Patent application title: CHIMERIC HIV FUSION PROTEINS AS VACCINES

Inventors: Chao-Wei Liao (Shin-Chu City, TW) Hsiu-Kang Chang (Taipei City, TW)
Assignees: HealthBanks Biotech Co., Ltd. HealthBanks USA Inc.
IPC8 Class: AC07K200FI
USPC Class: 530350
Class name: PROTEINS, I.E., MORE THAN 100 AMINO ACID RESIDUES
Publication date: 08/13/2009
Patent application number: 20090203884

Abstract:

A chimeric fusion protein useful as an immunogen for inducing HIV antigen-specific immune responses contains a first polypeptidyl region and a second polypeptidyl region. The first polypeptidyl region includes a Pseudomonas Exotoxin A (PE) binding domain and a PE translocation domain. The second polypeptidyl region includes (i) a first peptidyl segment containing a fragment of gp120 C1 domain, located at the N-terminus of the second peptidyl region; (ii) a second peptidyl segment containing a fragment of gp120 C5 domain, located at the C-terminus of the first peptidyl segment; and (iii) a third peptidyl segment containing a fragment of gp41, located at the C-terminus of the second peptidyl segment. The second polypeptidyl region contains an antigenic determinant specific to one subtype of HIV. An intermediate polypeptide containing a non-Env, HIV antigenic determinant selected from Gag24, Nef, Tat and Rev may be included.

Claims:

1. A chimeric fusion protein useful as an immunogen for inducing HIV antigen-specific immune responses comprising:a. a first polypeptidyl region containing a Pseudomonas Exotoxin A (PE) binding domain and a PE translocation domain, located at the N-terminus of the fusion protein; andb. a second polypeptidyl region located at the C-terminus of the fusion protein, including:i. a first peptidyl segment containing a fragment of gp120 C1 domain, located at the N-terminus of the second peptidyl region;ii. a second peptidyl segment containing a fragment of gp120 C5 domain, located at the C-terminus of the first peptidyl segment; andiii. a third peptidyl segment containing a fragment of gp41 amino acid sequence, located at the C-terminus of the second peptidyl segment,wherein the second polypeptidyl region contains an antigenic determinant which is specific to one subtype of HIV.

2. The fusion protein of claim 1 further comprising an endoplasmic reticulum retention sequence at the C-terminus of the fusion protein.

3. The fusion protein of claim 1, wherein the one subtype of HIV is at least one selected from the group consisting of HIV subtypes A, B, C, D, E, F, G, H, J and K.

4. The fusion protein of claim 1 further comprising an intermediate polypeptidyl region between the first and the second polypeptidyl regions, wherein the intermediate polypeptidyl region contains a non-Env, HIV antigenic determinant.

5. The fusion protein of claim 4, wherein the intermediate polypeptidyl region comprises an HIV protein, or a fragment thereof, which is at least one selected from the group consisting of Gag24, Nef, Tat and Rev.

6. The fusion protein of claim 4, wherein the intermediate polypeptidyl region comprises Gag24 amino acid sequence or a fragment thereof.

7. The fusion protein of claim 6, wherein the intermediate polypeptidyl region comprises an N- or C-terminus of Gag24 amino acid sequence.

8. The fusion protein of claim 4, wherein the intermediate polypeptidyl region comprises the amino acid sequence set forth by SEQ ID NO: 151.

9. A fusion protein useful as an immunogen for inducing HIV antigen-specific immune responses comprising:a. a first polypeptidyl region containing a PE binding domain and a PE translocation domain, located at the N-terminus of the fusion protein; andb. a second polypeptidyl region containing an HIV protein or a fragment thereof, located at the C-terminus of the fusion protein,wherein the second polypeptidyl region contains an antigenic determinant which is specific to one subtype of HIV.

10. The fusion protein of claim 9 further comprising an endoplasmic reticulum retention sequence at the C-terminus of the fusion protein.

11. The fusion protein of claim 9, wherein the second polypeptidyl region contains a fragment of HIV Env.

12. The fusion protein of claim 9, wherein the second polypeptidyl region contains one or more than one fragment of gp120 V3 domain.

13. The fusion protein of claim 12, wherein the second polypeptidyl region contains the amino acid sequence set forth by SEQ ID NO: 6.

14. The fusion protein of claim 9, wherein the second polypeptidyl region comprises an HIV protein, or a fragment thereof, which is at least one selected from the group consisting of Gag24, Nef, Tat and Rev.

15. The fusion protein of claim 14, wherein the second polypeptidyl region comprises HIV Gag24 amino acid sequence or a fragment thereof.

16. The fusion protein of claim 9, wherein the second polypeptidyl region is a chimeric protein comprising:i. a first peptidyl segment containing a fragment of gp120 C1 domain, located at the N-terminus of the second peptidyl region;ii. a second peptidyl segment containing a fragment of gp120 C5 domain, located at the C-terminus of the first peptidyl segment; andiii. a third peptidyl segment containing a fragment of gp41 amino acid sequence, located at the C-terminus of the second peptidyl segment.

17. The fusion protein of claim 16 further comprising an endoplasmic reticulum retention sequence at the C-terminus of the fusion protein.

18. The fusion protein of claim 16, wherein the second polypeptidyl region comprises the amino acid sequence set forth by SEQ ID NO: 7.

19. The fusion protein of claim 9 further comprising an intermediate polypeptidyl region between the first and the second polypeptidyl regions, wherein the intermediate polypeptidyl region contains a non-Env, HIV antigenic determinant.

20. The fusion protein of claim 9, wherein the intermediate polypeptidyl region comprises an HIV protein, or a fragment thereof, which is at least one selected from the group consisting of Gag24, Nef, Tat and Rev.

Description:

FIELD OF THE INVENTION

[0001]The present invention relates generally to HIV vaccines, and more specifically to chimeric HIV fusion proteins useful for inducing humoral and cell-mediated immune responses.

BACKGROUND OF THE INVENTION

[0002]The global epidemic of AIDS has created an urgent need for a vaccine against human immunodeficiency virus type 1 (HIV-1). It is likely that effective AIDS vaccines will need to generate efficient humoral and cellular immune responses. Virus-neutralizing antibodies and anti-HIV cytotoxic (CD8+) T lymphocytes (CTLs) mediated immunity are major requirements for protective immune responses elicited by HIV vaccines.

[0003]HIV has several major genes coding for viral proteins. The gag gene codes for p24, the viral capsid; p6 and p7, the nucleocapsid proteins; and p17, a matrix protein. The pol gene codes for reverse transcriptase, integrase, and protease which cleaves the proteins derived from gag and pol into functional proteins. The env gene codes for the precursor to gp120 and gp41, envelope proteins embedded in the viral envelope that enable the virus to attach to and fuse with target cells. The tat, rev, nef, vif, vpu genes each codes for a single protein with the same names, Tat, Rev, Nef, Vif, Vpr, Vpu, respectively.

[0004]Neutralizing antibodies have been shown to contribute to protection from virus infection in animal models of HIV-1 infection. The virus-specific targets on HIV-1 accessible to neutralizing antibodies are the envelope glycoproteins (Yang, X. et al. (2005) "Stoichiometry of Antibody Neutralization of Human Immunodeficiency Virus Type 1" Journal of Virology 79: 3500-3508). During the normal course of HIV-1 infections, virus-neutralizing antibodies are often generated but the titer of neutralizing is often low. Most neutralizing antibodies bind the gp120 envelope glycoprotein, which is the major exposed protein of the viral envelope glycoprotein trimer. The more conserved receptor-binding surfaces of the HIV-1 gp120 glycoprotein are also the targets for neutralizing antibodies. The CD4-binding site (CD4BS) antibodies recognize a conformational epitope composed of several segments of gp120 region that overlaps the binding site for CD4. CD4-induced (CD4i) antibodies bind a highly conserved gp120 element that is critical for the gp120-clemokine receptor interaction. The ability of CDBS and CD4i antibodies to interfere with receptor binding contributes to their neutralizing capability.

[0005]GP120 contains ten domains: conserved domains 1-5 (C1-C5) and variable domains 1-5 (V1-V5). The C1 and C5 domains are located at N- and C-terminals of gp120, respectively. Antibodies directed against the V3 loop, which determines chemokine receptor choice, can block the binding of gp120 to the receptors CCR5 and/or CXCR4. Neutralization by anti-V3 antibodies, although potent, is often limited to a small number of HIV-1 strains.

[0006]Gp120 is non-covalently associated with gp41. The gp41 subunit is anchored in the membrane and has a non-polar fusion peptide at its N-terminus. The gp120-gp41 complex forms oligomers on the surface of infected cells and on virions. The binding of gp120 to CD4 is thought to result in activation of the membrane fusion activity of gp41, leading to entry of the viral nucleocapsid into a cell. Antibodies to gp41 epitopes in the serum of HIV-infected individuals may play an important role in virus neutralization. Gp120-41 complex sequences of different HIV subtypes show a remarkably conserved N-terminal coiled-coil structures of gp41 as well as the C-terminal residues that interact with the N-terminal core structure of gp120.

[0007]Multiple immune effectors participate in prevention, containment and clearance of HIV infection. To prevent infection of host target cells, antibodies are required. After the first target cells have been infected with virus, it is important to have cytotoxic T lymphocytes (CTLs) as well as antibodies to reduce cell-to-cell spread and kill infected cells. An effective HIV vaccine should evoke antibodies that can bind to virus and prevent attachment of virus to target cells, as well as CTLs that can eliminate any cells that become infected.

[0008]It remains a difficult goal for vaccinologists to construct live-attenuated viruses that are both effective and safe, or to mimic the presentation of viral proteins observed in infection with recombinant antigens or with replicating or non-replicating vectors carrying appropriate genes or antigens. The large number of mutations in the V3 domain of gp120 has limited its usefulness as a target for HIV vaccine. It is still unclear how the trend of hypervariability in the variable domains is developing and how many domains are absolutely invariant in the evolving strains of HIV.

[0009]A previously unaddressed need exists in the art to address the deficiencies and inadequacies in HIV vaccine antigen production, especially in connection with the provision of efficacious, antigenic determinant peptides.

SUMMARY OF THE INVENTION

[0010]The importance of interaction between gp120 and gp41 for determination of the neutralization phenotype has been studied. One aspect of the invention relates to a chimeric fusion protein useful as an immunogen for inducing HIV antigen-specific immune responses. The chimeric contains: (a) a first polypeptidyl region containing a Pseudomonas Exotoxin A (PE) binding domain and a PE translocation domain, located at the N-terminus of the fusion protein; and (b) a second polypeptidyl region located at the C-terminus of the fusion protein, including: (i) a first peptidyl segment containing a fragment of gp120 C1 domain, located at the N-terminus of the second peptidyl region; (ii) a second peptidyl segment containing a fragment of gp120 C5 domain, located at the C-terminus of the first peptidyl segment; and (iii) a third peptidyl segment containing a fragment of gp41 amino acid sequence, located at the C-terminus of the second peptidyl segment, wherein the second polypeptidyl region contains an antigenic determinant which is specific to one subtype of HIV. The one subtype of HIV is at least one selected from the group consisting of HIV subtypes A, B, C, D, E, F, G, H, J and K.

[0011]In one embodiment of the invention, the fusion protein further includes an endoplasmic reticulum retention sequence, e.g., the amino acid sequence KDEL, at the C-terminus. In another embodiment of the invention, the chimeric fusion protein further includes an intermediate polypeptidyl region between the first and the second polypeptidyl regions, in which the intermediate polypeptidyl region contains a non-Env, HIV antigenic determinant. In one embodiment of the invention, the intermediate polypeptidyl region is at least one selected from the group consisting of Gag24, Nef, Tat and Rev. In another embodiment of the invention, the intermediate polypeptidyl region includes Gag24 amino acid sequence or a fragment thereof. Further in another embodiment of the invention, the intermediate polypeptidyl region contains an N- or C-terminus of Gag24 amino acid sequence. In one embodiment of the invention, the intermediate polypeptidyl region contains the amino acid sequence set forth by SEQ ID NO: 151.

[0012]Another aspect of the invention relates to a chimeric HIV fusion protein useful as an immunogen for inducing HIV antigen-specific immune responses, which includes: (a) a first polypeptidyl region containing a PE binding domain and a PE translocation domain, located at the N-terminus of the fusion protein; and (b) a second polypeptidyl region containing an HIV protein or a fragment thereof, located at the C-terminus of the fusion protein, wherein the second polypeptidyl region contains an antigenic determinant which is specific to one subtype of HIV.

[0013]In one embodiment of the invention, the second polypeptidyl region contains a fragment of HIV Env. In another embodiment of the invention, the second polypeptidyl region contains one or more than one fragment of gp120 V3 domain. In another embodiment of the invention, the second polypeptidyl region contains the amino acid sequence set forth by SEQ ID NO: 6. In one embodiment of the invention, the second polypeptidyl region comprises an HIV protein or a fragment thereof, which is selected from the group consisting of Gag24, Nef, Tat and Rev.

[0014]Further in another embodiment of the invention, the second polypeptidyl region contains HIV Gag24 amino acid sequence or a fragment thereof. Further in another embodiment of the invention, the second polypeptidyl region is a chimeric protein that includes: (i) a first peptidyl segment containing a fragment of gp120 C1 domain, located at the N-terminus of the second peptidyl region; (ii) a second peptidyl segment containing a fragment of gp120 C5 domain, located at the C-terminus of the first peptidyl segment; and (iii) a third peptidyl segment containing a fragment of gp41 amino acid sequence, located at the C-terminus of the second peptidyl segment. Yet in another embodiment of the invention, the second polypeptidyl region comprises the amino acid sequence set forth by SEQ ID NO: 7.

[0015]Yet another aspect of the invention relates to a method of inducing an HIV-antigen specific immune response. The method includes the step of administering an effective amount of the chimeric fusion protein as described above in a biocompatible carrier fluid suitable for carrying and delivering a predetermined aliquot of the fusion protein to a pre-chosen site in a living subject.

[0016]These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.

[0017]The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIGS. 1A-1F are the maps of plasmids encoding chimeric HIV fusion proteins.

[0019]FIG. 2A is a graph showing ELISA titers in the sera samples from animals immunized with various chimeric HIV envelope fusion proteins. Sera sample 1:2,500 dilution.

[0020]FIG. 2B is a graph showing ELISA titers in the sera samples from animals immunized with various chimeric HIV envelope fusion proteins. Sera sample 1:12,500 dilution.

[0021]FIG. 3 is a graph showing neutralizing antibodies against live viruses were induced in the mice immunized with HIV Env fusion protein vaccines.

[0022]FIG. 4 is a flow chart illustrating the construction of pPE(ΔIII)-HIV gag24-K3.

[0023]FIG. 5 is a flow chart illustrating the construction of pPE(ΔIII)-HIV gag24-gp120-41-K3.

[0024]FIG. 6 is a schematic drawing illustrating the fusion of an HIV antigenic determinant peptide to gp120 C1-C5-gp41 creates a chimeric that enhances the antigenic determinant peptide's cell-mediated immune response.

[0025]FIG. 7 is a flow chart illustrating the construction of pPE(ΔIII)-HIV nef-NC--K3.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

[0026]The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

[0027]Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.

[0028]As used herein, "around", "about" or "approximately" shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term "around", "about" or "approximately" can be inferred if not expressly stated.

[0029]As used herein, the term "carboxyl terminal moiety which permits retention of the fusion antigen to the endoplasmic reticulum (ER) membrane of a target cell" refers to a peptide fragment that enables the fusion antigen to bind to the ER membrane and to retain it in the ER lumen for glycosylation and make it appears to be more like foreign protein. In one embodiment of the invention, the carboxyl terminal moiety comprises, in a direction from the amino terminus to the carboxyl terminus, the following amino acid residues:

[0030]R¹--R²--R³--R⁴--(R⁵)_n

[0031]Wherein,

[0032]R¹ is a positively charged amino acid residue;

[0033]R² is a negatively charged amino acid residue;

[0034]R³ is a negatively charged amino acid residue;

[0035]R⁴is L;

[0036]R⁵ is a positively charged amino acid residue; and

[0037]n is 0 or 1.

[0038]Preferably, the carboxyl terminal moiety is a member of the KDEL family protein. As used herein, the term "KDEL family protein" refers to a group of proteins, which has a similar carboxyl end binding to the ER membrane of a cell and further has an ability for retention of such protein in the ER lumen. Generally, the length of the carboxyl end ranges from 4 to 16 residues. As discussed in U.S. Pat. No. 5,705,163 (which is incorporated herein by reference in its entirety), the amino residues at the carboxyl end of a KDEL family protein, particularly those in the last five amino acids, are important. As shown in the studies on the similar sequences present in different molecules and performing a specific biological function, a sequence that retains a newly formed protein within the endoplasmic reticulum is Lys Asp Glu Leu (KDEL). These findings suggest that the sequence at the carboxyl end of the fusion antigen according to the invention acts as some type of recognition sequence to assist translocation of the fusion antigen from an endocytic compartment into the ER and retains it in the lumen. The carboxyl terminal moiety comprises the sequence of KDEL. For example, the carboxyl terminal moiety may comprise the sequence of KKDLRDELKDEL (SEQ ID NO: 250), KKDELRDELKDEL (SEQ ID NO: 251), KKDELRVELKDEL (SEQ ID NO: 252), or KKDELRXELKDEL, in which R is D or V.

[0039]The terms "PE(ΔIII)-HIV gp120" and "PE(ΔIII)-HIV gp120 V3-V3" are interchangeable.

[0040]The terms "PE(ΔIII)-HIV gp120-41" and "PE(ΔIII)-HIV gp120 C1-C5-gp41" are interchangeable.

[0041]The terms "HIV subtype A gp120 C1-C5-gp41" and "chimera A" are interchangeable; the terms "HIV subtype B gp120 C1-C5-gp41" and "chimera B" are interchangeable; the terms "HIV subtype C gp120 C1-C5-gp41" and "chimera C" are interchangeable, and so on.

[0042]Immunogens. To be an immunogen, the formulation need only be a mixture of a fusion protein construct as described herein and a biocompatible carrier fluid suitable for carrying and delivering a predetermined aliquot of the fusion protein construct to a prechosen site in the body of a living subject. Immunogens embodying the invention can be administered in any appropriate carrier for intradermal, subcutaneous, intramuscular, parenteral, intranasal, intravaginal, intrarectal, oral or intragastric administration. They can be introduced by any means that effect antigenicity in humans. The dosage administered will vary and be dependent upon the age, health, and weight of the recipient; the kind of concurrent treatment, if any; the frequency of treatment; and the nature of the humoral antibody response desired. If the immunogens are to be given intradermally, subcutaneously, intramuscularly, intravenously or parenterally, they will be prepared in sterile form; in multiple or single dose formats; and dispersed in a fluid carrier such as sterile physiological saline or 5% dextrose solutions commonly used with injectables. In addition, other methods of administration can be advantageously employed as well.

[0043]Vaccines. To be a prepared vaccine, the minimal formulation comprises a predetermined quantity of a fusion protein construct as described herein; a biocompatible carrier suitable for carrying and delivering a predetermined aliquot of a fusion protein construct to a prechosen site in the body of a living subject; and at least one adjuvant composition dispersed in the carrier fluid or coupled to the fusion protein constrict. The vaccine, by definition, incorporates an immunogen and includes one or more adjuvants to facilitate or stimulate the immune response and to prolong the antigenic effect in-vivo over time. Among the useful adjuvant substances conventionally known are those compositions approved by the FDA (currently or pending for systemic and/or mucosal immunizations). Some are preferred for mucosally-administered vaccines and others are preferred for intragastric administered vaccines.

[0044]Modes of administration. Multiple modes of inoculation, the manner of introducing an immunogen or vaccine, are conventionally known and used. The systemic or parenteral forms of administration (introduction by injection or perfusion) typically include intraperitoneal, intravenous, intramuscular, subcutaneous, and subdermal inoculations. In contrast, mucosal modes of administration may include not only the intranasal and intragastric forms of introduction, but also oral, intravaginal, and intrarectal introductions.

EXAMPLES

[0045]Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.

1. HIV-1 gp120 and gp41 Fusion Proteins

Example 1

Selection of Truncated Segments From HIV Env Proteins, gp120 and gp41

[0046]The amino acid sequences of HIV gp120 and gp41 were retrieved from the National Center of Biotechnology Information (NCBI, USA) database and entered into software for evaluation of antigenic determinant (epitopes) of the target proteins, and candidate segments for synthesis displayed on an evaluation plot. Antigenic determinant regions of the target protein were chosen for synthesis by a reverse genetic engineering technique. Several peptide segments were selected as target peptides based on the results of the evaluation software. The software DNA strider v1.0 was used to analyze whether the nucleotide sequences of the target peptides contained restriction enzyme sites. If present in the DNA sequence in disadvantageous places, changes were made within the appropriate codons without altering the amino acid sequence. The software checked the newly created sequence, and designed restriction sites at both termini of the DNA sequence to facilitate cloning. Codons for some amino acid residues, such as Arg, Ile, Gln, Pro, were modified to increase the expression of proteins in E. coli expression systems. Table 1 lists the selected peptide segments and their corresponding amino acid sequences.

