US 2011 O305749A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0305749 A1 Duch et al. (43) Pub. Date: Dec. 15, 2011

(54) HIV-1 ENVELOPE POLYPEPTIDES FOR HIV Publication Classification VACCNE (51) Int. Cl. A6IR 39/2I (2006.01) A 6LX 9/27 (2006.01) (76) Inventors: Mogens Ryttergaard Duch, CI2N 5/071 (2010.01) Risskov (DK); Finn Skou CI2N I/O (2006.01) Pedersen, Arhus V (DK); Shervin C07K 6/10 (2006.01) Bahrami, Arhus C (DK); Palle A 6LX 39/395 (2006.01) C07K I4/6 (2006.01) Villesen Fredsted, Marslet (DK); A6IP37/04 (2006.01) Carsten Wiuf, Arhus N. (DK); Lars A6IP3 L/18 (2006.01) Jorgen Østergaard, Lystrup (DK); CI2N 7/00 (2006.01) Martin Tolstrup, Risskov (DK) CI2N L/20 (2006.01) B82Y5/00 (2011.01) (52) U.S. Cl...... 424/450: 424/188.1: 435/235.1; (21) Appl. No.: 13/060,564 435/325; 435/258.1; 435/252.1; 424/85.2: 530/387.9; 424/139.1:530/324; 530/350; 977/773; 977/906 (22) PCT Filed: Aug. 28, 2009 (57) ABSTRACT Immunogenic HIV-1 envelope polypeptides are provided, PCT/DK2O09/OSO221 wherein specific amino acid residues are mutated to repress (86). PCT No.: immunosuppression in GP41, thereby boosting the immune response against HIV-1. Specifically, mutation of those spe S371 (c)(1), cific residues does not affect the fusogenic properties of the (2), (4) Date: Sep. 1, 2011 viral particle and/or the overall protein structure of the viral envelope protein. The invention further provides peptides and antigens based on the immunogenic HIV-1 envelopes as well (30) Foreign Application Priority Data as nucleic acid sequences and vectors encoding those. More over, biological entities such as viral particles are provided, as Aug. 28, 2008 (DK) ...... PA 2008 O1184 well as use of the provided components for preparation of a Mar. 23, 2009 (DK) ...... PA 2009 OO398 vaccine against HIV-1/AIDS.

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HIV-1 ENVELOPE POLYPEPTIDES FOR HIV logical consequences of having HIV are huge. The situation VACCNE is, however, even more severe in developing countries, where more than 95% of those people infected with HIV/AIDS are FIELD OF INVENTION living. Worldwide more than 25 million people have died from AIDS in the last 25 years. 0001. The present invention relates to HIV-1 envelope 0005. Approximately 95% of the people who get infected polypeptides, which may be used in a vaccine for HIV. The today live in the developing countries, where expensive anti invention also encompasses biological entities comprising viral drugs are not available. Therefore, there is an urgent Such polypeptides or nucleic acids encoding those, in particu need for an effective vaccine—the only effective solution to lar retroviral particles. Moreover the invention relates to vac the uncontrolled HIV pandemic. During the last few years cine compositions, which comprise a polypeptide of the research has brought up new knowledge on the fundamental present invention and an adjuvant as well as kits comprising biology of HIV-virus which is leading to new antiviral drugs said vaccine compositions. and strategies for vaccine design. In spite of these substantial advances, an effective vaccine does not yet exist. Only attenu BACKGROUND OF INVENTION ated (that is live but weakened) HIV-strains has been able to 0002 Human immunodeficiency virus (HIV) is according provide immunity in primate studies even though they will to WHO one of the most serious health crisis the world faces never reach a required safety profile Suitable for mass vacci today. AIDS has killed more than 25 million people since nation. 1981. In the most severely affected countries, the average life 0006. The replication process for HIV-1 has an error rate expectancy is now declining to 49 years of age-13 years less of about one per 10,000 base pairs. Since the entire viral than in the absence of AIDS. According to UNAIDS an esti genome is just under 10,000 base pairs, it is estimated that on mated number of 39.5 million people were living with HIV average one erroris introduced into the HIV-1 genome at each virus in 2006 and 4.3 million were infected in 2006. In many viral replication cycle. This high mutation rate contributes to regions new infections are heavily concentrated in the extensive variability of the viruses inside any one person and younger generations (15-24 years of age). Access to treatment an even wider variability across populations. and care has greatly increased in recent years. Determining 0007. This variability has resulted in three HIV-1 variants real time trends to HIV incidence and in particular the impact being described, and the subspecies of virus called “clades.” of prevention programmes ideally requires long studies of a The distinctions are based on the structure of the envelope large number of people. Given the practical difficulties of proteins, which are especially variable. The M (for major) conducting Such studies focus has been placed on young variant is by far the most prevalent worldwide. Within the M women and their infants. Children living with HIV typically variant are clades A, B, C, D, E, F, G, H, I, Jand K, with clades acquire infection through a mother-to-child-transmission A through E representing the vast majority of infections glo (MTCT), which occur during pregnancy, delivery or during bally. Clades A, C and D are dominant in Africa, while clade breastfeeding. Renewed efforts are urgently required to B is the most prevalent in Europe, North and South America increase access to comprehensive and integrated programmes and Southeast Asia. Clades E and C are dominant in Asia. to prevent HIV infection in infants and young children, which These clades differ by as much as 35%. Another variant is will indicate a route to HIV-free generations. clade O, which is observed in Cameroun isolates of HIV-1. 0003. There are two known types of HIV: HIV-1 and The greatest variation in structure is seen in the envelope HIV-2 that infect humans. They belong to a group of retrovi proteins gp120 and gp41. ruses called Lentiviruses and a virus similar to HIV has been 0008. There are two important results from the very high found in African monkeys. Retroviruses transfer their genes mutation rate of HIV-1 that have profound consequences for from a producer cell to a target cell as a genomic RNA the epidemic. First, the high mutation rate is one of the transcript, which is reverse-transcribed after infection and mechanisms that allow the virus to escape from control by integrated into the DNA genome of the target cell. drug therapies. These new viruses represent resistant strains. 0004. The first person with a documented HIV-infection The high mutation rate also allows the virus to escape the died in 1959. In the early 1980s doctors in the US become patient's immune system by altering the structures that are aware, that more and more patients suffered from abnormal recognized by immune components. An added consequence infections and showed signs of immune failure. The Syn of this extensive variability is that the virus can also escape drome was named Acquired Immune Deficiency Syndrome from control by vaccines, and vaccines based on envelope (AIDS) and it was soon after discovered that HIV was the proteins will likely be non-effective. causative agent for the observed destruction of the immune system. Initially patients were offered a treatment based SUMMARY OF INVENTION solely on pain relief and almost all inevitably died. In mid 1990s there were two important breakthroughs in treatment. 0009. The present invention provides a new approach to Firstly, a new group of antiretroviral agents were discovered designing immunogenic HIV-1 envelope polypeptides and secondly it became possible to measure the amount of derived from HIV-1 envelope protein, GP41 and/or GP120. HIV virus in blood. These two advances made it possible to Specific amino acid residues are mutated to repress immuno treat patients with a combination of different agents and doc Suppression in GP41, thereby boosting the immune response tors were able to check, whether the treatment actually against HIV-1. Mutation of those specific residues however, worked. The result was that the immune system of infected does not affect the fusogenic properties of the viral particle patients gradually became normal and patients lived longer. and/or the overall protein structure of the viral envelope pro Today infected people in Western countries are having the tein. The polypeptides of the present invention can be inserted same level of quality of life as those not infected and they are in a suitable construct and expressed in an organism to pro able to have children, although the economical and psycho duce a vaccine or an immunogenic response against HIV. US 2011/0305749 A1 Dec. 15, 2011

0010. In one aspect, the present invention relates to an X(28): I, V, T, L, R, F, and M. and HIV-1 envelope polypeptide comprising an amino acid sequence selected from the group of amino acid sequences X(30): T. P.Y.A. N. S., I, V, R, L, M and H; and consisting of: X(1-22)-C(23)-X(24-28)-C(29)-X(30-50), or X(31): T. S. P.. N, M and I; and any part thereof, or functional homolog or any amino acid sequence with at least 90% identity to said amino acid X(32): A.N.T, S, D, R, F, Q, P, I, E. V. M. L., K, H, C, and B; sequence or part thereof. Thus, the claimed envelope and polypeptide comprise a 50 amino acid sequence, wherein X(33): V. A. L. M. G. R. and C; and X(1-22) indicates an unbranched sequence of 22 amino acid residues, selected from any known amino acid, while C(23) X(34): X; and denotes that amino acid residue number 23 is cysteine; fol X(35): W. R. G. L. M., and P; and lowed by 5 residues of any amino acid, X(24-28), a cysteine in position 29 (C(29)), and another unbranched sequence of X(36): N, S, D, B, K, E, R, Q, M, and G.; and 21 amino acid residues selected from any amino acid (X(30 X(37): S, T, A, N, D, V, I, E, Y, K, L, R, G, P. M. F. W. H., Q, 50)). B, and C.; and 0011. The 50 amino acid sequence is derived from any known HIV-1 envelope subtype, such as those identified by X(38): S, T, N., I, G, R, L, C, A, W, M and E; and SEQID NO: 74-119. Thus, the specific amino acid in position X(39): W. G. A. R. E. C. Y. V. S. M, and H; and X of the amino acid sequence above may be selected from an amino acid in the corresponding position of any of those and X(40): X; and other HV-1 subtypes. In a preferred embodiment, the present X(41): N, G, K, S, D, E,T, R, H, PA, B, V. Q, Y. M., and I; and invention relates to an HIV-1 envelope as defined in the aspect above, wherein the amino acid residues in the amino acid X(42): K, R, N, D, S, T, G, E., I, V.Y. Q, P, H, A, W. M., and C: sequence of the HIV-1 envelope polypeptide are selected and from the groups of residues consisting of: X(43): S.T., N, K, I, R, D, E, P. L. A. W. G. M. H. Y. F. V. and C; and X(1): L., S, R, P. F. A. V. M, and I; and X(44): L.Y. Q, F. E. H. S. V. K, M, T, I, W. N. D, R, P, A, and X(2): Q. R. K. H. L. M., and P; and G; and X(3): A, T, V, H, S, R, Q, G, M, and E; and X(45): D, E, N, S, T, K, G, L, A, Q, H, I,Y, B, R, V. P. M., F. W. X(4): R. K. G. E. T. S. C. M., and H; and Z, and C.; and X(5): V. I, L, D, A, S, F, M, and G.; and X(46): E, D, Q, Y. K. N. T. S. A. W. H. M., R., I, G, L, V, Z, F, B, and P; and X(6): L. Q. V. M. P. W. T. and I; and X(47): I, D, E, M, G, T, Q, S. W. L., N.Y. K. V. R. F. A. P. and X(7): A, S, T, V. L. G. F. D. M., and E; and H; and X(8): V. L. I. M. A. W. K. G. and E; and X(48): W.I.T., N, D, E, L. G., S.Y. R. V. K, H, A, Q, M, and F: X(9): E, K, G, D, A, V. M, and F; and and X(10): X; and X(49): D, N, E, G, W, Q, K, H, L, B, S.I.Y.T. A. R,M, Z, and X(11): Y. L. F. H. C., I, T, M, and N; and V; and X(12): L., I, V, M, Q, P. T.Y. and A; and X(50): N, D, T, K, S, H. L. G. E. W. I.Q. M. R. B.Y. P. and A: 0012 where the amino acids are designated by their con X(13): K, R, Q, G.S., E. H. W. T. V. M. N.Z.Y.A. P. and C; and ventional single letter code. X(14): D, N, G, E. Y. V. S., H, A, M, and I; and 0013. According to the present invention, the amino acids in position 10, 19, 24, 34, and 40 affect the immunogenic X(15): Q, R, H, K. P. L. M., and N; and properties of the HIV-1 envelope polypeptide. Thus, the X(16): Q, K, R. T. H. E. S. P. M., and L; and amino acid in those positions alone or in combination affect X(17): L. F., I, R, V. P. S. M, and H; and the immunogenicity of the envelope polypeptide. In a pre ferred embodiment, the amino acid residue in position X(10) X(18): L. M. P. I, H, and S; and is selected from the group consisting of R, S, T, K, G, A, N. X(19): X; and Q and I. In another preferred embodiment, the amino acid residue in position X(19) is selected from the group consist X(20): I, L. M. V. S., F.T. D, A, R, P, and J.; and ing of G. N. S. R. E. T. D. V. C and A. In another preferred X(21): W. R, G, F, L, M, and T; and embodiment, the amino acid residue in position X(24) is selected from the group consisting of S. K.T. R. A. P.Y. F, G, X(22): G, D, A. R. M., and C; and Q, I and H. In another preferred embodiment, the amino acid X(24): X; and residue in position X(34) is selected from the group consist ing of P. K. R. S. A. L. Q., E. H. T., I, V, and F. In another X(25): G. R. E., N, A, M, and D; and preferred embodiment, the amino acid residue in position X(40) is selected from the group consisting of S. N. G. T. R. X(26): K. R. N. E. Q, T. S. I. M., and G.; and I, V. K. W. A. P. Y. D, Q, H, E, and C. In one preferred X(27): L, H, I, T, V. F. R. Q. S. P. A. J. M., and Y; and embodiment, the amino acid residue in position X(10) is

US 2011/0305749 A1 Dec. 15, 2011

sequence as defined above or a fragment thereof. Another - Continued aspect of the present invention relates to a biological entity comprising at least one HIV-1 envelope as defined in the SEO ID NO : 50: present invention, and/or at least one antigen as defined in the LRARLLALETFIONOOLLRLWGCFGNLICYTSVKWNDTWKGNSDTSLENI present invention, and/or at least one nucleic acid as defined WDN in the present invention, and/or at least one vector as defined in the present invention. SEO ID NO: 51: 0018 Yet another aspect of the present invention relates to LRARLLALETFIONOOLLNLWGCFGNLICYTSVKWNDTWKGNSDTSLENI a vaccine composition comprising WDN 0019 a) an HIV-1 envelope polypeptide as defined herein, or a functional homologue thereof having at least 70% SEO ID NO: 52: identity to said peptide or an immunogenically active pep LOTRIOAVETFIRDOOLLGMWGCKGNLICTTAVPWNASWSNKSLEOIWNH tide fragment comprising a consecutive sequence of at least SEO ID NO: 53 : 10 residues of said peptide or said functional homologue LOTRIOAVETFIRDOOFMGIWGCKGNLICTTAVPWNASWSNKSLEOIWNH thereof, or a nucleic acid encoding said peptide or said SEO ID NO: 54: peptide fragment or said functional homologies thereof LOTRIOAVETFIRDOOLLNMWGCKGNLICTTAVPWNASWSNKSLEOIWNH and/or 0020 b) an antigen as defined herein, and/or SEO ID NO: 55: 0021 c) a nucleic acid as defined herein, and/or LOTRIQAVETFIRDQQ WGCKGNLICTTAVPWNASWSNKSLEOIWNH 0022 d) a vector as defined herein, and/or SEO ID NO: 56 : 0023 e) a biological entity as defined herein, and LOTRIQAVETFIRDQQ M R WGCFGNLICTTAVPWNASWSNKSLEOIWNH 0024 f) an adjuvant SEO ID NO: 57: for use as a medicament. LRTRVLALETLIONOOLLRIWGCFGKLICTTAVPWNASWSNKSLEOIWNH 0025. A further aspect relates to a kit-of-parts comprising the vaccine composition as defined above, and a second active SEO ID NO: 58: ingredient. LOTRIOAMETY WGCFGKLICTTAVPWNASWSNKSLEOIWNH 0026. Another aspect relates to a method of treating, pre SEO ID NO. 59: venting or ameliorating a clinical condition, said method LQTRIQAVETFIRDQQ M R WGCFGKLICTTAVPWNASWSNKSLEQIWNH comprising administering to an individual suffering from said SEO ID NO: 60: clinical condition an effective amount of an HIV-1 envelope SOARIQAVETFIRDQQ M R WGCFGKLICTTAVPWNASWSNKSLEOIWNH polypeptide or part thereofas defined in the present invention, an antigen as defined in the present invention, a nucleic acid SEO ID NO: 61: as defined in the present invention, a vector as defined in the LOTRIOAVETFIRDOOLLRMWGCFGKLICTTAVPWNASWSNKSLEOIWNH present invention, a biological entity as defined in the present SEO ID NO: 62: invention, a vaccine composition as defined in the present LOARILAMERYMKDOOLMRMWGCFGKLICTTAVPWNASWSNKSLEOIWNH invention, or a kit-of-parts as defined in the present invention. 0027. Yet another aspect of the present invention relates to SEO ID NO: 63: the use of an HIV-1 envelope polypeptide or part thereof as LRARILAMERYMKDOOLMRMWGCFGKLICTTAVPWNASWSNKSLEOIWNH defined in the present invention, an antigen as defined in the SEO ID NO: 64: present invention, a nucleic acid as defined in the present LOARILAVERYLKDOOLLRIWGCFGKLICTTAVPWNASWSNKSLEQIWNH invention, a vector as defined in the present invention, a SEO ID NO: 65 : biological entity as defined in the present invention, a vaccine LOARILAVETLIONOORLNLWGCKGKLICYTSVKWNTSWSNKSLEOIWNH composition as defined in the present invention, or a kit-of parts as defined in the present invention for the manufacture SEO ID NO: 66: ofa medicament for the treatment, amelioration or prevention LOARILAVETYLKDOOLLNIWGCKGKLICTTAVKWNASWSNKSLEQIWNH of a clinical condition. SEO ID NO: 67: 0028. A further aspect of the present invention relates to an LOARILAVETYLKDOOLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNH, HIV-1 envelope polypeptide or part thereofas defined in the and/or present invention, an antigen as defined in the present inven SEO ID NO: 68: tion, a nucleic acid as defined in the present invention, a LOARILAVKTYLKDOOLLNIWGCKGKLICTTAVKWNASWSNKSLEQIWNH vector as defined in the present invention, a biological entity 0015. In another aspect, the present invention relates to an as defined in the present invention, a vaccine composition as antigen comprising at least one peptide with an amino acid defined in the present invention, or a kit-of-parts as defined in sequence as defined herein, or a functional homolog thereof the present invention for treating, ameliorating or preventing having at least 70% identity to said peptide or an immuno a clinical condition. logical active fragment comprising a consecutive sequence of 0029. In another aspect the present invention relates to a at least 10amino acids selected from a region of said peptide. pharmaceutical composition comprising an HIV-1 envelope 0016. Another aspect of the present invention relates to a polypeptide or part thereofas defined in the present invention, nucleic acid sequence encoding at least one HIV-1 envelope an antigen as defined in the present invention, a nucleic acid polypeptide with an amino acid sequence as defined above or as defined in the present invention, a vector as defined in the a fragment thereof and/or a nucleic acid sequence encoding at present invention, a biological entity as defined in the present least one antigen according to the present invention. invention, a vaccine composition as defined in the present 0017. Yet another aspect relates to an isolated eukaryotic invention, or a kit-of-parts as defined in the present invention expression vector comprising at least one nucleic acid for treating, ameliorating or preventing a clinical condition. US 2011/0305749 A1 Dec. 15, 2011

0030. A further aspect of the present invention relates to a herein, a biological entity as defined herein, a vaccine method of reducing the risk of an individual encountering a composition as defined herein, and/or a kit-of-parts as clinical condition, said method comprising administration of defined herein, and an HIV-1 envelope polypeptide or part thereofas defined in 0046 c) determining whether said blood sample com the present invention, an antigen as defined in the present prises antibodies or T-cells comprising T-cell receptors invention, a nucleic acid as defined in the present invention, a specifically binding the protein or peptide vector as defined in the present invention, a biological entity 0047 d) thereby determining whether an immune as defined in the present invention, a vaccine composition as response to said protein or peptide has been raised in said defined in the present invention, or a kit-of-parts as defined in individual. the present invention to said individual in an amount Suffi cient to generate a protective immune response. DESCRIPTION OF DRAWINGS 0031. In yet another aspect, the present invention relates a 0048 FIG. 1. Genomic structure of HIV-1 showing the method of producing the vaccine composition of the present location of env and gp41 invention, comprising combining 0049 FIG. 2. HIV-1 gp41 alignment. 0032) a) an HIV-1 envelope polypeptide as defined in the 0050 FIG. 3. HIV-1 gp41 alignment. present invention, or a functional homologue thereof hav 0051 FIG. 4. Syncytia with wt HIV (both the visible and ing at least 70% identity to said peptide oran immunogeni fluorescence micrograf) cally active peptide fragment comprising a consecutive 0052 FIG. 5. Syncytia made by mutant R10T (both the sequence of at least 10 residues of said peptide or said visible and fluorescence micrograf) functional homologue thereof, or a nucleic acid encoding 0053 FIG. 6. Syncytia made by mutant Db mut (both the said peptide or said peptide fragment or said functional visible and fluorescence micrograf) homologies thereof and/or 0054 FIG. 7. Syncytia made by mutant O 10-40 (both the 0033 b) an antigen as defined in the present invention, visible and fluorescence micrograf) and/or 0055 FIG.8. Syncytia made by Pent mut (both the visible 0034 c) a nucleic acid as defined in the present invention, and fluorescence micrograf) and/or 0056 FIG. 9. Syncytia made by Pent +E9K (both the 0035 d) a vector as defined in any of the present invention, visible and fluorescence micrograf) and/or 0057 FIG.10: Proliferation inhibition of the four peptides 0036 e) a biological entity as defined in the present inven at 50 uM concentration tion, and 0058 FIG. 11: The proliferation of lymphocytes in 0037 f) an adjuvant. response to ConA (5 ug/mL) with a dose-dependence on 0038. In yet another aspect, the present invention relates to immunosuppressive peptides. an antibody, antigenbinding fragment or recombinant protein 0059 FIG. 12: Lymphocyte proliferation. The fluores thereof, which is specific for an HIV-1 envelope polypeptide cence level is proportional to lymphocyte proliferation. The or part thereofas defined in the present invention, and/or a figure illustrates PBMC lymphocyte proliferation in cells nucleic acid as defined in the present invention, and/or an treated as indicated. The column indicated jurkat' are non antigenas defined in the present invention, and/or a biological transduced jurkat cells. The other columns representlympho entity as defined in the present invention. cyte proliferation in the presence of jurkat cells transduced with a murine leukemia virus vector expressing either an 0039. In another aspect, the present invention relates to an eGFP marker gene alone (eGFP) or the eGFP marker gene in antibody obtainable by immunizing a host with an HIV-1 addition to a HIV envelope variant: HIV (WT), M/O, or HIV envelope polypeptide or part thereofas defined herein, and/or G19R. a nucleic acid as defined herein, and/or an antigen as defined 0060 FIG. 13: Normalized stimulation index. The graph herein, and/or a biological entity as defined herein, a vaccine shows a lymphocyte stimulation index, calculated as the pro composition as defined herein, and/or a kit-of-parts as defined liferation in question/proliferation in UT. The column labels herein. are the same as in FIG. 12. 0040. Furthermore, an aspect of the present invention 0061 FIG. 14: Cytokine secretion assay. Immunemodula relates to a method of producing an antibody as defined tory properties of different peptides. IFN-Y (grey bar) and herein, said method comprising the steps of TNF-C. (blackbar). Cytokine secretion is evaluated by ODso 0041 a) administering an HIV-1 envelope polypeptide or level for PBMC untreated (UT), and in the presence of HIV part thereofas defined herein, an antigen as defined herein, WT, HIV G19R, or HIV M/O peptide, as indicated. HIV WT a nucleic acid as defined herein, and/or a vector as defined significantly reduces both IFN-Y and TNF-C. secretion upon herein, a biological entity as defined herein, a vaccine ConA stimulation of PBMC's. The mutant G19R downregu composition as defined herein, and/or a kit-of-parts as lates TNF-C. but not IFN-Y. defined herein to an animal, and 0042 b) obtaining said antibody from said animal DETAILED DESCRIPTION OF THE INVENTION 0043. A further aspect of the present invention relates to a 0062. It is a major objective of the present invention to method of monitoring immunization, said method compris provide HIV-1 polypeptides and nucleic acids encoding said ing the steps of polypeptides for production of a vaccine against HIV-1. 0044 a) providing a blood sample from an individual 0063. The present invention provides immunogenic 0045 b) providing an HIV-1 envelope polypeptide or part HIV-1 envelope polypeptides, antigens comprising said thereofas defined herein, an antigen as defined herein, a HIV-1 envelope polypeptides or part thereof, and nucleic nucleic acid as defined herein, and/or a vector as defined acids encoding said polypeptides or part thereof, as well as US 2011/0305749 A1 Dec. 15, 2011

eukaryotic expression vectors comprising at least one said 0067. The term “a retroviral vector” comprises a retroviral nucleic acid or part thereof. Also provided are biological vector capable of being transcribed into RNA, which can be entities such as eukaryotic cells, prokaryotic cells and/or viral packaged into a retroviral particle, reverse transcribed into particles, in particular retroviral particles, which may be double stranded DNA and inserted into the host genome by infectious or non-infectious, said biological entities compris the retroviral enzymatic machinery. ing at least one HIV-1 envelope polypeptides and/or nucleic 0068. The term "heterologous is used hereinafter for any acid according to the present invention. The invention also combination of nucleic acid sequences that is not normally provides vaccine composition comprising at least one HIV-1 found intimately associated in nature. envelope polypeptide, antigen, nucleic acid, vector, and/or 0069. The terms “bicistronic' and “polycistronic' as used biological entity of the present invention, and an adjuvant. A herein, relates to a transcript encoding a transcript, which kit-of-parts is also provided comprising said vaccine compo comprise two or more open reading frames, respectively. In sition a second active ingredient, such as an immunostimu one embodiment the replication deficient vector of the lating composition, for example one or more interleukins present invention comprises one open reading frame, two and/or an antibiotic, such as amoxicillin, penicillin, acyclovir open reading frames, three, four, five or six open reading and/or vidarabine. Specifically, the invention also provides frames. for the use of the polypeptides, antigens, nucleic acids, vec (0070. The term “polynucleotide' or “nucleic acid tors and biological entities for the manufacture of a medica sequence” refers to a polymeric form of nucleotides at least 2 ment, and for methods of treating, preventing orameliorating bases in length. By "isolated nucleic acid sequence' is meant a clinical condition, said method comprising administering at a polynucleotide that is not immediately contiguous with least one HIV-1 envelope polypeptide, antigen, nucleic acid, either of the coding sequences with which it is immediately vector, and/or biological entity of the present invention to a contiguous (one on the 5' end and one on the 3' end) in the person in need thereof. The invention also provides methods naturally occurring genome of the organism from which it is for producing a vaccine and/or an antibody by administering derived. The term therefore includes, for example, a recom a polypeptide, antigen, nucleic acid, Vector and/or biological binant DNA or RNA which is incorporated into a viral vector. entity of the invention. The nucleotides of the invention can be ribonucleotides, 0064. The components of the present invention can be deoxyribonucleotides, or modified forms of either nucle used to induce an immune response against HIV, as well as to otide. The term includes single and double stranded forms of enhance the immune response in an immunized mammal DNA. relative to HIV antigen alone. Advantageously, the vaccine (0071. The term “polynucleotide(s)" generally refers to composition and/or components thereof of the invention can any polyribonucleotide or polydeoxyribonucleotide, which also induce HIV specific CD4 T helper cells and CD8+ T cells may be unmodified RNA or DNA or modified RNA or DNA. yielding potent Th1 immune responses against a broad spec Thus, for instance, polynucleotides as used herein refers to, trum of HIV epitopes, providing a strong HIV-specific cyto among others, single- and double-stranded DNA, DNA that is toxic T lymphocyte response. Thus, the vaccine compositions a mixture of single- and double-stranded regions, single- and and components thereof of the invention are useful for pre double-stranded RNA, and RNA that is mixture of single- and venting HIV infection and/or slowing progression to AIDS in double-stranded regions, hybrid molecules comprising DNA infected individuals. The compositions, components and and RNA that may be single-stranded or, more typically, methods can be used to elicit potent Th1 cellular and humoral double-stranded or a mixture of single- and double-stranded immune responses specific for conserved HIV epitopes, elicit regions. In addition, polynucleotide as used herein can also HIV-specific CD4 T helper cells, HIV-specific cytotoxic T refer to triple-stranded regions comprising RNA or DNA or lymphocyte activity, stimulate production of chemokines and both RNA and DNA. The strands in such regions may be from cyotokines such as beta-chemokines, interferon-gamma, the same molecule or from different molecules. The regions interleukin 2 (IL2), interleukin 7 (IL7), interleukin 15 (IL15), may include all of one or more of the molecules, but more alpha-defensin, and the like, and increase memory cells. The typically involve only a region of some of the molecules. One vaccine compositions and components thereof can be admin of the molecules of a triple-helical region often is an oligo istered via various routes of administration, and can be used to nucleotide. prevent maternal transmission of HIV, for vaccination of new borns, children and high-risk individuals, and for vaccination 0072. As used herein, the term “polynucleotide' includes of infected individuals. The components of the present inven DNAS or RNAS as described above that contain one or more tion can also be used in combination with other HIV thera modified bases. Thus, DNAS or RNAs with backbones modi pies, including antiretroviral therapy (ART) with various fied for stability or for other reasons are “polynucleotides’ as combinations of nuclease and protease inhibitors and agents that term is intended herein. Moreover, DNAS or RNAs com to block viral entry, such as T20. prising unusual bases, such as inosine, or modified bases, 0065. A main aspect of the present invention is to provide Such as tritylated bases, to name just two examples, are poly retroviral particles, which express HIV-1 envelope polypep nucleotides as the term is used herein. tide, or part thereof, and display the envelope on the surface of 0073. It will be appreciated that a great variety of modifi a retroviral particle, such as a lentiviral particle. The retroviral cations have been made to DNA and RNA that serve many particle may be fusogenic or non-fusogenic. A non-fusogenic useful purposes known to those of skill in the art. The term particle will promote an immunological response against polynucleotide as it is employed herein embraces Such HIV envelope on the particle surface. The fusogenic particle chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and is infectious and mediate fusion with target cells. RNA characteristic of viruses and cells, including simple and Terms and Definitions complex cells, interalia. 0066. To facilitate understanding of the invention, a num 0074 The term “amino acid and “amino acid sequence' ber of terms are defined below. refer to an oligopeptide, peptide, polypeptide, or protein US 2011/0305749 A1 Dec. 15, 2011

sequence, or a fragment of any of these, and to naturally 40, 50, 60, 75, 100, 150, 250, 500 or at least 700 contiguous occurring or synthetic molecules. Where “amino acid nucleotides or amino acid residues in length. Fragments may sequence' is recited to refer to a sequence of a naturally be preferentially selected from certain regions of a molecule. occurring protein molecule, "amino acid sequence' and like For example, a polypeptide fragment may comprise a certain terms are not meant to limit the amino acid sequence to the length of contiguous amino acids selected from the first 250 complete native amino acid sequence associated with the or 500 amino acids (or first 25% or 50%) of a polypeptide as recited protein molecule. shown in a certain defined sequence. Clearly these lengths are 0075 Thus, the term “amino acid comprises any syn exemplary, and any length that is supported by the specifica thetic or naturally occurringamino carboxylic acid, including tion, including the Sequence Listing, tables, and figures, may any amino acid occurring in peptides and polypeptides be encompassed by the present embodiments. including proteins and enzymes synthesized in vivo thus 0078. The term “Homology” refers to sequence similarity including modifications of the amino acids. The term amino or, interchangeably, sequence identity, between two or more acid is herein used synonymously with the term "amino acid polynucleotide sequences or two or more polypeptide residue' which is meant to encompass amino acids as stated Sequences. which have been reacted with at least one other species, such 0079 Methods of alignment of sequences for comparison as 2, for example 3. Such as more than 3 other species. The are well-known in the art. Various programs and alignment generic term amino acid comprises both natural and non algorithms are described and present a detailed consideration natural amino acids any of which may be in the "D' or “L” of sequence alignment methods and homology calculations, isomeric form. such as VECTOR NTI. The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is fre One-letter Three-letter quently measured in terms of percentage identity (or similar symbol symbol Amino acid ity or homology); the higher the percentage, the more similar A. Ala alanine the two sequences will be. B ASX aspartic acid or asparagine 0080. The NCBI Basic Local Alignment Search Tool C Cys cysteine D Asp aspartic acid (BLAST) is available from several sources, including the E Glu glutamic acid National Center for Biotechnology Information (NBCI, Phe phenylalanine Bethesda, Md.) and on the Internet, for use in connection with G Gly glycine the sequence analysis programs blastp, blastin, blastX, thlastin H His histidine and thlastx. It can be accessed at http://www.ncbi.nlm.nih. Ile isoleucine K Lys ysine gov/BLAST/. A description of how to determine sequence Leu eucine identity using this program is available at http://www.ncbi. M Met methionine nlm.nih.gov/BLAST/blast help.html. N ASn asparagine C Pro proline I0081 Homologs of the disclosed polypeptides are typi Q Gln glutamine cally characterised by possession of at least 94% sequence R Arg arginine identity counted over the full length alignment with the dis S Ser serine closed amino acid sequence using the NCBI Basic Blast 2.0, T Thr hreonine J* Sec Selenocysteine gapped blastp with databases such as the nr or Swissprot V Wal valine database. Alternatively, one may manually align the W Trp tryptophan sequences and count the number of identical amino acids. X Xaa unknown or other amino acid, This number divided by the total number of amino acids in i.e. X can be any of the conventional amino acids. your sequence multiplied by 100 results in the percent iden Y Tyr tyrosine tity. Z. Glx glutamic acid or glutamine (or Substances such I0082. The terms “percent identity” and “% identity,” as as 4-carboxyglutamic acid and applied to polynucleotide sequences, refer to the percentage 5-oxoproline that yield glutamic of residue matches between at least two polynucleotide acid on acid hydrolysis of peptides) sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, 0076. A “detectable label” refers to a reporter molecule or gaps in the sequences being compared in order to optimize enzyme that is capable of generating a measurable signal and alignment between two sequences, and therefore achieve a is covalently or noncovalently joined to a polynucleotide or more meaningful comparison of the two sequences. polypeptide. I0083) Nucleic acid sequences that do not show a high 0077. A “fragment” is a unique portion of the polynucle degree of identity may nevertheless encode similar amino otide encoding the HIV-1 envelope polypeptide of the present acid sequences due to the degeneracy of the genetic code. It is invention which is identical in sequence to but shorter in understood that changes in a nucleic acid sequence can be length than the parent sequence. Similarly the term frag made using this degeneracy to produce multiple nucleic acid ment refers to an HIV-1 envelope polypeptide of the present sequences that all encode Substantially the same protein. invention a fragment may comprise up to the entire length of I0084. The phrases “percent identity” and “96 identity, as the defined sequence, minus one nucleotide or amino acid applied to polypeptide sequences, refer to the percentage of residues. For example, a fragment may comprise from 5 to residue matches between at least two polypeptide sequences 2500 contiguous nucleotides or amino acid residues. A frag aligned using a standardized algorithm. Methods of polypep ment used as a probe, primer, antigen, therapeutic molecule, tide sequence alignment are well-known. Some alignment or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30. methods take into account conservative amino acid substitu US 2011/0305749 A1 Dec. 15, 2011

