to be updated to match the circulating influenza virus. DNA vaccination induces broader cross-reactivity against drifted strain and longer memory responses. It has been shown that a similar DNA vaccine may protect against the 1918 H1N1 recombinant strain (16). However, the results herein then suggest that the synthetic DNA vaccine based on the 1918 H1N1 sequences protects against extreme drifted variants represented by recent contemporary or seasonal circulating H1N1 strains. Thus it is likely that the suggested 1918 H1N1 DNA vaccine protects against H1N1 strains circulating for up to 89 years and therefore likely also future H1N1 variants. This is highly unexpected since traditional protein split vaccines only protects against the strain it is designed from and thus has to be produced from the actual circulating H1N1 strains sometimes as frequent as every year. Thus, a DNA vaccine encoding the HA and NA of 1918 H1N1 was not expected to protect against such a divergent strain as the present day H1N1, but it does.
[0028]Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029]FIG. 1A illustrates the mean serum specific IgG antibody response (ELISA) to influenza A of A/New Caledonia/20/99(H1N1) days after viral challenge. Six ferrets were in each group.
[0030]FIG. 1B illustrates the number of viral RNA copies (real time RTIPCR) in nasal wash in days after challenge. Six ferrets were in each group.
[0031]FIGS. 2A-2D provide results of 1918 pandemic H1N1 DNA vaccinated ferrets challenged with 2007 H1N1.
[0032]FIG. 2A shows fever at day 2 post challenge.
[0033]FIG. 2B shows body weight loss by day 4 post challenge.
[0034]FIG. 2C shows virus titer in nasal washings at day 7 post challenge.
[0035]FIG. 2D shows clinical score for illness based on a scoring table for sneezing, nasal discharge and activity level.
[0036]FIG. 3 is a bar chart showing hemadsorption as a measure of functional protein expression in mammalian cells of codon optimized HA from 1918 H1N1 (HA 1918), avian H5N7 (HA H5N7) and 1968 H3N2 (HA H3N2) compared to non-codon optimized 1918 H1N1 (HA NC).
DETAILED DESCRIPTION OF THE INVENTION
[0037]The present invention discloses the use of the naked DNA and/or RNA molecule encoding hemagglutinin (HA) from pandemic influenza, e.g. the 1918 H1N1 and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, as a vaccine component against present day and coming H1, H2, H3 containing influenza A infections in humans and/or swine. The naked DNA and/or RNA molecule encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus.
[0038]The naked DNA and/or RNA molecule encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus can optionally be included as vaccine components against present day and coming H1, H2, H3, N1, N2 containing influenza A infections in humans and/or swine.
[0039]The vaccine of the invention is believed to offer many of the advantages a DNA vaccine can provide over conventional vaccines. More particularly, it may be produced in high amounts in short time, abolishing the need for propagation in eggs; it is cost-effective, reproducible and the final product does not require cold storage conditions, because DNA is stable and resistant to the extremes of temperature. All currently licensed inactivated vaccines are efficient at inducing humoral antibody responses but only live attenuated virus vaccines efficiently induce a cytotoxic cellular response as well. However, DNA vaccines have the ability to induce a cytotoxic cellular response as well as a humoral antibody response. Therefore, these vaccines may better mimic the natural response to viral infection than inactivated vaccines in respect to specificity and antibodies isotypes.
[0040]The components in the vaccine are naked DNA and/or RNA coding for the hemagglutinin and/or the neuraminidase and /or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza strains, preferably with a mixture of such proteins from several pandemic strains.
[0041]In a preferred embodiment of the invention the DNA and/or RNA codons are humanized e.g. the DNA sequence for hemagglutinin and neuraminidase and Matrix and Nucleoprotein is changed so the sequence coding for said proteins is changed to be optimally expressed in mammalian cells.
[0042]The invention also discloses the vaccines against present day human and swine influenza A infection comprising the above-mentioned naked DNA and/or RNA coding hemagglutinin and/or neuraminidase and/or a matrix protein and/or the hemagglutinin protein and/or DNA or RNA from the pandemic influenza, e.g., the 1918 H1N1 and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, preferably with a mixture from various influenza strains.
[0043]In another embodiment the vaccine comprises naked DNA and/or RNA coding HA and/or NA from 1918 H1N1 strain plus HA and NA from 1957 H2N2 plus HA from 1968 H3N2 virus strains as DNA vaccines and/or proteins. The vaccine is intended to protect humans against circulating H1, H2, and H3 influenza A strains.
[0044]In another embodiment the vaccine comprises naked DNA and/or RNA molecules encoding HA and/or NA from the new circulating 2001 H5N7 low pathogenic (LP) Avian influenza virus (AIV) strain (A/Mallard/Denmark/64650/03(H5N7)) as DNA vaccines and/or proteins. The vaccine is intended to protect birds and humans and swine against H5, H7 and/or H2 containing influenza A strains.
[0045]In another embodiment the vaccine comprises naked DNA and/or RNA molecules encoding HA with or without NA and/or M and/or NP from the newly introduced and circulating March 2006 Denmark H5N1 high pathogenic Avian influenza A virus (AIV) strain (A/buzzard/Denmark/6370/06(H5N 1)), A/duck/Denmark/53-147-8/08 (H7N1) and A/widegeon/Denmark/66174/G18/04 (H2N3). The vaccine is intended to broadly protect birds and humans and swine against any H5 containing influenza A strains. Above mentioned vaccines can be used both prophylactically and therapeutically.
Definitions
Hemagglutinin:
[0046]The name hemagglutinin is derived from the viruses' ability to agglutinate red blood cells. The envelope glycoprotein HA is a rod-like shaped trimer of identical monomers. The HA protein is synthesised in the infected cell as a single polypeptide chain, HA0. This initial molecule has to be cleaved by the host cell proteases into disulfide linked HA1 (47 kDa) and HA2 (29 kDa) subunits in order for the virus to mediate membrane fusion and subsequent infection. The HA1 subunit is the globular domain of the HA molecule which comprises the receptor binding site, responsible for virus attachment to sialic acid receptors on the host cell. The five antigenic sites A, B, C, D and E at the globular head direct the host antibody response. The HA is the primary viral antigen and the only antigen inducing a virus neutralising response in the host. The HA main functions are virion-to-host cell membrane fusion and fusion of the endocytosed virion with the endosomal membrane allowing release of the genome into the cytoplasm. HA is a prototype 1 integral membrane protein that is targeted to the ER membrane through an N-terminal signal peptide sequence and cleaved by signal peptidase. The HA2 subunit forms the stem of the molecule. The N-terminus of HA2 (fusion peptide) is hydrophobic and is highly conserved in the HAs of different influenza virus strains, and it is essential in HA fusion activity. The HA is post translationally modified by addition of N-linked carbohydrates at asparagine residues (N) on each monomer and palmitic acid to cysteine (C) residues in the cytoplasmic tail region. HA binds to 5-N-acetyl neuramic acid (sialic acid) on the host cell surface and positions and are essential in determining preferred host cell tropism. Human infectious strains preferentially bind to sialic acid with α-(2,6) linkage to galactose, while avian influenza viruses (AIV) preferentially bind to α-(2,3),
Neuraminidase:
[0047]The neuraminidase (NA) is a class II membrane envelope glycoprotein with enzymatic activity. It is a tetramer of identical monomers forming a mushroom-like shape. The hydrophobic stalk region is membrane anchored and the globular head contains the enzyme active site and the three antigenic sites A, B and C of the molecule. The main function of the NA is to catalyse the cleavage of glycosidic linkages adjacent to sialic acid. The activity is essential for the progeny virion for efficient release from the surface of the infected cell. Like HA, NA is posttranslational modified with N-linked glycosylations. The NA molecule is target for antiviral drugs like zanamivir [5-acetamido-4-guanidino-6-(1,2,3-trihydroxypropyl)-5,6-dihydro-4H-pyran-- 2-carboxylic acid] and oseltamivir [(3R,4R,5S)-4-acetylamino-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carb- oxylic acid ethyl ester]. Inhibition of NA prevents virus release from the infected cell and delays virus propagation. Currently nine subtypes of NA have been recognised.
Matrix Proteins:
[0048]The matrix proteins consist of two proteins, the ion channel protein M2 and the structural protein M1 . The M1 protein is a matrix protein lining the interior side of the membrane derived from the infected host cell giving structure and rigidity to the membrane. The M1 protein contains a hydrophobic lipid binding domain and a RNP binding domain. Assembly of negative stranded RNA viruses requires localisation of M1 proteins to the plasma membrane. The M1 protein binds to the cytoplasmic tails of HA, NA and M2. NA stimulates the membrane binding by the M1 proteins. M1 together with NS2 is required for export of genomic RNPs from the nucleus, M1 also inhibits RNA synthesis. The M2 protein is a small homotetramer integral membrane protein, and ion channel, translated from a spliced mRNA in +1 reading frame. The ion channel is activated by the low pH of the endosome, allowing protons to enter the interior of the virus leading to conformational changes in M1 and disrupting the M1-RNP interactions. The M2 ion channel is a target for antiviral drugs like amantadine and rimantadine.
Nucleoprotein:
[0049]The Nucleoprotein (NP) is highly basic and binds the sugar-phosphate backbone of viral RNA in a non-sequence specific manner approximately every 25 nucleotides. NP interacts with both PB1 and PB2 and with a variety of other viral and cellular proteins. The interaction with M1 controls the transcriptional activity of RNPs and their intracellular trafficking. NP is mainly responsible for maintaining the structure of RNPs and in regulation of genome transcription and replication, the polymerase can not use naked viral RNA as template. NP associated with viral RNA is abundant in extracellular fluid and lung tissue during severe influenza A infection.
The 1918 Influenza Virus:
[0050]The most severe pandemic this century has been the 1918 H1N1 "Spanish flu". The virus killed between 40 and 50 million people worldwide during 1918 and 1919 (10). Based on preserved specimens, all genes have been genetically characterised and the entire virus has now been restored (27). This gives a unique opportunity to elucidate the mechanisms of immunopathogenesis of the pandemic strain.
[0051]The pandemic strains of 1957 (H2N2) and 1968 (H3N2) were both a result of genetic reassortment with avian viruses (11,17). The origin of the 1918 pandemic is debated. Taubenberger et al., (26) suggested based on phylogenetic analysis of the polymerase genes that the virus was entirely of avian origin. However, there are large disagreements about the actual origin of the virus and many still believe that this pandemic strain also was a reassortant between a mammalian and avian virus (1,26). The hemagglutinin (HA) and neuraminidase (NA) genes of the 1918 H1N1 strain did not possess known genetic indicators for high virulence that could have explained the severity observed in humans (19,20). However, the HA (and NA) protein on a backbone of recent human viruses conferred enhanced pathogenicity in mice (12,29). It might have been the combination of genes more than the HA itself that caused the lethal phenotype (27). The uncertainty about the origin and the mechanisms of high virulence of the 1918 H1N1 virus has raised questions if it is possible to develop protective immunity to this virus. Recently it has been published that a DNA vaccine encoding the HA of the 1918 H1N1 strain showed protection to a lethal challenge of the recombinant 1918 H1N1 virus strain in mice (Kong W, Hood C, Yang Z, Wei C, Xu L, Garcia-Sastre A, Tumpey T M, Nabel G J. Protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination. PNAS 103(43):15987-91 (2006)).
DNA Vaccines:
[0052]DNA vaccines are here defined as naked DNA or RNA, DNA or RNA in solution for direct intramuscular or subcutaneous injection with or without electroporation or coupled to particles, e.g., gold beads for gene gun administration. The DNA can be linear containing only a promoter, the influenza genes and polyadenylation signal or this expression cassette in an expression plasmid.
[0053]The administration of DNA vaccine can be by saline or buffered saline injection of naked DNA or RNA, or injection of DNA plasmid or linear gene expressing DNA fragments coupled to particles, or inoculated by gene gun.
[0054]The two most common types of DNA vaccine administration are saline injection of naked DNA and gene gun DNA inoculations (DNA coated on solid gold beads administrated with helium pressure). Saline intra muscular injections of DNA preferentially generate a Th1 IgG2a response while gene gun delivery tends to initiate a more Th2 IgG1 response. Intramuscular injected plasmids are at risk of being degraded by extracellular deoxyribonucleases, however, the responses induced are often more long-lived than those induced by the gene gun method. Vaccination by gene gun delivery of DNA to the epidermis has proven to be the most effective method of immunization, probably because the skin contains all the necessary cells types, including professional antigen presenting cells (APC), for eliciting both humoral and cytotoxic cellular immune responses (Langerhans and dendritic cells). Complete protection from a lethal dose of influenza virus has been obtained with as little as 1 μg DNA in mice. The standard DNA vaccine consists of a vector with a gene of interest cloned into a bacterial plasmid engineered for optimal expression in eukaryotic cells. In one embodiment, a vaccine vector includes an origin of replication allowing for production in bacteria, a bacterial antibiotic resistance gene allowing for plasmid selection in bacterial culture, a strong constitutive promoter for optimal expression in mammalian cells (eukaryotic promoters such as those derived from cytomegalovirus (CMV) or simian virus provide the highest gene expression), a polyadenylation sequence to stabilise the mRNA transcripts, such as bovine growth hormone (BHG) or simian virus polyadenylation, and a multiple cloning site for insertion of an antigen gene. An intron A sequence can be included to improve expression of genes. Many bacterial DNA vaccine vectors contain unmethylated cytidinephosphate-guanosine (CpG) dinucleotide motifs that may elicit strong innate immune responses in the host.
[0055]In one embodiment, a eukaryotic expression vector contains a constitutive eukaryotic promoter, an intron, a polylinker which allows for convenient insertion of a vaccine gene, and a polyadenylation signal is used. Such a vector may also
[0056]Suitable expression vectors are known the art and may be used as backbones engineered to contain the elements described herein. For example, commercially available vectors (e.g., the WRG7079 vector available form PowderJect Vaccines, Madison, Wis.) and/or vectors described in the literature (e.g., Corbet et al, 2000 (5)) can be used as a backbone to contain the elements described herein. For example, in one embodiment, an expression vector of the invention is designed to contain one or more of the secretory signal from an influenza virus (e.g., the influenza A 1918 HA or NA secretory signals).
[0057]In recent years there have been several approaches to enhance and customise the immune response to DNA vaccine constructs (2nd generation DNA vaccines). For instance dicistronic vectors or multiple gene expressing plasmids have been used to express two genes simultaneously. Specific promoters have been engineered that restrict gene expression to certain tissues, and cytokine/antigen fusion genes have been constructed to enhance the immune response. Furthermore, genes may be codon optimised for optimal gene expression in the host and naive leader sequences may be substituted with optimised leaders increasing translation efficiency.
Viral DNA Vaccines:
[0058]DNA can be delivered by a viral vector such as Adenovirus, Modified vaccinia virus Ankara (MVA), Vaccinia, Adeno-associated virus (AAV), Alphavirus etc. Viral DNA vaccines are not a part of the present study and are not encompassed by this invention.