Example 2

Construction of Chimeric Target Polypeptides gp120 V3-V3 and gp120 C1-C5-gp41

[0047]Two target peptides gp120 V3-V3 and gp120 C1-C5-gp41 were constructed using the selected peptide segments as follows: Three truncated peptide segments having the amino acid sequences of SEQ ID NO. 1 (from gp120 C1 domain), SEQ ID NO. 2 (from gp120 C5 domain) and SEQ ID NO. 5 (from gp41 region associated with gp 120), respectively, were ligated to form a chimeric target peptide gp120 C1-C5-gp41 (referred to as gp120-41). Two truncated segments having amino acid sequences of SEQ ID NOs: 3 and 4 (both from gp120 V3 domains), respectively, were fused to form polypeptide gp120 V3-V3 (referred to as gp120). One or more residues might be inserted in-between to link two peptide segments. The number of residues inserted in-between was about 1 to 15 amino acids, which might be selected from amino acid residues that would not alter the secondary structure of proteins, such as glycine, alanine, valine, and leucine. The amino acid residue cysteine in SEQ ID NO: 2 and in SEQ ID NO: 5 could form a disulfide bond so that the chimeric target peptide generated could possess a three-dimensional structure.

TABLE-US-00001 TABLE 1 HIV Env Target SEQ Proteins peptide segments Sequence of selected peptide segments ID No. gp120 C1 domain VEKLWVTVYYGVPVWK 1 C5 domain KVVKIEPLGVAPTKCKRRVVQREKR 2 V3 domain CTRPSNNTRKGIHMGPGGAFYTTGQIIRNIRQAHC 3 V3 domain CTRPNNNTRRSIHIEPEGAFYTTGEIIGDIRQAHC 4 gp41 gp 41 region in QARVIAVERYLKDQQLLGIWGGSGKLICCTTAV 5 association with PWNSSWSNKLDRIWNNMTWLE gp120

TABLE-US-00002 TABLE 2 Chimeric target SEQ peptide Amino acid sequence ID No. gp120 IGCTRPSNNTRKGIHMGPGGAFYTTGQIIRNIRQAHCGLLGGC 6 V3-V3 GLGLE gp120 VEKLWVTVYYGVPVWKKVVKIEPLGVAPTKCKRRVVQREKRGGGGGQ 7 C1-C5- gp41 LE

[0048]Table 2 lists the amino acid sequences of the chimeric target peptides gp120 V3-V3 and gp120 C1-C5-gp41. For chimeric peptide gp120 V3-V3 in table 2: the underlined letters denote the restriction sites; bold letters denote the first V3 domain segment, bold and italic letters denote the second V3 domain segment. For chimeric peptide gp120 C1-C5-gp41 in table 2: the bold letters denote the C1 domain segment; non-bold letters denote the C5 domain segment; non-bold and italic letters denote linkers; bold and italic letters denote the gp41 domain; and the underlined letters denote a restriction site.

[0049]The chimeric target peptide gp120 V3-V3 (SEQ ID NO: 6) was designed based on the construction of repeats in the V3 domain of gp120, and the chimera gp120 C1-C5-gp41 (SEQ ID NO: 7) was based on simulation of the gp41 region in association with gp120. The chimera gp120 V3-V3 (SEQ ID NO: 6) comprises amino acid sequences of SEQ ID NOs: 3 and 4 (both from gp120 V3 domains). The chimera gp120 C1-C5-gp41 comprises amino acid sequences of SEQ ID NOs: 1 (from gp120 C1 domain), 2 (from gp120 C5 domain), and 5 (gp41 region in junction with gp120) (Table 1). Disulfide bonds were formed due to cysteines in SEQ ID NO: 2 (from the C5 domain of gp120) and SEQ ID No. 5 (from gp41 region in junction with gp120).

[0050]Using the similar method as described above, the following chimeric target peptides were constructed: HIV subtype A gp120 C1-C5-gp41; HIV subtype B gp120 C1-C5-gp41; HIV subtype C gp120 C1-C5-gp41; HIV subtype D gp120 C1-C5-gp41; HIV subtype E gp120 C1-C5-gp41; HIV subtype F gp120 C1-C5-gp41; HIV subtype G gp120 C1-C5-gp41; HIV subtype H gp120 C1-C5-gp41; HIV subtype J gp120 C1-C5-gp41; and HIV subtype K gp120 C1-C5-gp41 (abbreviated as chimeric target peptides A, B, C, D, E, F, G, H, J, and K, respectively).

[0051]These chimeras A, B, C, D, E, F, G, H, J and K were each constructed from a combination of 3 peptide segments selected from each corresponding HIV-1 subtypes, A, B, C, D, F, G, H, J and K, respectively. The basic scheme of the construction was to link two peptide segments, each selected from the C1 and C5 domains of gp120, to another segment selected from the gp41 region in association with gp120 for each HIV-1 subtype. Thus, like the SEQ ID NO: 7, all these chimera target peptides have gp120-C1-C5-gp41-like structures.

[0052]Table 3 lists the amino acid sequences of these chimeras, in which the non-bold, italic letters denote a segment from the C1 domain, the bold letters denote a segment from the C5 domain, of HIV subtype A gp120 protein; the non-bold, non-italic letters GGGGG denote a linker, and the bold, italic letters denote a segment from HIV subtype A gp41 region in junction with gp120.

TABLE-US-00003 TABLE 3 Chimera for HIV SEQ subtype Amino Acid Sequence ID NO. A AENLWVTVYYGVPIWKKVVKEIPLGVAPTKARRRVVEREKRGGGGG 31 B TEKLWVTVYYGVPVWKKVVKEIPLGIAPTKAKRRVVQREKRGGGGG 32 C MGNLWVTVYYGVPVWKKYKVVEIKPLGVAPTKPKRRVVEREKRGGGGG 33 D ADNLWVTVYYGVPVWKKVVQIEPLGVAPTRAKRRVVEREKRGGGGG 34 E SXNLWVTVYYGVPVWRKVVQIEPLGIAPTRPKRRVVEREKRGGGGG 35 F ADNLWVTVYYGVPVWKKVVEIEPLGVAPTKAKRQVVQREKRGGGGG 36 G ASNNLWVTVYYGVPVWEDAKKVVKIKPLGVAPTKARRRVVGREKRGGGGG 37 H VVGNLWVTVYYGVPVWKKVVKIEPLGVAPTEARRRVVEREKRGGGGG 38 J AKEDLWVTVYYGVPVWKKVVEIEPLGVAPTKAKRRVVEREKRGGGGG 39 K IAANNLWVTVYYGVPVWKKVVQIEPLGIAPTRARRRVVQREKRGGGGG 40

Example 3

Synthesis of DNA Fragments Encoding Chimeric Target Polypeptides gp120 V3-V3 and gp120 C1-C5-gp41

[0053]The nucleotide sequences of tile DNA fragments encoding chimeric target peptides gp120 V3-V3 and gp120 C1-C5-gp41 were modified to increase translation efficiency without changing the amino acid sequences of the encoded proteins, using the method disclosed in Taiwan patent application No. 092126644, which is incorporated herein in its entirety by reference. The sequence modification allowed the encoded peptides or proteins to be efficiently expressed in E. coli pET plasmid expression system. Table 4 lists the modified sequences of the DNA fragments encoding the chimeric target peptides gp 120 V3-V3 and gp120 C1-C5-gp41, in which non-italic, capital letters denote restriction enzyme linkers for EcoRI, Nde1 and Sal1 cutting sites, and italic, capital letters denote a XhoI restriction enzyme site.

TABLE-US-00004 TABLE 4 Chimeric target SEQ peptide Nucleotide sequence ID No. gp120 GAATTCCATATGGTCGACatcggttgcacccgtc 8 V3-V3 cgagcaacaacacccgtaaaggtatccacatggg cccgggtggtgctttctacaccaccggtcagatc atccgtaacatccgtcaggctcactgtggtctgc tgggtggttgtacccgtccgaacaacaacacccg tcgtagcatccacatcgaaccggaaggtgctttc tacaccaccggtgaaatcatcggtgacatccgtc aggctcactgtggcctgggtCTCGAG gp120 C1- GAATTCCATATGGTCGACgttgaaaaactgtggg 9 C5-gp41 ttaccgtttactacggtgttccggtttggaaaaa agttgttaaaatcgaaccgctgggtgttgctccg accaaatgcaaacgtcgtgttgttcagcgtgaaa agcgtggtggcggtggcggtcaagctcgtgttat cgctgttgaacgttacctgaaagaccagcagctg ctgggtatctggggtggtagcggtaaactgatct gctgcaccaccgctgttccgtggaacagcagctg gagcaacaaactggaccgtatctggaacaacatg acctggCTCGAG

[0054]Table 5 lists SEQ ID NOs. of respective primer pairs used for PCR synthesis of the DNA fragments encoding chimeric target peptides gp120 V3-V3 (SEQ ID NO: 8) and gp120 C1-C5-gp41 (SEQ ID NO: 9). Non-DNA-template PCR reactions were performed by continuously using forward and reverse primer pairs to PCR synthesize DNA fragments. In the first-round PCR, the 3' end of the first forward primer (F1) had about 10-15 bases that were complementary to those in the pairing reversed primer (R1). The PCR profile was as follows: 5 min at 95° C., 1 min at 94° C., 0.5 min at 55° C., 1 min at 72° C. for 20 cycles, and 1 min at 72° C. Following the first-round PCR, the 3' ends of the primer pairs (such as F2 and R2, F3 and R3, F4 and R4, or F5 and R5) had about 10-15 bases that were complementary to the previous round PCR product as a DNA template. After the first-round PCR, 0.01-1 μl of the product was used as the DNA template for the second-round PCR. The second primer pair, F2 and R2, were added in a suitable amount together with dNTPs, reagents and Pfu polymerase, and the second round PCR was performed. Other primers were subsequently added in this manner so that the final extended DNA fragments were synthesized. All the DNA fragments synthesized from each primer pair and each round of PCR were analyzed for the size by gel electrophoresis. The DNA fragment encoding each individual chimeric target peptide was synthesized in this manner until the final PCR product was extended to the expected size, e.g., 264 bp in the case of gp120 V3-V3.

[0055]The DNA fragments (Table 6) encoding each individual chimeric target peptides gp120 C1-C5-gp⁴¹ for HIV subtype A, B, C, D, E, F, G, H, J and K were synthesized in a manner similar to the method described above using primer pairs listed in Table 6.

TABLE-US-00005 TABLE 5 Chimeric target primer Forward Reverse peptide pairs Primer SEQ ID NO. Primer SEQ ID NO. gp120 V3-V3 P1 F1 10 R1 15 P2 F2 11 R2 16 P3 F3 12 R3 17 P4 F4 13 R4 18 P5 F5 14 R5 19 gp120 C1-C5- P1 F1 20 R1 24 gp41 P2 F2 21 R2 25 P3 F3 22 R3 26 P4 F4 23 R4 27 P5 F4 23 R5 28 P6 F4 23 R6 29

Example 4

Construction of Plasmids for Expression of HIV Fusion Proteins PE(ΔIII)-gp120 V3-V3 and PE(ΔIII)-gp120 C1-C5-gp41

[0056]Various chimeric target polypeptides of HIV-1 Env proteins as described above were cloned and expressed as PE fusion proteins. Briefly, a polypeptide from Pseudomonas Exotoxin A, i.e., PE(ΔIII), which was devoid of cytotoxic domain III, was fused to respective chimeric target polypeptides. Plasmid pPE(ΔIII) was constructed by inserting a DNA fragment encoding the binding domain I and translocation domain II of Pseudomonas exotoxin A into vector pET15a. A DNA sequence coding for a carboxyl terminal peptide that comprises the amino acid sequence KDEL (SEQ ID NO. 30) was ligated to the carboxyl terminal portion of the PE(ΔIII) gene, and generated plasmid pPE(ΔIII)-KDEL3 (referred to as pPE(ΔIII)-K3). The isolated DNA fragments generated by the PCR reaction were respectively digested by restriction enzymes EcoRI and Xho I, and then ligated to the EcoRI, Xho I sites of the plasmid pPE(ΔIII)-KDEL3, resulting in chimeric genes that would expressed target peptides as fusion proteins (Hung, C. F. et al (2001) "Cancer Immunotherapy Using a DNA Vaccine Encoding the Translocation Domain of a Bacterial Toxin Linked to a Tumor Antigen" Cancer Research 61:3698-3703).

[0057]The plasmid pPE(ΔIII)-HIV gp120 (FIG. 1A) encodes a fusion protein PE(ΔIII)-HIV gp120 V3-V3 (referred to as PE(ΔIII)-HIV gp120). The plasmid pPE(ΔIII)-HIV gp120-K3 (FIG. 1B) encodes a fusion protein PE(ΔIII)-HIV gp120 V3-V3-K3 (referred to as PE(ΔIII)-HIV gp120-K3). The plasmid pPE(ΔIII)-HIV gp120-41 (FIG. 1C) encodes fusion protein PE(ΔIII)-HIV gp120 C1-C5-gp41 (referred to as PE(ΔIII)-HIV gp120-41). The plasmid pPE(ΔIII)-HIV gp120-41-K3 (FIG. 1D) encodes the fusion protein PE(ΔIII)-HIV gp120 C1-C5-gp41-K3 (referred to as PE(ΔIII)-HIV gp120-41-K3). The resulting plasmids were respectively transformed into E. coli Jam109 to obtain clones and maintain the clones therein. The Jam109 strain could stably maintain the plasmid inside bacteria cells without protein expression.

TABLE-US-00006 TABLE 6 Chimeric Nucleotide SEQ target Sequence Forward ID Reverse SEQ ID peptide ID NO. primer pairs Primer NO. Primer NO. A 41 P1 F1 51 R1 56 P2 F2 52 R2 57 P3 F3 53 R3 58 P4 F4 54 R4 59 P5 F5 55 R5 60 B 42 P1 F1 61 R1 66 P2 F2 62 R2 67 P3 F3 63 R3 68 P4 F4 64 R4 69 P5 F5 65 R5 70 C 43 P1 F1 71 R1 76 P2 F2 72 R2 77 P3 F3 73 R3 78 P4 F4 74 R4 79 P5 F5 75 R5 80 D 44 P1 F1 81 R1 86 P2 F2 82 R2 87 P3 F3 83 R3 88 P4 F4 84 R4 89 P5 F5 85 R5 90 E 45 P1 F1 91 R1 96 P2 F2 92 R2 97 P3 F3 93 R3 98 P4 F4 94 R4 99 P5 F5 95 R5 100 F 46 P1 F1 101 R1 106 P2 F2 102 R2 107 P3 F3 103 R3 108 P4 F4 104 R4 109 P5 F5 105 R5 110 G 47 P1 F1 111 R1 116 P2 F2 112 R2 117 P3 F3 113 R3 118 P4 F4 114 R4 119 P5 F5 115 R5 120 H 48 P1 F1 121 R1 126 P2 F2 122 R2 127 P3 F3 123 R3 128 P4 F4 124 R4 129 P5 F5 125 R5 130 J 49 P1 F1 131 R1 136 P2 F2 132 R2 137 P3 F3 133 R3 138 P4 F4 134 R4 139 P5 F5 135 R5 140 K 50 P1 F1 141 R1 146 P2 F2 142 R2 147 P3 F3 143 R3 148 P4 F4 144 R4 149 P5 F5 145 R5 150

Example 5

Expression and Analysis of Target Proteins

[0058]HIV-1 PE-fusion proteins were expressed in E. coli BL21 (DE3) plys cultures containing corresponding expression plasmids. Briefly, 5 ml of bacterial seeds (A600 of 1.0±0.3 O.D.) were inoculated into 250 ml of liquid broth (LB) supplemented with 500 μg/ml ampicillin and 50 ml of 10% glucose at 37° C. in a rotating incubator shaken at 150 rpm for 2-3 hours. Once O.D.₆₀₀ nm reached 0.3±0.1, the bacterial culture was induced with isopropylthio-β-D-galactoside (IPTG; Promega, USA) at a final concentration of 0.1 to 2 mM at 37° C. in a rotating incubator shaken at 150 rpm for 2 hours for protein expression.

[0059]Bacterial cells were pelleted after the protein induction was completed. After freezing and thawing of the pellet, bacterial cells were lysed with a solution containing in 10 ml: 0.3 mg/ml lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.06 mg/ml DNase I at room temperature for 20 minutes, followed by addition of 1 ml of 10% Triton X-100 and incubation at room temperature for 10 minutes. The lysed cells were centrifuged at 12,000 g for 10 minutes, and pellets were washed by 1 M and 2 M urea solutions. Insoluble inclusion bodies containing recombinant proteins were collected and dissolved in 8 ml of 8M urea solution or in an alkaline solution (pH 10 to 12) containing 1 to 3 M urea, and purified using a commercial pET His-Tag purification column system. The protein inclusion bodies dissolved in the urea solution were loaded onto a 4 ml Ni²+-nitrilotriacetic acid (Ni-NTA) resin affinity column, and the bound material eluted by different pH buffers (e.g. pH=8.0, 7.0, 6.5, 6.0, 5.4, and 3.5) containing 1 to 6 M urea with 0.1 to 0.3 M NaCl, 5 to 50 mM phosphate buffer, and 5 to 50 mM Tris.

[0060]The eluted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with coomassie blue. The optical densities of the bands in the gels were measured with a densitometer for evaluation of protein quantities. The concentration of fusion proteins, e.g., PE(ΔIII)-HIV gp120 C1-C5-gp41-K3 (referred to as gp120-41-K3) and PE(ΔIII)-HIV gp120 V3-V3-K3 (referred to as gp120-K3), in the eluted samples were about 0.8 mg/ml.

Example 6

Antibody Assay

[0061]Materials and methods. Five PE-HIV envelope peptides were used: (1) PE(ΔIII)-HIV gp120; (2) PE(ΔIII)-HIV gp120-KDEL; (3) PE(ΔIII)-HIV gp120-41; and (4) PE(ΔIII)-HIV gp120-41-KDEL. An oil adjuvant, ISA 206, was used with each of the peptide immunogens in emulsified preparations for injection into mice.

[0062]Animals. BALB/c mice were purchased form Harlan laboratories and housed in the Laboratory animal Resources Facility of KUMC. All mice were used in accordance with AAALAC and the KUMC Institutional Animal Care and Use Committee guidelines.

[0063]Immunization of animals with PE-HIV-Env fusion protein vaccines. Four- to six-week old BALB/c mice were divided into 5 groups, with 6 mice per group. The animals in groups 1 to 5 received (1) PE(ΔIII)-HIV gp120, (2) PE(ΔIII)-HIV gp120-K3, (3) PE(ΔIII)-HIV gp120-41, (4) PE(ΔIII)-HIV gp120-41-K3, and (5) PBS (control group), in adjuvant, respectively, following the immunization schedule shown in Table 7. Immunized mice were then exsanguinated following deep anesthesia and blood and spleens were collected for immunological assays.

TABLE-US-00007 TABLE 7 Immunization Schedule 1^st time 2^nd time 3^rd time 4^th time 5^th time No. of *(IM) (IM) (IM) (I/M) **(IP) Group mice Immunogen 0 week 2 weeks 4 weeks 6 weeks 8 weeks 1 6 PE(ΔIII)-HIV gp120 50 μg 25 μg 25 μg 25 μg 50 μg Adjuvant 50 μl 50 μl 50 μl 50 μl 0 2 6 PE(ΔIII)-HIV gp120-K3 50 μg 25 μg 25 μg 25 μg 50 μg Adjuvant 50 μl 50 μl 50 μl 50 μl 0 3 6 PE(ΔIII)-HIV gp120-41 50 μg 25 μg 25 μg 25 μg 50 μg Adjuvant 50 μl 50 μl 50 μl 50 μl 0 4 6 PE(ΔIII)-HIV gp120-41-K3 50 μg 25 μg 25 μg 25 μg 50 μg Adjuvant 50 μl 50 μl 50 μl 50 μl 0 5 6 PBS 0 0 0 0 50 μl Adjuvant 50 μl 50 μl 50 μl 50 μl 0 *IM denotes intramuscular injection **IP denotes intraperitoneal injection

[0064]Serum antibody binding assay. An ELISA test using a commercial kit was used to determine binding antibody titers. Briefly, two weeks after the last immunization, mice were anesthetized, sacrificed, and spleens and blood samples were collected. Sera prepared from the blood samples of the animals immunized with fusion proteins in groups 1 to 4 and animals in the placebo group were serially diluted 500×, 2500×, 12500×, and 62500×, respectively. Binding antibody titers induced by the four peptide groups were analyzed using ELISA kit (BioChain) for detection of anti-HIV antibodies. Plates were coated with HIV antigen. The manufacturer's protocol was modified for using mouse sera by substituting the anti-human antibody with a goat anti-mouse serum conjugated with Horseradish peroxidase (HRP). Results were expressed as OD absorbance at 450 nm against the blank. FIGS. 2A-2B illustrate the test results of sera from animals immunized with respective fusion proteins. The absorbance data reflects the amount of antibody titers in serum samples. The result indicates that each fusion protein vaccine after being injected into animals was able to induce antibodies against HIV with good titers in the dilution of 1:2,500 (FIG. 2A) and 12,500 (FIG. 2B).