tions. Such conservative Substitutions, explained in more a clinical condition as described herein. In a preferred detail above, generally preserve the charge and hydrophobic embodiment, the term treatment relates to prophylactic treat ity at the site of substitution, thus preserving the structure ment, i.e. a therapy to reduce the Susceptibility of a clinical (and therefore function) of the polypeptide. condition, a disorder or condition as defined herein. 0085 Percent identity may be measured over the length of 0091 Thus, “treatment,” “treating,” and the like, as used an entire defined polypeptide sequence, for example, as herein, refer to obtaining a desired pharmacologic and/or defined by a particular SEQID number, or may be measured physiologic effect, covering any treatment of a pathological over a shorter length, for example, over the length of a frag condition or disorder in a mammal, including a human. The ment taken from a larger, defined polypeptide sequence, for effect may be prophylactic interms of completely or partially instance, a fragment of at least 15, at least 20, at least 30, at preventing a disorder or symptom thereof and/or may be least 40, at least 50, at least 70 or at least 150 contiguous therapeutic in terms of a partial or complete cure for a disor residues. Such lengths are exemplary only, and it is under der and/or adverse affect attributable to the disorder. That is, stood that any fragment length Supported by the sequences “treatment includes (1) preventing the disorder from occur shown herein, in the tables, figures or Sequence Listing, may ring or recurring in a Subject, (2) inhibiting the disorder, Such be used to describe a length over which percentage identity as arresting its development, (3) stopping or terminating the may be measured. disorder or at least symptoms associated therewith, so that the I0086 Percent identity may be measured over the length of host no longer Suffers from the disorder or its symptoms. Such an entire defined sequence, for example, as defined by a as causing regression of the disorder or its symptoms, for particular SEQID number, or may be measured over a shorter example, by restoring or repairing a lost, missing or defective length, for example, over the length of a fragment taken from function, or stimulating an inefficient process, or (4) reliev a larger, defined sequence, for instance, a fragment of at least ing, alleviating, or ameliorating the disorder, or symptoms 20, at least 30, at least 40, at least 50, at least 70, at least 100, associated therewith, where ameliorating is used in a broad or at least 200 contiguous nucleotides. Such lengths are sense to refer to at least a reduction in the magnitude of a exemplary only, and it is understood that any fragment length parameter, Such as inflammation, pain, and/or immune defi Supported by the sequences shown herein, in the tables, fig ciency. ures, or Sequence Listing, may be used to describe a length 0092. The terms “prevent,” “preventing,” and “preven over which percentage identity may be measured. tion', as used herein, refer to a decrease in the occurrence of 0087. The phrases “nucleic acid and “nucleic acid pathological cells in an animal. The prevention may be com sequence” refer to a nucleotide, oligonucleotide, polynucle plete, e.g., the total absence of pathological cells in a subject. otide, or any fragment thereof. These phrases also refer to The prevention may also be partial, such that for example the DNA or RNA of genomic or synthetic origin which may be occurrence of pathological cells in a subject is less than that single-stranded or double-stranded and may represent the which would have occurred without the present invention. sense or the antisense Strand, to peptide nucleic acid (PNA), Prevention also refers to reduced susceptibility to a clinical other nucleic acid analog, or to any DNA-like or RNA-like condition. material. 0093. A “replication-deficient retroviral vector accord I0088. The term “operably linked” refers to the situation in ing to the present invention, is a vector, which does not com which a first nucleic acid sequence, amino acid sequence or prise all essential genes for viral propagation. The vector may ligand is placed in a functional relationship with a second comprise one or more nucleic acid sequences encoding ret nucleic acid sequence, amino acid sequence or ligand. For roviral components, such as an envelope polypeptide. How instance, a promoter is operably linked to a coding sequence ever, the replication-deficient retroviral vector is for example if the promoter affects the transcription or expression of the devoid of nucleic acids encoding one or more of the retroviral coding sequence. Operably linked DNA sequences or protein components gag, pol, and/or rev, which are normally required or ligands may be in close proximity or contiguous and, for retroviral lifecycle, and Rev/Rex for lentiviral lifecycel. where necessary to join two protein coding regions, in the Generation of retroviral particles derived from a replication same reading frame. deficient retroviral vector, thus, requires that the remaining I0089. The term “internal ribosome entry site' (IRES) components are provided in trans, for example encoded by a defines a sequence motif which promotes attachment of ribo nucleic acid sequence comprised in a producer cell. somes to that motif on internal mRNA sequences. Further 0094 Moreover, a “replication-competent retroviral vec more, all factors needed to efficiently start translation at the tor” according to the present invention, is a vector, which AUG-start-codon following said IRES attach to this sequence comprises all essential genes for viral propagation, i.e. gag, motive. Consequently, an mRNA containing a sequence pol and ENV. motive of a translation control element, e.g. IRES, results in 0.095 A “pharmaceutically acceptable carrier.” “pharma two translational products, one initiating from the 5' end of ceutically acceptable diluent, or “pharmaceutically accept the mRNA and the other by an internal translation mechanism able excipient’, or “pharmaceutically acceptable vehicle.” mediated by IRES. Accordingly, the insertion of a transla used interchangeably herein, refer to a non-toxic solid, semi tional control element, such as IRES, operably linked to an Solid or liquid filler, diluent, encapsulating material or formu ORF into a retroviral genome allows the translation of this lation auxiliary of any conventional type. A pharmaceutically additional ORF from a viral RNA transcript. Such RNA tran acceptable carrier is essentially non-toxic to recipients at the Scripts with the capacity to allow translation of two or more dosages and concentrations employed and is compatible with ORF are designated bi- or polycistronic RNA transcripts, other ingredients of the formulation. For example, the carrier respectively. for a formulation containing polypeptides would not nor 0090. The term “treatment”, as used anywhere herein mally include oxidizing agents and other compounds that are comprises any type of therapy, which aims at terminating, known to be deleterious to polypeptides. Suitable carriers preventing, ameliorating and/or reducing the Susceptibility to include, but are not limited to, water, dextrose, glycerol, US 2011/0305749 A1 Dec. 15, 2011

saline, ethanol, and combinations thereof. The carrier can is entry of the viral core into the cytoplasm of a target cell, contain additional agents such as wetting or emulsifying which is necessary for productive infection. agents, pH buffering agents, or adjuvants which enhance the 0101. As used herein, AIDS” refers to the symptomatic effectiveness of the formulation. Adjuvants of the invention phase of HIV infection, and includes both Acquired Immune include, but are not limited to Freunds's, Montanide ISA Deficiency Syndrome (commonly known as AIDS) and Adjuvants &Isqb; Seppic, Paris, France] Ribi's Adju “ARC.” or AIDS Related Complex. The immunological and vants (Ribi ImmunoChem Research, Inc., Hamilton, Mont.), clinical manifestations of AIDS are well known in the art and I Hunter's TiterMax (CytRX Corp., Norcross, Ga.), Alumi include, for example, opportunistic infections and cancers num Salt Adjuvants (Alhydrogel Superfos of Denmark/Ac resulting from immune deficiency. curate Chemical and Scientific Co., Westbury, N.Y.), Nitro 0102) The term “inhibiting AIDS” as used herein, refers to cellulose-Adsorbed Protein, Encapsulated Antigens, and a beneficial prophylactic or therapeutic effect of a composi Gerbu Adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Ger tion or component in relation to HIV infection or AIDS symp many/C-C Biotech, Poway, Calif.). Topical carriers include toms. Such beneficial effects include, for example, preventing liquid petroleum, isopropyl palmitate, polyethylene glycol, or delaying initial infection of an individual exposed to HIV; ethanol (95%), polyoxyethylene monolaurate (5%) in water, reducing viral burden in an individual infected with HIV; or sodium lauryl sulfate (5%) in water. Other materials such prolonging the asymptomatic phase of HIV infection; main as anti-oxidants, humectants, viscosity stabilizers, and simi taining low viral loads in HIV infected patients whose virus lar agents can be added as necessary. Percutaneous penetra levels have been lowered via anti-retroviral therapy (ART); increasing levels of CD4 T cells or lessening the decrease in tion enhancers such as AZone can also be included. CD4 T cells, both HIV-1 specific and non-specific, in drug 0096 “Pharmaceutically acceptable salts' include the naive patients and in patients treated with ART, increasing acid addition salts (formed with the free amino groups of the overall health or quality of life in an individual with AIDS: polypeptide) and which are formed with inorganic acids Such and prolonging life expectancy of an individual with AIDS. A as, for example, hydrochloric or phosphoric acids, or Such clinician can compare the effect of immunization with the organic acids as acetic, mandelic, oxalic, and tartaric. Salts patient's condition prior to treatment, or with the expected formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potas condition of an untreated patient, to determine whether the sium, ammonium, calcium, or ferric hydroxides, and Such treatment is effective in inhibiting AIDS. organic bases as isopropylamine, trimethylamine, 2-ethy 0103) As used herein, the term "enhances, with respect to an immune response is intended to mean that the immuno lamino ethanol, and histidine. genic agents and/or components elicits a greater immune 0097. Compositions for oral administration can form solu response than does a composition containing HIV antigen tions, Suspensions, tablets, pills, capsules, Sustained release alone. An enhanced immune response can be, for example, formulations, oral rinses, or powders. increased production of chemokines and/or cytokines that 0098. The term “unit dosage form,” as used herein, refers promote memory cells, an increase in memory cells, an to physically discrete units suitable as unitary dosages for increase in IgG2b production, in increase in cytotoxic T lym human and animal Subjects, each unit containing a predeter phocyte activity, an increase in beta-chemokine or IL 15 pro mined quantity of compounds of the present invention calcu duction, and the like. As an example of an enhanced immune lated in an “effective amount,” that is, a dosage sufficient to response, the immunogenic agents of the invention can produce the desired result or effect in association with a increase production of gamma-interferon by both CD4 cells pharmaceutically acceptable carrier. The specifications for (helper function) and CD8 cells (cytotoxic T lymphocytes; the novel unit dosage forms of the present invention depend CTLs). on the particular compound employed, the host, and the effect 0104. The term “beta-chemokine' refers to a member of a to be achieved, as well as the pharmacodynamics associated class of Small, chemoattractive polypeptides that includes with each compound in the host. RANTES, macrophage inflammatory protein-1.beta. (MIP 0099. The term “epitope' means a protein determinant 1.beta.) and macrophage inflammatory protein-1.alpha. capable of specific binding to an antibody. Epitopes usually (MIP-1.alpha.). The physical and functional properties of consist of chemically active Surface groupings of molecules beta-chemokines are well known in the art. In the case of Such as amino acids or Sugar side chains and usually have enhanced beta-chemokine production, the beta-chemokine specific three dimensional structural characteristics, as well production can be “HIV-specific, beta-chemokine produc as specific charge characteristics. Conformational and non tion.” which refers to production of a beta-chemokine in conformational epitopes are distinguished in that the binding response to stimulation of T cells with an HIV antigen. Alter to the former but not the latter is lost in the presence of natively, or additionally, the beta-chemokine production that denaturing solvents. is enhanced can be "non-specific beta-chemokine produc 0100. The term “fusion according to the present inven tion.” which refers to production of a beta-chemokine in the tion comprise cell-cell fusion as well as virus-cell fusion. absence of stimulation of T cells with an HIV antigen. Cell-cell Fusion or Syncytia formation is a process by which 0105. As used herein, the term “biological entity” relates the plasma membranes of two cells merge to form a single to any matter of biological origin, Such as a prokaryotic cell, continuous double lipid membrane. This process does not a eukaryotic cell, a virus, a liposome, a nanoparticle and/or a happen spontaneously and is often mediate by the Surface retroviral cell, and/or any components and/or compartments proteins of enveloped viruses Such as the envelope proteins of thereof. Such as for example a liposome. retroviruses. Virus cell fusion is process by which an envel 0106 The term “component of the present invention” as oped virus mediates merging of its lipid membrane with that used herein is meant to incorporate any aspect an embodiment of a target cell through interaction of the viral coat protein as of the present invention as defined herein. Thus, a compo with a cellular receptor. The result of viral cell fusion process nent of the present invention includes an HIV-1 envelope US 2011/0305749 A1 Dec. 15, 2011 polypeptide as defined herein, for example as identified by 0118 CTL: Cytotoxic T lymphocyte. A sub group of any of IGP1-7, SEQ ID NO: 1-337, and/or a functional T-cells expressing CD8 along with the T-cell receptor and homolog and/or fragment thereof. A component of the therefore able to respond to antigens presented by class I present invention also includes an antigen, a nucleic acid, a molecules. eukaryotic expression vector, a biological entity, a vaccine 0119 Cytokine: Growth or differentiation modulator, composition, a kit-of-parts, a method, a use, a compound, a used non-determinative herein, and should not limit the inter cell, and/or an antibody as defined or described in the present pretation of the present invention and claims. In addition to invention. the cytokines, adhesion or accessory molecules, or any com 0107 As used herein, the expression “immunogenic’ is bination thereof, may be employed alone or in combination used to describe an agent capable of eliciting at least one type with the cytokines. of immune response directed against an HIV envelope I0120 Delivery vehicle: An entity whereby a nucleotide polypeptide or fragment thereof. Thus, Such an immune sequence or polypeptide or both can be transported from at response may be any response, in particular a CTL response least one media to another. where CTLS are generated that are capable of recognising the I0121 DC: Dendritic cell. (DCs) are immune cells and HLA/polypeptide complex presented on cell Surfaces result form part of the mammalian immune system. Their main ing in cell lysis, i.e. the vaccine elicits the production in the function is to process antigen material and present it on the vaccinated subject of effector T-cells having a cytotoxic effect Surface to other cells of the immune system, thus functioning against the host cells harbouring the retroviral vector or RNA as antigen-presenting cells (APCs). thereof, as well as an antibody response giving rise to the 0.122 Envelope: Protein integrated in the membrane of the production of anti-HIV antibodies. retroviral particle. HIV-1 envelope proteins are gp120 and 0108. Adjuvant: Any substance whose admixture with an gp41, examples of gp41 is represented by FIGS. 2 and 3, as administered immunogenic polypeptide?peptide? antigen/ well as SEQID NO: 74-76. nucleic acid construct/biological entity increases or other I0123 Fragment: is used to indicate a non-full length part wise modifies the immune response to said determinant. of a nucleic acid or polypeptide. Thus, a fragment is itself also 0109 Antibody: Immunoglobulin molecules and active a nucleic acid or polypeptide, respectively. portions of immunoglobulin molecules. Antibodies are for 0.124 Functional homologue: A functional homologue example intact immunoglobulin molecules or fragments may be any nucleic acid/protein/polypeptide that exhibits at thereof retaining the immunologic activity. least Some sequence identity with a wild type version/se 0110 Antigen: Any substance that can bind to a clonally quence of a given gene/gene product/protein/polypeptide and distributed immune receptor (T-cell or B-cell receptor). Usu has retained at least one aspect of the original sequences ally a peptide, polypeptide or a multimeric polypeptide. Anti functionality. Herein a functional homologue of HIV-1 enve gens are preferably capable of eliciting an immune response. lope has the capability to induce an immune response to cells 0111 APC: Antigen-presenting cell. An APC is a cell that expressing HIV-1 envelope. displays foreign antigen complexed with MHC on its surface. 0.125 HIV: As used herein, the term “HIV refers to all T-cells may recognize this complex using their T-cell receptor forms, subtypes and variations of the 25 HIV virus, and is (TCR). APCs fall into two categories: professional, (of which synonymous with the older terms for HIV, such as HTLVIII there are three types: Dendritic cells, macrophages and and LAV. Various cell lines capable of propagating HIV or B-cells) or non-professional (does not constitutively express permanently infected with the HIV virus have been devel the Major histocompatibility complex proteins required for oped and deposited with the ATCC, including HuT 78 cells interaction with naive T cells; these are expressed only upon and the HuT 78 derivative H9, as well as those having acces stimulation of the non-professional APC by certain cytokines sion numbers CCL 214, TIB 161, CRL 1552 and CRL 8543, such as IFN-Y). which are described in U.S. Pat. No. 4,725,669 and Gallo, 0112 Boost: To boost by a booster shot or dose is to give Scientific 30 American 256:46 (1987). an additional dose of an immunizing agent. Such as a vaccine, 0.126 Immunostimulatory sequence: As used herein, the given at a time after the initial dose to Sustain the immune term “immunostimulatory sequence' or “ISS” refers to a response elicited by the previous dose of the same agent. genetic adjuvant, i.e. nucleotide sequence that is capable of 0113 Cancer: Herein any preneoplastic or neoplastic dis enhancing the immune response in a mammal when admin ease, benign or malignant, where "neoplastic' refers to an istered in combination with an antigen. An ISS id for example abnormal proliferation of cells. a nucleic acid sequence comprising at least one unmethylated 0114 Carrier: Entity or compound to which for example I0127. Individual: Generally any species or subspecies of antigens, polypeptides and/or biological entities are coupled bird, mammal, fish, amphibian, or reptile, preferably a mam to aid in the induction of an immune response. mal, most preferably a human being. 0115 Chimeric protein: A genetically engineered protein I0128 Infection: Herein the term “infection relates to any that is encoded by a nucleotide sequence made by a splicing kind of clinical condition giving rise to an immune response together of two or more complete or partial genes or a series and therefore includes infections, chronic infections, autoim of (non)random nucleic acids. mune conditions and allergic inflammations. In the context of 0116 Clinical condition: A condition that requires medi the present invention, infection predominantly relates to HIV cal attention, herein especially conditions associated with the infection and related infections. expression of HIV polypeptides such as envelope proteins. 0.129 Isolated: used in connection with nucleic acids, Examples of such conditions include: cancers and infections. polypeptides, and antibodies disclosed herein isolated 0117 Complement: A complex series of blood proteins refers to these having been identified and separated and/or whose action “complements' the work of antibodies. recovered from a component of their natural, typically cellu Complement destroys bacteria, produces inflammation, and lar, environment. Nucleic acids, polypeptides, and antibodies regulates immune reactions. of the invention are preferably isolated, and vaccines and US 2011/0305749 A1 Dec. 15, 2011

other compositions of the invention preferably comprise iso 0141 Variant: a variant of a given reference nucleic acid lated nucleic acids, polypeptides or isolated antibodies. or polypeptide refers to a nucleic acid or polypeptide that 0130 MHC: Major histocompatibility complex, two main displays a certain degree of sequence homology/identity to subclasses of MHC, Class I and Class II exist. said reference nucleic acid or polypeptide but is not identical 0131 Operative linker: A sequence of nucleotides or to said reference nucleic acid or polypeptide. amino acid residues that bind together two parts of a nucleic 0.142 Vector: a genetically engineered nucleic acid con acid constructor (chimeric) polypeptide in a manner securing struct. Typically comprising several elements such as genes the biological processing of the nucleic acid or polypeptide. or fragments of same, promoters, enhancers, terminators, 0132 Pathogen: a specific causative agent of disease, polyA tails, linkers, polylinkers, operative linkers, multiple especially a biological agent such as a virus, bacteria, prion or cloning sites (MCS), markers, STOP codons, other regulatory elements, internal ribosomal entry sites (IRES) or others. The parasite that can cause disease to its host, also referred to as an present invention comprises expression vectors, such as infectious agent. eukaryotic or prokaryotic expression vectors, as well as vec 0.133 Peptide: Plurality of covalently linked amino acid tors of retroviral origin. residues defining a sequence and linked by amide bonds. The 0.143 Virosome: A virosome is a fusion between a virus term is used analogously with oligopeptide and polypeptide. and a liposome, for example a liposome with HIV-1 envelope The natural and/or non-natural amino acids may be linked by polypeptides. peptide bonds or by non-peptide bonds. The term peptide also embraces post-translational modifications introduced by HIV-1 Envelope Polypeptide chemical or enzyme-catalyzed reactions, as are known in the art. The term can refer to a variant or fragment of a polypep 0144. Due to the high error rate during replication of the tide. HIV-1 genome, the genetic code for HIV-1 envelope is highly 0134 Pharmaceutical carriers: also termed excipients, or variable, both inside any one person and even more so across stabilizers are non-toxic to the cell or individual being populations. The present invention relates to any variant of exposed thereto at the dosages and concentrations employed. the HIV-1 envelope polypeptide, in particular any variant in Often the physiologically acceptable carrier is an aqueous pH which gp41, comprise an amino acid sequence of 50 amino buffered solution. Examples of physiologically acceptable acids, or part thereof, wherein the 50 amino acid sequence is carriers include buffers such as phosphate, citrate, and other defined as X(1-22)-C(23)-X(24-28)-C(29)-X(30-50). The organic acids; antioxidants including ascorbic acid; low number(s) in parentheses designate the number of each amino molecular weight (less than about 10 residues) polypeptide; acid in the 50 amino acid sequence, i.e. C(23) specifies that proteins, such as serum albumin, gelatin, or immunoglobu amino acid number 23 is a cystein, and X(1-22) designates lins; hydrophilic polymers such as polyvinylpyrrolidone; that amino acids 1 to 22 are any amino acids as defined above. amino acids such as glycine, glutamine, asparagine, arginine Note however, that the term X(1-22) does not imply that or lysine; monosaccharides, disaccharides, and other carbo amino acids 1-22 are the same amino acid; X merely indicates hydrates including glucose, mannose, or dextrins; chelating that each of the amino acids 1-22 are selected from all amino agents such as EDTA; Sugar alcohols such as mannitol or acids. Unless otherwise specified, the amino acids in the Sorbitol; salt-forming counterions such as Sodium; and/or present invention are designated by their conventional single nonionic surfactants such as TWEENTM, polyethylene glycol letter code, as defined above. The invariable cysteins in posi (PEG), and PLURONICSTM tions 23 and 29 of the 50 amino acid sequence may be used to align the amino acid sequence of the present invention with 0135 Plurality: At least two. HIV-1 envelope subtypes, see e.g. FIG. 2. 0.136 Promoter: A binding site in a DNA chain at which 0145 Due to the variability in the gp41 sequence among RNA polymerase binds to initiate transcription of messenger different HIV-1 subtypes and clades, the present invention RNA by one or more nearby structural genes. relates to any HIV-1 envelope polypeptide, which comprises 0.137 Signal peptide: A short sequence of amino acids that a 50 amino acid sequence of the present invention, or part determine the eventual location of a protein in the cell, also thereof. Non-limiting examples of sequences of HIV-1 enve referred to as Sorting peptide. lopes are provided in SEQ ID NO: 74-119, 120-124, and 0138 Surfactant: A surface active agent capable of reduc 330-333. Thus, any HIV-1 envelope including those defined ing the Surface tension of a liquid in which it is dissolved. A by SEQ ID NO: 74-119, 120-124, and 330-333 as well as Surfactant is a compound containing a polar group which is functional homologes and/or fragments thereof as well as hydrophilic and a nonpolar group which is hydrophobic and polypeptides with at least 70% identities to said homologes or often composed of a fatty chain. fragments, such as at least 80% identities, for example at least 0139 Treg: Regulatory T cells/T lymphocytes 90% identities, such as at least 94, 95, 96, 97,98, or at least 0140 Vaccine: A substance or composition capable of 99% identity thereto are within the scope of the present inven inducing an immune response in an animal. Also referred to tion. hereinas an immunogenic composition. An immune response 0146 The present invention, thus, relates to an HIV-1 being an immune response (humoral/antibody and/or cellu envelope polypeptide comprising an amino acid sequence lar) inducing memory in an organism, resulting in the infec selected from the group of amino acid sequences consisting tious agent, being met by a secondary rather than a primary of X(1-22)-C(23)-X(24-28)-C(29)-X(30-50) (identified as response, thus reducing its impact on the host organism. A IGP1), or any part thereof, or functional homolog or any vaccine of the present invention may be given as a prophy amino acid sequence with at least 90% identity to said amino lactic and/or therapeutic medicament. The composition may acid sequence or part thereof. The 50 amino acid sequence is comprise one or more of the following: HIV-1 envelope derived from any known HIV-1 envelope subtype, such as polypeptide(s), antigencs), nucleic acid(s), vector(s), biologi those identified by SEQ ID NO: 74-119. Thus, the specific cal entities, adjuvants and pharmaceutical carriers. amino acid in position X of the amino acid sequence above US 2011/0305749 A1 Dec. 15, 2011

may be selected from an amino acid in the corresponding X(37): S, T, A, N, D, V, I, E, Y, K, L, R, G, P. M. F. W. H., Q, position of any of those and other HV-1 subtypes. Thus, in B, and C.; and particular, the invention relates to an HIV-1 envelope polypeptide comprising an amino acid sequence selected X(38): S, T, N., I, G, R, L, C, A, W, M and E; and from the group of amino acid sequences consisting of X(1- X(39): W. G. A. R. E. C. Y. V. S. M, and H; and 22)-C(23)-X(24-28)-C(29)-X(30-50), or any part thereof, or functional homolog or any amino acid sequence with at least X(40): X; and 90% identity to said amino acid sequence or part thereof, X(41): N, G, K, S, D, E,T, R, H, PA, B, V. Q, Y. M., and I; and wherein the amino acid residues in the X(1-22)-C(23)-X(24 28)-C(29)-X(30-50) amino acid sequence are selected from X(42): K, R, N, D, S, T, G, E., I, V.Y. Q, P, H, A, W. M., and C: the groups of residues consisting of: and X(43): S.T., N, K, I, R, D, E, P. L. A. W. G. M. H. Y. F. V. and X(1): L., S, R, P. F. A. V. M, and I; and C; and X(2): Q. R. K. H. L. M., and P; and X(44): L.Y. Q, F. E. H. S. V. K, M, T, I, W. N. D, R, P, A, and X(3): A, T, V, H, S, R, Q, G, M, and E; and G; and X(4): R. K. G. E. T. S. C. M., and H; and X(45): D, E, N, S, T, K, G, L, A, Q, H, I,Y, B, R, V. P. M., F. W. Z, and C.; and X(5): V. I, L, D, A, S, F, M, and G.; and X(46): E, D, Q, Y. K. N. T. S. A. W. H. M., R., I, G, L, V, Z, F, X(6): L. Q. V. M. P. W. T. and I; and B, and P; and X(7): A, S, T, V. L. G. F. D. M., and E; and X(47): I, D, E, M, G, T, Q, S. W. L., N.Y. K. V. R. F. A. P. and X(8): V. L. I. M. A. W. K. G. and E; and H; and X(9): E, K, G, D, A, V. M, and F; and X(48): W.I.T., N, D, E, L. G., S.Y. R. V. K, H, A, Q, M, and F: X(10): X; and and X(49): D, N, E, G, W, Q, K, H, L, B, S.I.Y.T. A. R,M, Z, and X(11): Y. L. F. H. C., I, T, M, and N; and V; and X(12): L., I, V, M, Q, P. T.Y. and A; and X(50): N, D, T, K, S, H. L. G. E. W., I, Q, M, R, B, Y. P. and A. X(13): K, R, Q, G.S., E. H. W. T. V. M. N.Z.Y.A. P. and C; and 0147 According to the present invention, at least one of X(14): D, N, G, E. Y. V. S., H, A, M, and I; and the amino acids in position 10, 19, 24, 34, and/or 40 affect the immunogenic properties of the HIV-1 envelope polypeptide. X(15): Q, R, H, K. P. L. M., and N; and Thus, the amino acids in those positions alone or in combi X(16): Q, K, R. T. H. E. S. P. M., and L; and nation affects the immunogenicity of the envelope polypep tide. Thus in a primary aspect, the present invention relates to X(17): L. F., I, R, V. P. S. M, and H; and any HIV-1 envelope polypeptide, such as any HIV-1 envelope X(18): L. M. P. I, H, and S; and as defined herein, comprising an amino acid sequence with a mutation in any one of the positions corresponding to amino X(19): X; and acid residue position 10, 19, 24, 34 and/or 40 of any one of the X(20): I, L. M. V. S., F.T. D, A, R, P, and J.; and amino acid sequences identified herein as IGP1-7, SEQ ID NO: 1-73, 125-329 and/or 330-337, and/or any amino acid X(21): W. R, G, F, L, M, and T; and sequence at least 60% identical thereto, such as at least 70%, for example at least 80% identical, such as at least 90%, such X(22): G, D, A. R. M., and C; and at least 91, 92,93, 94, 95, 96, 97,98, such as at least 99% X(24): X; and identical to any of those amino acid sequences carrying muta tions in one or more of the amino acid residue positions 10, X(25): G. R. E., N, A, M, and D; and 19, 24, 34 and/or 40. Thus, any HIV-1 envelope polypeptide X(26): K. R. N. E. Q, T. S. I. M., and G.; and comprising an amino acid sequence identified herein, which carries a mutation in a position Such as positions 10, 19, 24, 34 X(27): L, H, I, T, V. F. R. Q. S. P. A. J. M., and Y; and and/or 40 of any one of IGP1-7, SEQID NO: 1-73, 125-329, X(28): I, V, T, L, R, F, and M. and and/or 334-337 are within the scope of the present invention, as well as any HIV-1 envelope comprising an amino acid X(30): T. P.Y.A. N. S., I, V, R, L, M and H; and sequence, which with respect to the otheramino acid residues X(31): T. S. P.. N, M and I; and (i.e. amino acid residues 1-9, 11-18, 20-23, 25-33, 35-39 and 41-50) of for example any one of IGP1-7, SEQID NO: 1-73, X(32): A.N.T, S, D, R, F, Q, P, I, E. V. M. L., K, H, C, and B; 125-329, and/or 334-337 is at least 60% identical thereto, and such as at least 70%, for example at least 80% identical, such as at least 90%, such at least 91, 92,93, 94, 95, 96, 97,98, such X(33): V. A. L. M. G. R. and C; and as at least 99% identical to any of those amino acid sequences. X(34): X; and 0.148. In a specific embodiment, the amino acid residue in positionX(10) is selected from the group consisting of R, S, X(35): W. R. G. L. M., and P; and T. K. G. A. N. Q and/or I. In another specific embodiment, the X(36): N, S, D, B, K, E, R, Q, M, and G.; and amino acid residue in position X(19) is selected from the US 2011/0305749 A1 Dec. 15, 2011 group consisting of G. N. S. R. E. T. D. V. C and/or A. In any amino acid sequence with at least 90% identity to said another specific embodiment, the amino acid residue in posi amino acid sequence or part thereof. tionX(24) is selected from the group consisting of S. K.T. R. 0156. In yet another embodiment, the amino acid residue A. P. Y. F, G, Q, I and/or H. In another specific embodiment, in position X(24) is lysine (K), such that the HIV-1 envelope the amino acid residue in position X(34) is selected from the of the present invention comprises an amino acids sequence group consisting of: P. K. R. S. A. L. Q., E. H. T.I.V. and/or F. selected from the group of amino acid sequences consisting In another specific embodiment, the amino acid residue in of X(1-22)-C(23)-K(24)-X(25-28)-C(29)-X(30-50), or any position X(40) is selected from the group consisting of S, N, part thereof, or functional homolog or any amino acid G, T, R., I, V. K. W. A. P. Y. D, Q, H, E, and/or C. sequence with at least 90% identity to said amino acid 0149 More specifically, in a further embodiment, the sequence or part thereof. amino acid residue in positionX(10) is arginine (R). Such that 0157. In a further embodiment, the amino acid residue in the HIV-1 envelope of the present invention comprises an position X(34) is lysine (K), such that the HIV-1 envelope of amino acids sequence selected from the group of amino acid the present invention comprises an amino acids sequence sequences consisting of X(1-9)-R(10)-X(11-22)-C(23)-X selected from the group of amino acid sequences consisting (24-28)-C(29)-X(30-50), or any part thereof, or functional of X(1-22)-C(23)-X(24)-28)-C(29)-X(30-33)-K(34)-X(35 homolog or any amino acid sequence with at least 90% iden 50), or any part thereof, or functional homolog or any amino tity to said amino acid sequence or part thereof. acid sequence with at least 90% identity to said amino acid 0150. In another embodiment, the amino acid residue in sequence or part thereof. position X(19) is glycine (G), such that the HIV-1 envelope of 0158. In another embodiment, the amino acid residue in the present invention comprises an amino acids sequence position X(40) is serine (S), such that the HIV-1 envelope of selected from the group of amino acid sequences consisting the present invention comprises an amino acids sequence of X(1-18)-G(19)-X(20-22)-C(23)-X(24-28)-C(29)-X(30 selected from the group of amino acid sequences consisting 50), or any part thereof, or functional homolog or any amino of X(1-22)-C(23)-X(24-28)-C(29)-X(30-39)-K(40)-X(41 acid sequence with at least 90% identity to said amino acid 50), or any part thereof, or functional homolog or any amino sequence or part thereof. acid sequence with at least 90% identity to said amino acid 0151. In yet another embodiment, the amino acid residue sequence or part thereof. in position X(24) is serine (S), such that the HIV-1 envelope 0159. In a specific embodiment, the HIV-1 envelope of the present invention comprises an amino acids sequence polypeptide of the present invention comprises an amino acid selected from the group of amino acid sequences consisting sequence selected from the group of amino acid sequences of X(1-22)-C(23)-S(24)-X(25-28)-C(29)-X(30-50), or any consisting of IGP1-7 and SEQ ID NO: 1-337, such as the part thereof, or functional homolog or any amino acid group consisting of IGP1-7 and SEQID NO: 1-73, 125-329, sequence with at least 90% identity to said amino acid or 334-337, any part thereof, functional homolog and/or any sequence or part thereof. amino acid sequence with at least 90% identity to said amino acid sequence or part thereof. Each of those amino acids 0152. In a further embodiment, the amino acid residue in sequences, e.g. IGP1-7, SEQID NO: 1-73, 125-329, or 334 position X(34) is proline (P), such that the HIV-1 envelope of 337 are also within the scope of the present invention and, the present invention comprises an amino acids sequence consequently, an HIV-1 envelope polypeptide of the present selected from the group of amino acid sequences consisting invention, comprising any amino acid sequence selected from of X(1-22)-C(23)-X(24)-28)-C(29)-X(30-33)-P(34)-X(35 IGP1-7 or SEQ ID NO: 1-337, such as SEQ ID NO: 1-73, 50), or any part thereof, or functional homolog or any amino 125-329, or 334-337 are claimed as individual embodiments. acid sequence with at least 90% identity to said amino acid Also, among HIV-1 envelopes a frequent polymorphism exist sequence or part thereof. at the position corresponding to position 5 in SEQ ID NO: 0153. In another embodiment, the amino acid residue in 4-68. For example the first 10 amino acids of SEQID NO: 5 position X(40) is serine (S), such that the HIV-1 envelope of are LQARILAVETYLKDQQLLNI, but the I in position 5 is the present invention comprises an amino acids sequence replaced by Vin many naturally found envelopes, and also the selected from the group of amino acid sequences consisting L in position 17 is replaced by R in some clades or subtypes. of X(1-22)-C(23)-X(24-28)-C(29)-X(30-39)-S(40)-X(41 Thus, the present invention comprise HIV-1 envelope 50), or any part thereof, or functional homolog or any amino polypeptide of the present invention comprising either I or V acid sequence with at least 90% identity to said amino acid in a position corresponding to position 5 and L or R in a sequence or part thereof. position corresponding to position 17 of SEQID NO: 5, and 0154 More specifically, in a further embodiment, the I and V may replace each other in a position corresponding to amino acid residue in position X(10) is threonine (T), such position 5 and L and R may replace each other in a position that the HIV-1 envelope of the present invention comprises an corresponding to position 17 of SEQID NO. 5 in any of the amino acids sequence selected from the group of amino acid amino acid sequences provided in the present invention, sequences consisting of X(1-9)-T(10)-X(11-22)-C(23)-X including any one of IGP1-7 and SEQID NO: 1-337. (24-28)-C(29)-X(30-50), or any part thereof, or functional 0160. In a specific embodiment, the HIV-1 envelope homolog or any amino acid sequence with at least 90% iden polypeptide of the present invention comprises an amino acid tity to said amino acid sequence or part thereof. sequence selected from the group consisting of IGP1-7, SEQ O155 In another embodiment, the amino acid residue in ID NO: 1-19, or any part thereof, or functional homolog or position X(19) is Asparagine (N), such that the HIV-1 enve any polypeptide with at least 90% identity to said polypeptide lope of the present invention comprises an amino acids or part thereof. In another embodiment, the HIV-1 envelope sequence selected from the group of amino acid sequences polypeptide of the present invention comprises an amino acid consisting of X(1-18)-N(19)-X(20-22)-C(23)-X(24-28)-C sequence selected from the group consisting of SEQID NO: (29)-X(30-50), or any part thereof, or functional homolog or 20-63, or any part thereof, or functional homolog or any US 2011/0305749 A1 Dec. 15, 2011 polypeptide with at least 90% identity to said polypeptide or comprising an HIV-1 envelope polypeptide of the present part thereof. In another embodiment, the HIV-1 envelope invention that has a sequence that is at least 36% identical to polypeptide of the present invention comprises an amino acid the amino acid sequence of any polypeptide of the present sequence selected from the group consisting of SEQID NO: invention, or is a fragment of a sequence that is at least 36% 64-68, or any part thereof, or functional homolog or any thereto. polypeptide with at least 90% identity to said polypeptide or 0.167 However, in other embodiments of the present part thereof. invention the HIV-1 envelope polypeptide or a fragment 0161. As non-limiting examples of a specific embodiment thereof has a sequence that is for example at least 40%. Such of the present invention, is an HIV-1 envelope polypeptide as at least 45%, for example at least 50%, such as at least 55%, comprising or consisting of the amino acid sequence LRAR for example at least 60%, such as at least 65%, for example at LLALETFIQNQQLLNLWGCKGNLICYTS least 67%, such as at least 70%, for example at least 72%, VKWNDTWKGNSDTSLENIWDN (SEQID NO:4), LQA such as at least 75%, for example at least 77%, such as at least RIMAVERYLKDQQLLGIWGCSGKLICTTAVPWNASW 80%, for example at least 81%, such as at least 82%, for SNKSLEQIWNH (SEQ ID NO: 20), QARILAVERY example at least 83%, such as at least 84%, for example at LKDQQLLRIWGCFGKLICTTAVPW least 85%, such as at least 86%, for example at least 87%, NASWSNKSLEQIWNH (SEQ ID NO: 64), HIV G19R: such as at least 88%, for example at least 89%, such as at least LQARVLAVERYLKDQQLLRIWGC (SEQ ID NO: 132), 90%, for example at least 91%, such as at least 92%, for HIV M/O chimera: LQARILAVETLIQNQQLLNLWGC example at least 93%, such as at least 94%, for example at (SEQ ID NO: 133) and/or any part thereof, or functional least 95%, such as at least 96%, for example at least 97%, homolog or any polypeptide with at least 70%, such as at least such as at least 98%, for example at least 99% identical to the 80%, for example at least 90% identity to said polypeptide or amino acid sequence of any polypeptide of the present inven part thereof. tion. 0162 Specific examples of HIV-1 envelope polypeptides according to the present invention are represented by SEQID Production of Peptides NO: 120-124, 325, and 330-333. Thus in one embodiment, the HIV-1 envelope of the present invention is selected from 0.168. The polypeptides of the invention can be produced the group consisting of SEQID NO: 120-124, 325, and 330 by any known method of producing a linear amino acid 333. sequence either being synthesized as a synthetic polypeptide 0163 The polypeptides of the present invention may be or produced by recombinant DNA techniques. A nucleic acid free carboxyl- or amino groups, amides, acyls, acetyls or salts sequence which encodes a polypeptide of the invention or a thereof, two or more of the Cys residues may form part of an multimer of the said polypeptides is introduced into an intrachain- or interchain disulphide binding, a —S—(CH2) expression vector. Suitable expression vectors are for p-S or a —(CH2)p-bridge wherein p=1-8, optionally inter instance plasmids, cosmids, viruses and YAC (yeast artificial vened by one or more heteroatoms such as O, N or S. The C chromosome) which comprise necessary control regions for and N-terminals ends of the polypeptide sequences could replication and expression. The expression vector may be deviate from the natural sequences by modification of the stimulated to expression in a host cell. Suitable host cells are terminal NH2-group and/or COOH-group. They may, for for example bacteria, yeast cells and mammal cells. Such techniques are well known in the art and described for instance, be acylated, acetylated, amidated or modified to instance by Sambrook et al., Molecular Cloning: A Labora provide a binding site for a carrier or another molecule. tory Manual, Cold Spring Harbor Laboratory Press, Cold 0164. The polypeptides may be lyophilized, or dissolved Spring Harbor, 1989. Other well-known techniques are deg or suspended in a suitable liquid buffer. Moreover, the HIV-1 radation or synthesis by coupling of one amino acid residue to envelope may be comprised in a composition, or integrated in the next one in liquid phase or preferably on a solid phase a biological entity, for example in the membrane of a cell, or (resin) for instance by the so-called Merrifield synthesis. See on the surface of a viral particle. Thus, the polypeptide or for instance Barany and Merrifield in the Peptides, Analysis, polypeptide antigen according to the invention is either in a Synthesis, Biology, Vol. 2, E. Gross and Meinhofer, Ed. free or in a carrier-bound form. The carrier or solid phase to (Acad. Press, N.Y., 1980), Kneib-Coronier and Mullen Int. J. which the peptide is optionally bound can be selected from a Peptide Protein Res., 30, p. 705-739 (1987) and Fields and wide variety of known carriers. It should be selected with Noble Int. J. Peptide Protein Res., 35, p. 161-214 (1990). In regard to the intended use of the immobilized polypeptide as case a linked or cyclic peptide is desired, the amino acid a diagnostic antigen or as an immunizing component in a sequence is subjected to a chemical oxidation step in order to vaccine. cyclize or link the two cysteine residues within one or 0.165 Examples of carriers that can be used for diagnostic between two peptide sequences, when the appropriate linear purposes, for example, are magnetic beads or latex of co polymers such as styrene-divinyl benzene, hydroxylated Sty amino acid sequences are synthesized, see Akaji et al., Tetra rene-divinylbenzene, polystyrene, carboxylated polystyrene, hedron Letter, 33, 8, p. 1073-1076, 1992. beads of carbon black, non-activated or polystyrene or poly Antigen vinyl chloride activated glass, epoxy-activated porous mag netic glass, gelatine or polysaccharide particles or other pro 0169. One aspect of the present invention relates to an tein particles, red blood cells, mono- or polyclonal antibodies antigen, which comprises a peptide derived from an HIV-1 or fab fragments of Such antibodies, liposomes and/or nano envelope polypeptide of the present invention. In particular, particles. the antigen incorporates a part of HIV-1 envelope polypep 0166 Further embodiments of the invention include any tide, including any region of gp41 and/or gp120. Such as for vector, method, provirus, retroviral particle, use, composi example the transmembrane domain (TM-domain)) or Sur tion, vaccine, Vaccine composition or kit as described herein, face subunit (SU). Preferably, the antigen comprises at least US 2011/0305749 A1 Dec. 15, 2011