Codon Optimization:
[0059]Codon optimization is the complete exchange of the virus codons to those of highly expressed human genes and therefore also mammalian genes that include swine. Codon optimization does not change the encoded amino acids of the protein antigens encoded but may increase the eukaryotic protein expression in mammalian cells. Since genes of highly expressed human proteins has a high content of C and G there are an increased possibility of generating both immune stimulatory GpG motifs but also immune inhibitory GC sequences. Genes engineered using codon optimization are called "humanized" genes and are frequently used in DNA vaccines to enhance expression.
[0060]The DNA or RNA sequence for hemagglutinin and neuraminidase and Matrix and Nucleoprotein is changed so the sequence coding for said proteins is changed to be optimally expressed in humans.
[0061]In one embodiment, the invention provides the use of the 1918 HA and/or NA codon-optimized genes in a DNA vaccine against all seasonal circulating H1N1 influenza A strains including the A/New Caledonia/20/99(H1N1) like virus.
TABLE-US-00001 TABLE 1 nucleotide and amino acid sequences of the codon optimized genes and the proteins they express. HA 1918 synthetic gene 0607838, Based on acc. No.: AF117241: A/South Carolina/1/18 SEQ ID NO: 1: Nucleotide ATGGAGGCCAGGCTGCTGGTGCTGCTGTGCGCCTTCGCCGCCACCAACGCCGACACCATCTGCAT CGGCTACCACGCCAACAACAGCACCGACACCGTGGATACCGTGCTGGAGAAGAACGTGACCGTG ACCCACAGCGTGAACCTGCTGGAGGACAGCCACAACGGCAAGCTGTGCAAGCTGAAGGGAATC GCTCCCCTGCAGCTGGGCAAGTGCAACATCGCCGGCTGGCTGCTGGGCAACCCCGAGTGCGACC TGCTGCTGACCGCCAGCAGCTGGTCCTACATCGTGGAGACCAGCAACAGCGAGAACGGCACCTG CTACCCCGGCGACTTCATCGACTACGAGGAGCTGCGGGAGCAGCTGTCCAGCGTGAGCAGCTTC GAGAAGTTCGAGATCTTCCCCAAGACCAGCTCCTGGCCCAACCACGAGACCACCAAGGGCGTGA CCGCCGCCTGTAGCTACGCCGGAGCCAGCAGCTTCTACAGAAACCTGCTGTGGCTGACCAAGAA GGGCAGCAGCTACCCCAAGCTGTCCAAGAGCTACGTGAACAACAAGGGCAAGGAAGTGCTGGT GCTGTGGGGCGTGCACCACCCCCCTACCGGCACCGACCAGCAGAGCCTGTACCAGAACGCCGAC GCCTACGTGAGCGTGGGCAGCAGCAAGTACAACAGAAGGTTCACCCCCGAGATCGCCGCCAGGC CCAAGGTGCGCGACCAGGCCGGCAGGATGAACTACTACTGGACCCTGCTGGAGCCCGGCGACAC CATCACCTTCGAGGCCACCGGCAACCTGATCGCCCCTTGGTACGCCTTCGCCCTGAACAGGGGCA GCGGCAGCGGCATCATCACCAGCGACGCCCCCGTGCACGACTGCAACACCAAGTGCCAGACCCC CCACGGAGCCATCAACAGCAGCCTGCCCTTCCAGAACATCCACCCCGTGACCATCGGCGAGTGC CCCAAGTACGTGAGAAGCACCAAGCTGAGGATGGCCACCGGCCTGAGGAACATCCCCAGCATCC AGAGCAGGGGCCTGTTCGGAGCCATCGCCGGATTCATCGAGGGCGGCTGGACCGGCATGATCGA CGGCTGGTACGGCTACCACCACCAGAACGAGCAGGGCAGCGGCTACGCCGCCGACCAGAAGAG CACCCAGAACGCCATCGACGGCATCACCAACAAGGTGAACAGCGTGATCGAGAAGATGAACAC CCAGTTCACCGCCGTGGGCAAGGAGTTCAACAACCTGGAGAGGCGGATCGAGAACCTGAACAA GAAGGTGGACGACGGCTTCCTGGACATCTGGACCTACAACGCCGAGCTGCTGGTGCTGCTGGAG AACGAGAGGACCCTGGACTTCCACGACAGCAACGTGAGGAACCTGTACGAGAAGGTGAAGAGC CAGCTGAAGAACAACGCCAAGGAGATCGGCAACGGCTGCTTCGAGTTCTACCACAAGTGCGACG ACGCCTGCATGGAGAGCGTGAGAAACGGCACCTACGACTACCCCAAGTACAGCGAGGAGAGCA AGCTGAACCGGGAGGAGATCGACGGCGTGAAGCTGGAGAGCATGGGCGTGTACCAGATCCTGG CCATCTACAGCACCGTGGCCAGCAGCCTGGTGCTGCTGGTGTCCCTGGGAGCCATCAGCTTTTGG ATGTGCAGCAACGGCAGCCTGCAGTGCAGGATCTGCATCTGA SEQ ID NO: 2 Amino acid MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCKLKGIAPL QLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEELREQLSSVSSFEKFEIFPK TSSWPNHETTKGVTAACSYAGASSFYRNLLWLTKKGSSYPKLSKSYVNNKGKEVLVLWGVHHPPT GTDQQSLYQNADAYVSVGSSKYNRRFTPEIAARPKVRDQAGRMNYYWTLLEPGDTITFEATGNLIAP WYAFALNRGSGSGIITSDAPVHDCNTKCQTPHGAINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLR NIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGITNKVNSVIEKM NTQFTAVGKEFNNLERRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVRNLYEKVKSQL KNNAKEIGNGCFEFYHKCDDACMESVRNGTYDYPKYSEESKLNREEIDGVKLESMGVYQILAIYSTV ASSLVLLVSLGAISFWMCSNGSLQCRICI. NA 1918 synthetic gene 0607869, Based on acc. No.: AF250356: A/Brevig mission/1/1918 SEQ ID NO: 3 Nucleotide ATGAACCCCAACCAGAAGATCATCACCATCGGCAGCATCTGCATGGTGGTGGGCATCATCAGCC TGATCCTGCAGATCGGCAACATCATCAGCATCTGGGTGTCCCACAGCATCCAGACCGGCAACCA GAACCACCCCGAGACCTGCAACCAGTCCATCATCACCTACGAGAACAACACCTGGGTGAACCAG ACCTACGTGAACATCAGCAACACCAACGTGGTGGCCGGCCAGGACGCCACCTCCGTGATCCTGA CAGGCAACAGCAGCCTGTGCCCCATCAGCGGCTGGGCCATCTACAGCAAGGACAACGGCATCAG GATCGGCAGCAAGGGCGACGTGTTCGTGATCAGAGAGCCCTTCATCAGCTGCAGCCACCTGGAA TGCAGGACCTTCTTCCTGACCCAAGGAGCCCTGCTGAACGACAAGCACAGCAACGGCACCGTGA AGGACAGAAGCCCCTACAGGACCCTGATGAGCTGCCCCGTGGGCGAGGCTCCCAGCCCCTACAA CAGCAGATTCGAGAGCGTGGCCTGGTCCGCCAGCGCCTGCCACGACGGCATGGGCTGGCTGACC ATCGGCATCAGCGGCCCTGACAACGGGGCCGTGGCCGTGCTGAAGTACAACGGAATCATCACCG ACACCATCAAGAGCTGGCGGAACAACATCCTGAGGACCCAGGAAAGCGAGTGCGCCTGCGTGA ACGGCAGCTGCTTCACCATCATGACCGACGGCCCCAGCAACGGCCAGGCCAGCTACAAGATCCT GAAGATCGAGAAGGGCAAGGTGACCAAGAGCATCGAGCTGAACGCCCCCAACTACCACTACGA GGAATGCAGCTGCTACCCCGACACCGGCAAGGTCATGTGCGTGTGCAGGGACAACTGGCACGGC AGCAACAGGCCCTGGGTGTCCTTCGACCAGAACCTGGACTACCAGATCGGATACATCTGCAGCG GCGTGTTCGGCGACAACCCCAGGCCCAACGACGGCACCGGCAGCTGCGGCCCTGTGAGCAGCAA CGGGGCCAATGGCATCAAGGGCTTCAGCTTCAGATACGACAACGGCGTGTGGATCGGCCGCACC AAGAGCACCAGCAGCAGATCCGGCTTCGAGATGATCTGGGACCCCAACGGCTGGACCGAGACC GACAGCAGCTTCAGCGTGAGGCAGGACATCGTGGCCATCACCGACTGGTCCGGCTACAGCGGCA GCTTCGTGCAGCACCCCGAGCTGACCGGCCTGGACTGCATGAGGCCCTGTTTCTGGGTGGAGCTG ATCAGAGGCCAGCCCAAGGAGAACACCATCTGGACCAGCGGCAGCAGCATCAGCTTTTGCGGCG TGAACAGCGACACCGTGGGCTGGTCCTGGCCCGACGGGGCCGAGCTGCCCTTCAGCATCGATAA GTGA SEQ ID NO: 4: Amino acid MNPNQKIITIGSICMVVGIISLILQIGNIISIWVSHSIQTGNQNHPETCNQSIITYENNTWVNQTYVNISNT NVVAGQDATSVILTGNSSLCPISGWAIYSKDNGIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLND KHSNGTVKDRSPYRTLMSCPVGEAPSPYNSRFESVAWSASACHDGMGWLTIGISGPDNGAVAVLKY NGIITDTIKSWRNNILRTQESECACVNGSCFTIMTDGPSNGQASYKILKIEKGKVTKSIELNAPNYHYE ECSCYPDTGKVMCVCRDNWHGSNRPWVSFDQNLDYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGA NGIKGFSFRYDNGVWIGRTKSTSSRSGFEMIWDPNGWTETDSSFSVRQDIVAITDWSGYSGSFVQHPE LTGLDCMRPCFWVELIRGQPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFSIDK. NP 1918 synthetic gene 0607866, Based on acc. No.: AY44935: A/Brevig mission/1/1918 SEQ ID NO: 5: Nucleotide ATGGCCAGCCAGGGCACCAAGAGAAGCTACGAGCAGATGGAAACCGACGGCGAGAGGCAGAAC GCCACCGAGATCAGGGCCAGCGTGGGCAGGATGATCGGCGGCATCGGCAGGTTCTACATCCAGA TGTGCACCGAGCTGAAGCTGTCCGACTACGAGGGCAGGCTGATCCAGAACAGCATCACCATCGA GAGGATGGTGCTGTCCGCCTTCGACGAGAGAAGAAACAAGTACCTGGAAGAGCACCCCAGCGC CGGCAAGGACCCCAAGAAAACCGGCGGACCCATCTACAGAAGGATCGACGGCAAGTGGATGAG AGAGCTGATCCTGTACGACAAGGAGGAAATCAGAAGGATCTGGCGGCAGGCCAACAACGGCGA GGACGCCACAGCCGGCCTGACCCACATGATGATCTGGCACAGCAACCTGAACGACGCCACCTAC CAGAGGACCAGGGCCCTCGTCAGAACCGGCATGGACCCCCGGATGTGCAGCCTGATGCAGGGCA GCACACTGCCCAGAAGAAGCGGAGCTGCTGGAGCCGCCGTGAAGGGCGTGGGCACCATGGTGA TGGAACTGATCAGGATGATCAAGAGGGGCATCAACGACAGGAACTTTTGGAGGGGCGAGAACG GCAGAAGGACCAGGATCGCCTACGAGAGGATGTGCAACATCCTGAAGGGCAAGTTCCAGACAG CCGCCCAGAGGGCCATGATGGACCAGGTCCGGGAGAGCAGGAACCCCGGCAACGCCGAGATCG AGGACCTGATCTTCCTGGCCAGAAGCGCCCTGATCCTGAGGGGCAGCGTGGCCCACAAGAGCTG CCTGCCCGCCTGCGTGTACGGACCCGCCGTGGCCAGCGGCTACGACTTCGAGAGAGAGGGCTAC AGCCTGGTCGGCATCGACCCCTTCAGGCTGCTGCAGAACTCCCAGGTGTACTCTCTGATCAGGCC CAACGAGAACCCCGCCCACAAGTCCCAGCTGGTCTGGATGGCCTGCCACAGCGCCGCCTTCGAG GATCTGAGAGTGAGCAGCTTCATCAGGGGCACCAGAGTGGTGCCCAGGGGCAAGCTGTCCACCA GGGGCGTGCAGATCGCCAGCAACGAGAACATGGAAACCATGGACAGCAGCACCCTGGAACTGA GAAGCAGGTACTGGGCCATCAGGACCAGAAGCGGCGGCAACACCAACCAGCAGAGGGCCAGCG CCGGACAGATCAGCGTGCAGCCCACCTTCTCCGTGCAGAGGAACCTGCCCTTCGAGAGGGCCAC CATCATGGCCGCCTTCACCGGCAACACCGAGGGCAGGACCAGCGACATGAGGACCGAGATCATC AGAATGATGGAAAGCGCCAGGCCCGAGGACGTGAGCTTCCAGGGCAGGGGCGTGTTCGAGCTG TCCGATGAGAAGGCCACCTCCCCCATCGTGCCCAGCTTCGACATGAGCAACGAGGGCAGCTACT TCTTCGGCGACAACGCCGAGGAATACGACAACTGA SEQ ID NO: 6: Amino acid MASQGTKRSYEQMETDGERQNATEIRASVGRMIGGIGRFYIQMCTELKLSDYEGRLIQNSITIERMVL SAFDERRNKYLEEHPSAGKDPKKTGGPIYRRIDGKWMRELILYDKEEIRRIWRQANNGEDATAGLTH MMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKR GINDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALIL RGSVAHKSCLPACVYGPAVASGYDFEREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMAC HSAAFEDLRVSSFIRGTRVVPRGKLSTRGVQIASNENMETMDSSTLELRSRYWAIRTRSGGNTNQQR ASAGQISVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSD EKATSPIVPSFDMSNEGSYFFGDNAEEYDN. M 1918 synthetic gene 0607868, Based on acc. No.: AY130766: A/Brevig mission/1/1918 SEQ ID NO: 7: Nucleotide ATGAGTCTTTTAACCGAGGTCGAAACGTACGTTCTCTCTATCGTCCCGTCAGGCCCCCTCAAAGC CGAGATCGCGCAGAGACTTGAAGATGTCTTTGCAGGGAAGAACACCGATCTTGAGGCTCTCATG GAATGGCTAAAGACAAGACCAATCCTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCAC GCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGG AACGGGGATCCAAATAACATGGACAGAGCAGTTAAACTGTACAGGAAGCTTAAGAGGGAGATA ACATTCCATGGGGCCAAAGAAGTAGCACTCAGTTATTCCGCTGGTGCACTTGCCAGTTGTATGGG CCTCATATACAACAGGATGGGGACTGTGACCACTGAAGTGGCATTTGGCCTGGTATGCGCAACC TGTGAACAGATTGCTGATTCCCAGCATCGGTCTCACAGGCAAATGGTGACAACAACCAATCCAC TAATCAGACATGAGAACAGAATGGTACTGGCCAGCACTACGGCTAAGGCTATGGAGCAAATGGC TGGATCGAGTGAGCAAGCAGCAGAGGCCATGGAGGTTGCTAGTCAGGCTAGGCAAATGGTGCA GGCGATGAGAACCATTGGGACTCATCCTAGCTCCAGTGCTGGTCTGAAAGACGATCTTATTGAA AATTTGCAGGCCTACCAGAAACGAATGGGGGTGCAGATGCAACGATTCAAGTGATCCTCTCGTT ATTGCCGCAAGTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTCTTTTTTTCAAA TGCATTTATCGTCGCCTTAAATACGGTTTGAAAAGAGGGCCTTCTACGGAAGGAGTGCCGGAGT CTATGAGGGAAGAATATCGAAAGGAACAGCAGAGTGCTGTGGATGTTGACGATGGTCATTTTGT CAACATAGAGCTGGAGTAAGGCGCC Amino acid SEQ ID NO: 8: M1 protein MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTLTVP SERGLQRRRFVQNALNGNGDPNNMDRAVKLYRKLKREITFHGAKEVALSYSAGALASCMGLIYNR MGTVTTEVAFGLVCATCEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQA AEAMEVASQARQMVQAMRTIGTHPSSSAGLKDDLIENLQAYQKRMGVQMQRFK. SEQ ID NO: 9: M2 protein MSLLTEVETPTRNEWGCRCNDSSDPLVIAASIIGILHLILWILDRLFFKCIYRRLKYGLKRGPSTEGVPE SMREEYRKEQQSAVDVDDGHFVNIELE.