Example 7

Immunized Mouse Sera Neutralize HIV-Attenuated Live Vaccine Virus

[0065]Neutralization assays were performed using mouse sera against an attenuated live SHIV vaccine virus that was developed in the MMD Lab. The assays were performed in X4 GHOST cells using a plaque reduction assay. X4 GHOST cells were capable of harboring HIV virus plaques after being infected by an HIV-attenuated live vaccine virus. Briefly, serum samples collected from immunized mice were tested for their contents of HIV specific neutralizing antibodies. Briefly, quadruplicates of serial twofold dilutions of sera in RPMI 1640 medium were prepared in 96-well plates. Twenty plaque-forming units of virus were incubated with the twofold dilutions of serum samples from each immunized mouse and a normal serum for two hours at 37 C, respectively. The suspensions were then inoculated onto monolayers of X4 GHOST cells that constitutively expressed the HIV LTR linked to GFP. These cultures were incubated for three days and then examined by immunofluorescence for determination of the numbers of fluorescence spots that represented successful virus hits. Neutralization titers of the serum samples were scored as the highest dilution of the immune serum sample that prevented development of 50% of the plaques induced with the control nonimmune serum. Mice developed neutralizing antibodies against the live vaccine virus (FIG. 3). The highest serum neutralization titers were obtained with sera from mice immunized with PE(ΔIII)-HIV gp120-41-KDEL.

Example 8

Immunized Mouse Sera Neutralize Simian-Human Immunodeficiency Virus (SHIV_KU2)

[0066]Immunization of animals. Mice were immunized with higher dosages of PE-fusion protein vaccines using a similar protocol described above. Briefly, mice were divided into 5 groups (groups 6-10), with 6 mice per group, and received PE(ΔIII)-HIV gp120, PE(ΔIII)-HIV gp120-K3, PE(ΔIII)-HIV gp120-41, PE(ΔIII)-HIV gp120-41-K3, and PBS (control group), in adjuvant, respectively. The immunization schedule is shown in Table 8.

TABLE-US-00008 TABLE 8 Immunization First Second No. time time Third time of *(IM) (IM) **(IP) Group mice Immunogen 0 week 2 weeks 6 weeks 6 6 PE(ΔIII)-HIVgp120 100 μg 50 μg 50 μg Adjuvant 100 μl 50 μl 0 7 6 PE(ΔIII)-HIVgp120-K3 100 μg 50 μg 50 μg Adjuvant 100 μl 50 μl 0 8 6 PE(ΔIII)-HIVgp120-41 100 μg 50 μg 50 μg Adjuvant 100 μl 50 μl 0 9 6 PE(ΔIII)-HIVgp120- 100 μg 50 μg 50 μg 41-K3 Adjuvant 100 μl 50 μl 0 10 6 PBS 100 μl 50 μl 50 μl Adjuvant 100 μl 50 μl 0 *IM: intramuscular injection with adjuvant ISA 206. **IP: intraperitoneal injection with adjuvant ISA 206.

TABLE-US-00009 TABLE 9 No. of live mice/No. of Neutralization Group Vaccine challenged mice titer 6 PE(ΔIII)-HIV gp120 4/6 1:20 7 PE(ΔIII)-HIV gp120-K3 1/6 1:20 8 PE(ΔIII)-HIV gp120-41 5/6 1:20 9 PE(ΔIII)-HIV gp120-41-K3 3/6 1:20-40 10 PBS/adjuvant* 0/6 -- *Adjuvant: ISA 206.

[0067]Two weeks after the third immunization, blood samples were collected from each group of animals and processed to obtain serum samples for assay of antibody titers. ELISA antibody assays showed similar titers in this experiment as those in Example 6.

[0068]To perform neutralization assays using SHIV_KU2, mice from each group were challenged by pathogenic SHIV_KU2 vaccine (constructed by Dr. Narayan, University of Kansas Medical Center, US Pat. No. 5,849,994). All four groups of the immunized animals developed neutralizing antibody titers of approximate 1:20 against SHIV_KU2. Table 9 shows the survival data. PE(III) gp120-41 was the best antigen preparation for induction of neutralizing antibodies against SHIV_KU2 since 5 out of 6 mice developed these antibodies.

Example 9

HIV Gag24, Nef, Tat, and Rev Fusion Proteins

[0069]Materials and methods. Four HIV fusion proteins were tested for their immunogenicity: (I) HIV Gag24 fusion protein vaccines, PE(ΔIII)-HIV Gag24-K3 and PE(ΔIII)-HIV Gag24-gp120-41-K3; (II) HIV Nef fusion protein vaccines comprising PE(ΔIII)-HIV Nef-N--K3and PE(ΔIII)-HIV Nef-C--K3; (III) HIV Tat fusion protein vaccine comprising PE(ΔIII)-HIV tat-K3; and (IV) HIV Rev fusion protein vaccine comprising PE(ΔIII)-HIV Rev-K3. The above (I) to (IV) HIV fusion proteins were constructed using similar methods described in Examples 2 to 4. Briefly, various polypeptide segments (Table 10) were selected from HIV proteins Gag24, Nef, Tat and Rev, respectively.

TABLE-US-00010 TABLE 10 Targeted HIV peptide SEQ Proteins segments Amino acid sequence ID No. Gag24 Full length VDRDELKGIGMTNNPPIPVGEIYK 151 Gag24 RWIILGLNKIVRMYSPTMTNNPPI PVGEIYRWIILGLNKIVRMYSPT Nef Nef- N PTVRQRMDRTEPAAEGVGAVSRDL 152 terminus EKHGAITSSNTAATNADCAWLEAQ EEEEVGFPVRPQVPLRPMTYKAAV DISHFLKEKGGLEGLIYSQKRQEI LDLWIYHTQGYFPDWQNYTPGPGI RYPLTFGWCFKL Nef-C FLKVPVDPEQVEKANEGDNNCLLH 153 terminus PISQHGMDDPEKEVLMWKFDSRLA FQHIAREKHPEYYKDCLG Tat Full length RDELKGIGMEPVDPRLEPWKHPGS 154 Tat QPRTACNNCYCKKCCFHCPVCFIS KGLGISYGRKKRRQRRRAPQDSET HQVSLSKQPTSQLRGDPTGPKESK KKVERETETDPNV Rev Full length LLAVRIIKTLYQSNPYPKPEGYRR 155 Rev VRRNRRRRWRARQRQIHSISERIL ITCLGRPTEPVPLQLPPIERLNIN CSESGGTSGTQRVGNP

TABLE-US-00011 TABLE 11 Nucleotide SEQ Target Sequence Forward ID Reverse SEQ ID peptide ID NO. primer pairs Primer NO. Primer NO. Gag24 156 P1 F1 161 R1 165 P2 F2 162 R2 166 P3 F3 163 R3 167 P4 F4 164 R4 168 Nef-N 157 P1 F1 169 R1 176 terminus P2 F2 170 R2 177 P3 F3 171 R3 178 P4 F4 172 R4 179 P5 F5 173 R5 180 P6 F6 174 R6 181 P7 F7 175 R7 182 P8 F7 175 R8 183 P9 F7 175 R9 184 Nef-C 158 P1 F1 185 R1 189 terminus P2 F2 186 R2 190 P3 F3 187 R3 191 P4 F4 188 R4 192 Tat 159 P1 F1 193 R1 200 P2 F2 194 R2 201 P3 F3 195 R3 202 P4 F4 196 R4 203 P5 F5 197 R5 204 P6 F6 198 R6 205 P7 F7 199 R7 206 Rev 160 P1 F1 207 R1 213 P2 F2 208 R2 214 P3 F3 209 R3 215 P4 F4 210 R4 216 P5 F5 211 R5 217 P6 F6 212 R5 217

[0070]The DNA fragments (Table 11) encoding respective polypeptide segments were synthesized using primers listed in Table 11 by multi-round PCR synthesis method as described previously. Gel electrophoresis experiments were performed to examine the PCR products generated by each primer pair in the multiple-round PCR synthesis of DNA fragments, e.g., 410, 412, 414, 416 (FIG. 4). The PCR synthesized DNA fragments were fused to a PE fragment, cloned and expressed, respectively, using a similar method described in Example 4. The PE fragment is a polypeptide called PE (ΔIII) that contains a binding domain and a translocation domain from Pseudomonas Exotoxin A but lacks a cytotoxic domain. For example, the DNA fragment encoding gag24 generated by PCR was digested by restriction enzymes EcoRI and Xho I to isolate a 225 bp fragment, 402, which was ligated to a PE(ΔIII) fragment within an EcoRI and Xho I-digested plasmid pPE(ΔIII)-KDEL3, 401, to generate pPE(ΔIII)-HIV-gag24-K3 (FIG. 4).

[0071]A 1.7 Kb gp120-41 fragment, isolated from Sal I and Pst I-digested pPE(ΔIII)-HIV gp120-41-K3, 502, was fused to gag24 by inserting downstream, or C-terminal, to the gag24 gene within a Pst I, Xho I-digested plasmid pPE(ΔIII)-HIV gag24-K3, 501, to generate plasmid pPE(ΔIII)-HIV gag24-gp120-41-K3 (FIG. 5). Plasmid pPE(ΔIII)-HIV nef-C--K3 was digested by Sal I and Pst I to isolate a 1.6 Kb fragment, 702, followed by ligation to a nef-N fragment within a Pst I, Xho I-digested plasmid pPE(ΔIII)-HIV nef-N-KDEL, 701, to generate pPE(ΔIII)-HIV nef-NC-KDEL (FIG. 7).

[0072]The DNA fragment encoding Tat or Rev generated by PCR was digested by restriction enzymes EcoRI and Xho I to isolate fragment, which was ligated to a PE(ΔIII) fragment within an EcoRI and Xho I-digested plasmid pPE(ΔIII)-KDEL3. By employing DNA recombinant method, plasmids pPE(ΔIII)-HIV Tat-K3 (FIG. 1F), and pPE(ΔIII)-HIV Rev-K3 (FIG. 1E) were generated.

[0073]The sequence KDEL3 (i.e., K3) is an endoplasmic reticulum (ER) retention peptide located at the carboxyl terminal portion of the chimeric fusion protein. The sequence listing illustrates the nucleotide sequences of PE(ΔIII)-HIV Gag24-K3, PE(ΔIII)-HIV gag24-gp120-41-K3, PE-(ΔIII)-HIV nef-N--K3, pPE(ΔIII)-HIV nef-C--K3, pPE(ΔIII)-HIV-nef-NC--K3, pPE(III)-HIV rev-K3, pPE(ΔIII)-HIV-tat-K3 as SEQ ID NOs: 236, 238, 240, 242, 244, 246 and 248, and the corresponding amino acid sequences as SEQ ID NOs: 237, 239, 241, 243, 245, 247, and 249, respectively. For clinical applications, any undesired sequence such as oncogen sequences, if present in the bridge between PE(ΔIII) and HIV target peptide (e.g., between EcoRI and AatII), may be deleted without affecting the HIV target antigenic determinants.

[0074]Immunization of animals with fusion proteins. Chimeric fusion proteins as described above were expressed for vaccination. Female mice C57BL/6J aged 6- to 8-week old were purchased from National Taiwan University (Taipei, Taiwan) and bred in Animal Center of National Taiwan University Hospital. Mice were divided into groups and injected three times at two-week intervals with respective fusion proteins or PBS (control group) in ISA 206 oil adjuvant (Table 12). Two weeks after the last immunization, mice were exsanguinated under deep anesthesia and blood and spleens were collected for immunological assays. An ELISA test using a commercial kit was used to determine binding antibody titers.

TABLE-US-00012 TABLE 12 Immunization Schedule HIV No. of First time Second time Third time target peptides Vaccine* Mice 0 week 2 weeks 4 weeks Placebo PBS/Adjuvant 3 100 μl 100 μl 100 μl Gag24 PE(ΔIII)-HIV Gag24-K3 4 100 μg 100 μg 100 μg gp120 PE(ΔIII)-HIV gp120-41-K3 4 100 μg 100 μg 100 μg Gag24-gp120- PE(ΔIII)-HIV Gag24- 3 100 μg 100 μg 100 μg 41 gp120-41-K3 Nef PE(ΔIII)-HIV Nef-N-K3 4 100 μg 100 μg 100 μg PE(ΔIII)-HIV Nef-C-K3 (50 μg each) (50 μg each) (50 μg each) Tat PE(ΔIII)-HIV Tat-K3 4 100 μg 100 μg 100 μg Rev PE(ΔIII)-HIV Rev-K3 4 100 μg 100 μg 100 μg *Vaccines were prepared by mixing fusion proteins or PBS with 50 μl of adjuvant ISA 206 before intramuscular (IM) injection.

[0075]Serum Antibody Test. Sera from mice were prepared and serially diluted 500×, 2500×, 12500×, and 62500× using the method described in Example 5. Antibody titers were measured using indirect ELISA analysis. ELISA plates were prepared and coated with corresponding peptides in Tables 13 and 14.

[0076]Cytokine release assay. Splenocyte cytokine levels in the medium of cultured cells were examined by ELISA to measure levels of cytokines TNF-α, γ-IFN, IL-4, IL-10 and IL-12. Briefly, spleens were aseptically collected from mice and dissociated to harvest splenocytes. Cells were resuspended in RPMI, and mononuclear cells were counted in a hemocytomer. Splenocytes were diluted to an optimal density and cultured in 5×10⁶ cells/well in 6-well plates in 5 ml of RPMI. Immunogen inducer peptides, e.g., Gag24-N and Gag24-C, etc., were respectively added to splenocytes in triplicate. On the second day after the addition of immunogens, supernatants were collected. The amounts of TNF-α, γ-IFN, IL-4, IL-10 and IL-12 produced by splenocyte CD8+ T cells were assayed using quantitative ELISA assay kits (Invitrogen BioSource) by following the manufacturer's protocol with slight modifications.

TABLE-US-00013 TABLE 13 SEQ Length Antigen peptide Peptide sequence ID NO. (a.a.) gp120-41-N1 VEKLWVTVYYGVPVWK 218 16 gp120-41-N2 KVVKIEPLGVAPTKCK 219 16 gp120-41-N3 APTKCKRRVVQREKR 220 15 gp120-41-C1 QARVWRYLKDQQLL 221 14 gp120-41-C2 GIWGCSGKLICCTTAV 222 17 P gp120-41-C3 AVPWNASSWSNKLDR 223 15

TABLE-US-00014 TABLE 14 SEQ Length Antigen peptide Peptide sequence ID NO. (a.a.) HIV-Tat-N PVDPRLEPWHKPGSQPRTAC 224 20 HIV-Tat-C QLRGDPTGPKESKKKVERET 225 20 HIV-Tat-M SYGRKKRRQRRRAPQDSETH 226 20 HIV-Rev-N QSNPYPKPEGYRRVRRNRRR 227 20 HIV-Rev-C NCSESGGTSGTQRVGNPLEK 228 20 HIV-Nef-n1-N SKLKKGWPTVRQRMDRTE 229 18 HIV-Nef-n1-C TQGYFPDWQNYTPGPGIR 230 18 HIV-Nef-c1-N VDPEQVEKANEGDNN 231 15 HIV-Nef-c1-M ISQHGMDDPEKEVLM 232 15 HIV-Nef-c1-C QHIAREKHPEYYKDCLGLEK 233 20 HIV-Gag24-N PEFHMVDRDELKGIGMTN 234 18 HIV-Gag24-C RMYSPTMTNNPPIPV 235 15

[0077]Spleen lymphoid cell proliferation CMI assay. Cell proliferation ELISA BrdU (colorimetric) assays for CMI reactions were performed. The steps for culturing splenocytes were similar to those used in cytokine release assay except that cells were cultured in 96-well plates. Briefly, immunogens or antigen peptides, e g., Gag24-N, Gag24-C, etc., were respectively added to cell culture on day-2 to stimulate cell proliferation. ConA (10 μg/ml), as a positive control, was added to stimulate cell2s for one day. Cells were pulse-labeled with BrdU on day-3 at 37° C. for 12-24 hr. Only proliferating cells incorporated BrdU into their DNA. Cells were fixed with FixDenat solution. The FixDenat solution also denatured the genomic DNA, exposing the incorporated BrdU to immunodetection. The BrdU label in the DNA was located with a peroxidase-conjugated anti-BrdU antibody (anti-BrdU-POD). The bound anti-BrdU-POD was quantitated with a peroxidase substrate TMB by measuring absorbance at OD650 using ELISA plate reader.

[0078]Table 15 shows Gag24-specific antibodies titers in immunized mouse Sera The antibody titer assay indicated that Gag24-N antigenic determinant or epitope peptide was stronger in inducing antibody reactions than the Gag24-C epitope peptide. The ability of Gag24-N peptide in inducing antibody titers was, however, weak when it was in the fusion protein PE(ΔIII)-Gag24-K3. Once the Gag24-N antigenic determinant peptide was modified to include polypeptide gp120 and gp41 α-helix to form fusion protein PE(ΔIII)-Gag24-gp120-41-K3, its ability of inducing Gag24-N-specific IgG increased significantly. Thus, the peptide Gag24-N could elicit a Th2 cell-dependent, antigenic determinant (or epitope)-specific humoral immune response.

TABLE-US-00015 TABLE 15** Antibody IgG IgA IgE Mouse No. Vaccine Coated Antigen #1 #2 #3 Pl* #1 #1 #2 #3 Pl #1 #1 #2 #3 Pl #1 PE(ΔIII)- Gag24-N 10 10 10 1 3 3 3 1 1 1 3 1 Gag24-K3 Gag24-C 3 3 3 1 10 3 3 1 1 3 1 1 PE(ΔIII)- Gag24-N 100 100 100 1 3 3 3 1 3 3 3 1 Gag24- Gag24-C 3 3 10 1 3 3 3 1 1 1 1 1 gp120-41- K3 *The term "Pl" denotes "placebo," in which mice were injected with PBS/adjuvant. **The data represented here were endpoints of serum semi-log serial dilution. The experiments were repeated in three mice per immunogen inducer group.

[0079]The results from the cell proliferation CMI assay indicated that both fusion proteins, PE(ΔIII)-Gag24-K3 and PE(ΔIII)-Gag24-gp120-41-K3, after being injected into mice could induce cell-mediated immune response to Gag24 antigen (Table 16). The Gag24 antigen, however, had a low efficacy in inducing cell-mediated immune responses in the fusion protein PE(ΔIII)-Gag24-K3. Once it was modified to fuse with gp120 C1 and C5 domains and gp41 α-helix to form PE(ΔIII)-Gag24-gp120 C1-C5-gp41-K3, Gag24 antigen's ability in inducing cell-mediated immune responses significantly increased. Thus, PE(ΔIII)-Gag24-gp120 C1-C5-gp41-K3 is much stronger than PE(ΔIII)-Gag24-K3 in inducing Gag24-specific, cell-mediated responses and cytokine release. As shown in FIG. 6, chimeric polypeptide HIV PE(ΔIII)-gp120 C1-C5-gp41 600 can act as a building unit for connecting other HIV antigenic determinant peptide 602 and thereby markedly enhance cell-mediated immune responses of the inserted HIV antigenic peptide 602, such as Gag24. Chimeric polypeptide HIV PE(ΔIII)-gp120 C1-C5-gp41 600 includes PE(ΔIII) 604, HIV gp120 C1-C5-gp41 608, an endoplasmic reticulum retention sequence 610, with a bridge or linker 606 in-between. The fusion of an HIV antigenic determinant polypeptide 602 with weak CMI responses and HIV gp120 C1-C5-gp41 608 results in a chimeric PE-HIV fusion protein 620 that exhibits enhanced CMI responses specific to the antigenic determinant 602.

TABLE-US-00016 TABLE 16 CMI assay on immunized mouse splenocytes* Animal Group Vaccine Placebo Fusion Protein Vaccine Immunogen Inducer (n = 3) (n = 3) PE(ΔIII)-Gag24-K3 Gag24-N 0.32 0.19 Gag24-C 0.65 0.26 ConA 1.56 0.24 PE(ΔIII)-Gag24-gp120-41-K3 Gag24-N 1.31 0.19 Gag24-C 1.25 0.26 ConA 1.42 0.20

[0080]The data from the cytokine induction test (Table 17) showed that both vaccines PE(ΔIII)-Gag24-K3 and PE(ΔIII)-Gag24-gp120-41-K3 after being injected into mice did not induce detectable IL-4, which indicated that they would be better vaccine candidates for HIV. Of the two fusion protein vaccines, PE(ΔIII)-Gag24-gp120-41-K3 was much more effective than PE(ΔIII)-Gag24-K3 in inducing splenocytes to produce large amounts of IL-10 and IL-12. A comparison of Gag24-N and Gag24-C peptides in their cytokine inducing effects showed that in the PE(ΔIII)-Gag24-K3 vaccine group, Gag24-C peptide appeared to have a stronger T-cell-dependent epitope effect than Gag24-N peptide. In the PE(ΔIII)-Gag24-gp120-41-K3 vaccine group, both Gag24-N and Gag24-C peptides were capable of inducing cell-mediated immune responses and had no difference in their effects in inducing cytokine release. The data indicated that fusion protein PE(ΔIII)-Gag24-K3 had a low efficacy in inducing Gag24-specific cytokine release; however, fusion protein PE(ΔIII)-Gag24-gp120-41-K3 had a strong effect in eliciting Gag24-specific immune responses.

