AU2009238330A1 - Generation of a broad T-cell response in humans against HIV - Google Patents

Description

Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT APPLICANT: BAVARIAN NORDIC A/S Invention Title: GENERATION OF A BROAD T-CELL RESPONSE IN HUMANS AGAINST HIV The following statement is a full description of this invention, including the best method of performing it known to me: FIELD OF THE INVENTION [001] The invention relates to compositions and methods for the generation of a T cell response against HIV proteins in vivo. BACKGROUND OF THE INVENTION [002] The Human Immunodeficiency virus (HIV) is the causative agent of the Acquired Immunodeficiency Syndrome (AIDS). Like all retroviruses, the HIV genome encodes the Gag, Pol and Env proteins. In addition, the HIV genome encodes further regulatory proteins, for example, Tat and Rev, as well as accessory proteins, such as Vpr, Vpx, Vpu, Vif and Nef. [003] Despite public health efforts to control the spread of the AIDS epidemic, the number of new infections is still increasing. The World Health Organization estimated the global epidemic at 37.8 million infected individuals at the end of the year 2003, and 36.1 million infected individuals at the end of the year 2000, 50% higher than what was predicted on the basis of the data about a decade ago (WHO & UNAIDS. UNAIDS, 2004). Without further improvements on comprehensive prevention mechanisms, the number of new HIV infections to occur, globally, this decade is projected to be 45 million (2004 Report on The Global AIDS Epidemic, UNAIDS and WHO). [004] HIV infection is a chronic infectious disease that can be partially controlled, but not yet cured. There are effective means of preventing complications and delaying progression to AIDS. At the present time, not all persons infected with HIV have progressed to AIDS, but it is generally believed that the majority will. [005] A combination of several antiretroviral agents, termed Highly Active Anti Retroviral Therapy (HAART), has been highly effective in reducing viral load, which can improve T-cell counts. This is not a cure for HIV, and people on HAART with suppressed 1 levels of HIV can still transmit the virus to others. However, there is good evidence that if the levels of HIV remain suppressed and the CD4 count remains greater than 200, then the quality and length of life can be significantly improved and prolonged. [006] Although drugs used in HAART regimens are able to reduce the viral titres, there are several concerns about antiretroviral regimens. One main problem concerning HAART is (long-term) side effects and thereby compliance of the patient. If patients miss doses, drug resistance can develop (Sethi et al., 2003, "Association between adherence to antiretroviral therapy and human immunodeficiency virus drug resistance", Clin.Infect.Dis., vol. 37, no. 8, pp. 1112-1118.) Also, anti-retroviral drugs are costly, and the majority of the world's infected individuals do not have access to medications and treatments for HIV and AIDS. [007] Given the steady spread of the epidemic, a number of different HIV-1 vaccine delivery strategies, such as novel vectors or adjuvant systems, have now been developed and evaluated in different pre-clinical settings, as well as in clinical trials. Ideally, a preventive HIV vaccine would abort HIV infection by providing sterilizing immunity through stimulation of high titres of broadly neutralizing antibodies. Most licensed vaccines against viruses depend on such responses (Plotkin, S. A. 2001, "Immunologic correlates of protection induced by vaccination", Pediatr.Infect.Dis.J, vol. 20, no. 1, pp. 63-75). Thus, the HIV vaccines that were tested for efficacy in the past were usually based on single HIV proteins, such as Env. The first vaccine candidate that entered a phase-Ill clinical trial is based on envelope gp 120 protein in alum formulations (Francis et al., AIDS Res. Hum. Retroviruses 14 (Suppl 3)(5): S325-31 [1998]). The results of the first clinical studies were discouraging. 2 [008] In phase III trials envelope-based (protein) vaccines did not prevent infection or ameliorate post-infection course. (Flynn et al., 2005, "Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection", J Infect.Dis., vol. 191, no. 5, pp. 654-665; Gupta et al., 2002, "Safety and immunogenicity of a high-titered canarypox vaccine in combination with rgp120 in a diverse population of HIV-1-uninfected adults: AIDS Vaccine Evaluation Group Protocol 022A", J Acquir./mmune.Defic.Syndr., vol. 29, no. 3, pp. 254-261.) [009] Even when an immune response was generated against such a single protein, for example, Env, the immune response proved ineffective. One reason for the ineffectiveness is the high mutation rate of HIV, in particular with respect to the Env protein, reportedly resulting in viruses, in which the proteins are not recognized by the immune response induced by the vaccine. Since no effective prophylactic treatment is available, there is still a need to bring an effective vaccine to the clinic. [010] Therefore, much emphasis is currently put on the induction of cellular immune responses against HIV, bolstered by the observation the HIV-infected long-term non progressors (LNTP) control their infection by cellular immunity (Harrer et al., 2005, "Therapeutic vaccination of HIV-1 -infected patients on HAART with a recombinant HIV-1 nef-expressing MVA: safety, immunogenicity and influence on viral load during treatment interruption", Antivir. Ther., vol. 10, no. 2, pp. 285-300. (0111 There are many different strategies that may lead to an effective HIV vaccine. The vaccines being tested should produce antibodies and/or or cytotoxic T cells (CTLs) to fight infection. Some of the current approaches to an HIV vaccine are discussed below. 3 Recombinant subunit protein vaccines [012] Recombinant proteins are produced by genetically engineering yeast, insect cells, or mammalian cells to produce one or more foreign proteins that serve as immunogen. Envelope-based (gpl20 or gpl 40) as well as non-structural protein subunit vaccines (like Tat or Tat-Nef fusion proteins) have been and are tested in different clinical phases. Examples are: * AIDSVAX B/B (VaxGen), * Clade C Env subunit (Chiron), * gp120 MN (VaxGen), AIDSVAX B/B (VaxGen) gp140 SF-162 - oligomeric, V2 deleted (Chiron), " gpl20W61D (GlaxoSmithKline) " NefTat (GlaxoSmithKline) * LFn-p24 (Anthrax-derived Lethal factor + p24; WRAIR/AVANT) [013] These systems are often not an effective manner to produce vaccines because some proteins are not produced with their native glycosylatation patterns or in their native structure. Synthetic peptide vaccine [014] It has been shown that the immune system will mount a response to very short peptide sections of a protein antigen when they are presented appropriately. Synthetic peptides can be given alone, linked to lipid molecules or combined as multi peptide vaccines to broaden the induced immune response. Examples are: * Multiepitope CTL peptide vaccines (Wyeth) - LIPO-5 (Sanofi-Pasteur/ ANRS) 4 " lipopeptide HIV vaccine BioQuest * two peptide-based HIV vaccines, Vacc-4x and Vacc-5q (Bionor Immuno) [015] Furthermore, Phase I trials are underway exploring LIPO-5 as a booster for DNA or viral vector (ALVAC) priming. Inactivated HIV vaccine [016] The first and most advanced therapeutic vaccine approach in clinical development is Remune*, a whole inactivated gp120-stripped HIV-1. While studies have demonstrated that Remune can spark the production of HIV-specific T-cells, clinical trials did not show that Remune is of therapeutic benefit to people with HIV (Kahn et al. 2000). The developer is now focusing on the development of IR103, which contains Remune and an adjuvant (Amplivax), an immune system stimulant being used to increase the body's immune response to Remune and HIV. DNA vaccines [017] The primary advantages of DNA vaccines are the manufacturing process, which does not require cell substrates, and their stability under a variety of conditions. [018] DNA vaccines are being investigated in several clinical trials as a prime for either protein or viral vector boosting. - Genevax-HIV (Wyeth) alone or as prime for Canarypox based vaccine e multiple epitopes from HIV + the PADRE helper T cell epitopes (Pharmexa Epimmune) " GTU-MultiHIV (DNA plasmid vaccine consisting of 6 different genes) (FIT Biotech) 5 [019] However, thus far these vaccines have not reproduced the high levels of CD8+ responses observed in animals. Several strategies to enhance the immunogenicity of DNA vaccines are currently tested in Phase I clinical trials, such as e.g. * the addition of "minigenes" (Epimmune), * PLG microparticles (Chiron), * IL-2 or IL-12 (Wyeth). Viral vector vaccines [020] The advantage of this strategy is to mimic as closely as possible the efficacy of vaccines developed using similar live-attenuated organisms to the disease causing agent while at the same time offering much greater safety. Several different viral vectors are currently under investigation like: * Adeno and adeno-associated vectors * Alphavirus vectors (VEE) " Canarypox vectors * Fowlpox vector " Modified Vaccinia Ankara (MVA) vectors [021] MVA-BN* based recombinant vaccines are tolerated very well. The observed adverse events are primarily mild to moderate injection site reactions (erythema, pain, etc) and to a lesser extend mild to moderate systemic reactions like myalgia and headache. An MVA BN@-based recombinant HIV vaccine administered trice in two phase I/Il clinical trials showed no safety signals. Among the subjects vaccinated to date with the vaccine, mostly mild and transient local as well as general systemic reactions were observed. 6 [022] Modified Vaccinia Ankara (MVA) virus is a member of the Orthopoxvirus family and has been generated by about 570 serial passages on chicken embryo fibroblasts of the Ankara strain of Vaccinia virus (CVA) (for review see Mayr, A., et al., Infection 3, 6-14 [1975}). As a consequence of these passages, the resulting MVA virus contains 31 kilobases less genomic information compared to CVA, and is highly host-cell restricted (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038 [1991]). MVA is characterized by its extreme attenuation, namely, by a diminished virulence or infectious ability, but still holds an excellent immunogenicity. When tested in a variety of animal models, MVA was proven to be avirulent, even in immuno-suppressed individuals. [023] Several excellent properties of the MVA strain pertinent to its use in vaccine development have been demonstrated in extensive clinical trials (Mayr et al., Zbl. Bakt. Hyg. 1, Abt. Org. B 167, 375-390 [1987]). During these studies, performed in over 120,000 humans, including high-risk patients, no side effects were seen (Stickl et al., Dtsch. med. Wschr. 99, 2386-2392 (1974]). [024] It has been further found that MVA is blocked in the late stage of the virus replication cycle in mammalian cells (Sutter, G. and Moss, B., Proc. Nati. Acad. Sci. USA 89, 10847-10851 [1992]). Accordingly, MVA fully replicates its DNA, synthesizes early, intermediate, and late gene products, but is not able to assemble mature infectious virions, which could be released from an infected cell. For this reason, namely, its replication-restricted nature, MVA serves as a gene expression vector. [025] HIV infection and disease are widespread. The overwhelming majority of new HIV infections occur in developing countries that lack the economic resources and 7 infrastructure to acquire and successfully deliver effective antiretroviral therapy. Thus, a need exists for a safe, effective, and easily administered HIV/AIDS vaccine. SUMMARY OF THE INVENTION [026] Accordingly, this invention provides methods for inducing a T cell response in a human patient. In a preferred embodiment, the T cell response is to at least three HIV-1 proteins. Preferrably, the method comprises administering an MVA vector encoding at least three HIV-1 proteins selected from HIV-1 Gag, Pol, Vpr, Vpu, Rev, and Nef. Most preferably, the MVA vector induces a T cell response in the patient to at least three of the HIV-1 proteins. [027] In one embodiment, one of the proteins is an HIV-1 Gag protein. In one embodiment, one of the proteins is an HIV-1 Pol protein. In one embodiment, one of the proteins is an HIV-1 Nef protein. In one embodiment, the HIV-1 proteins are Gag, Pol, and Nef. In a preffered embodiment, one of the proteins is a truncated HIV-1 Nef protein. [028] In one embodiment, one of the proteins is an HIV-1 Vpr protein. In one embodiment, one of the proteins is an HIV-1 Vpu protein. In one embodiment, one of the proteins is an HIV-1 Rev protein. [029] In a preferred embodiment, the vector comprises a coding sequence for HIV-1 Gag-Pol protein. In a preferred embodiment, vector also comprises a coding sequence for a truncated HIV-1 Nef protein. In a preferred embodiment, the vector further comprises a coding sequence for HIV-1 Vif, Vpr, Vpu, and Rev proteins. In a preferred embodiment, the vector further comprises a coding sequence for HIV-1 Tat protein. [030] In one embodiment, a dosage of 10 7 to 10 9