one peptide with an amino acid sequence of an HIV-1 enve acid sequence according to the present invention or a func lope polypeptide of the present invention, or a functional tional homolog or a fragment thereof, or a nucleic acid encod homolog thereofhaving at least 70%, such as at least 80%, for ing a polypeptide with at least 70% identity thereto. The example at least 90% identity to said envelope polypeptide or vector of the present invention is a prokaryotic expression an immunological active fragment comprising or consisting vector or a eukaryotic expression vector, preferably a mam of a consecutive sequence of at least 10 amino acids selected malian expression vector. Thus, in one embodiment, the from a region of said envelope polypeptide. In another present invention relates to an isolated eukaryotic expression embodiment, the antigen of the present invention comprises or consists of at least one peptide with an amino acid sequence vector comprising at least one nucleic acid sequence encod comprising an immunological active fragment comprising or ing at least one HIV-1 envelope polypeptide of the present consisting of a consecutive sequence of at least 12 amino invention, or a fragment thereof and/or a nucleic acid acids, such as at least 15 amino acids, for example at least 20 sequence encoding at least one antigen as defined herein. amino acids, such as 30 amino acids, such as at least 40 amino 0.173) Numerous vectors are available and the skilled per acids, for example at least 50 amino acids, Such as 60 amino son will be able to select a useful vector for the specific acids, such as at least 70 amino acids, for example at least 80 purpose. The vector may, for example, be in the form of a amino acids, such as 90 amino acids, such as at least 100 plasmid, cosmid, viral particle or artificial chromosome. The amino acids, such as at least 110 amino acids, for example at appropriate nucleic acid sequence may be inserted into the least 120 amino acids, such as 130 amino acids, Such as at vector by a variety of procedures, for example, DNA may be least 140 amino acids, for example at least 150 amino acids, inserted into an appropriate restriction endonuclease site(s) Such as 160 amino acids, such as at least 170 amino acids, for using techniques well known in the art. Apart from the nucleic example at least 180 amino acids, such as 190 amino acids, acid sequence according to the invention, the vector may such as at least 200 amino acids, such as at least 300 amino furthermore comprise one or more of a signal sequence, an acids, for example at least 400 amino acids, such as 500 origin of replication, one or more marker genes, an enhancer amino acids, such as at least 600 amino acids, for example at element, a promoter, and a transcription termination least 700 amino acids, such as 800 amino acids, such as at sequence. The vector may also comprise additional least 900 amino acids, for example at least 1000 amino acids sequences. Such as enhancers, poly-A tails, linkers, polylink selected from a region of an HIV-1 envelope polypeptide of ers, operative linkers, multiple cloning sites (MCS), STOP the present invention, for example selected from a sequence codons, internal ribosomal entry sites (IRES) and host according to any of SEQID NO: 1-326, IGP1-7 or SEQ ID homologous sequences for integration or other defined ele NO: 327-337. ments. Methods for engineering nucleic acid constructs are 0170 The present invention also relates to an antigen com well known in the art (see, e.g., Molecular Cloning: A Labo prising or consisting of at least one peptide having at least ratory Manual, Sambrook et al., eds., Cold Spring Harbor 50%, such as at least 60%, for example at least 70% such as at Laboratory, 2nd Edition, Cold Spring Harbor, N.Y., 1989). least 80% for example at least 90%, such as at least 95%, such The vector is preferably an expression vector, comprising the as at least 98%, for example at least 99% identity to any nucleic acid operably linked to a regulatory nucleic acid polypeptide according to the present invention, for example a sequence directing expression thereof in a Suitable cell. sequence according to any of SEQID NO: 1-124 or SEQID Within the scope of the present invention said regulatory NO: 125-326, IGP1-7 or SEQID NO:327-337 or any part or nucleic acid sequence should in general be capable of direct functional homolog or immunological active fragment ing expression in a mammalian cell, preferably a human cell, thereof, for example any part or functional homolog or immu more preferably in an antigen presenting cell or a T-cell. nological active fragment thereof comprising or consisting of 0.174. In one preferred embodiment the vector is a viral at least 3, Such as at least 5, Such as at least 10, or Such as at vector. The vector may also be a bacterial vector, Such as an least 15 consecutive amino acid residues. attenuated bacterial vector. Attenuated bacterial vectors may Nucleic Acid be used in order to induce lasting mucosal immune responses 0171 In one aspect, the present invention relates to a at the sites of infection and persistence. Different recombi nucleic acid sequence, e.g. DNA and/or RNA, encoding at nant bacteria may be used as vectors, for example the bacte least one HIV-1 envelope polypeptide of the present inven rial vector may be selected from the group consisting of tion, or a fragment thereof and/or a nucleic acid sequence Salmonella, Lactococcus, and Listeria. In general, induction encoding at least one antigen as defined herein. Thus in a of immunity to the heterologous antigen HPV 16 L1 or E7 specific embodiment, the present invention relates to a could be shown, with strong CTL induction and tumor regres nucleic acid sequence encoding an HIV-1 envelope polypep sion in mice. The vector may furthermore comprise a nucleic tide comprising or consisting of an amino acid sequence acid encoding a T-cell stimulatory polypeptide. selected from the group consisting of IGP1-7, SEQID NO:1- 0.175. The vector of the present invention may be any 124, SEQ ID NO: 125-326 or SEQ ID NO:327-337 and/or eukaryotic expression vector, for example a mammalian any part or functional homolog thereof. It is understood that a expression vector, or a yeast vector. The vector may comprise nucleic acid encoding any sequence selected from any of at least one intron, which will facilitate the transport from the IGP1-7, SEQID NO: 1-124, SEQID NO: 125-326 or SEQID nucleus to the cytoplasma of the vector encoded RNA, for NO:327-337 or part thereof, constitute a single embodiment, example in packaging cells. In another embodiment, the vec and is consequently claimed individually. In one embodi tor is capable of expressing RNA in the cytoplasm by cyto ment, the nucleic acid sequence has been optimized by codon plasmic transcription, which can be translated into envelope optimization, as explained elsewhere herein. polypeptide. The vector is also, in one embodiment, capable of expressing high levels of vector encoded RNA, which is Vectors transported to the cytoplasma to be translated into envelope 0172. In one aspect, the present invention relates to an polypeptide as encoded in the vector. Thus, in one embodi isolated expression vector comprising at least one nucleic ment the vector of the present invention is transcribed in the US 2011/0305749 A1 Dec. 15, 2011 nucleus, thereby producing high levels of transcript, which after transport to the cytoplasm can be translated into enve TABLE 3-continued lope polypeptide. The vector of the present invention may be transfected into a packaging cell which is capable of produc List of gammaretroviruses ing viral particles comprising said lentiviral envelope Mammalian virus group polypeptide. Murine endogenous retrovirus Murine leukemia-related retroviruses 0176). In one embodiment, the vector is a retroviral vector. Epicrionops marmoratus retrovirus The retroviral vector may be either replication deficient or Ichthyophis kohtaoensis retrovirus replication competent. Osteolaemus tetraspis retrovirus 0177. The retroviral vector can be derived from any spe Sericulus bakeri retrovirus cies of retroviridae. In one embodiment, the retroviral vector Terdus iliacus retrovirus Tomistoma Schlegelii retrovirus is derived from Orthoretrovirinae, comprising Alpharetrovi Viper berus retrovirus rus, Betaretrovirus, and Gammaretrovirus. In a specific Xenotropic Mul V-related virus embodiment, the retroviral vector is derived from Avian car Monodelphis sp. retrovirus cinoma Mill Hill virus 2, Avian leukosis virus, Avian myelo Replication competent viruses blastosis virus, Avian myelocytomatosis virus 29, Avian sar Feline leukemia virus Gibbon ape leukemia virus (GALV) coma virus CT10, Fujinami sarcoma virus, Rous sarcoma Murine leukemia virus virus, UR2 sarcoma virus orY73 sarcoma virus. The alphavi Porcine type-Concovirus ruses are listed in table 1. Each of the alphaviruses specified Replication defective viruses above is intended to be an individual embodiment. Conse Abelson murine leukemia virus quently, a retroviral vector according to the present invention Gardner-Arnstein feline sarcoma virus derived from each of them is claimed individually. Hardy-Zuckerman feline sarcoma virus Harvey murine sarcoma virus Kirsten murine sarcoma virus TABLE 1. Moloney murine sarcoma virus Murine Osteosarcoma virus List of alpharetroviruses Snyder-Theilen feline sarcoma virus Spleen focus-forming virus Alpharetrovirus Avian carcinoma Mill Hill virus 2 Woolly monkey sarcoma virus Alpharetrovirus Avian leukosis virus unclassified Gammaretrovirus Alpharetrovirus Avian myeloblastosis virus Baboon endogenous virus Alpharetrovirus Avian myelocytomatosis virus 29 Baboon endogenous virus strain M7 Alpharetrovirus Avian sarcoma virus CT10 Feline endogenous virus Alpharetrovirus Fujinami sarcoma virus Feline endogenous virus ECE1 Alpharetrovirus Rous sarcoma virus Feline endogenous virus RD114 Alpharetrovirus UR2 sarcoma virus Koala retrovirus Alpharetrovirus Y73 sarcoma virus Macaca mulatta type C retrovirus Macaca endogenous retrovirus MLV-related retrovirus 0178. In another specific embodiment, the retroviral vec Ratleukemia virus tor is derived from Jaagsiektesheep retrovirus, Langur virus, Ratsarcoma virus Mason-Pfizer monkey virus, Mouse mammary tumor virus or RD114 retrovirus Recombinant M-MuLVRaLV retrovirus Squirrel monkey retrovirus. The betaviruses are listed in table Mammalian virus group 2. Each of the betaviruses specified herein is intended to be an Murine endogenous retrovirus individual embodiment. Consequently, a retroviral vector Murine leukemia-related retroviruses according to the present invention derived from each of them Epicrionops marmoratus retrovirus chthyophis kohtaoensis retrovirus may be claimed individually. Osteolaemus tetraspis retrovirus Sericulus bakeri retrovirus TABLE 2 Terdus iliacus retrovirus Tomistoma Schlegelii retrovirus List of beta.retroviruses Viper berus retrovirus Xenotropic MuLV-related virus Beta.retrovirus Jaagsiektesheep retrovirus Xenotropic Mul V-related virus VP35 Beta.retrovirus Langur virus Xenotropic Mul V-related virus VP42 Beta.retrovirus Mason-Pfizer monkey virus Xenotropic Mul V-related virus VP62 Beta.retrovirus Mouse mammary tumor virus Monodelphis sp. retrovirus Beta.retrovirus Squirrel monkey retrovirus Replication competent viruses Feline leukemia virus Feline leukemia provirus (clone CFE-16) 0179. In another embodiment the retroviral vector accord Feline leukemia provirus (clone CFE-6) ing to the present invention is derived from gammaretrovi Feline leukemia provirus fit Feline leukemia virus strain AGlasgow-1 ruses as shown in table 3 below. Feline leukemia virus strain Blambda-B1 Feline leukemia virus strain CFA27 TABLE 3 Feline leukemia virus strain CFS246 Feline leukemia virus strain CSarma List of gammaretroviruses Feline sarcoma virus Gardner-Arnstein feline leukemia oncovirus B Avian (Reticuloendotheliosis) virus group Gibbon ape leukemia virus (GALV) Chick syncytial virus Simian sarcoma-associated virus Reticuloendotheliosis virus Murine leukemia virus Avian spleen necrosis virus AKR (endogenous) murine leukemia virus Spleen necrosis virus Friend murine leukemia virus US 2011/0305749 A1 Dec. 15, 2011 17

virus, Woolly monkey sarcoma virus, unclassified Gam TABLE 3-continued maretrovirus, Baboon endogenous virus, Baboon endog enous virus strain M7, Feline endogenous virus, Feline List of gammaretroviruses endogenous virus ECE1, Feline endogenous virus RD114, Moloney murine leukemia virus Koala retrovirus, Macaca mulatta type C retrovirus, Macaca Murine leukemia virus isolates endogenous retrovirus, MLV-related retrovirus, Ratleukemia unclassified Murine leukemia virus virus, Rat sarcoma virus, RD114 retrovirus, Recombinant Porcine type-Concovirus M-MuV/Ral V retrovirus, Murine endogenous retrovirus, Porcine endogenous retrovirus Porcine endogenous type Cretrovirus Murine leukemia-related retroviruses, Epicrionops marmora Replication defective viruses tus retrovirus, Ichthyophis kohtaoensis retrovirus, Osteolae Abelson murine leukemia virus mus tetraspis retrovirus, Sericulus bakeri retrovirus, Terdus Gardner-Arnstein feline sarcoma virus iliacus retrovirus, Tomistoma schlegelii retrovirus, Viper Hardy-Zuckerman feline sarcoma virus berus retrovirus, Xenotropic Mul V-related virus, Xenotropic Feline sarcoma virus (STRAIN HARDY-ZUCKERMAN 2) MuDV-related virus VP35, Xenotropic MuDV-related virus Feline sarcoma virus (STRAIN HARDY-ZUCKERMAN 4) Harvey murine sarcoma virus VP42, Xenotropic Mul V-related virus VP62, Monodelphis Kirsten murine sarcoma virus sp. retrovirus, Replication competent viruses, Feline leuke Moloney murine sarcoma virus mia virus, Feline leukemia provirus (clone CFE-16), Feline Cas-NS-1 murine sarcoma virus leukemia provirus (clone CFE-6), Feline leukemia provirus FBJ murine osteosarcoma virus fit, Feline leukemia virus strain A/Glasgow-1, Feline leuke Moloney murine sarcoma virus (STRAIN HT-1) Moloney murine sarcoma virus (STRAINM1) mia virus strain B/lambda-B1, Feline leukemia virus strain Moloney murine sarcoma virus (strain TS110) C/FA27, Feline leukemia virus strain C/FS246, Feline leuke Murine sarcoma virus 3611 mia virus strain C/Sarma, Feline sarcoma virus, Gardner Myeloproliferative sarcoma virus Arnstein feline leukemia oncovirus B. Gibbon ape leukemia NS.C58 murine sarcoma virus virus (GALV), Simian sarcoma-associated virus, Murine leu Murine osteosarcoma virus FBR murine osteosarcoma virus kemia virus, AKR (endogenous) murine leukemia virus, Snyder-Theilen feline sarcoma virus Friend murine leukemia virus, Moloney murine leukemia Spleen focus-forming virus virus, Murine leukemia virus isolates, unclassified Murine Friend spleen focus-forming virus leukemia virus, Porcine type-C oncovirus, Porcine endog Rauscher spleen focus-forming virus enous retrovirus, Porcine endogenous type Cretrovirus, Rep Woolly monkey sarcoma virus lication defective viruses, Abelson murine leukemia virus, Gardner-Arnstein feline sarcoma virus, Hardy-Zuckerman feline sarcoma virus, Feline sarcoma virus (STRAIN 0180 Thus, the retroviral vector is in one embodiment HARDY-ZUCKERMAN 2), Feline sarcoma virus (STRAIN derived from Chick syncytial virus, Feline leukemia virus, HARDY-ZUCKERMAN 4), Harvey murine sarcoma virus, Finkel-Biskis-Jinkins murine sarcoma virus, Gardner-Arn Kirsten murine sarcoma virus, Moloney murine sarcoma Stein feline sarcoma virus, Gibbon ape leukemia virus, virus, Cas-NS-1 murine sarcoma virus, FBJ murine osteosa Guinea pig type-C oncovirus, Hardy-Zuckerman feline sar rcoma virus, Moloney murine sarcoma virus (STRAIN coma virus, Harvey murine sarcoma virus, Kirsten murine HT-1), Moloney murine sarcoma virus (STRAIN M1), Molo sarcoma virus, Moloney murine sarcoma virus, Murine leu ney murine sarcoma virus (strain TS110), Murine sarcoma kemia virus (MLV), Porcine type-C oncovirus, Reticuloen virus 3611, Myeloproliferative sarcoma virus, N.S.C58 dotheliosis virus, Snyder-Theilen feline sarcoma virus, murine sarcoma virus, Murine osteosarcoma virus, FBR Trager duck spleen necrosis virus, Viper retrovirus or Woolly murine osteosarcoma virus, Snyder-Theilen feline sarcoma monkey sarcoma virus. See table 3 for a list of gammaviruses. virus, Spleen focus-forming virus, Friend spleen focus-form Each of the gammaviruses specified herein is intended to be ing virus, Rauscher spleen focus-forming virus or Woolly an individual embodiment. Consequently, a retroviral vector monkey sarcoma virus. Each of the gammaviruses mentioned according to the present invention derived from each of them above is intended to be an individual embodiment. Conse may be claimed individually. In a particularly preferred quently, a retroviral vector according to the present invention embodiment, the retroviral vector is derived from Murine derived from each of them is claimed individually. Leukemia Virus (MLV) or Moloney Murine Leukemia Virus 0182. In a further embodiment, the retroviral vector is (MoMLV) or Akv MLV. derived from Bovine leukemia virus, Primate T-lymphotropic 0181. In a specific embodiment, the retroviral vector is virus 1, Primate T-lymphotropic virus 2 or Primate T-lym derived from Avian (Reticuloendotheliosis) virus group such photropic virus 3. The deltaviruses are listed in table 4. Each as Chick syncytial virus, Reticuloendotheliosis virus, Avian of the deltaviruses mentioned herein is intended to be an spleen necrosis virus, Spleen necrosis virus, Mammalian individual embodiment. Consequently, a retroviral vector virus group, Murine endogenous retrovirus, Murine leuke according to the present invention derived from each of them mia-related retroviruses, Epicrionops marmoratus retrovirus, is claimed individually. Ichthyophis kohtaoensis retrovirus, Osteolaemus tetraspis retrovirus, Sericulus bakeri retrovirus, Terdus iliacus retrovi rus, Tomistoma schlegelii retrovirus, Viperberus retrovirus, TABLE 4 Xenotropic MuDV-related virus, Monodelphis sp. retrovirus, List of delta retroviruses Replication competent viruses, Feline leukemia virus, Gib Delta retrovirus Bovine leukemia virus bon ape leukemia virus (GALV), Murine leukemia virus, Delta retrovirus Primate T-lymphotropic virus 1 Porcine type-C oncovirus, Replication defective viruses, Delta retrovirus Primate T-lymphotropic virus 2 Abelson murine leukemia virus, Gardner-Arnstein feline sar Delta retrovirus Primate T-lymphotropic virus 3 coma virus, Hardy-Zuckerman feline sarcoma virus, Harvey murine sarcoma virus, Kirsten murine sarcoma virus, Molo ney murine sarcoma virus, Murine osteosarcoma virus, Sny 0183 In yet a further embodiment, the retroviral vector is der-Theilen feline sarcoma virus, Spleen focus-forming derived from Walleye dermal sarcoma virus, Walleye epider US 2011/0305749 A1 Dec. 15, 2011 mal hyperplasia virus 1 or Walleye epidermal hyperplasia selected from the group consisting of the enhanced green virus 2. The epsilonViruses are listed in table 5. Each of the fluorescent protein (eGFP), lac Z, dsRed, enhanced yellow epsilonViruses mentioned herein is intended to be an indi fluorescent protein (eyFP), enhanced cyan fluorescent pro vidual embodiment. Consequently, a retroviral vector accord tein (eCFP), enhanced blue fluorescent protein (eBFP) and ing to the present invention derived from each of them may is the human alpha-1-antitrypsin (ha AT). It is understood that individually. any of the enhanced green fluorescent protein (eGFP), lac Z. dsRed, enhanced yellow fluorescent protein (eyFP), TABLE 5 enhanced cyan fluorescent protein (eCFP), enhanced blue List of epsilon retroviruses fluorescent protein (eBFP) or the human alpha-1-antitrypsin (ha AT) there are also claimed in separate embodiments. In a Epsilonretrovirus Walleye dermal sarcoma virus Epsilonretrovirus Walleye epidermal hyperplasia virus 1 preferred embodiment the eGFP gene is used. Epsilonretrovirus Walleye epidermal hyperplasia virus 2 0187. In another embodiment of the present invention, the vectors and/or nucleic acids of the present invention comprise 0184. In a primary aspect of the present invention is pro a Suicide gene and/or a selection gene encoding a selective vided a replication deficient retroviral vector. The vector marker. The selection gene of the present invention may be comprises at least one heterologous nucleic acid sequence any gene Suitable for example for selecting cells harbouring encoding an HIV-1 envelope polypeptide of the present the vector constructs of the present invention. Typically the invention or a fragment thereof. In one embodiment, the selection gene is a gene that confers resistance to antibiotics lentiviral envelope polypeptide or fragment thereof is derived or drugs. Examples of such selection genes are the puromycin from Bovine immunodeficiency virus, Caprine arthritis resistance gene (Puro), the tetracycline resistance gene, the encephalitis virus, Equine infectious anemia virus, Feline streptomycin resistance gene, the hygromycin B resistance immunodeficiency virus, Human immunodeficiency virus 1. gene (Hygro), the Zeocin resistance gene (Zeo), the neomycin Human immunodeficiency virus 2, Puma lentivirus, Simian resistance gene (neo), and the blasticidin resistance gene immunodeficiency virus, Visna/maedi virus or hepatitis C. (Bst). Therefore, the selection gene of the present invention is The lentiviruses, wherefrom the lentiviral envelope or frag selected from the group consisting of puromycin resistance ment thereof can be derived are listed in table 6. gene (Puro), the tetracycline resistance gene, the Streptomy cin resistance gene, the hygromycin B resistance gene (Hy TABLE 6 gro), the Zeocin resistance gene (Zeo), the neomycin resis List of lentiviruses from which a nucleic acid sequence encoding tance gene (neo) and the blasticidin resistance gene (Bst). In an envelope polypeptide or fragment thereof is used according a preferred embodiment the selection gene is selected from to the present invention. the group consisting of puromycin resistance gene (Puro), the Lentivirus Bovine immunodeficiency virus hygromycin B resistance gene (Hygro), the Zeocin resistance Lentivirus Caprine arthritis encephalitis virus gene (Zeo), the neomycin resistance gene (neo) and the blas Lentivirus Equine infectious anemia virus ticidin resistance gene (Bst). It is appreciated that the resis Lentivirus Feline immunodeficiency virus Lentivirus Human immunodeficiency virus 1 tance gene is selected from any of puromycin resistance gene Lentivirus Human immunodeficiency virus 2 (Puro), the tetracycline resistance gene, the streptomycin Lentivirus Puma lentivirus resistance gene, the hygromycin B resistance gene (Hygro), Lentivirus Simian immunodeficiency virus the Zeocin resistance gene (Zeo), the neomycin resistance Lentivirus Visnamaedi virus gene (neo) or the blasticidin resistance gene (Bst). In a pre ferred embodiment the resistance gene is the neomycin resis 0185. Each of the lentiviruses mentioned above is tance gene. In another preferred embodiment, the selective intended to be an individual embodiment. Consequently, a marker is neomycin phosphotransferase II. retroviral vector comprising at least one heterologous nucleic acid sequence encoding an HIV-1 envelope polypeptide of the 0188 The Suicide gene and/or a selection gene encoding a present invention or a fragment thereof derived from each of selective marker according to the present invention is in them is claimed individually. another embodiment Herpes simplex virus thymidine kinase (HSV-TK). Reporter Gene, Suicide Gene and Selectable Markers 0189 In a specific embodiment, the genes of the nucleic acids and/or vectors of the present invention are under the 0186 The vector of the present invention may comprise at control of a constitutive promoter, however in another least one additional nucleic acid sequence, in addition to a embodiment, the promoter is non-constitutive and may be nucleic acid encoding an HIV-1 envelope. In a particular activated. embodiment of the present invention, the vectors and/or nucleic acids of the present invention comprise a reporter Constitutive Transport Element (CTE) and Rev Responsive gene. Thus, in one embodiment, the at least one additional Element (RRE) nucleic acid sequence of a vector of the present invention encodes a reporter gene. In the present context the term 0190. The nucleic acids, including vectors and genomes of “reporter gene’ refers to any reporter gene that can be used to biological entities, of the present invention, in one embodi evaluate whether a host cell harbours the vector, provirus, ment, further comprise a constitutive transport element retroviral particle, composition and/or kit of the present (CTE) that is characterised in that it serves as a signal of invention. A number of reporter genes and systems for detec nuclear export of unspliced viral RNAs. The CTE of the tion exist which will be appreciated by a person skilled in the present invention may be selected from the CTEs listed below art. For example the reporter gene of the present invention is in table 6A. US 2011/0305749 A1 Dec. 15, 2011 19

at least two nucleic acid sequences, such as at least three TABLE 6A nucleic acid sequences. Such as at least four nucleic acid sequences. Such as at least five nucleic acid sequences. The CTE elements additional nucleic acid sequences preferably comprise a translatable open reading frame. The nucleic acid sequences Element Origin Ref and/or vectors of the present invention are thus in one CTE Mason-Pfizer Monkey Virus Bray et al 1994 PNAS embodiment bi- or multicistronic. Therefore, in a preferred RTE Rodent intracisternal type Wodrich et al 2001 JVirol embodiment, the nucleic acids and/or vectors of the present A particle element (LAPE) CTE Simian type D retrovirus Tabernero et al 1996 J invention further comprise at least one internal ribosomal Virol entry site. Thus, the vector according to the present invention WPRE Woodchuck Hepatitis virus Zufferey et al 1999 J Virol comprises, in one embodiment, two additional nucleic acid RTEm26 Rodent intracisternal type Smulewitch et all 2005 J sequences, three, four, five or six additional nucleic acid A particle element (LAPE) Virol sequences and at least two IRES elements, three, four, five or DR Rous SarcomaVirus Paca et al 2000JVirol six IRES elements. In one embodiment, the at least one IRES are of different origin. Specifically, the nucleic acid sequence 0191 In a particular embodiment the CTE is derived from encoding an HIV-1 envelope polypeptide of the present for Mason-Pfizer monkey virus, in another preferred embodi invention or fragment thereof may be preceded by an IRES. ment the CTE is derived from the Woodchuck Hepatitis virus, Similarly, any of the at least one additional nucleic acid for example the Woodchuck Hepatitis virus posttranscrip sequences encoding an HIV-1 envelope polypeptide or a part tional regulatory element (WPRE). thereof, as mentioned herein, may be preceded by an IRES. In 0.192 Moreover, the vector may comprise a Rev respon one embodiment of the present invention the HIV-1 envelope sive element (RRE). The presence of an RRE in the RNA polypeptide or fragment thereof is not translated under the transcript facilitates its transport from the nucleus to the cyto control of IRES.. An IRES preceding a nucleic acid sequence plasma, for example in a semipackaging cell, when REV/ results in the translation of said sequence under the control of REX is coexpressed. In one specific embodiment, the vector the IRES. The IRES elements according to the present inven is transcribed in the cytoplasm, thereby producing high levels tion may be derived from picornaviridae, retroviridae or ret of transcript, which can be translated into envelope polypep rotransposons, mammalia or combinations thereof. In a spe tide. This envelope polypeptide may then be incorporated into cific embodiment, the IRES is selected from the IRES viral particles in a packaging/producer cell. elements ofencephalomyocarditis (ECMV) or another picor navirus. Specifically, the IRES according to the present inven Internal Ribosomal Entry Site (IRES) tion can be derived from the IRES element of eIF4G. In further embodiments of the present invention, the vector.com 0193 The present invention also relates to a vector com prises an IRES element, which is selected from any of the prising at least one additional nucleic acid sequence. Such as IRES elements listed in the tables below.