TABLE-US-00017 TABLE 17 Cytokines release assay* Cytokine TNF-α γ-IFN IL-4 IL-10 IL-12 Immunogen Animal Group Vaccine Inducer Vac** Plac** Vac** Plac** Vac** Plac** Vac** Plac** Vac** Plac** PE(ΔIII)- Gag24-N 8.6 3.4 6.1 5.6 0 0 2 0 53.6 31.0 Gag24- Gag24-C 12.9 9.6 12.8 12.8 0 0 12 8 36.5 35.6 K3 ConA 143.1 53.4 206.4 81.2 9 0 9 4 60.0 36.1 PE(ΔIII)- Gag24-N 70.0 3.4 17.5 5.6 0 0 136 0 87.6 31.0 Gag24- Gag24-C 86.6 9.6 24.4 12.8 0 0 144 8 67.2 35.6 gp120- ConA 314 53.4 765.4 81.2 13.8 0 196 4 87.6 36.1 41-K3 *The standard deviation is not shown here. The unit of the concentration is in pg/ml; n = 3 in both vaccine and placebo groups. **The term "Vac" refers to "Vaccine," and "Plac" refers to "Placebo."

[0081]The data from the immunized mice Sera ELISA test indicated that the farthest C-terminal portion of Nef-C antigen determinant peptide had the strongest antibody reaction (Table 18). Based on the antibody-inducing reactions by fusion protein vaccine PE(ΔIII)-Nef-K3, it was concluded that peptide Nef-C--C was one of the Th2 cell-dependent, HIV antigenic determinant sites (Table 18).

TABLE-US-00018 TABLE 18** Nef-specific antibody titers in HIV-Nef fusion protein-immunized mice Antibody IgG IgA IgE Mouse No. Vaccine Coated Antigen #1 #2 #3 P1* #1 #2 #3 P1 #1 #2 #3 P1 PE(ΔIII)- Nef-N-N 30 30 30 1 3 10 10 10 1 1 1 1 Nef-N- Nef-N-C 30 30 30 1 30 30 30 1 1 3 1 1 K3 PE(ΔIII)- Nef-C-N 30 30 30 1 10 10 10 1 1 1 1 1 Nef-C- Nef-C-M 30 30 30 1 10 10 10 1 1 1 1 1 K3 Nef-C-C 10⁴ 10³ 300 1 10² 10 10 1 1 1 1 1 *The term "P1" denotes "placebo #1." Mice in the placebo group were injected with PBS/adjuvant. **The data represent the vaccinated mouse serum titers/placebo serum titers of the endpoints of serum semi-log serial dilution. The experiments were repeated in three mice per each vaccinated group.

TABLE-US-00019 TABLE 19 CMI assay on HIV-Nef fusion protein-immunized mouse splenocytes* Animal Groups Immunogen Vaccine Placebo Vaccine Inducer (n = 3) (n = 3) PE(ΔIII)-Nef-N-K3 Nef-N-N 0.7 0.3 and Nef-N-C 0.8 0.2 PE(ΔIII)-Nef-C-K3 Nef-C-N 0.5 0.3 Nef-C-M 0.7 0.2 Nef-C-C 1.0 0.3 ConA 0.7 0.3

[0082]The cell-mediated immune responses in immunized mice indicated that both PE(ΔIII)-Nef-N--K3 and PE(ΔIII)-Nef-C--K3 had Nef-antigen-specific CMI reactions, and among which the Nef-N--C and Nef-C--C antigenic determinant portions induced stronger CMI responses (Table 19).

TABLE-US-00020 TABLE 20* Cytokines release assay from HIV-Nef fusion protein-immunized mouse splenocytes Cytokine TNF-α γ-IFN IL-4 IL-10 IL-12 Immunogen Animal Group Vaccine Inducer Vac*** Plc*** Vac Plc Vac Plc Vac Plc Vac Plc Mixture of Nef-N-N 46.8 2.1 38.9 9.2 0 0 PE(ΔIII)- Nef-N-C 42.8 4.9 34.4 26.7 0 0 HIV-Nef-N- Nef-C-N 65.2 12.7 61.7 30.9 0 0 143 0 55 38 K3 Nef-C-M 47.8 6.9 27.0 17.4 0 0 and Nef-C-C 51.2 2.9 25.1 10.6 0 0 PE(ΔIII)- ConA 80.8 14.7 84.6 15.3 0 0 135 0 69 50 HIV-Nef-C- BK** 61.2 10.5 46.3 22.5 K3 *conc. (pg/ml); n = 3 in both vaccine and placebo group. **BK refers to "Blank," in which no immunogen inducer was added into splenocytes. ***Vac refers to "Vaccine," and Plc refers to "Placebo."

[0083]The data from the cytokine induction test showed that Nef fusion proteins did not induce detectable Nef-specific IL-4, which was an indication that Nef fusion proteins would be better vaccine candidates against HIV. The data also indicated that a vaccine composition comprising fusion proteins PE(ΔIII)-HIV-Nef-N--K3 and PE(ΔIII)-HIV Nef-C could stimulate splenocytes to produce a higher amount of IL-10 (Table 20).

[0084]The results from serum antibody assay indicated that the N-terminal portion of HIV-1 Tat protein could induce remarkable antibody responses, while the mid-segment and C-terminal portions were weak in inducing antibody responses. Thus, the N-terminal portion of HIV Tat protein could elicit Th2 cell-dependent, antigenic determinant-specific humoral immunity (Table 21).

[0085]The results from the cell immune response indicated that PE(ΔIII)-Tat-K3 could induce cell mediated immune responses to all Tat protein segments, among which the N-terminus of Tat protein was stronger than mid- and C-terminal segments in inducing cell immune responses (Table 22).

TABLE-US-00021 TABLE 21 Antibody titers in HIV-Tat-fusion protein-immunized mice Antibody IgG IgA IgE Coated Mouse No. Vaccine Antigen #1 #2 #3 #4 P1 #1 #2 #3 #4 P1 #1 #2 #3 #4 P1 PE Tat-N 10³ 10³ 10³ 10³ 1 10³ 10³ 10³ 10³ 1 ? ? 10 10 1 (ΔIII)- Tat-M 10 10 10 10 1 3 3 3 3 1 3 3 3 3 1 HIV- Tat-C 10 1 3 3 1 3 3 3 3 1 1 1 1 1 1 Tat- K3 *The term "P1" refers to "placebo #1." The data represent vaccinated mouse serum titers/placebo serum titers of the endpoints of serum semi-log serial dilution.

TABLE-US-00022 TABLE 22 CMI assay on HIV-Tat-fusion protein immunized mouse splenocytes* Animal Groups Immunogen Vaccine Placebo Vaccine Inducer (n = 4) (n = 4) PE(ΔIII)-HIV- Tat-N 1.1 0.2 Tat-K3 Tat-M 1.0 0.2 Tat-C 0.5 0.2 ConA 1.3 0.2

[0086]The results from cytokine release assay indicated that HIV Tat fusion protein was not able to induce a detectable level of Tat-specific IL-4. Its effects in inducing γ-IFN and TNF-α release were not obvious, either. The fusion protein PE-(ΔIII)-Tat-K3, however, was able to stimulate splenocytes to produce Tat-N terminus-specific IL-12. Thus, PE-(ΔIII)-Tat-K3 was still effective in inducing a cell immune response that was specific to the N terminal portion of Tat and therefore the N-terminus of Tat could evoke Th1 cell-dependent, antigenic determinant-specific cell mediated immune responses in the PE delivery system of the present invention (Table 23).

[0087]The antibody data from PE(III)-Rev-K3 fusion protein-immunized mice model indicated that the antibody responses to HIV-Rev antigenic determinant peptide was not strong enough to confirm the locus of Th2 cell-dependent, antigenic determinant in Rev protein (Table 24).

TABLE-US-00023 TABLE 23 Cytokines release assay on HIV-Tat fusion protein immunized-mouse splenocytes* Cytokine TNF-α γ-IF2N IL-4 IL-10 IL-12 Immunogen Animal Group Vaccine Inducer Vac Plac Vac Plac Vac Plac Vac Plac Vac Plac PE(ΔIII)- Tat-N 10 5 0 18 0 1 6 0 41 10 HIV- Tat-M 18 5 7 4 -- -- -- -- -- -- Tat-K3 Tat-C 32 9 21 17 -- -- -- -- -- -- BK 10 18 2 27 -- -- -- -- -- -- ConA 155 27 243 81 33 0 48 25 55 27 *conc. (pg/ml); n = 3 in both vaccine (Vac) and placebo (Plac) group.

TABLE-US-00024 TABLE 24 Antibody titers in HIV-Rev fusion protein-immunized mice Antibody IgG IgA IgE Mouse No. Vaccine Immunogen #1 #2 #3 #4 P1* #1 #2 #3 #4 P1 #1 #2 #3 #4 P1 PE(III)- HIV- 10 10 10 3 1 3 10 3 10 1 1 1 1 1 1 HIV- Rev-N Rev- HIV- 3 3 1 1 1 3 3 3 3 1 1 1 1 1 1 K3 Rev-C *The term "P1" denotes "placebo #1." Mice in the placebo group were injected with PBS/adjuvant. The data represent vaccinated mouse serum titers/placebo serum titers of the endpoints of serum semi-log serial dilution.

[0088]The data from cell immune responses in Table 25 indicated that PE(III)-Rev-K3 fusion protein vaccine was not able to induce cell immune response to Rev antigen. The cytokine release inducing test also gave the similar result. It showed no obvious effects in inducing TNF-α and γ-IFN release (Table 26). Thus, the fusion protein vaccine PE(ΔIII)-Rev-K3 might not be a good component in an HIV vaccine. However, whether Rev is able to elicit a CMI response under the condition of fusion with gp120-41 in a PE delivery system remains to be investigated. A plasmid pPE(ΔIII)-HIV-Rev-gp120-41-K3 was constructed for investigation.

TABLE-US-00025 TABLE 25 CMI assay on HIV-Rev fusion protein-immunized mouse Splenocytes Animal Groups Immunogen Vaccine Placebo Vaccine Inducer (n = 3) (n = 3) PE(ΔIII)-HIV-Rev-K3 Rev-N 0.39 0.25 Rev-C 0.39 0.24 ConA 0.40 0.24

TABLE-US-00026 TABLE 26 Cytokines release assay on HIV-Rev fusion protein-immunized mouse Splenocytes* Cytokine TNF-α γ-IFN Animal Group Immunogen Vaccine Placebo Vaccine Placebo Vaccine Inducer (n = 4) (n = 4) (n = 4) (n = 4) PE(ΔIII)-HIV- Rev-N 29 3 6 4 Rev-K3 Rev-C 30 3 7 5 BK 38 4 9 7 ConA 50 6 16 5 *conc. (pg/ml)

[0089]The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.

[0090]The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in tie art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.

[0091]Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is "prior art" to the invention described herein. The application claims the priority of U.S. provisional application No. 61/025,094, filed Jan. 31, 2008, which is incorporated herein in its entirety by reference. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.

Sequence CWU 1

252116PRTHuman immunodeficiency virus 1Val Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys1 5 10 15225PRTHuman immunodeficiency virus 2Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Cys Lys1 5 10 15Arg Arg Val Val Gln Arg Glu Lys Arg20 25335PRTHuman immunodeficiency virus 30Cys Thr Arg Pro Ser Asn Asn Thr Arg Lys Gly Ile His Met Gly Pro1 5 10 15Gly Gly Ala Phe Tyr Thr Thr Gly Gln Ile Ile Arg Asn Ile Arg Gln20 25 30Ala His Cys35435PRTHuman immunodeficiency virus 4Cys Thr Arg Pro Asn Asn Asn Thr Arg Arg Ser Ile His Ile Glu Pro1 5 10 15Glu Gly Ala Phe Tyr Thr Thr Gly Glu Ile Ile Gly Asp Ile Arg Gln20 25 30Ala His Cys35554PRTHuman immunodeficiency virus 5Gln Ala Arg Val Ile Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu1 5 10 15Leu Gly Ile Trp Gly Gly Ser Gly Lys Leu Ile Cys Cys Thr Thr Ala20 25 30Val Pro Trp Asn Ser Ser Trp Ser Asn Lys Leu Asp Arg Ile Trp Asn35 40 45Asn Met Thr Trp Leu Glu50682PRTArtificial sequenceChimera HIV gP120 V3-V3 6Ile Gly Cys Thr Arg Pro Ser Asn Asn Thr Arg Lys Gly Ile His Met1 5 10 15Gly Pro Gly Gly Ala Phe Tyr Thr Thr Gly Gln Ile Ile Arg Asn Ile20 25 30Arg Gln Ala His Cys Gly Leu Leu Gly Gly Cys Thr Arg Pro Asn Asn35 40 45Asn Thr Arg Arg Ser Ile His Ile Glu Pro Glu Gly Ala Phe Tyr Thr50 55 60Thr Gly Glu Ile Ile Gly Asp Ile Arg Gln Ala His Cys Gly Leu Gly65 70 75 80Leu Glu7100PRTArtificial sequenceChimera HIV gP120-C1-C5-gp41 7Val Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys1 5 10 15Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Cys Lys20 25 30Arg Arg Val Val Gln Arg Glu Lys Arg Gly Gly Gly Gly Gly Gln Ala35 40 45Arg Val Ile Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly50 55 60Ile Trp Gly Gly Ser Gly Lys Leu Ile Cys Cys Thr Thr Ala Val Pro65 70 75 80Trp Asn Ser Ser Trp Ser Asn Lys Leu Asp Arg Ile Trp Asn Asn Met85 90 95Thr Trp Leu Glu1008264DNAArtificial sequencechimeric gene HIV gP120 V3-V3 8gaattccata tggtcgacat cggttgcacc cgtccgagca acaacacccg taaaggtatc 60cacatgggcc cgggtggtgc tttctacacc accggtcaga tcatccgtaa catccgtcag 120gctcactgtg gtctgctggg tggttgtacc cgtccgaaca acaacacccg tcgtagcatc 180cacatcgaac cggaaggtgc tttctacacc accggtgaaa tcatcggtga catccgtcag 240gctcactgtg gcctgggtct cgag 2649318DNAArtificial sequencechimeric gene HIV gP120 C1-C5-gp41 9gaattccata tggtcgacgt tgaaaaactg tgggttaccg tttactacgg tgttccggtt 60tggaaaaaag ttgttaaaat cgaaccgctg ggtgttgctc cgaccaaatg caaacgtcgt 120gttgttcagc gtgaaaagcg tggtggcggt ggcggtcaag ctcgtgttat cgctgttgaa 180cgttacctga aagaccagca gctgctgggt atctggggtg gtagcggtaa actgatctgc 240tgcaccaccg ctgttccgtg gaacagcagc tggagcaaca aactggaccg tatctggaac 300aacatgacct ggctcgag 3181040DNAArtificial sequenceForward primer F1 for chimeric gene HIV gP120 V3-V3 10catccgtaac atccgtcagg ctcactgtgg tctgctgggt 401139DNAArtificial sequenceForward primer F2 for chimeric gene HIV gP120 V3-V3 11gctttctaca ccaccggtca gatcatccgt aacatccgt 391242DNAArtificial sequenceForward primer F3 for chimeric gene HIV gP120 V3-V3 12aaaggtatcc acatgggccc gggtggtgct ttctacacca cc 421339DNAArtificial sequenceForward primer F4 for chimeric gene HIV gP120 V3-V3 13acccgtccga gcaacaacac ccgtaaaggt atccacatg 391446DNAArtificial sequenceForward primer F5 for chimeric gene HIV gP120 V3-V3 14cccgaattcc atatggtcga catcggttgc acccgtccga gcaaca 461539DNAArtificial sequenceReverse primer R1 for chimeric gene HIV gP120 V3-V3 15gttgttgttc ggacgggtac aaccacccag cagaccaca 391639DNAArtificial sequenceReverse primer R2 for chimeric gene HIV gP120 V3-V3 16ttcgatgtgg atgctacgac gggtgttgtt gttaggacg 391739DNAArtificial sequenceReverse primer R3 for chimeric gene HIV gP120 V3-V3 17ggtggtgtag aaagcacctt ccggttcgat gtggatgct 391839DNAArtificial sequenceReverse primer R4 for chimeric gene HIV gP120 V3-V3 18acggatatca ccgatgattt caccggtggt gtagaaagc 391964DNAArtificial sequenceReverse primer R5 for chimeric gene HIV gP120 V3-V3 19aaaaaaattc ccattatttt tctcgagacc caggccacag tgagcctgac ggatgtcacc 60gatg 642044DNAArtificial sequenceForward primer F1 for chimeric gene HIV gP120 C1-C4-gp41 20gttgctccga ccaaatgcaa acgtcgtgtt gttcagcgtg aaaa 442147DNAArtificial sequenceForward primer F2 for chimeric gene HIV gP120 C1-C4-gp41 21aaaaaagttg ttaaaatcga accgctgggt gttgctccga ccaaatg 472249DNAArtificial sequenceForward primer F3 for chimeric gene HIV gP120 C1-C4-gp41 22gtgggttacc gtttactacg gtgttccggt ttggaaaaaa gttgttaaa 492349DNAArtificial sequenceForward primer F4 for chimeric gene HIV gP120 C1-C4-gp41 23cccgaattcc atatggtcga cgttgaaaaa ctgtgggtta ccgtttact 492454DNAArtificial sequenceReverse primer R1 for chimeric gene HIV gP120 C1-C4-gp41 24aacagcgata acacgagctt gaccgccacc gccaccacgc ttttcacgct gaac 542546DNAArtificial sequenceReverse primer R2 for chimeric gene HIV gP120 C1-C4-gp41 25cagcagctgc tggtctttca ggtaacgttc aacagcgata acacga 462644DNAArtificial sequenceReverse primer R3 for chimeric gene HIV gP120 C1-C4-gp41 26gatcagttta ccgctaccac cccagatacc cagcagctgc tggt 442745DNAArtificial sequenceReverse primer R4 for chimeric gene HIV gP120 C1-C4-gp41 27gctgttccac ggaacagcgg tggtgcagca gatcagttta ccgct 452844DNAArtificial sequenceReverse primer R5 for chimeric gene HIV gP120 C1-C4-gp41 28ccagatacgg tccagtttgt tgctccagct gctgttccac ggaa 442939DNAArtificial sequenceReverse primer R6 for chimeric gene HIV gP120 C1-C4-gp41 29aaactcgagc caggtcatgt tgttccagat acggtccag 39304PRTArtificial sequenceCarboxyl terminal moiety KDEL 30Lys Asp Glu Leu13198PRTArtificial sequenceChimera for HIV subtype A gp120 C1-C5-gp41 31Ala Glu Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Ile Trp Lys1 5 10 15Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Arg20 25 30Arg Arg Val Val Glu Arg Glu Lys Arg Gly Gly Gly Gly Gly Gln Ala35 40 45Arg Val Leu Ala Val Glu Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly50 55 60Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Pro Thr Asn Val Pro Trp65 70 75 80Asn Ser Ser Trp Ser Asn Lys Ser Leu Asp Glu Ile Trp Glu Asn Met85 90 95Thr Trp3298PRTArtificial sequenceChimera for HIV subtype B gp120 C1-C5-gp41 32Thr Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys1 5 10 15Lys Val Val Lys Ile Glu Pro Leu Gly Ile Ala Pro Thr Lys Ala Lys20 25 30Arg Arg Val Val Gln Arg Glu Lys Arg Gly Gly Gly Gly Gly Gln Ala35 40 45Arg Val Leu Ala Leu Glu Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly50 55 60Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Thr Val Pro Trp65 70 75 80Asn Ala Ser Trp Ser Asn Arg Ser Leu Asp Glu Ile Trp Asp Asn Met85 90 95Thr Trp33100PRTArtificial sequenceChimera for HIV subtype C gp120 C1-C5-gp41 33Met Gly Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys1 5 10 15Lys Tyr Lys Val Val Glu Ile Lys Pro Leu Gly Val Ala Pro Thr Lys20 25 30Pro Lys Arg Arg Val Val Glu Arg Glu Lys Arg Gly Gly Gly Gly Gly35 40 45Gln Thr Arg Val Leu Ala Ile Glu Arg His Leu Arg Asp Gln Gln Leu50 55 60Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val65 70 75 80Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser Gln Glu Glu Ile Trp Asp85 90 95Asn Met Thr Trp1003498PRTArtificial sequenceChimera for HIV subtype D gp120 C1-C5-gp41 34Ala Asp Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys1 5 10 15Lys Val Val Gln Ile Glu Pro Leu Gly Val Ala Pro Thr Arg Ala Lys20 25 30Arg Arg Val Val Glu Arg Glu Lys Arg Gly Gly Gly Gly Gly Gln Ala35 40 45Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly50 55 60Ile Trp Gly Cys Ser Gly Lys His Ile Cys Thr Thr Asn Val Pro Trp65 70 75 80Asn Ser Ser Trp Ser Asn Arg Ser Leu Asn Glu Ile Trp Gln Asn Met85 90 95Thr Trp3598PRTArtificial sequenceChimera for HIV subtype E gp120 C1-C5-gp41 35Ser Xaa Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Arg1 5 10 15Lys Val Val Gln Ile Glu Pro Leu Gly Ile Ala Pro Thr Arg Pro Lys20 25 30Arg Arg Val Val Glu Arg Glu Lys Arg Gly Gly Gly Gly Gly Gln Ala35 40 45Arg Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Lys Phe Leu Gly50 55 60Leu Trp Gly Cys Ser Gly Lys Ile Ile Cys Thr Thr Ala Val Pro Trp65 70 75 80Asn Ser Ser Trp Ser Asn Arg Ser Phe Glu Glu Ile Trp Asn Asn Met85 90 95Thr Trp3698PRTArtificial sequenceChimera for HIV subtype F gp120 C1-C5-gp41 36Ala Asp Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys1 5 10 15Lys Val Val Glu Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys20 25 30Arg Gln Val Val Gln Arg Glu Lys Arg Gly Gly Gly Gly Gly Gln Ala35 40 45Arg Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly50 55 60Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Asn Val Pro Trp65 70 75 80Asn Ser Ser Trp Ser Asn Lys Ser Gln Glu Glu Ile Trp Asn Asn Met85 90 95Thr Trp37102PRTArtificial sequenceChimera for HIV subtype G gp120 C1-C5-gp41 37Ala Ser Asn Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp1 5 10 15Glu Asp Ala Lys Lys Val Val Lys Ile Lys Pro Leu Gly Val Ala Pro20 25 30Thr Lys Ala Arg Arg Arg Val Val Gly Arg Glu Lys Arg Gly Gly Gly35 40 45Gly Gly Gln Ala Arg Val Leu Ala Leu Glu Arg Tyr Leu Arg Asp Gln50 55 60Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr65 70 75 80Asn Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Thr Tyr Asn Asp Ile85 90 95Trp Asp Asn Met Thr Trp10038102PRTArtificial sequenceChimera for HIV subtype H gp120 C1-C5-gp41 38Val Val Gly Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp1 5 10 15Lys Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Glu Ala20 25 30Arg Arg Arg Val Val Glu Arg Glu Lys Arg Gly Gly Gly Gly Gly Gln35 40 45Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu50 55 60Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Asn Val Pro65 70 75 80Trp Asn Ser Ser Trp Ser Asn Lys Ser Leu Asp Glu Ile Trp Asp Asn85 90 95Met Thr Trp Met Glu Trp1003999PRTArtificial sequenceChimera for HIV subtype J gp120 C1-C5-gp41 39Ala Lys Glu Asp Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp1 5 10 15Lys Lys Val Val Glu Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala20 25 30Lys Arg Arg Val Val Glu Arg Glu Lys Arg Gly Gly Gly Gly Gly Gln35 40 45Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu50 55 60Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Asn Val Pro65 70 75 80Trp Asn Ala Ser Trp Ser Asn Lys Ser Tyr Glu Asp Ile Trp Glu Asn85 90 95Met Thr Trp4099PRTArtificial sequenceChimera for HIV subtype K gp120 C1-C5-gp41 40Ala Ala Asn Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp1 5 10 15Lys Lys Val Val Gln Ile Glu Pro Leu Gly Ile Ala Pro Thr Arg Ala20 25 30Arg Arg Arg Val Val Gln Arg Glu Lys Arg Gly Gly Gly Gly Gly Arg35 40 45Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Arg Asp Gln Gln Leu Leu50 55 60Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Asn Val Pro65 70 75 80Trp Asn Ser Ser Trp Ser Asn Lys Ser Gln Ser Glu Ile Trp Glu Asn85 90 95Met Thr Trp41294DNAArtificial sequenceChimeric gene for HIV subtype A gp120 C1-C5-gp41 41gctgaaaacc tgtgggttac cgtttactac ggtgttccaa tctggaaaaa agttgttaaa 60atcgaaccac tgggtgttgc tccaaccaaa gctcgtcgtc gtgttgttga acgtgaaaaa 120cgtggtggtg gtggtggtca ggctcgtgtt ctggctgttg aacgttacct gcgtgaccag 180cagctgctgg gtatctgggg ttgttccggt aaactgatct gtccaaccaa cgttccatgg 240aactcctcct ggtccaacaa atccctggac gaaatctggg aaaacatgac ctgg 29442294DNAArtificial sequenceChimeric gene for HIV subtype B gp120 C1-C5-gp41 42accgaaaaac tgtgggttac cgtttactac ggtgttccag tgtggaaaaa agttgttaaa 60atcgaaccac tgggtatcgc tccaaccaaa gctaaacgtc gtgttgttca gcgtgaaaaa 120cgtggtggtg gtggtggtca ggctcgtgtt ctggctctgg aacgttacct gcgtgaccag 180cagctgctgg gtatctgggg ttgttccggt aaactgatct gtaccaccac cgttccatgg 240aacgcttcct ggtccaaccg ttccctggac gaaatctggg acaacatgac ctgg 29443300DNAArtificial sequenceChimeric gene for HIV subtype C gp120 C1-C5-gp41 43atgggtaacc tgtgggttac cgtttactac ggtgttccag tgtggaaaaa atacaaagtt 60gttgaaatca aaccactggg tgttgctcca accaaaccaa aacgtcgtgt tgttgaacgt 120gaaaaacgtg gtggtggtgg tggtcagacc cgtgttctgg ctatcgaacg tcacctgcgt 180gaccagcagc tgctgggtat ctggggttgt tccggtaaac tgatctgtac caccgctgtt 240ccatggaact cctcctggtc caacaaatcc caggaagaaa tctgggacaa catgacctgg 30044294DNAArtificial sequenceChimeric gene for HIV subtype D gp120 C1-C5-gp41 44gctgacaacc tgtgggttac cgtttactac ggtgttccag tgtggaaaaa agttgttcag 60atcgaaccac tgggtgttgc tccaacccgt gctaaacgtc gtgttgttga acgtgaaaaa 120cgtggtggtg gtggtggtca ggctcgtatt ctggctgttg aacgttacct gaaagaccag 180cagctgctgg gtatctgggg ttgttccggt aaacacatct gtaccaccaa cgttccatgg 240aactcctcct ggtccaaccg ttccctgaac gaaatctggc agaacatgac ctgg 29445294DNAArtificial sequenceChimeric gene for HIV subtype E gp120 C1-C5-gp41 45tccaacaacc tgtgggttac cgtttactac ggtgttccag tgtggcgtaa agttgttcag 60atcgaaccac tgggtatcgc tccaacccgt ccaaaacgtc gtgttgttga acgtgaaaaa 120cgtggtggtg gtggtggtca ggctcgtgtt ctggctgttg aacgttacct gaaagaccag 180aaattcctgg gtctgtgggg ttgttccggt aaaatcatct gtaccaccgc tgttccatgg 240aactcctcct ggtccaaccg ttccttcgaa gaaatctgga acaacatgac ctgg 29446294DNAArtificial sequenceChimeric gene for HIV subtype F gp120 C1-C5-gp41 46gctgacaacc tgtgggttac cgtttactac ggtgttccag tgtggaaaaa agttgttgaa 60atcgaaccac tgggtgttgc tccaaccaaa gctaaacgtc aggttgttca gcgtgaaaaa 120cgtggtggtg gtggtggtca ggctcgtgtt ctggctgttg aacgttacct gaaagaccag 180cagctgctgg gtatctgggg ttgttccggt aaactgatct gtaccaccaa cgttccatgg 240aactcctcct ggtccaacaa atcccaggaa gaaatctgga acaacatgac ctgg 29447306DNAArtificial sequenceChimeric gene for HIV subtype G gp120 C1-C5-gp41 47gcttccaaca acctgtgggt taccgtttac tacggtgttc cagtgtggga agacgctaaa 60aaagttgtta aaatcaaacc actgggtgtt gctccaacca aagctcgtcg tcgtgttgtt 120ggtcgtgaaa aacgtggtgg tggtggtggt caggctcgtg ttctggctct ggaacgttac 180ctgcgtgacc agcagctgct gggtatctgg ggttgttccg gtaaactgat ctgtaccacc 240aacgttccat ggaacgcttc ctggtccaac aaaacctaca acgacatctg ggacaacatg 300acctgg 30648306DNAArtificial sequenceChimeric gene for HIV subtype H gp120 C1-C5-gp41 48gttgttggta acctgtgggt taccgtttac tacggtgttc cagtgtggaa aaaagttgtt 60aaaatcgaac cactgggtgt tgctccaacc gaagctcgtc gtcgtgttgt tgaacgtgaa 120aaacgtggtg gtggtggtgg tcaggctcgt gttctggctg ttgaacgtta cctgaaagac 180cagcagctgc tgggtatctg gggttgttcc ggtaaactga tctgtaccac caacgttcca 240tggaactcct cctggtccaa