TCID

5 o of the vector is administered to the patient. In one embodiment, a dosage of 108 to 10 9

TCID

50 of the 8 vector is administered to the patient. In one embodiment, a dosage of 2 x 108 TCID 50 of the vector is administered to the patient. [031] In one embodiment, the MVA vector induces a T-cell response in the patient to 4 HIV-1 proteins. [032] In one embodiment, the MVA is MVA-BN. [033] In one embodiment, the patient is infected with HIV-1. [034] Additional objects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. [035] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. BRIEF DESCRIPTION OF THE DRAWINGS [036] The invention is more fully understood through reference to the drawings. [037] Figure 1 depicts HIV-BN*-MAG construct (MVA-mBN120B): MVA-BN* expressing multiple full length or truncated HIV-1 proteins. [038] Figure 2 depicts Vaccinations of 2 x 108 TCID 50 of the MVA-BN@-MAG vaccine were given at Weeks 0, 4 and 12. Blood samples for antibody and T cell responses were taken at weeks 0, 1, 5, 12, 13 and 20. FU - follow up. [039] Figure 3 depicts HIV-specific T cell responder rates. a) Percentage of responders calculated from n (number of subjects who were responders) and based on a 9 group size of N = 15. RT = reverse transcriptase, CTL = cytotoxic T lymphocytes, HTL helper T lymphocytes. - 86.7% (13/15) HIV vaccine responder rate. - The highest proportion of subjects responded to gag (73.3%, 11/15). 0 Within gag, p24 resulted in higher responder rates (40.0% and 46.7%) than did p 17 (20.0%). * Responder rates to pol and the mixed protein pool were equal (53.3%, 8/15) * Within pol, the protease and RT had equal responder rates (26.7%) and were followed by integrase with a 20.0% responder rate. * Since the mixed pool contained peptides from multiple proteins it is not possible to determine the immunogenic proteins. * Nef responder rate was 40.0% (6/15) * 7 subjects (46.7%) and 1 subject (6.7%) responded to HTL and CTL polytope peptides * No responders were detected for both tat and vif. [040] Figure 4 a-d depict median SFU/1 x106 PBMC for the indicated HIV-1 proteins. Arrows indicate vaccinations. * Gag responders (11/15 subjects): Median peak of 437 SFU/1x106 PBMC at Week 13, one week following the third immunization. - Pol responders (8/15 subjects): Median peak of 80 SFU/1x106 PBMC at Week 13, one week following the third immunization. 10 * Nef responders (6/15 subjects): Median peak of 276 SFU/1x106 PBMC at Week 12, eight weeks following the second immunization. * Mixed pool (p2p7, vpr, rev) responders (8/15 subjects): Median peak of 65 SFU/1 x1 06 PBMC at Week 12, eight weeks following the second immunization. * Gag, pol, nef and mixed responsive IFN-y secreting PBMCs remained higher than baseline 20 weeks after the first immunization. DETAILED DESCRIPTION OF THE INVENTION [041] An MVA-BN@-Multiantigen vector was constructed and tested preclincally in mice. In mice, the HIV-Multiantigen MVA-construct is immunogenic. Also, the immune response was directed against at least 2 proteins encoded in the recombinant MVA product (i.e. Nef and Gag specific responses were detected), the CD8 T cell restricted immune responses were not limited to a single CD8 T cell molecule (since both H2-Kd and H2-Dd responses were induced), and both CD8 and CD4 T cell restricted responses were induced by the MVA-construct. The vector was then tested in humans and was able to induce a broad immune response to multiple HIV-1 proteins and to vaccinia, and the responses were still higher than baseline 20 weeks after receiving the first immunization. Thus, the invention provides methods for inducing a T cell response in a human patient. The T cell response can be induced against HIV proteins using an MVA vector. [042] MVA-BN@-Multiantigen was well tolerated in HIV-1 infected subjects. [043] Three sc immunizations with 2x10 8