TABLE 7 IRES elements which can be used according to the present invention. RNA. 2006 Oct; 12(10): 1755-85 TABLE 1. Reported cellular IRES Gene name Function Organism (2) seqpos Reference C-Casc) II CSubunit Rat 203203 (200-431) Pir(2) et al., 2001 Ca(2)-dependent kinase II ABETA Amolaid (2) precursor protein Human 341201 (899-1049) Qin and Saman 2004 AMURUNA1 (2) factor Human 2944212 (8496-10040) (2)ner et al., 2000 Antip AC) Drosophila 6648961 (1-1209) Oh et al., 1992 Apaf-1 Pro-apoptosis factor Human 2330014 (1-200) Coldwell et al., 2000 APC Adenomatosis peptides coli gene Human 82396 (486-520) Heppaner Goss et al., 2002 ARC Cytoskeleton association protein Rat 834413 (1-200) PirC2) et al., 2001 ATIR Angiotensin II type I Human 8490885 (1-273) Mertin et al., 2003 receptor-Coprotein cocpied receptor BAC-1 p36 Anti-apoptosis factor Human 143475 (1-413) Coldwell et al., 2001 Bcl-2 Anti-apoptotic factor Human 29366 (313-1463) Sherrilet al., 2004 (2) ER protein chaperone Human 143393 (1-225) Macerak and Samow 1991 BPix-b Bak-interacting exchange factor Mouse 32288334 (1-375) Rhes et al., 2004 isoform b Cat-1 Cationic amino acid transporter Rat 8542233 (1-273) Femander et al., 2001 c-fos Transcription factor Chicken 232221 (500-815) Sehgal et al., 2000 c-Myb Transcription factor Human 45502012 (1-202) Mitchell et al., 2005 c-Myc Transcription factor Human 1493193 (2501-2881, Stoneley et al., 1998 4506-4523) Connexin26 Gap junction protein Human 762120 (1472-1631, Lahlou et al., 2005 4780-4804) Connexin32 Gap junction protein Human 974140 (404-529,884–903) Hudder and Werner 2000 Connexin43 Gap junction protein Rat 43393193 (1-232) SchC2) et al., 1999 CCND1 Cyclin D1 Human 22788593 (1380-3591) S(2) et al., 2005 DAP3 Translation initiation factor Human 1903413 (1-357) Henis-KonnenbC2) et al., 2000 US 2011/0305749 A1 Dec. 15, 2011 20

TABLE 7-continued IRES elements which can be used according to the present invention. RNA. 2006 Oct; 12(10): 1755-85 TABLE 1. Reported cellular IRES Gene name Function Organism (2) seqpos Reference Dendrin Putative modulator of the Rat 1252674(1-151) Pinkstaffet al., 2001 post-synaptic cytoskeleton Translation initiation factor 21655145 (143-538) Johnnes and SaG) 1998 Estrogen receptor C. 182192 (293-814) Barraille et al., 1999 RNA binding protein 1668.818 (13698-13964) Chiang et al., 2001 Fibroblast growth factor 178226 (1-360) Martineau et al., 2004 A- angiogenic factor Fibroblast growth factor Mouse 4321921 (865-1236) Martineau et al., 2004 A- angiogenic factor Fibroblast growth factor 9125828 (35-185) Martineau et al., 2004 B - angiogenic factor Fibroblast growth factor PocC2) defined Martineau et al., 2004 C - angiogenic factor Fibroblast growth factor 21595686 (10-93) Martineau et al., 2004 D- angiogenic factor Fibroblast growth factor 31363 (486-809) Martineau et al., 2004 (2) et al., 2003. HAP4 Transcriptional activator Yeast 3762 (228-500) (2)ka et al., 1994 Haides Transcription repressor 157621 (686-1072) Maier et al., 2002 Htap2 Anti-apoptosis protein 34367137 (1313-1465) Wamaku(?) et al., 2004 HIF-1C Trancription factor Mouse 12837319(1-287) Ling et al., 2002 HRNPAFB Heterogenesis nuclear 33872827 (1-227) Qin and Sanmow 2004 riboprotein AB Hsp70 Heat shock protein 184419 (274-491) Rub(2) et al., 2003(2) Yuch and Schneder 2000 Hsp70 eat shock protien FlyO) 157720 (1514-1757) Hemandez et al., 2004 Hsp101 eat shock protein Plant 4584956 (290-438) Dinkova et al., 2005 GF-II leader 1 insulin-like growth factor 8 26190552 (130580-130698, Teerinket al., 1995 -UTR leader 1 139619-139838, 141156-141399, 153434-153443) GF-II leader 2 insulin-like growth factor 6453816 (125-735) Pedersen et al., 2002 -UTR leader 2 GF-IE Growth factor receptor 204227 (413-1358) Cd et al., 2003 Kv1.4 Voltage-gated potassium channel Mouse 26331157 (1-1201) Negulescu et al., 1998 La RNA binding protein--more 511006 (249-345, Canter and Samow 2000 abudant trancript 2329-2340) La RNA binding protein-less 511006 (698-885, Cader and Samow 2000 abudant trancript 2329-2340) LE 1 ymphoid enhancer factor 8 2285703 (1523-2703) Limener et al., 2005 L-myc Lung myc 8 88906 (224-431, lopling et al., 2004 796-802) MAP2 Cytoskeleton-associated protein 98.7493 (1-120) Pinkstaffet al., 2001 MC) MAX binding protein-trancriptional 8 O41919 (33-215) Soneiey et al., 2001 receptor M5 Methionine synthase 8 763268 (126-397) Oltean and Banerjee 2005 MTGSa (RUNX133) Trancription factor 8 940399 (1-411) Mitchell et al., 2005 MYCHEX1 Upstream open reading 8 1493393 (2223-2306) NanbO2) et al., 2001 rame on c-Myc trancript My12 Myelin trancription factor 2 2246660 (997-1158) Kim et al., 1998 Nap11.1 Nucleosome assembly 461207 (16-142) Qin and Samow 2004 protein 1-like 1 NBS1 Nijimegan breakage syndrome allele Ondefined Moiser et al., 2001 Neurogranin (RC3) Neural-specific regulator of CIMKII 924645 (4217-4470) Pinstaffet al., 2001 activity Homodomain trancription factor 3338686 (2988-1959) Wisade et al., 2000 Neuronal myc-trancription factor 1692795 (1-323) lopling and Wills 2001 intercellular signaling receptor 622786 (1-238) Lauding and Overbaugh 2000 Nucleophosmin 2745708 (1222-1320) Qin and Samow 2004 NF-all repressing 24.06601 (1-656) OuC2) et al., 2000 actor-trancription factor ODC 205803 (1-426) Pyronnet et al., 2000 EC) Translation interaction factor 1323279 (14130-14641) Zhou et al., 2001 homolog at etF4G P2C2) Cyclin-dependant kinase inhibitor Mouse 532221 (1-221) Miskimins et al., 2001 PKC8 Protein kinase C delta Rat 206180 (2-363) Monish and Rum(2) 2002 PITSC)RE Cyclin-dependant kinase Human 507159 (946-1120) Cornelis et al., 2000, Tinten et al., 2003 PP2CB Protein phosphotase 2CB Rat 12666526 (1-400) Se(2) et al., 2001 Reaper Pro-apoptosis protein Fly, Dmit 476009 (1-174) Hermandez et al., 2004 US 2011/0305749 A1 Dec. 15, 2011 21

TABLE 7-continued IRES elements which can be used according to the present invention. RNA. 2006 Oct; 12(10): 1755-85 TABLE 1. Reported cellular IRES

Gene name Function Organism (2) seqpos Reference Runx2 Type I Runt-related transcription Mouse 391766 (1-1018) Kiao et al., 2003 actor 2-UTR2 Runx2 Type II Runt-related transcription Mouse 390766 (1-207 Kiao et al., 2003 actor 2-UTR1 Scampes Calcium channel Dog 21553346 (263-368) DeC). Toneil et al., 2003 Smadi Mediator of bone 8 4433529 (259-360) S(2) et al., 2002 morphogenetic protein signaling SNM1 Homolog of 2) sensitivity to 8 522302(1-921) Zhang et al., 2002 mitrogene mustand gene Tyrosine kinase with 8 20411198. (2) et al., 2003 immunoglobulin like and (28326-27857) EGF-like domains 1 Transcriptional activator Yeast 122898 (2)-275) (2)zoka et al., 1994 I Neurotropsin receptor-tropomysin 8 18369867 (3761-1940) CC2) et al., 2003 (2) tyrosine kinase Ultrabinding-hometic gene Fly 8294 (3518-4135) Hart and Bienz 1996 X Upstream of MC2) 8 32220548 (1-468) CO2) et al., 2005 Utropsin Mouse 74.144053 (195-704) Miuta et al., 2005 Vasopressin V1b receptor Rat 945.040 (35-544) Kahadan-Dield et al., 2003 Vascular endothelial Human/mouse 4154290 (2864-3403) Stein et al., 1998: growth factor-A 1134964 (1218-2234) Huex et al., 2001 V Voscular endotheial growth factor-A 8 415290 (2363-2863) Stein et al., 1990; Huex et al., 2003 Vinkentin Stuctural protein 8. 2437837 (1758-1982) Qusand Sarpow 2004 AP Apoposis inhibtor 8 28250426 (306-409) Bolcket al., 1999 YAPI Yes-as-ocefed Protein transcriptional Yeast 4792 (297-333) Zhou et al., 2001 activator

Sequences are either the minimal sequence of the fully functioning DRES of the begining of the known 5'-UTR. The sequences 3' end includes the stafG) position(2). Some (2) in the published positions have been(2) upon comminications with the authors where possible, (2) indicates text missing or illegible when filed

TABLE 8 Reported viral IRES elements which can be used according to the present invention. RNA. 2006 Oct; 12(10): 1755-85. TABLE 2 Reported viral IRES Virus Viral name product Viral host GI (IRES seq. pos.) Reference Dioistro(2) () CrPy Cricket paralysis virus Insect 8895.586 (1-211) Wilson et al., 2008 ORF1-cons(2) protein CrPy OSf2-stuctural proteins Insect (2) (6025-6216) Wilson et al., 2008 contains (2) stan codon needed ior pseudokina DCV Drosaphila C virus IRES1 Insect 2388672 (1-661) Johnson and Christian 1998 DCV Drosaphila C virus IRES2 Insect 2388672 (6080-6266) Johnson and Christian 1998: contains (2) stan codon Kanamed and Nakashuma 2001 needed for pseudokina PSIV Plau) stall intentine virus- Insect 234.4756 (5949-6195) (2) and Nakahima 1999 capsod protein RhPV Rhops(2)phum padi ORF2 Insect 2.911238 (6327-7112) Domier et al., 2000 gca start codon TRV Triaticc(2) virus-5'-UTR Insect 6003484 (1-631) Cabener et al., 2005 TRV-IGR Triaticc) virus-intergenic region Insect 6003484 (5934-6311) Cabener et al., 2005 TSV TC2) syndrome SC) Contains reu start codon Hafakeyama et al., 2004 virus-capsid protein needed for pseudokina FlaenvinicG) (2) HCV Hepatitis C virus Human 126311192-(1-144) Tsukiyama-katara et al., 1992 US 2011/0305749 A1 Dec. 15, 2011 22

TABLE 8-continued Reported viral IRES elements which can be used according to the present invention. RNA. 2006 Oct; 12(10): 1755-85. TABLE 2 Reported viral IRES Virus Viral name product Viral host GI (IRES seq. pos.) Reference Flav C2) () Bovine viral diaC2) virus Cow (2.36067 O-383 (2) et al., 1995 Classical Swine leves Pig 1256482 (1-376) Riphomad et al., 1997 virusbog() virus Flavg.) Onclassified GSV-B Hepaditis virus(2) Primates 13162367 (23-448) Grace et al., 1999 8 inom patient GB Herpesvaidae, (2) (dsDNA) KSHV Kernnel-(2-associated Human 2O63326 Bieieki and (2) 2000; (2) (22.3206-122709) Gaundhoji and Garram 2001; (2) et al., 2008) Retravirible

F-MutV riend murine leutamis virus Mouse 61544 (1-157) 61544 (2) and Darlix 1995 (2), and sag polyprotein (1-621) F-mutV Friend murine lukemia Mouse 61544 (5492-5760) Deiski and Darlix 2008a. virus-envelope gene HaMEV Harvey murine() gat 207622 (25-533) HertioZetal, 1995 virus-Vt30 HTIV-1 Human T-cell Lympho(2) Human 221866 (354-621) Attai et al., 1995 virus 1-R and portial US region No staff codon MoMutV Moitery murine leukemia virus Mouse 333923 (812-1041) Vagnea et al., 1995b RSV Raus sarcoma virus-gag Chicken 2603459 (210-382) Defauki and Darlix 2000b RSV-Src. Rous sarcoma virus-src Chicken 28O1439 (7086-7131) Defauki and Darlix 2000b NoG) thanC2) spliced UTR exists HIV Human immunnodefency virus 14657 (607-1041) CC2)mann et al., 2000 Piomavirides Aphthovirus

FMDV Font and motiff disease virus Mammalis 61070 (1252-716) Nohn et al., 1990 Piocommavirides Candiovirus

EMCV Encephaicmmy(2) virus Human 9626692 (260-836) lang et al., 1998 TMEV Theier's murind Mouse 6205528-(876) Pilipenko et al., 1994 encephafocmyetis, virus PiccC) (2)virus

CC2) b3 Human 5439992O11-2433 Yang et al. 1987 EC) 71 strain E(2) Human 11711 2011-7460 BC2) and SiC) 2003 PV Pediovirus Human 611234-25O1 (2) and Sonenberg 1966 Picomavadae Hep:(2)

HAV Hepatitis A virus Human 329585 (150-720) Brown et al. 1994 PiccC2) Rhinovirus

HRV Human rhinovirus Human 220708 (20-625) Borman and Jackson 1993 PiccC) Techovirus

PPV-1 10111645 (146–414) (2) et al. 2001 1 stair(2) Leu62) prima (2) group

HIV Human Immunodeficiency Human 433852O 838-808 Backet al. 2001 virus type t gag (2) resides in CDS US 2011/0305749 A1 Dec. 15, 2011 23

TABLE 8-continued

Reported viral IRES elements which can be used according to the present invention. RNA. 2006 Oct; 12(10): 1755-85. TABLE 2 Reported viral IRES

Virus Viral name product Viral host GI (IRES seq. pos.) Reference

(2) PoC) PLRV (2) virus Plate (2)) 222301 (1513-1728) Jaag et al., 2009 includes 21 Potyviridae bases of CDS Podyvirus

CPMV Cowpea mouse virus Plant 58910 (161-514) Thoma et al. 1991 PVY Potato virus Y Plant 61450 (1-187) Lewis and Astict Manfacter 1993 TEV Tobacco etch virus Plant 335207 (2-147) Carringon and (2) 1990 Potyviridae Avipoxodis (dsDNA)

REV Aviar(2) Bird 28927608 (400-974) Lopez Eastra et al. 1997 virus type A (2) Tobaco mosaic virus Plant 486713 (34.56-5600) (2) et al. 1997 criMV () (2) Gladiavirus (dsDNA virus)

GLV Glandia lamidia virus Glandis lambida 1352866 (8-368) Garlapati and Wang 2005

Surgenece and either the munimal sequence of the fully(?) DRES or the beginning of the 5' UTR. The sequence's 3'-end includes the start codon. position(2). Some error in the published positions have been corrected upon communications with the?) where possible, (2) indicates text missing or illegible when filed

TABLE 9

Reported minimal IRES modules. RNA. 2006 Oct; 12 (10) : 1755-85 TABLE O Reported minimal IRES modules. (2) Full name and product (2) Sequence Reference ARC-1 Active iRNA complementary Plant AUACCCCCCC () sequence ()

(2) (2) Human ACUACAACACCCGA (2) (minimal GCAAGGACGCG IRES) ACTCT

(2) (2) Mouse CCGGCGGGT (2) et al. 2000

() (2) RNA-binding protein Mouse UUUAGAAUGUCUUCU (2) et al. 2001. UCGAGAA (2) and (2) 2003 synthetic Short synthetic nucleotides Human/yeast Five positive (2) and () (2) transcribed within IRES (2) (2)-(2)/56 positive (2) et al. (2) 18-(2)

() (2) simplex virus (2) Human CCGUGCUGGCGUCO2)G (2) and CO2) kinase mutant (2C2) codon

(2) indicates text missing or illegible when filed US 2011/0305749 A1 Dec. 15, 2011 24

TABLE 10 list of viral IRES elements, which can be used according to the present invention. Curr Gene Ther. 2004 Mar; 4(1): 15-31. Table 1A. List of Viral IRESe(2) Discovered Up to Date Viral IRES Virus 5'UTR Length IRES Length Reference PicorrC2) Family (2) PV Poliovirus Hellen et al., 1994 CV-B (BB)* Cossackievirus B (B3) Ray and Das, 2002 EV (12)* Echovirus (12) Bradricket al., 2001 SVDV Swine Vesiculor Disease Virus Chen et al., 1993) () HRV (14)* Human Rhinovirus (14) 636n SS On Rojao-Eiening et al., 1995 Apthovirus FMDV Foot and Maxb Disease Virus 438m Belcham, 1992 Cardiovirus EMCV Encepholomyocattis Virus 833n 431n Jang and Winser, 1990 TMEV (GDVII) Thiller's Morine Encepholomyocattis Virus 1067m Piligenko et al., 2000 Heostovirus HAV Hepenitie A Virus 388m Glass and Gammers, 1993 Tesciavirus PTV (Taltiue)* Puncine Eesclovirus-1 (Taltiue) 412n Kake et al., 2002 Erbovirus ERBV Eqnire Plasmids B Virus 92On 71.On Hinton and Crobb, 2001) Other Viruses

HCV Hepatitis C Virus 341n Wong et al., 1994 MHV Morine Hepatitis Virus 286m Jesdrack et al., 1994 MMLV Moloney Morine Leukemia Virus (gag)** 126n Vagner et al., 1995 FrMILV Friend Morine Leukemia Virus (gag)* SOOn Deffsud and Ostlux, 2000 HTLV-1 Human T Cell Leukemia Virus 1 284n Attal et al., 1995) RPV R(2) pediVirus 579n Woodway et al., 2001 CSFV Cladical Swine Fever Virus 374 35On Sizone et al., 1998 PSTV Plonin Stat Inovessine Virus 25On Sseski and (2), 1998) BVDV Bovine Virus Disease Virus 385 Chen et al., 1998) CrPV Cricker Pazolysis Virus 204n Jan and Semonce, 2002 TEV Tabacco Etch Virus 143n Gellie, 2001) KSHV Kaposile Sarcoma-associated Herpissina 233n BieC2) and Tailbox, 2001) (voyclic)* *Strain number or name indicated in parenthes. **Gene name indicated in parantheses. Table 1B. List of Cellular IRESes Discovered Up to Date Cellular IRES Gene Product 5'UTR Length IRES Length References

Growth Factors

VEGF Vascular Endothelial Growth Factor 1014n 163nt Sreia et al. 1998) FGF2 Fibroblost Growth Factor 2 31.8m 16Snt Vagner et al., 1995a) IGF II (5'UTR 1) Insulin-like Growth Factor II 598 (2) et al., 1995) IGF II (5'UTR2) Insulin-like Growth Factor II 40On 2OOnt Peterson et al., 2002 Cyr81 (A(2)-induced secreted protein) 25On (2) et al., 1999) Probe-oncogenes c-myc (A member of myc family) 27 Ont (2) et al., 1997) N-myc (A member of myc family) Jopling and Willis, 2001 PDGF-2/cC2) Platelet-derived Growth Factor 2 396nt Barnett et al., 1997 ODC (2) Decarbonylate 153nt Pyronet et al., 2000 (A serise-threonine protein kinase) JCC2) et al., 1999) c.(2) Transcription Factor Seigal et al., 2000 BAG-1 Anti-apoptosis factor Coldwell et al., 2001

PITSLRE (A Protein Nisase) 1 OOn 382nt (80%) Cornells et al., 200 US 2011/0305749 A1 Dec. 15, 2011 25

TABLE 10-continued Recestory/Channels.(2)

IGF-IR Insulin-like Growth Factor Receptor 943n Gizaud et al., 2003 Norcb2 (A cell Surface transmembrane receptors 214n 23Snt Lasting and protein) Overbaugh, 2000 (A Cardiac Voltage-gated PoC) 12OOn 2OOnt Negolesca et al., 1998 Channel) Connexid43 (Agap(2) protein) 2O8m 164nt Schdevi et al., 1999 Connexid32 (Agap (2) protein) 15On Hudder and Warner, 2000 Cat-1 Cationic Amino Acid Transporte 234n Fernandez et al. 2001 Scoper (A Sphingcaylphosphobolize Receptor) 361m De Pletti Tonelli et al., 2003) Transcriptional Translational Factors (Ahemeodomain transcription factor) 973 Wstads, et al., 2000 (A mut decausan transcription factors) 163n Pozzier et al., 2000) Hypossis-inducible Factor 1C. 265m Lund et al., 2002 (A DNA-binding protein) 1155 Kruez et al., 1998 LOAuttestige 115m 8Ont Carner and Sannow, 2000 LOAuttestige 483n 109nt Carner and Sannow, 2000 Eukaryotic) Factor 4G 357 101 nt Gau et al., 1993 (ARNA-binding protein) 257n 204nt Chiang et al., 2009 (ARNA-binding protein) 22On Chappell et al., 2001 Transcriptional Repressors (Mad family-related Transcriptional 18On (2) et. al., 2001) repressor) MEF NP-XB Repressing Facter 652n CC2)det. al., 2000) Gitz (A homeodomine protein) 196n Clet. al., 2000) Signaling Molerbis SC25 (Intracellular(or of RMP signaling 32On 1OOnt SH2)ki et al., 2002) (2)ay) Ar(2)-natutent Facters Apaf-1 Apoptonic Prc(2erse Activiting Factor (Pto-) 578 233nt ColcC2)e(2) et al., 2000 DAFC2)NAT1 Death-associated protein 5 (pro-) 306m H(2)-Kc(2) et al., 2000 XIAP X-linked Inhibitor of Apoptosis (2-) 1007 162nt H(2) et al., 1999) Others Hsp70 Heas Shock Protein 216n R(2) et al., 2003) p27 (A cell cycle IEC2)) 217n Mis?) et al., 2001) B-mRNA B Subunit of Mitoxiondrial R-ATP 15On Iequients and Caé2s. Syntase 2000 Eip Inc(2)ogloG) Heavy Clasic E(2)ling 22On 91nt Yang and S(2), 1997 Protein A3) (A d2) gene) 1735 Yeet. al., 1997 ETC) U(2) (A frosophils gene) 268m Yeet. al., 1997 HeiO2)ess (MG2)h R(2)gc(2)) 13On Mrier et. al., 2003) Table 3B. Cellular IRESee and Signals that Regulate the IRES Activity

Cell lines Cellular IRES origin Tested in Regulation of activity References

Growth Factors

VEGF Brain C6, Neuro-24 Hypoxia Axin et al., 1998, Enez et al., 1998; Stain et Kidney 2P3, DO57 Hypoglycernar al., 1998, Wong et al., 2002 Cervix HeLa, NB32 Brain SK-N-AS, SK-N-BE Transformation Vagner et al., 1995a. Vagner et al., 1996 Kidney COS7, 293 Heat Shock Liver SK-Hep-5 Serum starvasion & oxidative StreSS Skin Harmone skin fibroblast Bone Sros2 Ovary CHO IGF II (5'UTRI) Cervix HeLa Teesicket al., 1995 IGF II (5'UTR II) RD Pedersen et al., 2002 Cyrol Cervix HeLa Jakannes et al., 1999 US 2011/0305749 A1 Dec 15, 2011 26

TABLE 10-continued Proto-oncogenes Brain Neuro-2a, SF539 Cell cycle G2G2M phase (2) et al., 1997, Swedey et al., 1998) Lung MRCS Apoptosis PyrcC2) et al., 2000; Stoneley et al., 2000 Liver HepG2 HeLA-Kerebilt et al., 2002: Nerve et al., Kidney 295, 29ST, COST 2002, Wong et al., 2002 Bladder T24 Cervix HeLa, KR31 Ovary CHO Brain SH-SYSY, NB2a. Neuroual cell differentiation Jopling and Willis. 2001 Breast MCF7 Kidney 293 Cervix Testicle PDGF-2k-bis Blood Differentiation Berostein et al., 1997: Sells et al., 1999) Cervia Cell cycle G2/M phase Pyroceet et al., 2000 Pim-3 Cervia Jouannes et al., 1999 c-jun Seligal et al., 2000

BAG-1 Cervix Heat Shock Coldiwell et al., 2001 Tumor Suporersor PITGLRE Kidney Cell cycle G2M phase Coraeits et al., 2000 Blood Seropter Charrodels/Transports Kidney Girsodet al., 2001 Cervix Lauring and Overbsogh 2000 Kv1.4, KCNA4 Negulearn et al., 1999 Connexin43 Brain Schiavi et al., 1999 Cervix Connexin32 Brain Hudder and Wemer. 2000 Cervix Car-1 Brain Amino acid starvation Fermodel et al., 2001 Scamper Kidney De Pietri Touelli et al., 2003) Transcriptional Transmitional Factors Nrx6.1 Process B cells (BTC3, INS1) - Wetada et al., 2000 high activity Kidney C. cell (CTC1.6) - Suboptimal COS7–low activity AMLL RUNX1 Blood K582,02 SUP-TI Megskeryoxylik differentiation Pomer et al., 2000 high activity Bjah, SXY6.4 - low activity 293 HeLa HIF-1C. Kidney 293 Hypxia Lang et al., 2002 Cerviz HeLa Serun starvasion MYT2 Brain SVG Kim et al., 1998 Cervix HeLa Lal Cervix HeLa Cartex and Serum, 2000 LaP Cervix HeLa Cartex and Serum, 2000 elF4G Brain Genet al., Wong et al., 2002 Liver Kidney Blood Cervix

Cellular IRES Gene Product 5'UTR Length IRES Length References Transcriptional Processess

Max (Msd family-relsted transcriptional 18Ont Stoneley et al., 2001 repressor) MRF WF-kB Repressing Psctor 653nt (2) et al., 2000) Gts (A homeodomsin protein) 190nt Chappess et al., 2000 Signaling Molecule

Starts (Intracellular issedistor of ESIP signaling 321nt 1 OOnt Shireld et al., 2002 pathway) US 2011/0305749 A1 Dec. 15, 2011 27

TABLE 10-continued Apoprosis-related Factor Apaf-1 Apoprotic Presses Actionsing Factor (pro-) S78t 233nt Colorell et al., 2000 DAP5pG7NAT1 Deals-associated protein 5 (pro-) 306t Heinis-KoreMix et al., 2000 XIAP X-linked Sadictor of Apoprosis (pro-) 1 OOnt 16nt Holrik et al., 1999) Others Hsp70 Hear Shory Protein 216t Enbrssors et al., 2003 p27 (A cell cycle registory) 217t Maximim et al., 2001 BmRNA B Submit of Mitchondrial H-ATP 15Ont Irqiends and Caervs, Synthase 2000 Bip Immoglobulin Heavy Chain Binding 12Ont 91nt Yang and Sarnow, 1997 Protein Amp A pedia (A drosphila gene) 1735t Yeet al., 1997) () (2) (A drosphila gene) 968t Yeet al., 1997) Hairless (Notch anagonist) 139t Maiez et al., 2002

Cell line Cellular IRES origin Tested in Regulation of activity References

Growth Factors VEGF Brain C6, Neuro-24 Hypoxia Axin et al., 1998, Enez et al., 1998; Stain et Kidney 293, COST Hypoglycemia al., 1998, Wong et al., 2002 Cervix HeLa, NB32 FGF2 Brain SK-N-AS, SK-N-BE Transformation Vagner et al., 1995a. Vagner et al., 1996 Kidney COS7, 293 Heat Shock Liver SK-Hep-5 Serum starvasion & oxidative StreSS Skin Harmone skin fibroblast Bone Sros2 Ovary CHO IGF II (5'UTRI) Cervix HeLa Teesicket al., 1995 IGF II (5'UTR II) RD Pedersen et al., 2002 Cyrol Cervix HeLa Jakannes et al., 1999 Proto-oncogenes c-myc Brain Neuro-2a, SF539 Cell cycle G2G2M phase (2) et al., 1997, Swedey et al., 1998) Lung MRCS Apoptosis PyrcC2) et al., 2000; Stoneley et al., 2000 Liver HepG2 HeLA-Kerebilt et al., 2002: Nerve et al., Kidney 295, 29ST, COST 2002, Wong et al., 2002 Bladder T24 Cervix HeLa, KR31 Ovary CHO N-myc Brain SH-SYSY, NB2a. Neuroual cell differentiation Jopling and Willis. 2001 Breast MCF7 Kidney 293 Cervix HeLa Testicle NT2 PDGF-2k-sis Blood KS62 Differentiation Bernstesis et al., 1997, Setta et al., 1999 ODC Cervix HeLa Cell cycle G12d phase FC2) et al., 2000) Pim-3 Cervix HeLa Johanses et al., 1999 c-Jan (DCEF Sekgal et al., 2000 RAT1 NTHSTS RAG-1 Cervix HeLa Heat Stock Coldwell et. al., 2001 Tumor Suppressor PITSLRE Kidney 93T Cell cycle GIM phase Cornelle et al., 2000 Blood BSF3 Receptors/CEC2)/Transporters GF-IR Kidney COST Gitsel et al., 2001 Norcb2 Cervix HeLa L:(2) and Ove(2), 2000) Kv1.4KCNA4 NIH3T3 Ne?) et al., 1998) Connexed43 Brain Mem2a Schiavi et al., 1999 Cervix HeLa Connexed 2 Brain Neuro-2a. Holder and Werner, 2000) Cervix HeLa Cat-I Brain C6 Amino acic(2) Fernander et al., 2000 Scamper Kidney MDCK De Pieri Tenei et al., 2003) US 2011/0305749 A1 Dec. 15, 2011 28

TABLE 10-continued Transcriptional Transcriptional Factors Nax6.1 Pancreas B cells (BTC3, INSI)- Wetada et al., 2000 high activity Kidney C. cell (CTC1.6)- Suboptional COS7 - low activity AMLIRUNX: Blood K562,02 SUP-T1- Meg(2) differentiation Pomer et al., 2000 high activity Kidney Bixb. SKW6.4 - low Cervix activity 283 HeLa HIF-1C. Kidney 293 Hyposis Lang et al., 2002 Cervix HeLa Serum(2) MYT2 Brain SVG Kim et al., 1998 Cervix HeLa LSF Cervix HeLa Caster and Se2), 2000) LSF Cervix HeLa Caster and Se(2), 2000) eIF4G Brain Mem-2a. Gan et al., 1998, Wang et al., 2002 Liver HepG2 Kidney 293 Blood N562 Cervix KBI3 FMR1 Brain SK-N-MC Chinmuget al., 2001 Rbm3 Brain SK-Hemo-2a, C6 Hypothema Chappellet. al., 2001 Transcription at Restressors Mur Cervix HeLa Stertey et al., 2001 MEF Kidney BHK Ornament et al., 2010 Cervis HeLa Ovary CHO Gre Brain SK-N-SH, Neuro-2a, Co He et al., 1999; Chappellet al., 2009 Kidney NRK Muscle CCu Pindsory AiT-20 gland KS63 Blood Signaling Malenole Stands Embrysek ATDC3 Stusoket. al., 2002 Muscle CCu Bone MC3T3BI Brain Ghoma - low activity Liver HepG2 - low activity Kidney 293,2993, COS7 - low Cervex activity HeLa - low activity Apoptosis-related Factors Apef-3 Cervix HeLa - higher activity Apoptosis Coldwell et al., 2000; Heris Kerenbilt et al., Breast MCF7 2002, Mevine et al., 2002 Lung MRC3 Liver HeG2 Kidney 293,29ST, COST DAFC2)p97NAF1 Kidney 293,293T Apoptosis Heria-Kerenblit et al., 2000; Henis-Ketebilt Cervix HeLa, HFB et al., 2002; Nevises et al., 2002 Ovary CHO MIAP Brain SP539 Y-irradiation Holcik et al., 1999; Holicket al., 2000; Lung H661, H520 Serun starvasion Helis-Nore(2) et al., 2002: Hevina et al., 2002: Cervix C8 Apoptosis Holicket al., 2003 Ovary CHO, SHOV3 Kidney 293,29ST Bladder T24 CC) NIH3T3 Hsp20 Kidney 2O3 (2) et al., 2003) Brain TE571 p27 Brain DOPIT Cell cycle MC) et al., 2001) B-mRNA In vitro translation legrierdo and C(2), 2000) Bip Brain Neuro-2a. Heat Shock Yang and 2, 1997: Kiim and Jang, 2002; Kidney 2O3 Nevins et al., 2002 Bladder T24 Cervix HeLa, KB31 US 2011/0305749 A1 Dec. 15, 2011 29

TABLE 10-continued Amp Drosophilis Schneider SL2 Yeet al., 1997 UC Drosophilis Schneider SL2 Yeet al., 1997 Hairless Drosophilis S2 Cell cycle G2/M phase Meier et al., 2002

(2) indicates text missing or illegible when filed

0194 In a specific embodiment of the present invention, acid sequence encoding an HIV-1 envelope polypeptide of the the retroviral vector or nucleic acid comprises an IRES, present invention or a fragment thereof is selected from the wherein said IRES is located in the 3'-LTR or the 5'-LTR, or nucleic acid sequences encoding envelope polypeptides in a region flanked by the 3'-LTR and the 5'-LTR. In particular, derived from HIV-1, HIV-2, or SIV. In a specific embodiment, said IRES may be located in the R region of both the 5'-LTR the at least one heterologous nucleic acid sequence encoding and/or 3'-LTR. In another embodiment, the IRES is located in an HIV-1 envelope polypeptide of the present invention or a the U3 region of the 3'-Long Terminal Repeat or the U5 region fragment thereof encodes HIV-1 envelope polypeptide, for of the 5'-LTR. In yet another specific embodiment of the example an HOV envelope polypeptide as defined in any of present invention, said IRES is located in the U3 region SEQID NO: 1-326,327-337 or a fragment thereof. In another between the inverted repeats and the transcription regulatory specific embodiment, the at least one heterologous nucleic elements. acid sequence encoding an HIV-1 envelope polypeptide of the present invention or a fragment thereof encodes a C-termi Preferred Vectors nally truncated HIV-1 envelope, or a fragment thereof. In yet another specific embodiment, the at least one heterologous 0.195 A central aspect of the present invention relates to a nucleic acid sequence encoding an HIV-1 envelope polypep eukaryotic expression vector comprising at least one heter tide of the present invention or a fragment thereof encodes an ologous nucleic acid sequence encoding an HIV-1 envelope SIV envelope. polypeptide of the present invention or a fragment thereof. In 0197) The vectors of the present invention, also encom one embodiment, said vector comprises an intron. The intron pass vectors as defined above, wherein said at least one addi is deleted by splicing, thus, facilitating the transport from the tional nucleic acid sequence is a reporter gene. Different nucleus to the cytoplasm of said vector encoded RNA in embodiments of reporter genes according to the present packaging cells. As an alternative to an intron, the vectors of invention include reporter genes encoding enhanced green the present invention may also comprise a constitutive trans fluorescent protein (eGFP), lac Z, dsRed, enhanced yellow port element (CTE). The CTE facilitates the transport of the fluorescent protein (eyFP), enhanced cyan fluorescent pro vector encoded RNA from the nucleus to the cytoplasm of the tein (eCFP), enhanced blue fluorescent protein (eBFP) and semipackaging cell. In another approach, the vector may the human alpha-1-antitrypsin (ha AT). It is understood that comprise a Rev responsive element (RRE). The presence of any of the enhanced green fluorescent protein (eGFP), lac Z. an RRE in the RNA transcript facilitates its transport from the dsRed, enhanced yellow fluorescent protein (eyFP), nucleus to the cytoplasma of the semipackaging cell, when enhanced cyan fluorescent protein (eCFP), enhanced blue REV/REX is coexpressed. In one specific embodiment, the fluorescent protein (eBFP) or the human alpha-1-antitrypsin vectoris transcribed in the cytoplasm, thereby producing high (ha AT). However, the at least one additional nucleic acid levels of transcript, which can be translated into envelope sequence may also encode a selective marker, Such as neo polypeptide. This envelope polypeptide may then be incor mycin phosphotransferase II, and/or a Suicide gene. More porated into viral particles in a packaging/producer cell. over, the at least one additional nucleic acid sequence may 0196. In a specific embodiment, the vector of the present encode an immunomodulating polypeptide or peptide. Such invention is a retroviral vector. Retroviral vectors include as an immunostimulating polypeptide, a genetic adjuvant, both replication deficient retroviral vectors, and replication cytokines and/or hormones. competent retroviral vectors. In one embodiment, the present 0198 Important embodiments of the present invention invention relates to a vector, which comprises at least one include vectors, wherein said IRES is selected from the IRES heterologous nucleic acid sequence encoding an HIV-1 enve elements of picornaviridae, retroviridae or retrotransposons, lope polypeptide of the present invention or a fragment mammalia or combinations thereof. In a specific embodi thereof. In one embodiment, the nucleic acid sequence has ment, said IRES is selected from the IRES elements of picor been modified codon optimization to eliminate RNA ele navirus. In another specific embodiment, said IRES is ments, which may reduce the RNA expression level. Such selected from the IRES elements of encephalomyocarditis codon optimization are known to those of skill within the art. (ECMV). The IRES may be inserted at different locations in The vector may further comprise at least one additional the retroviral vector. In one embodiment, the IRES is located nucleic acid sequence and/or at least one internal ribosomal in a region flanked by the 3'-LTR and the 5'-LTR. In another entry site (IRES). In one embodiment, at least one heterolo embodiment, the IRES is located in the 3'-Long Terminal gous nucleic acid sequence encoding an HIV-1 envelope Repeat (LTR) or the 5'-LTR. In yet another embodiment, the polypeptide of the present invention or fragment thereof is IRES is located in the U3 region of the 3' LTR. In a further preceded by an IRES, and in another embodiment, the at least embodiment, the IRES is located in the U3 region between one additional nucleic acid sequence is preceded by an IRES. the inverted repeats and the transcription regulatory elements. The vectors of the present invention are preferably derived 0199 The design of the vectors of the present invention from gamma-retroviruses, for example Murine Leukemia allows the vectors to be used for vaccination purposes. Thus, Virus (MLV), Moloney Murine Leukemia Virus (MoMLV), in one embodiment, the lentiviral envelope encoded by the or AkV MLV. Preferably, the at least one heterologous nucleic vectors as defined by the present invention is capable of US 2011/0305749 A1 Dec. 15, 2011 30 inducing an immunogenic response in a host animal. For cific embodiment, the biological entity of the present inven example, said immunogenic response is an antibody response tion is capable of mediating fusion of said biological entity and/or cytotoxic T Lymphocyte (CTL) response. and cells expressing receptors for HIV. In another embodi 0200. In one embodiment, the immunogenic response of a ment, the biological entity of the present invention is capable vector according to the present invention is directed against a of infecting a CD4 positive cell. Moreover, the biological retroviral particle expressing said lentiviral envelope. In a entity is in one embodiment capable of inducing an immuno specific embodiment, the lentiviral envelope is incorporated genic response in a host animal. In particular, said immuno in said retroviral particle. The immunogenic response may genic response may be directed towards said biological entity thus be directed against the retroviral particle in the host in said host animal. The host animal may be any animal, animal. Thus, the retroviral particle is not required to be however, preferably a mammal, more preferably a human infectious and/or fusogenic. In a preferred embodiment, the being. However, in one preferred embodiment, the biological envelope polypeptide as described herein is expressed and entity of the present invention is capable of infecting mam displayed on the surface of said retroviral particle. The malian cells, such as preferably human cells. In the present present invention, thus relates to a vector, wherein said enve context, the term "human cells' comprise any sort of human lope is incorporated in said retroviral particle. cell including mutated human cells pathogenic cells as well as 0201 In another embodiment, the immunogenic response any type of human stem cell. is a CTL response, wherein said vector is integrated into the 0206. Further embodiments of the invention include any genome of a host cell. biological entity comprising an HIV-1 envelope polypeptide 0202. In one embodiment, the retroviral vector of the that has a sequence that is at least 70% identical to the amino present invention is derived from AkV MLV, and comprises a acid sequence shown in any one of IGP1-7, SEQ ID NO.: first nucleic acid sequence encoding eGFP and an additional 1-124 or SEQID NO: 125-326,327-337 or is a fragment of a nucleic acid sequence encoding an HIV-1 envelope polypep sequence that is at least 70%, such as at least 80%, for tide (e.g. any of SEQID NO: 1-326,327-338, IGP1-7 or part example 90%, such as at least 95%, such as 100% identical to thereof) preceded by an ECMV IRES element. In another the amino acid sequence shown in SEQ ID NO.: 1-124 or preferred embodiment, the retroviral vector of the present SEQID NO: 125-326,327-337, IGP1-7 or a part thereof, for invention is derived from AkV MLV, and comprises a first example a part thereof comprising or consisting of at least 3, nucleic acid sequence encoding neomycin phosphotrans such as 4, 5, 6, 7,8,9, such as at least 10, for example at least ferase II and an additional nucleic acid sequence encoding an 15 amino acids. In another aspect, the present invention HIV-1 envelope polypeptide (e.g. any of SEQID NO: 1-326, relates to a biological entity comprising an RNA transcribed 327-337, IGP1-7 or part thereof), preceded by an ECMV from any of the embodiments of the retroviral vectors IRES element. described herein, or an RNA encoding any of the HIV enve lope polypeptides of the present invention. Biological Entity 0207. In a preferred embodiment, the HIV-1 envelope polypeptide as described herein is expressed on the Surface of 0203. In a primary aspect, the present invention provides a the biological entity. Cell surface expression of envelope biological entity Such as a eukaryotic cell, a prokaryotic cell, polypeptides may be detected by persons skilled in the art. a viral particle, and/or a retroviral particle, wherein said bio One method of detecting surface expression of ENV is pro logical entity comprises at least one HIV-1 envelope polypep vided in example 3 below. Moreover, incorporation of ENV tide as defined by the present invention and/or nucleic acid into retroviral particles may in one example be detected by the according to the present invention. In a preferred embodi method shown in example 4 ment, the biological entity of the present invention is a retro 0208. In a specific embodiment, the biological entity of the viral particle and/or a provirus. However, in another preferred present invention comprise an HIV-1 envelope polypeptide as embodiment the biological entity is a prokaryotic cell and/or defined elsewhere herein, wherein said envelope polypeptide a eukaryotic cell. Such as a mammalian cell. In yet another is glycosylated. In a preferred embodiment, said envelope embodiment the biological entity is a liposome, a nanopar polypeptide comprises alpha-gal. In a specific embodiment, ticle, a viroSome, a protzoa, and/or enterobacteria. Specifi the biological entity of the present invention comprises an cally, the biological entity may be prokaryotic and/or eukary HIV-1 envelope polypeptide or fragment thereofas defined otic cells in the intestinal tract and/or other parts of the elsewhere herein, which comprises gal-alfa 1-3Galbeta1 digestive system. Moreover, the biological entity may be a 4GlcNAc-R epitopes on the envelope protein. Thus, in a nanoparticle or a liposome with synthetic envelope polypep preferred embodiment, the packaging/producer cells of the tides. present invention comprise alpha-galactosidase enzyme, 0204 Thus, in one embodiment, the present invention pro which make them capable of producing biological entities, vides a biological entity comprising at least one HIV-1 enve Such as retroviral particles comprising alpha-gal labeled lope as defined elsewhere herein or part thereof, and/or at envelope polypeptides. least one antigen as defined elsewhere herein, and/or at least 0209. In a specific embodiment, the biological entity is a one nucleic acid as defined elsewhere herein, and/or at least nanoparticle, for example a nanoparticle with synthetic enve one vector as defined elsewhere herein. lope peptides, a liposome, for example a viroSome or any 0205. In a preferred embodiment the HIV-1 envelope or combination thereof. Thus, in a specific embodiment, the part thereof is expressed on the surface of the biological entity present invention relates to a liposome comprising any com of the present invention. Specifically, the HIV-1 envelope ponent of the present invention, including an HIV-1 envelope polypeptide or part thereof of said biological entity comprises polypeptide as defined herein and/or fragments thereof. gal-alfa 1-3Galbeta1-4GlcNAc-R epitopes. In a specific embodiment, the biological entity of the present invention is Retroviral Particles and Producer Cells not capable of mediating fusion of said biological entity and 0210. The retroviral particle of the present invention is cells expressing receptors for HIV. However in another spe derived from any retrovirus, including any of the retroviruses, US 2011/0305749 A1 Dec. 15, 2011

wherefrom the retroviral vectors of the present invention may infectious viral particles. Accordingly, the transduction effi be derived, as describes above. In a preferred embodiment, ciency directly correlates with the amount of infectious viral the retroviral particle is a gamma-retroviral particle. particles produced. 0211. In one embodiment, the retroviral particles of the 0216. However, a problem of low transduction efficiency present invention are fusogenic and/or infectious. However, is overcome by the present invention. A replication-defective in another embodiment, the retroviral particles of the present retroviral vector is provided, said vector comprising a gene invention are non-fusogenic and/or non-infectious. Specifi encoding an HIV-1 envelope polypeptide of the present cally, in one embodiment, the retroviral particle of the present invention or fragment thereof. According to one embodiment invention is not capable of mediating fusion of said retroviral of the present invention, a nucleic acid sequence encoding an particle and cells expressing receptors for HIV. However, in HIV-1 envelope polypeptide of the present invention or frag another embodiment, the retroviral particle of the present ment thereof is under translational control of a heterologous invention is capable of mediating fusion of said retroviral IRES. Alternatively, a nucleic sequence encoding an HIV-1 particle and cells expressing receptors for HIV. A number of envelope polypeptide of the present invention or fragment different assays are known for the skilled practitioner for thereof may be present as in the vector backbone and is detecting fusogenicity and/or infectiousness of a retroviral expressed in the transcript of the viral RNA and translated by particle. Example 5 provides one such methodology for the host cell machinery. Since the vector according to one detecting fusogenicity of retroviral particles. embodiment of the present invention encodes itself an enve 0212. The retroviral vector of the present invention is in lope, a packaging cell needs only to provide the proteins one embodiment constructed as a replication-defective vector encoded by gag and/or pol. Such packaging cells comprising based on any of the retroviruses mentioned elsewhere herein. a gag and/or pol encoding DNA construct, but no env encod A replication-defective retroviral vector is characterised in ing DNA construct, are known as semi-packaging cell. that, one or more genes essential for virus replication, pack Advantageously, this semi-packaging cell is not resistant to aging of viral RNA and/or formation of infective particle, Superinfection since these cells do not express envelope pro have been deleted from the retroviral vector. Thus, to recon tein prior to transfection with the retroviral particle. Conse stitute the viral life cycle and generate viral particles com quently, no envelope protein binds to the cellular receptor and prising such replication defective vectors a specialised pro thus, no resistance is mediated in said cell. Consequently, ducer cell providing the deleted genes is needed. Such viral particles are only generated and released after transfec producer cell are constructed by transducing a cell with DNA tion of the semi-packaging cell with the vector. The released constructs encoding the genetic information of the retroviral retroviral particles comprising a gene of a functional lentivi proteins, which are essential for packaging a retroviral vector ral envelope polypeptide or fragment thereofare able to infect genome and generating viral particles. further semi-packaging cells in culture. Thus, the vector of 0213. In the presence of a retroviral vector genome (the the present invention is in one embodiment, replication-com process known as transduction) a producer cell will generate petent in the semi-packaging cells and thus allows for an easy infectious viral particles which comprise the retroviral and highly efficient production of the retroviral particles in genome derived from the vector. The viral particles produced high titers. Infection of target cells (also known as host cells) in this manner will be released and can infect another pro lacking gag and/or pol, by infectious particles produced in ducer cell. Such producer cells are also designated as pack these semi-packaging cells result in the transfer of the genetic aging cells. In one embodiment, the retroviral particle of the information of the vector only. Since this vector according to present invention is obtained by transfection of a producer this embodiment does not comprise the gag and/or pol, no cell with a retroviral vector or part thereof, oran RNA or part further replication of the vector in the target cell is observed. thereof according to the present invention. In another embodi Accordingly, said replication-defective vector is a safe vector ment, the retroviral particle of the present invention is e.g. for use in genetherapy or for use as a vaccine as described obtained by transfection of a semipackaging cell with a vector herein. However, the present invention also encompasses or part thereof, or an RNA or part thereof according to the semi-packaging cells, which are capable of producing non present invention. infectious retroviral particles. 0214. In one aspect, the present invention relates to a pro 0217. In a particular embodiment of the invention, the ducer cell comprising a vector as provided by the present producer cell does not comprise lentiviral tat or rev for invention. In one example, a producer cell of the present example HIV-1 tat or rev. Also the rex gene originating from invention does not comprise lentiviral tat or rev and/or rex HTLV is in one embodiment not present in the producer cell. originating from HTLV. 0218. The semi-packaging cells of the present invention 0215 Packaging/producer cells are often produced by are selected from all types of eukaryotic cells e.g. mammalian transfecting cells with genetic information and/or genes cells, any type of eukaryotic cell that will survive in the colon essential for retroviral particle formation. The culture of and/or digenstive system, yeast cells. In a further embodi packaging cells is Subsequently Supertransfected with the ment, cells from old world monkeys and humans for produc vector DNA. However, the genetic information and/or genes tion of infectious viral particles. For viral particle production essential for retroviral particle formation and the vector DNA capable of activating the innate immune system, all eukary may also be introduced in a single round of transfection. otic cells except cells derived from old world monkeys and Supertransfection here describes another or a second trans humans may be used in the as semi-packaging cells. fection event, namely the transfection of the packaging cell Examples of cells used are Human embryonic Kidney cells with the vector. The resulting Supertransfected packaging cell HEK 293, HEK 293T, Human rhabdomysarcoma cells will Subsequently produce infectious viral particles compris TE671, Canine osteosarcome cells D17, murine fibroblast ing the vector RNA genome. Said particles, which will be cells NIH3T3. In a particular embodiment the semi-packag released from the packaging cell, can be isolated. It should be ing cells are 293T cells comprising Mo-MV GagPol DNA noted that only Supertransfected packaging cells produce and/or protozoa. US 2011/0305749 A1 Dec. 15, 2011 32

0219. In another embodiment, the retroviral particle 0226 CD4 is also a primary receptor used by HIV-1 to according to the present invention comprises vesicular sto gain entry into host T cells. The HIV-1 envelope gp120 pro matitis virus envelope (VSV-G), the amphotropic murine leu tein attaches to CD4, creating a shift in the conformation of kemia virus envelope, Mutated SL3-2 envelope, Xenotropic the viral gp120 protein, which allows HIV-1 to bind to two murine leukaemia virus envelope, 10A1 virus envelope, other cell surface receptors on the host cell (the chemokine Hepatitis virus C envelope, gibbon ape leukaemia virus. receptors CCR5 and CXCR4). Following another change in Human T-cell lymphotropic virus, or envelopes of endog shape of a different HIV-1 envelope protein (gp41), HIV inserts a fusion peptide into the host T cell that allows the enous human retroviruses. In one embodiment, the retroviral outer membrane of the virus to fuse with the T-cell mem particle comprises the G glycoprotein of the vesicular stoma brane. HIV infection leads to a progressive reduction in the titis virus envelope (VSV-G). number of T cells possessing CD4 receptors and, therefore, 0220. The retroviral particles are preferably capable of the CD4 count is used as an indicator to help physicians infecting animal cells, such as mammalian cells, preferably decide when to begin treatment in HIV-infected patients. human cells. In a specific embodiment, the retroviral particles 0227. The terms “CD4-positive cell' or “CXCR4/CCR5 are capable of infecting stem cells. In another specific positive cell as used herein, relates to any cells expressing embodiment, the retroviral particles are capable of infecting a CD4 receptor or CXCR4/CCR5 receptor, respectively. The CD4 positive cell. Moreover, the retroviral particles of the CD4 receptor and/or CXCR/CCR5 receptor may be acces present invention are capable of inducing an immunogenic sible from the extracellular space at the cellular membrane. response in a host animal, preferably a human being. In a 0228. In one embodiment, the retroviral particle, wherein preferred embodiment, the immunogenic response is directed an HIV-1 envelope polypeptide of the present invention is towards a retroviral particle of the present invention in said expressed from a retroviral vector, comprises the G glycopro host animal. Thus, it is not required that the retroviral particle tein of the vesicular stomatitis virus envelope (VSV-G), the is infectious and/or fusogenic. The immunogenic response amphotropic murine leukemia virus envelope, Mutated may directed against the retroviral particle it self. In one SL3-2 envelope, Xenotropic murine leukaemia virus enve embodiment, the host animal is a human being. lope, 10A1 virus envelope, Hepatitis virus C envelope, gib 0221. In a specific embodiment, the retroviral packaging bonape leukaemia virus, human T-cell lymphotropic virus or cells or semipackaging cells producing retroviral particles are envelopes of endogenous human retroviruses. encapsulated and/or any other biological entity of the present 0229. In a particular embodiment of the present invention, invention, which express an HIV envelope polypeptide as the producer cell does not comprise lentiviral tat or rev, for defined herein is encapsulated, wherein the capsules have a example HIV-1 tat or rev. Also the rex gene originating from porous capsule wall which is permeable to said retroviral HTLV is not present in the host cell. particles and/or said biological entity. Infections Target Cells (Host Cells) 0230. The word infection as used herein relates to any kind of clinical condition giving rise to an immune response. Such 0222 Aprimary aspect of the present invention relates to as an inflammation, and therefore includes infectious dis a biological entity, Such as a retroviral particle, as defined eases, chronic infections, autoimmune conditions and aller herein, capable of infecting specific target cells. Infection of gic inflammations. Thus, infections, such as infectious dis a target cell can occur by receptor mediated endocytosis and/ eases, chronic infections, autoimmune conditions and or membrane fusion, as described elsewhere. allergic inflammations are all clinical conditions of relevance 0223) One aspect of the present invention relates to a host for the present invention, and are dealt within turn hereunder. cell comprising a vector or part thereof of the present inven Furthermore, the terms infection and inflammation are used tion. Another aspect of the present invention relates to a host interchangeably herein. cell comprising a biological entity, such as a retroviral par 0231. Inflammation is the complex biological response of ticle, of the present invention. vascular tissues to harmful stimuli. Such as pathogens, dam 0224 One embodiment of the present invention relates to aged cells, or irritants. It is a protective attempt by the organ any component of the present invention including a vector, a ism to remove the injurious stimuli as well as initiate the nucleic acid, a biological entity, Such as a retroviral particle, healing process for the tissue. Inflammation can be classified a composition, and/or a kit-of-parts described herein com as either acute or chronic. Acute inflammation is the initial prising a retroviral envelope polypeptide, capable of mediat response of the body to harmful stimuli and is achieved by the ing infection of a cell, by use of the CD4-CXCR4/CCR5 increased movement of plasma and leukocytes from the blood receptor pathway. Thus, embodiments of the present inven into the injured tissues. A cascade of biochemical events tion comprise any components of the present invention com propagates and matures the inflammatory response, involving prising an HIV-envelope polypeptide as defined herein, the local vascular system, the immune system, and various capable of mediating infection of a CD4-positive cell, and/or cells within the injured tissue. Prolonged inflammation, a CXCR4/CCR5-positive cell. known as chronic inflammation, leads to a progressive shift in 0225 CD4 (cluster of differentiation 4) is a glycoprotein the type of cells which are present at the site of inflammation that is found primarily on the surface of helper T cells, as well and is characterized by simultaneous destruction and healing as regulatory T cells and dendritic cells. CD4 is a member of of the tissue from the inflammatory process. the immunoglobulin Superfamily. It has four immunoglobulin 0232 An infectious diseases may becaused by a virus, and domains (D1 to D4) that are exposed on the extracellular viral diseases against which the vaccine composition of the surface of the cell: D1 and D3 resemble immunoglobulin present invention may be administered in the treatment of variable (IgV) domains, while D2 and D4 resemble immuno HIV, AIDS, AIDS Related Complex (ARC); thus it is an globulin constant (IgC) domains. On T cells, CD4 is the object of the present invention to administer a component if co-receptor for the T cell receptor (TCR) and recruits the the present invention as the treatment of or as part of the tyrosine kinase Ick. treatment of these viral infections. US 2011/0305749 A1 Dec. 15, 2011

Infectious Disease Combination Treatment preventing a clinical disorder associated with HIV infection 0233. It is further provided for that a treatment of any in an individual; in other words the pharmaceutical compo infectious disease by the administration of the vaccine com sition of the present invention is a vaccine composition, and is position according to the present invention may be given in accordingly referred to as such. However, the vaccine com conjunction with a further (second) active ingredient or in position of the present invention may also be referred to as a combination with a further treatment such as antibiotic treat pharmaceutical composition. The vaccine compositions of ment, chemotherapy, treatment with immunostimulating Sub the present invention may be “traditional vaccine composi stances, treatment using dendritic cells, antiviral agents anti tions comprising antigens such as proteins, polypeptides and/ parasitic agents and so forth. or nucleic acid molecules. They may also be in the form of 0234 Examples of a second active ingredient that may be compositions comprising cells, such as modified cells origi used in the treatment of an infectious disease in combination nating from the individual and later processed, or to compo with the vaccine of the present invention include, and are not sitions comprising complex molecules Such as antibodies or limited to antibiotics. The term antibiotics herein refers to TCRS. In particular, the vaccine compositions of the present Substances with anti-bacterial, anti-fungal, anti-viral and/or invention may comprise viral particles such as retroviral par anti-parasitical activity; examples of relevance to the present ticle as defined elsewhere herein. All vaccine compositions of invention include, but are not limited to: Amikacin, Genta the present invention are claimed for use as a medicament. mycin, Kanamycin, Neomycin, Netilmicin, Paromomycin, 0237 Generally, a vaccine is a substance or composition Streptomycin, Tobramycin, Ertapenem, Imipenem, Mero capable of inducing an immune response in an individual. The penem, Chloramphenicol, Fluoroquinolones, Ciprofloxacin, composition may comprise one or more of the following: an Gatifloxacin, Gemifloxacin, Grepafloxacin, Levofloxacin, “active component'. Such as an antigen?s) (e.g. protein, Lomefloxacin, Moxifloxacin, Norfloxacin, Ofloxacin, Spar polypeptides, peptides, nucleic acids and the like), nucleic floxacin, Trovafloxacin, Glycopeptides, Vancomycin, Lin acid constructs comprising one or more antigens amongst cosamides, Clindamycin, Macrollides/Ketolides, Azithromy other elements, cells, (e.g. loaded APC, T cells for adoptive cin, Clarithromycin, Dirithromycin, Erythromycin, transfer aso.), complex molecules (Antibodies, TCRs and Cefadroxil, Cefazolin, Cephalexin, Cephalothin, Cephapirin, MHC complexes and more), carriers, adjuvants and pharma Cephradine, Cefaclor, Cefamandole, Cefonicid, Cefotetan, ceutical carriers. The various components of a vaccine com Cefoxitin, Cefprozil, Cefuroxime, Loracarbef, Cefdinir, position according to the present invention are disclosed in Cefditoren, Cefixime, Cefoperazone, Cefotaxime, Ce?po more detail elsewhere herein. doxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, 0238. In a broadest aspect, the present invention relates to Cefepime, Monobactams, Aztreonam, Nitroimidazoles, Met a vaccine composition comprising ronidazole, Oxazolidinones, Linezolid, Penicillins, Amox 0239 a) an HIV-1 envelope polypeptide as defined else icillin, Amoxicillin/Clavulanate, Ampicillin, Sulbactam, where herein, or a functional homologue thereofhaving at Bacampicillin, Carbenicillin, Cloxacillin, Dicloxacillin, least 70% identity to said peptide or an immunogenically Methicillin, Mezlocillin, Nafcillin, Oxacillin, Penicillin G, active peptide fragment comprising a consecutive Penicillin V. Piperacillin, Piperacillin/Tazobactam, Ticarcil sequence of at least 10 residues of said peptide or said lin, Ticarcillin/Clavulanate, Streptogramins, Quinupristin, functional homologue thereof, or a nucleic acid encoding Dalfopristin, Sulfonamide/Sulfamethoxazole, Trimethop said peptide or said peptide fragment or said functional rim, Tetracyclines, Demeclocycline, Doxycycline, Minocy homologies thereof and/or cline, Tetracycline, AZole antifungals Clotrimazole Flucona 0240 b) an antigen as defined elsewhere herein, and/or Zole, Itraconazole, Ketoconazole, Miconazole, Voriconazole, Amphotericin B. Nystatin, Echinocandin, Caspofungin, 0241 c) a nucleic acid as defined elsewhere herein, and/or Micafungin, Ciclopirox, Flucytosine, Griseofulvin, and Ter 0242 d) a vector as defined elsewhere herein, and/or binafine. Of further relevance are antivirals such as Vidara 0243 e) a biological entity as defined elsewhere herein, bine, Acyclovir, Gancyclovir and Valcyte (valganciclovir), and Nucleoside-analog reverse transcriptase inhibitors (NRTI): 0244 f) an adjuvant AZT (Zidovudine), ddI (Didanosine), ddC (Zalcitabine), d4T 0245. In one embodiment, the immunologically active (Stavudine), 3TC (Lamivudine), Non-nucleoside reverse peptide fragment of the vaccine composition above consists transcriptase inhibitors (NNRTI): Nevirapine, Delavirdine, of a consecutive sequence of in the range of from 10 to 50 Protease Inhibitors Saquinavir, Ritonavir, Indinavir, Nelfi amino acids. However in another embodiment, the immuno navir, Ribavirin, Amantadine/Rimantadine, Relenza and logically active peptide fragment of the vaccine composition Tamiflu, Pleconaril, Interferons. above consists of a consecutive sequence of at least 10amino 0235. In an embodiment, the present invention regards a acids, such as at least 20 amino acids, such as at least 30, Such vaccine composition comprising at least one HIV-1 envelope as at least 40, such as at least 50, for example at least 60, such polypeptide and/or a functional homologs or fragment as at least 70, for example at least 80, such as at least 90, for thereofas defined elsewhere herein, for the treatment of an example at least 100 amino acids. In another embodiment, the infectious disease, such as HIV, AIDS and/or ARC in combi immunologically active peptide fragment of the vaccine com nation with at least one antibiotic. Preferably, the vaccine position above consists of 8 to 10 or 18 to 25 consecutive composition of the present invention is used for the treatment amino acids of a polypeptide as defined herein, for example of chronic infections e.g. HIV, AIDS and/or ARC and there any peptide selected from any of SEQID NO: 1-124, or SEQ fore is used in combination with any of the above listed ID NO: 125-326,327-337. antibiotics such as anti-viral agents. 0246. In another embodiment of the vaccine composition of the present invention, the HIV-1 envelope polypeptide or Vaccine Composition immunologically active peptide fragment thereof consists of 0236. The present invention regards pharmaceutical com the vaccine composition above consists at the most 50 amino positions capable of treating, reducing the risk of and/or acid residues, for example at the most 45 amino acid residues, US 2011/0305749 A1 Dec. 15, 2011 34

Such as at the most 40 amino acid residues, for example at the 0250 One embodiment combines any one of the compo most 35 amino acid residues, such as at the most 30 amino nents of the present invention, including an HIV-1 envelope acid residues, for example at the most 25 amino acid residues, polypeptide, an antigen, a nucleic acid, a eukaryotic expres such as 20 to 25 amino acid residues. However, in another sion vector, and/or a biological entity of the present invention embodiment, the HIV-1 envelope polypeptide or fragment with various at least one adjuvant to produce a vaccine com position. thereof consists of at the most 20 amino acid residues, for 0251 Examples of adjuvants and immunomodulating example at the most 19 amino acid residues. Such as at the peptides are described elsewhere herein. Adjuvants, broadly most 18 amino acid residues, for example at the most 17 defined, are Substances which promote immune responses. amino acid residues. Such as at the most 16 amino acid resi Frequently, the adjuvant of choice is Freund's complete or dues, for example at the most 15 amino acid residues, such as incomplete adjuvant, or killed B. pertussis organisms, used at the most 14 amino acid residues, for example at the most 13 e.g. in combination with alum precipitated antigen. A general amino acid residues. Such as at the most 12 amino acid resi discussion of adjuvants is provided in Goding, Monoclonal dues, for example at the most 11 amino acid residues, such as Antibodies: Principles & Practice (2nd edition, 1986) at 8 to 10 amino acid residues. pages 61-63. Goding notes, however, that when the antigen of 0247 The vaccine composition of the invention is capable interest is of low molecular weight, or is poorly immuno of eliciting an immune response against an HIV-1 envelope genic, coupling to an immunogenic carrier is recommended. polypeptide of the present invention, or a functional homo Examples of such carrier molecules include keyhole limpet logue thereof having at least 70% identity to any sequence haemocyanin, bovine serum albumin, ovalbumin and fowl selected from SEQ ID NO 1-124 or SEQ ID NO: 125-326, immunoglobulin. Various Saponin extracts have also been 327-337, when administered to an individual. In another Suggested to be useful as adjuvants in immunogenic compo embodiment, the vaccine composition of the invention is sitions. Recently, it has been proposed to use granulocyte capable of eliciting an immune response against an antigen macrophage colony stimulating factor (GM-CSF), a well presenting cell expressing an HIV-1 envelope of the present known cytokine, as an adjuvant (WO 97/28816). invention, and/or against an antigen as defined in the present 0252. The vaccine compositions according to the inven invention, and/or against a biological entity of the present tion preferably comprise an adjuvant and/or a carrier and/or at invention, when administered to an individual. In one least one immunomodulating peptide. Examples of useful embodiment the individual is infected with HIV. The vaccine adjuvants and carriers are given elsewhere herein. Thus, the composition of the invention is in one embodiment capable of biological components of the present invention, Such as ret eliciting a cellular immune response in the individual. roviral particles present in the composition can be associated 0248 For example, the vaccine composition is capable of with a carrier Such as e.g. a protein or an antigen-presenting eliciting the production in a vaccinated individual of effector cell to a T cell. T-cells having a cytotoxic effect against HIV-1 infected cells 0253) In particular the use of adjuvants is desired when the in a subject. In another embodiment, the vaccine composition immunogenic agents of the present invention are used to is capable of eliciting the production in a vaccinated indi vidual of regulatory T-cells having a cytotoxic effect against boost the immune response due to the ability of the biological cells expressing HIV-1 envelope polypeptide or part thereof, entities of the present invention, Such as retroviral particles and/or antigen presenting cells expressing HIV-1 envelope or are able to infect, integrate and display a lentiviral envelope or part thereof. In another embodiment, the vaccine composition fragments thereof on the surface of a host cell, thereby boost of the present invention is capable of initiating an antibody ing or enhancing the immune response, in particular the CTL response in an individual and/or a biological entity. response as discussed elsewhere herein. 0249. In a particular embodiment the vaccine composition 0254 Adjuvants are any substance whose admixture into is to be given against infection with HIV, in particular HIV-1. the vaccine composition increases or otherwise modifies the The present invention therefore also pertains to a vaccine immune response of the biological entity coated with the composition which is administered to an animal including a HIV-1 envelope polypeptide or fragment thereofas defined human being, in which the vaccine is capable of eliciting an elsewhere herein. Carriers are scaffold structures, for immune response against a disease caused by a lentivirus, in example a polypeptide or a polysaccharide, to which the particular HIV-1. Thus, a vaccine composition of the present biological entity coated with the HIV-1 envelope polypeptide invention is capable of eliciting a clinical response in a Sub or fragment thereof is capable of being associated. ject, wherein the clinical response is characterised by a 0255. Desirable functionalities of adjuvants capable of reduced Susceptibility, resistance, stabilisation, remission or being used in accordance with the present invention are listed curing/recovery of an HIV infection and/or AIDS. in the below table.

TABLE 11

Modes of adiuvantaction

Action Adjuvant type Benefit 1. Immunomodulation Generally Small molecules or Upregulation of immune proteins which modify the response. Selection of Th1 or cytokine network Th2 2. Presentation Generally amphipathic molecules Increased neutralizing antibody or complexes which interact with response. Greater duration of immunogen in its native response conformation US 2011/0305749 A1 Dec. 15, 2011 35

TABLE 1 1-continued

Modes of adiuvantaction Action Adjuvant type Benefit 3. CTL Particles which can bind or Cytosolic processing of protein induction enclose immunogen and yielding correct class 1 which can fuse with or disrupt restricted peptides cell membranes wo emulsions for direct Simple process if promiscuous attachment of peptide to cell peptide(s) known surface MHC-1 4. Targeting Particulate adjuvants which Efficient use of adjuvant and bind immunogen. Adjuvants immunogen which saturate Kupffer cells Carbohydrate adjuvants which As above. May also determine target lectin receptors on type of response if targeting macrophages and DCs selective 5. Depot wo emulsion for short term Efficiency Generation Microspheres or nanospheres for Potential for single-dose long term vaccine Source: Cox, J. C., and Coulter, A. R. (1997). Vaccine 15, 248-56,

0256 A vaccine composition according to the present 0261 Accordingly, the invention encompasses atherapeu invention may comprise more than one different adjuvant. tic composition further comprising an adjuvant Substance Furthermore, the invention encompasses a therapeutic com including any of the above or combinations thereof. It is also position further comprising any adjuvant Substance including contemplated that for example an antigen or a retroviral par any of the above or combinations thereof. It is also contem ticle of the invention and the adjuvant can be administered plated that the retroviral particle coated with the lentiviral simultaneously or separately in any appropriate sequence. envelope polypeptide or fragment thereof, and the adjuvant 0262 The pharmaceutical compositions may be prepared can be administered separately in any appropriate sequence. and administered using any conventional protocol known by 0257. A carrier may be present independently of an adju a person skilled in the art. It will be appreciated by the person vant. In particular, the inclusion of a carrier is relevant in connection with using a biological entity of the present inven skilled in the art that the protocol may be easily adapted to any tion are used to boost the immune response due to the ability of the vaccine compositions described herein. of the retroviral particles of the present invention to infect, 0263. In on embodiment of the invention, the vaccine integrate and display the HIV-1 envelope or fragments thereof compositions of the invention are useful for the prophylaxis on the Surface of a host cell, thereby boosting or enhancing the of HIV infection or for treatment of HIV infection in a human immune response, in particular the CTL response as dis being, where the human being is receiving treatment for the cussed elsewhere herein. infection. In a further embodiment, the pharmaceutical com 0258. The function of a carrier can for example be to positions, vaccines and vaccine compositions of the invention increase the molecular weight of in particular peptide frag are suitable for the treatment, amelioration and/or prevention ments in order to increase their activity or immunogenicity, to of a lentiviral infection, such as HIV infection, including confer stability, to increase the biological activity, or to HIV-1 infection, and AIDS. increase serum half-life. The carrier may be any suitable 0264. The choice of components in the vaccine composi carrier known to the person skilled in the art, for example a tion of the invention will depend on parameters determinable protein or an antigen presenting cell. A carrier protein could by the person of skill in the art. The composition of the be but is not limited to keyhole limpet hemocyanin, serum invention may also contain a combination of two or more proteins such as transferrin, bovine serum albumin, human HIV-1 envelope polypeptides, antigens, nucleic acids, mam serum albumin, thyroglobulin or ovalbumin, immunoglobu malian vectors and/or biological entities. Thus, as examples, lins, or hormones, such as insulin or palmitic acid. For immu the vaccine composition may contain any combination of nization of humans, the carrier must be a physiologically those components of the present invention. Also, the compo acceptable carrier acceptable to humans and safe. However, sition may comprise a combination of a peptide restricted by tetanus toxoid and/or diptheria toxoid are suitable carriers in a HLA-A molecule and a peptide restricted by a HLA-B one embodiment of the invention. Alternatively, the carrier molecule, e.g. including those HLA-A and HLA-B molecules may be dextrans, for example Sepharose. that correspond to the prevalence of HLA phenotypes in the 0259. In one embodiment, the vaccine composition of the target population, such as e.g. HLA-A2 and HLA-B35. Addi present invention, further comprises an immunogenic tionally, the composition may comprise a peptide restricted polypeptide or peptide fragment selected from a polypeptide by an HLA-C molecule. or peptide fragment, which is not derived from HIV-1 enve 0265. In the case of peptide-based vaccines, epitopes can lope. be administered in an MHC-ready form, which enables 0260. In another specific embodiment, the vaccine com presentation through exogenous loading independently of position of the present invention comprises antigen present antigen uptake and processing by host antigen-presenting ing biological entity of the present invention, as specified cells. The peptides of the present invention comprise both elsewhere herein. In yet another embodiment, the vaccine peptides in a short MHC-ready form and in a longer form composition comprises a eukaryotic expression vector of the requiring processing by the proteasome thus providing a present invention, as defined elsewhere. more complex vaccine composition that can target multiple US 2011/0305749 A1 Dec. 15, 2011 36 tumor antigens. The more different HLA groups are targeted Angel J B, et al. CPG 7909 adjuvant improves hepatitis by a vaccine, the higher likelihood of the vaccine functioning B virus vaccine seroprotection in antiretroviral-treated in diverse populations. HIV-infected adults. AIDS 2005; 19(14): 1473-9)) 0266 The present invention regards in a preferred 0278. Accelerate seroconversion, possibly permitting embodiment a vaccine composition comprising any HIV-1 post-exposure vaccination envelope polypeptide or a functional homologue thereofhav 0279 Reduce non-responders rate ing at least 70% identity to any of SEQID NOs: 1-124, SEQ ID NO: 125-326,327-337 or an immunogenically active pep 0280 Reduce amount of antigen required tide fragment comprising or consisting of a consecutive 0281 Increase antibody avidity and protective activity sequence of at least 3, Such as at least 5, Such as at least 10 0282 More sustained antibody levels amino acids of said HIV-1 envelope or said functional homo 0283 Thus in one embodiment, the vectors, methods, logue thereof or a nucleic acid encoding said HIV-1 envelope proviruses, retroviral particles, uses, compositions, vaccines, or said peptide fragment; in combination with an adjuvant for vaccine compositions and kits according to the present inven use as a medicament. The vaccine composition may be tion comprise an adjuvant and/or a nucleic acid sequence administered to treat, prevent, or reduce the risk associated encoding an adjuvant. with a clinical condition in an individual. 0284. In one embodiment, the adjuvant of the present invention is an immunostimulatory adjuvant. Examples of Adjuvants Such adjuvants are toll-like receptoragonists, such as agonists for TLR9, including CpG ODNs. 0267 A vaccine composition according to the present 0285 TLRagonist may in one embodiment be utilized as invention may comprise one or more than one adjuvant. a mean of attracting and activating antigen presenting cells Immunostimulatory adjuvants augment antigen-specific (APC: primarily Monocyte/macrophages and dendritic immune responses by physical localization and improved cells). This allows to selectively targeting the infection of presentation of antigen, and by provocation of inflammatory pseudotyped MLV particles to the activated APC and thus or innate immune responses (Petrovsky N. Aguilar J C. Vac promoting immunological cross-talk. cine adjuvants: current state and future trends. Immunol Cell 0286. In yet another embodiment, the components of the Biol 2004; 8205):488-96). A key feature in the innate immune present invention, including the vectors, nucleic acids, bio system is its capability to detect foreign organisms using a set logical entities, compositions and kits, comprises at least one of cell receptors termed pattern-recognition receptors (PRR). immunomodulating peptide and/or at least one nucleic acid One family of PRRs are Toll-Like Receptors, such as Toll sequence encoding an immunomodulating peptide. For Like Receptor 9 (TLR9). TLR9 detects unmethylated CpG example, the at least one additional nucleic acid sequence of dinucleotides, which are relative common in the genomes of a vector of the present invention encodes an immunomodu most bacteria and DNA viruses. lating polypeptide. Specifically, said immunomodulating 0268 Use of CpG oligodeoxynucleotides (ODNs) as peptide may be an immunostimulating polypeptide, an adjuvants has been tested in several vaccine trials. Cooper immunodominant polypeptide and/or a genetic adjuvant. (2005) used CpG 7909 as an adjuvant to a hepatitis B vacci Such embodiments include components of the present inven nation schedule in HIV patients and after 12 months seropro tion including vectors, nucleic acids, retroviral particles, tective titres were found in 100% of subjects in the CpG group compositions, and kit-of-parts comprising at least one cytok compared to 63% in the control group (p=0.008). In a recent ine and/or hormone, and/or at least one nucleic acid sequence study immunotherapy with a ragweed-toll-like receptor Ö encoding a cytokine and/or a hormone. Examples of cytok agonist vaccine for allergic rhinitis appeared to offer long ines include without restriction Interleukin-2 (IL-2), Interleu term clinical efficacy in the treatment of ragweed allergic kin-4 (IL-4), Interleukin-10 (IL-10), Granulocyte-macroph rhinitis (Creticos PS, Schroeder JT and Hamilton RG, et al. age colony stimulating factor (GM-CSF), Vascular Immunotherapy with a ragweed-toll-like receptor 6 agonist endothelial growth factor (VEGF), Interleukin-12 (IL-12), vaccine for allergic rhinitis. N Engl J Med 2006; 355(14): Fibroblast growth factor (FGF), Interleukin-7 (IL-7), Inter 1445-55). TLR9-receptor agonists are also currently being leukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-a), evaluated as adjuvant to novel malaria vaccine candidates but Tumor Necrosis Factor-beta (TNF-b), Lymphotactin, Inter they are also being used in a number of cancer trials. feron-alpha (IFN-a). Interferon-beta (IFN-b), Interferon 0269. Some of the shortcomings of regular vaccination gamma (IFN-g), Tumor Necrosis Factor (TNF), Interleukin a. 15 (IL-15), Interleukin-5 (IL-5), Interleukin-13 (IL-13), 0270 need for several boosts to achieve protection Interleukin-1a (IL-1 alfa), Interleukin-1b (IL-1beta), Inter 0271 delay in rise of protective antibodies leukin-18 (IL-18), MCP-1, MIP-1a, MIP-1b, RANTES, 0272 prevalence of vaccine non-responders (as out TCA-3, CD80, CD86, CD40L, CCL3, CCL4, CCL5, Lym lined above-this is particularly a problem for immune phocyte Chemotactic Factor (LCF), Erythropoietin (EPO), compromised individuals) Prothymosin-alpha, Thymopoietin, Thymosin-alpha-1. Each of the cytokines mentioned herein is intended to be an indi 0273 cost of antigen and vaccine production which is a vidual embodiment. Consequently, a vector, method, provi very significant limitation in the development of new rus, use, composition, vaccine, vaccine composition and/or conjugated pneumococcal vaccines. kit comprising at least one heterologous nucleic acid 0274 poorly protective antibodies with low affinity— sequence encoding a cytokine polypeptide or a fragment this has been observed in a number of trials with pneu thereof derived from each of them are claimed individually. mococcal vaccines in HIV-infected individuals. 0287 Immunomodulating polypeptides may also be (0275 Fall in antibody titre over time. expressed by the vector by including at least one nucleic acid 0276. These shortcomings can be overcome by the effects sequence encoding an immunomodulating polypeptide. A of TLR9-agonists: number of immunomodulating peptides are known to the 0277 Reduce number of vaccinations required to person skilled in the art. The immunomodulating polypep achieve Seroprotection (this was demonstrated in the tides may be immunostimulatory adjuvants, immunostimu Engerix and CpG 7909 trial (Cooper CL. Davis H L and latory cytokines or immunostimulant polypeptides other than US 2011/0305749 A1 Dec. 15, 2011 37 cytokines. Non-limiting examples of immunomodulating polypeptides according to the present invention are shown in TABLE 6a-continued table 6a, table 6b and table 6c. List of preferred immunostimulatory adjuvants which can be used as TABLE 6a immunostimulatory adjuvants according to the present invention. Protein and peptides List of preferred immunostimulatory adjuvants which can be used as immunostimulatory adjuvants according to the present invention. Mycobacterium vaccae Cancer Immunol. Immunother. 2002 Protein and peptides (SRL 172) Nov; 51(10): 521-31. Epub 2002 Sep 20. Adjuvants and the promotion of Th1-type Alpha-1,3- J. Virol. 2006 Jul: 80(14): 6943-51. cytokines in tumour immunotherapy. galactosyltransferase Increased immunogenicity of human Dredge K. Marriott JB, Todryk SM, immunodeficiency virus gp120 Dalgleish AG. engineered to express Galalpha1 Flagellin (ligand to Hum. Gene Ther. 2006 Nov; 17(11): 1051-61. 3Galbeta1-4GlcNAc-Repitopes. Abdel TLR5) DNA vaccines: recent developments Motel U, Wang S, Lu S, Wigglesworth K, and future possibilities. Liu MA, Wahren B, Galili U. Karlsson Hedestam GB. bacilius Calmette- Cancer Immunol. Immunother. 2002 Guérin (BCG) Nov; 51(10): 521-31. Epub 2002 Sep 20. Antrax proteins Hum. Gene Ther. 2006 Nov; 17(11): 1051-61. Adjuvants and the promotion of Th1-type DNA vaccines: recent developments cytokines in tumour immunotherapy. and future possibilities. Liu MA, Wahren B, Dredge K. Marriott JB, Todryk SM, Karlsson Hedestam GB. Dalgleish AG.

TABLE 6b List of immunostimulatory cytokines, which can be used as immunostimulatory adjuvants according to the present invention. Genetic adjuvants other than cytokines and conventional adjuvants. Category Classification Name References Genetic adjuvants Confirmulatory molecules CD80 22, 23 CD86 22, 23 CD154 (CD4OL) 25, 26 Chemokines TCA-3 24 RANTES 34 MIP1-a 33 Complement C3d 28 Heat shock protein Hsp70 29 Apoplosis inducer Fas 27 Caspase 30, 32 Transcriptional factors TRFs 31 Conventional adjuvants Mineral salts Aluminum phosphate Aluminum hydoxide 38 Bacteria-derived adjuvants Monophosphoryl lipid A 41, 43 Cholers foxin 44 Muramyl peptides Lipid particles Cationic liposomes 35-37 Mannan-coated liposomes 39 Emulsifier-based adjuvants QS-21 42 Synthetic adjuvants Ubenimex 40

(From Methods. 2003 November; 31(3):243-54. Adjuvant formulations and delivery systems for DNA vaccines. Sasaki S, Takeshita F, Xin KQ, Ishii N. Okuda K.)

TABLE 6c List of immunostimulatory cytokines, which can be used as immunostimulatory adjuvants according to the present invention. (Vaccine. 2001 Mar. 21; 19(17 19): 2647-56. Genetic adiuvants for DNA vaccines. Scheerlinck JY. Species', Effects Cytokines Ag/model route Protection Ab/IgG CTL IgG1/IgG2a DTH Reference MCP-1 BABNo Ag R, i.m. e n.d. n.d. n.d. 51, 52 MIP-1C. BABNo Ag R, i.m. e n.d. n.d. n.d. 51, 52 MIP-1C HIV-1 M, i.m. n.d. n.d. n.d. 55 MIP-1C HIV-1 M, i.m.fi.in. n.d. 56) MIP-1B BABNo Ag R, i.m. e n.d. n.d. n.d. (51) US 2011/0305749 A1 Dec. 15, 2011 38

TABLE 6c-continued List of immunostimulatory cytokines, which can be used as immunostimulatory adjuvants according to the present invention. (Vaccine. 2001 Mar. 21; 19(17 19): 2647-56. Genetic adiuvants for DNA vaccines. Scheerlinck JY. Species', Effects Cytokines Ag/model route Protection Ab/IgG CTL IgG1/IgG2a DTH Reference RANTES HIV-1 M, i.m. n.d. n.d. n.d. n.d. 55 RANTES BAB/No Ag R, i.m. : fe n.d. n.d. n.d. (51) TCA-3 HIV-1 M, i.m. n.d. 57 CD8O HSV-2 M, i.m. : : n.d. : = 54) CD8O HSV-2 M, i.m. : n.d. : 54) CD8O HIV-1 M, i.m. n.d. : : n.d. = 58,59 CD8Of Influenza M, i.m. n.d. n.d. : n.d. n.d. 29 CD86 HSV-3 M, i.m.i.d. : : n.d. : = 54) CD86 HIV-1 M, i.m. n.d. : : 58,60,53,59 CD86 HIV-1 C, i.m. n.d. n.d. n.d. n.d. 60 CD86/ Influenza M, i.m. n.d. n.d. n.d. n.d. 29 CD4OL 3-gal M, i.m. n.d. 46

0288 Adjuvants could for example be selected from the a mixture of the two. They are prepared for use as an emulsion group consisting of AlK(SO), AlNaCSO), AlNH. (SO). with an aqueous solution comprising HIV-1 envelope silica, alum, Al(OH), Cas (PO4), kaolin, carbon, aluminum polypeptide or peptide fragment thereof. The Surfactant is hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threo mannide oleate. QS-21 (Antigenics; Aquila Biopharmaceuti nyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-ala cals, Framingham, Mass.) is a highly purified, water-soluble nyl-D-isoglutamine (CGP 1 1687, also referred to as nor saponin that handles as an aqueous solution. QS-21 and Mon MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L- tanide ISA-51 adjuvants can be provided in sterile, single-use alanine-2-(1'2'-dipalmitoyl-sn-glycero-3- vials. hydroxphosphoryloxy)-ethylamine (CGP 19835A, also 0290. The well-known cytokine GM-CSF is another pre referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2% ferred adjuvant of the present invention. GM-CSF has been squalene/Tween-80(R) emulsion, lipopolysaccharides and its used as an adjuvant for a decade and may preferably be various derivatives, including lipid A, Freund's Complete GM-CSF as described in WO 97/2881 6. Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck 0291. In a preferred embodiment, the vaccine composition Adjuvant 65, polynucleotides (for example, poly IC and poly adjuvant is selected from the group consisting of bacterial AU acids), wax D from Mycobacterium, tuberculosis, sub DNA based adjuvants, oil/surfactant based adjuvants, viral stances found in Corynebacterium parvum, Bordetella per dsRNA based adjuvants, imidazochinilines, and/or incom tussis, and members of the genus Brucella, Titermax, plete Freund's adjuvant (IFA). In another preferred embodi ISCOMS, Quil A, ALUN (see US 58767 and U.S. Pat. No. ment, the vaccine composition adjuvant is a Montanide ISA 5,554.372), Lipid A derivatives, choleratoxin derivatives, adjuvant. In another preferred embodiment the adjuvant is HSP derivatives, LPS derivatives, synthetic peptide matrixes Montanide ISA 51 or Montanide ISA 720. In another pre or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51 ferred embodiment, the adjuvant is Montanide ISA51. In yet and QS-21. Preferred adjuvants to be used with the invention another embodiment, the adjuvant is GM-CSF. include oil/surfactant based adjuvants such as Montanide adjuvants (available from Seppic, Belgium), preferably Mon Carriers tanide ISA-51. Other preferred adjuvants are bacterial DNA 0292. The vaccine composition of to the present invention based adjuvants. Such as adjuvants including CpG oligo may comprise any adjuvant Substance and/or carrier includ nucleotide sequences. Yet other preferred adjuvants are viral ing any of the above or combinations thereof. It is also con dsRNA based adjuvants, such as poly I:C. Imidazochinilines templated that any of the immunogenic agents of the vaccine are yet another example of preferred adjuvants. The most composition and the adjuvant and/or carrier can be adminis preferred adjuvants are adjuvants suitable for human use. tered simultaneously or separately and/or repetitively in any 0289 Montanide adjuvants (all available from Seppic, appropriate sequence. Belgium), may be selected from the group consisting of Mon 0293. A carrier may be present independently of an adju tanide ISA-51, Montanide ISA-50, Montanide ISA-70, Mon vant. The function of a carrier can for example be to increase tanide ISA-206, Montanide ISA-25, Montanide ISA-720, the molecular weight of in particular peptide fragments in Montanide ISA-708, Montanide ISA-763A, Montanide ISA order to increase their activity or immunogenicity, to confer 207, Montanide ISA-264, Montanide ISA-27, Montanide stability, to increase the biological activity, or to increase ISA-35, Montanide ISA51F, Montanide ISA 016D and Mon serum half-life. Furthermore, a carrier may aid in presenting tanide IMS, preferably from the group consisting of Mon the HIV-1 envelope polypeptide, variant or peptide fragments tanide ISA-51, Montanide IMS and Montanide ISA-720, thereof to T-cells. The carrier may be any suitable carrier more preferably from the group consisting of Montanide known to a person skilled in the art, for example a protein or ISA-51. Montanide ISA-51 (Seppic, Inc.) is oil/surfactant an antigen presenting cell. A carrier protein could be, but is based adjuvants in which different surfactants are combined not limited to, keyhole limpet hemocyanin, serum proteins with a non-metabolizable mineral oil, a metabolizable oil, or Such as transferrin, bovine serum albumin, human serum US 2011/0305749 A1 Dec. 15, 2011 39 albumin, thyroglobulin or ovalbumin, immunoglobulins, or 0300. The pharmaceutical compositions may be provided hormones. Such as insulin or palmitic acid. For immunization to the individual by a variety of routes such as Subcutaneous, of humans, the carrier must be a physiologically acceptable topical, oral and intramuscular. Administration of pharma carrier acceptable to humans and safe. However, tetanus tox ceutical compositions is accomplished orally or parenterally. oid and/or diptheria toxoid are suitable carriers in one Methods of parenteral delivery include topical, intra-arterial embodiment of the invention. Alternatively, the carrier may (directly to the tissue), intramuscular, Subcutaneous, be dextrans for example Sepharose. intramedullary, intrathecal, intraventricular, intravenous, 0294 Thus it is an aspect of the present invention that the intraperitoneal, or intranasal administration. The present immunogenic agent, such as an HIV-1 envelope polypeptide invention also has the objective of providing Suitable topical, or fragment or variant thereof or peptide derived here from present in the vaccine composition is associated with a carrier oral, systemic and parenteral pharmaceutical formulations Such as e.g. a protein of the above or an antigen-presenting for use in the methods of prophylaxis and treatment with the cell such as e.g. a dendritic cell (DC). vaccine composition. 0295 Accordingly, the invention encompasses atherapeu 0301 For example, the vaccine compositions can be tic composition further comprising an adjuvant Substance administered in Such oral dosage forms as tablets, capsules including any of the above or combinations thereof. It is also (each including timed release and Sustained release formula contemplated that the antigen, i.e. the peptide of the invention tions), pills, powders, granules, elixirs, tinctures, Solutions, and the adjuvant can be administered simultaneously or sepa Suspensions, syrups and emulsions, or by injection. Likewise, rately in any appropriate sequence. they may also be administered in intravenous (both bolus and infusion), intraperitoneal, Subcutaneous, topical with or with Dosis and Administration out occlusion, or intramuscular form, all using forms well 0296. The amount of the immunogenic HIV1-envelope knownto those of ordinary skill in the pharmaceutical arts. An polypeptide, biological entity, and/orantigen of the invention effective but non-toxic amount of the vaccine, comprising any in the vaccine composition may vary, depending on the par of the herein described compounds can be employed as a ticular application. However, a single dose of the HIV1-en prophylactic or therapeutic agent. Also any and all conven velope polypeptide, biological entity, and/or antigen is pref tional dosage forms that are known in the art to be appropriate erably anywhere from about 1 lug to about 5000 ug, more for formulating injectable immunogenic peptide composition preferably from about 50 ug to about 2500 lug such as about are encompassed, such as lyophilized forms and Solutions, 100 g to about 1000 g. Modes of administration include Suspensions or emulsion forms containing, if required, con intradermal, Subcutaneous and intravenous administration, ventional pharmaceutically acceptable carriers, diluents, pre implantation in the form of a time release formulation, etc. servatives, adjuvants, buffer components, etc. Any and all forms of administration known to the art are encompassed herein. Also any and all conventional dosage 0302 Preferred modes of administration of the vaccine forms that are known in the art to be appropriate for formu composition according to the invention include, but are not lating injectable immunogenic peptide composition are limited to systemic administration, such as intravenous or encompassed. Such as lyophilized forms and Solutions, Sus Subcutaneous administration, intradermal administration, pensions or emulsion forms containing, if required, conven intramuscular administration, intranasal administration, oral tional pharmaceutically acceptable carriers, diluents, preser administration, rectal administration, vaginal administration, Vatives, adjuvants, buffer components, etc. pulmonary administration and generally any form of mucosal 0297. The vaccine compositions may be prepared and administration. Furthermore, it is within the scope of the administered using any conventional protocol known by a present invention that the means for any of the administration person skilled in the art. In examples 3-5 non-limiting forms mentioned in the herein are included in the present examples of preparation of a vaccine composition according invention. to the invention is given as well as a non-limiting example of 0303) A vaccine according to the present invention can be administration of Such as a vaccine. It will be appreciated by administered once, or any number of times Such as two, three, the person skilled in the art that the protocol may be easily four or five times. Administering the vaccine more than once adapted to any of the vaccine compositions described herein. has the effect of boosting the resulting immune response. The In a further embodiment of the invention, the pharmaceutical vaccine can further be boosted by administering the vaccine composition of the invention is useful for treating an indi in a form or body part different from the previous adminis vidual suffering from a clinical condition characterized by tration. The booster shot is either a homologous or a heter expression of HIV-1 envelope, such as HIV, AIDS and/or ologous booster shot. A homologous booster shot is a where ARC. the first and Subsequent vaccinations comprise the same con 0298. The immunoprotective effect of the composition of structs and more specifically the same delivery vehicle espe the invention can be determined using several approaches cially the same viral vector. A heterologous booster shot is known to those skilled in the art. A Successful immune where identical constructs are comprised within different response may also be determined by the occurrence of DTH viral vectors. reactions after immunization and/or the detection of antibod 0304. The present invention also in one aspect, relates to a ies specifically recognizing the peptide(s) of the vaccine com method of producing a vaccine composition of the present position. invention as defined elsewhere herein, comprising combining 0299 Vaccine compositions according to the invention 0305 a) an HIV-1 envelope polypeptide as defined else may be administered to an individual in therapeutically effec where herein, or a functional homologue thereofhaving at tive amounts. The effective amount may vary according to a least 70% identity to said peptide or an immunogenically variety of factors such as the individual's condition, weight, active peptide fragment comprising a consecutive sex and age. Other factors include the mode of administration. sequence of at least 10 residues of said peptide or said US 2011/0305749 A1 Dec. 15, 2011 40

functional homologue thereof, or a nucleic acid encoding Laboratory Press, 1988. Polyclonal antibodies may be said peptide or said peptide fragment or said functional derived from any Suitable mammalian species, for example homologies thereof and/or from mice, rats, rabbits, donkeys, goats, sheeps, cows or 0306 b) an antigen as defined elsewhere herein, and/or camels. The antibody is preferably not derived from a non 0307 c) a nucleic acid as defined elsewhere herein, and/or mammalian species, i.e. the antibody is for example prefer 0308 d) a vector as defined elsewhere herein, and/or ably not a chicken antibody. The antibody may also for 0309 e) a biological entity as defined elsewhere herein, example be an artificial polyclonal antibody as for example and described in U.S. Pat. No. 5,789,208 or U.S. Pat. No. 6,335, 0310 f) an adjuvant 163, both patent specifications are hereby incorporated by reference into the application in their entirety. Antibody 0315. The antibodies according to the present invention 0311. It is one aspect of the present invention to provide may also be recombinantantibodies. Recombinant antibodies antibodies or functional equivalents thereof. Such as antigen are antibodies or fragments thereof or functional equivalents binding fragments or recombinant proteins specifically rec thereof produced using recombinant technology. For example ognising and binding an HIV-1 envelope polypeptide, Such as recombinant antibodies may be produced using a synthetic an HIV envelope polypeptide encoded by a gene selected library or by phage display. Recombinant antibodies may be from the group consisting of SEQ ID NO: 74-119. In one produced according to any conventional method for example embodiment, the antibody, antigenbinding fragment, recom the methods outlined in "Recombinant Antibodies', Frank binant protein or functional homologue thereof is capable of Breitling, Stefan Dibel, Jossey-Bass, September 1999. inhibiting binding of said polypeptide to a native cellular 0316 Human monoclonal antibodies of the invention can interaction partner. The antibody or functional homologue be produced by a variety of techniques, including conven thereof specifically recognizes an epitope or a functional tional monoclonal antibody methodology, e.g., the standard homologue thereof. The epitope may be any of the epitopes somatic cell hybridization technique of Kohler and Milstein, mentioned herein. Nature 256:495 (1975). Although somatic cell hybridization 0312 The antibody or functional equivalent thereof may procedures are preferred, in principle, other techniques for be any antibody known in the art, for example a polyclonal or producing monoclonal antibody can be employed, e.g., viral a monoclonal antibody derived from a mammal or a synthetic or oncogenic transformation of B-lymphocytes or phage dis antibody, such as a single chain antibody or hybrids compris play techniques using libraries of human antibody genes. ing antibody fragments. Furthermore, the antibody may be Antibody Targets mixtures of monoclonal antibodies or artificial polyclonal antibodies. In addition functional equivalents of antibodies 0317. The antibody, antigen binding fragment or recom may be antibody fragments, in particular epitope binding binant protein thereof is specific for a translational gene prod fragments. Furthermore, antibodies or functional equivalent uct of polypeptide encoded by HIV-1, such as an envelope thereof may be small molecule mimetics, mimicking an anti polypeptide encoded by a gene selected from the group con body. Naturally occurring antibodies are immunoglobulin sisting of SEQID NO: 74-119, 120-124,330-333 or a part or molecules consisting of heavy and light chains. In preferred functional homolog of said polypeptide, and/or any envelope embodiments of the invention, the antibody is a monoclonal polypeptide comprising a nucleic acid sequence selected antibody. from the group consisting of SEQ ID NO: 1-337 or part 0313 Monoclonal antibodies (Mab's) are antibodies, thereof. In one embodiment, the antibody, antigen binding wherein every antibody molecule are similar and thus recog fragment or recombinant protein thereof is capable of specifi nises the same epitope. Monoclonal antibodies are in general cally inhibiting binding of a native protein interaction partner produced by a hybridoma cell line. Methods of making mono to a polypeptide or part thereof encoded by a gene of the clonal antibodies and antibody-synthesizing hybridoma cells present invention. Said inhibition of binding is suitable for are well known to those skilled in the art. Antibody producing treatment of a clinical condition as defined elsewhere herein, hybridomas may for example be prepared by fusion of an Such as HIV-1 or AIDS. antibody producing B lymphocyte with an immortalized 0318. In one embodiment of the present invention, the B-lymphocyte cell line. Monoclonal antibodies according to antibody, antigen binding fragment or recombinant protein the present invention may for example be prepared as thereof is capable of specifically recognizing and binding an described in Antibodies: A Laboratory Manual. By Ed Har HIV-1 envelope polypeptide, such as an HIV-1 envelope low and David Lane, Cold Spring Harbor Laboratory Press, polypeptide selected from any one of SEQID NO: 74-119, 1988. Said monoclonal antibodies may be derived from any 120-124, 330-333. Suitable mammalian species, however frequently the mono 0319 Specifically, the antibody, antigen binding fragment clonal antibodies will be rodent antibodies for example or recombinant protein thereof according to the present murine or rat monoclonal antibodies. It is preferred that the invention, is capable of specifically recognizing and binding antibodies according to the present invention are monoclonal to an HIV-1 derived peptide as defined herein, such as any one antibodies or derived from monoclonal antibodies. of SEQ ID NO: 1-329, 330-337. In particular, the antibody, 0314 Polyclonal antibodies is a mixture of antibody mol antigen binding fragment or recombinant protein thereof ecules recognising a specific given antigen, hence polyclonal according to the present invention, is capable of specifically antibodies may recognise different epitopes within said anti recognizing and binding to an epitope consisting of 3 to 10 gen. In general polyclonal antibodies are purified from serum amino acid residues, such as 3 to 8 amino acid residues, such of a mammal, which previously has been immunized with the as 3 to 6 amino acid residues selected from an HIV-1 envelope antigen. Polyclonal antibodies may for example be prepared polypeptide sequence such as SEQID NO: 74-119. However, by any of the methods described in Antibodies: A Laboratory even more specifically the antibody, antigen binding frag Manual. By Ed Harlow and David Lane, Cold Spring Harbor ment or recombinant protein thereof according to the present US 2011/0305749 A1 Dec. 15, 2011

invention, is capable of specifically recognizing and binding ing to any one of SEQID NO: 1-329,330-337 for producing to an epitope consisting of 3 to 10 amino acid residues. Such an antibody. In one example said use relates to the use for as 3 to 8 amino acid residues, such as 3 to 6 amino acid producing an antibody for the treatment of a clinical condi residues of an HIV-1 derived peptide selected from SEQID tion such as HIV-1 and/or AIDS. NO: 1-73 and/or 120-329 and/or 330-337. 0320 Another aspect of the present invention relates to Functional Homologues antibodies and functional homologues thereof, which are able 0330 Functional homologues of polypeptides according to specifically recognize and bind, and modulate the activity to the present invention is meant to comprise any polypeptide of a polypeptide encoded by a gene of the present invention, sequence which is capable of associating with an envelope preferably an HIV-1 envelope polypeptide sequence Such as receptor protein and/or which renders a cell fusogenic when SEQ ID NO: 74-119, 120-124, 330-333, or a functional expressing said functional homolog, and/or which is capable homolog or part thereof. of eliciting an immune response, when said functional 0321) Importantly, the present invention encompasses use homolog is presented on the Surface of a cell, particle and/or of an antibody as defined herein, for the manufacture of a other physical entity. Examples 1-5 provides different medicament for the treatment of a clinical condition as examples of methods for testing immunosuppression/immu defined herein, such as HIV-1 and/or AIDS. nogenicity, Surface expression, and fusogenicity between two 0322. Also, the present invention encompasses methods of human cell lines. treatment of a clinical condition as defined herein, Such as 0331 Functional homologues according to the present HIV-1 and/or AIDS comprising administration of an antibody invention comprise polypeptides with an amino acid as described herein to a person in need thereof. The invention sequence, which are sharing at least some homology with the also relates to an antibody as defined herein for treatment of predetermined polypeptide sequences as outlined herein. For said clinical condition. example such polypeptides are at least about 40 percent, Such 0323 Thus, one aspect of the present invention relates to as at least about 50 percent homologous, for example at least an antibody, antigenbinding fragment or recombinant protein about 60 percent homologous, such as at least about 70 per thereof, which is specific for an HIV-1 envelope polypeptide cent homologous, for example at least about 75 percent or part thereof as defined herein, and/or a nucleic acid as homologous, Such as at least about 80 percent homologous, defined herein, and/or an antigen as defined herein, and/or a for example at least about 85 percent homologous, such as at biological entity as defined herein. In one embodiment, the least about 90 percent homologous, for example at least 92 antibody, antigen binding fragment or recombinant protein percent homologous, such as at least 94 percent homologous, thereof is capable of initiating an immune response against for example at least 95 percent homologous, such as at least HIV-1 retroviral particles. 96 percent homologous, for example at least 97 percent 0324. The HIV-1 envelope polypeptide or part thereof is homologous, Such as at least 98 percent homologous, for any HIV-1 envelope polypeptide, or any polypeptide derived example at least 99 percent homologous with the predeter therefrom described herein above, for example selected from mined polypeptide sequences as outlined herein above. The any one of SEQID NO: 1-329, 330-337, or part thereof. homology between amino acid sequences may be calculated 0325 In another aspect, the present invention relates to an using well known algorithms such as for example any one of antibody obtainable by immunizing a host with an HIV-1 BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, envelope polypeptide or part thereofas defined herein, Such BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, as a polypeptide identified by SEQID NO: 1-329,330-337 or BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, part thereof, and/or a nucleic acid as defined herein, and/oran and BLOSUM 90. antigenas defined herein, and/or a biological entity as defined 0332 Functional homologues may comprise an amino herein, a vaccine composition as defined herein, and/or a acid sequence that comprises at least one substitution of one kit-of-parts as defined herein. amino acid for any other amino acid. For example Such a 0326. The present invention also relates to a method of Substitution may be a conservative amino acid substitution or producing antibody of the present invention, said method it may be a non-conservative substitution. A conservative comprising the steps of amino acid Substitution is a Substitution of one amino acid 0327 a) administering an antigen, HIV-1 envelope within a predetermined group of amino acids for another polypeptide or part thereof as defined herein, and/or a amino acid within the same group, wherein the amino acids nucleic acid as defined herein, and/oran antigen as defined within predetermined groups exhibit similar or substantially herein, and/or a biological entity as defined herein, a vac similar characteristics. Within the meaning of the term “con cine composition as defined herein, and/or a kit-of-parts as servative amino acid Substitution” as applied herein, one defined herein to an animal amino acid may be substituted for another within groups of 0328 b) obtaining said antibody from said animal amino acids characterised by having Specific methodologies for the production of antibodies are 0333 i) polar side chains (Asp, Glu, Lys, Arg, His, Asn. described herein above. The antigen, HIV-1 envelope Gln, Ser, Thr, Tyr, and Cys) polypeptide or part thereofused for producing an antibody is 0334 ii) non-polar side chains (Gly, Ala, Val, Leu, Ile, preferably a polypeptide selected from any one of SEQ ID Phe, Trp, Pro, and Met) NO: 1-73 and/or SEQ ID NO: 120-329, 330-337. In a pre 0335 iii) aliphatic side chains (Gly, Ala Val, Leu, Ile) ferred embodiment, the antigen, HIV-1 envelope polypeptide 0336 iv) cyclic side chains (Phe, Tyr, Trp, His, Pro) or part thereof comprise one or more epitopes of the present 0337 v) aromatic side chains (Phe, Tyr, Trp) invention. 0338 vi) acidic side chains (Asp, Glu) 0329. Thus in one aspect, the present invention relates to 0339 vii) basic side chains (Lys, Arg, His) the use of an antigen, HIV-1 envelope polypeptide or part 0340 viii) amide side chains (ASn, Gln) thereof of the present invention, such as a polypeptide accord 0341 ix) hydroxy side chains (Ser. Thr) US 2011/0305749 A1 Dec. 15, 2011 42

0342 x) sulphor-containing side chains (Cys, Met), and within +/-0.25, such as within +/-0.2 of the value of the 0343 xi) amino acids being monoamino-dicarboxylic amino acid it has substituted. The importance of the hydro acids or monoamino-monocarboxylic-monoamidocar philic and hydropathic amino acid indices in conferring inter boxylic acids (Asp, Glu, ASn, Gln). active biologic function on a protein is well understood in the 0344) Non-conservative substitutions are any other substi art (Kyte & Doolittle, 1982 and Hopp, U.S. Pat. No. 4,554, tutions. A non-conservative Substitution leading to the forma 101, each incorporated herein by reference). tion of a functional homologue would for example i) differ 0348. The amino acid hydropathic index values as used substantially in hydrophobicity, for example a hydrophobic herein are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); residue (Val, Ile, Leu, Phe or Met) substituted for a hydro phenylalanine (+2.8); cysteine/cystine (+2.5); methionine philic residue Such as Arg, Lys, Trp or ASn, or a hydrophilic (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); residue such as Thr, Ser. His, Gln, Asn. Lys, Asp, Glu or Trp serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1. substituted for a hydrophobic residue; and/or ii) differ sub 6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); stantially in its effect on polypeptide backbone orientation aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and argin such as substitution of or for Pro or Gly by another residue. ine (-4.5) (Kyte & Doolittle, 1982). and/or iii) differ substantially in electric charge, for example 0349 The amino acid hydrophilicity values are: arginine Substitution of a negatively charged residue such as Glu or (+3.0); lysine (+3.0); aspartate (+3.0.+-0.1); glutamate (+3. Asp for a positively charged residue such as Lys, His or Arg 0.+-0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2): (and vice versa); and/or iv) differ substantially in steric bulk, glycine (O); threonine (-0.4); proline (-0.5.+-0.1); alanine for example substitution of a bulky residue such as His, Trp, (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); Phe or Tyrfor one having a minor side chain, e.g. Ala, Gly or valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2. Ser (and vice versa). 3); phenylalanine (-2.5); tryptophan (-3.4) (U.S. Pat. No. 0345 Functional homologues according to the present 4,554,101). invention may comprise more than one such substitution, 0350 Substitution of amino acids can therefore in one Such as e.g. two amino acid Substitutions, for example three or embodiment be made based upon their hydrophobicity and four amino acid Substitutions, such as five or six amino acid hydrophilicity values and the relative similarity of the amino Substitutions, for example seven or eight amino acid substi acid side-chain Substituents, including charge, size, and the tutions, such as from 10 to 15 amino acid substitutions, for like. Exemplary amino acid Substitutions which take various example from 15 to 25 amino acid substitution, such as from of the foregoing characteristics into consideration are well 25 to 30 amino acid substitutions, for example from 30 to 40 known to those of skill in the art and include: arginine and amino acid substitution, such as from 40 to 50 amino acid lysine; glutamate and aspartate; serine and threonine; substitutions, for example from 50 to 75 amino acid substi glutamine and asparagine; and valine, leucine and isoleucine. tution, such as from 75 to 100 amino acid substitutions, for 0351. In addition to the polypeptide compounds described example more than 100 amino acid substitutions. The addi herein, Sterically similar compounds may be formulated to tion or deletion of an amino acid may be an addition or mimic the key portions of the peptide structure and that Such deletion of from 2 to 5 amino acids, such as from 5 to 10 compounds may also be used in the same manner as the amino acids, for example from 10 to 20 amino acids, such as peptides of the invention. This may beachieved by techniques from 20 to 50 amino acids. However, additions or deletions of of modelling and chemical designing known to those of skill more than 50 amino acids, such as additions from 50 to 200 in the art. For example, esterification and other alkylations amino acids, are also comprised within the present invention. may be employed to modify the amino terminus of, e.g., a The polypeptides according to the present invention, includ di-arginine peptide backbone, to mimic a tetra peptide struc ing any variants and functional homologues thereof, may in ture. It will be understood that all such sterically similar one embodiment comprise more than 5 amino acid residues, constructs fall within the scope of the present invention. Such as more than 10 amino acid residues, for example more 0352 Peptides with N-terminal alkylations and C-termi than 20 amino acid residues, such as more than 25 amino acid nal esterifications are also encompassed within the present residues, for example more than 50 amino acid residues. Such invention. Functional equivalents also comprise glycosylated as more than 75 amino acid residues, for example more than and covalent or aggregative conjugates, including dimers or 100 amino acid residues, such as more than 150 amino acid unrelated chemical moieties. Such functional equivalents are residues, for example more than 200 amino acid residues. prepared by linkage of functionalities to groups which are 0346 Additional factors may be taken into consideration found in fragment including at any one or both of the N- and when determining functional homologues according to the C-termini, by means known in the art. meaning used herein. For example functional homologues 0353 Functional equivalents may thus comprise frag may be capable of associating with antisera which are specific ments conjugated to aliphatic or acyl esters or amides of the for the polypeptides according to the present invention. carboxyl terminus, alkylamines or residues containing car 0347 In a further embodiment the present invention boxyl side chains, e.g., conjugates to alkylamines at aspartic relates to functional equivalents which comprise Substituted acid residues; O-acyl derivatives of hydroxyl group-contain amino acids having hydrophilic or hydropathic indices that ing residues and N-acyl derivatives of the amino terminal are within +/-2.5, for example within +/-2.3, such as within amino acid or amino-group containing residues, e.g. conju +/-2.1, for example within +/-2.0, such as within +/- 1.8, for gates with Met-Leu-Phe. Derivatives of the acyl groups are example within +/-1.6, Such as within +/-1.5, for example selected from the group of alkyl-moieties (including C3 to within +/- 1.4, such as within +/- 1.3 for example within +/-1. C10 normal alkyl), thereby forming alkanoyl species, and 2. Such as within +/- 1.1, for example within +/- 1.0, Such as carbocyclic or heterocyclic compounds, thereby forming within +/-0.9, for example within +/-0.8, such as within aroyl species. The reactive groups preferably are difunctional +/-0.7, for example within +/-0.6, such as within +/-0.5, for compounds known perse for use in cross-linking proteins to example within +/-0.4, such as within +/-0.3, for example insoluble matrices through reactive side groups. US 2011/0305749 A1 Dec. 15, 2011

0354 Homologues of nucleic acid sequences within the a composition eliciting at least one type of immune response Scope of the present invention are nucleic acid sequences, directed against HIV-1 envelope expressing biological enti which encodes an RNA and/or a protein with similar biologi ties, such as mammalian cells, APCs or DCs and/or retroviral cal function, and which is either particles. Thus, such an immune response may be any of the 0355 a) at least 50% identical, such as at least 60% following: A CTL response where CTLs are generated that identical, for example at least 70% identical, such as at are capable of recognizing the HLA/peptide complex pre least 75% identical, for example at least 80% identical, sented on cell Surfaces resulting in cell lysis, i.e. the vaccine such as at least 85% identical, for example at least 90% elicits the production in the vaccinated subject of effector identical, such as at least 95% identical T-cells having a cytotoxic effect against the cancer cells; a 0356 b) or able to hybridise to the complementary B-cell response giving rise to the production of anti-HIV strand of said nucleic acid sequence under Stringent antibodies; and/or a DTH type of immune response. It is on conditions. object of the present invention to monitor the immunization 0357 Stringent conditions as used herein shall denote of an individual by monitoring any of the above reactions stringency as normally applied in connection with Southern Subsequent to administering the composition of the present blotting and hybridisation as described e.g. by Southern E. invention to said individual. M., 1975, J. Mol. Biol. 98:503-517. For such purposes it is 0362. In one aspect the invention relates to methods of routine practise to include steps of prehybridization and monitoring immunization, said method comprising the steps hybridization. Such steps are normally performed using Solu of tions containing 6xSSPE, 5% Denhardt's, 0.5% SDS, 50% 0363 a) providing a blood sample from an individual, formamide, 100 g/ml denaturated salmontestis DNA (incu 0364 b) providing an HIV-1 envelope polypeptide or a bation for 18 hrs at 42° C.), followed by washings with fragment thereof, an antigen, a nucleic acid, a biological 2xSSC and 0.5% SDS (at room temperature and at 37° C.), entity, and/or a vaccine composition, and/or a kit-of-parts and a washing with 0.1 xSSC and 0.5% SDS (incubation at to an animal, wherein said HIV-1 envelope polypeptide or 68°C. for 30 min), as described by Sambrook et al., 1989, in a fragment thereof, an antigen, a nucleic acid, a biological “Molecular Cloning/A Laboratory Manual, Cold Spring entity, and/or a vaccine composition, and/or a kit-of-parts Harbor), which is incorporated herein by reference. may be any of the HIV-1 envelope polypeptides or frag 0358 Homologous of nucleic acid sequences also encom ments thereof, antigens, a nucleic acid, biological entities, pass nucleic acid sequences which comprise additions and/or and/or vaccine compositions, and/or kits-of-parts deletions. Such additions and/or deletions may be internal or described herein, and at the end. Additions and/or deletions may be of 1-5 nucle 0365 c) determining whether said blood sample com otides, such as 5 to 10 nucleotide, for example 10 to 50 prises antibodies or T-cells comprising T-cell receptors nucleotides, such as 50 to 100 nucleotides, for example at specifically binding an HIV-1 envelope polypeptide or a least 100 nucleotides. fragment thereof, an antigen and/or biological entity as defined be the present invention, and Second Active Ingredient 0366 d) thereby determining whether an immune response to said protein or peptide has been raised in said 0359. It is an aspect of the present invention that the vac individual. cine composition herein provided is used in combination with 0367 The individual is preferably a human being, for a second active ingredient. The administration of the vaccine example a human being that has been immunized with an composition and the second active ingredient may be sequen HIV-1 envelope polypeptide or a fragment thereof, an anti tial or combined. Examples of second active ingredients are gen, a nucleic acid, a biological entity, and/or a vaccine com given elsewhere herein. It is a further aspect that the vaccine position, and/or a kit-of-parts of the present invention. composition may be used in combination with other therapy of relevance for the given clinical condition to be treated. CTL Response Such therapy may include Surgery and/or gene therapy, immunostimulating Substances or antibodies; a person 0368. The components of the present invention including HIV-1 envelope polypeptides and/or fragments thereof, vec skilled in the art is able to determine the appropriate combi tors, nucleic acids, biological entities, compositions, and kit nation treatment for a given scenario. of-parts is capable of inducing an immunogenic response in a 0360. In some cases it will be appropriate to combine the host animal, for example in a human. The immunogenic treatment method of the invention with a further medical response may be divided into to two types of responses, the treatment Such as treatment with immunostimulating Sub antibody response and the cytotoxic T lymphocyte (CTL) stances, gene therapy, treatment with antibodies and/or anti response. biotics and treatment using dendritic cells. 0369. In the antibody response, specific antibodies are important in and may protect against viral infections. The Monitoring Immunization most effective type of antiviral antibody is “neutralizing 0361. In preferred embodiments, the pharmaceutical com antibody—this is antibody which binds to the virus, usually to position of the invention is a vaccine composition. It is there the viral envelope of the virus particle or capsid proteins, and fore of interest, and an aspect of the present invention to which blocks the virus from binding and gaining entry to the monitor the immunization in an individual to whom the vac host cell. Virus specific antibodies may also act as opsonins in cine composition of the present invention is administered. enhancing phagocytosis of virus particles—this effect may be The pharmaceutical composition may thus be an immuno further enhanced by complement activation by antibody genic composition or vaccine capable of eliciting an immune coated virus particles e.g. through production of the viral response to HIV infection and/or AIDS. As used herein, the particles in eukaryotic cells e.g. mouse cells that ads gal expression “immunogenic composition or vaccine” refers to alfa 1-3Galbeta1-4GlcNAc-R epitopes on the envelope pro US 2011/0305749 A1 Dec. 15, 2011 44 tein. In addition, in the case of some viral infections, viral a CTL response. In a specific embodiment, the immunogenic proteins are expressed on the surface of the infected cell. response is a CTL response, wherein said vector, RNA, These may act as targets for virus-specific antibodies, and mRNA of the present invention is integrated into the genome may lead to complement-mediated lysis of the infected cell, of a host cell. or may directa subset of natural killer cells to lyse the infected cell through a process known as antibody-directed cellular 0374. In one embodiment, the vector or retroviral particle cytotoxicity (ADCC). At mucosal Surfaces (such as the res is able to infect, integrate and display the HIV-1 envelope or piratory and gastrointestinal tracts), virus infection may fragments thereof on the surface of a host cell the lentiviral induce the production of specific antibodies of the IgA iso envelope polypeptide, thus providing a means of boosting or type, which may be protective against infection at these Sur enhancing the immune response as compared to viral vac faces. Not all antibodies to viruses are protective, however, cines that are not able to infect human cells. In a specific and in certain cases an antibody to the virus may facilitate its embodiment of the present invention, however, is provided entry into a cell through Fc receptor-mediated uptake of the non-infectious retroviral particles, which express an HIV-1 antibody coated particle. Such antibodies are called enhanc envelope as described herein and display said envelope on its ing antibodies. Surface, thereby allowing the immune system to recognize 0370. During the course of a viral infection, antibody is said envelope or fragment thereof. most effective at an early stage, before the virus has gained 0375. The components of the present invention including entry to its target cell. In this respect, antibody is relatively HIV-1 envelope polypeptides and/or fragments thereof, vec ineffective in primary viral infections, due mainly to the lag tors, nucleic acids, biological entities, retroviral particles, phase in antibody production. Preformed antibody, particu compositions, and kits-of-parts can thus be used alone or in larly neutralising antibody, however, is an effective form of combination with other vaccines directed against lentiviruses protective immunity against viral infections, as witnessed by as defined elsewhere herein, for example HIV. the Success of many viral vaccines, which work by stimulat 0376. During the course of a viral infection with for ing virus-neutralising antibody responses. example HIV, antibody is most effective at an early stage, 0371. The principal effector cells which are involved in before the virus has gained entry to its target cell. In this clearing established viral infections are the virus specific respect, antibody is relatively ineffective in primary viral cytotoxic T lymphocytes (CTL), for example the CD8+ cyto infections, due mainly to the lag phase in antibody produc toxic T lymphocytes. These cells recognise (viral) antigens tion. Preformed antibody, particularly neutralising antibody, which have been synthesised within cell's nucleus or cytosol, however, is an effective form of protective immunity against and which have been degraded. They are presented at the viral infections, as witnessed by the Success of many viral cell's surface as short peptides associated with self class I vaccines, which work by stimulating virus-neutralising anti MHC molecules. The recognition of antigen by CD8+ T cells body responses. In contrast to existing vaccines against len is, therefore, distinct from that of CD4+ T cells in several tiviruses, the present invention thus provides an additional respects. It requires synthesis of the target antigen within the feature which renders the vaccine capable for eliciting a CTL cell (and is therefore restricted largely to virally infected or response. tumour cells); it is “restricted by class I MHC molecules (as 0377. An immunogenic composition of the invention is opposed to MHC class II restriction for CD4+ T cells); MHC effective in enhancing an immune response, for example, class I molecules are expressed on almost all somatic cells, so enhanced beta-chemokine and/or IL 15, IFN, IL2, TNFa pro virtually any cell, on infection with virus, can act as a “target' duction, increased HIV-specific CD4 helper cells, IgG2b anti cell for antigen specific CTL (contrasts with the limited tissue body production, HIV specific cytotoxic T lymphocyte distribution of class II MHC); recognition of an antigen pre (CTL) production, IFNy production by CD4+ cells and CD8 senting cell (APC) by an antigen-specific CTL usually results T cells, and the like, in a mammal administered the compo in the destruction of the APC. sition. As described in U.S. application Ser. No. 09/565,906, 0372. In the context of the present invention the immune filed May S. 2000, and WO 00/67787, each of which is response is produced against the HIV-1 envelope polypeptide incorporated herein by reference, and in Examples I and III, or fragment thereof which is displayed on the host cells or the below, production of the beta-chemokine RANTES can be virus particles carrying the envelope. The virus particles car detected and quantitated using an ELISA assay of Superna rying an HIV envelope may be given to an animal, including tants of T cells (such as lymph node cells or peripheral blood a human, as a vaccine. The vaccine is produced as described cells) from mammals administered the composition. In order herein using a vector of the present invention and/or a retro to determine antigen-specific beta-chemokine production, T viral particle and given to an animal for example a human cells from an immunized mammal can be stimulated with being. The retroviral particle produced according to the HIV antigen in combination with antigen-presenting thy present invention may infect a host cell and upon integration mocytes, and the beta-chemokine levels measured in the of the vector of the present invention into the genome of the Supernatant. In order to determine non-specific beta-chemok host cell, transcription and translation by the host cell, the ine production, either T cell Supernatant or a blood or plasma HIV envelope polypeptide is presented on the surface of the sample from an immunized mammal can be assayed. Simi host cell. The host cell targeted in this manner will be subject larly, production of other beta-chemokines, such as MIP-1C. to a CTL response. and MIP-1.B. can be detected and quantitated using commer 0373) In one embodiment of the present invention a com cially available ELISA assays, according to the manufactur ponent, such as a vector and/or a biological entity described er's instructions. Methods of measuring cytokine production, herein is able to induce an immune response. The response including inteferon, ILLS, IL2, TNFa. IL10 and IL7, by may be an antibody response following vaccination with ret ELISPOT, ELISA, or intracellular cytokine staining are well roviral particles using the vector of the present invention. known to those skilled in the art (see, for example, Robbins et However, in yet another embodiment the immune response is al., AIDS 17:1121-1126 (2003)). US 2011/0305749 A1 Dec. 15, 2011

0378. An immunogenic composition of the invention can which aids in the presentation of especially the peptides of the further be capable of enhancing HIV-specific IgG2b antibody present invention. Examples of carriers are provided else production in a mammal administered the composition. High where herein. Some of the peptide fragments of the invention levels of IgG2b antibodies, which are associated with a Th1 are relatively small molecules and it may therefore be type response, are correlated with protection against HIV required in compositions as described herein to combine the infection and progression to AIDS. Thus, the invention pro peptides with various materials such as adjuvants and/or car vides compositions that can increase a TH1 response. An riers, to produce vaccines, immunogenic compositions, etc. immunogenic composition of the invention can further be Adjuvants, broadly defined, are substances which promote capable of enhancing HIV-specific cytotoxic T lymphocyte immune responses. (CTL) responses in a mammal administered the composition. An immunogenic composition of the invention can increase 0381. A carrier may be present independently of an adju IFN-y production by both CD4+ T cells and CD8+ T cells. vant. The function of a carrier can for example be to increase IFN-Y production by CD4+ T cells is characterized as a classic the molecular weight of in particular peptide fragments in CD4 helper 25 response important to cell-mediated immu order to increase their activity or immunogenicity, to confer nity. CD4+ T cells producing both IFN and IL2 may be most stability, to increase the biological activity, or to increase effective. IFN-production by CD8+ T cells is representative serum half-life. Furthermore, a carrier may aid in presenting of a cytotoxic--T lymphocyte (CTL) response, and is highly the HIV-1 envelope polypeptide, variant or peptide fragments correlated with cytolytic activity. Cells producing both IFN thereof to T-cells. The carrier may be any suitable carrier and TNFamay be most effective. CTL activity is an important known to a person skilled in the art, for example a protein or component of an effective prophylactic or therapeutic anti an antigen presenting cell. A carrier protein could be, but is HIV immune response. Methods of determining whether a not limited to, keyhole limpet hemocyanin, serum proteins CTL response is enhanced following administration of an Such as transferrin, bovine serum albumin, human serum immunogenic composition of the invention are well known in albumin, thyroglobulin or ovalbumin, immunoglobulins, or the art, and include cytolytic assays and LPA assays (de hormones. Such as insulin or palmitic acid. For immunization scribed, for example, in Deml et al. Supra (1999); see of humans, the carrier must be a physiologically acceptable Example III), and ELISA and ELISPOT assays for CD8 carrier acceptable to humans and safe. However, tetanus tox specific IFN-y production (see U.S. application Ser. No. oid and/or diptheria toxoid are suitable carriers in one 09/565,906 and WO 00/67787 and Examples I and II below), embodiment of the invention. Alternatively, the carrier may intracellular staining and FACS analysis using a myriad of be dextrans for example Sepharose. antibodies against cell Surface markers. 0382. Thus it is an aspect of the present invention that the HIV-1 envelope polypeptide, fragment, or variant derived Kit of Parts here from present in the composition is associated with a 0379. As used herein, the term "kit-of-parts” refers to carrier Such as e.g. a protein of the above or an antigen components packaged or marked for use together. For presenting cell Such as e.g. a dendritic cell (DC). example, a kit-of-parts can contain any component of the 0383. One aspect of the present invention relates to a kit present invention, including HIV-1 envelope polypeptides of-parts comprising a therapeutically effective amount of a and/or fragments thereof, vectors, nucleic acids, biological vector, provirus, retroviral particle, composition, Vaccine, entities, retroviral particles, and/or vaccine compositions. and/or vaccine composition of the present invention. Alternatively, a kit-of-parts can contain any two components 0384. In one embodiment, the second active ingredient in in one container, and a third component and any additional the kit-of-parts of the present invention is an immunostimu components in one or more separate containers. Optionally, a lating composition. Examples of immunostimulatory agents kit-of-parts further contains instructions for combining the are provided elsewhere herein, however, in one preferred components so as to formulate an immunogenic composition embodiment, the immunostimulating composition comprises Suitable for administration to a mammal. The components of one or more interleukins. For example, the interleukins are the kit-of-parts are preferably comprised in individual com selected from the group consisting of IL-2 and/or IL-21. positions, it is however within the scope of the present inven 0385. In another embodiment, the second active ingredi tion that the components of the kit-of-parts all are comprised ent in the kit-of-parts of the present invention is an antibiotic, within the same composition. The components of the kit-of Such as an antibiotic is selected from the group consisting of parts may thus be administered simultaneously or sequen amoxicillin, penicillin, acyclovir and/or Vidarabine. tially in any order. 0386 The compositions provided by the kits-of-parts of 0380. One aspect of the present invention, relates to a the present invention are to be administered simultaneously kit-of-parts comprising the vaccine composition as defined or sequentially. previously herein, and a second active ingredient. The kit-of 0387. The invention also relates to a kit-of-parts compris parts preferably comprises an adjuvant and/or a carrier. ing Examples of useful adjuvants are given elsewhere herein. Thus, the vaccine composition may in a kit-of-parts of the 0388 any of the vaccine compositions described herein present invention be associated with an adjuvant and/or a and/or carrier. As specified previously, adjuvants are any Substance 0389) an HIV-1 envelope polypeptide or variant hereof whose admixture into the vaccine composition increases or and/or otherwise modifies the immune response to an HIV-1 enve 0390 any of the HIV-1 envelope polypeptides or frag lope polypeptide or a peptide fragment thereof, as defined ments or variant thereof, and/or peptides derived there herein. Carriers are scaffold structures, for example a from as described herein, for example as defined by any polypeptide or a polysaccharide, to which the HIV-1 envelope of SEQID NO: 1-124 or SEQID NO: 125-326,327-337 or peptide fragment thereof is capable of being associated and and/or US 2011/0305749 A1 Dec. 15, 2011 46

0391 any of the nucleic acids encoding the proteins of intradermal, intravenous, Subcutaneous, intraperitoneal, the above two bullet points and/or eukaryotic expression intranasal, oral or other mucosal routes. vectors comprising said nucleic acid and/or 0392 any biological entity as defined herein, compris Medical Applications ing a polypeptide or nucleic acid of the present invention 0393. The invention also relates to a kit-of-parts compris 0407. The present invention provides a number of thera ing peutical applications. All components of the present inven 0394 any of the vaccine compositions described herein tion including HIV-1 envelope polypeptides and/or fragments and/or thereof, antigens, vectors, nucleic acids, biological entities, 0395 an HIV-1 envelope polypeptide or variant hereof Such as retroviral particles, compositions, and kit-of-parts are and/or claimed for use as a medicament. It is understood that the 0396 any of the HIV-1 envelope polypeptides or frag components of the present invention including HIV-1 enve ments or variant thereof, and/or peptides derived there lope polypeptides and/or fragments thereof, antigens, vec from as described herein, for example as defined by any tors, nucleic acids, biological entities, such as retroviral par of SEQID NO: 1-124 or SEQID NO: 125-326,327-337 ticles, compositions, and kit-of-parts may be used for treating and/or a medical condition. Thus, one aspect of the present invention 0397 any of the nucleic acids encoding the proteins of relates to the use of any component of the present invention, the above two bullet points and/or eukaryotic expression including an HIV-1 envelope polypeptide and/or fragments vectors comprising said nucleic acid and/or thereof, an antigen, a vector, a nucleic acid, a vector, a bio 0398 any biological entity as defined herein, compris logical entity, such as a retroviral particle, a vaccine compo ing a polypeptide or nucleic acid of the present invention sition and/or a kit-of-parts for the manufacture of a medica and instructions on how to use the kit of parts. ment for the treatment, prevention and/or amelioration of a 0399. The invention also relates to a kit-of-parts compris clinical condition. ing 0408. Another aspect relates to any component of the 04.00 any of the vaccine compositions described herein present invention, including an HIV-1 envelope polypeptide and/or and/or fragments thereof, an antigen, a vector, a nucleic acid, 04.01 an HIV-1 envelope polypeptide or variant hereof a vector, a biological entity, such as a retroviral particle, a and/or vaccine composition and/or a kit-of-parts for treating, ame 0402 any of the HIV-1 envelope polypeptides or frag liorating and/or preventing a clinical condition. ments or variant thereof, and/or peptides derived there 04.09. In a preferred embodiment, said clinical condition is from as described herein, for example as defined by any infection, such as HIV infection, such as HIV-1 infection of SEQID NO: 1-124 or SEQID NO: 125-326,327-337 and/or AIDS and/or ARC. In another preferred embodiment, and/or said treatment is prophylactic treatment, for example by 0403 any of the nucleic acids encoding the proteins of reducing the susceptibility of lentiviral infection, such as HIV the above two bullet points and/or eukaryotic expression infection and/or AIDS and/or ARC. vectors comprising said nucleic acid and/or 0410 Thus, one aspect of the present invention relates to 0404 any biological entity as defined herein, compris the use of any component of the present invention, including ing a polypeptide or nucleic acid of the present invention an HIV-1 envelope polypeptide and/or fragments thereof, an and a second active ingredient. antigen, a vector, a nucleic acid, a vector, a biological entity, 04.05 Preferably, the second active ingredient is chosen in Such as a retroviral particle, a vaccine composition and/or a correspondence with the clinical condition to be treated so kit-of-parts for the manufacture of a medicament for the that the second active ingredient is chosen among other clini treatment, prevention and/or amelioration of an infection, cal agents suitable for treatment of the specific clinical con such as lentiviral infection, such as HIV infection and/or dition, for example HIV, AIDS and/or ARC, which is known AIDS and/or ARC. Also, the present invention relates to any the person skilled in the art. For example, if treating a micro component of the present invention, including an HIV-1 enve bial/viral infection, the second active ingredient is preferably lope polypeptide and/or fragments thereof, an antigen, a vec an anti-biotic and/or an anti-viral agent. tor, a nucleic acid, a vector, a biological entity, Such as a 0406. The components of the kit can be combined ex viva retroviral particle, a vaccine composition and/or a kit-of-parts to produce an immunogenic composition, or alternatively, for treating, ameliorating and/or preventing lentiviral infec any two components can be combined ex vivo, and adminis tion, including HIV infection and/or AIDS and/or ARC. The tered with a third component, such that an immunogenic present invention also relates to use of any component of the composition forms in vivo. For example, an HIV envelope present invention, including an HIV-1 envelope polypeptide polypeptide can be emulsified in, dissolved in, mixed with, or and/or fragments thereof, an antigen, a vector, a nucleic acid, adsorbed to an adjuvant and injected into a mammal, pre a vector, a biological entity, such as a retroviral particle, a ceded or followed by injection of a second component, Such vaccine composition and/or a kit-of-parts for the manufacture as an adjuvant and/or a carrier. Likewise, each component of of a medicament for lentiviral vaccination, such as HIV vac the kit can be administered separately. Those skilled in the art cination, such as HIV-1 vaccination. understand that there are various methods of combining and 0411 Another aspect of the present invention relates to administering an the components of the kit-of-parts, so as to any component of the present invention, including an HIV-1 enhance the immune response in a mammal. The kit-of-parts envelope polypeptide and/or fragments thereof, an antigen, a can be administered by the same routes of administration as a vector, a nucleic acid, a vector, a biological entity, Such as a vaccine composition of the present invention, for example it retroviral particle, a vaccine composition and/or a kit-of-parts can be administered locally or systemically by methods well for use as a medicament. In one embodiment, the compo known in the art, including, but not limited to, intramuscular, nents, when administered to an animal including a human US 2011/0305749 A1 Dec. 15, 2011 47 being, is capable of eliciting an immune response against a logical entity, such as a retroviral particle, a vaccine compo disease caused by lentivirus, for example HIV, such as HIV-1 sition and/or a kit-of-parts for treating, ameliorating or pre or HIV-2, or SIV. venting a clinical condition. 0412. In one aspect, the present invention relates to the use 0419. In one embodiment, any component of the present of any component of the present invention, including an invention or treating, ameliorating or preventing a clinical HIV-1 envelope polypeptide and/or fragments thereof, an condition, as well as a pharmaceutical composition compris antigen, a vector, a nucleic acid, a vector, a biological entity, ing any of said components for treating, ameliorating or pre Such as a retroviral particle, a vaccine composition and/or a venting a clinical condition, the clinical condition is an infec kit-of-parts for the manufacture of a medicament for gene tion. In another embodiment, the clinical condition is HIV therapy. Another aspect relates to the use of any component of infection and/or acquired immunodeficiency syndrome the present invention for the manufacture of a medicament for (AIDS) and/or ARC. immune therapy. 0420. In another aspect, the present invention relates to a 0413. One aspect of the present invention relates to a method of reducing the risk of an individual encountering a method of treating, preventing or ameliorating a clinical con clinical condition, said method comprising administration of dition, said method comprising administering to an individual at least one component of the present invention, including an Suffering from said clinical condition an effective amount of HIV-1 envelope polypeptide and/or fragments thereof, an any component of the present invention, including an HIV-1 antigen, a vector, a nucleic acid, a vector, a biological entity, envelope polypeptide and/or fragments thereof, an antigen, a Such as a retroviral particle, a vaccine composition and/or a vector, a nucleic acid, a vector, a biological entity, Such as a kit-of-parts to said individual in an amount Sufficient togen retroviral particle, a vaccine composition and/or a kit-of erate a protective immune response. In one embodiment, the parts. In one embodiment, the clinical condition is an infec clinical condition is infection with HIV. tion, and/or more specifically HIV infection and/or AIDS and/or ARC. Moreover, the individual suffering from HIV EXAMPLES infection and/or AIDS is preferably a human being. In one embodiment, said human being is HIV seronegative. How Example 1 ever, in another embodiment, said human being is HIV serop Cells and Cell Based Assays for Immunosuppression ositive. In one embodiment of the methods of treating, pre venting or ameliorating a clinical condition according to the Ref: present invention, a component of the present invention, 0421) Immunology Letters. 19 (1988) 7-14 including an HIV-1 envelope polypeptide and/or fragments 0422 Human retrovirus-related synthetic peptides inhibit thereof, an antigen, a vector, a nucleic acid, a vector, a bio T lymphocyte proliferation logical entity, Such as a retroviral particle, a vaccine compo 0423 George J. Cianciolol, Hal Bogerd and Ralph Sny sition and/or a kit-of-parts and/or components thereof is derman administered to said organism two or more times. 0414. Any use of a component of the present invention for Virus Preparation the manufacture of a medicament, and/or methods of treating, preventing and/or ameliorating a clinical condition compris 0424 Supernatant from virus producing cultures will ing administering a component of the present invention may either be purified by chromatography and inactivated by pso be combined with a further treatment. In one example, the ralene/UV light treatment or purified by sucrose gradient further treatment is selected from the group consisting of centrifugation. The partickles purified by Sucrose gradient treatment with immunostimulating Substances, gene therapy, centrifugation will either be used directly, inactivated by UV treatment with antibodies, treatment using dendritic cells treatment or disrupted by 0.6M KCL and 0.5% triton X-100 and/or treatments against infections. and clarified by centrifugation at 60,000 to 100,000 g for 1 0415. Another aspect of the present invention relates to a hour. pharmaceutical composition comprising a therapeutically 0425 Human mononuclear cells will be isolated from effective amount of the vector, the producer cell, a retroviral healthy laboratory Volunteers by density gradient centrifuga particle and/or a host cell according to the present invention. tion of heparinized (10 units/ml) blood using lymphocyte 0416) The invention also relates to a method for introduc separation medium (LSM; Litton Bionetics, Charleston, ing a nucleotide sequence into target cells, said method com S.C.). The cells will be resuspended to 2x10 lymphocytes/ml prising infection of target cells with a retroviral particle as in RPMI 1640 (Hazelton, Denver, Pa.) supplemented with defined elsewhere herein. This method may for example be 100 units/ml of penicillin, 100 g/ml of streptomycin, 2 mM used for the production of transgenic animals, said method L-glutamine, 1% non-essential amino acids, 1 mM sodium comprising infection or transduction of embryonic stem cells pyruvate, and 2% fetal bovine serum (FBS; Hyclone) for with a retroviral particles or a vector of the present invention. assays of stimulated blastogenesis. Murine CTLL-2 cells 0417. One aspect of the present invention relates to any were obtained from the American Type Culture Collection component of the present invention, including an HIV-1 enve and maintained in RPM1 1640 containing 5% fetal bovine lope polypeptide and/or fragments thereof, an antigen, a vec serum and 5% human interleukin 2 (IL-2) (Electronucleon tor, a nucleic acid, a vector, a biological entity, Such as a ics, Silver Spring, Md.). retroviral particle, a vaccine composition and/or a kit-of-parts for treating, ameliorating or preventing a clinical condition. Murine Cytotoxic T Lymphocyte (CTL) Proliferation Assay 0418. Another aspect relates to a pharmaceutical compo 0426 CTL-2 cells (5x10s cells per well in 96-well culture sition comprising any component of the present invention, plates) will be incubated in the presence or absence of the including an HIV-1 envelope polypeptide and/or fragments indicated amount of peptide for 20 hat 37° C. in RPMI 1640 thereof, an antigen, a vector, a nucleic acid, a vector, a bio culture medium supplemented with 100 units/ml penicillin, US 2011/0305749 A1 Dec. 15, 2011 48