caaatccctg gacgaaatct gggacaacat gacctggatg 300gaatgg 30649297DNAArtificial sequenceChimeric gene for HIV subtype J gp120 C1-C5-gp41 49gctaaagaag acctgtgggt taccgtttac tacggtgttc cagtgtggaa aaaagttgtt 60gaaatcgaac cactgggtgt tgctccaacc aaagctaaac gtcgtgttgt tgaacgtgaa 120aaacgtggtg gtggtggtgg tcaggctcgt gttctggctg ttgaacgtta cctgaaagac 180cagcagctgc tgggtatctg gggttgttcc ggtaaactga tctgtaccac caacgttcca 240tggaacgctt cctggtccaa caaatcctac gaagacatct gggaaaacat gacctgg 29750297DNAArtificial sequenceChimeric gene for HIV subtype K gp120 C1-C5-gp41 50gctgctaaca acctgtgggt taccgtttac tacggtgttc cagtgtggaa aaaagttgtt 60cagatcgaac cactgggtat cgctccaacc cgtgctcgtc gtcgtgttgt tcagcgtgaa 120aaacgtggtg gtggtggtgg tcgtgctcgt gttctggctg ttgaacgtta cctgcgtgac 180cagcagctgc tgggtatctg gggttgttcc ggtaaactga tctgtaccac caacgttcca 240tggaactcct cctggtccaa caaatcccag tccgaaatct gggaaaacat gacctgg 2975145DNAArtificial sequenceForward primer F1 for chimeric gene HIV subtype A gp120 C1-C5-gp41 51gttgaacgtg aaaaacgtgg tggtggtggt ggtcaggctc gtgtt 455245DNAArtificial sequenceForward primer F2 for chimeric gene HIV subtype A gp120 C1-C5-gp41 52gttgctccaa ccaaagctcg tcgtcgtgtt gttgaacgtg aaaaa 455349DNAArtificial sequenceForward primer F3 for chimeric gene HIV subtype A gp120 C1-C5-gp41 53ctggaaaaaa gttgttaaaa tcgaaccact gggtgttgct ccaaccaaa 495446DNAArtificial sequenceForward primer F4 for chimeric gene HIV subtype A gp120 C1-C5-gp41 54gtgggttacc gtttactacg gtgttccaat ctggaaaaaa gttgtt 465547DNAArtificial sequenceForward primer F5 for chimeric gene HIV subtype A gp120 C1-C5-gp41 55cccgaattcc atatggtcga cgctgaaaac ctgtgggtta ccgttta 475647DNAArtificial sequenceReverse primer R1 for chimeric gene HIV subtype A gp120 C1-C5-gp41 56tgctggtcac gcaggtaacg ttcaacagcc agaacacgag cctgacc 475746DNAArtificial sequenceReverse primer R2 for chimeric gene HIV subtype A gp120 C1-C5-gp41 57tttaccggaa caaccccaga tacccagcag ctgctggtca cgcagg 465846DNAArtificial sequenceReverse primer R3 for chimeric gene HIV subtype A gp120 C1-C5-gp41 58agttccatgg aacgttggtt ggacagatca gtttaccgga acaacc 465948DNAArtificial sequenceReverse primer R4 for chimeric gene HIV subtype A gp120 C1-C5-gp41 59tttcgtccag ggatttgttg gaccaggagg agttccatgg aacgttgg 486049DNAArtificial sequenceReverse primer R5 for chimeric gene HIV subtype A gp120 C1-C5-gp41 60atttttctcg agccaggtca tgttttccca gatttcgtcc agggatttg 496145DNAArtificial sequenceForward primer F1 for chimeric gene HIV subtype B gp120 C1-C5-gp41 61gttcagcgtg aaaaacgtgg tggtggtggt ggtcaggctc gtgtt 456246DNAArtificial sequenceForward primer F2 for chimeric gene HIV subtype B gp120 C1-C5-gp41 62atcgctccaa ccaaagctaa acgtcgtgtt gttcagcgtg aaaaac 466349DNAArtificial sequenceForward primer F3 for chimeric gene HIV subtype B gp120 C1-C5-gp41 63gtggaaaaaa gttgttaaaa tcgaaccact gggtatcgct ccaaccaaa 496446DNAArtificial sequenceForward primer F4 for chimeric gene HIV subtype B gp120 C1-C5-gp41 64gtgggttacc gtttactacg gtgttccagt gtggaaaaaa gttgtt 466547DNAArtificial sequenceForward primer F5 for chimeric gene HIV subtype B gp120 C1-C5-gp41 65cccgaattcc atatggtcga caccgaaaaa ctgtgggtta ccgttta 476647DNAArtificial sequenceReverse primer R1 for chimeric gene HIV subtype B gp120 C1-C5-gp41 66tgctggtcac gcaggtaacg ttccagagcc agaacacgag cctgacc 476746DNAArtificial sequenceReverse primer R2 for chimeric gene HIV subtype B gp120 C1-C5-gp41 67tttaccggaa caaccccaga tacccagcag ctgctggtca cgcagg 466848DNAArtificial sequenceReverse primer R3 for chimeric gene HIV subtype B gp120 C1-C5-gp41 68agcgttccat ggaacggtgg tggtacagat cagtttaccg gaacaacc 486944DNAArtificial sequenceReverse primer R4 for chimeric gene HIV subtype B gp120 C1-C5-gp41 69atttcgtcca gggaacggtt ggaccaggaa gcgttccatg gaac 447047DNAArtificial sequenceReverse primer R5 for chimeric gene HIV subtype B gp120 C1-C5-gp41 70atttttctcg agccaggtca tgttgtccca gatttcgtcc agggaac 477145DNAArtificial sequenceForward primer F1 for chimeric gene HIV subtype C gp120 C1-C5-gp41 71gttgaacgtg aaaaacgtgg tggtggtggt ggtcagaccc gtgtt 457245DNAArtificial sequenceForward primer F2 for chimeric gene HIV subtype C gp120 C1-C5-gp41 72gttgctccaa ccaaaccaaa acgtcgtgtt gttgaacgtg aaaaa 457352DNAArtificial sequenceForward primer F3 for chimeric gene HIV subtype C gp120 C1-C5-gp41 73aaaaaataca aagttgttga aatcaaacca ctgggtgttg ctccaaccaa ac 527451DNAArtificial sequenceForward primer F4 for chimeric gene HIV subtype C gp120 C1-C5-gp41 74ctgtgggtta ccgtttacta cggtgttcca gtgtggaaaa aatacaaagt t 517545DNAArtificial sequenceForward primer F5 for chimeric gene HIV subtype C gp120 C1-C5-gp41 75cccgaattcc atatggtcga catgggtaac ctgtgggtta ccgtt 457647DNAArtificial sequenceReverse primer R1 for chimeric gene HIV subtype C gp120 C1-C5-gp41 76tgctggtcac gcaggtgacg ttcgatagcc agaacacggg tctgacc 477746DNAArtificial sequenceReverse primer R2 for chimeric gene HIV subtype C gp120 C1-C5-gp41 77tttaccggaa caaccccaga tacccagcag ctgctggtca cgcagg 467846DNAArtificial sequenceReverse primer R3 for chimeric gene HIV subtype C gp120 C1-C5-gp41 78agttccatgg aacagcggtg gtacagatca gtttaccgga acaacc 467948DNAArtificial sequenceReverse primer R4 for chimeric gene HIV subtype C gp120 C1-C5-gp41 79tttcttcctg ggatttgttg gaccaggagg agttccatgg aacagcgg 488049DNAArtificial sequenceReverse primer R5 for chimeric gene HIV subtype C gp120 C1-C5-gp41 80atttttctcg agccaggtca tgttgtccca gatttcttcc tgggatttg 498145DNAArtificial sequenceForward primer F1 for chimeric gene HIV subtype D gp120 C1-C5-gp41 81gttgaacgtg aaaaacgtgg tggtggtggt ggtcaggctc gtatt 458245DNAArtificial sequenceForward primer F2 for chimeric gene HIV subtype D gp120 C1-C5-gp41 82gttgctccaa cccgtgctaa acgtcgtgtt gttgaacgtg aaaaa 458349DNAArtificial sequenceForward primer F3 for chimeric gene HIV subtype D gp120 C1-C5-gp41 83gtggaaaaaa gttgttcaga tcgaaccact gggtgttgct ccaacccgt 498446DNAArtificial sequenceForward primer F4 for chimeric gene HIV subtype D gp120 C1-C5-gp41 84gtgggttacc gtttactacg gtgttccagt gtggaaaaaa gttgtt 468547DNAArtificial sequenceForward primer F5 for chimeric gene HIV subtype D gp120 C1-C5-gp41 85cccgaattcc atatggtcga cgctgacaac ctgtgggtta ccgttta 478647DNAArtificial sequenceReverse primer R1 for chimeric gene HIV subtype D gp120 C1-C5-gp41 86tgctggtctt tcaggtaacg ttcaacagcc agaatacgag cctgacc 478746DNAArtificial sequenceReverse primer R2 for chimeric gene HIV subtype D gp120 C1-C5-gp41 87tttaccggaa caaccccaga tacccagcag ctgctggtct ttcagg 468846DNAArtificial sequenceReverse primer R3 for chimeric gene HIV subtype D gp120 C1-C5-gp41 88agttccatgg aacgttggtg gtacagatgt gtttaccgga acaacc 468948DNAArtificial sequenceReverse primer R4 for chimeric gene HIV subtype D gp120 C1-C5-gp41 89tttcgttcag ggaacggttg gaccaggagg agttccatgg aacgttgg 489049DNAArtificial sequenceReverse primer R5 for chimeric gene HIV subtype D gp120 C1-C5-gp41 90atttttctcg agccaggtca tgttctgcca gatttcgttc agggaacgg 499145DNAArtificial sequenceForward primer F1 for chimeric gene HIV subtype E gp120 C1-C5-gp41 91gttgaacgtg aaaaacgtgg tggtggtggt ggtcaggctc gtgtt 459246DNAArtificial sequenceForward primer F2 for chimeric gene HIV subtype E gp120 C1-C5-gp41 92atcgctccaa cccgtccaaa acgtcgtgtt gttgaacgtg aaaaac 469349DNAArtificial sequenceForward primer F3 for chimeric gene HIV subtype E gp120 C1-C5-gp41 93gtggcgtaaa gttgttcaga tcgaaccact gggtatcgct ccaacccgt 499446DNAArtificial sequenceForward primer F4 for chimeric gene HIV subtype E gp120 C1-C5-gp41 94gtgggttacc gtttactacg gtgttccagt gtggcgtaaa gttgtt 469544DNAArtificial sequenceForward primer F5 for chimeric gene HIV subtype E gp120 C1-C5-gp41 95cccgaattcc atatggtcga ctccaacctg tgggttaccg ttta 449648DNAArtificial sequenceReverse primer R1 for chimeric gene HIV subtype E gp120 C1-C5-gp41 96tttctggtct ttcaggtaac gttcaacagc cagaacacga gcctgacc 489746DNAArtificial sequenceReverse primer R2 for chimeric gene HIV subtype E gp120 C1-C5-gp41 97tttaccggaa caaccccaca gacccaggaa tttctggtct ttcagg 469846DNAArtificial sequenceReverse primer R3 for chimeric gene HIV subtype E gp120 C1-C5-gp41 98agttccatgg aacagcggtg gtacagatga ttttaccgga acaacc 469948DNAArtificial sequenceReverse primer R4 for chimeric gene HIV subtype E gp120 C1-C5-gp41 99tttcttcgaa ggaacggttg gaccaggagg agttccatgg aacagcgg 4810049DNAArtificial sequenceReverse primer R5 for chimeric gene HIV subtype E gp120 C1-C5-gp41 100atttttctcg agccaggtca tgttgttcca gatttcttcg aaggaacgg 4910145DNAArtificial sequenceForward primer F1 for chimeric gene HIV subtype F gp120 C1-C5-gp41 101gttcagcgtg aaaaacgtgg tggtggtggt ggtcaggctc gtgtt 4510245DNAArtificial sequenceForward primer F2 for chimeric gene HIV subtype F gp120 C1-C5-gp41 102gttgctccaa ccaaagctaa acgtcaggtt gttcagcgtg aaaaa 4510349DNAArtificial sequenceForward primer F3 for chimeric gene HIV subtype F gp120 C1-C5-gp41 103gtggaaaaaa gttgttgaaa tcgaaccact gggtgttgct ccaaccaaa 4910446DNAArtificial sequenceForward primer F4 for chimeric gene HIV subtype F gp120 C1-C5-gp41 104gtgggttacc gtttactacg gtgttccagt gtggaaaaaa gttgtt 4610547DNAArtificial sequenceForward primer F5 for chimeric gene HIV subtype F gp120 C1-C5-gp41 105cccgaattcc atatggtcga cgctgacaac ctgtgggtta ccgttta 4710647DNAArtificial sequenceReverse primer R1 for chimeric gene HIV subtype F gp120 C1-C5-gp41 106tgctggtctt tcaggtaacg ttcaacagcc agaacacgag cctgacc 4710746DNAArtificial sequenceReverse primer R2 for chimeric gene HIV subtype F gp120 C1-C5-gp41 107tttaccggaa caaccccaga tacccagcag ctgctggtct ttcagg 4610846DNAArtificial sequenceReverse primer R3 for chimeric gene HIV subtype F gp120 C1-C5-gp41 108agttccatgg aacgttggtg gtacagatca gtttaccgga acaacc 4610948DNAArtificial sequenceReverse primer R4 for chimeric gene HIV subtype F gp120 C1-C5-gp41 109tttcttcctg ggatttgttg gaccaggagg agttccatgg aacgttgg 4811049DNAArtificial sequenceReverse primer R5 for chimeric gene HIV subtype F gp120 C1-C5-gp41 110atttttctcg agccaggtca tgttgttcca gatttcttcc tgggatttg 4911145DNAArtificial sequenceForward primer F1 for chimeric gene HIV subtype G gp120 C1-C5-gp41 111gttggtcgtg aaaaacgtgg tggtggtggt ggtcaggctc gtgtt 4511245DNAArtificial sequenceForward primer F2 for chimeric gene HIV subtype G gp120 C1-C5-gp41 112gttgctccaa ccaaagctcg tcgtcgtgtt gttggtcgtg aaaaa 4511351DNAArtificial sequenceForward primer F3 for chimeric gene HIV subtype G gp120 C1-C5-gp41 113gacgctaaaa aagttgttaa aatcaaacca ctgggtgttg ctccaaccaa a 5111452DNAArtificial sequenceForward primer F4 for chimeric gene HIV subtype G gp120 C1-C5-gp41 114gtgggttacc gtttactacg gtgttccagt gtgggaagac gctaaaaaag tt 5211550DNAArtificial sequenceForward primer F5 for chimeric gene HIV subtype G gp120 C1-C5-gp41 115cccgaattcc atatggtcga cgcttccaac aacctgtggg ttaccgttta 5011647DNAArtificial sequenceReverse primer R1 for chimeric gene HIV subtype G gp120 C1-C5-gp41 116tgctggtcac gcaggtaacg ttccagagcc agaacacgag cctgacc 4711746DNAArtificial sequenceReverse primer R2 for chimeric gene HIV subtype G gp120 C1-C5-gp41 117tttaccggaa caaccccaga tacccagcag ctgctggtca cgcagg 4611848DNAArtificial sequenceReverse primer R3 for chimeric gene HIV subtype G gp120 C1-C5-gp41 118agcgttccat ggaacgttgg tggtacagat cagtttaccg gaacaacc 4811944DNAArtificial sequenceReverse primer R4 for chimeric gene HIV subtype G gp120 C1-C5-gp41 119atgtcgttgt aggttttgtt ggaccaggaa gcgttccatg gaac 4412046DNAArtificial sequenceReverse primer R5 for chimeric gene HIV subtype G gp120 C1-C5-gp41 120tttctcgagc caggtcatgt tgtcccagat gtcgttgtag gttttg 4612145DNAArtificial sequenceForward primer F1 for chimeric gene HIV subtype H gp120 C1-C5-gp41 121gttgaacgtg aaaaacgtgg tggtggtggt ggtcaggctc gtgtt 4512245DNAArtificial sequenceForward primer F2 for chimeric gene HIV subtype H gp120 C1-C5-gp41 122gttgctccaa ccgaagctcg tcgtcgtgtt gttgaacgtg aaaaa 4512349DNAArtificial sequenceForward primer F3 for chimeric gene HIV subtype H gp120 C1-C5-gp41 123gtggaaaaaa gttgttaaaa tcgaaccact gggtgttgct ccaaccgaa 4912448DNAArtificial sequenceForward primer F4 for chimeric gene HIV subtype H gp120 C1-C5-gp41 124ctgtgggtta ccgtttacta cggtgttcca gtgtggaaaa aagttgtt 4812548DNAArtificial sequenceForward primer F5 for chimeric gene HIV subtype H gp120 C1-C5-gp41 125cccgaattcc atatggtcga cgttgttggt aacctgtggg ttaccgtt 4812647DNAArtificial sequenceReverse primer R1 for chimeric gene HIV subtype H gp120 C1-C5-gp41 126tgctggtctt tcaggtaacg ttcaacagcc agaacacgag cctgacc 4712746DNAArtificial sequenceReverse primer R2 for chimeric gene HIV subtype H gp120 C1-C5-gp41 127tttaccggaa caaccccaga tacccagcag ctgctggtct ttcagg 4612846DNAArtificial sequenceReverse primer R3 for chimeric gene HIV subtype H gp120 C1-C5-gp41 128agttccatgg aacgttggtg gtacagatca gtttaccgga acaacc 4612948DNAArtificial sequenceReverse primer R4 for chimeric gene HIV subtype H gp120 C1-C5-gp41 129tttcgtccag ggatttgttg gaccaggagg agttccatgg aacgttgg 4813055DNAArtificial sequenceReverse primer R5 for chimeric gene HIV subtype H gp120 C1-C5-gp41 130tttctcgagc cattccatcc aggtcatgtt gtcccagatt tcgtccaggg atttg 5513145DNAArtificial sequenceForward primer F1 for chimeric gene HIV subtype J gp120 C1-C5-gp41 131gttgaacgtg aaaaacgtgg tggtggtggt ggtcaggctc gtgtt 4513245DNAArtificial sequenceForward primer F2 for chimeric gene HIV subtype J gp120 C1-C5-gp41 132gttgctccaa ccaaagctaa acgtcgtgtt gttgaacgtg aaaaa 4513349DNAArtificial sequenceForward primer F3 for chimeric gene HIV subtype J gp120 C1-C5-gp41 133gtggaaaaaa gttgttgaaa tcgaaccact gggtgttgct ccaaccaaa 4913448DNAArtificial sequenceForward primer F4 for chimeric gene HIV subtype J gp120 C1-C5-gp41 134ctgtgggtta ccgtttacta cggtgttcca gtgtggaaaa aagttgtt 4813548DNAArtificial sequenceForward primer F5 for chimeric gene HIV subtype J gp120 C1-C5-gp41 135cccgaattcc atatggtcga cgctaaagaa gacctgtggg ttaccgtt 4813647DNAArtificial sequenceReverse primer R1 for chimeric gene HIV subtype J gp120 C1-C5-gp41 136tgctggtctt tcaggtaacg ttcaacagcc agaacacgag cctgacc 4713746DNAArtificial sequenceReverse primer R2 for chimeric gene HIV subtype J gp120 C1-C5-gp41 137tttaccggaa caaccccaga tacccagcag ctgctggtct ttcagg 4613848DNAArtificial sequenceReverse primer R3 for chimeric gene HIV subtype J gp120 C1-C5-gp41 138agcgttccat ggaacgttgg tggtacagat cagtttaccg gaacaacc 4813944DNAArtificial sequenceReverse primer R4 for chimeric gene HIV subtype J gp120 C1-C5-gp41 139atgtcttcgt aggatttgtt ggaccaggaa gcgttccatg gaac 4414046DNAArtificial sequenceReverse primer R5 for chimeric gene HIV subtype J gp120 C1-C5-gp41 140tttctcgagc caggtcatgt tttcccagat gtcttcgtag gatttg 4614145DNAArtificial sequenceForward primer F1 for chimeric gene HIV subtype K gp120 C1-C5-gp41 141gttcagcgtg aaaaacgtgg tggtggtggt ggtcgtgctc gtgtt 4514246DNAArtificial sequenceForward primer F2 for chimeric gene HIV subtype K gp120 C1-C5-gp41 142atcgctccaa cccgtgctcg tcgtcgtgtt gttcagcgtg aaaaac 4614349DNAArtificial sequenceForward primer F3 for chimeric gene HIV subtype K gp120 C1-C5-gp41 143gtggaaaaaa gttgttcaga tcgaaccact gggtatcgct ccaacccgt 4914448DNAArtificial sequenceForward primer F4 for chimeric gene HIV subtype K gp120 C1-C5-gp41 144ctgtgggtta ccgtttacta cggtgttcca gtgtggaaaa aagttgtt 4814548DNAArtificial sequenceForward primer F5 for chimeric gene HIV subtype K gp120 C1-C5-gp41 145cccgaattcc atatggtcga cgctgctaac