TCID

50 MVA-BN@-MAG resulted in increased or new HIV and vaccinia specific cellular and humoral immune responses. For 11 T cell responses, the HIV-specific responder rate was 86.7% and the vaccinia specific responder rate was 100%. These response rates were achieved at maximal levels following 1-3 immunizations for HIV proteins and 1 immunization for vaccinia specific T cell response. Median responses remained higher than baseline 20 weeks after the first immunization for all HIV proteins which induced responses as well as for vaccinia specific T cell response. [044] Although no responses were observed with the HIV proteins tat and vif, a broad cellular immune response against the remaining four HIV proteins/polyprotein pools (gag, pol, nef and mixed [p2p7-vpr-rev]) was observed in 20% of the subjects. In total, 67% of all subjects responded to at least two HIV-specific proteins. T cell responses [045] The invention encompasses generating T cell reponses against one or more HIV proteins, especially against 2, 3, 4, 5, 6, or more HIV proteins. In one embodiment, a vector (MVA) or insert (HIV protein) specific T cell response can be measured as a T cell response sufficient to exhibit either the occurrence of a specific signal in a subject who had no signal at Baseline, or a relative increase by a factor of at least 1.7 over the Baseline value in subjects who had a specific signal at Baseline using the techniques set forth below. [046] In one embodiment, a vector (MVA) or insert (HIV protein) specific signal can have a frequency of at least 50 SFU per 1x106 cells after correction for background (subtraction of SFU/1x1 06 non-stimulated cells/ two-fold above background) using the techniques set forth below. In one embodiment, a T cell response can be measured using an Enzyme Linked Immunospot (ELISPOT) assay used for the quantitative in vitro 12 determination of Interferon-gamma (IFN-y) producing cells in cryopreserved Peripheral Blood Mononuclear Cells (PBMC). [047] Briefly, 96 well filter plates (HTS plates, Millipore) are coated with a capture antibody (against IFN-y according to the manufacturer's instructions (BD Biosciences, IFN-y ELISPOT pair) at 4 0 C overnight. Subsequently, resuscitated PBMC in X-VIVO 15 supplemented with 9% Human AB Serum, from LONZA, Belgium, are added to the wells in a concentration of 200,000 cells/200pl final volume and, to assess the HIV specific cellular response, cells are stimulated with peptide pools at a final concentration of 5pg/ml per peptide. Following an incubation period (overnight at 37 0 C/5% CO:), the wells are washed and a biotin-labelled detection antibody in PBS/9%FCS is added. After washing with PBS/0.05% Tween 20, streptavidin-coupled horse radish peroxidase (HRP, BD Biosciences) is added to the wells, followed by a washing step and the addition of a precipitating substrate (AEC, 3-Amino-9-Ethylcarbazole, BD Biosciences). The number of cytokine producing cells is determined by counting the spots using a CTL S5 Microanalyzer. Reported values are background corrected and normalized to 1x106 PBMC. [048] Of course, other techniques to measure T cell responses are known to the skilled artisan and can be used instead of the above technique and still fall within the scope of the invention. HIV proteins and derivatives of full-length HIV proteins [049] The T cell response can be to at least 1, 2, 3, 4, 5, or 6 HIV, preferably HIV-1, proteins. Preferably, the response is to HIV-1 HIV-1 Gag, Pol, Vpr, Vpu, Vif, or 13 Nef protein. Preferrably, the method comprises administering an MVA vector encoding at least three HIV-1 proteins selected from HIV-1 Gag, Pol, Vpr, Vpu, Vif, and Nef proteins. [050] According to this invention, an "HIV protein" is defined as a full-length HIV protein or an HIV protein that retains at least 70% of the amino acids of a full-length HIV protein. The HIV protein can be truncated and/or mutated. In some embodiments, the HIV protein retains at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the amino acids of a full-length HIV protein. Preferably, these amino acids are consecutive amino acids. Thus, an "HIV protein" can have full activity, reduced activity, no activity, or transdominant activity. [051] In one embodiment, the HIV protein comprises all or part of the amino acid sequence of the HIV-1 isolate HXB2R having the GenBank accession number K03455. Preferably, the HIV protein retains at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the amino acids of a full-length HIV protein of the HIV-1 isolate HXB2R having the GenBank accession number K03455. [052] The term "derivative of the amino acid sequence of a full-length HIV protein," as used in the present specification, refers to HIV proteins that have an altered amino acid sequence compared to the corresponding naturally occurring full-length HIV protein. An altered amino acid sequence may be a sequence in which one or more amino acids of the sequence of the full-length HIV protein are substituted, inserted, or deleted. For example, the derivative can have one or more conservative amino acid substitutions. More particularly, a "derivative of the amino acid sequence of a full-length HIV protein" is an amino acid sequence showing an identity of at least 50%, such as of at least 60%, of at least 70%, of at least 80%, or even of at least 90%, when the 14 corresponding part of the amino acid sequence in the protein is compared to the amino acid sequence of the respective full-length HIV protein of known HIV isolates. [053] According to the invention, an amino acid sequence is regarded as having the above indicated sequence identity even if the identity is found for the corresponding protein of only one HIV isolate, irrespective of the fact that there might be corresponding proteins in other isolates showing a lower identity. By way of example, if a Vpr derivative in the fusion protein shows an identity of 95% to the Vpr sequence of one HIV isolate, but only an identity of 50-70% to (all) other HIV isolates, the identity of said Vpr derivative is regarded as being of at least 90%. [054] In a preferred embodiment, the term "derivative of a full-length HIV protein" refers to an amino acid sequence showing an identity of at least 50%, 60%, 70%, 80%, or 90% to the respective HIV protein in the HIV-1 isolate HXB2R (GenBank accession number K03455). [055] The percent identity may be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Apple. Math 2:482, 1981). The default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; 15 (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Other programs used by one skilled in the art of sequence comparison and which give similar results may also be used. [056] In a preferred embodiment, the invention relates to recombinant MVA viruses comprising one or a plurality of exogenous sequences in the viral genome, selected from a group consisting of expression cassettes comprising one or more HIV proteins and expression cassettes comprising one or more derivatives of full-length HIV proteins. [057] According to one embodiment, the exogenous sequences comprise HIV peptides selected from a group consisting of the full-length proteins Gag (capsid protein), Pol (polymerase protein), Env (envelope protein), Tat, Vif, Vpu, Vpr, Rev and Nef; and parts, mutants, and/or derivatives thereof. [058] In certain embodiments, the recombinant MVA virus, in particular MVA-BN and its derivatives, comprises regulatory/accessory proteins of HIV. The protein can exhibit full biological activity or be inactive. [059] The regulatory/accessory proteins of HIV proteins have a biological activity that can have undesired side effects. Thus, it is also within the scope of the invention that, in certain other embodiments, one or more HIV proteins expressed from the recombinant MVA virus has a reduced biological activity compared to the wild-type protein, and thus is said to have reduced activity. [060] One skilled in the art is familiar with tests suitable to determine whether an HIV protein has reduced activity. 16 [061] The molecular mechanism of the Vif protein, which is essential for viral replication in vivo, remains unknown, but Vif possesses a strong tendency toward self association. This multimerization was shown to be important for Vif function in viral life cycle (Yang S. et al., J Biol Chem 276: 4889-4893 [2001]). Additionally, vif was shown to be specifically associated with the viral nucleoprotein complex, and this might be functionally significant (Khan M.A. et al., J Virol. 75 (16): 7252-65 [2001]). [062] Thus, in a preferred embodiment, the exogenous sequence expresses a vif with reduced activity, and the vif shows a reduced multimerization and/or association to the nucleoprotein complex. [063] The Vpr protein plays an important role in the viral life cycle. Vpr regulates the nuclear import of the viral pre-integration complex and facilitates infection of non dividing cells such as macrophages (Agostini et al., AIDS Res Hum Retroviruses 18(4):283-8 [2002]). Additionally, it has transactivating activity mediated by interaction with the LTR (Vanitharani R. et al., Virology 289 (2):334-42 [2001]). [064] Thus, in a preferred embodiment, the exogenous sequence expresses a Vpr with reduced activity, and the Vpr shows either decreased or no transactivation and/or interaction, with the viral preintegration complex. [065] Vpx, which is highly homologous to Vpr, is also critical for efficient viral replication in non-dividing cells. Vpx is packaged in virus particles via an interaction with the p6 domain of the gag precursor polyprotein. Like Vpr, Vpx is involved in the transportation of the preintegration complex into the nucleus (Mahalingam et al., J. Virol. 75 (1):362-74 [2001]). 17 [066] Thus, in a preferred embodiment, the exogenous sequence expresses a Vpx with reduced activity, and the Vpx has a decreased ability to associate to the preintegration complex via the gag precursor. [067] The Vpu protein is known to interact with the cytoplasmic tail of the CD4 and causes CD4 degradation (Bour et al., Virology 69 (3):1510-20 [1995]). [068] Therefore, in a preferred embodiment, the exogenous sequence expresses a Vpu with reduced activity, and the Vpu has a reduced ability to trigger CD4 degradation [069] The relevant biological activity of the well-characterized Tat protein is the transactivation of transcription via interaction with the transactivation response element (TAR). It was demonstrated that Tat is able to transactivate heterologous promoters lacking HIV sequences other than TAR (Han P. et al., Nucleic Acid Res 19 (25):7225-9 [1991]). [070] Thus, in a preferred embodiment, the exogenous sequence expresses a Tat with reduced activity, and the Tat shows reduced transactivation of promoters via the TAR element. [071] In another preferred embodiment, the exogenous sequence expresses a transdominant Tat, and the transdominant Tat can be obtained by making the following substitutions: 22 (Cys > Gly) and 37 (Cys>Ser). [072] Nef protein is essential for viral replication responsible for disease progression by inducing the cell surface downregulation of CD4 (Lou T et al., J Biomed Sci 4(4):132 [1997]). This downregulation is initiated by direct interaction between CD4 18 and Nef (Preusser A. et al., Biochem Biophys Res Commun 292 (3):734-40 [2002]). Thus, Nef protein with reduced function shows reduced interaction with CD4. [073] Thus, in preferred embodiments, the exogenous sequence expresses a Nef with reduced activity, and the Nef is truncated at the amino terminus such as, for example, a Nef in which the 19 N-terminal amino acids are deleted. [074] The relevant function of Rev is the posttranscriptional transactivation initiated by interaction with the Rev-response element (RRE) of viral RNA (lwai et al., Nucleic Acids Res 20 (24):6465-72 [1992]). [075] Thus, in a preferred embodiment, the exogenous sequence expresses a Rev with reduced activity, and the Rev shows a reduced interaction with the RRE. [076] In certain embodiments, the structural and/or accessory/regulatory proteins are expressed individually. [077] In other embodiments, multiple polypeptides are expressed and some or all of the polypeptides are expressed as fusion proteins. In this context reference is made to WO 03/097675, the content of which is herewith incorporated by reference. In a preferred embodiment, the MVA vector comprises an expression cassette encoding a Vif-Vpr-Vpu-Rev fusion protein, such as that described in U.S. Patent No. 7,501,127, which is hereby incorporated by reference.. [078] In a preferred embodiment, the recombinant MVA virus according to the invention, such as MVA-BN and its derivatives, expresses a fusion protein comprising at least four HIV polypeptides selected from the group consisting of the HIV proteins Vif, Vpr, Vpu, Vpx, Rev, Tat, and Nef and derivatives of the full-length HIV proteins. In a preferred embodiment, the MVA vector expresses HIV-lVif, Vpr, Vpu, and Rev proteins. 19 [079] In another embodiment, the recombinant MVA virus according to the invention, such as MVA-BN and its derivatives, expresses at least eight HIV polypeptides selected from the group consisting of the HIV proteins Vif, Vpr, Vpu, Rev, Tat, Nef, Gag and Pol, and derivatives of the full-length HIV proteins. [080] In another embodiment, a recombinant MVA virus according to the invention, such as MVA-BN and its derivatives, expresses one or more polypeptides selected from the group consisting of: (i) a fusion protein comprising Vif-Vpu-Vpr-Rev proteins, ligated in this order, or in a different order, or comprising derivatives of the full-length proteins; (ii) a Nef protein, or a derivative of the full-length protein, in particular a Nef protein in which N-terminal amino acids are deleted, such as the first 19 amino acids; (iii) a Tat, protein, or a derivative of the full-length protein, in particular a transdominant Tat; and (iv) a Gag-Pol fusion protein, or a derivative of the full-length protein arranged in the exemplified order, or in the reverse order (i.e., Pol-Gag). [081] The number of expression cassettes from which the HIV polypeptides are expressed is not critical. In one embodiment, the HIV polypeptides are expressed from two to five expression cassettes comprising a plurality of the following: (i) an expression cassette expressing a fusion protein comprising Vif-Vpu Vpr-Rev proteins, ligated in this order, or in a different order, or comprising derivatives of the full-length proteins; (ii) an expression cassette expressing a Nef protein, or a derivative of the full-length protein, in particular a Nef protein in which N-terminal amino acids are deleted, 20 such as the first 19 amino acids; (iii) a Tat, protein, or a derivative of the full-length protein, in particular a transdominant Tat; and (iv) a Gag-Pol fusion protein, or a derivative of the full-length protein; arranged in the exemplified order, or in the reverse order (i.e., Pol-Gag). Recombinant MVA Viruses [082] In a preferred embodiment, the recombinant MVA virus of the invention is replication incompetent in humans and non-human primates. The terms MVA virus that is "replication incompetent" in humans and/or non-human primates, and the synonymous term virus that is "not capable of being replicated to infectious progeny virus" in humans and/or non-human primates, both refer preferably to MVA viruses that do not replicate at all in the cells of the human and/or non-human primate vaccinated with said virus. However, also within the scope of the present application are those viruses that show a minor residual replication activity that is controlled by the immune system of the human and/or non-human primate to which the recombinant MVA virus is administered. [083] In one embodiment, the replication incompetent recombinant MVA viruses may be viruses that are capable of infecting cells of the human and/or non-human primate in which the virus is used as vaccine. Viruses that are "capable of infecting cells" are viruses that are capable of interacting with the host cells to such an extent that the virus, or at least the viral genome, becomes incorporated into the host cell. Although the viruses used according to the invention are capable of infecting cells of the vaccinated human and/or non human primate, they are not capable of being replicated to infectious progeny virus in the cells of the vaccinated human and/or non-human primate. 21 [084] According to the invention, it is to be understood, that a virus that is capable of infecting cells of a first animal species, but is not capable of being replicated to infectious progeny virus in said cells, may behave differently in a second animal species. For example, MVA-BN and its derivatives (see below) are viruses that are capable of infecting cells of the human, but that are not capable of being replicated to infectious progeny virus in human cells. However, the same viruses are efficiently replicated in chickens; i.e., in chickens, MVA-BN is a virus that is both capable of infecting cells and capable of being replicated to infectious progeny virus in those cells. [085] A suitable test that allows one to predict whether a virus is capable or not capable of being replicated in humans is disclosed in WO 02/42480 (incorporated herein by reference) and uses the severely immune compromised AGR129 mice strain. Furthermore, instead of the AGR129 mice, any other mouse strain can be used that is incapable of producing mature B and T cells, and as such is severely immune compromised and highly susceptible to a replicating virus. The results obtained in this mouse model reportedly are indicative for humans and, thus, according to the present application, a virus that is replication incompetent in said mouse model is regarded as a virus that is "replication incompetent in humans." [086] In other embodiments, the viruses according to the invention are preferably capable of being replicated in at least one type of cells of at least one animal species. Thus, it is possible to amplify the virus prior to its administration to the animal that is to be vaccinated and/or treated. By way of example, reference is made to MVA-BN that can be amplified in CEF (chicken embryo fibroblasts) cells, but that is a virus that is not capable of being replicated to infectious progeny virus in humans. 22 [087] In further embodiments, Modified Vaccinia virus Ankara (MVA) is suitable for use in humans and several animal species such as mice and non-human primates. MVA is known to be exceptionally safe. MVA has been generated by long-term serial passages of the Ankara strain of Vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H. Infection 3, 6-14 [1975]; Swiss Patent No. 568,392). Examples of MVA virus strains that have been deposited in compliance with the requirements of the Budapest Treaty, and that are useful for the generation of recombinant viruses according to the invention, are strains MVA 572 deposited at the European Collection of Animal Cell Cultures (ECACC), Salisbury (UK) with the deposition number ECACC 94012707 on January 27, 1994; MVA 575 deposited under ECACC 00120707 on December 7, 2000; and MVA-BN deposited with the number 00083008 at the ECACC on August 30, 2000. [088] In one embodiment, the MVA strain used in generating a recombinant MVA is MVA-575, or a derivative thereof. [089] In a preferred embodiment, the MVA strain used in generating a recombinant MVA is MVA-572, or a derivative thereof. [090] In another preferred embodiment, the MVA strain used in generating a recombinant MVA is MVA-Vero, or a derivative thereof. MVA-Vero strains have been deposited at the European Collection of Animal Cell Cultures under the deposition numbers ECACC V99101431 and 01021411. The safety of the MVA-Vero is reflected by its biological, chemical and physical characteristics as described in the International Patent Application PCT/EP01/02703, incorporated herein by reference in its entirety. In 23 comparison to other MVA strains, the Vero-MVA includes one additional genomic deletion. [091] In a more preferred embodiment, the MVA strain is MVA-BN, or a derivative thereof. MVA-BN has been deposited at the European Collection of Animal Cell Cultures with the deposition number ECACC V00083008. MVA-BN virus is an extremely attenuated virus also derived from Modified Vaccinia Ankara virus. A definition of MVA-BN and its derivatives is given in PCT/EP01/13628, incorporated herein by reference in its entirety. [092] The term "derivatives" of a virus according to the invention refers to progeny viruses showing the same characteristic features as the parent virus, but showing differences in one or more parts of its genome. The term "derivative of MVA" describes a virus which has the same functional characteristics compared to MVA. For example, a derivative of MVA-BN has the characteristic features of MVA-BN. One of these characteristics of MVA-BN, or of derivatives thereof, is its attenuation and lack of replication in human cell lines, such as the human keratinocyte cell line HaCaT, the human embryo kidney cell line 293, the human bone osteosarcoma cell line 143 B, and the human cervix adenocarcinoma cell line HeLa. [093] In a preferred embodiment, the virus according to the invention is a virus that has been produced and/or passaged under serum free conditions to reduce the risk of infections with agents contained in serum. One skilled in the art of the invention is familiar with methods for producing and/or passaging virus under serum free conditions. [094] In one embodiment, the expression cassette is inserted into a naturally occurring deletion site (e.g., site 1, 11, 111, IV, V, or VI) of the MVA viral genome. 