[0062]The 1918 HA and NA amino sequences are publicly available (GenBank A/south Carolina/1/18 AF117241, A/Brevig Mission/1/18 AF250356) and can be translated into DNA using standard optimal codons for eukaryotic mammalian expression using standard expression vectors (key features: CMV promoter, intron A, Kozak sequence, vaccine gene inclusive of its secretion sequence, stop codon, Polyadenylation A, kanamycin resistance gene) are included for growing and selection of transfected E. coli for plasmid DNA production.
[0063]DNA vaccination with the 1918 H1N1HA and NA synthetic codon optimized genes using gene gun standard conditions induces protective immunity to present day circulating influenza A virus as exemplified using A/New Caledonia/20/99(H1N1) virus challenge in DNA vaccinated ferrets (Mustela Putorius Furo). This is highly surprising since the two viruses are separated by more than 80 years of antigenic drift and show about 21% difference in the HA1 protein. Normally a protective protein vaccine must be based upon the amino acid sequence of the circulating seasonal influenza A strain to induce protection. Moreover the protection by the 1918 DNA vaccine against 2007 circulating strain is more consistent than the traditional protein vaccine based on the homologous circulating strain (New Caledonia). This suggest that the 1918 based DNA vaccine induces a much broader protective immunity that protects against influenza A H1N1 strains from 1918 to present time and perhaps beyond.
[0064]The unusually broad protection may be due to a unique amino acid sequence in the 1918 HA and/or NA proteins inducing broader protective antibodies to special epitopes or cellular immunity or immune adjuvants effect, or a particular gene expression or particular immune induction by the optimized nucleotide sequence of the particular 1918 H1N1 genes, or some or all of these factors in combination.
[0065]The advantages are that a limited number of vaccine components delivered as a DNA vaccine either as naked DNA or RNA as plasmid or linear encoding sequences or incorporated into recombinant virus provide for more efficient delivery.
[0066]The discovery of a broad protection induced by the pandemic influenza A strain 1918 H1N1 may suggest that a similar good protection may be obtained against circulating H2 strains using DNA vaccines based on HA and/or NA from the 1958 H2N2 pandemic strain and against circulating H3 strains using DNA vaccines based in HA and/or NA from the 1968 pandemic strain.
[0067]The unusually broad and/or efficient protection obtained using a pandemic influenza A strain instead of the present day circulating strains may be due to special features in the sequence of the first new pathogenic and spreading virus. These features may gradually wane by accumulation of sequence changes during years of adaptation to the human and swine population.
[0068]If the protective feature is contained in the encoded amino acid sequence of the HA and/or NA 1918 and not the nucleotide sequence then the HA and/or NA protein(s) from 1918 may be used alone as an alternative to DNA or in combination with the DNA vaccine for immunization or vaccinations.
[0069]The use of the DNA vaccine components may serve as an adjuvant for the protein components and thus the protein and the DNA can be preferentially administered together as a mixed vaccine.
[0070]As a more universal DNA and/or protein vaccine against contemporary influenza in humans and/or swine a mixture may be used of HA and NA from the 1918 H1N1 pandemic strain plus HA and/or NA from the 1957 H2N2 pandemic strain plus HA from the 1968 H3N2 pandemic strain, where the N2 component is similar to the NA of the preferred earlier 1957 H2N2 strain.
TABLE-US-00002 TABLE 2 nucleotide and amino acid sequences of the codon optimized genes and the proteins they express (not codon optimized). HA H3N2 Acc. No.: AB295605: A/Aichi/2/1968(H3N2) SEQ ID NO: 10: Nucleotide ATAATTCTATTAATCATGAAGACCATCATTGCTTTGAGCTACATTTTCTGTCTGGCTCTCGGCCAAGACCTTCC- A GGAAATGACAACAGCACAGCAACGCTGTGCCTGGGACATCATGCGGTGCCAAACGGAACACTAGTGAAAACAAT- C ACAGATGATCAGATTGAAGTGACTAATGCTACTGAGCTAGTTCAGAGCTCCTCAACGGGGAAAATATGCAACAA- T CCTCATCGAATCCTTGATGGAATAGACTGCACACTGATAGATGCTCTATTGGGGGACCCTCATTGTGATGTTTT- T CAAAATGAGACATGGGACCTTTTCGTTGAACGCAGCAAAGCTTTCAGCAACTGTTACCCTTATGATGTGCCAGA- T TATGCCTCCCTTAGGTCACTAGTTGCCTCGTCAGGCACTCTGGAGTTTATCACTGAGGGTTTCACTTGGACTGG- G GTCACTCAGAATGGGGGAAGCAATGCTTGCAAAAGGGGACCTGGTAGCGGTTTTTTCAGTAGACTGAACTGGTT- G ACCAAATCAGGAAGCACATATCCAGTGCTGAACGTGACTATGCCAAACAATGACAATTTTGACAAACTATACAT- T TGGGGGGTTCACCACCCGAGCACGAACCAAGAACAAACCAGCCTGTATGTTCAAGCATCAGGGAGAGTCACAGT- C TCTACCAGGAGAAGCCAGCAAACTATAATCCCGAATATCGAGTCCAGACCCTGGGTAAGGGGTCTGTCTAGTAG- A ATAAGCATCTATTGGACAATAGTTAAGCCGGGAGACGTACTGGTAATTAATAGTAATGGGAACCTAATCGCTCC- T CGGGGTTATTTCAAAATGCGCACTGGGAAAAGCTCAATAATGAGGTCAGATGCACCTATTGATACCTGTATTTC- T GAATGCATCACTCCAAATGGAAGCATTCCCAATGACAAGCCCTTTCAAAACGTAAACAAGATCACATATGGAGC- A TGCCCCAAGTATGTTAAGCAAAACACCCTGAAGTTGGCAACAGGGATGCGGAATGTACCAGAGAAACAAACTAG- A GGCCTATTCGGCGCAATAGCAGGTTTCATAGAAAATGGTTGGGAGGGAATGATAGACGGTTGGTACGGTTTCAG- G CATCAAAATTCTGAGGGCACAGGACAAGCAGCAGATCTTAAAAGCACTCAAGCAGCCATCGACCAAATCAATGG- G AAATTGAACAGGGTAATCGAGAAGACGAACGAGAAATTCCATCAAATCGAAAAGGAATTCTCAGAAGTAGAAGG- G AGAATTCAGGACCTCGAGAAATACGTTGAAGACACTAAAATAGATCTCTGGTCTTACAATGCGGAGCTTCTTGT- C GCTCTGGAGAATCAACATACAATTGACCTGACTGACTCGGAAATGAACAAGCTGTTTGAAAAAACAAGGAGGCA- A CTGAGGGAAAATGCTGAAGACATGGGCAATGGTTGCTTCAAAATATACCACAAATGTGACAACGCTTGCATAGA- G TCAATCAGAAATGGGACTTATGACCATGATGTATACAGAGACGAAGCATTAAACAACCGGTTTCAGATCAAAGG- T GTTGAACTGAAGTCTGGATACAAAGACTGGATCCTGTGGATTTCCTTTGCCATATCATGCTTTTTGCTTTGTGT- T GTTTTGCTGGGGTTCATCATGTGGGCCTGCCAGAGAGGCAACATTAGGTGCAACATTTGCATTTGAGTGTATTA- G TAATTA SEQ ID NO: 11: Amino acid MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKT ITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDL FVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPG SGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRV TVSTRRSQQTIIPNIESRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKM RTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLAT GMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQ INGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQ HTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYR DEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQRGNIRCNICI NA H3N2 Acc. No.: AB295606: A/Aichi/2/1968(H3N2) SEQ ID NO: 12: Nucleotide GAAAATGAATCCAAATCAAAAGATAATAACAATTGGCTCTGTCTCTCTCACCATTGCAACAGTATGCTTCCTCA- T GCAGATTGCCATCCTGGTAACTACTGTAACATTGCATTTTAAGCAATATGAGTGCGACTCCCCCGCGAGCAACC- A AGTAATGCCGTGTGAACCAATAATAATAGAAAGGAACATAACAGAGATAGTGTATTTGAATAACACCACCATAG- A GAAAGAGATATGCCCCAAAGTAGTGGAATACAGAAATTGGTCAAAGCCGCAATGTCAAATTACAGGATTTGCAC- C TTTTTCTAAGGACAATTCAATCCGGCTTTCTGCTGGTGGGGACATTTGGGTGACGAGAGAACCTTATGTGTCAT- G CGATCATGGCAAGTGTTATCAATTTGCACTCGGGCAGGGGACCACACTAGACAACAAACATTCAAATGACACAA- T ACATGATAGAATCCCTCATCGAACCCTATTAATGAATGAGTTGGGTGTTCCATTTCATTTAGGAACCAGGCAAG- T GTGTATAGCATGGTCCAGCTCAAGTTGTCACGATGGAAAAGCATGGCTGCATGTTTGTATCACTGGGGATGACA- A AAATGCAACTGCTAGCTTCATTTATGACGGGAGGCTTGTGGACAGTATTGGTTCATGGTCTCAAAATATCCTCA- G AACCCAGGAGTCGGAATGCGTTTGTATCAATGGGACTTGCACAGTAGTAATGACTGATGGAAGTGCTTCAGGAA- G AGCCGATACTAGAATACTATTCATTGAAGAGGGGAAAATTGTCCATATTAGCCCATTGTCAGGAAGTGCTCAGC- A TGTAGAAGAGTGTTCCTGTTATCCTAGATATCCTGGCGTCAGATGTATCTGCAGAGACAACTGGAAAGGCTCTA- A TAGGCCCGTCGTAGACATAAATATGGAAGATTATAGCATTGATTCCAGTTATGTGTGCTCAGGGCTTGTTGGCG- A CACACCTAGAAACGACGACAGATCTAGCAATAGCAATTGCAGGAATCCTAATAATGAGAGAGGGAATCAAGGAG- T GAAAGGCTGGGCCTTTGACAATGGAGATGACGTGTGGATGGGAAGAACGATCAGCAAGGATTTACGCTCAGGTT- A TGAAACTTTCAAAGTCATTGGTGGTTGGTCCACACCTAATTCCAAATCGCAGATCAATAGACAAGTCATAGTTG- A CAGCGATAATCGGTCAGGTTACTCTGGTATTTTCTCTGTTGAGGGCAAAAGCTGCATCAATAGGTGCTTTTATG- T GGAGTTGATAAGGGGAAGGAAACAGGAGACTAGAGTGTGGTGGACCTCAAACAGTATTGTTGTGTTTTGTGGCA- C TTCAGGTACCTATGGAACAGGCTCATGGCCTGATGGGGCGAACATCAATTTCATGCCTATATAAGCTTTCGCAA- T TTTAGA SEQ ID NO: 13: Amino acid MNPNQKIITIGSVSLTIATVCFLMQIAILVTTVTLHFKQYECDS PASNQVMPCEPIIIERNITEIVYLNNTTIEKEICPKVVEYRNWSKPQCQITGFAPFSK DNSIRLSAGGDIWVTREPYVSCDHGKCYQFALGQGTTLDNKHSNDTIHDRIPHRTLLM NELGVPFHLGTRQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYDGRLVDSIGSW SQNILRTQESECVCINGTCTVVMTDGSASGRADTRILFIEEGKIVHISPLSGSAQHVE ECSCYPRYPGVRCICRDNWKGSNRPVVDINMEDYSIDSSYVCSGLVGDTPRNDDRSSN SNCRNPNNERGNQGVKGWAFDNGDDVWMGRTISKDLRSGYETFKVIGGWSTPNSKSQI NRQVIVDSDNRSGYSGIFSVEGKSCINRCFYVELIRGRKQETRVWWTSNSIVVFCGTS GTYGTGSWPDGANINFMPI HA H2N2 Acc. No: CY022013: A/Albany/20/1957(H2N2) SEQ ID NO: 14: Nucleotide ATAGACAACCAAAAGCAAAACAATGGCCATCATTTATCTCATTCTCCTGTTCACAGCAGTGAGAGGGGACCAGA- T ATGCATTGGATACCATGCCAATAATTCCACAGAGAAGGTCGACACAATTCTAGAGCGGAACGTCACTGTGACTC- A TGCCAAGGACATTCTTGAGAAGACCCATAACGGAAAGTTATGCAAACTAAACGGAATCCCTCCACTTGAACTAG- G GGACTGTAGCATTGCCGGATGGCTCCTTGGAAATCCAGAATGTGATAGGCTTCTAAGTGTGCCAGAATGGTCCT- A TATAATGGAGAAAGAAAACCCGAGAGACGGTTTGTGTTATCCAGGCAGCTTCAATGATTATGAAGAATTGAAAC- A TCTCCTCAGCAGCGTGAAACATTTCGAGAAAGTAAAGATTCTGCCCAAAGATAGATGGACACAGCATACAACAA- C TGGAGGTTCACGGGCCTGCGCGGTGTCTGGTAATCCATCATTCTTCAGGAACATGATCTGGCTGACAAAGAAAG- G ATCAAATTATCCGGTTGCCAAAGGATCGTACAACAATACAAGCGGAGAACAAATGCTAATAATTTGGGGGGTGC- A CCATCCCAATGATGAGACAGAACAAAGAACATTGTACCAGAATGTGGGAACCTATGTTTCCGTAGGCACATCAA- C ATTGAACAAAAGGTCAACCCCAGACATAGCAACAAGGCCTAAAGTGAATGGACTAGGAAGTAGAATGGAATTCT- C TTGGACCCTATTGGATATGTGGGACACCATAAATTTTGAGAGTACTGGTAATCTAATTGCACCAGAGTATGGAT- T CAAAATATCGAAAAGAGGTAGTTCAGGGATCATGAAAACAGAAGGAACACTTGGGAACTGTGAGACCAAATGCC- A AACTCCTTTGGGAGCAATAAATACAACATTGCCTTTTCACAATGTCCACCCACTGACAATAGGTGAGTGCCCCA- A ATATGTAAAATCGGAGAAGTTGGTCTTAGCAACAGGACTAAGGAATGTTCCCCAGATTGAATCAAGAGGATTGT- T TGGGGCAATAGCTGGTTTTATAGAAGGAGGATGGCAAGGAATGGTTGATGGTTGGTATGGATACCATCACAGCA- A TGACCAGGGATCAGGGTATGCAGCGGACAAAGAATCCACTCAAAAGGCATTTGATGGAATCACCAACAAGGTAA- A TTCTGTGATTGAAAAGATGAACACCCAATTTGAAGCTGTTGGGAAAGAATTCAGTAACTTAGAGAGAAGACTGG- A GAACTTGAACAAAAAGATGGAAGACGGGTTTCTAGATGTGTGGACATACAATGCTGAGCTTCTAGTTCTGATGG- A