100 g/ml streptomycin, 2 mM L-glutamine, 1 mM sodium Calculation of Inhibition pyruvate, 1% non-essential amino acids, 2% fetal bovine 0430 serum, and 1% partially purified human IL-2 (Electronucle Percent inhibition will be calculated as:% Inh. =con onics). One uCi of 3H thymidine was added to each well and trol (Stim.)-control (unstim.)-exper. (stim.)-exper. the incubation continued for an additional 4 h. Cultures were (unstim.)control (Stim.)-control (unstim.)x100. harvested onto glass fiber filters and incorporated 3H thymi dine will be measured by liquid Scintillation counting. Each Example 2 sample will be tested in quadruplicate and Standard errors were less than 5% of the mean. The average incorporation by Immunogenicity of Retroviral Particles untreated cells was 50000-75000 cpm/well. 0431. The development of a broad and neutralising anti body response is vital for a protective HIV-1 vaccine (Mas Anti-CD3 Stimulated Proliferation colaetal 2000, Nat Medicine. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric 0427 Human mononuclear cells will be isolated from virus by passive infusion of neutralizing antibodies). In addi healthy Volunteers by density gradient centrifugation of tion, the induction of specific and effective cytotoxic T lym blood using LSM. The cells were resuspended to 2x10' lym phocytes has been shown to be required for infection control phocytes/ml in supplemented RPMI 1640 with 2% fetal (Schmitz et al 1999, Science. Control of Viremia in Simian bovine serum. One-tenth ml of cell suspension was added to Immunodeficiency Virus Infection by CD8+ Lymphocytes). each well of a 96-well tissue culture plate with 50 ul of either 0432. Thus, the development of vaccine strategies that media or the indicated amount of peptide and 50ll containing encompass both arms of the immune system are thus impor 2ng of anti-CD3 (OKT3) antibody (Ortho Pharmaceutical) tant. Differential MHC I and II antigen presentation is a key factor for initiation of a potent immune response. There and the plate incubated for ca. 68 hat 37°C. Fifty ul of media appear to be short-comings in antigen presentation when containing 1.0 uCi 3Hthymidine (New England Nuclear; antigens are administered solely as peptides or DNA. In con 6-7 Ci/mmole) will be added for an additional 4h at 37°C., trast cross-talk between antigen presenting cells and T helper the cells harvested by filtration onto glass fiber filters, and lymphocytes are promoted in vaccine strategies based on incorporated radioactivity determined by liquid scintillation either infectious or non-infectious viral particles. A potent spectrophotometry. All samples will be run in quadruplicate. induction of cell mediated immunity can be achieved even Anti-CD3 stimulated human mononuclear cells in the pres with whole-killed viral particles (McBurney etal 2007, Virol ence of media alone. ogy. Membrane embedded HIV-1 envelope on the surface of a virus-like particle elicits broader immune responses than Human Two-Way Mixed Lymphocyte Reactions (MLR) soluble envelopes). This is likely a consequence of improved antigen uptake and systemic immunestimulation in macroph 0428 Human mononuclear cells will be isolated and sus ages and dendritic cells (Buonaguro et al 2006, J. Virol. pended in media as described for anti-CD3 stimulated prolif Baculovirus-derived human immunodeficiency virus type i eration. Fifty ul (containing 1x10 cells) of cell suspension virus-like particle activate dendritic cels and induce ex vivo from each of two individuals was added to each well of a t-cell responses). 96-well tissue culture plate, an additional 50 ul of media 0433) To achieve potent immunogenic retroviral particles, added, and the cultures incubated for 120 hat 37° C. Fifty ul Y-retroviral particles may be produced with functional HIV-1 of media containing the indicated amount of peptide will be envelope trimers on the Surface. These particles can function added, the cultures incubated an additional 20 h and then as a Superior immunogenic particle avoiding any risk associ pulsed with 1.0 uCi 3H thymidine and incorporated radio ated with viral inactivation procedures. activity determined as described above. All samples will be tested in quadruplicate. Example 3 Detection of Cell Surface Expression of ENV by Human B-Cell Proliferation Flow Cytometry 0429 Human mononuclear cells will be isolated as 0434. The cells are labeled with anti HIV-ENV antibodies described above and resuspended at 1x10 V lymphocytes/ml through incubation of the cells with the Ab for 45 min on ice. in supplemented RPMI 1640 media. Fifty ul of cell suspen Followed by washing of the unbound Ab with PBS. The sion will be added to each of quadruplicate cultures in 96-well cell-anti envelope Ab complex is Subsequently incubated flat-bottom plates. Fifty ul of media or media containing with a fluorescentlabled Abagainst the primary ENV-binding peptide or BSA control will be added to each well. An Ab for 45 min. on ice followed by a second PBS wash. A flow additional 50/Zl of BCGF (B cell growth factor, purified from cytometer will be used to detect fluorescence associated with pbytohemagglutinin-stimulated human T lymphocytes and the cells, which is indicative of ENV expression. free of inducing agent as well as immune interferon and T cell Example 4 growth factor; Cellular Products, I no. Buffalo, N.Y.) was added to give a final concentration of 10% (v/v). After addi Detection of Incorporation of ENV into Retroviral tion of 50 ul of rabbit anti-human IgG (IgG fraction; Cooper Particles by Flow Cytometry Biomedical, Inc., Malvern, Pa.; 1:100 v/v in media) the 0435 Supernatant containing retroviral particles is incu cultures will be incubated for 72 h at 37°C. in humidified 5% bated with cells expressing the CD4 receptor for 45 min. on CO2 and DNA synthesis measured by 3H thymidine incor ice followed by PBS wash. The cells are subsequently labeled poration as described above. with anti HIV-ENV antibodies through incubation of the cells US 2011/0305749 A1 Dec. 15, 2011 49 with the Ab for 45 min on ice. followed by washing of the 0440 This peptide is derived from Murine Leukemia unbound Ab with PBS. The cell-anti envelope Ab complex is viruses and contains immunosuppressive activity associated subsequently incubated with a fluorescent labled Ab against with the envelope protein. the primary ENV-binding Ab for 45 min. on ice followed by a second PBS wash. A flow cytometer will be used to detect fluorescence associated with the cells, which is indicative of CS-3 : LOARVLAVERYLKDQOLLGIWGC, (SEQ ID NO: 131) ENV expression. 0441 This peptide is derived from HIV-1 group M and contains immunosuppressive activity associated with the Example 5 envelope protein. Detection of Fusogenicity of the ENV by Syncytia Assay HIV G19R: LOARVLAVERYLKDQOLLRIWGC (SEQ ID NO: 132) 0436 An Env-expressing plasmid is transfected into 293T cells. Two days later, the ENV-expressing cells are co-culti 0442. This peptide corresponds to CS-3 with a single point vated with D17 cells expressing CD4 (10,000 cells pr. square mutation at position 19 (indicated in bold). centimeter) and one or more of the HIV co-receptors (ie. CXCR4, CCR5 etc. 10,000 cells pr. square centimeter). Fuso (SEQ ID NO: 133) genicity of the ENV protein can be detected by examination HIV MAO chimera: LQARILAVETLIONOOLLNLWGC of the level of cell-cell fusion in a microscope. 0443) This peptide is a chimera between the CS3 peptide Example 6 and the corresponding peptide in the envelope protein of HIV-1 clade O, that is the first 10 amino acid residues are Detection of Fusogenicity of the ENV by Syncytia derived from clade M sequence (CS-3, in bold) and the rest Assay from Clade O. 0437. An Env-expressing plasmid (which also expresses 0444 The peptides are dimerized via the C-terminal Cys egfp marker) is transfected into 293T cells. Two days later, the teines and added at the concentrations indicated below. ENV-expressing cells are co-cultivated with D17 cells 0445. In FIG. 10 the inhibition of lymphocyte prolifera expressing CD4 (10,000 cells pr. square centimeter) and one tion is depicted as percentage relative to the ConA stimulated or more of the HIV co-receptors (ie. CXCR4, CCR5 etc. cells without peptide addition. With the two reference pep 10,000 cells pr. square centimeter). Fusogenicity of the ENV tides CKS-17 (from MLV) and CS-3 (from HIV-1 group M) protein can be detected by examination of the level of cell-cell inhibition of 93% and 82% respectively is observed at peptide fusion in a microscope, either in visible light or by green concentration of 50 uM. In contrast the two variants HIV fluorescence found in the cells., se FIG. 4 to FIG.9 G19R and HIVM/O chimera inhibits the lymphocyte prolif eration 15 and 22% respectively. This demonstrates that the peptides HIV G19R (SEQ ID NO: 132) and HIV M/O chi mera (SEQID NO: 133) of the present invention are signifi Wt ------cantly less immunosuppressive than the wild type HIV R1OT (#3) SEQID NO: 127 ------Db mut (#4) SEQID NO: 128 ------derived CS-3 and MLV derived CKS-17 peptides. O 10-40 (#1) SEQID NO: 125 ------0446 FIG. 11 depicts the proliferation at various peptide Pent mut (#2) SEQID NO: 126 ------concentrations. The inhibition profiles for the two variants Pent + E9K (#5) SEQID NO: 129 (+) (HIV G19R and HIV M/O chimera) are less steep indicating a less pronounced immunosuppressive effect. 0447 These assays clearly demonstrate that an HIV enve Example 7 lope, an antigen, vector, retroviral particle, entity, vaccine composition or kit-of-parts of the present invention compris Immunosuppression of Selected Peptides ing or consisting of HIV G19R and/or HIV M/O chimera Experiment Design peptides or nucleic acid molecules encoding said peptides are particularly Suitable for prophylactic treatment and vaccina 0438. The experiment utilizes Human Peripheral Blood tion, since the peptides are less immunosuppressive than wild Mononuclear Cells (PBMC) prepared fresh from healthy type HIV envelope peptides. donors. These are stimulated by Con A (5ug/mL) concomi tant to peptide addition at the indicated concentrations. Cul Example 8 tures are maintained and lymphocyte proliferation is mea sured 72 hrs later by EdU incorporation and Click-iT Mixed Leukocyte Reaction labelling with Oregon Green (Invitrogen, Denmark) as rec ommended by the manufacturer. The degree of activatedlym Experiment Design phocytes is proportional to the fluorescence detection. 0448. The experiment utilizes Human PBMC prepared fresh from healthy donors. These are stimulated by adding a Results human T cell line (Jurkat) at a 1:1 ratio. The jurkat cells have 0439. The peptides employed are: been irreversible arrested by incubating them with 50 ug/mL Mitomycin C for 1 hour prior to PBMC stimulation. Cultures are maintained and lymphocyte proliferation is measured 96 CKS-17 : LQNRRGLDLLFLKEGGLC (SEQ ID NO: 130) hrs later by EdU incorporation and Click-iT labelling with Oregon Green (Invitrogen, Denmark) as recommended by the US 2011/0305749 A1 Dec. 15, 2011 50 manufacturer. The degree of activatedlymphocytes is propor TNF-C. secretion upon Con A stimulation of PBMC's. The tional to the flourescence detection. mutant G19R downregulates TNF-C. but not IFN-Y. Thus, the 0449 The jurkat cells have been transduced by a murine results in FIG. 14 clearly identify HIV WT peptide as a potent leukemia virus vector expressing either an eGFP marker gene blocker of both IFN-gamma and TNF-alpha, while the point alone (eGFP in FIG. 12 and FIG. 13) or the eGFP marker gene mutation G19R only blocks TNF-alpha. in addition to a HIV envelope variant: HIV (WT), M/O, or HIV G19R. Thus, Jurkat cells indicated by eGFP in FIG. 12 Sequences and FIG. 13 contain a retroviral vector without HIV envelope. 0458 In the amino acid sequences below, the amino acids Results are designated by their conventional single letter code. 0450. The mutations within the immunosuppressive IGP1 domain in an HXB2 envelope background are indicated in General Immunogenic Peptide 1 (IGP1): bold and underlined: 0459. An HIV-1 envelope polypeptide comprising an amino acid sequence selected from the group of amino acid HIW WT-HXB2 : sequences consisting of: X(1-22)-C(23)-X(24-28)-C(29)-X (SEO ID NO: 327) (30-50): WGIKOLOARVLAVERYLKDOOLLGIWGCSGKLICTTAVPWNASWSNKSLE.

HIW G19R-HXB2: (SEQ ID NO: 328) XXXXXXXXXXXXXXXXXXXXXXCXXXXXCXXXXXXXXXXXXXXXXXXXXX WGIKOLOARVLAVERYLKDOOLLRIWGCFGKLICTTAVPWNASWSNKSLE. HIV M/O-HXB2 : IGP2 (SEQ ID NO: 329) WGIKQLQARVLAVELIONOORLNLWGCKGKLICYTSWKWNSWSNKSLE. General Immunogenic Peptide 2 (IGP2): 0451. The column indicated jurkat' in FIG. 12 and FIG. 0460. The amino acid sequence as defined by IGP1, 13 are non-transduced jurkat cells. wherein the amino acid residues in said amino acid sequence 0452. In FIG.12 lymphocyte proliferation is depicted. The are selected from the groups of residues consisting of: fluorescence is proportional to proliferation. 0453 Activation of PBMC is examined in the presence of X(1): L., S, R, P. F. A. V. M., and I; and jurkat stimulator cells. The “eGFP column contain no HIV X(2): Q, R. K. H. L. M., and P; and envelope have a higher activation level. When jurkat cells express the HIV wt envelope, a decrease in proliferation is X(3): A, T, V, H, S, R, Q, G, M, and E; and observed. This decrease is relieved by introducing either the X(4): R. K. G. E. T. S. C. M., and H; and point mutation G19R or having an HIV M/O chimera, thus indicating that an envelope polypeptide carrying the point X(5): V, I, L, D, A, S, F, M, and G.; and mutation G19R or the HIV M/O chimera display an immu X(6): L. Q. V. M. P. W. T. and I; and nostimulatory effect. X(7): A, S, T, V. L. G. F. D. M., and E; and 0454 FIG. 13 depicts the stimulation index which is cal culated as the proliferation in question/proliferation in UT. X(8): V. L. I. M. A. W. K. G. and E; and Example 9 X(9): E, K, G, D, A, V. M, and F; and X(10): X; and Cytokine Modulation X(11): Y. L. F. H. C., I, T, M, and N; and 0455 To verify the effect of immunosuppressive peptides of the present invention stimulated PBMC culture superna X(12): L., I, V, M, Q, P. T.Y. and A; and tant was analysed for cytokine secretion, as a measure of the X(13): K, R, Q, G.S., E. H. W. T. V. M. N.Z.Y.A. P. and C; and cells immune response. 0456 Donor PBMC's were maintained at 10 cells/well in X(14): D, N, G, E. Y. V. S., H, A, M, and I; and a 96-well. Stimulated by +/-Concanavalin A (ConA) and X(15): Q, R, H, K. P. L. M., and N; and +/-50 uM peptide (HIV WT, HIV G19R, or HIV M/O). After 72 hrs, lymphocyte proliferation was measured and Superna X(16): Q, K, R. T. H. E. S. P. M., and L; and tants analysed by ELISA for levels of the proinflammatory cytokines, IFN-gamma (FIG. 14 grey bar) and TNF-alpha X(17): L. F., I, R, V. P. S. M, and H; and (FIG.14 blackbar). The phenotypic effect verified in example X(18): L. M. P. I, H, and S; and 8 is Supported by data of inflammatory cytokines secreted from these stimulated PBMC cultures. X(19): X; and 0457. The present example shows the immunemodulatory X(20): I, L. M. V. S., F.T. D, A, R, P, and J.; and properties of different peptides, and the results are shown in FIG. 14 for IFN-Y (grey bar) and TNF-C. (blackbar). Cytokine X(21): W. R, G, F, L, M, and T; and secretion is evaluated for PBMC untreated (UT), and in the X(22): G, D, A. R. M., and C; and presence of HIV WT (CS-3 (SEQID NO: 131)), HIV G19R (SEQID NO: 132), or HIV M/O peptide (SEQID NO: 133), X(24): X; and as indicated. HIV WT significantly reduces both IFN-Y and X(25): G. R. E., N, A, M, and D; and

US 2011/0305749 A1 Dec. 15, 2011

X(9): E, K, G, D, A, V. M, and F; and X(48): W.I.T., N, D, E, L. G., S.Y. R. V. K, H, A, Q, M, and F: X(10): X; and and X(49): D, N, E, G, W, Q, K, H, L, B, S.I.Y.T. A. R,M, Z, and X(11): Y. L. F. H. C., I, T, M, and N; and V; and X(12): L., I, V, M, Q, P. T.Y. and A; and X(50): N, D, T, K, S, H. L. G. E. W. I.Q. M. R. B.Y. P. and A: X(13): K, R, Q, G.S., E. H. W. T. V. M. N.Z.Y.A. P. and C; and 0470 wherein the amino acids are designated by their X(14): D, N, G, E. Y. V. S., H, A, M, and I; and conventional single letter code. 0471. Item 3. The HIV-1 envelope polypeptide according X(15): Q, R, H, K. P. L. M., and N; and to any of the preceding, wherein the amino acid residue in X(16): Q, K, R. T. H. E. S. P. M., and L; and positionX(10) is selected from the group consisting of R, S, T. K. G. A. N. Q and I. X(17): L. F., I, R, V. P. S. M, and H; and 0472. Item 4. The HIV-1 envelope polypeptide according X(18): L. M. P. I, H, and S; and to any of the preceding, wherein the amino acid residue in position X(19) is selected from the group consisting of: G. N. X(19): X; and S, R, E, T, D, V, C and A. 0473 Item 5. The HIV-1 envelope polypeptide according X(20): I, L. M. V. S., F.T. D, A, R, P, and J.; and to any of the preceding, wherein the amino acid residue in X(21): W. R, G, F, L, M, and T; and positionX(24) is selected from the group consisting of S. K. T. R. A. P.Y. F, G, Q, I and H. X(22): G, D, A. R. M., and C; and 0474 Item 6. The HIV-1 envelope polypeptide according X(24): X; and to any of the preceding, wherein the amino acid residue in position X(34) is selected from the group consisting of P. K. X(25): G. R. E., N, A, M, and D; and R. S. A. L. Q., E. H. T., I, V, and F. X(26): K. R. N. E. Q, T. S. I. M., and G.; and 0475 Item 7. The HIV-1 envelope polypeptide according to any of the preceding, wherein the amino acid residue in X(27): L, H, I, T, V. F. R. Q. S. P. A. J. M., and Y; and position X(40) is selected from the group consisting of S, N, X(28): I, V, T, L, R, F, and M. and G, T, R., I, V. K. W. A. P. Y. D, Q, H, E, and C. 0476. Item 8. The HIV-1 envelope polypeptide according X(30): T. P.Y.A. N. S., I, V, R, L, M and H; and to any of the preceding, wherein the amino acid residue in position X(10) is threonine (T). X(31): T. S. P.. N, M and I; and 0477. Item 9. The HIV-1 envelope polypeptide according X(32): A.N.T, S, D, R, F, Q, P, I, E. V. M. L., K, H, C, and B; to any of the preceding, wherein the amino acid residue in and position X(19) is asparagine (N). 0478. Item 10. The HIV-1 envelope polypeptide according X(33): V. A. L. M. G. R. and C; and to any of the preceding, wherein the amino acid residue in X(34): X; and position X(24) is lysine (K). 0479. Item 11. The HIV-1 envelope polypeptide according X(35): W. R. G. L. M., and P; and to any of the preceding, wherein the amino acid residue in X(36): N, S, D, B, K, E, R, Q, M, and G.; and position X(34) is lysine (K). 0480 Item 12. The HIV-1 envelope polypeptide according X(37): S, T, A, N, D, V, I, E, Y, K, L, R, G, P. M., F. W. H., Q, to any of the preceding, wherein the amino acid residue in B, and C.; and position X(40) is serine (S). X(38): S, T, N., I, G, R, L, C, A, W, M and E; and 0481 Item 13. The HIV-1 envelope polypeptide according to any of the preceding, wherein the amino acid sequence is X(39): W. G. A. R. E. C. Y. V. S. M, and H; and selected from the group consisting of SEQ ID NO: 2-68 or X(40): X; and SEQ ID NO: 130-326, or any part thereof, or functional homolog or any polypeptide with at least 80% identity to said X(41): N, G, K, S, D, E,T, R, H, PA, B, V. Q, Y. M., and I; and polypeptide or part thereof. X(42): K, R, N, D, S, T, G, E., I, V, Y, Q, P, H, A, W. M., and C: 0482 Item 14. The HIV-1 envelope polypeptide according and to any of the preceding, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 4-19, or X(43): S, T, N, K, I, R, D, E, P. L. A. W. G. M. H. Y. F. V. and any part thereof, or functional homolog or any polypeptide C; and with at least 90% identity to said polypeptide or part thereof. 0483 Item 15. The HIV-1 envelope polypeptide according X(44): L.Y. Q, F. E. H. S. V. K, M, T, I, W. N. D, R, PA, and to any of the preceding, wherein the amino acid sequence is G; and selected from the group consisting of SEQID NO:20-63, or X(45): D, E, N, S, T, K, G, L, A, Q, H, I,Y, B, R, V. P. M., F. W. any part thereof, or functional homolog or any polypeptide Z, and C.; and with at least 90% identity to said polypeptide or part thereof. 0484. Item 16. The HIV-1 envelope polypeptide according X(46): E, D, Q, Y, K, N. T. S. A. W. H. M., R., I, G, L, V, Z, F, to any of the preceding, wherein the amino acid sequence is B, and P; and selected from the group consisting of SEQID NO: 64-68, or X(47): I, D, E, M, G, T, Q, S. W. L., N.Y. K. V. R. F. A. P. and any part thereof, or functional homolog or any polypeptide H; and with at least 90% identity to said polypeptide or part thereof. US 2011/0305749 A1 Dec. 15, 2011

0485. Item 17. The HIV-1 envelope polypeptide according 0496. Item 28. A nucleic acid sequence encoding at least to any of the preceding, wherein the amino acid sequence is one HIV-1 envelope polypeptide with an amino acid sequence selected from the group consisting of SEQID NO: 130-133, as defined in any of Item 1 to Item 26 or a fragment thereof or any part thereof, or functional homolog or any polypeptide and/or a nucleic acid sequence encoding at least one antigen with at least 90% identity to said polypeptide or part thereof. as defined in Item 27. 0486 Item 18. The HIV-1 envelope polypeptide according 0497. Item 29. The nucleic acid according to Item 28, to any of the preceding, wherein the amino acid sequence is wherein the nucleic acid sequence has been modified by selected from the group consisting of SEQID NO: 134-323, codon optimization. or any part thereof, or functional homolog or any polypeptide 0498. Item 30. An isolated eukaryotic expression vector with at least 90% identity to said polypeptide or part thereof. comprising at least one nucleic acid sequence as defined in 0487. Item 19. The HIV-1 envelope polypeptide according any of Item 28 and/or Item 29 or a fragment thereof. to any of the preceding, wherein the amino acid sequence is 0499. Item 31. The vector according to Item 30, wherein LRARLLALETFIQNQQLLNLWGCKGNLI said is a mammalian expression vector. CYTSVKWNDTWKGNSDTSLENIWDN (SEQID NO:4), (0500 Item 32. The vector according to any of Item 30 to or any part thereof, or functional homolog or any polypeptide Item 31, wherein said vector comprise at least one intron. with at least 90% identity to said polypeptide or part thereof. (0501) Item 33. The vector according to any of Item 30 to 0488. Item 20. The HIV-1 envelope polypeptide according Item 32, which is transcribed in the nucleus, thereby produc to any of the preceding, wherein the amino acid sequence is ing high levels of transcript, which after transport to the LQARIMAVERYLKDQQLLGIWGCS cytoplasm can be translated into envelope polypeptide. GKLICTTAVPWNASWSNKSLEQIWNH (SEQ ID NO: 0502. Item 34. The vector according to any of Item 30 to 20) or any part thereof, or functional homolog or any polypep Item 32, which is transcribed in the cytoplasm, thereby pro tide with at least 90% identity to said polypeptide or part ducing high levels of transcript, which can be translated into thereof. envelope polypeptide. 0489. Item 21. The HIV-1 envelope polypeptide according (0503 Item 35. The vector according to any of Item 30 to to any of the preceding, wherein the amino acid sequence is Item 34, wherein said vector comprises a constitutive trans LQARILAVERYLKDQQLLRIWGCF port element (CTE). GKLICTTAVPWNASWSNKSLEQIWNH (SEQ ID NO: 0504. Item 36. The vector according to any of Item 30 to 64) or any part thereof, or functional homolog or any polypep Item 35, wherein said vector comprises a Rev responsive tide with at least 90% identity to said polypeptide or part element (RRE). thereof. (0505. Item 37. The vector according to Item 30, wherein 0490 Item 22. The HIV-1 envelope polypeptide according said vector is a retroviral vector. to any of the preceding, wherein the amino acid sequence is (0506. Item 38. The vector according to Item 37, wherein HIV G19R: LQARVLAVERYLKDQQLLRIWGC (SEQ ID said vector is a replication deficient retroviral vector. NO: 132) or any part thereof, or functional homolog or any (0507. Item 39. The vector according to Item 37, wherein polypeptide with at least 90% identity to said polypeptide or said vector is a replication competent retroviral vector. part thereof. 0508. Item 40. The isolated vector according to any of 0491 Item 23. The HIV-1 envelope polypeptide according Item 30 to Item 39, wherein the vector is derived from to any of the preceding, wherein the amino acid sequence is gamma-retroviruses. HIV M/O chimera: LQARILAVETLIQNQQLLNLWGC (SEQID NO: 133) or any part thereof, or functional homolog 0509. Item 41. The vector according to Item 40, wherein or any polypeptide with at least 90% identity to said polypep the vector is derived from Murine Leukemia Virus (MLV). tide or part thereof. 0510 Item 42. The vector according to Item 41, wherein 0492. Item 24. The HIV-1 envelope polypeptide according the vector is derived from Moloney Murine Leukemia Virus to any of the preceding with an amino acid sequence selected (MoMLV). from the group consisting of SEQID NO: 120-124 or SEQID 0511. Item 43. The vector according to Item 42, wherein NO: 324-326, or any part thereof, or functional homolog or the vector is derived from AkV MLV. any amino acid sequence with at least 90% identity to said 0512. Item 44. The vector according to any of Item 30 to amino acid sequence or part thereof. Item 43, further comprising at least one additional nucleic 0493 Item 25. The HIV-1 envelope polypeptide according acid sequence. to any of the preceding with an amino acid sequence of SEQ 0513 Item 45. The vector according to Item 44, further ID NO: 120, or any part thereof, or functional homolog or any comprising at least one internal ribosomal entry site (IRES). amino acid sequence with at least 90% identity to said amino 0514. Item 46. The vector according to Item 45, wherein acid sequence or part thereof. said IRES is selected from the IRES elements of picornaviri 0494. Item 26. The HIV-1 envelope polypeptide according dae, retroviridae or retrotransposons, mammalia or combina to any of the preceding, wherein said envelope polypeptide tions thereof. has been produced synthetically. 0515. Item 47. The vector according to Item 46, wherein 0495. Item 27. An antigen comprising at least one peptide said IRES is selected from the IRES elements of picornavirus. with an amino acid sequence of an HIV-1 envelope polypep 0516. Item 48. The vector according to Item 46, wherein tide as defined in any of Item 1 to Item 26, or a functional said IRES is selected from the IRES elements of encepha homolog thereofhaving at least 70% identity to said envelope lomyocarditis (ECMV). polypeptide oran immunological active fragment comprising 0517. Item 49. The vector according to any of the preced a consecutive sequence of at least 10 amino acids selected ing Item 45 to Item 48, wherein said IRES is located in a from a region of said envelope polypeptide. region flanked by the 3'-LTR and the 5'-LTR. US 2011/0305749 A1 Dec. 15, 2011

0518) Item 50. The vector according to any of the preced 0537 Item 69. The biological entity according to any of ing Item 49, wherein said IRES is located in the 3'-Long Item 62 to Item 68, wherein said immunogenic response is Terminal Repeat (LTR) or the 5'-LTR. directed towards said biological entity in said host animal. 0519. Item 51. The vector according to Item 50, wherein 0538 Item 70. The biological entity according to any of Item 62 to Item 69, wherein said host animal is a human said IRES is located in the U3 region of the 3' LTR. being. 0520 Item 52. The vector according to Item 50, wherein 0539. Item 71. The biological entity according to any of said IRES is located in the U3 region between the inverted Item 62 to Item 70, which is capable of infecting human cells. repeats and the transcription regulatory elements. 0540 Item 72. The biological entity according to any of 0521. Item 53. The vector according to any of Item 44 to Item 62 to Item 71, which is capable of infecting stem cells. Item 52, wherein said at least one additional nucleic acid 0541. Item 73. The biological entity according to any of sequence is a reporter gene. Item 62 to Item 72, wherein said biological entity is selected 0522. Item 54. The vector according to Item 53, wherein from the group consisting of a viral particle, a retroviral said reporter gene encodes enhanced green fluorescent pro particle, a eukaryotic cell, a prokaryotic cell, a liposome tein (eGFP), lac Z, dsRed, enhanced yellow fluorescent pro and/or a nanoparticle. tein (eyFP), enhanced cyan fluorescent protein (eCFP), 0542. Item 74. The biological entity according to any of enhanced blue fluorescent protein (eBFP) and the human Item 62 to Item 73, wherein said biological entity is a lipo alpha-1-antitrypsin (haAT). It is understood that any of the Some, a nanoparticle, a viroSome, a protzoa, an enterobacte enhanced green fluorescent protein (eGFP), lac Z, dsRed, ria, and/or a nanoparticle or a liposome with synthetic enve enhanced yellow fluorescent protein (eyFP), enhanced cyan lope polypeptides. fluorescent protein (eCFP), enhanced blue fluorescent protein 0543. Item 75. The biological entity according to any of (eBFP) or the human alpha-1-antitrypsin (ha AT). Item 62 to Item 74, wherein said biological entity biological 0523 Item 55. The vector according to any of Item 44 to entity is a nanoparticle or a liposome with at least one syn Item 52, wherein said at least one additional nucleic acid thetic HIV-1 envelope polypeptide as defined in Item 26. sequence encodes a selective marker. 0544. Item 76. The biological entity according to any of 0524. Item 56. The vector according to Item 55, wherein Item 62 to Item 73, wherein said biological entity is a retro said selective marker is neomycin phosphotransferase II. viral particle. 0525) Item 57. The vector according to Item 55, wherein 0545. Item 77. The biological entity according to any of said at least one additional nucleic acid sequence encodes a Item 62 to Item 76, wherein said biological entity is a provi Suicide gene. U.S. 0526. Item 58. The vector according to any of Item 44 to (0546) Item 78. The biological entity according to Item 76, Item 52, wherein said at least one additional nucleic acid wherein said retroviral particle is a gammaretroviral particle. sequence encodes an immunomodulating polypeptide. 0547. Item 79. A vaccine composition comprising 0527. Item 59. The vector according to Item 58, wherein a) an HIV-1 envelope polypeptide as defined in any of Item 1 said immunomodulating polypeptide is an immunostimulat to Item 26, or a functional homologue thereofhaving at least ing polypeptide. 70% identity to said peptide or an immunogenically active 0528. Item 60. The vector according to Item 58, wherein peptide fragment comprising a consecutive sequence of at said immunomodulating polypeptide is an genetic adjuvant. least 10 residues of said peptide or said functional homologue 0529) Item 61. The vector according to Item 59, wherein thereof, or a nucleic acid encoding said peptide or said pep said immunostimulating polypeptide is selected from the tide fragment or said functional homologies thereof and/or group consisting of cytokines or hormones. b) an antigen as defined in Item 27, and/or 0530 Item 62. A biological entity comprising at least one c) a nucleic acid as defined in any of Item 28 to Item 29, and/or HIV-1 envelope as defined in any of Item 1 to Item 26, and/or d) a vector as defined in any of Item 30 to Item 61, and/or at least one antigen as defined in Item 27, and/or at least one e) a biological entity as defined in any of Item 62 to Item 78, nucleic acid as defined in any of Item 28 to Item 29, and/or at and least one vector as defined in any of Item 30 to Item 61. f) an adjuvant 0531. Item 63. The biological entity according to Item 62, 0548 for use as a medicament. wherein said HIV-1 envelope or part thereof is expressed on 0549. Item 80. The vaccine composition according to Item the surface of the biological entity. 79, wherein said immunogenically active peptide fragment 0532. Item 64. The biological entity according to any of consists of a consecutive sequence of in the range of from 10 Item 62 to Item 63, wherein said HIV-1 envelope or part to 50 amino acids. thereof comprises gal-alifa1-3Galbeta1-4GlcNAc-R 0550 Item 81. The vaccine composition according to any epitopes. of Item 79 to Item 80, wherein the adjuvant is selected from the group consisting of bacterial DNA based adjuvants, oil/ 0533. Item 65. The biological entity according to any of surfactant based adjuvants, viral dsRNA based adjuvants and Item 62 to Item 64, which is not capable of mediating fusion imidazochinilines, incomplete Freund's adjuvant (IFA). of said biological entity and cells expressing receptors for 0551. Item 82. The vaccine composition according to any HIV. of Item 79 to Item 80, wherein said adjuvant is selected from 0534. Item 66. The biological entity according to any of the group consisting of AlK(SO4)2, AlNaCSO4)2, AlNH4 Item 62 to Item 64, which is capable of mediating fusion of (SO4), silica, alum, Al(OH)3. Ca3(PO4)2, kaolin, carbon, said biological entity and cells expressing receptors for HIV. aluminum hydroxide, muramyl dipeptides, N-acetyl-mu 0535 Item 67. The biological entity according to any of ramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nor Item 62 to Item 66, wherein said biological entity is capable nuramyl-L-alanyl-D-isoglutamine (CGP 1 1687, also referred of infecting a CD4 positive cell. to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutami 0536. Item 68. The biological entity according to any of nyl-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxphos Item 62 to Item 67, wherein said biological entity is capable phoryloxy)-ethylamine (CGP 19835A, also referred to as of inducing an immunogenic response in a host animal. MTP-PE), RIBI (MPL+TDM+CWS) in a 2% squalene/ US 2011/0305749 A1 Dec. 15, 2011

Tween-80(R) emulsion, lipopolysaccharides and its various protein or peptide fragment selected from a protein or peptide derivatives, including lipid A, Freund's Complete Adjuvant fragment, which is not derived from HIV-1 envelope. (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, 0565. Item 96. The vaccine composition according to any polynucleotides (for example, poly IC and poly AU acids), of Item 79 to Item 95, wherein the vaccine composition com poly GC, wax D from Mycobacterium, tuberculosis, sub prises antigen presenting biological entity according to any of stances found in Corynebacterium parvum, Bordetella per Item 62 to Item 78. tussis, and members of the genus Brucella, liposomes or other 0566 Item 97. The vaccine composition according to any lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see US of Item 79 to Item 96, comprising a vector according to any of 58767 and U.S. Pat. No. 5,554.372), Lipid A derivatives, Item 30 to Item 61. choleratoxin derivatives, HSP derivatives, LPS derivatives, 0567 Item 98. A kit-of-parts comprising the vaccine com synthetic peptide matrixes or GMDP. Interleukin 1, Interleu position as defined in any of Item 79 to Item 97, and a second kin 2, Montanide ISA-51 and QS-21. Preferred adjuvants to active ingredient. be used with the invention include oil/surfactant based adju 0568. Item 99. The kit-of-parts according to Item 98, vants such as Montanide adjuvants (available from Seppic, wherein the second active ingredient is an immunostimulat Belgium), preferably Montanide ISA-51. ing composition. 0552) Item 83. The vaccine composition according to any 0569. Item 100. The kit-of-parts according to any of Item of Item 79 to Item 81, wherein the adjuvant is a Montanide 98 or Item 99, wherein the further immunostimulating com ISA adjuvant. position comprises one or more interleukins. 0553 Item 84. The vaccine composition according to Item 0570 Item 101. The kit-of-parts according to any of Item 83, wherein the adjuvant is Montanide ISA 51 or Montanide 98 to Item 100, wherein the interleukins are selected from ISA 720. IL-2 and or IL-21. 0554. Item 85. The vaccine composition according to Item 0571. Item 102. The kit-of-parts according to Item 98, 84, wherein the adjuvant is Montanide ISA 51. wherein the second active ingredient is an antibiotic. 0555 Item 86. The vaccine composition according to any 0572 Item 103. The kit-of-parts according to Item 102, of Item 79 to Item 81, wherein the adjuvant is GM-CSF. wherein the antibiotic is selected from: amoxicillin, penicil 0556. Item 87. The vaccine composition according to any lin, acyclovir and/or Vidarabine. of Item 79 to Item 86, wherein the vaccine composition is 0573 Item 104. The kits-of-parts according to any of Item capable of eliciting an immune response against HIV enve 98 to Item 103, where the provided compositions are to be lope and/or against an antigen presenting cell expressing an administered simultaneously or sequentially. HIV-1 envelope as defined in any of Item 1 to Item 25, and/or 0574) Item 105. A method of treating, preventing orame against an antigen as defined in Item 27, and/or against a liorating a clinical condition, said method comprising admin biological entity as defined in any of Item 62 to Item 78 when istering to an individual Suffering from said clinical condition administered to an individual. an effective amount of an HIV-1 envelope polypeptide or part 0557. Item 88. The vaccine composition according to any thereofas defined in any of Item 1 to Item 26, an antigen as of Item 79 to Item 87, wherein said vaccine composition is defined in Item 27, a nucleic acid as defined in any of Item 28 capable of eliciting a cellular immune response in the indi to Item 29, a vector as defined in any of the preceding Item 30 vidual. to Item 61, a biological entity as defined in any of Item 62 to 0558 Item 89. The vaccine according to Item 88, wherein Item 78, a vaccine composition as defined in any of Item 79 to said individual is infected with HIV. Item 97, or a kit-of-parts as defined in any of Item 98 to Item 0559) Item 90. The vaccine composition according to any 104. of Item 79 to Item 89, comprising a peptide fragment, which (0575 Item 106. The method according to Item 105, is restricted by a MHC Class I molecule. wherein said clinical condition is an infection. 0560 Item 91. The vaccine composition according to any (0576. Item 107. The method according to Item 105, of Item 79 to Item 90, comprising a peptide fragment, which wherein said clinical condition is HIV infection and/or AIDS. is restricted by a MHC Class II molecule. (0577. Item 108. The method according to Item 107, 0561. Item 92. The vaccine composition according to any wherein said individual suffering from HIV infection and/or of Item 79 to Item 91, wherein the HIV-1 polypeptide or AIDS is a human being. fragment thereof consists of at the most 50 amino acid resi (0578. Item 109. The method according to Item 108, dues, for example at the most 45 amino acid residues, such as wherein said human being is HIV seronegative. at the most 40 amino acid residues, for example at the most 35 (0579. Item 110. The method according to Item 108, amino acid residues. Such as at the most 30 amino acid resi wherein said human being is HIV seropositive. dues, for example at the most 25 amino acid residues, such as 0580 Item 111. The method according to any of Item 105 20 to 25 amino acid residues. to Item 110, wherein said an HIV-1 envelope polypeptide or 0562 Item 93. The vaccine composition according to any part thereofas defined in any of Item 1 to Item 26, an antigen of Item 79 to Item 92, where the vaccine elicits the production as defined in Item 27, a nucleic acid as defined in any of Item in a vaccinated individual of regulatory T-cells having a cyto 28 to Item 29, a vector as defined in any of the preceding Item toxic effect against cells expressing HIV-1 envelope polypep 30 to Item 61, a biological entity as defined in any of Item 62 tide or part thereof, and/orantigen presenting cells expressing to Item 78, a vaccine composition as defined in any of Item 79 HIV-1 envelope or part thereof. to Item 97, or a kit-of-parts as defined in any of Item 98 to Item 0563 Item 94. The vaccine composition according to any 104 is administered to said organism two or more times. of Item 79 to Item 93, wherein the vaccine composition is 0581. Item 112. The method of to any of Item 105 to Item capable of eliciting a clinical response in a subject, wherein 111, which is combined with a further treatment. the clinical response is characterised by a reduced suscepti 0582. Item 113. The method of Item 112, wherein the bility, resistance, stabilisation, remission or curing/recovery further treatment is selected from the group consisting of of an HIV infection and/or AIDS. chemotherapy, treatment with immunostimulating Sub 0564) Item 95. The vaccine composition according to any stances, gene therapy, treatment with antibodies and treat of Item 79 to Item 94, further comprising an immunogenic ment using dendritic cells.