24 [095] In one embodiment, the expression cassette is inserted into an intergenic region of the MVA viral genome. The term "intergenic regions" (IGRs) of the viral genome refers to regions of the MVA viral genome, wherein the regions are, in turn, located between or, in certain embodiments, are flanked by two adjacent open reading frames (ORFs) of the MVA genome. For example, if the IGR comprises a sequence that is not part of either ORF but exists between the two adjacent ORFs, then the IGR is said to be "located between" two adjacent ORFs. On the other hand, there are situations in which there is no additional sequence that exists between the two adjacent ORFs. In other words, the two adjacent ORFs abut. In the latter situations then, the IGR does not have a corresponding sequence in itself; rather, the IGR refers to the site, or genomic position, whereby an heterologous sequence can be inserted between the two ORFs, thus enlarging the distance separating the otherwise abutting ORFs. In this case, the IGR is said to be "flanked by" two adjacent ORFs. [096] "ORFs of the MVA genome" occur in two coding directions: forward and reverse (for detailed description, see for example, Antoine et al., Virology 244, 365-396 [1998]), incorporated herein by reference). Consequently, the polymerase activity occurs from left to right, i.e., forward direction and, correspondingly, from right to left, reverse direction. In certain embodiments of the invention, ORFs occuring in the forward coding direction are referred to as 5'ORF3', whereas ORFs occurring in the reverse coding direction are referred to as 3'FRO5' to facilitate the understanding of their orientation in the MVA genome. [097] It is common practice in poxvirology, and it became a standard classification for Vaccinia viruses, to identify ORFs by their orientation and their position 25 on the different Hindlll restriction digest fragments of the genome (see for example, Goebel et al., Virology 179, 247-266 and 517-563, [1990]; and Massung, R.F. et al., Virology 201, 215-240 [1994], incorporated herein by reference). For the common practice nomenclature, the different Hindill fragments are named by descending capital letters corresponding with their descending size. The ORFs are numbered from left to right on each Hindlll fragment and the orientation of the ORF is indicated by a capital L (standing for transcription from right to Left) or R (standing for transcription from left to Right). [098] Additionally, there is a more recent publication of the MVA genome structure, which uses a different nomenclature, simply numbering the ORF from the left to the right end of the genome, and indicating their orientation with a capital L or R (Antoine et al., Virology 244, 365-396 [1998]). As an example the 14L ORF, according to the old nomenclature, corresponds to the 064L ORF according to Antoine et al. If not indicated differently, the invention uses the nomenclature according to Antoine et al. [099] Accordingly, herein, the IGRs are referred to in one of two ways, depending on the nomenclature used to name the ORFs. For example, an IGR located between the two adjacent ORFs, ORF 001 L and ORF 002L, is said to be IGR 001 L-002L. An IGR located between the two adjacent ORFs, ORF 14L and ORF 15L, is said to be IGR 14L 15L. [0100] Herein, and according to the old nomenclature, ORF 006L corresponds to C10L, 019L corresponds to C6L, 020L to N1L, 021L to N2L, 023L to K2L, 028R to K7R, 029L to F1 L, 037L to F8L, 045L to F1 5L, 050L to E3L, 052R to E5R, 054R to E7R, 055R to E8R, 056L to E9L, 062L to 11L, 064L to 14L, 065L to 15L, 081R to L2R, 082L to L3L, 26 086R to J2R, 088R to J4R, 089L to J5L, 092R to H2R, 095R to H5R, 107R to D1OR, 108L to D11L, 122R to A11R, 123L to A12L, 125L to A14L, 126L to A15L, 135R to A24R, 136L to A25L, 137L to A26L, 141L to A30L, 148R to A37R, 149L to A38L, 152R to A4OR, 153L to A41L, 154R to A42R, 157L to A44L, 159R to A46R, 160L to A47L, 165R to A56R, 166R to A57R, 167R to B1 R, 170R to B3R, 176R to B8R, 180R to B12R, 184R to B16R, 185L to B17L, and 187R to B19R. [0101] Accordingly, IGR 14L-15L (old nomenclature) corresponds to IGR 064L 065L (new nomenclature), and refers to the intergenic region located between, or flanked by, ORFs 14L and 15L. [0102] Furthermore, unless immediately preceeded by the term "IGR" and thus specified as referring to an IGR, the use of the term 14L-15L refers to the pair of ORFs 14L and 15L; it is not to be confused with IGR 14L-15L, which refers to the region located in between, or flanked by, the ORFs 14L and 15L. By analogy, the use of the expression, for example, "a group of ORFs selected from 001 L-002L, 002L-003L, 005R-006R," is synonymous with the use of the expression, and refers to, "a group of pairs of ORFs selected from the pair 001 L and 002L; the pair 002L and 003L; and the pair 005R and 006R. [0103] Accordingly, in certain embodiments, the invention relates to recombinant MVA viruses comprising one or more HIV DNA sequences inserted into one or more of IGRs. [0104] While the nucleotide sequence of an ORF encodes an amino acid sequence forming a peptide, polypeptide, or protein, the IGRs between two ORFs have no coding capacity. Accordingly, in certain embodiments, the IGRs may comprise 27 regulatory elements, binding sites, promoter and/or enhancer sequences essential for, or involved in, the transcriptional control of the viral gene expression. Thus, the IGR may be involved in the regulatory control of the viral life cycle. [0105] According to the invention, the nucleotide sequence of an ORF should start with a start codon and end with a stop codon. Depending on the orientation of the two adjacent ORFs, the IGR, i.e., the region in between these ORFs, is flanked by one of the following: the two stop codons of the two adjacent ORFs; the two start codons of the two adjacent ORFs; the stop codon of the first ORF and the start codon of the second ORF; or the start codon of the first ORF and the stop codon of the second ORF. [0106] Accordingly, in one embodiment, the site for insertion of the exogenous DNA sequence into the IGR is downstream, i.e. 3', of the stop codon of a first ORF; and, in case the adjacent ORF, also termed second ORF, has the same orientation as the first ORF, the insertion site further lies upstream, i.e. 5', of the start codon of the second ORE. This arrangement can be represented as: 5'ORF3'-lGR-5'ORF3'. [0107] In a further embodiment, the site for insertion of the exogenous DNA sequence into the IGR is downstream, i.e. 3', of the stop codon of a first ORF; and, in case the second ORF has an opposite orientation relative to the first ORF, which means the orientation of the two adjacent ORFs points to each other, the insertion site further lies downstream of the stop codons of both ORFs. This arrangement can be represented as: 5'ORF3'-IGR-3'FRO5'. [0108] In yet a further embodiment, in case the two adjacent ORFs read in the same direction from right to left of the viral genome, which is synonymous with a positioning that is characterized in that the start codon of the first ORF is adjacent to the 28 stop codon of the second ORF, then the exogenous DNA is inserted upstream (or 5') of one start codon and downstream (or 3') from the other. This arrangement can be represented as: 3'FRO5'-IGR-3'FRO5'. [0109] In yet a further embodiment, in case the two adjacent ORFs read in opposite direction, but the orientation of the two adjacent ORFs points away from each other, which is synonymous with a positioning that is characterized in that the start codons of the two ORFs are adjacent to each other, then the exogenous DNA is inserted upstream relative to both start codons. This arrangement can be represented as: 3'FRO5'-IGR-5'ORF3'. [0110] In one embodiment, heterologous DNA sequences can be inserted into one or more IGRs in between two adjacent ORFs, said IGR selected from the group comprising: 001L-002L, 002L-003L, 005R-006R, 006L-007R, 007R-008L, 008L-009L, 017L-018L, 018L-019L, 019L-020L, 020L-021L, 023L-024L, 024L-025L, 025L-026L, 028R-029L, 030L-031L, 031L-032L, 032L-033L, 035L-036L, 036L-037L, 037L-038L, 039L-040L, 043L-044L, 044L-045L, 046L-047R, 049L-050L, 050L-051 L, 051 L-052R, 052R-053R, 053R-054R, 054R-055R, 055R-056L, 061L-062L, 064L-065L, 065L-066L, 066L-067L, 077L-078R, 078R-079R, 080R-081R, 081R-082L, 082L-083R, 085R-086R, 086R-087R, 088R-089L, 089L-090R, 092R-093L, 094L-095R, 096R-097R, 097R-098R, 101R-102R, 103R-104R, 105L-106R, 107R-108L, 108L-109L, 109L-110L, 11OL-111L, 113L-114L, 114L-115L, 115L-116R, 117L-118L, 118L-119R, 122R-123L, 123L-124L, 124L-125L, 125L-126L, 133R-134R, 134R-135R, 136L-137L, 137L-138L, 141L-142R, 143L-144R, 144R-145R, 145R-146R, 146R-147R, 147R-148R, 148R-149L, 152R-153L, 153L-154R, 29 154R-155R, 156R-157L, 157L-158R, 159R-160L, 160L-161R, 162R-163R, 163R-164R, 164R-165R, 165R-166R, 166R-167R, 167R-168R, 170R-171R, 173R-174R, 175R 176R, 176R-177R, 178R-179R, 179R-180R, 180R-181R, 183R-184R, 184R-185L, 185L-186R, 186R-187R, 187R-188R, 188R-189R, 189R-190R, 192R-193R. [0111] In a preferred embodiment, the heterologous sequence is inserted into an IGR flanked by two adjacent ORFs selected from the group comprising 007R-008L, 018L-019L, 044L-045L, 064L-065L, 136L-137L, 148R-149L. Generation of Recombinant MVA [0112] Methods suitable to generate a plasmid vector according to the invention are familiar to those skilled in the art of the invention. For example, to generate a plasmid vector with the sequences of the invention, the sequences can be isolated and cloned into a standard cloning vector, such as pBluescript (Stratagene), wherein they flank the exogenous DNA to be inserted into the MVA genome. Optionally, such a plasmid vector comprises a selection- or reporter-gene cassette, which can be deleted from the final recombinant virus, due to a repetitive sequence included into said cassette. [0113] Methods to introduce exogenous DNA sequences by a plasmid vector into an MVA genome and methods to obtain recombinant MVA are well known to the person skilled in the art and, additionally, can be deduced from the following references: (i) Molecular Cloning, A Laboratory Manual, Second Edition, by J. Sambrook, E.F. Fritsch and T. Maniatis. Cold Spring Harbor Laboratory Press, 1989: describes techniques and know how for standard molecular biology techniques such cloning of DNA, RNA isolation, western blot analysis, RT-PCR and PCR amplification techniques; 30 (ii)Virology Methods Manual, edited by Brian W.J. Mahy and Hillar 0. Kangro, Academic Press, 1996: describes techniques for the handling and manipulation of viruses; (iii) Molecular Virology: A Practical Approach, edited by AJ Davison and RM Elliott, The Practical Approach Series, IRL Press at Oxford University Press, Oxford 199, Chapter 9, Expression of genes by Vaccinia virus vectors; and (iv) Current Protocols in Molecular Biology, Publisher: John Wiley and Son Inc., 1998, Chapter 16, section IV: Expression of proteins in mammalian cells using Vaccinia viral vector: describes techniques and know-how for the handling, manipulation and genetic engineering of MVA. [0114] In one embodiment, the MVA and derivatives thereof, according to the invention, preferably the MVA deposited at ECACC under deposition number V00083008, is produced by transfecting a cell with a plasmid vector according to the invention, infecting the transfected cell with an MVA and, subsequently, identifying, isolating and, optionally, purifying the MVA according to the invention. [0115] According to a further embodiment of the invention, the exogenous DNA sequence comprises a spacer or spacing sequence, which separates poxviral transcription control element and/or coding sequence in the exogenous DNA sequence from the stop codon and/or the start codon of the adjacent ORFs. [0116] According to the invention, this spacer or spacing sequence, located between the stop/start codon of the adjacent ORF and the coding sequence inserted in the exogenous DNA, has the advantage of stabilizing the inserted exogenous DNA and, 31 thus, any resulting recombinant virus. The size of a suitable spacer sequence is variable, as long as the sequence is without its own coding or regulatory function. [0117] According to a further embodiment, the spacer sequence, separating the poxviral transcription control element and/or the coding sequence in the exogenous DNA sequence from the stop codon of the adjacent ORF, is at least one nucleotide long. [0118] According to yet another embodiment, the spacing sequence, separating the poxviral transcription control element and/or the coding sequence in the exogenous DNA sequence from the start codon of the adjacent ORF, is at least 30 nucleotides. [0119] In one embodiment, if a typical Vaccinia virus promoter element is upstream of a start codon, the insertion of exogenous DNA might separate the promoter element from the start codon of the adjacent ORF. A spacing sequence of about 30 nucleotides is the preferred distance to secure that, a poxviral promoter located upstream of the start codon of the ORF, is not influenced. [0120] Additionally, according to a further preferred embodiment, the distance between the inserted exogenous DNA and the start codon of the adjacent ORF comprises about 50 nucleotides, and more preferably about 100 nucleotides. [01211 "A typical Vaccinia promoter" element can be identified by scanning for, for example, the sequence "TAAAT" for late promoters (Davison & Moss, J. Mol. Biol. 210: 771-784 [1989]), and for an A/T rich domain for early promoters. [0122] According to a further embodiment, the spacing sequence comprises an additional poxviral transcription control element, which is capable of controlling the transcription of the adjacent ORF. 32 (0123] In a preferred embodiment, the expression of heterologous nucleic acid sequence is preferably, but not exclusively, under the transcriptional control of a poxvirus promoter. An example of a suitable poxvirus promoter is the cowpox ATI promoter (see WO 03/097844, incorporated herein by reference). In certain embodiments, the expression of each expression cassette is controlled by a different promoter. In other embodiments, all expression cassettes are controlled by a copy of the same promoter. [0124] In one embodiment, the promoter is an early promoter. In one embodiment, the promoter is a late promoter. In one embodiment, the promoter is a hybrid early/late promoter. [0125] In one embodiment, the invention relates to a recombinant virus in which all HIV expression cassettes, are controlled by a cowpox ATI promoter or a derivative thereof, as defined in WO 03/097844. [01261 In one embodiment, the expression cassettes can be inserted into 1 to 10 insertion sites in the genome of a recombinant MVA according to the invention, such as MVA-BN and its derivatives. [0127] Recombinant MVA viruses, in particular MVA-BN and its derivatives, used for the expression of at least six HIV proteins, or six parts or derivatives thereof, can be easily obtained if not all expression cassettes are inserted into the same insertion side. [0128] Thus, in certain embodiment, the different expression cassettes are inserted into 2 to 8, or 3 to 5, or into 3 insertion sites in the MVA genome. [0129] The insertion of heterologous nucleic acid sequence can be done into a non-essential region of the virus genome. According to one embodiment, the 33 heterologous nucleic acid sequence is inserted at a naturally occurring deletion site of the MVA genome (disclosed in PCT/EP96/02926, incorporated herein by reference). [0130] According to a further embodiment, one or more heterologous sequences can be inserted into one or more intergenic regions (IGRs) of the MVA genome as described herein. [0131] Methods on how to insert heterologous sequences into the poxviral genome are known to a person skilled in the art. [0132] In a preferred embodiment, the expression cassettes comprising one or more HIV proteins, parts, or derivatives thereof, can be inserted into one or more intergenic regions selected from the group consisting of IGR 07-08, IGR 14L-15L, and IGR 136-137 of the MVA genome, in particular the genome of MVA-BN and its derivatives. [0133] In another embodiment, the recombinant poxvirus is MVA-BN, or a derivative thereof, comprising all the following expression cassettes inserted into the specified insertion sites: (i) an expression cassette expressing Vif-Vpu-Vpr-Rev, or derivatives of the full-length proteins, as a fusion protein in this or a different order; inserted into the intergenic region IGR 07-08; (ii) a second expression cassette expressing Nef, or a derivative of the full-length protein, in particular a Nef protein in which N-terminal amino acids are deleted, such as Nef lacking the first 19 amino acids; inserted into IGR 14L-15L; (iii) a third expression cassette that expresses Tat, or a derivative of the full-length protein, in particular a transdominant Tat, inserted into IGR 136-137; and 34 (iv) a fourth expression cassette that express a Gag-Pol fusion protein, or derivatives of the full-length proteins; inserted into IGR 136-137. [0134] Thus, in the latter embodiment, the third and the fourth expression cassettes are inserted into the same integration site. It is to be taken into account that IGR 14L-15L on the one side, and IGR 136-137 and IGR 07-08 on the other side, belong to two different numbering systems; these numbering or nomenclature systems are explained above. Vaccines [0135] In a preferred embodiment, the recombinant virus according to the invention can induce a protective immune response. The term "protective immune response" as used herein is intended to mean that the vaccinated subject is able to control in some way an infection with the pathogenic agent against which the vaccination was done. Usually, the animal or subject having developed a "protective immune response" develops milder clinical symptoms than an unvaccinated subject, and/or the progression of the disease is slowed down. [0136] The invention further relates to medicaments and vaccines comprising the recombinant MVA virus of the invention. [0137] In other embodiments, the invention further relates to pharmaceutical compositions and vaccines comprising a recombinant MVA virus as defined above. [0138] In further embodiments, the invention further relates to the use of a recombinant MVA virus as defined above for the preparation of a medicament and/or vaccine for the treatment and/or prevention of AIDS. 35 [0139] In another embodiment, the invention further relates to a method of prevention AIDS comprising the step of administration of a MVA virus as defined above. [0140] It is pointed out that the term "prevention of AIDS" as used in the context of the invention does not mean that the recombinant MVA virus prevents AIDS in all subjects under all conditions. To the contrary, this term as used herein refers to any statistically significant protective effect, even if this effect is considered low. [0141] Possible concentrations and modes of administration are indicated herein. [0142] Numerous ways to prepare recombinant MVA formulations are known to the skilled artisan, as are modes of storage. In this context, reference is made to WO 03053463, incorporated herein by reference. [0143] In a further embodiment, the invention relates to a host cell infected with a recombinant MVA virus as defined above. The host cell can be a cell that is not part of an entire living organism. In another embodiment, the invention further relates to the genome of a recombinant MVA virus according to the invention. Pharmaceutical compositions [0144] Since the MVA is highly growth-restricted and, thus, highly attenuated, it is useful for the treatment of a wide range of mammals including humans, and particularly immune-compromised animals or humans. Thus, in one embodiment, the invention also provides pharmaceutical compositions and vaccines for inducing an immune response in a living animal body, including a human. [0145] In one embodiment, the pharmaceutical composition may generally include one or more pharmaceutical acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Non-limiting examples of such 36 auxiliary substances are water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically selected from the group comprising large, slowly metabolized molecules such as, for example, proteins, polysaccharides, polylactic acids, polyglycolitic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like. [0146] In another embodiment, for the preparation of vaccines, the recombinant MVA virus according to the invention is converted into a physiologically acceptable form. This can be done based on the experience in the preparation of poxvirus vaccines used for vaccination against smallpox (as described by Stickl, H. et al. Dtsch. med. Wschr. 99, 2386-2392 [1974] ). For example, the purified virus is stored at -80'C with a titer of 5x10 8