aacctgtggg ttaccgtt 4814647DNAArtificial sequenceReverse primer R1 for chimeric gene HIV subtype K gp120 C1-C5-gp41 146tgctggtcac gcaggtaacg ttcaacagcc agaacacgag cacgacc 4714746DNAArtificial sequenceReverse primer R2 for chimeric gene HIV subtype K gp120 C1-C5-gp41 147tttaccggaa caaccccaga tacccagcag ctgctggtca cgcagg 4614846DNAArtificial sequenceReverse primer R3 for chimeric gene HIV subtype K gp120 C1-C5-gp41 148agttccatgg aacgttggtg gtacagatca gtttaccgga acaacc 4614948DNAArtificial sequenceReverse primer R4 for chimeric gene HIV subtype K gp120 C1-C5-gp41 149tttcggactg ggatttgttg gaccaggagg agttccatgg aacgttgg 4815046DNAArtificial sequenceReverse primer R5 for chimeric gene HIV subtype K gp120 C1-C5-gp41 150tttctcgagc caggtcatgt tttcccagat ttcggactgg gatttg 4615171PRTHuman immunodeficiency virus 151Val Asp Arg Asp Glu Leu Lys Gly Ile Gly Met Thr Asn Asn Pro Pro1 5 10 15Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn20 25 30Lys Ile Val Arg Met Tyr Ser Pro Thr Met Thr Asn Asn Pro Pro Ile35 40 45Pro Val Gly Glu Ile Tyr Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile50 55 60Val Arg Met Tyr Ser Pro Thr65 70152132PRTHuman immunodeficiency virus 152Pro Thr Val Arg Gln Arg Met Asp Arg Thr Glu Pro Ala Ala Glu Gly1 5 10 15Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser20 25 30Ser Asn Thr Ala Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln35 40 45Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg50 55 60Pro Met Thr Tyr Lys Ala Ala Val Asp Ile Ser His Phe Leu Lys Glu65 70 75 80Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser Gln Lys Arg Gln Glu Ile85 90 95Leu Asp Leu Trp Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln100 105 110Asn Tyr Thr Pro Gly Pro Gly Ile Arg Tyr Pro Leu Thr Phe Gly Trp115 120 125Cys Phe Lys Leu13015366PRTHuman immunodeficiency virus 153Phe Leu Lys Val Pro Val Asp Pro Glu Gln Val Glu Lys Ala Asn Glu1 5 10 15Gly Asp Asn Asn Cys Leu Leu His Pro Ile Ser Gln His Gly Met Asp20 25 30Asp Pro Glu Lys Glu Val Leu Met Trp Lys Phe Asp Ser Arg Leu Ala35 40 45Phe Gln His Ile Ala Arg Glu Lys His Pro Glu Tyr Tyr Lys Asp Cys50 55 60Leu Gly65154109PRTHuman immunodeficiency virus 154Arg Asp Glu Leu Lys Gly Ile Gly Met Glu Pro Val Asp Pro Arg Leu1 5 10 15Glu Pro Trp Lys His Pro Gly Ser Gln Pro Arg Thr Ala Cys Asn Asn20 25 30Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Pro Val Cys Phe Ile Ser35 40 45Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg50 55 60Arg Ala Pro Gln Asp Ser Glu Thr His Gln Val Ser Leu Ser Lys Gln65 70 75 80Pro Thr Ser Gln Leu Arg Gly Asp Pro Thr Gly Pro Lys Glu Ser Lys85 90 95Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro Asn Val100 10515588PRTHuman immunodeficiency virus 155Leu Leu Ala Val Arg Ile Ile Lys Thr Leu Tyr Gln Ser Asn Pro Tyr1 5 10 15Pro Lys Pro Glu Gly Tyr Arg Arg Val Arg Arg Asn Arg Arg Arg Arg20 25 30Trp Arg Ala Arg Gln Arg Gln Ile His Ser Ile Ser Glu Arg Ile Leu35 40 45Ile Thr Cys Leu Gly Arg Pro Thr Glu Pro Val Pro Leu Gln Leu Pro50 55 60Pro Ile Glu Arg Leu Asn Ile Asn Cys Ser Glu Ser Gly Gly Thr Ser65 70 75 80Gly Thr Gln Arg Val Gly Asn Pro85156207DNAHuman immunodeficiency virus 156cgtgacgaac tgaaaggtat cggtatgacc aacaacccgc cgatcccggt tggtgaaatc 60tacaaacgtt ggatcatcct gggtctgaac aaaatcgttc gtatgtacag cccgaccatg 120accaacaacc cgccaattcc agtaggcgag atttaccgtt ggatcatcct gggtctgaac 180aaaatcgttc gcatgtacag cccgact 207157396DNAHuman immunodeficiency virus 157ccgaccgttc gtcagcgtat ggaccgtacc gaaccggctg ctgagggtgt tggtgctgtt 60agccgtgacc tggaaaaaca cggtgctatc acctcttcta acaccgctgc taccaacgct 120gactgcgctt ggctggaagc tcaggaagaa gaagaagttg gttttccggt tcgtccgcag 180gttccgctgc gtccgatgac ctacaaagct gctgttgaca tcagccactt cctgaaagaa 240aaaggcggcc tggaaggtct gatctacagc cagaaacgtc aggaaatcct ggacctgtgg 300atctaccaca cccagggcta cttcccggac tggcagaact acaccccggg tccgggtatc 360cgttacccgc tgaccttcgg ctggtgtttc aaactg 396158198DNAHuman immunodeficiency virus 158ttcctgaaag ttccggttga cccggaacag gttgaaaaag ctaacgaagg tgataacaac 60tgcctgctgc acccgatcag ccagcacggt atggacgacc cggaaaaaga agttctgatg 120tggaaattcg acagccgtct ggctttccag cacatcgctc gtgaaaaaca cccggaatac 180tacaaagact gcctgggt 198159327DNAHuman immunodeficiency virus 159cgtgacgaac tgaaaggtat cggtatggaa ccggttgacc cgcgtctgga accgtggaaa 60cacccgggta gccagccgcg taccgcttgc aacaactgct actgcaaaaa atgttgtttc 120cactgcccgg tttgctttat ctctaaaggt ctgggtatca gctacggtcg taaaaagcgt 180cgccagcgcc gccgcgctcc gcaggactcc gaaacccacc aggttagcct gagcaagcaa 240ccgaccagcc agctgcgtgg tgacccgacc ggtccgaaag aaagcaaaaa aaaagttgaa 300cgtgaaaccg aaaccgaccc gaacgtt 327160264DNAHuman immunodeficiency virus 160ctgctggctg ttcgtatcat caaaaccctg taccagtcta acccgtaccc gaaaccggaa 60ggttaccgtc gtgtccgtcg taaccgccgc cgccgctggc gtgctcgtca gcgtcagatc 120cactccatca gcgaacgtat cctgatcacc tgcctgggtc gtccgaccga accggttccg 180ctgcaactgc cgccgatcga acgtctgaac atcaactgta gcgaaagcgg tggtaccagc 240ggtacccagc gtgttggtaa cccg 26416139DNAArtificial sequenceForward Primer F1 for gag24 161tggatcatcc tgggtctgaa caaaatcgtt cgtatgtac 3916239DNAArtificial sequenceForward Primer F2 for gag24 162ccggttggtg aaatctacaa acgttggatc atcctgggt 3916341DNAArtificial sequenceForward Primer F3 for gag24 163tcggtatgac caacaacccg ccgatcccgg ttggtgaaat c 4116456DNAArtificial sequenceForward Primer F4 for gag24 164cccgaattcc atatggtcga ccgtgacgaa ctgaaaggta tcggtatgac caacaa 5616539DNAArtificial sequenceReverse Primer R1 for gag24 165cgggttgttg gtcatggtcg ggctgtacat acgaacgat 3916639DNAArtificial sequenceReverse Primer R2 for gag24 166gtaaatctcg cctactggaa ttggcgggtt gttgttcat 3916740DNAArtificial sequenceReverse Primer R3 for gag24 167tgttcagacc caggatgatc caacggtaaa tctcgcctac 4016869DNAArtificial sequenceReverse Primer R4 for gag24 168aaaaaaattc ccattatttt tctcgagagt cgggctgtac atgcgaacga ttttgttcag 60acccaggat 6916939DNAArtificial sequenceForward Primer F1 for nef-N terminus 169gcttggctgg aagctcagga agaagaagaa gttggtttt 3917040DNAArtificial sequenceForward Primer F2 for nef-N terminus 170caccgctgct accaacgctg actgcgcttg gctggaagct 4017142DNAArtificial sequenceForward Primer F3 for nef-N terminus 171aaacacggtg ctatcacctc ttctaacacc gctgctacca ac 4217240DNAArtificial sequenceForward Primer F4 for nef-N terminus 172tggtgctgtt agccgtgacc tggaaaaaca cggtgctatc 4017342DNAArtificial sequenceForward Primer F5 for nef-N terminus 173cgtaccgaac cggctgctga gggtgttggt gctgttagcc gt 4217440DNAArtificial sequenceForward Primer F6 for nef-N terminus 174gaccgttcgt cagcgtatgg accgtaccga accggctgct 4017544DNAArtificial sequenceForward Primer F7 for nef-N terminus 175cccgaattcc atatggtcga cccgaccgtt cgtcagcgta tgga 4417642DNAArtificial sequenceReverser Primer R1 for nef-N terminus 176acgcagcgga acctgcggac gaaccggaaa accaacttct tc 4217739DNAArtificial sequenceReverser Primer R2 for nef-N terminus 177aacagcagct ttgtaggtca tcggacgcag cggaacccg 3917843DNAArtificial sequenceReverser Primer R3 for nef-N terminus 178ctttttcttt caggaagtgg ctgatgtcaa cagcagcttt gta 4317939DNAArtificial sequenceReverser Primer R4 for nef-N terminus 179gtagatcaga ccttccaggc cgcctttttc tttcaggaa 3918041DNAArtificial sequenceReverser Primer R5 for nef-N terminus 180tccaggattt cctgacgttt ctggctgtag atcagacctt c 4118140DNAArtificial sequenceReverser Primer R6 for nef-N terminus 181agccctgggt gtggtagatc cacaggtcca ggatttcctg 4018239DNAArtificial sequenceReverser Primer R7 for nef-N terminus 182gtagttctgc cagtccggga agtagccctg ggtgtggta 3918344DNAArtificial sequenceReverser Primer R8 for nef-N terminus 183agcgggtaac ggatacccgg acccggggtg tagttctgcc agtc 4418466DNAArtificial sequenceReverser Primer 9 for nef-N terminus 184aaaaaaattc ccattatttt tctcgagcag tttgaaacac cagccgaagg tcagcgggta 60acggat 6618539DNAArtificial sequenceForward Primer F1 for nef-C terminus 185ctgcacccga tcagccagca cggtatggac gacccggaa 3918639DNAArtificial sequenceForward Primer F2 for nef-C terminus 186aacgaaggtg ataacaactg cctgctgcac ccgatcagc 3918740DNAArtificial sequenceForward Primer F3 for nef-C terminus 187tgacccggaa caggttgaaa aagctaacga aggtgataac 4018854DNAArtificial sequenceForward Primer F4 for nef-C terminus 188cccgaattcc atatggtcga cttcctgaaa gttccggttg acccggaaca ggtt 5418939DNAArtificial sequenceReverse Primer R1 for nef-C terminus 189gaatttccac atcagaactt ctttttccgg gtcgtccat 3919040DNAArtificial sequenceReverse Primer R2 for nef-C terminus 190tgtgctggaa agccagacgg ctgtcgaatt tccacatcag 4019139DNAArtificial sequenceReverse Primer R3 for nef-C terminus 191ttccgggtgt ttttcacgag cgatgtgctg gaaagccag 3919263DNAArtificial sequenceReverse Primer R4 for nef-C terminus 192aaaaaaattc ccattatttt tctcgagacc caggcagtct ttgtagtatt ccgggtgttt 60ttc 6319340DNAArtificial sequenceForward Primer F1 for tat 193ctttatctct aaaggtctgg gtatcagcta cggtcgtaaa 4019446DNAArtificial sequenceForward Primer 2 for tat 194caaaaaatgt tgtttccact gcccggtttg ctttatctct aaaggt 4619539DNAArtificial sequenceForward Primer F3 for tat 195accgcttgca acaactgcta ctgcaaaaaa tgttgtttc 3919639DNAArtificial sequenceForward Primer F4 for tat 196aaacacccgg gtagccagcc gcgtaccgct tgcaacaac 3919743DNAArtificial sequenceForward Primer F5 for tat 197accggttgac ccgcgtctgg aaccgtggaa acacccgggt agc 4319839DNAArtificial sequenceForward Primer F6 for tat 198gaactgaaag gtatcggtat ggaaccggtt gacccgcgt 3919945DNAArtificial sequenceForward Primer F7 for tat 199cccgaattcc atatggtcga ccgtgacgaa ctgaaaggta tcggt 4520039DNAArtificial sequenceReverse Primer R1 for tat 200gagcgcggcg gcgctggcga cgctttttac gccgtagct 3920142DNAArtificial sequenceReverse Primer R2 for tat 201aacctggtgg gtttcggagt cctgcggagc gcggcggcgc tg 4220241DNAArtificial sequenceReverse Primer R3 for tat 202tggctggtcg gttgcttgct caggctaacc tggtgggttt c 4120340DNAArtificial sequenceReverse Primer R4 for tat 203gaccggtcgg gtcaccacgc agctggctgg tcggttgctt 4020439DNAArtificial sequenceReverse Primer 5 for tat 204aacttttttt ttgctttctt tcggaccggt cgggtcacc 3920539DNAArtificial sequenceReverse Primer R6 for tat 205cgggtcggtt tcggtttcac gttcaacttt ttttttgct 3920651DNAArtificial sequenceReverse Primer R7 for tat 206aaaaaaattc tcattatttt tctcgagaac gttcgggtcg gtttcggttt c 5120740DNAArtificial sequenceForward Primer F1 for rev 207tcagatccac tccatcagcg aacgtatcct gatcacctgc 4020848DNAArtificial sequenceForward Primer F2 for rev 208aaccgccgcc gccgctggcg tgctcgtcag cgtcagatcc actccatc 4820945DNAArtificial sequenceForward Primer F3 for rev 209aaaccggaag gttaccgtcg tgtccgtcgt aaccgccgcc gccgc 4521042DNAArtificial sequenceForward Primer F4 for rev 210accctgtacc agtctaaccc gtacccgaaa ccggaaggtt ac 4221139DNAArtificial sequenceForward Primer F5 for rev 211ctgctggctg ttcgtatcat caaaaccctg taccagtct 3921248DNAArtificial sequenceForward Primer F6 for rev 212ccccccgaat tccatatggt cgacctgctg gctgttcgta tcatcaaa 4821340DNAArtificial sequenceReverse Primer R1 for rev 213gaaccggttc ggtcggacga cccaggcagg tgatcaggat 4021439DNAArtificial sequenceReverse Primer R2 for rev 214ttcgatcggc ggcagttgca gcggaaccgg ttcggtcgg 3921539DNAArtificial sequenceReverse Primer R3 for rev 215ttcgctacag ttgatgttca gacgttcgat cggcggcag 3921639DNAArtificial sequenceReverse Primer R4 for rev 216ctgggtaccg ctggtaccac cgctttcgct acagttgat 3921759DNAArtificial sequenceReverse Primer R5 for rev 217aaaaaaattc ccattatttt tctcgagcgg gttaccaaca cgctgggtac cgctggtac 5921816PRTArtificial sequenceFusion protein gp120-41-N1 218Val Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys1 5 10 1521916PRTArtificial sequenceFusion protein gp120-41-N2 219Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Cys Lys1 5 10 1522015PRTArtificial sequenceFusion protein gp120-41-N3 220Ala Pro Thr Lys Cys Lys Arg Arg Val Val Gln Arg Glu Lys Arg1 5 10 1522114PRTArtificial sequenceFusion protein gp120-41-C1 221Gln Ala Arg Val Trp Arg Tyr Leu Lys Asp Gln Gln Leu Leu1 5 1022217PRTArtificial sequenceFusion protein gp120-41-C2 222Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Cys Thr Thr Ala Val1 5 10 15Pro22315PRTArtificial sequenceFusion protein gp120-41-C3 223Ala Val Pro Trp Asn Ala Ser Ser Trp Ser Asn Lys Leu Asp Arg1 5 10 1522420PRTHuman immunodeficiency virus 224Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro1 5 10 15Arg Thr Ala Cys2022520PRTHuman immunodeficiency virus 225Gln Leu Arg Gly Asp Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val1 5 10 15Glu Arg Glu Thr2022620PRTHuman immunodeficiency virus 226Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp1 5 10 15Ser Glu Thr His2022720PRTHuman immunodeficiency virus 227Gln Ser Asn Pro Tyr Pro Lys Pro Glu Gly Tyr Arg Arg Val Arg Arg1 5 10 15Asn Arg Arg Arg2022820PRTHuman immunodeficiency virus 228Asn Cys Ser Glu Ser Gly Gly Thr Ser Gly Thr Gln Arg Val Gly Asn1 5 10 15Pro Leu Glu Lys2022918PRTHuman immunodeficiency virus 229Ser Lys Leu Lys Lys Gly Trp Pro Thr Val Arg Gln Arg Met Asp Arg1 5 10 15Thr Glu23018PRTHuman immunodeficiency virus 230Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly1 5 10 15Ile Arg23115PRTHuman immunodeficiency virus 231Val Asp Pro Glu Gln Val Glu Lys Ala Asn Glu Gly Asp Asn Asn1 5 10 1523215PRTHuman immunodeficiency virus 232Ile Ser Gln His Gly Met Asp Asp Pro Glu Lys Glu Val Leu Met1 5 10 1523320PRTHuman immunodeficiency virus 233Gln His Ile Ala Arg Glu Lys His Pro Glu Tyr Tyr Lys Asp Cys Leu1 5 10 15Gly Leu Glu Lys2023418PRTHuman immunodeficiency virus 234Pro Glu Phe His Met Val Asp Arg Asp Glu Leu Lys Gly Ile Gly Met1 5 10 15Thr Asn23515PRTHuman immunodeficiency virus 235Arg Met Tyr Ser Pro Thr Met Thr Asn Asn Pro Pro Ile Pro Val1 5 10 152361563DNAArtificial sequenceFusion gene PE(delta III)-HIV-Gag24-K3 236atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atggccgaag aagctttcga cctctggaac gaatgcgcca