AAATGAGAGGACACTTGACTTTCATGATTCTAATGTCAAGAATCTGTATGATAAAGTCAGAATGCAGCTGAGAG- A CAACGTCAAAGAACTAGGAAATGGATGTTTTGAATTTTATCACAAATGTGATGATGAATGCATGAATAGTGTGA- A AAACGGGACGTATGATTATCCCAAGTATGAAGAAGAGTCTAAACTAAATAGAAATGAAATCAAAGGGGTAAAAT- T GAGCAGCATGGGGGTTTATCAAATCCTTGCCATTTATGCTACAGTAGCAGGTTCTCTGTCACTGGCAATCATGA- T GGCTGGGATCTCTTTCTGGATGTGCTCCAACGGGTCTCTGCAGTGCAGGATCTGCATATGATTATAAGTCATTT- T ATAATTAA SEQ ID NO: 15: Amino acid MAIIYLILLFTAVRGDQICIGYHANNSTEKVDTILERNVTVTHA KDILEKTHNGKLCKLNGIPPLELGDCSIAGWLLGNPECDRLLSVPEWSYIMEKENPRD GLCYPGSFNDYEELKHLLSSVKHFEKVKILPKDRWTQHTTTGGSRACAVSGNPSFFRN MIWLTKKGSNYPVAKGSYNNTSGEQMLIIWGVHHPNDETEQRTLYQNVGTYVSVGTST LNKRSTPDIATRPKVNGLGSRMEFSWTLLDMWDTINFESTGNLIAPEYGFKISKRGSS GIMKTEGTLGNCETKCQTPLGAINTTLPFHNVHPLTIGECPKYVKSEKLVLATGLRNV PQIESRGLFGAIAGFIEGGWQGMVDGWYGYHHSNDQGSGYAADKESTQKAFDGITNKV NSVIEKMNTQFEAVGKEFSNLERRLENLNKKMEDGFLDVWTYNAELLVLMENERTLDF HDSNVKNLYDKVRMQLRDNVKELGNGCFEFYHKCDDECMNSVKNGTYDYPKYEEESKL NRNEIKGVKLSSMGVYQILAIYATVAGSLSLAIMMAGISFWMCSNGSLQCRICI NA H2N2 Acc. No.: CY022015: A/Albany/20/1957(H2N2) SEQ ID NO: 16: Nucleotide TGAAAATGAATCCAAATCAAAAGATAATAACAATTGGCTCTGTCTCTCTCACCATTGCAACAGTATGCTTCCTC- A TGCAGATTGCCATCCTGGCAACTACTGTGACATTGCATTTTAAACAACATGAGTGCGACTCCCCCGCGAGCAAC- C AAGTAATGCCATGTGAACCAATAATAATAGAAAGGAACATAACAGAGATAGTGTATTTGAATAACACCACCATA- G AGAAAGAGATTTGCCCCGAAGTAGTGGAATACAGAAATTGGTCAAAGCCGCAATGTCAAATTACAGGATTTGCA- C CTTTTTCTAAGGACAATTCAATCCGGCTTTCTGCTGGTGGGGACATTTGGGTGACGAGAGAACCTTATGTGTCA- T GCGATCCTGGCAAGTGTTATCAATTTGCACTCGGGCAAGGGACCACACTAGACAACAAACATTCAAATGGCACA- A TACATGATAGAATCCCTCACCGAACCCTATTAATGAATGAGTTGGGTGTTCCATTTCATTTAGGAACCAAACAA- G TGTGTGTAGCATGGTCCAGCTCAAGTTGTCACGATGGAAAAGCATGGTTGCATGTTTGTGTCACTGGGGATGAT- A GAAATGCGACTGCCAGCTTCATTTATGACGGGAGGCTTGTGGACAGTATTGGTTCATGGTCTCAAAATATCCTC- A GGACCCAGGAGTCGGAATGCGTTTGTATCAATGGGACTTGCACAGTAGTAATGACTGATGGAAGTGCATCAGGA- A GAGCCGATACTAGAATACTATTCATTAAAGAGGGGAAAATTGTCCATATCAGCCCATTGTCAGGAAGTGCTCAG- C ATATAGAGGAGTGTTCCTGTTACCCTCGATATCCTGACGTCAGATGTATCTGCAGAGACAACTGGAAAGGCTCT- A ATAGGCCCGTTATAGACATAAATATGGAAGATTATAGCATTGATTCCAGTTATGTGTGCTCAGGGCTTGTTGGC- G ACACACCCAGGAACGACGACAGCTCTAGCAATAGCAATTGCAGGGATCCTAACAATGAGAGAGGGAATCCAGGA- G TGAAAGGCTGGGCCTTTGACAATGGAGATGATGTATGGATGGGAAGAACAATCAACAAAGATTCACGCTCAGGT- T ATGAAACTTTCAAAGTCATTGGTGGTTGGTCCACACCTAATTCCAAATCGCAGGTCAATAGACAGGTCATAGTT- G ACAACAATAATTGGTCTGGTTACTCTGGTATTTTCTCTGTTGAGGGCAAAAGCTGCATCAATAGGTGCTTTTAT- G TGGAGTTGATAAGGGGAAGGCCACAGGAGACTAGAGTATGGTGGACCTCAAACAGTATTGTTGTGTTTTGTGGC- A CTTCAGGTACTTATGGAACAGGCTCATGGCCTGATGGGGCGAACATCAATTTCATGCCTATATAAGCTTTCGCA- A TTTTAGAAAA SEQ ID NO: 17: Amino acid MNPNQKIITIGSVSLTIATVCFLMQIAILATTVTLHFKQHECDS PASNQVMPCEPIIIERNITEIVYLNNTTIEKEICPEVVEYRNWSKPQCQITGFAPFSK DNSIRLSAGGDIWVTREPYVSCDPGKCYQFALGQGTTLDNKHSNGTIHDRIPHRTLLM NELGVPFHLGTKQVCVAWSSSSCHDGKAWLHVCVTGDDRNATASFIYDGRLVDSIGSW SQNILRTQESECVCINGTCTVVMTDGSASGRADTRILFIKEGKIVHISPLSGSAQHIE ECSCYPRYPDVRCICRDNWKGSNRPVIDINMEDYSIDSSYVCSGLVGDTPRNDDSSSN SNCRDPNNERGNPGVKGWAFDNGDDVWMGRTINKDSRSGYETFKVIGGWSTPNSKSQV NRQVIVDNNNWSGYSGIFSVEGKSCINRCFYVELIRGRPQETRVWWTSNSIVVFCGTSGTY GTGSWPDGANINFMPI
[0071]The following examples are illustrative of the compositions and methods of the invention. It will be readily understood by one of skill in the art that the specific conditions described herein can be varied without departing from the scope of the present invention. It will be further understood that other compositions not specifically illustrated are within the scope of the invention as defined herein.
EXAMPLES
Example 1: Construction of Expression Vectors
[0072]The 1918 pandemic H1N1 genes were designed from nucleotide sequences published in GenBank (HA: A/South Carolina/1/18 AF117241, and NA, NP and M: A/Brevig Mission/1/18 AF250356, AY744035 and AY130766, respectively). The genes were made synthetically and designed to include the appropriate restriction enzymes and Kozak sequence (GCCACC), -1 base upstream from the start codon, for efficient cloning and transcription in the WRG7079 expression vector (PowderJect, Madison, Wis.). The genes were synthesised using only codons from highly expressed human genes (5) (codon optimised). By this the nucleotide codons are altered (humanised), but the encoded amino acids are identical to those encoded by the viral RNA. The genes were further cloned individually into the WRG7079 expression vector. Key elements in the expression vector are a kanamycin resistance gene, cytomegalovirus immediate-early promoter, intron A, and polyadenylation signal. The tissue plasminogen activator (tPA) signal sequence in the original WRG7079 expression vector, used to target proteins to a secretory pathway, was excised in favour of the influenza signal sequence located in the 1918 HA and NA genes. The same vector was also applied for expression of the internal genes NP and M1 that do not have secretory signals, and which are naturally located inside the virus and inside the infected cells; therefore the tPA secretory signal of the WRG7079 was removed. The WRG7079 was further modified to remove an unwanted app109 nucleotide sequence from the SIV nef gene.
[0073]Viral RNA from the A/New Caledonia/20/99(H1N1) MDCK cell cultivated virus was isolated by QIAamp® Viral RNA Mini Kit (QIAGEN, Hilden, Germany) and RT-PCR was performed as previously described (2) by OneStep® RT-PCR Kit (QIAGEN). The primers were designed to amplify the coding gene of HA and NA. The same restriction sites and Kozak sequence were included in the primers as for the 1918 H1N1 constructs (HA NC F: 5'-caacgcgtgccaccatgaaagcaaaactactgg-3' (SEQ ID NO:18), HA NC R: 5'-tcggcgcctcagatgcatattctacactgc-3' (SEQ ID NO:19), NA NC F: 5'-caacgcgtgccaccatgaatccaaatc-3' (SEQ ID NO:20), NA NC R: 5'-tcg gcgccctacttgtcaatggtgaa cggc-3' (SEQ ID NO:21)). The RT-PCR products were purified from an agarose gel by the GFX® PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Piscataway, USA) prior to sequencing.
[0074]Purified PCR products were sequenced directly. The sequencing reaction was performed by ABI PRISM® BigDye® Terminators v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, Calif., USA) as described previously (2). The development of the sequences was performed on an automatic ABI PRISM® 3130 genetic analyzer (Applied Biosystems) with 80 cm capillaries. Consensus sequences were generated in SeqScape® Software v2.5 (Applied Biosystems). Sequence assembly, multiple alignment and alignment trimming were performed with the BioEdit software v.7.0.5 9. The PCR products were further restriction enzyme digested and cloned into the WRG7079 expression vector in DH5α bacteria. Endotoxin free DNA purification of the vaccine clones were prepared by EndoFree Plasmid Giga Kit (QIAGEN). All inserts and vaccine clones were control sequenced.
Example 2: Immunisations
[0075]A total of 24 ferrets (Mustela Putorius Furo), approximately seven months old, were divided in four groups by using a chip-tag identification for dogs (E-vet, pet-id, Haderslev, Denmark), six animals in each group. All animals were kept together and fed a standard diet with food and water ad libitum. The animals were housed according to the Danish Animal Experimentation Act and kept at level II biosecurity facilities at the Faculty of Life Sciences, Copenhagen. The acclimatisation period was nine days.
[0076]Four groups of six ferrets were vaccinated as follows; (1) HA (codon optimised gene) and NA (codon optimised gene) 1918 H1N1 plasmid DNA vaccinated, (2) HA, NA, NP and M (all codon optimised) 1918 H1N1 plasmid DNA vaccinated, (3) empty plasmid vaccinated (negative vaccine control) and (4) HA and NA (not codon optimised) A/New Caledonia/20/99(H1N1) plasmid DNA vaccinated (positive vaccine control). All ferrets received four standard gene gun shots onto shaved abdomen. HA and NA DNA mixed vaccines were given in two shots and NP and M DNA mixed vaccines were given in two shots. Therefore groups 1 and 4 receiving only HA and NA DNA vaccine were additionally shot twice with empty plasmid DNA, ensuring that all animals had received the same amount of DNA and the same number of shots. The ferrets were gene gun (Helios, Bio-Rad, Hercules, Calif.) inoculated (400 psi compressed helium) on shaved abdominal skin, using 2 μg plasmid DNA-coated gold particles (1.6 μm-sized particles), 80-95% coating efficiency each shot. Each ferret received four shots, three times biweekly. Ferrets were challenged ten days after the third immunisation by 1×107 50% egg infectious dose (EID50) of A/New Caledonia/20/99(H1N1) virus in 100 μl PBS administrated into the nostrils with a syringe. Blood serum was collected at day -2, 3, 5 and 7 post-challenge from vena jugularis of anesthetised animals (tiletamine/zolazepam (zoletil-mix for cats)). Animals were terminated with pentobarbital.
Example 3: Quantitative Real Time RT-PCR Assay for Influenza A
[0077]At the day of blood serum collection the nostrils of each ferret were flushed with 1 ml PBS and the flushing were frozen down immediately for real-time RT-PCR analysis. Two hundred micro litres of nasal wash were extracted on an automated MagNA Pure LC Instrument applying the MagNa Pure LC Total Nucleic Acid Isolation Kit (Roche diagnostics, Basel, Switzerland). The extracted material was eluated in 200 μl Milli-Q H2O. The RT-PCR reactions were performed with oligonucleotide sequences as described by Spackman et al.,(23). Extracted material (5 μl) was added to 20 μl of master mix consisting of 10 nM of each primer and 2 nM of the Taqman probe labelled with FAM in the 5' end and black hole quencher 1 in the 3' end together with reagents from the OneStep® RT-PCR Kit (QIAGEN, Hilden, Germany) according to the manufacturer. Target sequences were amplified on the MX3005 system from Stratagene with the following program: 20 min 50° C., 15 min 95° C. and 40 cycles of 15 sec 95° C. and 60 sec at 55° C. The content of viral genomes in the samples was determined using a standard curve developed by amplifying dilution of H1N1 with known concentration.