TCID

50 /ml formulated in 10 mM Tris, 140 mM NaCI pH 7.4. [0147] In one embodiment, the MVA virus according to the invention is used for the preparation of vaccine shots. For example, about 102 to about 108 particles of the virus are lyophilized in 100 ml of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule. In another non-limiting example, the vaccine shots are produced by stepwise freeze-drying of the virus in a formulation. In certain embodiments, this formulation can contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other aids, such as antioxidants or inert gas, stabilizers or recombinant proteins (for example, human serum albumin) suitable for in vivo administration. The glass ampoule is then sealed and can be stored between 40C and room temperature for several months. However, as long as no immediate need exists, the ampoule is stored preferably at temperatures below -20'C. 37 [0148] In a further embodiment, for vaccination or therapy, the lyophilisate can be dissolved in 0.1 to 0.5 ml of an aqueous solution, preferably physiological saline or Tris buffer, and administered either systemically or locally, i.e. parenterally, subcutaneously, intramuscularly, by scarification or any other path of administration know to the skilled practitioner. The mode of administration, the dose and the number of administrations can be optimized by those skilled in the art in a known manner. However, most commonly, a patient is vaccinated with a second shot about one month to six weeks after the first vaccination shot. A third and subsequent shots can be given, preferably 4-12 weeks after the previous shot. Administration to Humans [0149] In certain embodiments, recombinant MVA, or a derivative thereof, according to the invention, is administered in a concentration range of about 104 to about 109 TCID 5 o/ml, preferably in a concentration range of about 10 5 to about 5x10 8

TCID

50 /ml, most preferably in a concentration range of about 106 to about 108 TCID 50 /ml. [0150] A typical vaccination dose for humans comprises from 10 7