aagcctgcgt gctcgacctc 120aaggacggcg tgcgttccag ccgcatgagc gtcgacccgg ccatcgccga caccaacggc 180cagggcgtgc tgcactactc catggtcctg gagggcggca acgacgcgct caagctggcc 240atcgacaacg ccctcagcat caccagcgac ggcctgacca tccgcctcga aggcggcgtc 300gagccgaaca agccggtgcg ctacagctac acgcgccagg cgcgcggcag ttggtcgctg 360aactggctgg taccgatcgg ccacgagaag ccctcgaaca tcaaggtgtt catccacgaa 420ctgaacgccg gcaaccagct cagccacatg tcgccgatct acaccatcga gatgggcgac 480gagttgctgg cgaagctggc gcgcgatgcc accttcttcg tcagggcgca cgagagcaac 540gagatgcagc cgacgctcgc catcagccat gccggggtca gcgtggtcat ggcccagacc 600cagccgcgcc gggaaaagcg ctggagcgaa tgggccagcg gcaaggtgtt gtgcctgctc 660gacccgctgg acggggtcta caactacctc gcccagcaac gctgcaacct cgacgatacc 720tgggaaggca agatctaccg ggtgctcgcc ggcaacccgg cgaagcatga cctggacatc 780aaacccacgg tcatcagtca tcgcctgcac tttcccgagg gcggcagcct ggccgcgctg 840accgcgcacc aggcttgcca cctgccgctg gagactttca cccgtcatcg ccagccgcgc 900ggctgggaac aactggagca gtgcggctat ccggtgcagc ggctggtcgc cctctacctg 960gcggcgcggc tgtcgtggaa ccaggtcgac caggtgatcc gcaacgccct ggccagcccc 1020ggcagcggcg gcgacctggg cgaagcgatc cgcgagcagc cggagcaggc ccgtctggcc 1080ctgaccctgg ccgccgccga gagcgagcgc ttcgtccggc agggcaccgg caacgacgag 1140gccggcgcgg ccaacgccga cgtggtgagc ctgacctgcc cggtcgccgc cggtgaatgc 1200gcgggcccgg cggacagcgg cgacgccctg ctggagcgca actatcccac tggcgcggag 1260ttcctcggcg acggcggcga cgtcgaattc catatggtcg accgtgacga actgaaaggt 1320atcggtatga ccaacaaccc gccgatcccg gttggtgaaa tctacaaacg ttggatcatc 1380ctgggtctga acaaaatcgt tcgtatgtac agcccgacca tgaccaacaa cccgccaatt 1440ccagtaggcg agatttaccg ttggatcatc ctgggtctga acaaaatcgt tcgcatgtac 1500agcccgactc tcgagtacct caaaaaagac gaactgcgtg tagaactgaa agacgaactg 1560taa 1563237520PRTArtificial sequenceFusion protein PE(delta III)-HIV Gag24-K3 237Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys20 25 30Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg35 40 45Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu50 55 60His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala65 70 75 80Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu85 90 95Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg100 105 110Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His115 120 125Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly130 135 140Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp145 150 155 160Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala165 170 175His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly180 185 190Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp195 200 205Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp210 215 220Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr225 230 235 240Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His245 250 255Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro260 265 270Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu275 280 285Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln290 295 300Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu305 310 315 320Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala325 330 335Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu340 345 350Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser355 360 365Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala370 375 380Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys385 390 395 400Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro405 410 415Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe His Met420 425 430Val Asp Arg Asp Glu Leu Lys Gly Ile Gly Met Thr Asn Asn Pro Pro435 440 445Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn450 455 460Lys Ile Val Arg Met Tyr Ser Pro Thr Met Thr Asn Asn Pro Pro Ile465 470 475 480Pro Val Gly Glu Ile Tyr Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile485 490 495Val Arg Met Tyr Ser Pro Thr Leu Glu Tyr Leu Lys Lys Asp Glu Leu500 505 510Arg Val Glu Leu Lys Asp Glu Leu515 5202381863DNAArtificial sequenceFusion gene PE(delta III)-HIV Gag24-gp120-41-K3 238atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atggccgaag aagctttcga cctctggaac gaatgcgcca aagcctgcgt gctcgacctc 120aaggacggcg tgcgttccag ccgcatgagc gtcgacccgg ccatcgccga caccaacggc 180cagggcgtgc tgcactactc catggtcctg gagggcggca acgacgcgct caagctggcc 240atcgacaacg ccctcagcat caccagcgac ggcctgacca tccgcctcga aggcggcgtc 300gagccgaaca agccggtgcg ctacagctac acgcgccagg cgcgcggcag ttggtcgctg 360aactggctgg taccgatcgg ccacgagaag ccctcgaaca tcaaggtgtt catccacgaa 420ctgaacgccg gcaaccagct cagccacatg tcgccgatct acaccatcga gatgggcgac 480gagttgctgg cgaagctggc gcgcgatgcc accttcttcg tcagggcgca cgagagcaac 540gagatgcagc cgacgctcgc catcagccat gccggggtca gcgtggtcat ggcccagacc 600cagccgcgcc gggaaaagcg ctggagcgaa tgggccagcg gcaaggtgtt gtgcctgctc 660gacccgctgg acggggtcta caactacctc gcccagcaac gctgcaacct cgacgatacc 720tgggaaggca agatctaccg ggtgctcgcc ggcaacccgg cgaagcatga cctggacatc 780aaacccacgg tcatcagtca tcgcctgcac tttcccgagg gcggcagcct ggccgcgctg 840accgcgcacc aggcttgcca cctgccgctg gagactttca cccgtcatcg ccagccgcgc 900ggctgggaac aactggagca gtgcggctat ccggtgcagc ggctggtcgc cctctacctg 960gcggcgcggc tgtcgtggaa ccaggtcgac caggtgatcc gcaacgccct ggccagcccc 1020ggcagcggcg gcgacctggg cgaagcgatc cgcgagcagc cggagcaggc ccgtctggcc 1080ctgaccctgg ccgccgccga gagcgagcgc ttcgtccggc agggcaccgg caacgacgag 1140gccggcgcgg ccaacgccga cgtggtgagc ctgacctgcc cggtcgccgc cggtgaatgc 1200gcgggcccgg cggacagcgg cgacgccctg ctggagcgca actatcccac tggcgcggag 1260ttcctcggcg acggcggcga cgtcgaattc catatggtcg accgtgacga actgaaaggt 1320atcggtatga ccaacaaccc gccgatcccg gttggtgaaa tctacaaacg ttggatcatc 1380ctgggtctga acaaaatcgt tcgtatgtac agcccgacca tgaccaacaa cccgccaatt 1440ccagtaggcg agatttaccg ttggatcatc ctgggtctga acaaaatcgt tcgcatgtac 1500agcccgactc tcgacgttga aaaactgtgg gttaccgttt actacggtgt tccggtttgg 1560aaaaaagttg ttaaaatcga accgctgggt gttgctccga ccaaatgcaa acgtcgtgtt 1620gttcagcgtg aaaagcgtgg tggcggtggc ggtcaagctc gtgttatcgc tgttgaacgt 1680tacctgaaag accagcagct gctgggtatc tggggtggta gcggtaaact gatctgctgc 1740accaccgctg ttccgtggaa cagcagctgg agcaacaaac tggaccgtat ctggaacaac 1800atgacctggc tcgagtacct caaaaaagac gaactgcgtg tagaactgaa agacgaactg 1860taa 1863239620PRTArtificial sequenceFusion protein PE(delta III)-HIV Gag24-gp120-41-K3 239Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys20 25 30Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg35 40 45Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu50 55 60His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala65 70 75 80Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu85 90 95Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg100 105 110Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His115 120 125Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly130 135 140Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp145 150 155 160Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala165 170 175His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly180 185 190Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp195 200 205Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp210 215 220Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr225 230 235 240Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His245 250 255Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro260 265 270Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu275 280 285Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln290 295 300Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu305 310 315 320Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala325 330 335Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu340 345 350Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser355 360 365Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala370 375 380Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys385 390 395 400Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro405 410 415Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe His Met420 425 430Val Asp Arg Asp Glu Leu Lys Gly Ile Gly Met Thr Asn Asn Pro Pro435 440 445Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn450 455 460Lys Ile Val Arg Met Tyr Ser Pro Thr Met Thr Asn Asn Pro Pro Ile465 470 475 480Pro Val Gly Glu Ile Tyr Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile485 490 495Val Arg Met Tyr Ser Pro Thr Leu Asp Val Glu Lys Leu Trp Val Thr500 505 510Val Tyr Tyr Gly Val Pro Val Trp Lys Lys Val Val Lys Ile Glu Pro515 520 525Leu Gly Val Ala Pro Thr Lys Cys Lys Arg Arg Val Val Gln Arg Glu530 535 540Lys Arg Gly Gly Gly Gly Gly Gln Ala Arg Val Ile Ala Val Glu Arg545 550 555 560Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Gly Ser Gly Lys565 570 575Leu Ile Cys Cys Thr Thr Ala Val Pro Trp Asn Ser Ser Trp Ser Asn580 585 590Lys Leu Asp Arg Ile Trp Asn Asn Met Thr Trp Leu Glu Tyr Leu Lys595 600 605Lys Asp Glu Leu Arg Val Glu Leu Lys Asp Glu Leu610 615 6202401749DNAArtificial sequenceFusion gene PE(delta III)-HIV nef-N-K3 240atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atggccgaag aagctttcga cctctggaac gaatgcgcca aagcctgcgt gctcgacctc 120aaggacggcg tgcgttccag ccgcatgagc gtcgacccgg ccatcgccga caccaacggc 180cagggcgtgc tgcactactc catggtcctg gagggcggca acgacgcgct caagctggcc 240atcgacaacg ccctcagcat caccagcgac ggcctgacca tccgcctcga aggcggcgtc 300gagccgaaca agccggtgcg ctacagctac acgcgccagg cgcgcggcag ttggtcgctg 360aactggctgg taccgatcgg ccacgagaag ccctcgaaca tcaaggtgtt catccacgaa 420ctgaacgccg gcaaccagct cagccacatg tcgccgatct acaccatcga gatgggcgac 480gagttgctgg cgaagctggc gcgcgatgcc accttcttcg tcagggcgca cgagagcaac 540gagatgcagc cgacgctcgc catcagccat gccggggtca gcgtggtcat ggcccagacc 600cagccgcgcc gggaaaagcg ctggagcgaa tgggccagcg gcaaggtgtt gtgcctgctc 660gacccgctgg acggggtcta caactacctc gcccagcaac gctgcaacct cgacgatacc 720tgggaaggca agatctaccg ggtgctcgcc ggcaacccgg cgaagcatga cctggacatc 780aaacccacgg tcatcagtca tcgcctgcac tttcccgagg gcggcagcct ggccgcgctg 840accgcgcacc aggcttgcca cctgccgctg gagactttca cccgtcatcg ccagccgcgc 900ggctgggaac aactggagca gtgcggctat ccggtgcagc ggctggtcgc cctctacctg 960gcggcgcggc tgtcgtggaa ccaggtcgac caggtgatcc gcaacgccct ggccagcccc 1020ggcagcggcg gcgacctggg cgaagcgatc cgcgagcagc cggagcaggc ccgtctggcc 1080ctgaccctgg ccgccgccga gagcgagcgc ttcgtccggc agggcaccgg caacgacgag 1140gccggcgcgg ccaacgccga cgtggtgagc ctgacctgcc cggtcgccgc cggtgaatgc 1200gcgggcccgg cggacagcgg cgacgccctg ctggagcgca actatcccac tggcgcggag 1260ttcctcggcg acggcggcga cgtcgaattc catatggtcg acccgaccgt tcgtcagcgt 1320atggaccgta ccgaaccggc tgctgagggt gttggtgctg ttagccgtga cctggaaaaa 1380cacggtgcta tcacctcttc taacaccgct gctaccaacg ctgactgcgc ttggctggaa 1440gctcaggaag aagaagaagt tggttttccg gttcgtccgc aggttccgct gcgtccgatg 1500acctacaaag ctgctgttga catcagccac ttcctgaaag aaaaaggcgg cctggaaggt 1560ctgatctaca gccagaaacg tcaggaaatc ctggacctgt ggatctacca cacccagggc 1620tacttcccgg actggcagaa ctacaccccg ggtccgggta tccgttaccc gctgaccttc 1680ggctggtgtt tcaaactgct cgagtacctc aaaaaagacg aactgcgtgt agaactgaaa 1740gacgaactg 1749241583PRTArtificial sequencefusion protein PE(delta III)-HIV Nef-N-K3 241Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys20 25 30Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg35 40 45Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu50 55 60His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala65 70 75 80Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu85 90 95Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg100 105 110Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His115 120 125Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly130 135 140Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp145 150 155 160Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala165 170 175His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly180 185 190Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp195 200 205Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp210 215 220Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr225 230 235 240Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His245 250 255Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro260 265 270Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu275 280 285Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln290 295 300Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu305 310 315 320Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala325 330 335Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu340 345 350Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser355 360 365Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala370 375 380Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys385 390 395 400Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro405 410 415Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe His Met420 425 430Val Asp Pro Thr Val Arg Gln Arg Met Asp Arg Thr Glu Pro Ala Ala435 440 445Glu Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile450 455 460Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu465 470 475 480Ala Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro485 490 495Leu Arg Pro Met Thr Tyr Lys Ala Ala Val Asp Ile Ser His Phe Leu500 505 510Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser Gln Lys Arg Gln515 520 525Glu Ile Leu Asp Leu Trp Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp530