Example 4: Serum Antibody Determined by ELISA
[0078]ELISA plates (96 wells) were coated with 100 μl, split influenza vaccine (Vaxigrip, Sanofi Pasteur, Belgium) diluted 1:100 in 35 mM NaHCO3 pH 9.6 and 15 mM Na2CO3 overnight at 4° C. Wells were blocked with 1% PBS/BSA for 30 minutes at room temperature. Plates were washed with 0.05% PBS/tween (PBST). Sera 1:100 were diluted in 0.1% BSA/PBST two-folds in the plate and incubated for one hour at room temperature. The plates were washed and incubated with 100 μl biotinylated rabbit anti-ferret IgG diluted 1:250 for one hour in room temperature, washed, and incubated with 100 μl 1:1,000 horseradish peroxidase (HRP) streptavidin (DakoCytomation, Glostrup, Denmark). After 30 minutes the plates were washed and 100 μl of hydrogen peroxide with OPD was added. The reaction was stopped by adding 50 μl 0.5 M H2SO4 and read at OD492 nm.
Example 5: Results
[0079]Ferrets were negative for influenza specific antibodies seven days before start of immunisations as measured by ELISA.
[0080]High IgG specific serum antibodies (to A/New Caledonia/20/99(H1N1) in ELISA) were observed at day seven post-challenge in ferrets vaccinated with both HA+NA 1918 (two plasmids) and HA+NA+NP+M 1918 DNA vaccines (four plasmids) (FIG. 1). Ferrets vaccinated with HA+NA DNA A/New Caledonia/20/99(H1N1) induced lower specific serum antibody titre on day seven. It is possible that higher antibody response could have been observed at later time points if the experiment had not been terminated at day seven after challenge for practical reasons.
[0081]At day five post-challenge, the ferrets vaccinated with empty plasmid (negative vaccine control) showed high viral load in nasal washing measured as viral RNA copies in the nasal washings, indicating no protection against the viral challenge. However, ferrets vaccinated with HA+NA 1918 and HA+NA, NP+M 1918 DNA vaccines were completely protected from infection with an A/New Caledonia/20/99(H1N1) like virus (FIG. 1). Partial protection was observed in ferrets vaccinated with HA+NA A/New Caledonia/20/99(H1N1) DNA plasmids.
[0082]The data clearly show that DNA gene gun immunisations based on genes from the 1918 H1N1 pandemic strain induce strong specific antibody response and protect ferrets completely against infection with a H1N1 strain that has drifted by 89 years. No negative or positive effects on the humoral immune response or protection was observed by including the NP and M genes in the HA+NA DNA vaccination since the protection from infection already was already 100%.
[0083]The A/South Carolina/1/18 and A/New Caledonia/20/99 are 21.2% different in the HA1 protein and possess eight substitutions at residues involved in antigenic sites 3 (1918 to New Caledonia); Cb S83P, Sa T128V and K160N, Sb S156G, Q193H and D196N, Cal N207S and A224E.
[0084]DNA vaccines do have the ability of immune stimulatory mechanisms. This might be one reason why such good cross reactivity and protection was induced against challenge infection. Cross-protection and cross-reactivity induced by DNA vaccines of strains differing by 11-13% in HA1 has been demonstrated by others (13-15) but not as high as the 21.2% observed in the present studies.
Example 6: 1918 Pandemic H1N1 DNA Vaccinated Ferrets were Challenged with 2007 H1N1
[0085]Vaccine production and vaccinations and assays were carried out as described above.
[0086]A total of 10 ferrets (Mustela Putorius Furo), approximately seven months old, were divided in two groups by using a chip-tag identification for dogs (E-vet, pet-id, Haderslev, Denmark), five animals in each group. All animals were kept together and fed a standard diet with food and water ad libitum. The animals were housed according to the Danish Animal Experimentation Act and kept at level II biosecurity facilities at the Faculty of Life Sciences, Copenhagen. The acclimatisation period was one day. Two groups of five ferrets were vaccinated as follows; (1) HA (codon optimised gene) and NA (codon optimised gene) 1918 H1N1 plasmid DNA vaccinated, (2) non-vaccinated, naive animals. HA and NA DNA mixed vaccines were given in four shots. The ferrets were gene gun (Helios, Bio-Rad, Hercules, Calif.) inoculated (400 psi compressed helium) on shaved abdominal skin, using 2 μg plasmid DNA-coated gold particles (1.6 μm-sized particles), 80-95% coating efficiency each shot. Vaccinated ferrets received four shots, three times biweekly. Ferrets were challenged ten days after third immunisation by 1×107 50% egg infectious dose (EID50) of A/New Caledonia/20/99(H1N1) virus in 1000 μl PBS administrated into the nostrils with a syringe. Blood serum was collected at day -48, 0, 5, 7 and 12 post-challenge from vena jugularis of anesthetised animals (tiletamine/zolazepam (zoletil-mix for cats)). Animals were terminated with pentobarbital. The 1918 DNA vaccinated ferrets had a lower temperature rise than the unvaccinated group (p=0.2) at the day of maximal temperature rise, day 2 post challenge (FIG. 2A). No difference in weight loss between the vaccinated and the unvaccinated animals was observed at the day of maximal weight loss, day 4 post challenge (FIG. 2B). Vaccinated animals displayed fewer influenza symptoms than unvaccinated animals measured by sneezing, nasal discharge and activity level (p=0.065) (FIG. 2C.). Ferrets in both groups had high virus load post infection measured by quantitative real-time RT-PCR, however by day 7 post infection the 1918 DNA vaccinated ferrets better cleared their virus infection than the unvaccinated ferrets (p=0.63) (FIG. 2D).
Example 7: Challenge with New Caledonia H1N1 in Ferrets
[0087]Traditional protein H1N1 New Caledonia vaccine plus/minus DDA/TDB adjuvants versus 1918 H1N1 HA plus NA DNA vaccine (versus empty DNA vaccine vector) using two DNA immunizations (instead or the usual 3 DNA immunizations)
[0088]Traditional protein H1N1 split vaccine (two immunizations) versus 1918 H1N1 HA plus NA codon optimized DNA vaccine versus codon optimized and non-codon-optimized New Caledonia H1N1 HA and NA versus codon optimized M and NP from 1918 H1N1 virus (versus empty DNA vaccine vector) using three immunizations.
[0089]Ferrets are challenged with H1N1 New Caledonia-like virus intra nasally and virus quantitated in basal washings by real-time RT/PCR assay. Ferret antibodies will be examined for ELISA antibodies and H1 antibody reactions to H1N1, H2N2, H3N2, H5N7, and/or H5N1.
Example 8: Mouse Antibody Experiments
[0090]Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Caledonia H1N1 (shows the difference between codon optimization and non-optimization) versus codon optimized HA and NA from 1918 H1N1 strain is inoculated in mice. Antibody titers and epitope mapping of induced antibodies is done by overlapping peptides in ELISA and cross-reactions measured to other influenza A virus.
Example 9: Protein Expression Experiments
[0091]Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Calidonia H1N1 (shows the difference between codon optimization and non-optimization) versus codon optimized HA and NA from 1918 H1N1 strain is expressed in mammalian cell lines in vitro and standard radio immuno precipitation (RIPA) are done with polyclonal influenza A antibodies to examine the improved protein expression obtained by codon optimization. Codon optimized from 1918 H1N1, H5N7 and H3N2 strain versus non-codon optimized HA DNA vaccines from 1918 H1N1 strain is expressed in mammalian cell lines in vitro and hemadsorption is measured. This shows that the H1 is functionally expressed better when codons are optimized (FIG. 3).
Example 10: Cytokine Induction Experiments
[0092]Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Caledonia H1N1 (shows the difference between codon optimization and non-optimization) versus codon optimized HA and NA from 1918 H1N1 strain is added onto mammalian peripheral blood monocytes (PBMCs) in vitro and measurements of resulting cytokine production is measured in the cell supernatant to examine the innate immune induction (adjuvant effect) obtained by codon optimization and by the codon optimised H1N1 1918 HA and NA as compared to the codon optimised H1N1 New Caledonia HA and NA to examine special cytokine induction by the 1918 genes.
Example 11: 1918 HA and NA Protein Vaccine Experiments
[0093]Proteins are produced by the DNA vaccine plasmids and used as a protein vaccine in mice or ferrets as compared to DNA vaccination and to traditional protein split vaccine to measure the immune induction of 1918 proteins versus DNA vaccine.
Example 12: Mouse DNA Vaccine Delivery Experiments
[0094]Codon optimized HA and/or NA DNA vaccines from 1918 H1N1 strain is inoculated in mice as expression plasmids or as a linear piece of DNA containing the necessary components for vaccine gene expression but without the rest of the plasmid to rule out any effect of the rest of the plasmid.
Example 13: Swine DNA Vaccine Delivery Experiments
[0095]Codon optimized HA and/or NA DNA vaccines from 1918 H1N1 strain is inoculated in pigs as expression plasmids and challenge with a present day New Caledonia-like H1N1 strain and protection against disease and immune induction are measured.
REFERENCES
[0096]1. Antonovics, J., M. E. Hood, and C. H. Baker. 2006. Molecular virology: Was the 1918 flu avian in origin? Nature 440:E9. [0097]2. Bragstad, K., P. H. Jorgensen, K. J. Handberg, S. Mellergaard, S. Corbet, and A. Fomsgaard. 2005. New avian influenza A virus subtype combination H5N7 identified in Danish mallard ducks. Virus. Res. 109:181-190. [0098]3. Caton, A. J., G. G. Brownlee, J. W. Yewdell, and W. Gerhard. 1982. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell 31:417-427. [0099]4. Chen, Z., S. e. Kadowaki, Y. Hagiwara, T. Yoshikawa, K Matsuo, T. Kurata, and S. I. Tamura. 2000. Cross-protection against a lethal influenza virus infection by DNA vaccine to neuraminidase. Vaccine 18:3214-3222. [0100]5. Corbet, S., L. Vinner, D. M. Hougaard, K Bryder, H. V. Nielsen, C. Nielsen, and A. Fomsgaard. 2000. Construction, biological activity, and immunogenicity of synthetic envelope DNA vaccines based on a primary, CCR5-tropic, early HIV type 1 isolate (BX08) with human codons. AIDS Res. Hum. Retroviruses 16:1997-2008. [0101]6. Davis, H. L., B. A. Demeneix, B. Quantin, J. Coulombe, and R. G. Whalen. 1993. Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. Hum. Gene Ther. 4:733-740. [0102]7. Donnelly, J. J., A. Friedman, D. Martinez, D. L. Montgomery, J. W. Shiver, S. L. Motzel, J. B. Ulmer, and M. A. Liu. 1995. Preclinical efficacy of a prototype DNA vaccine: enhanced protection against antigenic drift in influenza virus. Nat. Med 1:583-587. [0103]8. Epstein, S. L., W. p. Kong, J. A. Misplon, C. Y. Lo, T. M. Tumpey, L. Xu, and G. J. Nabel. 2005. Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein. Vaccine 23:5404-5410. [0104]9. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser 41:95-98. [0105]10. Johnson, N. P. and J. Mueller. 2002. Updating the accounts: global mortality of the 1918-1920 "Spanish" influenza pandemic. Bull. Hist Med 76:105-115. [0106]11. Kawaoka, Y., S. Krauss, and R. G. Webster. 1989. Avian-to-human transmission of the PBI gene of influenza A viruses in the 1957 and 1968 pandemics. J Virol 63:4603-4608. [0107]12. Kobasa, D., A. Takada, K. Shinya, M. Hatta, P. Halfmann, S. Theriault, H. Suzuki, H. Nishimura, K. Mitamura, N. Sugaya, T. Usui, T. Murata, Y. Maeda, S. Watanabe, M. Suresh, T. Suzuki, Y. Suzuki, H. Feldmann, and Y. Kawaoka. 2004. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature 431:703-707. [0108]13. Kodihalli, S., H. Goto, D. L. Kobasa, S. Krauss, Y. Kawaoka, and R. G. Webster. 1999. DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice. J. Virol. 73:2094-2098. [0109]14. Kodihalli, S., J. R. Haynes, H. L. Robinson, and R. G. Webster. 1997. Cross-protection among lethal H5N2 influenza viruses induced by DNA vaccine to the hemagglutinin. J. Virol. 71:3391-3396. [0110]15. Kodihalli, S., D. L. Kobasa, and R. G. Webster. 2000. Strategies for inducing protection against avian influenza A virus subtypes with DNA vaccines. Vaccine 18:2592-2599. [0111]16. Kong, W. p., C. Hood, Z. y. Yang, C. J. Wei, L. Xu, A. Garcia-Sastre, T. M. Tumpey, and G. J. Nabel. 2006. Protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination. PNAS 103:15987-15991. [0112]17. Lindstrom, S. E., N. J. Cox, and A. Klimov. 2004. Genetic analysis of human H2N2 and early H3N2 influenza viruses, 1957-1972: evidence for genetic divergence and multiple reassortment events. Virology 328:101-119. [0113]18. Ljungberg, K., C. Kolmskog, B. Wahren, G. van Amerongen, M. Baars, A. Osterhaus, A. Linde, and G. Rimmelzwaan. 2002. DNA vaccination of ferrets with chimeric influenza A virus hemagglutinin (H3) genes. Vaccine 20:2045-2052. [0114]19. Reid, A. H., T. G. Fanning, J. V. Hultin, and J. K. Taubenberger. 1999. Origin and evolution of the 1918 "Spanish" influenza virus hemagglutinin gene. Proc. Natl. Acad. Sci. U.S.A. 96:1651-1656. [0115]20. Reid, A. H., T. G. Fanning, T. A. Janczewski, and J. K. Taubenberger. 2000. Characterization of the 1918 "Spanish" influenza virus neuraminidase gene. Proc. Natl. Acad. Sci U.S.A. 97:6785-6790. [0116]21. Seo, S. H., E. Hoffmann, and R. G. Webster. 2002. Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nat. Med. 8:950-954. [0117]22. Smith, H. and C. Sweet. 1988. Lessons for human influenza from pathogenicity studies with ferrets. Rev. Infect Dis 10:56-75. [0118]23. Spackman, E., D. A. Senne, T. J. Myers, L. L. Bulaga, L. P. Garber, M. L. Perdue, K. Lohman, L. T. Daum, and D. L. Suarez. 2002. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin. Microbiol. 40:3256-3260. [0119]24. Talon, J., C. M. Horvath, R. Polley, C. F. Basler, T. Muster, P. Palese, and A. Garcia-Sastre. 2000. Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein. J. Virol. 74:7989-7996. [0120]25. Tamura, S., T. Tanimoto, and T. Kurata. 2005. Mechanisms of broad cross-protection provided by influenza virus infection and their application to vaccines. Jpn. J Infect Dis 58:195-207. [0121]26. Taubenberger, J. K., A. H. Reid, R M. Lourens, R. Wang, G. Jin, and T. G. Fanning. 2005. Characterization of the 1918 influenza virus polymerase genes. Nature 437:889-893. [0122]27. Tumpey, T. M., C. F. Basler, P. V. Aguilar, H. Zeng, A. Solorzano, D. E. Swayne, N. J. Cox, J. M. Katz, J. K. Taubenberger, P. Palese, and A. Garcia-Sastre. 2005. Characterization of the reconstructed 1918 spanish influenza pandemic virus. Science 310:77-80. [0123]28. Tumpey, T. M., A. Garcia-Sastre, J. K Taubenberger, P. Palese, D. E. Swayne, and C. F. Basler. 2004. Pathogenicity and immunogenicity of influenza viruses with genes from the 1918 pandemic virus. Proc. Natl. Acad. Sci U.S.A. 101:3166-3171. [0124]29. Tumpey, T. M., A. Garcia-Sastre, J. K Taubenberger, P. Palese, D. E. Swayne, M. J. Pantin-Jackwood, S. Schultz-Cherry, A. Solorzano, N. Van Rooijen, J. M. Katz, and C. F. Basler. 2005. Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice. J Virol 79:14933-14944. [0125]30. Ulmer, J. B., T. M. Fu, R. R. Deck, A. Friedman, L. Guan, C. DeWitt, X. Liu, S. Wang, M. A. Liu, J. J. Donnelly, and M. J. Caulfield. 1998. Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA. J Virol 72:5648-5653. [0126]31. Wang, X., M. Li, H. Zheng, T. Muster, P. Palese, A. A. Beg, and A. Garcia-Sastre. 2000. Influenza A Virus NS1 Protein Prevents Activation of NF-kappa B and Induction of Alpha/Beta Interferon. J. Virol. 74:11566-11573. [0127]32. Webster, R. G., E. F. Fynan, J. C. Santoro, and H. Robinson. 1994. Protection of ferrets against influenza challenge with a DNA vaccine to the haemagglutinin. Vaccine 12:1495-1498.
[0128]All publications cited in this specification are incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.
Sequence CWU
1
2111701DNAArtificialHA 1918 synthetic gene 0607838; A/South
Carolina/1/18 1atggaggcca ggctgctggt gctgctgtgc gccttcgccg ccaccaacgc
cgacaccatc 60tgcatcggct accacgccaa caacagcacc gacaccgtgg ataccgtgct
ggagaagaac 120gtgaccgtga cccacagcgt gaacctgctg gaggacagcc acaacggcaa
gctgtgcaag 180ctgaagggaa tcgctcccct gcagctgggc aagtgcaaca tcgccggctg
gctgctgggc 240aaccccgagt gcgacctgct gctgaccgcc agcagctggt cctacatcgt
ggagaccagc 300aacagcgaga acggcacctg ctaccccggc gacttcatcg actacgagga
gctgcgggag 360cagctgtcca gcgtgagcag cttcgagaag ttcgagatct tccccaagac
cagctcctgg 420cccaaccacg agaccaccaa gggcgtgacc gccgcctgta gctacgccgg
agccagcagc 480ttctacagaa acctgctgtg gctgaccaag aagggcagca gctaccccaa
gctgtccaag 540agctacgtga acaacaaggg caaggaagtg ctggtgctgt ggggcgtgca
ccacccccct 600accggcaccg accagcagag cctgtaccag aacgccgacg cctacgtgag
cgtgggcagc 660agcaagtaca acagaaggtt cacccccgag atcgccgcca ggcccaaggt
gcgcgaccag 720gccggcagga tgaactacta ctggaccctg ctggagcccg gcgacaccat
caccttcgag 780gccaccggca acctgatcgc cccttggtac gccttcgccc tgaacagggg
cagcggcagc 840ggcatcatca ccagcgacgc ccccgtgcac gactgcaaca ccaagtgcca
gaccccccac 900ggagccatca acagcagcct gcccttccag aacatccacc ccgtgaccat
cggcgagtgc 960cccaagtacg tgagaagcac caagctgagg atggccaccg gcctgaggaa
catccccagc 1020atccagagca ggggcctgtt cggagccatc gccggattca tcgagggcgg
ctggaccggc 1080atgatcgacg gctggtacgg ctaccaccac cagaacgagc agggcagcgg
ctacgccgcc 1140gaccagaaga gcacccagaa cgccatcgac ggcatcacca acaaggtgaa
cagcgtgatc 1200gagaagatga acacccagtt caccgccgtg ggcaaggagt tcaacaacct
ggagaggcgg 1260atcgagaacc tgaacaagaa ggtggacgac ggcttcctgg acatctggac
ctacaacgcc 1320gagctgctgg tgctgctgga gaacgagagg accctggact tccacgacag
caacgtgagg 1380aacctgtacg agaaggtgaa gagccagctg aagaacaacg ccaaggagat
cggcaacggc 1440tgcttcgagt tctaccacaa gtgcgacgac gcctgcatgg agagcgtgag
aaacggcacc 1500tacgactacc ccaagtacag cgaggagagc aagctgaacc gggaggagat
cgacggcgtg 1560aagctggaga gcatgggcgt gtaccagatc ctggccatct acagcaccgt
ggccagcagc 1620ctggtgctgc tggtgtccct gggagccatc agcttttgga tgtgcagcaa
cggcagcctg 1680cagtgcagga tctgcatctg a
17012566PRTArtificialHA 1918 synthetic gene 0607838; A/South
Carolina/1/18 2Met Glu Ala Arg Leu Leu Val Leu Leu Cys Ala Phe Ala
Ala Thr Asn1 5 10 15Ala
Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr20
25 30Val Asp Thr Val Leu Glu Lys Asn Val Thr Val
Thr His Ser Val Asn35 40 45Leu Leu Glu
Asp Ser His Asn Gly Lys Leu Cys Lys Leu Lys Gly Ile50 55
60Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp
Leu Leu Gly65 70 75
80Asn Pro Glu Cys Asp Leu Leu Leu Thr Ala Ser Ser Trp Ser Tyr Ile85
90 95Val Glu Thr Ser Asn Ser Glu Asn Gly Thr
Cys Tyr Pro Gly Asp Phe100 105 110Ile Asp
Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe115
120 125Glu Lys Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp
Pro Asn His Glu130 135 140Thr Thr Lys Gly
Val Thr Ala Ala Cys Ser Tyr Ala Gly Ala Ser Ser145 150
155 160Phe Tyr Arg Asn Leu Leu Trp Leu Thr
Lys Lys Gly Ser Ser Tyr Pro165 170 175Lys
Leu Ser Lys Ser Tyr Val Asn Asn Lys Gly Lys Glu Val Leu Val180
185 190Leu Trp Gly Val His His Pro Pro Thr Gly Thr
Asp Gln Gln Ser Leu195 200 205Tyr Gln Asn
Ala Asp Ala Tyr Val Ser Val Gly Ser Ser Lys Tyr Asn210
215 220Arg Arg Phe Thr Pro Glu Ile Ala Ala Arg Pro Lys
Val Arg Asp Gln225 230 235
240Ala Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr245
250 255Ile Thr Phe Glu Ala Thr Gly Asn Leu
Ile Ala Pro Trp Tyr Ala Phe260 265 270Ala
Leu Asn Arg Gly Ser Gly Ser Gly Ile Ile Thr Ser Asp Ala Pro275
280 285Val His Asp Cys Asn Thr Lys Cys Gln Thr Pro
His Gly Ala Ile Asn290 295 300Ser Ser Leu
Pro Phe Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys305
310 315 320Pro Lys Tyr Val Arg Ser Thr
Lys Leu Arg Met Ala Thr Gly Leu Arg325 330
335Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly340
345 350Phe Ile Glu Gly Gly Trp Thr Gly Met
Ile Asp Gly Trp Tyr Gly Tyr355 360 365His
His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser370
375 380Thr Gln Asn Ala Ile Asp Gly Ile Thr Asn Lys
Val Asn Ser Val Ile385 390 395
400Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn
Asn405 410 415Leu Glu Arg Arg Ile Glu Asn
Leu Asn Lys Lys Val Asp Asp Gly Phe420 425
430Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn435
440 445Glu Arg Thr Leu Asp Phe His Asp Ser
Asn Val Arg Asn Leu Tyr Glu450 455 460Lys
Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly465
470 475 480Cys Phe Glu Phe Tyr His
Lys Cys Asp Asp Ala Cys Met Glu Ser Val485 490
495Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys
Leu500 505 510Asn Arg Glu Glu Ile Asp Gly
Val Lys Leu Glu Ser Met Gly Val Tyr515 520
525Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu530
535 540Val Ser Leu Gly Ala Ile Ser Phe Trp
Met Cys Ser Asn Gly Ser Leu545 550 555
560Gln Cys Arg Ile Cys Ile56531410DNAArtificialHA 1918
synthetic gene 0607869; A/Brevig mission/1/1918 3atgaacccca
accagaagat catcaccatc ggcagcatct gcatggtggt gggcatcatc 60agcctgatcc
tgcagatcgg caacatcatc agcatctggg tgtcccacag catccagacc 120ggcaaccaga
accaccccga gacctgcaac cagtccatca tcacctacga gaacaacacc 180tgggtgaacc
agacctacgt gaacatcagc aacaccaacg tggtggccgg ccaggacgcc 240acctccgtga
tcctgacagg caacagcagc ctgtgcccca tcagcggctg ggccatctac 300agcaaggaca
acggcatcag gatcggcagc aagggcgacg tgttcgtgat cagagagccc 360ttcatcagct
gcagccacct ggaatgcagg accttcttcc tgacccaagg agccctgctg 420aacgacaagc
acagcaacgg caccgtgaag gacagaagcc cctacaggac cctgatgagc 480tgccccgtgg
gcgaggctcc cagcccctac aacagcagat tcgagagcgt ggcctggtcc 540gccagcgcct
gccacgacgg catgggctgg ctgaccatcg gcatcagcgg ccctgacaac 600ggggccgtgg
ccgtgctgaa gtacaacgga atcatcaccg acaccatcaa gagctggcgg 660aacaacatcc
tgaggaccca ggaaagcgag tgcgcctgcg tgaacggcag ctgcttcacc 720atcatgaccg
acggccccag caacggccag gccagctaca agatcctgaa gatcgagaag 780ggcaaggtga
ccaagagcat cgagctgaac gcccccaact accactacga ggaatgcagc 840tgctaccccg
acaccggcaa ggtcatgtgc gtgtgcaggg acaactggca cggcagcaac 900aggccctggg
tgtccttcga ccagaacctg gactaccaga tcggatacat ctgcagcggc 960gtgttcggcg
acaaccccag gcccaacgac ggcaccggca gctgcggccc tgtgagcagc 1020aacggggcca
atggcatcaa gggcttcagc ttcagatacg acaacggcgt gtggatcggc 1080cgcaccaaga
gcaccagcag cagatccggc ttcgagatga tctgggaccc caacggctgg 1140accgagaccg
acagcagctt cagcgtgagg caggacatcg tggccatcac cgactggtcc 1200ggctacagcg
gcagcttcgt gcagcacccc gagctgaccg gcctggactg catgaggccc 1260tgtttctggg
tggagctgat cagaggccag cccaaggaga acaccatctg gaccagcggc 1320agcagcatca
gcttttgcgg cgtgaacagc gacaccgtgg gctggtcctg gcccgacggg 1380gccgagctgc
ccttcagcat cgataagtga
14104469PRTArtificialHA 1918 synthetic gene 0607869; A/Brevig
mission/1/1918 4Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys
Met Val1 5 10 15Val Gly
Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile20
25 30Trp Val Ser His Ser Ile Gln Thr Gly Asn Gln Asn
His Pro Glu Thr35 40 45Cys Asn Gln Ser
Ile Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln50 55
60Thr Tyr Val Asn Ile Ser Asn Thr Asn Val Val Ala Gly Gln
Asp Ala65 70 75 80Thr
Ser Val Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly85
90 95Trp Ala Ile Tyr Ser Lys Asp Asn Gly Ile Arg
Ile Gly Ser Lys Gly100 105 110Asp Val Phe
Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu115
120 125Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu
Asn Asp Lys His130 135 140Ser Asn Gly Thr
Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser145 150
155 160Cys Pro Val Gly Glu Ala Pro Ser Pro
Tyr Asn Ser Arg Phe Glu Ser165 170 175Val
Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr180
185 190Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val
Ala Val Leu Lys Tyr195 200 205Asn Gly Ile
Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu210
215 220Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly
Ser Cys Phe Thr225 230 235
240Ile Met Thr Asp Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Leu245
250 255Lys Ile Glu Lys Gly Lys Val Thr Lys
Ser Ile Glu Leu Asn Ala Pro260 265 270Asn
Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val275
280 285Met Cys Val Cys Arg Asp Asn Trp His Gly Ser
Asn Arg Pro Trp Val290 295 300Ser Phe Asp
Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly305
310 315 320Val Phe Gly Asp Asn Pro Arg
Pro Asn Asp Gly Thr Gly Ser Cys Gly325 330
335Pro Val Ser Ser Asn Gly Ala Asn Gly Ile Lys Gly Phe Ser Phe Arg340
345 350Tyr Asp Asn Gly Val Trp Ile Gly Arg
Thr Lys Ser Thr Ser Ser Arg355 360 365Ser
Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp370
375 380Ser Ser Phe Ser Val Arg Gln Asp Ile Val Ala
Ile Thr Asp Trp Ser385 390 395
400Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu
Asp405 410 415Cys Met Arg Pro Cys Phe Trp
Val Glu Leu Ile Arg Gly Gln Pro Lys420 425
430Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val435
440 445Asn Ser Asp Thr Val Gly Trp Ser Trp
Pro Asp Gly Ala Glu Leu Pro450 455 460Phe
Ser Ile Asp Lys46551497DNAArtificialHA 1918 synthetic gene 0607866;
A/Brevig mission/1/1918 5atggccagcc agggcaccaa gagaagctac
gagcagatgg aaaccgacgg cgagaggcag 60aacgccaccg agatcagggc cagcgtgggc
aggatgatcg gcggcatcgg caggttctac 120atccagatgt gcaccgagct gaagctgtcc
gactacgagg gcaggctgat ccagaacagc 180atcaccatcg agaggatggt gctgtccgcc
ttcgacgaga gaagaaacaa gtacctggaa 240gagcacccca gcgccggcaa ggaccccaag
aaaaccggcg gacccatcta cagaaggatc 300gacggcaagt ggatgagaga gctgatcctg
tacgacaagg aggaaatcag aaggatctgg 360cggcaggcca acaacggcga ggacgccaca
gccggcctga cccacatgat gatctggcac 420agcaacctga acgacgccac ctaccagagg
accagggccc tcgtcagaac cggcatggac 480ccccggatgt gcagcctgat gcagggcagc
acactgccca gaagaagcgg agctgctgga 540gccgccgtga agggcgtggg caccatggtg
atggaactga tcaggatgat caagaggggc 600atcaacgaca ggaacttttg gaggggcgag
aacggcagaa ggaccaggat cgcctacgag 660aggatgtgca acatcctgaa gggcaagttc
cagacagccg cccagagggc catgatggac 720caggtccggg agagcaggaa ccccggcaac
gccgagatcg aggacctgat cttcctggcc 780agaagcgccc tgatcctgag gggcagcgtg
gcccacaaga gctgcctgcc cgcctgcgtg 840tacggacccg ccgtggccag cggctacgac
ttcgagagag agggctacag cctggtcggc 900atcgacccct tcaggctgct gcagaactcc
caggtgtact ctctgatcag gcccaacgag 960aaccccgccc acaagtccca gctggtctgg
atggcctgcc acagcgccgc cttcgaggat 1020ctgagagtga gcagcttcat caggggcacc
agagtggtgc ccaggggcaa gctgtccacc 1080aggggcgtgc agatcgccag caacgagaac
atggaaacca tggacagcag caccctggaa 1140ctgagaagca ggtactgggc catcaggacc
agaagcggcg gcaacaccaa ccagcagagg 1200gccagcgccg gacagatcag cgtgcagccc
accttctccg tgcagaggaa cctgcccttc 1260gagagggcca ccatcatggc cgccttcacc
ggcaacaccg agggcaggac cagcgacatg 1320aggaccgaga tcatcagaat gatggaaagc
gccaggcccg aggacgtgag cttccagggc 1380aggggcgtgt tcgagctgtc cgatgagaag
gccacctccc ccatcgtgcc cagcttcgac 1440atgagcaacg agggcagcta cttcttcggc
gacaacgccg aggaatacga caactga 14976498PRTArtificialHA 1918 synthetic
gene 0607866; A/Brevig mission/1/1918 6Met Ala Ser Gln Gly Thr Lys
Arg Ser Tyr Glu Gln Met Glu Thr Asp1 5 10
15Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val
Gly Arg Met20 25 30Ile Gly Gly Ile Gly
Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys35 40
45Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile
Glu50 55 60Arg Met Val Leu Ser Ala Phe
Asp Glu Arg Arg Asn Lys Tyr Leu Glu65 70
75 80Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr
Gly Gly Pro Ile85 90 95Tyr Arg Arg Ile
Asp Gly Lys Trp Met Arg Glu Leu Ile Leu Tyr Asp100 105
110Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly
Glu Asp115 120 125Ala Thr Ala Gly Leu Thr
His Met Met Ile Trp His Ser Asn Leu Asn130 135
140Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met
Asp145 150 155 160Pro Arg
Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser165
170 175Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr
Met Val Met Glu180 185 190Leu Ile Arg Met
Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg195 200
205Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met
Cys Asn210 215 220Ile Leu Lys Gly Lys Phe
Gln Thr Ala Ala Gln Arg Ala Met Met Asp225 230
235 240Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala
Glu Ile Glu Asp Leu245 250 255Ile Phe Leu
Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His260
265 270Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Pro Ala
Val Ala Ser Gly275 280 285Tyr Asp Phe Glu
Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe290 295
300Arg Leu Leu Gln Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro
Asn Glu305 310 315 320Asn
Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala325
330 335Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile
Arg Gly Thr Arg Val340 345 350Val Pro Arg
Gly Lys Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn355
360 365Glu Asn Met Glu Thr Met Asp Ser Ser Thr Leu Glu
Leu Arg Ser Arg370 375 380Tyr Trp Ala Ile
Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg385 390
395 400Ala Ser Ala Gly Gln Ile Ser Val Gln
Pro Thr Phe Ser Val Gln Arg405 410 415Asn
Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn420
425 430Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu
Ile Ile Arg Met Met435 440 445Glu Ser Ala
Arg Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe450
455 460Glu Leu Ser Asp Glu Lys Ala Thr Ser Pro Ile Val
Pro Ser Phe Asp465 470 475
480Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr485
490 495Asp Asn7988DNAArtificialHA 1918
synthetic gene 0607868; A/Brevig mission/1/1918 7atgagtcttt
taaccgaggt cgaaacgtac gttctctcta tcgtcccgtc aggccccctc 60aaagccgaga
tcgcgcagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120gctctcatgg
aatggctaaa gacaagacca atcctgtcac ctctgactaa ggggatttta 180ggatttgtgt
tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240caaaatgccc
ttaatgggaa cggggatcca aataacatgg acagagcagt taaactgtac 300aggaagctta
agagggagat aacattccat ggggccaaag aagtagcact cagttattcc 360gctggtgcac
ttgccagttg tatgggcctc atatacaaca ggatggggac tgtgaccact 420gaagtggcat
ttggcctggt atgcgcaacc tgtgaacaga ttgctgattc ccagcatcgg 480tctcacaggc
aaatggtgac aacaaccaat ccactaatca gacatgagaa cagaatggta 540ctggccagca
ctacggctaa ggctatggag caaatggctg gatcgagtga gcaagcagca 600gaggccatgg
aggttgctag tcaggctagg caaatggtgc aggcgatgag aaccattggg 660actcatccta
gctccagtgc tggtctgaaa gacgatctta ttgaaaattt gcaggcctac 720cagaaacgaa
tgggggtgca gatgcaacga ttcaagtgat cctctcgtta ttgccgcaag 780tatcattggg
atcttgcact tgatattgtg gattcttgat cgtctttttt tcaaatgcat 840ttatcgtcgc
cttaaatacg gtttgaaaag agggccttct acggaaggag tgccggagtc 900tatgagggaa
gaatatcgaa aggaacagca gagtgctgtg gatgttgacg atggtcattt 960tgtcaacata
gagctggagt aaggcgcc
9888252PRTArtificialHA 1918 synthetic gene 0607868; A/Brevig
mission/1/1918 8Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile
Val Pro1 5 10 15Ser Gly
Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe20
25 30Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu
Trp Leu Lys Thr35 40 45Arg Pro Ile Leu
Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe50 55
60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg
Phe Val65 70 75 80Gln
Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala85
90 95Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile
Thr Phe His Gly Ala100 105 110Lys Glu Val
Ala Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met115
120 125Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr
Glu Val Ala Phe130 135 140Gly Leu Val Cys
Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg145 150
155 160Ser His Arg Gln Met Val Thr Thr Thr
Asn Pro Leu Ile Arg His Glu165 170 175Asn
Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met180
185 190Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met
Glu Val Ala Ser Gln195 200 205Ala Arg Gln
Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser210
215 220Ser Ser Ala Gly Leu Lys Asp Asp Leu Ile Glu Asn
Leu Gln Ala Tyr225 230 235
240Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys245
250997PRTArtificialHA 1918 synthetic gene 0607868; A/Brevig
mission/1/1918 9Met Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Glu
Trp Gly1 5 10 15Cys Arg
Cys Asn Asp Ser Ser Asp Pro Leu Val Ile Ala Ala Ser Ile20
25 30Ile Gly Ile Leu His Leu Ile Leu Trp Ile Leu Asp
Arg Leu Phe Phe35 40 45Lys Cys Ile Tyr
Arg Arg Leu Lys Tyr Gly Leu Lys Arg Gly Pro Ser50 55
60Thr Glu Gly Val Pro Glu Ser Met Arg Glu Glu Tyr Arg Lys
Glu Gln65 70 75 80Gln
Ser Ala Val Asp Val Asp Asp Gly His Phe Val Asn Ile Glu Leu85
90 95Glu101731DNAArtificialHA H3N2
A/Aichi/2/1968(H3N2) 10ataattctat taatcatgaa gaccatcatt gctttgagct
acattttctg tctggctctc 60ggccaagacc ttccaggaaa tgacaacagc acagcaacgc
tgtgcctggg acatcatgcg 120gtgccaaacg gaacactagt gaaaacaatc acagatgatc
agattgaagt gactaatgct 180actgagctag ttcagagctc ctcaacgggg aaaatatgca
acaatcctca tcgaatcctt 240gatggaatag actgcacact gatagatgct ctattggggg
accctcattg tgatgttttt 300caaaatgaga catgggacct tttcgttgaa cgcagcaaag
ctttcagcaa ctgttaccct 360tatgatgtgc cagattatgc ctcccttagg tcactagttg
cctcgtcagg cactctggag 420tttatcactg agggtttcac ttggactggg gtcactcaga
atgggggaag caatgcttgc 480aaaaggggac ctggtagcgg ttttttcagt agactgaact
ggttgaccaa atcaggaagc 540acatatccag tgctgaacgt gactatgcca aacaatgaca
attttgacaa actatacatt 600tggggggttc accacccgag cacgaaccaa gaacaaacca
gcctgtatgt tcaagcatca 660gggagagtca cagtctctac caggagaagc cagcaaacta
taatcccgaa tatcgagtcc 720agaccctggg taaggggtct gtctagtaga ataagcatct
attggacaat agttaagccg 780ggagacgtac tggtaattaa tagtaatggg aacctaatcg
ctcctcgggg ttatttcaaa 840atgcgcactg ggaaaagctc aataatgagg tcagatgcac
ctattgatac ctgtatttct 900gaatgcatca ctccaaatgg aagcattccc aatgacaagc
cctttcaaaa cgtaaacaag 960atcacatatg gagcatgccc caagtatgtt aagcaaaaca
ccctgaagtt ggcaacaggg 1020atgcggaatg taccagagaa acaaactaga ggcctattcg
gcgcaatagc aggtttcata 1080gaaaatggtt gggagggaat gatagacggt tggtacggtt
tcaggcatca aaattctgag 1140ggcacaggac aagcagcaga tcttaaaagc actcaagcag
ccatcgacca aatcaatggg 1200aaattgaaca gggtaatcga gaagacgaac gagaaattcc
atcaaatcga aaaggaattc 1260tcagaagtag aagggagaat tcaggacctc gagaaatacg
ttgaagacac taaaatagat 1320ctctggtctt acaatgcgga gcttcttgtc gctctggaga
atcaacatac aattgacctg 1380actgactcgg aaatgaacaa gctgtttgaa aaaacaagga
ggcaactgag ggaaaatgct 1440gaagacatgg gcaatggttg cttcaaaata taccacaaat
gtgacaacgc ttgcatagag 1500tcaatcagaa atgggactta tgaccatgat gtatacagag
acgaagcatt aaacaaccgg 1560tttcagatca aaggtgttga actgaagtct ggatacaaag
actggatcct gtggatttcc 1620tttgccatat catgcttttt gctttgtgtt gttttgctgg
ggttcatcat gtgggcctgc 1680cagagaggca acattaggtg caacatttgc atttgagtgt
attagtaatt a 173111566PRTArtificialHA H3N2
A/Aichi/2/1968(H3N2) 11Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys
Leu Ala Leu Gly1 5 10
15Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly20
25 30His His Ala Val Pro Asn Gly Thr Leu Val
Lys Thr Ile Thr Asp Asp35 40 45Gln Ile
Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr50
55 60Gly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp
Gly Ile Asp Cys65 70 75
80Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Val Phe Gln85
90 95Asn Glu Thr Trp Asp Leu Phe Val Glu Arg
Ser Lys Ala Phe Ser Asn100 105 110Cys Tyr
Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val115
120 125Ala Ser Ser Gly Thr Leu Glu Phe Ile Thr Glu Gly
Phe Thr Trp Thr130 135 140Gly Val Thr Gln
Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro Gly145 150
155 160Ser Gly Phe Phe Ser Arg Leu Asn Trp
Leu Thr Lys Ser Gly Ser Thr165 170 175Tyr
Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys180
185 190Leu Tyr Ile Trp Gly Val His His Pro Ser Thr
Asn Gln Glu Gln Thr195 200 205Ser Leu Tyr
Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Arg Arg210
215 220Ser Gln Gln Thr Ile Ile Pro Asn Ile Glu Ser Arg
Pro Trp Val Arg225 230 235
240Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly245
250 255Asp Val Leu Val Ile Asn Ser Asn Gly
Asn Leu Ile Ala Pro Arg Gly260 265 270Tyr
Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser Asp Ala275
280 285Pro Ile Asp Thr Cys Ile Ser Glu Cys Ile Thr
Pro Asn Gly Ser Ile290 295 300Pro Asn Asp
Lys Pro Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala305
310 315 320Cys Pro Lys Tyr Val Lys Gln
Asn Thr Leu Lys Leu Ala Thr Gly Met325 330
335Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Leu Phe Gly Ala Ile Ala340
345 350Gly Phe Ile Glu Asn Gly Trp Glu Gly
Met Ile Asp Gly Trp Tyr Gly355 360 365Phe
Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys370
375 380Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly
Lys Leu Asn Arg Val385 390 395
400Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe
Ser405 410 415Glu Val Glu Gly Arg Ile Gln
Asp Leu Glu Lys Tyr Val Glu Asp Thr420 425
430Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu435
440 445Asn Gln His Thr Ile Asp Leu Thr Asp
Ser Glu Met Asn Lys Leu Phe450 455 460Glu
Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn465
470 475 480Gly Cys Phe Lys Ile Tyr
His Lys Cys Asp Asn Ala Cys Ile Glu Ser485 490
495Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala
Leu500 505 510Asn Asn Arg Phe Gln Ile Lys
Gly Val Glu Leu Lys Ser Gly Tyr Lys515 520
525Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys530
535 540Val Val Leu Leu Gly Phe Ile Met Trp
Ala Cys Gln Arg Gly Asn Ile545 550 555
560Arg Cys Asn Ile Cys Ile565121431DNAArtificialHA H3N2
A/Aichi/2/1968(H3N2) 12gaaaatgaat ccaaatcaaa agataataac aattggctct
gtctctctca ccattgcaac 60agtatgcttc ctcatgcaga ttgccatcct ggtaactact
gtaacattgc attttaagca 120atatgagtgc gactcccccg cgagcaacca agtaatgccg
tgtgaaccaa taataataga 180aaggaacata acagagatag tgtatttgaa taacaccacc
atagagaaag agatatgccc 240caaagtagtg gaatacagaa attggtcaaa gccgcaatgt
caaattacag gatttgcacc 300tttttctaag gacaattcaa tccggctttc tgctggtggg
gacatttggg tgacgagaga 360accttatgtg tcatgcgatc atggcaagtg ttatcaattt
gcactcgggc aggggaccac 420actagacaac aaacattcaa atgacacaat acatgataga
atccctcatc gaaccctatt 480aatgaatgag ttgggtgttc catttcattt aggaaccagg
caagtgtgta tagcatggtc 540cagctcaagt tgtcacgatg gaaaagcatg gctgcatgtt
tgtatcactg gggatgacaa 600aaatgcaact gctagcttca tttatgacgg gaggcttgtg
gacagtattg gttcatggtc 660tcaaaatatc ctcagaaccc aggagtcgga atgcgtttgt
atcaatggga cttgcacagt 720agtaatgact gatggaagtg cttcaggaag agccgatact
agaatactat tcattgaaga 780ggggaaaatt gtccatatta gcccattgtc aggaagtgct
cagcatgtag aagagtgttc 840ctgttatcct agatatcctg gcgtcagatg tatctgcaga
gacaactgga aaggctctaa 900taggcccgtc gtagacataa atatggaaga ttatagcatt
gattccagtt atgtgtgctc 960agggcttgtt ggcgacacac ctagaaacga cgacagatct
agcaatagca attgcaggaa 1020tcctaataat gagagaggga atcaaggagt gaaaggctgg
gcctttgaca atggagatga 1080cgtgtggatg ggaagaacga tcagcaagga tttacgctca
ggttatgaaa ctttcaaagt 1140cattggtggt tggtccacac ctaattccaa atcgcagatc
aatagacaag tcatagttga 1200cagcgataat cggtcaggtt actctggtat tttctctgtt
gagggcaaaa gctgcatcaa 1260taggtgcttt tatgtggagt tgataagggg aaggaaacag
gagactagag tgtggtggac 1320ctcaaacagt attgttgtgt tttgtggcac ttcaggtacc
tatggaacag gctcatggcc 1380tgatggggcg aacatcaatt tcatgcctat ataagctttc
gcaattttag a 143113469PRTArtificialHA H3N2
A/Aichi/2/1968(H3N2) 13Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser
Val Ser Leu Thr1 5 10
15Ile Ala Thr Val Cys Phe Leu Met Gln Ile Ala Ile Leu Val Thr Thr20
25 30Val Thr Leu His Phe Lys Gln Tyr Glu Cys
Asp Ser Pro Ala Ser Asn35 40 45Gln Val
Met Pro Cys Glu Pro Ile Ile Ile Glu Arg Asn Ile Thr Glu50
55 60Ile Val Tyr Leu Asn Asn Thr Thr Ile Glu Lys Glu
Ile Cys Pro Lys65 70 75
80Val Val Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Gln Ile Thr Gly85
90 95Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile
Arg Leu Ser Ala Gly Gly100 105 110Asp Ile
Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp His Gly Lys115
120 125Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu
Asp Asn Lys His130 135 140Ser Asn Asp Thr
Ile His Asp Arg Ile Pro His Arg Thr Leu Leu Met145 150
155 160Asn Glu Leu Gly Val Pro Phe His Leu
Gly Thr Arg Gln Val Cys Ile165 170 175Ala
Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val180
185 190Cys Ile Thr Gly Asp Asp Lys Asn Ala Thr Ala
Ser Phe Ile Tyr Asp195 200 205Gly Arg Leu
Val Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile Leu Arg210
215 220Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr
Cys Thr Val Val225 230 235
240Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Arg Ile Leu Phe245
250 255Ile Glu Glu Gly Lys Ile Val His Ile
Ser Pro Leu Ser Gly Ser Ala260 265 270Gln
His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg275
280 285Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn
Arg Pro Val Val Asp290 295 300Ile Asn Met
Glu Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser Gly305
310 315 320Leu Val Gly Asp Thr Pro Arg
Asn Asp Asp Arg Ser Ser Asn Ser Asn325 330
335Cys Arg Asn Pro Asn Asn Glu Arg Gly Asn Gln Gly Val Lys Gly Trp340
345 350Ala Phe Asp Asn Gly Asp Asp Val Trp
Met Gly Arg Thr Ile Ser Lys355 360 365Asp
Leu Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser370
375 380Thr Pro Asn Ser Lys Ser Gln Ile Asn Arg Gln
Val Ile Val Asp Ser385 390 395
400Asp Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys
Ser405 410 415Cys Ile Asn Arg Cys Phe Tyr
Val Glu Leu Ile Arg Gly Arg Lys Gln420 425
430Glu Thr Arg Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly435
440 445Thr Ser Gly Thr Tyr Gly Thr Gly Ser
Trp Pro Asp Gly Ala Asn Ile450 455 460Asn
Phe Met Pro Ile465141733DNAArtificialHA H2N2 A/Albany/20/1957(H2N2)
14atagacaacc aaaagcaaaa caatggccat catttatctc attctcctgt tcacagcagt
60gagaggggac cagatatgca ttggatacca tgccaataat tccacagaga aggtcgacac
120aattctagag cggaacgtca ctgtgactca tgccaaggac attcttgaga agacccataa
180cggaaagtta tgcaaactaa acggaatccc tccacttgaa ctaggggact gtagcattgc
240cggatggctc cttggaaatc cagaatgtga taggcttcta agtgtgccag aatggtccta
300tataatggag aaagaaaacc cgagagacgg tttgtgttat ccaggcagct tcaatgatta
360tgaagaattg aaacatctcc tcagcagcgt gaaacatttc gagaaagtaa agattctgcc
420caaagataga tggacacagc atacaacaac tggaggttca cgggcctgcg cggtgtctgg
480taatccatca ttcttcagga acatgatctg gctgacaaag aaaggatcaa attatccggt
540tgccaaagga tcgtacaaca atacaagcgg agaacaaatg ctaataattt ggggggtgca
600ccatcccaat gatgagacag aacaaagaac attgtaccag aatgtgggaa cctatgtttc
660cgtaggcaca tcaacattga acaaaaggtc aaccccagac atagcaacaa ggcctaaagt
720gaatggacta ggaagtagaa tggaattctc ttggacccta ttggatatgt gggacaccat
780aaattttgag agtactggta atctaattgc accagagtat ggattcaaaa tatcgaaaag
840aggtagttca gggatcatga aaacagaagg aacacttggg aactgtgaga ccaaatgcca
900aactcctttg ggagcaataa atacaacatt gccttttcac aatgtccacc cactgacaat
960aggtgagtgc cccaaatatg taaaatcgga gaagttggtc ttagcaacag gactaaggaa
1020tgttccccag attgaatcaa gaggattgtt tggggcaata gctggtttta tagaaggagg
1080atggcaagga atggttgatg gttggtatgg ataccatcac agcaatgacc agggatcagg
1140gtatgcagcg gacaaagaat ccactcaaaa ggcatttgat ggaatcacca acaaggtaaa
1200ttctgtgatt gaaaagatga acacccaatt tgaagctgtt gggaaagaat tcagtaactt
1260agagagaaga ctggagaact tgaacaaaaa gatggaagac gggtttctag atgtgtggac
1320atacaatgct gagcttctag ttctgatgga aaatgagagg acacttgact ttcatgattc
1380taatgtcaag aatctgtatg ataaagtcag aatgcagctg agagacaacg tcaaagaact
1440aggaaatgga tgttttgaat tttatcacaa atgtgatgat gaatgcatga atagtgtgaa
1500aaacgggacg tatgattatc ccaagtatga agaagagtct aaactaaata gaaatgaaat
1560caaaggggta aaattgagca gcatgggggt ttatcaaatc cttgccattt atgctacagt
1620agcaggttct ctgtcactgg caatcatgat ggctgggatc tctttctgga tgtgctccaa
1680cgggtctctg cagtgcagga tctgcatatg attataagtc attttataat taa
173315562PRTArtificialHA H2N2 A/Albany/20/1957(H2N2) 15Met Ala Ile Ile
Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp1 5
10 15Gln Ile Cys Ile Gly Tyr His Ala Asn Asn
Ser Thr Glu Lys Val Asp20 25 30Thr Ile
Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu35
40 45Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn
Gly Ile Pro Pro50 55 60Leu Glu Leu Gly
Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro65 70
75 80Glu Cys Asp Arg Leu Leu Ser Val Pro
Glu Trp Ser Tyr Ile Met Glu85 90 95Lys
Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp100
105 110Tyr Glu Glu Leu Lys His Leu Leu Ser Ser Val
Lys His Phe Glu Lys115 120 125Val Lys Ile
Leu Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly130
135 140Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser
Phe Phe Arg Asn145 150 155
160Met Ile Trp Leu Thr Lys Lys Gly Ser Asn Tyr Pro Val Ala Lys Gly165
170 175Ser Tyr Asn Asn Thr Ser Gly Glu Gln
Met Leu Ile Ile Trp Gly Val180 185 190His
His Pro Asn Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val195
200 205Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu
Asn Lys Arg Ser Thr210 215 220Pro Asp Ile
Ala Thr Arg Pro Lys Val Asn Gly Leu Gly Ser Arg Met225
230 235 240Glu Phe Ser Trp Thr Leu Leu
Asp Met Trp Asp Thr Ile Asn Phe Glu245 250
255Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys260
265 270Arg Gly Ser Ser Gly Ile Met Lys Thr
Glu Gly Thr Leu Gly Asn Cys275 280 285Glu
Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro290
295 300Phe His Asn Val His Pro Leu Thr Ile Gly Glu
Cys Pro Lys Tyr Val305 310 315
320Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro
Gln325 330 335Ile Glu Ser Arg Gly Leu Phe
Gly Ala Ile Ala Gly Phe Ile Glu Gly340 345
350Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn355
360 365Asp Gln Gly Ser Gly Tyr Ala Ala Asp
Lys Glu Ser Thr Gln Lys Ala370 375 380Phe
Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn385
390 395 400Thr Gln Phe Glu Ala Val
Gly Lys Glu Phe Ser Asn Leu Glu Arg Arg405 410
415Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val
Trp420 425 430Thr Tyr Asn Ala Glu Leu Leu
Val Leu Met Glu Asn Glu Arg Thr Leu435 440
445Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met450
455 460Gln Leu Arg Asp Asn Val Lys Glu Leu
Gly Asn Gly Cys Phe Glu Phe465 470 475
480Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn
Gly Thr485 490 495Tyr Asp Tyr Pro Lys Tyr
Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu500 505
510Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu
Ala515 520 525Ile Tyr Ala Thr Val Ala Gly
Ser Leu Ser Leu Ala Ile Met Met Ala530 535
540Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile545
550 555 560Cys
Ile161435DNAArtificialHA H2N2 A/Albany/20/1957(H2N2) 16tgaaaatgaa
tccaaatcaa aagataataa caattggctc tgtctctctc accattgcaa 60cagtatgctt
cctcatgcag attgccatcc tggcaactac tgtgacattg cattttaaac 120aacatgagtg
cgactccccc gcgagcaacc aagtaatgcc atgtgaacca ataataatag 180aaaggaacat
aacagagata gtgtatttga ataacaccac catagagaaa gagatttgcc 240ccgaagtagt
ggaatacaga aattggtcaa agccgcaatg tcaaattaca ggatttgcac 300ctttttctaa
ggacaattca atccggcttt ctgctggtgg ggacatttgg gtgacgagag 360aaccttatgt
gtcatgcgat cctggcaagt gttatcaatt tgcactcggg caagggacca 420cactagacaa
caaacattca aatggcacaa tacatgatag aatccctcac cgaaccctat 480taatgaatga
gttgggtgtt ccatttcatt taggaaccaa acaagtgtgt gtagcatggt 540ccagctcaag
ttgtcacgat ggaaaagcat ggttgcatgt ttgtgtcact ggggatgata 600gaaatgcgac
tgccagcttc atttatgacg ggaggcttgt ggacagtatt ggttcatggt 660ctcaaaatat
cctcaggacc caggagtcgg aatgcgtttg tatcaatggg acttgcacag 720tagtaatgac
tgatggaagt gcatcaggaa gagccgatac tagaatacta ttcattaaag 780aggggaaaat
tgtccatatc agcccattgt caggaagtgc tcagcatata gaggagtgtt 840cctgttaccc
tcgatatcct gacgtcagat gtatctgcag agacaactgg aaaggctcta 900ataggcccgt
tatagacata aatatggaag attatagcat tgattccagt tatgtgtgct 960cagggcttgt
tggcgacaca cccaggaacg acgacagctc tagcaatagc aattgcaggg 1020atcctaacaa
tgagagaggg aatccaggag tgaaaggctg ggcctttgac aatggagatg 1080atgtatggat
gggaagaaca atcaacaaag attcacgctc aggttatgaa actttcaaag 1140tcattggtgg
ttggtccaca cctaattcca aatcgcaggt caatagacag gtcatagttg 1200acaacaataa
ttggtctggt tactctggta ttttctctgt tgagggcaaa agctgcatca 1260ataggtgctt
ttatgtggag ttgataaggg gaaggccaca ggagactaga gtatggtgga 1320cctcaaacag
tattgttgtg ttttgtggca cttcaggtac ttatggaaca ggctcatggc 1380ctgatggggc
gaacatcaat ttcatgccta tataagcttt cgcaatttta gaaaa
143517469PRTArtificialHA H2N2 A/Albany/20/1957(H2N2) 17Met Asn Pro Asn
Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr1 5
10 15Ile Ala Thr Val Cys Phe Leu Met Gln Ile
Ala Ile Leu Ala Thr Thr20 25 30Val Thr
Leu His Phe Lys Gln His Glu Cys Asp Ser Pro Ala Ser Asn35
40 45Gln Val Met Pro Cys Glu Pro Ile Ile Ile Glu Arg
Asn Ile Thr Glu50 55 60Ile Val Tyr Leu
Asn Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Glu65 70
75 80Val Val Glu Tyr Arg Asn Trp Ser Lys
Pro Gln Cys Gln Ile Thr Gly85 90 95Phe
Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly100
105 110Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser
Cys Asp Pro Gly Lys115 120 125Cys Tyr Gln
Phe Ala Leu Gly Gln Gly Thr Thr Leu Asp Asn Lys His130
135 140Ser Asn Gly Thr Ile His Asp Arg Ile Pro His Arg
Thr Leu Leu Met145 150 155
160Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gln Val Cys Val165
170 175Ala Trp Ser Ser Ser Ser Cys His Asp
Gly Lys Ala Trp Leu His Val180 185 190Cys
Val Thr Gly Asp Asp Arg Asn Ala Thr Ala Ser Phe Ile Tyr Asp195
200 205Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser
Gln Asn Ile Leu Arg210 215 220Thr Gln Glu
Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val225
230 235 240Met Thr Asp Gly Ser Ala Ser
Gly Arg Ala Asp Thr Arg Ile Leu Phe245 250
255Ile Lys Glu Gly Lys Ile Val His Ile Ser Pro Leu Ser Gly Ser Ala260
265 270Gln His Ile Glu Glu Cys Ser Cys Tyr
Pro Arg Tyr Pro Asp Val Arg275 280 285Cys
Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Ile Asp290
295 300Ile Asn Met Glu Asp Tyr Ser Ile Asp Ser Ser
Tyr Val Cys Ser Gly305 310 315
320Leu Val Gly Asp Thr Pro Arg Asn Asp Asp Ser Ser Ser Asn Ser
Asn325 330 335Cys Arg Asp Pro Asn Asn Glu
Arg Gly Asn Pro Gly Val Lys Gly Trp340 345
350Ala Phe Asp Asn Gly Asp Asp Val Trp Met Gly Arg Thr Ile Asn Lys355
360 365Asp Ser Arg Ser Gly Tyr Glu Thr Phe
Lys Val Ile Gly Gly Trp Ser370 375 380Thr
Pro Asn Ser Lys Ser Gln Val Asn Arg Gln Val Ile Val Asp Asn385
390 395 400Asn Asn Trp Ser Gly Tyr
Ser Gly Ile Phe Ser Val Glu Gly Lys Ser405 410
415Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro
Gln420 425 430Glu Thr Arg Val Trp Trp Thr
Ser Asn Ser Ile Val Val Phe Cys Gly435 440
445Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn Ile450
455 460Asn Phe Met Pro
Ile4651833DNAArtificial1918 H1N1 construct 18caacgcgtgc caccatgaaa
gcaaaactac tgg 331930DNAArtificial1918
H1N1 construct 19tcggcgcctc agatgcatat tctacactgc
302027DNAArtificial1918 H1N1 construct 20caacgcgtgc
caccatgaat ccaaatc
272130DNAArtificial1918 H1N1 construct 21tcggcgccct acttgtcaat ggtgaacggc
30
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