TCID

50 to 10 9 TCID50, preferably about 108 to 10 9

TCID

50 , especially about 2 x 108 TCID 50 , administered subcutaneously. [0151] In one embodiment, an immune response is induced with a single administration of the recombinant poxvirus as defined above, in particular with an MVA strain, such as MVA-BN and its derivatives. Accordingly, one may use the MVA virus according to the invention, in particular an MVA strain, such as MVA-BN and its derivatives in homologous prime boost regimes. In these regimes, it is possible to use a recombinant poxvirus such as a recombinant MVA for a first vaccination, and to boost 38 the immune response generated in the first vaccination by further administration of the same virus, or of a related recombinant MVA virus, than the one used in the first vaccination. Preferrably, 2, 3, 4, 5, or 6 vaccinations are administered. [0152] In another embodiment, the recombinant poxvirus according to the invention, in particular an MVA strain, such as MVA-BN and its derivatives, may also be used in heterologous prime-boost regimes; these regimes are those in which one or more of the vaccinations is done with a MVA virus as defined above, and one or more of the additional vaccinations is done with another type of vaccine, for example, another virus vaccine, a protein or a nucleic acid vaccine. [0153] According to the invention, the mode of administration may be intramuscular, intravenously, intradermal, intranasal, or subcutaneously. Preferred embodiments are subcutaneous and intramuscular administration. However, any other mode of administration may be used such as, for example, scarification. [0154] In one embodiment, the recombinant MVA according to the invention is useful as a medicament or vaccine. [0155] According to a preferred embodiment, the recombinant MVA is used for the introduction of an exogenous coding sequence into a target cell, said sequence being either homologous or heterologous to the genome of the target cell. [0156] In one embodiment, the introduction of an exogenous coding sequence into a target cell is done in vitro to produce proteins, polypeptides, peptides, antigens or antigenic epitopes. [0157] In a preferred embodiment, the method of introduction of an exogenous coding sequence into a target cell in vitro to produce proteins, polypeptides, peptides, 39 antigens or antigenic epitopes comprises the infection of a host cell with the recombinant MVA according to the invention; cultivation of the infected host cell under suitable conditions; and isolation and/or enrichment of the polypeptide, peptide, protein, antigen, epitope and/or virus produced by said host cell. [01581 In a further embodiment, the method for introduction of exogenous sequences into cells is applied for in vitro and/or in vivo therapy. [01591 In one embodiment, for in vitro therapy, isolated cells that have been previously (ex vivo) infected with the recombinant MVA according to the invention are administered to the living animal body for affecting, preferably for inducing, an immune response. [0160] In another embodiment, for in vivo therapy, the recombinant MVA virus according to the invention is directly administered to the living animal body for affecting, preferably for inducing, an immune response. [0161] In a preferred embodiment, the cells surrounding the site of inoculation, and also cells where the virus is transported to via, for example, the blood stream, are directly infected in vivo by the recombinant MVA according to the invention. After infection, these cells synthesize the proteins, peptides or antigenic epitopes of the therapeutic genes, which are encoded by the exogenous coding sequences, and subsequently, present them or parts thereof on the cellular surface. Subsequently, specialized cells of the immune system recognize the presentation of such heterologous proteins, peptides or epitopes and launch a specific immune response. [0162] The MVA vector can be administered to a human patient that is not infected with HIV. In one embodiment, the patient is infected with HIV-1. In further 40 embodiments, the patient's CD4 count is below or above 200 CD4 cells/mi. In one embodiment, the patient's CD4 count is above 350 CD4 cells/mi. In another embodiment, the patient is on HAART. [0163] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the appended claims. EXAMPLE 1 Generation of a Recombinant MVA-BN Comprising in the Viral Genome a Truncated nef Gene, a gag-pol Fusion Gene, a Transdominant Tat Gene and a Vif Vpr-Vpu-Rev Fusion Gene, each Under the Control of the ATI Promoter [0164] An MVA vector, mBN87, was generated as described in U.S. Patent No. 7,501,127, which is hereby incorporated by reference. Briefly, the gag-pol fused gene was obtained by PCR from DNA from HXB2 infected cells. The nef gene was amplified by PCR from DNA of MVA-nef (LAI) to obtain a truncated version. The first 19 aa were deleted resulting in Nef-truncated. The vif and vpu genes were generated by RT-PCR from HIV RNA from a primary isolate MvP-899, while the vpr, rev and tat genes were synthesized by oligo annealing based on the sequence of HXB2. The protein Tat mutated was created by introducing two mutations in Tat, which are not localized in important epitopes but lead to the loss of transactivating activity. The mutations are the following substitutions: 22 (Cys > Gly) and 37 (Cys>Ser). The DNA constructs were cloned into recombinant vectors. [0165] After 5 rounds of plaque purification, the insertion of the foreign DNA (truncated nef gene, a gag-pol fusion gene, a transdominant tat gene, and a vif-vpr-vpu 41 rev fusion gene) and absence of wild-type virus was confirmed by PCR. The resulting recombinant virus clone was named mBN87A. After 5 plaque-purifications under non selective conditions the recombinant virus MVA-mBN87 B devoid of the selection cassette could be isolated. The identity of the recombinant vector was confirmed by standard methods. [01661 In MVA-mBN87B, the vif-vpr-vpu-rev gene doesn't have a stop codon at the end which results in the addition of 31 non-specific amino acids. Thus, a stop codon was added to the fusion gene and by cloning of the new recombinant virus MVA mBN120B. This construct was used in preclinical studies in mice and clinical stucies in humans. EXAMPLE 2 Preclinical studies in mice [0167] Whether MVA-mBN120B is able to mount a HIV-specific cellular immune response in adult non-transgenic mice (BALB/c) was investigated. The most promising epitopes were selected and for each protein two CD4 and two CD8 T cell peptides were synthesized. [0168] On Days 0 and 21, mice were administered subcutaneously (s.c.) with 500pl of either TBS (Group 1) as reference item or approximately 4x10 8

TCID

50

MVA

mBN120B (Group 2). On Day 35, blood samples were collected from all animals by retro-orbital puncture and processed to serum for potential future analysis. Following blood sampling, the animals were sacrificed by cervical dislocation and spleens necropsied for subsequent analysis of the cellular immune responses by restimulation of 42 splenocytes with the HIV specific peptides encoded in the vaccine inserts using an IFNy ELISpot assay. [0169] The HIV-protein specific cellular immune responses were determined by restimulation of splenocytes with specific peptides and subsequent detection of IFNy release from the splenocytes by ELISpot assay. The peptides are as follows, showing peptide denomination, T cell restriction, and peptide sequence: Nef-1 CD4 FHHVARELHPEYFKNC (SEQ ID NO:1) Nef-2 CD4 DPEREVLEWRFDSRLA (SEQ ID NO:2) Nef-3 CD8 HTQGYFDP (SEQ ID NO:3) Nef-4 CD8 RYPLTFGWC (SEQ ID NO:4) Gag-1 CD4 IYKRWIILGLNK (SEQ ID NO:5) Gag-2 CD4 GLNKIVRMYSPT (SEQ ID NO:6) Gag-3 CD8 AMQMLKETI (SEQ ID NO:7) Gag-4 CD8 EIYKRWIIL (SEQ ID NO:8) Pol-1 CD4 VQNANPDCK (SEQ ID NO:9) Pol-2 CD4 TIKIGGQLK (SEQ ID NO:10) Pol-3 CD8 IFQSSMTKI (SEQ ID NO:1 1) Pol-4 CD8 QPDKSESEL (SEQ ID NO:12) Tat-1 CD4 FITKALGISYGRK (SEQ ID NO:13) Tat-2 CD4 RQRRRAHQN (SEQ ID NO:14) Tat-3 CD8 QPKTAGTNC (SEQ ID NO:15) Tat-4 CD8 SFITKALGI (SEQ ID NO:16) Vif-1 CD4 KKAKGWMYK (SEQ ID NO:17) Vif-2 CD4 RCEYQAGHN (SEQ ID NO:18) Vif-3 CD8 QYLALAALI (SEQ ID NO:19) Vif-4 CD8 AGHNKVGSL (SEQ ID NO:20) Vpu-1 CD4 KPQKTKGHR (SEQ ID NO:21) Vpu-2 CD4 WAGVEAIIR (SEQ ID NO:22) Vpu-3 CD8 TYGDTWAGV (SEQ ID NO:23) Vpu-4 CD8 AGVEAIIRI (SEQ ID NO:24) Vpr-1 CD4 IVLIEYRKI (SEQ ID NO:25) Vpr-2 CD4 EEALAALVD (SEQ ID NO:26) Vpr-3 CD8 TQPIPIVAI (SEQ ID NO:27) Vpr-4 CD8 VLIEYRKIL (SEQ ID NO:28) Rev-1 CD4 RQARRNRRR (SEQ ID NO:29) Rev-2 CD4 SPQILVESP (SEQ ID NO:30) Rev-3 CD8 SGDSDEELI (SEQ ID NO:31) Rev-4 CD8 LPPLERLTL (SEQ ID NO:32). 43 [01701 Briefly, aliquots containing 500pg of each peptide were first dissolved in a small volume of dimethyl sulfoxide followed by further dilution with RPMI medium to obtain a stock solution of 1mg/ml (volumes between 5 and 25pi of acetic acid was additionally required to ensure complete reconstitution of peptides # 4, 14, 20, and 21; volumes between 5 and 25pl were additionally required to ensure complete reconstitution of peptides # 20, 22, 23 and 24). [01711 Spleen homogenisation was performed in Dispomix tubes using the "Saw 03" program. Following homogenisation, cell suspensions were transferred into 50ml tubes, centrifuged, and the erythrocytes were lysed for 5 minutes with red blood cell lysis buffer. Following two washing steps, a small aliquot of the cell suspension was mixed with trypan blue and the cell concentration was calculated by manual counting with a counting chamber (from Madaus). The cell density was adjusted for the individual splenocyte suspensions. Following plating the cells into the ELISpot plate (pre-coated with anti-IFNy antibody), the peptides were added at a final concentration of 2.5pg/ml. Duplicate incubations of 2.5x10 5 splenocytes per well were performed on the horizontally oriented ELISpot plates with splenocytes from different mice being plated horizontally and with different stimuli being plated vertically (i.e. plates 1 + 5, 2+ 6, 3 + 7, 4 + 8 covered stimulation with peptides # 1 - 8, 9 - 16, 17 - 24, 25 - 32, respectively). On plates 5 and 10, incubations with final concentrations of 0.5pg Concanavalin A (ConA) and 0.5pg/ml staphylococcus enterotoxin B (SEB) as positive controls, or with medium control as negative control was performed in row number B, E, or H, respectively. Following an overnight incubation of 19 h, the ELISpot plates were developed as recommended by the supplier. 44 TABLE 1 Group 1 Group 2 TBS MVA-mBN120B Stimulation Peptide Group SEM N Group SEM N Designation mean mean Peptide # 1 Nef-CD4-1 7.6 1.9 5 21.6 6.7 5 Peptide # 2 Nef-CD4-2 8.8 0.8 5 49.6 13.0 5 Peptide # 3 Nef-CD8-1 8.0 2.4 5 20.8 7.0 5 Peptide # 4 Nef-CD8-2 8.8 2.1 5 27.2 11.1 5 Peptide # 5 Gag-CD4-1 8.8 3.0 5 30.8 4.8 5 Peptide # 6 Gag-CD4-2 9.6 3.5 5 28.4 7.6 5 Peptide # 7 Gag-CD8-1 10.0 2.7 5 304.8 84.2 5 Peptide # 8 Gag-CD8-2 6.0 2.5 5 158.0 47.3 5 Peptide # 9 Pol-CD4-1 6.8 1.4 5 22.0 7.3 5 Peptide # 10 Pol-CD4-2 8.4 1.9 5 20.4 5.4 5 Peptide # 11 Pol-CD8-1 9.6 1.7 5 26.8 11.0 5 Peptide # 12 Pol-CD8-2 7.6 2.6 5 20.8 6.1 5 Peptide # 13 Tat-CD4-1 8.4 2.6 5 20.8 2.7 5 Peptide # 14 Tat-CD4-2 10.4 2.3 5 20.8 2.1 5 Peptide # 15 Tat-CD8-1 6.0 1.4 5 19.6 2.5 5 Peptide # 16 Tat-CD8-2 8.8 2.9 5 16.0 7.3 5 Peptide # 17 Vif-CD4-1 8.0 3.0 5 21.2 5.3 5 Peptide # 18 Vif-CD4-2 9.2 2.2 5 31.2 6.5 5 Peptide # 19 Vif-CD8-1 7.2 1.6 5 27.6 6.0 5 Peptide # 20 Vif-CD8-2 6.4 3.5 5 25.2 8.5 5 Peptide # 21 Vpu-CD4-1 4.8 1.7 5 22.4 6.0 5 Peptide # 22 Vpu-CD4-2 7.2 2.1 5 17.2 3.2 5 Peptide # 23 Vpu-CD8-1 11.2 2.4 5 21.6 3.3 5 Peptide # 24 Vpu-CD8-2 7.2 1.9 5 24.0 7.3 5 Peptide # 25 Vpr-CD4-1 6.8 2.0 5 20.0 6.9 5 Peptide # 26 Vpr-CD4-2 6.0 2.6 5 18.8 4.5 5 Peptide # 27 Vpr-CD8-1 8.8 2.1 5 24.0 4.3 5 Peptide # 28 Vpr-CD8-2 6.4 3.1 5 24.8 6.6 5 Peptide # 29 Rev-CD4-1 7.6 2.6 5 20.8 6.5 5 Peptide # 30 Rev-CD4-2 8.0 2.8 5 27.2 6.4 5 Peptide # 31 Rev-CD8-1 8.8 3.0 5 20.4 6.4 5 Peptide # 32 Rev-CD8-2 7.2 1.6 5 19.6 4.6 5 Con A n. a. 443.6 30.0 5 212.0* 59.8 5 SEB n.a. 247.6 38.7 5 154.8* 56.1 5 Medium n.a. 6.8 2.6 5 20.8 7.0 5 n.a. = not applicable 45 [0172] Three peptides were identified to be able to mount an HIV-specific cellular response. From these peptides, the highest IFN1 responses were determined following stimulation with the H2-Kd restricted CD8 T cell specific peptide "Gag-CD8-1". This is not surprising, since this peptide is frequently cited in the literature (e.g. Liu et al., Vaccine, 2006, 24, page 3332). The second Gag-specific CD8 T cell restricted peptide "Gag-CD8 2" was also able to induce a good specific IFN1 release in all mice. This peptide was so far only described by Shinoda et al. (Vaccine, 2004, 22, page 3676) to induce a specific CTL response in the context of a longer peptide. However, the longer peptide described in the literature not only contains the CD8 T cell epitope, but also additional predictable (and therefore potential) CD8 but also CD4 T cell epitopes. Thus, it is shown for the first time that "Gag-CD8-2" is able to induce a specific IFN1 release. Based on epitope prediction, "Gag-CD8-2" is an H2-Dd restricted CD8 T cell epitope. The third responsive peptide "Nef-CD4-2" was able to induce a specific IFN1 release in the majority of BALB/c mice. This peptide was already described by Mitchel et al. (AIDS Research and Human Retroviruses, 1992, 8, page 469) as a peptide to which a proliferative response and a cytolytic activity could be determined. From the epitope prediction, this peptide was identified as being primarily restricted to CD4 T cells (scores of 9.6 for the I-Ed molecule and 9.1 for the lAd molecule were identified in the PredBALB/C data base, whereas scores for the CD8 T cell restricted H2d molecules were below 7.9). Surprisingly, peptides other than the three responsive ones, e.g. "Nef-CD4-1" or "Tat-CD8-1", which had been selected based on published literature results were not found to be able to induce a specific IFN1 release. The reason for this discrepancy is not known. 46 [0173] In summary, the immunogenicity study in BALB/c mice with MVA mBN120B demonstrated not only that the HIV-Multiantigen MVA-construct is immunogenic, but revealed also that the immune response is directed against at least 2 proteins encoded in the recombinant MVA product (i.e. Nef and Gag specific responses were detected), that the CD8 T cell restricted immune responses are not limited to a single CD8 T cell molecule (since both H2-Kd and H2-Dd responses are induced), and that both CD8 and also CD4 T cell restricted responses were induced by the MVA construct. Furthermore, these results indicate that, at least, the Nef-gene and the Gag gene are expressed from the vector in vivo. EXAMPLE3 Clinical studies in humans [0174] In a Phase I study safety, reactogenicity and immunogenicity of a recombinant MVA-BN* vaccine expressing 8 out of 9 genes from HIV-1 clade B subgroup, (including a gag-pol fusion, vpr, vpu, vif, rev, tat, and nef) was evaluated in 15 HIV-1 infected subjects. This safety testing encompassed an analysis of solicited and unsolicited local and systemic adverse reactions. Furthermore, cellular and humoral immune responses to the vector were assessed. The collected specimens were also used to develop assays to specifically analyze the HIV-specific immune responses induced by the study vaccine MVA-mBN120B in order to establish the potential of such a homologous prime-boost vaccine approach to induce a broad cell-mediated response to different HIV antigens. [0175] In this Phase I trial, 15 HIV-1 infected patients stable on HAART with CD4 counts > 3 50/pl received three vaccinations with 2 x 108 MVA-BN*-MAG at Weeks 0, 4, 47 and 12. Solicited Adverse events (AE) were documented on diary cards, unsolicited AEs and cardiac signs and symptoms were captured throughout the study until the follow up visit at Week 20. Disease specific parameters such as plasma HI-viral load and CD4 counts of the patients were determined. Vaccinia specific humoral immune responses were measured by ELISA; cellular immune responses to the HIV-1 inserts as well as to vaccinia were assessed by an Interferon-y (IFN-y) ELISPOT assay using 15-mer peptides with an 11 amino acids overlap as a stimulant (for inducing HIV responses) and MVA-BN* at an multiplicity of infection (MOI) of 1 (for inducing vaccinia responses) respectively in a batched analysis. [0176] The study was a mono-centric, open-label, Phase I study conducted to assess safety and reactogenicity of the recombinant MVA HIV multiantigen vaccine in HIV-infected subjects with CD4 counts > 350 cells/pl. [0177] Subjects received immunizations at Day 0 and after 4 and 12 weeks with a dose of 2 x 108 tissue culture infectious dose 50 (TCID 50 ) MVA-mBN120B. The vaccine was administered subcutaneously. [0178] The study consisted of a screening period of up to three weeks and an active study period (a 12-week priming phase and an 8-week boosting phase) of up to 20 weeks. The total duration of the study per subject was up to 23 weeks. [0179] Eligible subjects entered the active study phase starting with Visit 1. At Visit 1, all subjects received the first MVA-BN120B vaccination, administered subcutaneously. All subjects received a second vaccination four weeks later at Visit 3 and a third vaccination 12 weeks later (after Visit 1) at Visit 5. Each immunization consisted of two administrations of MVA-mBN120B each with a dose of 1 x 108 TCID 50 per administration. 48 The vaccine was administered subcutaneously by injecting 0.5 ml of MVA-mBN120B in the deltoid region of each arm. Subjects received three immunizations: one at Week 0, one after four weeks and one after 12 weeks. Any adverse event (AE) that occurred during or after the vaccination was recorded. [0180] The following procedures and investigations were performed at the respective scheduled visit. The Enzyme Linked Immunospot (ELISPOT) assay used for the quantitative in vitro determination of Interferon-gamma (IFN-y) producing cells in cryopreserved Peripheral Blood Mononuclear Cells (PBMC) was performed after stimulation with live Vaccinia virus (VV): Modified Vaccinia Virus Ankara-Bavarian Nordic (MVA-BN*) or Vaccinia Virus Western Reserve (VV-WR). [0181] Briefly, 96 well filter plates (HTS plates, Millipore) were coated with a capture antibody (against IFN-y according to the manufacturer's instructions (BD Biosciences, IFN-y ELISPOT pair) at 4'C overnight. Subsequently resuscitated PBMC were added to the wells in a concentration of 200,000 cells/200pl final volume in combination with MVA-BN* at an MOI of 1. Following an incubation period (overnight at 37*C/5% CC 2 ) the wells were washed and a biotin-labelled detection antibody in PBS/9%FCS was added. After washing with PBS/0.05% Tween 20, streptavidin-coupled horse radish peroxidase (HRP, BD Biosciences) was added to the wells, followed by a washing step and the addition of a precipitating substrate (AEC, 3-Amino-9 Ethylcarbazole, BD Biosciences). The number of cytokine producing cells was determined by counting the spots using a CTL S5 Microanalyzer. Reported values are background corrected and normalized to 1x106 PBMC. 49 [0182] To assess the vaccinia specific cellular response, cells were stimulated with live MVA-BNO at an MOI of 1. [0183] To assess the HIV specific cellular response, cells were stimulated with peptide pools (15-mers with 11 aa overlap) at a final concentration of 5pg/ml per peptide. [0184] Fifteen peptide pools were used for stimulation and are depicted in Table 2. TABLE 2 protein Pool # Peptides Number of Peptides p1 7 (Gag) I 100% coverage 31 2 N terminal part, 100% coverage 28 p24 (Gag) 3 C terminal part, 100% coverage 27 Protease (Pol) 4 Immunogenic regions only 11 RT (Pol) 5 Immunogenic regions only 22 Integrate (Pol) 6 Immunogenic regions only 10 7 Predicted poorly immunogenic 14 Nef 8 Predicted poorly immunogenic I1 9 Predicted highly immunogenic 10 10 Predicted highly immunogenic 9 Tat II 100% coverage 10 Vif 12 Immunogenic regions only 16 p2p7 100% coverage 11+8+6= Vpr 13 Immunogenic regions only 25 Rev Immunogenic regions only POLYTOPE HTL 14 15 POLYTOPE CTL 15 A2, A3, B7 restricted 13 [0185] Peptides were synthesized at more than 90% purity as confirmed by high performance liquid chromatography (Metabion, Martinsried, Germany and Proimmune, UK). [0186] A vector or HIV-MAG-specific signal was defined by a frequency of at least 50 SFU per 1x106 cells after correction for background (subtraction of SFU/1x106 non 50 stimulated cells/ > two-fold above background). The number of SFU/1x1 06 cells after correction for background was reported. [0187] Vector or HIV-MAG-specific T cell responses were defined as either the occurrence of a signal in a subject who had no signal at Baseline, or a relative increase by a factor of at least 1.7 over the Baseline value in subjects who had a signal at Baseline. [0188] Subjects who had responses at one or more post-Baseline visits were classified as responders. [0189] A specific signal was defined (for each subject, visit and stimulation condition) by subtracting the numbers of spot-forming cells in background (non stimulated) wells from those appearing in corresponding experimental (stimulated) wells. Specific signals of less than 50 spot forming units (SFU) were returned to zero for the calculation of responses. [0190] A positive specific response was defined when either there was the appearance of a positive specific signal equal to or above the assay cut-off of 50 SFU per 1x106 PBMC in subjects who were previously below the assay cut off at baseline (V1); or a rise of a factor of at least 1.7 in the number of SFU from the baseline (V1) signal for subjects who had a baseline (V1) signal equal to or above the assay cut-off value. Otherwise the response was defined as negative, except in the case that either the respective post-baseline or the baseline values were missing; then the response status was defined as missing. 51 [0191] Subjects could have more than one response over the multiple post baseline visits but only one response was required to be considered a specific responder. [0192] HIV peptide stimulation was analyzed at three levels for all subjects and a separate analysis for responders only, by stimulating pool (1-15), by protein/polyprotein (gag, pol, nef, tat, vif, mixed), and by vaccine (i.e. including all HIV proteins). [0193] Descriptive statistics were derived by stimulation condition (including stimulation with HIV-MAG peptides and live MVA-BN*) for all sampling points and included the number of observations, arithmetic mean and standard deviation (SD), median and range of the number of SFU. This was performed for all subjects and a separate analysis was performed for responders only at all three levels of analysis (i.e. for responder on the pool level, for responder on the protein/polyprotein and responder on the vaccine level). [0194] The number and percentage of positive specific responders (responder rate) along with the 95% Clopper-Pearson confidence interval was tabulated for each pool, each protein/polyprotein and for the overall HIV-MAG vaccine as well as for MVA BN*. The percentage was calculated based on the number of subjects included in the specific analysis. A subject only needed to respond to one pool at the protein/polyprotein and vaccine level to be considered a responder. The same was true for vector-specific responder rates which were tested using only one stimulating condition (stimulation by MVA-BN*). [0195] The breadth of the HIV specific response was represented by a cumulative depiction of subject protein/polyprotein responses using the following categories; number 52 of subjects with a response to 1 or more, 2 or more, 3 or more, 4 or more 5 or more, and 6 proteins/polyproteins. [0196] Prior to vaccination as determined by ELISPOT, 87% of the subjects generated cellular immune responses to gag, 60% to nef, 53% to CTL epitopes, 40% to mixed proteins and HTL epitopes, 33% to pol and only 7% to tat and vif. [0197] Responder rates for each peptide pool, protein/polyprotein and HIV vaccine (HIV-MAG) are summarized in Table 3 and also reveal the time points at which responses were detected. The use of single responses to define a responder was defined in the SAP and is a higher sensitivity method for examining responses; however, this method may be prone to higher false positive rates. For this reason, a supplementary analysis has been performed using a higher stringency definition of responder; two responses are required to be defined as a responder. [0198] Response rates, which imply new responses or increased responses over Baseline values, were high to the HIV proteins coded within the MVA-BN* vaccine-vector with 87% (13/15) of the subjects responding. Even using the higher stringency definition of responder, 80% of the subjects were responders to the HIV components. The highest proportion of subjects responded to gag (73%, 11/15). Within gag, p24 resulted in higher responder rates (40% and 47% for the two gag-p24 pools respectively) than did p17 (20%). Even using a higher stringency definition of responder, 60% of the subjects responded to gag. Responder rates to pol and the mixed protein pool were similar (53%, 8/15) and within pol, the protease and RT had the highest and equal responder rates (27%) and was followed by integrase with a 20% responder rate. Since the mixed pool contained peptides from multiple proteins, the most immunogenicpeptides could not be 53 detrminmed. Using the higher stringency definition of responder rate still resulted in high responder rates to both pol (33%) and mixed (40%). Nef responder rate was 40% (6/15) with responses to pool 4 being the highest (27%). Nef response was 27% using the higher stringency definition of responder rate. A total of 7 subjects (46.7%) and 1 subject (6.7%) responded to HTL and CTL polytope peptides and no responders were detected for both Tat and Vif. [0199] Responder rates for each peptide pool, protein/polyprotein and HIV vaccine (HIV-MAG) are summarized in Table 3, which also reveals the time points at which responses were detected. In addition the table shows responder rates using a higher stringency definition of responder where two responses are required within one pool to be defined as a responder. [0200] Responder rates were high to the HIV proteins coded within the MVA-BN* vaccine-vector with 87% (13/15) of the subjects responding to the HIV insert. Even using the higher stringency definition of responder, 80% of the subjects were responders to the HIV components. The highest proportion of subjects responded to gag (73%, 11/15). Within gag, p24 resulted in higher responder rates (40% and 47% for the two gag-p24 pools respectively) than did p17 (20%). Even using the higher stringency definition of responder, 60% of the subjects responded to gag. Responder rates to pol and the mixed protein pool were similar (53%, 8/15) and within pol, the protease and RT had equal responder rates (27%) and were followed by integrase with a 20% responder rate. Since the mixed pool contained peptides from multiple proteins it is not possible to distinguish the most immunogenic peptides. Using the higher stringency definition of responder rate still resulted in high responder rates to both pol (33%) and mixed (40%). Nef responder 54 rate was 40% (6/15) with responses to pool 4 being the highest (27%). Nef response was 27% using the higher stringency definition of responder rate. A total of 7 subjects (46.7%) and 1 subject (6.7%) responded to HTL and CTL polytope peptides and no responders were detected for both tat and vif. TABLE 3 Pool Pool Protein Protein Responders Responders Responders Responders (1 or more (2 or more (I or more (2 or more Protein/ Responses responses) responses) responses) responses) Pool Polyprotein Subject (Weeks) n (%) n (%) n (%) n (%) 1 Gag-p17 006 5, 13 008 12 013 1, 12 3 (20.0) 2 (13.3) 2 Gag-p24- 001 12

NH

3 002 20 008 12, 13,20 010 1,5,12,13,20 011 1,5, 12, 13,20 015 1, 5 6 (40.0) 4 (26.7) 3 Gag-p24- 004 1,5, 12, 13 COOH 006 13 008 12,20 009 5,12 011 5,13,20 014 1,5, 12,13,20 015 1 7 (46.7) 5 (33.3) 11 (73.3) 9 (60.0) 4 Pol- 004 5 Protease 006 1,5, 13 014 12, 13 015 1, 5 4(26.7) 3 (20.0) 5 Pol-RT 001 1, 12, 13 006 1, 5, 12, 13,20 008 12 014 13 4(26.7) 2(13.3) 6 Pol- 005 12 Integrase 006 5, 12, 13, 20 010 1, 13,20 3 (20.0) 2(13.3) 8 (53.3) 5 (33.3) 7 Nef-1 006 12 1 (6.7) 0 (0.0) 8 Nef-2 005 1, 13 015 1 2(13.3) 1 (6.7) 9 Nef-3 015 1,5 1 (6.7) 1(6.7) 10 Nef-4 001 12, 13 004 1 013 1, 5, 12, 13 015 1, 5 4 (26.7) 3 (20.0) 6 (40.0) 4(26.7) 55 I1 Tat None None 0 (0.0) 0(0.0) 0 (0.0) 0 (0.0) 12 Vif None None 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 13 Mixed 001 5, 12,13,20 004 1 006 1,12,13,20 008 12, 13,20 009 1, 5, 12, 13, 20 010 1 012 1,5 013 1, 12 8 (53.3) 6 (40.0) 8 (53.3) 6 (40.0) 14 HTL 004 1 006 1, 5, 12, 13,20 008 1, 12,20 009 20 012 1,5, 13 014 5, 13,20 015 1 7 (46.7) 4 (26.7) 7 (46.7) 4 (26.7) 15 CTL 006 1, 12, 20 1 (6.7) 1 (6.7) 1 (6.7) 1 (6.7) Responders (1 or more responses) Responders (2 or more responses) n (%) n (%) HIV-MAG' 13 (86.7) 12 (80.0) Table 3: ELISPOT Responder Rates by Visit and Stimulating Condition (Pool, Protein & HIV-MAG; N = 15) a Response to Protein. A subject was a protein-specific responder if he had at least one positive response for at least one pool for the protein at a post-baseline visit. b Response to HIV-MAG. A subject was a HIV-MAG-specific responder if he had at least one positive response for at least one HIV protein (gag, pol, nef, tat, vif or mixed [p 2 p 7 , rev, vpr]) at a post baseline visit. N =number of subjects in the specified group, n = number of subjects who were responders, % = percentage based on N. Immunizations were given at Week 0, Week 4 and Week 12. [0201] The breadth of HIV-specific T cell response refers to the numbers of proteins/polyproteins for which subjects generated new or increased T cell responses. Table 4 shows the breath of response to HIV proteins. Vaccination resulted in the generation of responses to several proteins in most subjects. Responses to up to four different proteins/polyproteins including gag, pol, tat, vif, nef and mixed (p2p7, vpr, rev) are shown in Table 4. 66.7% of all subjects responded to at least two and 46.7% to at least three proteins/polyproteins. 56 TABLE 4 N= 15 Number of proteinslpolyproteins R+ (%) 95% Cl At least I protein/polyprotein 13 (86.7) 59.5, 98.3 At least 2 proteins/polyproteins 10 (66.7) 38.4, 88.2 At least 3 proteins/polyproteins 7 (46.7) 21.3, 73.4 At least 4 proteins/polyproteins 3 (20.0) 4.3, 48.1 N = number of subjects in the specified group, R+ = number of subjects who were responders to any protein mentioned above), % = percentage based on N, 95% Cl = Clopper-Pearson confidence interval, lower limit and upper limit. [0202] All 15 subjects received 3 vaccinations and were followed up until end of the study. No serious AEs were reported and no study subject was withdrawn due to a related AE. All subjects reported general (mostly nausea) and local reactions (mostly induration) with one grade 3 event (injection site pain). Thus, administration of MVA BN*-MAG was well tolerated in HIV-1 infected subjects. [0203] All subjects responded to vaccinia. Median SFU values for vaccinia-specific responders reached a peak of 350 SFU/1 x1 06 PBMC at Week 12, eight weeks following the second immunization, and was not further increased following the third vaccination. As observed for HIV responses the number of vaccinia responsive IFN-y secreting PBMCs remained higher than baseline 20 weeks after the first immunization. Anti vaccinia antibody seroconversion rate reached 100.0% at Week 5 (one week after the second vaccination) and remained at 100% for the duration of the study. ELISA GMT's revealed a slight increase 1 week after the first vaccination and strong booster responses. Vaccinia-specific antibody titers reached a peak of 876 one week after the third immunization and remained much higher than baseline 20 weeks after the first immunization. 57 [0204] The MVA-BN@-MAG HIV vaccine candidate was well tolerated in HIV-1 infected subjects. HIV-specific T cell responder rate was 86.7% and the vaccinia-specific responder rate was 100%. A broad cellular immune response against the four HIV protein/polyprotein pools (gag, pol, nef and mixed [p2p7-vpr-rev]) was observed; 66.7% of all subjects responded to at least two and 46.7% to at least three HIV-1 proteins/polyproteins. Median T cell responses remained higher than baseline 20 weeks after the first immunization for all HIV proteins which induced responses. This was also true for the vaccinia-specific T cell response. Thus, the MVA-BN@-MAG vaccine was able to induce a broad immune response to multiple HIV-1 proteins and to vaccinia and the responses were still higher than baseline 20 weeks after receiving the first immunization. 58

Claims (19)

1. A method for inducing a T-cell response to at least three HIV-1 proteins in a human patient comprising administering an MVA vector encoding at least three HIV-1 proteins; wherein the proteins are selected from HIV-1 Gag, Pol, Vpr, Vpu, Rev, and Nef; and wherein the MVA vector induces a T-cell response in the patient to at least three of the HIV-1 proteins.

2. The method of claim 1, wherein the HIV-1 proteins are Gag, Pol, and Nef.

3. The method of claim 1, wherein one of the proteins is an HIV-1 Gag protein.

4. The method of claim 1, wherein one of the proteins is an HIV-1 Pol protein.

5. The method of claim 1, wherein one of the proteins is an HIV-1 Nef protein.

6. The method of claim 5, wherein the protein is a truncated HIV-1 Nef protein.

7. The method of claim 1, wherein one of the proteins is an HIV-1 Vpr protein.

8. The method of claim 1, wherein one of the proteins is an HIV-1 Vpu protein.

9. The method of claim 1, wherein one of the proteins is an HIV-1 Rev protein.

10. The method of claim 1, wherein the vector comprises a coding sequence for HIV 1 Gag-Pol protein.

11. The method of claim 10, wherein the vector comprises a coding sequence for a truncated HIV-1 Nef protein.

12. The method of claim 11, wherein the vector comprises a coding sequence for HIV-1 Vif, Vpr, Vpu, and Rev proteins. 59

13. The method of claim 12, wherein the vector further comprises a coding sequence for HIV-1 Tat protein.

14. The method of claim 1, wherein a dosage of 107 to 10 9 TCID 50 of the vector is administered to the patient.

15. The method of claim 14, wherein a dosage of 108 to 109 TCID 50 of the vector is administered to the patient.

16. The method of claim 15, wherein a dosage of 2 x 108 TCID 5 o of the vector is administered to the patient.

17. The method of claim 1, wherein the MVA vector induces a T-cell response in the patient to 4 HIV-1 proteins.

18. The method of claim 1, wherein the MVA is MVA-BN.

19. The method of claim 1, wherein the patient is infected with HIV-1. 60