535 540Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg Tyr Pro Leu Thr Phe545 550 555 560Gly Trp Cys Phe Lys Leu Leu Glu Tyr Leu Lys Lys Asp Glu Leu Arg565 570 575Val Glu Leu Lys Asp Glu Leu5802421881DNAArtificial sequenceFusion gene PE(delta III)-HIV nef-C-K3 242atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atggccgaag aagctttcga cctctggaac gaatgcgcca aagcctgcgt gctcgacctc 120aaggacggcg tgcgttccag ccgcatgagc gtcgacccgg ccatcgccga caccaacggc 180cagggcgtgc tgcactactc catggtcctg gagggcggca acgacgcgct caagctggcc 240atcgacaacg ccctcagcat caccagcgac ggcctgacca tccgcctcga aggcggcgtc 300gagccgaaca agccggtgcg ctacagctac acgcgccagg cgcgcggcag ttggtcgctg 360aactggctgg taccgatcgg ccacgagaag ccctcgaaca tcaaggtgtt catccacgaa 420ctgaacgccg gcaaccagct cagccacatg tcgccgatct acaccatcga gatgggcgac 480gagttgctgg cgaagctggc gcgcgatgcc accttcttcg tcagggcgca cgagagcaac 540gagatgcagc cgacgctcgc catcagccat gccggggtca gcgtggtcat ggcccagacc 600cagccgcgcc gggaaaagcg ctggagcgaa tgggccagcg gcaaggtgtt gtgcctgctc 660gacccgctgg acggggtcta caactacctc gcccagcaac gctgcaacct cgacgatacc 720tgggaaggca agatctaccg ggtgctcgcc ggcaacccgg cgaagcatga cctggacatc 780aaacccacgg tcatcagtca tcgcctgcac tttcccgagg gcggcagcct ggccgcgctg 840accgcgcacc aggcttgcca cctgccgctg gagactttca cccgtcatcg ccagccgcgc 900ggctgggaac aactggagca gtgcggctat ccggtgcagc ggctggtcgc cctctacctg 960gcggcgcggc tgtcgtggaa ccaggtcgac caggtgatcc gcaacgccct ggccagcccc 1020ggcagcggcg gcgacctggg cgaagcgatc cgcgagcagc cggagcaggc ccgtctggcc 1080ctgaccctgg ccgccgccga gagcgagcgc ttcgtccggc agggcaccgg caacgacgag 1140gccggcgcgg ccaacgccga cgtggtgagc ctgacctgcc cggtcgccgc cggtgaatgc 1200gcgggcccgg cggacagcgg cgacgccctg ctggagcgca actatcccac tggcgcggag 1260ttcctcggcg acggcggcga cgtccgtcac cactttaccc cgagtgagcg tcaattgtgt 1320ttgtcgtcaa tccagactgc ctttaatcaa ggcgctggta cttgcatcct gtcagattct 1380gggcgtatca gttacactgt ggagtttagt ttgcctacgc atcatactgt gcgcctgatc 1440cgcgttacag caccaccgtc agcactcgac gcgaccgtct acaacggtag cagtaagtac 1500ggtgacacca gcactagcaa cgtgcgtggt gaccttcaag tgttagctca gaaggcagaa 1560cgtactctgc ctacctcctt caacttcggt gccatcaagg caactcgtgt tactgaattc 1620catatggtcg acttcctgaa agttccggtt gacccggaac aggttgaaaa agctaacgaa 1680ggtgataaca actgcctgct gcacccgatc agccagcacg gtatggacga cccggaaaaa 1740gaagttctga tgtggaaatt cgacagccgt ctggctttcc agcacatcgc tcgtgaaaaa 1800cacccggaat actacaaaga ctgcctgggt ctcgagtacc tcaaaaaaga cgaactgcgt 1860gtagaactga aagacgaact g 1881243627PRTArtificial sequenceFusion protein PE(delta III)-HIV Nef-C-K3 243Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys20 25 30Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg35 40 45Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu50 55 60His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala65 70 75 80Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu85 90 95Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg100 105 110Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His115 120 125Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly130 135 140Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp145 150 155 160Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala165 170 175His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly180 185 190Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp195 200 205Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp210 215 220Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr225 230 235 240Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His245 250 255Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro260 265 270Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu275 280 285Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln290 295 300Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu305 310 315 320Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala325 330 335Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu340 345 350Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser355 360 365Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala370 375 380Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys385 390 395 400Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro405 410 415Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Arg His His Phe420 425 430Thr Pro Ser Glu Arg Gln Leu Cys Leu Ser Ser Ile Gln Thr Ala Phe435 440 445Asn Gln Gly Ala Gly Thr Cys Ile Leu Ser Asp Ser Gly Arg Ile Ser450 455 460Tyr Thr Val Glu Phe Ser Leu Pro Thr His His Thr Val Arg Leu Ile465 470 475 480Arg Val Thr Ala Pro Pro Ser Ala Leu Asp Ala Thr Val Tyr Asn Gly485 490 495Ser Ser Lys Tyr Gly Asp Thr Ser Thr Ser Asn Val Arg Gly Asp Leu500 505 510Gln Val Leu Ala Gln Lys Ala Glu Arg Thr Leu Pro Thr Ser Phe Asn515 520 525Phe Gly Ala Ile Lys Ala Thr Arg Val Thr Glu Phe His Met Val Asp530 535 540Phe Leu Lys Val Pro Val Asp Pro Glu Gln Val Glu Lys Ala Asn Glu545 550 555 560Gly Asp Asn Asn Cys Leu Leu His Pro Ile Ser Gln His Gly Met Asp565 570 575Asp Pro Glu Lys Glu Val Leu Met Trp Lys Phe Asp Ser Arg Leu Ala580 585 590Phe Gln His Ile Ala Arg Glu Lys His Pro Glu Tyr Tyr Lys Asp Cys595 600 605Leu Gly Leu Glu Tyr Leu Lys Lys Asp Glu Leu Arg Val Glu Leu Lys610 615 620Asp Glu Leu6252441953DNAArtificial sequenceFusion gene PE(delta III)-HIV nef-NC-K3 244atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atggccgaag aagctttcga cctctggaac gaatgcgcca aagcctgcgt gctcgacctc 120aaggacggcg tgcgttccag ccgcatgagc gtcgacccgg ccatcgccga caccaacggc 180cagggcgtgc tgcactactc catggtcctg gagggcggca acgacgcgct caagctggcc 240atcgacaacg ccctcagcat caccagcgac ggcctgacca tccgcctcga aggcggcgtc 300gagccgaaca agccggtgcg ctacagctac acgcgccagg cgcgcggcag ttggtcgctg 360aactggctgg taccgatcgg ccacgagaag ccctcgaaca tcaaggtgtt catccacgaa 420ctgaacgccg gcaaccagct cagccacatg tcgccgatct acaccatcga gatgggcgac 480gagttgctgg cgaagctggc gcgcgatgcc accttcttcg tcagggcgca cgagagcaac 540gagatgcagc cgacgctcgc catcagccat gccggggtca gcgtggtcat ggcccagacc 600cagccgcgcc gggaaaagcg ctggagcgaa tgggccagcg gcaaggtgtt gtgcctgctc 660gacccgctgg acggggtcta caactacctc gcccagcaac gctgcaacct cgacgatacc 720tgggaaggca agatctaccg ggtgctcgcc ggcaacccgg cgaagcatga cctggacatc 780aaacccacgg tcatcagtca tcgcctgcac tttcccgagg gcggcagcct ggccgcgctg 840accgcgcacc aggcttgcca cctgccgctg gagactttca cccgtcatcg ccagccgcgc 900ggctgggaac aactggagca gtgcggctat ccggtgcagc ggctggtcgc cctctacctg 960gcggcgcggc tgtcgtggaa ccaggtcgac caggtgatcc gcaacgccct ggccagcccc 1020ggcagcggcg gcgacctggg cgaagcgatc cgcgagcagc cggagcaggc ccgtctggcc 1080ctgaccctgg ccgccgccga gagcgagcgc ttcgtccggc agggcaccgg caacgacgag 1140gccggcgcgg ccaacgccga cgtggtgagc ctgacctgcc cggtcgccgc cggtgaatgc 1200gcgggcccgg cggacagcgg cgacgccctg ctggagcgca actatcccac tggcgcggag 1260ttcctcggcg acggcggcga cgtcgaattc catatggtcg acccgaccgt tcgtcagcgt 1320atggaccgta ccgaaccggc tgctgagggt gttggtgctg ttagccgtga cctggaaaaa 1380cacggtgcta tcacctcttc taacaccgct gctaccaacg ctgactgcgc ttggctggaa 1440gctcaggaag aagaagaagt tggttttccg gttcgtccgc aggttccgct gcgtccgatg 1500acctacaaag ctgctgttga catcagccac ttcctgaaag aaaaaggcgg cctggaaggt 1560ctgatctaca gccagaaacg tcaggaaatc ctggacctgt ggatctacca cacccagggc 1620tacttcccgg actggcagaa ctacaccccg ggtccgggta tccgttaccc gctgaccttc 1680ggctggtgtt tcaaactgct cgacttcctg aaagttccgg ttgacccgga acaggttgaa 1740aaagctaacg aaggtgataa caactgcctg ctgcacccga tcagccagca cggtatggac 1800gacccggaaa aagaagttct gatgtggaaa ttcgacagcc gtctggcttt ccagcacatc 1860gctcgtgaaa aacacccgga atactacaaa gactgcctgg gtctcgagta cctcaaaaaa 1920gacgaactgc gtgtagaact gaaagacgaa ctg 1953245651PRTArtificial sequenceFusion protein PE(delta III)-HIV Nef-NC-K3 245Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys20 25 30Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg35 40 45Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu50 55 60His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala65 70 75 80Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu85 90 95Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg100 105 110Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His115 120 125Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly130 135 140Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp145 150 155 160Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala165 170 175His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly180 185 190Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp195 200 205Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp210 215 220Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr225 230 235 240Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His245 250 255Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro260 265 270Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu275 280 285Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln290 295 300Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu305 310 315 320Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala325 330 335Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu340 345 350Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser355 360 365Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala370 375 380Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys385 390 395 400Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro405 410 415Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe His Met420 425 430Val Asp Pro Thr Val Arg Gln Arg Met Asp Arg Thr Glu Pro Ala Ala435 440 445Glu Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile450 455 460Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu465 470 475 480Ala Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro485 490 495Leu Arg Pro Met Thr Tyr Lys Ala Ala Val Asp Ile Ser His Phe Leu500 505 510Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser Gln Lys Arg Gln515 520 525Glu Ile Leu Asp Leu Trp Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp530 535 540Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg Tyr Pro Leu Thr Phe545 550 555 560Gly Trp Cys Phe Lys Leu Leu Asp Phe Leu Lys Val Pro Val Asp Pro565 570 575Glu Gln Val Glu Lys Ala Asn Glu Gly Asp Asn Asn Cys Leu Leu His580 585 590Pro Ile Ser Gln His Gly Met Asp Asp Pro Glu Lys Glu Val Leu Met595 600 605Trp Lys Phe Asp Ser Arg Leu Ala Phe Gln His Ile Ala Arg Glu Lys610 615 620His Pro Glu Tyr Tyr Lys Asp Cys Leu Gly Leu Glu Tyr Leu Lys Lys625 630 635 640Asp Glu Leu Arg Val Glu Leu Lys Asp Glu Leu645 6502461947DNAArtificial sequenceFusion gene PE(delta III)-HIV rev-K3 246atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atggccgaag aagctttcga cctctggaac gaatgcgcca aagcctgcgt gctcgacctc 120aaggacggcg tgcgttccag ccgcatgagc gtcgacccgg ccatcgccga caccaacggc 180cagggcgtgc tgcactactc catggtcctg gagggcggca acgacgcgct caagctggcc 240atcgacaacg ccctcagcat caccagcgac ggcctgacca tccgcctcga aggcggcgtc 300gagccgaaca agccggtgcg ctacagctac acgcgccagg cgcgcggcag ttggtcgctg 360aactggctgg taccgatcgg ccacgagaag ccctcgaaca tcaaggtgtt catccacgaa 420ctgaacgccg gcaaccagct cagccacatg tcgccgatct acaccatcga gatgggcgac 480gagttgctgg cgaagctggc gcgcgatgcc accttcttcg tcagggcgca cgagagcaac 540gagatgcagc cgacgctcgc catcagccat gccggggtca gcgtggtcat ggcccagacc 600cagccgcgcc gggaaaagcg ctggagcgaa tgggccagcg gcaaggtgtt gtgcctgctc 660gacccgctgg acggggtcta caactacctc gcccagcaac gctgcaacct cgacgatacc 720tgggaaggca agatctaccg ggtgctcgcc ggcaacccgg cgaagcatga cctggacatc 780aaacccacgg tcatcagtca tcgcctgcac tttcccgagg gcggcagcct ggccgcgctg 840accgcgcacc aggcttgcca cctgccgctg gagactttca cccgtcatcg ccagccgcgc 900ggctgggaac aactggagca gtgcggctat ccggtgcagc ggctggtcgc cctctacctg 960gcggcgcggc tgtcgtggaa ccaggtcgac caggtgatcc gcaacgccct ggccagcccc 1020ggcagcggcg gcgacctggg cgaagcgatc cgcgagcagc cggagcaggc ccgtctggcc 1080ctgaccctgg ccgccgccga gagcgagcgc ttcgtccggc agggcaccgg caacgacgag 1140gccggcgcgg ccaacgccga cgtggtgagc ctgacctgcc cggtcgccgc cggtgaatgc 1200gcgggcccgg cggacagcgg cgacgccctg ctggagcgca actatcccac tggcgcggag 1260ttcctcggcg acggcggcga cgtccgtcac cactttaccc cgagtgagcg tcaattgtgt 1320ttgtcgtcaa tccagactgc ctttaatcaa ggcgctggta cttgcatcct gtcagattct 1380gggcgtatca gttacactgt ggagtttagt ttgcctacgc atcatactgt gcgcctgatc 1440cgcgttacag caccaccgtc agcactcgac gcgaccgtct acaacggtag cagtaagtac 1500ggtgacacca gcactagcaa cgtgcgtggt gaccttcaag tgttagctca gaaggcagaa 1560cgtactctgc ctacctcctt caacttcggt gccatcaagg caactcgtgt tactgaattc 1620catatggtcg acctgctggc tgttcgtatc atcaaaaccc tgtaccagtc taacccgtac 1680ccgaaaccgg aaggttaccg tcgtgtccgt cgtaaccgcc gccgccgctg gcgtgctcgt 1740cagcgtcaga tccactccat cagcgaacgt atcctgatca cctgcctggg tcgtccgacc 1800gaaccggttc cgctgcaact gccgccgatc gaacgtctga acatcaactg tagcgaaagc 1860ggtggtacca gcggtaccca gcgtgttggt aacccgctcg agtacctcaa aaaagacgaa 1920ctgcgtgtag aactgaaaga cgaactg 1947247649PRTArtificial sequenceFusion protein PE(delta III)-HIV Rev-K3 247Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys20 25 30Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg35 40 45Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu50 55 60His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala65 70 75 80Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu85 90 95Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg100 105 110Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His115 120 125Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly130 135 140Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp145 150 155 160Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala165 170 175His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly180 185 190Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp195 200 205Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp210 215 220Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr225 230

235 240Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His245 250 255Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro260 265 270Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu275 280 285Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln290 295 300Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu305 310 315 320Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala325 330 335Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu340 345 350Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser355 360 365Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala370 375 380Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys385 390 395 400Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro405 410 415Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Arg His His Phe420 425 430Thr Pro Ser Glu Arg Gln Leu Cys Leu Ser Ser Ile Gln Thr Ala Phe435 440 445Asn Gln Gly Ala Gly Thr Cys Ile Leu Ser Asp Ser Gly Arg Ile Ser450 455 460Tyr Thr Val Glu Phe Ser Leu Pro Thr His His Thr Val Arg Leu Ile465 470 475 480Arg Val Thr Ala Pro Pro Ser Ala Leu Asp Ala Thr Val Tyr Asn Gly485 490 495Ser Ser Lys Tyr Gly Asp Thr Ser Thr Ser Asn Val Arg Gly Asp Leu500 505 510Gln Val Leu Ala Gln Lys Ala Glu Arg Thr Leu Pro Thr Ser Phe Asn515 520 525Phe Gly Ala Ile Lys Ala Thr Arg Val Thr Glu Phe His Met Val Asp530 535 540Leu Leu Ala Val Arg Ile Ile Lys Thr Leu Tyr Gln Ser Asn Pro Tyr545 550 555 560Pro Lys Pro Glu Gly Tyr Arg Arg Val Arg Arg Asn Arg Arg Arg Arg565 570 575Trp Arg Ala Arg Gln Arg Gln Ile His Ser Ile Ser Glu Arg Ile Leu580 585 590Ile Thr Cys Leu Gly Arg Pro Thr Glu Pro Val Pro Leu Gln Leu Pro595 600 605Pro Ile Glu Arg Leu Asn Ile Asn Cys Ser Glu Ser Gly Gly Thr Ser610 615 620Gly Thr Gln Arg Val Gly Asn Pro Leu Glu Tyr Leu Lys Lys Asp Glu625 630 635 640Leu Arg Val Glu Leu Lys Asp Glu Leu6452481680DNAArtificial sequenceFusion gene PE(delta III)-HIV tat-K3 248atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atggccgaag aagctttcga cctctggaac gaatgcgcca aagcctgcgt gctcgacctc 120aaggacggcg tgcgttccag ccgcatgagc gtcgacccgg ccatcgccga caccaacggc 180cagggcgtgc tgcactactc catggtcctg gagggcggca acgacgcgct caagctggcc 240atcgacaacg ccctcagcat caccagcgac ggcctgacca tccgcctcga aggcggcgtc 300gagccgaaca agccggtgcg ctacagctac acgcgccagg cgcgcggcag ttggtcgctg 360aactggctgg taccgatcgg ccacgagaag ccctcgaaca tcaaggtgtt catccacgaa 420ctgaacgccg gcaaccagct cagccacatg tcgccgatct acaccatcga gatgggcgac 480gagttgctgg cgaagctggc gcgcgatgcc accttcttcg tcagggcgca cgagagcaac 540gagatgcagc cgacgctcgc catcagccat gccggggtca gcgtggtcat ggcccagacc 600cagccgcgcc gggaaaagcg ctggagcgaa tgggccagcg gcaaggtgtt gtgcctgctc 660gacccgctgg acggggtcta caactacctc gcccagcaac gctgcaacct cgacgatacc 720tgggaaggca agatctaccg ggtgctcgcc ggcaacccgg cgaagcatga cctggacatc 780aaacccacgg tcatcagtca tcgcctgcac tttcccgagg gcggcagcct ggccgcgctg 840accgcgcacc aggcttgcca cctgccgctg gagactttca cccgtcatcg ccagccgcgc 900ggctgggaac aactggagca gtgcggctat ccggtgcagc ggctggtcgc cctctacctg 960gcggcgcggc tgtcgtggaa ccaggtcgac caggtgatcc gcaacgccct ggccagcccc 1020ggcagcggcg gcgacctggg cgaagcgatc cgcgagcagc cggagcaggc ccgtctggcc 1080ctgaccctgg ccgccgccga gagcgagcgc ttcgtccggc agggcaccgg caacgacgag 1140gccggcgcgg ccaacgccga cgtggtgagc ctgacctgcc cggtcgccgc cggtgaatgc 1200gcgggcccgg cggacagcgg cgacgccctg ctggagcgca actatcccac tggcgcggag 1260ttcctcggcg acggcggcga cgtcgaattc catatggtcg accgtgacga actgaaaggt 1320atcggtatgg aaccggttga cccgcgtctg gaaccgtgga aacacccggg tagccagccg 1380cgtaccgctt gcaacaactg ctactgcaaa aaatgttgtt tccactgccc ggtttgcttt 1440atctctaaag gtctgggtat cagctacggt cgtaaaaagc gtcgccagcg ccgccgcgct 1500ccgcaggact ccgaaaccca ccaggttagc ctgagcaagc aaccgaccag ccagctgcgt 1560ggtgacccga ccggtccgaa agaaagcaaa aaaaaagttg aacgtgaaac cgaaaccgac 1620ccgaacgttc tcgagtacct caaaaaagac gaactgcgtg tagaactgaa agacgaactg 1680249560PRTArtificial sequenceFusion protein PE(delta III)-HIV Tat-K3 249Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys20 25 30Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg35 40 45Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu50 55 60His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala65 70 75 80Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu85 90 95Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg100 105 110Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His115 120 125Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly130 135 140Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp145 150 155 160Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala165 170 175His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly180 185 190Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp195 200 205Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp210 215 220Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr225 230 235 240Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His245 250 255Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro260 265 270Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu275 280 285Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln290 295 300Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu305 310 315 320Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala325 330 335Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu340 345 350Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser355 360 365Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala370 375 380Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys385 390 395 400Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro405 410 415Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe His Met420 425 430Val Asp Arg Asp Glu Leu Lys Gly Ile Gly Met Glu Pro Val Asp Pro435 440 445Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro Arg Thr Ala Cys450 455 460Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Pro Val Cys Phe465 470 475 480Ile Ser Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln485 490 495Arg Arg Arg Ala Pro Gln Asp Ser Glu Thr His Gln Val Ser Leu Ser500 505 510Lys Gln Pro Thr Ser Gln Leu Arg Gly Asp Pro Thr Gly Pro Lys Glu515 520 525Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro Asn Val Leu530 535 540Glu Tyr Leu Lys Lys Asp Glu Leu Arg Val Glu Leu Lys Asp Glu Leu545 550 555 56025012PRTArtificial sequenceCarboxyl terminal moiety 250Lys Lys Asp Leu Arg Asp Glu Leu Lys Asp Glu Leu1 5 1025113PRTArtificial sequenceCarboxyl terminal moiety 251Lys Lys Asp Glu Leu Arg Asp Glu Leu Lys Asp Glu Leu1 5 1025213PRTArtificial sequenceCarboxyl terminal moiety 252Lys Lys Asp Glu Leu Arg Val Glu Leu Lys Asp Glu Leu1 5 10

Patent applications by Chao-Wei Liao, Shin-Chu City TW

Patent applications by Hsiu-Kang Chang, Taipei City TW

Patent applications by HealthBanks Biotech Co., Ltd.

Patent applications by HealthBanks USA Inc.

Patent applications in class PROTEINS, I.E., MORE THAN 100 AMINO ACID RESIDUES

Patent applications in all subclasses PROTEINS, I.E., MORE THAN 100 AMINO ACID RESIDUES

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Patent application title: CHIMERIC HIV FUSION PROTEINS AS VACCINES

Abstract:

Claims:

Description: