LIVE VIRUS VACCINES TO PROTECT PRIMATES FROM HIV-1 INFECTION AND DISEASE

Abstract: The instant invention teaches pathogenic SHIV which cause AIDS in monkeys, the construction of virus for use in vaccines against pathogenic SHIV and HIV-1, and methods for prophylactic and therapeutic vaccination to prevent or inhibit HIV-1 infection and/or disease. The instant invention teaches the construction of modified live virus for use in vaccines against HIV-1, and methods for prophylactic and therapeutic vaccination to prevent or inhibit HIV-1 infection and/or disease. The instant invention teaches the use of viral DNA vaccines for the treatment and prevention of HIV infection and/or disease.

Description: Live Virus Vaccines to Protect Primates from HIV-1 Infection and Disease Related Applications This application is a Continuation-in-part of U.S. Provisional Patent Application Serial number 60/102,143, filed September 28, 199808/850,492; and U.S. Patent Application Serial No. 09/071,066 filed April 30, 1998, which is a continuation in part of 08/958,410 filed October 29, 1997, which is a continuation in part of 08/850,492 filed May 2, 1997, which is a continuation in part of 08/442,010 filed May 16, 1995, now U.S. Patent No. 5,849,994, all hereby incorporated by reference in its entirety. Statement of Government Rights This work was supported in part by NIH grant numbers AI38492 and RR06753, the Government of the United States of America may have rights in this invention. Field of the Invention This invention relates to the field of prophylactic and therapeutic vaccines for generating protection from HIV-1 induced disease and infection, and compositions and methods for such vaccination, and compositions and methods for the therapeutic treatment of HIV- 1 infected individuals. Background of the Invention It is recognized that effective vaccines which will inhibit or prevent HIV-1 infection or induced disease in humans will be useful for the treatment of certain high-risk populations, and as a general prophylactic vaccination for the general population that may risk HIV-1 infection or HIV-1 induced disease. A vaccine that will confer long-term protection against the transmission of HIV-1 would be most useful. Unfortunately, numerous problems stand in the way of developing effective vaccines for the prevention of HIV-1 infection and disease. Certain problems are most likely the result of the unique nature of the HIV-1 virus and its functional properties, and as yet no effective vaccine has been developed (for review see: Berzofsky et al., Developing Synthetic Peptide Vaccines for HIV-1, Vaccines 95, pps. 135- 142, 1995; Cease and Berzofsky, Toward a Vaccine for AIDS: The Emergence of Immunobiology-Based Vaccine Design, Annual Review of Immunology 12:923-989, 1994; Berzofsky, Progress towards Artificial Vaccines for HIV, Vaccines 92, pps. 41-50, 1992). There is a Need for an Effective Vaccine The theoretical basis for the development of an effective HIV-1 vaccine is the fact that in nearly all viral infections, certain segments of an infected population recover and become immune to future viral infection by the same pathogen. Examples of typical viral pathogens include measles, poliomyelitis, chicken pox, hepatitis B, small pox, etc. Thus, it is assumed that protective immunity against HIV-1 virus can be conferred on primate populations via a properly designed vaccine. The high mortality rate of HIV-1 infection, and the apparently extremely rare incidence of recovery and protective immunity against HIV-1 infection has cast doubt on the ability of primates to generate natural immunity to HIV- 1 infection when the pathogenic HIV-1 is the un-modified wild-type viral pathogen. Thus, significant resistance remains as to the possibility of creating an effective HIV-1 vaccine via human- modification of pathogenic virus. A hallmark measure of resistance to future viral infection, is the generation of "neutralizing antibodies" to the virus pathogen, and cellular immunity against infected cells. In typical viral infections, the generation of neutralizing antibodies and cellular immunity by the infected individual's immune system heralds recovery from infection. However, in HIV-1 infection, neutralizing antibodies and cellular immunity appear very early during the infection, and have been associated with only a transient decrease in viral burden. In HIV-1 infection, in spite of the generation of neutralizing antibodies and cellular immunity, viral replication rebounds and AIDS (acquired immune deficiency syndrome) develops. Thus in HIV-1 infected people, neutralizing antibodies and cellular immunity are not accurate measures of protective immunity. However, induction and maintenance of these arms of the immune response by vaccine may prevent infection by the virus under natural conditions. A further problem in developing an effective vaccine for HIV-1 that has concerned researchers is the antigenic diversity of the wild-type virus. It has been believed to be a strong possibility that vaccines generated via recombinant HIV-1 coat proteins will confer resistance only to specific phenotypes of virus, and not provide broad spectrum immunity. Currently, vaccine development using recombinant HIV-1 gpl20 peptide, a viral coat protein of HIV-1, has passed phase-one clinical trials showing no toxicity. However, data indicated that neutralizing antibodies appeared only transiently. Thus, recombinant HIV-1 gpl20 peptide vaccines may only act in the short-term, with subsequent reversion to susceptibility of infection. The Fear of Live Virus Vaccines In general, it is accepted that live-virus vaccines induce better immunity against pathogenic viruses than isolated viral proteins (see for example Putkonen et al., Immuization with Live Attenuated SIVmac Can Protect Macaques against Mucosal Infection with SIVsm, Vaccines 96, pps. 20-210, 1996; Dimmock and Primrose Introduction to Modern Virology, 4 th Ed., Blackwell Science, 1994). However, the use of live lentivirus vaccines, such as HIV-1 vaccine, is resisted because of great concern that the vaccine virus will persist indefinitely in the inoculated population because of the integration of viral DNA into the host DNA of the inoculated individuals (see for example ten Haaft et al., Evidence of Circulating Pathogenic SIV following Challenge of Macaques Vaccinated with Live Attenuated SIV, Vaccines 96, pps. 219-224, 1996). Thus, a safe, and effective vaccine against HIV-1 will encompass modifications to prevent the development of virulent pathogenic infection that could occur by either random mutation or other change to the initially non-pathogenic vaccine virus. Model Systems for Testing Vaccines Since testing of vaccine efficacy requires vaccine inoculation then challenge of the subject with infectious virus, it is ethically and practically difficult to attempt preliminary studies using human subjects. The use of model systems for preliminary design and testing of candidate vaccines has been hampered by various species specific features of the viruses. Apart from humans, HIV-1 itself is currently known to replicate only in certain rare and endangered chimpanzees. The feasibility of obtaining sufficient numbers of such endangered animals for a full preliminary study of HIV-1 virus vaccines is quite low. It is much more preferable to prepare initial studies using validated analogous animal model systems. One analogous model system for HIV-1 infection has been the SIV mac system (Simian Immunodeficiency Virus, macaque) and infection of macaque monkeys. Deletion of the regulatory gene nef rendered SIV mac a virulent. However, SIV nιac is only an analogous model system for human HIV-1 infection, and does not replicate all of the salient features of HIV-1 infection/disease of humans (see generally Hu et al., Transmembrane Protein and Core Antigens in Protection against SIV Infection, Vaccines 95, pps. 167-173, 1995; Lewis and Johnson, Developing animal models for AIDS research - progress and problems, TIBTECH 13:142-150, 1995; Desrosiers, The Simian Immunodeficiency Viruses, Annual Review of Immunology 8:557-578, 1990). Researchers have also developed chimeric SIV-HIV chimeric virus, placing the envelope proteins of HIV-1 on a background of SIV mac , which proved infectious to monkeys, but did not result in full-blown AIDS or an accurate model to mimic HIV-1 infection in monkeys (see generally Shibata and Adachi, SIV/HIV recombinants and their use in studying biological properties, AIDS Research and Human Retroviruses 8(3):403-409, 1992; Sakuragi et al., Infection of macaque monkeys with a chimeric human and simian immunodeficiency virus, J. General Virology 73:2983-2987, 1992). The recent development, by the present inventor, of highly pathogenic strains of SHIV, SHIV KLM , and SHIV KU _ 2 , provided a new model for the study of HIV infection and disease which corresponds to the human disease much more accurately than any other previous model. U.S. Patent 5,849,994 (incorporated by reference in its entirety) describes this development and validation of a monkey model for human HIV infection and AIDS. This development of a new truly pathogenic virus which induces full-blown AIDS in monkeys, and the identification of the genetic basis for certain aspects of pathogenicity, allows for the full scale efficient preparation and evaluation of HIV-1 vaccines, supported by in vivo data. As described below, the present invention teaches specific methods and virus constructs which are effective in generating an immune response in a vaccinated host to virus. Thus the present invention teaches constructs and methods for the effective in vivo therapeutic and/or prophylactic vaccination of mammals to inhibit or prevent HIV infection and/or related disease. Brief Description of the Drawings Figure 1 is a diagram of the genetic organization of HIV-1 /HIV-2 virus for vaccine. Figure 2 is a diagram of the genetic organization of SHIV. Figure 3 is a diagram of the genetic organization of SHIV-4 and Δ2 SHIV. Figure 4 is a diagram of the genetic organization of PPc SHIV and Δvj_>u PPc SHIV Figure 5 is a diagram of the generation of pathogenic SHIV: SHIV KU . ] and SHIV KU.2 . Figure 6 depicts the scheme used to construct recombinant pDSDvpu. The details of the plasmid manipulations are described in the text. Figure 7 is a diagram of the genomic structure (Figure 7 A) of 3= SHIV-4, (Figure 7B) 3=Avpu SHIV-4, and (Figure 7C) 3 =AvpuAnef SHIV-4. Open bar - HIV-1 sequences; Solid bar - SIVmac239 sequences. Figure 8 is a graph of neutralizing antibody titers against SHIV KU _, in vaccinated macaques. Macaques were challenged with SHIV KL at weeks 0 and 5. The * indicates terminal plasma obtained from macaque 42107, which died at week 24. Neither control animal developed neutralizing antibodies to SHIV^.,. Brief Summary of the Invention The present invention teaches an avirulent HIV-1 virus, wherein the vpu gene of the HIV-1 virus has been rendered non-functional or deleted. In addition to the non-functional vpu gene, the HIV-1 virus may have a non-functional or deleted nef gene. The instant invention also encompasses a HIV-1 /HIV-2 chimeric virus wherein the chimeric DNA comprises HIV-2 LTR, gag, pol, and nef ' genes and HIV-1 env, tat, rev, vpu genes, where said vpu gene is rendered non-functional or is deleted. In a further embodiment the nef gene is optionally rendered non-functional or deleted. The instant invention also encompasses an avirulent SIV/HIV-1 chimeric virus, having a SIV LTR, gag, pol, and nef genes and HIV-1 env, tat, rev, vpu genes, where said vpu gene is rendered non-functional or deleted. In a further embodiment the nef gene is optionally rendered non-functional or deleted. The instant invention embodies vaccines which inhibit the infection of cells by HIV-1, comprising a virus wherein the DNA of said virus comprises DNA consisting of HIV-2 LTR, gag, pol, and nef genes and HIV-1 env, tat, rev, vpu genes, where said vpu gene is rendered non- functional. In one embodiment the vpu gene is rendered non-functional by deletion. In a further embodiment the nef gene is optionally rendered non-functional or deleted. In a further embodiment, the instant invention teaches vaccines which inhibits the infection of cells by HIV-1, comprising a virus wherein the DNA of said virus comprises DNA consisting of HIV-2 LTR, gag, pol, and nef genes and HIV-1 env, tat, rev genes, and no HIV-1 vpu gene. A further embodiment of the invention encompasses vaccines which inhibit the infection of cells by HIV- 1 , comprising a virus wherein the DNA of said virus comprises HIV-1 DNA wherein the vpu gene of HIV-1 has been rendered non-functional. In one form, the vpu gene is rendered non-functional by deletion. Thus the instant invention teaches vaccines which inhibit the infection of cells by HIV-1, comprising a virus wherein the DNA of said virus comprises HIV-1 DNA, and no vpu gene of HIV-1. Another embodiment of the invention encompasses a vaccine which inhibits infection of cells by such a SIV/HIV-1 chimeric virus such as SHIN KU .„ which comprises said virus wherein the vpu gene is rendered non-functional or deleted, in a suitable carrier. In another embodiment of the invention, such a vaccine contains said virus wherein the nef gene is optionally rendered non-functonal or deleted. In accordance to the teaching of the instant invention, the disclosure teaches methods for inoculating a subject to inhibit virus infection by chimeric SHIV/HIV-1 virus such as SHIV KU _,, comprising administering a vaccine containing a modified virus, the DNA of said virus comprising DNA consisting of SIV LTR, gag, pol, and nef genes and HIV-1 env, tat, rev, vpu genes, where said vpu gene is non-functional. In another embodiment of the invention, such a vaccine contains said virus wherein the nef gene is optionally rendered non- functonal or deleted. Further, the instant invention teaches methods for inoculating a subject to inhibit virus infection by HIV-1 comprising administering a vaccine containing a modified virus, the DNA of said virus comprising DNA consisting of HIV-2 LTR, gag, pol, and nef genes and HIV-1 env, tat, rev, vpu genes, where said vpu gene is non-functional. In a preferred embodiment, the vpu gene is deleted. In another embodiment of the invention, such a vaccine contains said virus wherein the nef gene is optionally rendered non-functonal or deleted. Also encompassed by the instant invention are methods for inoculating a subject to inhibit virus infection by HIV-1 comprising administering a vaccine containing a modified virus, the DNA of said virus comprising DNA consisting of HIV-1 DNA, where the HIV-1 vpu gene is non-functional. In a preferred embodiment the vpu gene is deleted. In another embodiment of the invention, such a vaccine contains said virus wherein the nef gene is optionally rendered non- functional or deleted. The present invention also embodies the administration of viral DNA of the present invention directly to the subject to be treated. Such administration being made with DNA suspended in saline or other suitable carrier or packaging. In a broad aspect, the instant invention provides for a general method for the creation of effective vaccines for conveying immunity to HIV-1 virus, wherein the virus is manipulated to impede its ability to effectively replicate and/or accumulate in the infected/inoculated host. Wherein one embodiment of this method is the perturbation of the activity of the vpu gene or gene product in the virus, or during host-virus interaction. In a further embodiment of the invention, the instant invention embodies the treatment of currently infected HIV-1 positive patients to prevent further HIV-1 infection and/or continuing HIV- 1 infection. Thus the present invention teaches constructs and methods for the effective in vivo therapeutic and/or prophylactic vaccination of mammals to inhibit or prevent HIV infection and/or related disease. Description of the Invention Development of a truly pathogenic virus that generates full-blown AIDS in monkeys is described in U.S. Patent No. 5,849,994. This pathogenic virus, named SHIV KU _, (originally isolated as PPc and PNb) is the first virus bearing the envelope of HIV-1 which can cause AIDS in a non-human primate, as indicated by a) all animals lost CD4 " T cells during the first three weeks after inoculation with the virus (an excellent marker for virus pathogenicity); b) the virus is predictably pathogenic, with 70% of inoculated animals developing AIDS within six months (and thus vaccine efficacy can be evaluated in a short time using this monkey model system); and c) the virus invades across mucosal surfaces and causes AIDS after deposition in the mouth or in the vagina (thus allowing for evaluation of testing for efficacy against sexual transmission). This is truly a pathogenic, virulent virus which infects cells and causes HIV-1 like infection and disease in a monkey. The DNA of the virus was constructed of SIV LTR, gag, pol, and nef genes and HIV-1 env, tat, rev, vpu genes. Development of pathogenic SHIV KU _, was accomplished by sequential passage of virus through bone marrow using an SHIV construct containing the tat, rev, vpu, and env genes derived from a laboratory strain of HIV-1 (see also Joag et al., Chimeric Simian/Human Immunodeficiency Virus That Causes Progressive Loss of CD4+ T Cells and AIDS in Pig-Tailed Macaques, J. Virology 70(5):3189-3197, 1996). The virulent variant of SHIV was obtained after four passages as described in Narayan in U.S. Patent No. 5,849,994, and is summarized in Figure 5. Briefly, SHIV passaged through Rhesus macaque bone marrow was then further passaged through pig-tailed macaque bone marrow, resulting in a pathogenic SHIV, isolated from animal PNb and now named SHIV KU .,. Virus passaged an additional time through a Rhesus macaque resulted in a pathogenic SHIV, isolated from animal 14A, now named SHIV KU _ 2 . Virus isolated from animal PPc, a pig-tailed macaque was not pathogenic. Examination of the genes of the virulent variant yielded information as to the genetic basis of the acquired virulence and pathogenicity. It was found that three genes had undergone several mutations during the passages and acquisition of virulence. Manipulation of genes can be routinely perfoπned using standard molecular biology and virology techniques (see for example Short Protocols in Molecular Biology. 2 nd Edition, A Compendium of Methods from Current Protocols in Molecular Biology. (Eds. Ausubel et al., John Wiley & Sons, New York, 1992); Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual 2 nd . Ed., CSH Laboratory Press, New York). It has been found that the vpu gene, present in the original SHIV as a non-functional gene, became functional in the SHIV KL . The vpu gene is an accessory gene found only in HIV-1. (for review see Vaishnav and Wong-Staal, The Biochemistry of AIDS, Annual Review of Biochemistry 60:577-630, 1991). It has been found that the env gene of HIV-1 in the pathogenic SHIV contained ten consensus mutations, and the nef gene of SIV in the SHIV had five mutations. The results indicate that the vaccine virus of the invention are somehow impeded in the ability to replicate or accumulate efficiently, and as such the immune system of the inoculated host is able to respond to this viral challenge as if to any other virus, by mounting an appropriate, effective and thus immunity-conferring response. Thus the treatment of currently infected HIV-1 positive patients with agents that will interfere with the HIV-1 vpu gene or gene product can be effective therapeutics for modulating the effects of HIV-1 infection and/or continuing HIV-1 infection. Such agents can be either chemical, antibody based, or some other form of bioactive molecule. The vpu gene of HIV-1 and the nef gene of SIV mac are thought to encode for nonstructural regulatory proteins. Deletion of both these genes from the SHIV KU _, molecular construct resulted in a first vaccine virus called Δ2 SHIV (Δ2 SHIV -4). A second vaccine was made using a virus which incorporated the env and nef genes taken from the SHIV KU _, virus, and deleted only the vpu gene, this virus construct being named Avpu SHIV KU _, (Avpu SHIV-PPc) It has been found that Avpu SHIV KU _, (Avpu SHIV-PPc) replicates more vigorously than Δ2 SHIV in macaque tissue cell cultures and in macaques. In the past, the vpu gene has not been considered as an important gene for pathogenesis because of lack of experimental evidence. Examination of the genetic mutations which occurred during the course of successive passages of non-pathogenic SHIV through monkey bone marrow, and the onset of pathogenicity reveals important information as to the genetic changes which lead to pathogenic SHIV such as SHIV KU _,. This examination has shown that while a functional vpu is most likely not the sole determinant of viral pathogenicity/virulence, it is however required that the vpu gene must become functional before other mutations could occur in either the e«v or nef genes and the subsequent development of pathogenic SHIV. Recently it has been shown that the vpu gene product may play an important role in down-regulation of the expression of MHC class I molecules, and thereby contribute to the inability of CD8 + T cells to eradicate HIV-1 from infected individuals (Kerkau et al., J. Exp. Medicine 185(7): 1295-1305, 1997). Certain aspects of the present invention will be better understood by the illustrated embodiments of the following examples. The following examples are meant by way of illustration and not by limitation. One of ordinary skill in the art will be able to understand and use the teaching of the present invention to make modifications and further embodiments which are still within the teaching, spirit and scope of the invention. Example 1 Resistance to replication of SIV mac or avirulent SHIV, results in resistance to infection by SHIV KU _, Twelve macaques who have developed immunological control over replication of SIV-... or avirulent SHIV, unexpectedly demonstrated resistance to SHIV KU _, infection. These animals had developed sterilizing immunity which prevented the establishment of infection by SHIV KL . Thus these SIV-infected, and now resistant animals, resisted challenge by SHIV KLM virus which has human HIV-1 envelope. Since the envelope of SIV mac (which is closely related to the env of HIV-2) is distinct from the env of HIV-1, the resistance of these animals to infection by SHIV KU _, suggests that questions about effectiveness of vaccines being compromised by the inherent variability in wild-type HIV viruses is in operation a non- issue. TABLE 1: Virus and Host Response after inoculation with SHIV-4 Week post CD4' count infectious cells Neutralizing inoculation per μl per million cells Ab titer Example 2 SHIV KU _, infects across mucosal membranes Sexually mature macaques inoculated intravaginally with SHIV KU _, rapidly developed systemic and CNS infection and disease manifested by intense proliferation of the virus, precipitous and permanent loss of CD4 + T cells, and development of AIDS. Analysis of the viral infection indicates that the rapid kinetics of the virus invasion across mucosal surfaces shows that local post-exposure prophylactic measures, such as anti-viral douches, must be implemented before two days post infection, since by 2 days post infection virus has reached the regional lymph nodes. In a similar vein, it is found that systemic dissemination of virus has occurred by day 4 post infection, and CNS infection has occurred by day 7, thus antiviral therapeutic intervention after day 4 may not prevent the establishment of systemic infection. TABLE 2: Course of Infection - Blood assay of Macaques infected with SHIV^ .] Test post infection Intravenous route Intravaginal route Assay Week animal 15A animal 15B animal 15C animal 15D infectious 1 10000 10000 5 3 cells per 10 6 2 100 100 10000 10000 3 100 1000 100 Plasma 1 1 1 0 0 infectivity 2 1 2 2 2 (log TCID/ml) 3 0 0 1 2 Plasma p27 1 925 7064 0 0 (pg/ml) 2 0 1 12 2067 3138 3 0 0 0 0 CD4 + cells 0 121 1 994 929 762 per μl 1 588 470 688 907 Example 3 Δ2 SHIV Vaccine The Δ2 SHIV virus was constructed (with the nef and vpu genes deleted from SHIV- 4) and characterized in macaque cell cultures as to the genetic identity, confirmed by sequence analysis of genomic DNA. The nef anά vpu genes could be completely deleted or only partially deleted, or rendered non-functional by insertion of DNA into the gene, with the objective being that no functional nef or vpu gene product is produced. The Δ2 SHIV virus was inoculated subcutaneously into six adult female macaques. All six animals produced antibodies detectable by immunoprecipitation with SHIV KL virus by three months after initial inoculation. By five months after initial inoculation, all six animals were producing neutralizing antibodies to SHIV KU „, virus measuring with a response of 1 in 10 to 1 in 20. At six months after initial inoculation, all six animals were healthy and showed no manifestations of AIDS pathology. Monthly analysis of blood cells via FACS (Fluorescence activated cell sorting) showed that the six animals were free from AIDS pathology. Virus was no longer detectable in blood mononuclear cells by six months after initial inoculation, as detectable by infectivity assays and by PCR analysis. Subsequently, at about six months after initial inoculation, the six inoculated animals and two un-vaccinated control animals were challenged by intravaginal administration of SHIV KU .| virus. The animals were evaluated for presence of infectious cells, and for their CD4 + cell count. Generally, as an assay for virus infectivity the frequency of infectious cells was measured by inoculation of serial 10-fold dilutions of PBMC (peripherial bone marrow cells, in particular blood mononuclear cells) or other cells onto indicator cells in 24-well tissue culture plates, which were then observed for development of syncytial CPE (cytopathic effects) during a 7-day period. Then lOOμl from each well was transferred to another plate, fresh indicator cells added (human T cell line C8166) , and the plates were then observed for a further 7 days. Routinely 10 6 cells from each animal were used for estimation of infectivity, and reported as number of infectious cells per 10 6 cells. Cell counts were generally determined using FACS (fluorescence-activated cell sorting) where cells were reacted with mAb to CD4 (SIM.4, NIH AIDS research and Reagents repository), or CD8 (Dako T8, Dako). After washing, the cells were stained with FITC-conjugated goat anti-mouse IgG (Dako), fixed in 1% buffered formalin, and analyzed on a fluorescence-activated cell counter. Evaluation of the efficacy of the vaccine is summarized by the data in Table 3 below. TABLE 3: Evaluation of Virus and Host Response after Δ2 SHIV (AvpuAnef HW) Vaccine Week Post Vaccine: Δ2 SHIV (AvpuAne SUW) PHA culture assay B CD8 + cell depletion then PHA culture assay Neutralizing Antibody titer The immune response of the host monkeys to the inoculation was monitored via standard immunological assays for immune response to the vaccine virus, including PHA, CD8 depleted PHA and assay for neutralizing antibody titer (see Joag et al., supra). For virus recovery from PBMC cultured with PHA and IL-2, a total of 2 x 10 6 PBMC were cultured in medium containing 1 μg of PHA-P (Wellcome) for 2 days. Cultures were then rinsed once, the cell pellets resuspended in medium with 100 U of recombinant human IL-2 (Cetus) per ml, cultured for 5 days, and then centrifuged again, and the cell-free supernatant fluids were assayed for virus infectivity as above. For virus recovery from CD8 + -depleted PBMC, a total of 2 x 10 6 PBMC were incubated with a monoclonal antibody to CD8 (Dako-T8; Dako) for lhr at 4°C and washed once, and sheep anti-mouse immunoglobulin G-coated paramagnetic beads (Dynal Laboratories) were added. The beads were removed with a magnet. This procedure routinely resulted in the removal of 75 to 85% of the CD8 + T cells. Aliquots (2 x 10 6 cells each) of CD8 + -depleted and control undepleted PBMC were cultured in PHA medium for 2 days and in IL-2 medium for 5 days as described above, after which the supernatant fluids were assayed for virus infectivity. For neutralizing antibody assays, serial doubling dilutions of plasma in RPMI were prepared in duplicate of quadruplicate in 96-well plates, 10 to 20 TCID 50 of virus was added to each well, plates were incubated for lhr at 37°C, and 10 4 indicator cells were added to each well. Plates were observed for CPE 7 days later, wells were scored individually, and the 50% neutralization endpoint was calculated by the Karber method. Data summarizing further evaluation of the Δ2 SHIV vaccine is shown below in Table 4. TABLE 4: Evaluation of challenge of Δ2 SHIV (ΔvpwΔπe/SHIV) Vaccinated Animals Week Post Infection Control Vaccine: Δ2 SHIV (ΔvpuΔπe ' SHIV) animal animal animal animal animal animal animal animal PFy PLy 42105 42107 PNa PLk PDj PPm o 835 A 1729 1249 1 152 1217 630 1507 1 197 0 B 0 0 0 0 0 0 0 3 796 26 538 449 792 592 1728 1254 0 10 10 100 0 0 0 0 Data reported as Number of Infectious cells/million cells c Week 0 is Time of Challenge Example 4 Avpu PPc SHIV Vaccine SHIV PPc is a non-pathogenic SHIV generated from animal PPc during the course of sequential passage through bone marrow as descibed previously (see Figure 5). Modification of the parental SIV virus, was done to incorporate HIV-1 vpu, nef and env to create a SHIV. It was found that this modified parental SHIV was actually not pathogenic. Inoculation of this modified parental SHIV into two macaques demonstrated that the virus was avirulent, and could result in the generation of immunity to the virus. However, this virus retained the full potential to become a pathogenic virus. Thus in order to prevent this type of mutation and create a safer vaccine, deletion of the vpu gene was made which resulted in Avpu PPc SHIV. The inactivation of the vpu gene can be accomplished by complete deletion of the vpu gene, partial deletion of portions of the vpu gene, or it could be rendered non- functional by insertion of DNA, the objective being to inactivate the gene such that no functional vpu gene product is present. In order to prevent mutation of the altered vpu gene back into a functional gene, complete deletion of the vpu gene may be preferred. The Avpu PPc SHIV was inoculated into six adult pigtailed macaques, and a summary of the data evaluating the vaccine is given below in Table 5. TABLE 5: Evaluation of Avpu PPc SHIV Vaccine Control: PPc SHIV Vaccine: Avpu PPc SHIV Data reported as Number of CD4 cells/μl B Data reported as Number of Infectious cells/million cells c Week 0 is the week of challenge Example 5 HIV-1 Vaccine Because of their SIV mac content (LTR, gag, pol, and nef), Avpu PPc SHIV and Δ2 SHIV do not replicate efficiently in human cells, and thus while useful as vaccines in humans, may not generate complete immunity. Using the teachings of the results from Avpu PPc SHIV and Δ2 SHIV, it is proposed that a human HIV-1 vaccine be constructed by using HIV-2 as the substitute for the SHIV component of the validated model vaccines. In one embodiment, construction of an effective human vaccine would involve combining the LTR, gag, pol, and nef genes of HIV-2 in the same arrangement as in SIN^., with the genes of HIV-1 to construct a purely human HIV counterpart to the Avpu SHIV KU ., and Δ2 SHIV virus vaccines. In one embodiment the genes of HIV-2 is combined in the same arrangement as in SIV mac , of course it is also possible to combine the genes in the natural order as found in HIV-2, or in different order or orientations. However, the genes should be expressed in a fashion as to allow for the generation of an effective immune response in the inoculated subject. Thus in one form of this embodiment a HIV-2 virus is used to provide the background of genes, and the HIV-1 env, tat, rev, vpu genes are substituted into this background of HIV-2 genes. As taught above, it may be preferred not to transfer the vpu gene, or at minimum to transfer and render non-functional the vpu gene. The human HIV vaccines can undergo preliminary testing in human cell cultures of lymphocytes and macrophages for evidence of infection and any retained pathogenicity. These virus constructs can also be tested in monkeys, such as chimpanzees and macaques, for evidence of replication. In one embodiment, construction of an effective human vaccine would involve rendering non-functional the vpu gene in an HIV-1 virus. Optionally, the nef gene can also be rendered non-functional or deleted. Interestingly, it has been found that the vpu gene in many HIV-1 cultured virus is inactivated (Vaishnav & Wong-Staal, 1991, supra.), but as shown by the results here, activation of vpu in these HIV-1 culture stocks will create pathogenic virulent HIV-1. Thus it is preferred, to make a safe vaccine, that the vpu gene be rendered inactive by deletion of some or all of the gene from the virus DNA, preferably where the vpu gene has been deleted completely. Example 6 In Vivo vaccination against sexually-transmitted HIV disease SHIV KL , bearing the envelope of HIV-1, causes fulminant infection with sub-total loss of CD4 + T cells and AIDS in intravaginally-inoculated macaques. This virus and route of inoculation was used to challenge six adult macaques immunized seven months previously by subcutaneous injection with attenuated AvpuAnef SHIV-4. The animals developed neutralizing antibodies to SHIV, but only minimally-detectable cell mediated immune responses. Two unvaccinated controls and one vaccinate succumbed to challenge, but five of six vaccinates developed only transient infection with the virulent virus and have remained healthy, without CD4 * T cell loss. This is the first demonstration of protection against virulent SHIV administered by the intravaginal route. These results support the concept that sexually-transmitted HIV disease can be prevented by immunization. HIV-1 is primarily a sexually transmitted virus that causes persistent systemic infection and loss of CD4 + T cells. The infection culminates in loss of immunocompetence and development of AIDS. Although several effective anti-HIV drugs, notably viral protease inhibitors, are now available, their widespread use is hampered by their high cost and the need for multiple treatments daily for an indefinite period. Their use is thus not feasible in less developed countries where the bulk of HIV infections occur. Under these conditions, development of a safe and effective vaccine is a priority. Moreover, such a vaccine should protect against the sexually transmitted virus. The inability of HIV-1 to infect animals other than chimpanzees has meant that macaque models using non-human primate lentiviruses have become the only practical alternative for testing proof-of-concept approaches to vaccines against HIV-1. SIV mac , which is closely related to HIV-2 and SIVsm, causes AIDS in macaques and has been extensively used in vaccine studies. Macaques vaccinated with attenuated strains of SIV mac , produced by deleting auxiliary genes, particularly nef, resisted infection after challenge with virulent strains of SIVmac (Daniel, M. D., Kirchhoff, F., Czajak, S. C, Sehgal, P. K. & Desrosiers, R. C. Science 258, 1938-1941 (1992); Almond, N., Kent, K., Cranage, M., Rud, E., Clarke, B. & Stott, E. J. Lancet 345, 1342-1344 (1995); Stahl Hennig, C, Dittmer, U., Nisslein, T., et al. J. Gen. Virol. 77, 2969-2981 (1996); Wyand, M. S., Manson, K. H., Garcia Moll, M., Montefiori, D. & Desrosiers, R. C. J. Virol. 70, 3724-3733 (1996)). A major limitation of the SIV model of AIDS in macaques is that this virus is only distantly related to HIV-1 genetically, and although SIV mac does cause AIDS, the biological properties of SIV mac vary greatly from those of HIV-1. Therefore, vaccine-induced responses which protect against virulent SIV nιac in macaques may or may not be directly applicable to HIV-1. Since some of the differences in the biological properties of these viruses are attributable to their envelope glycoproteins, chimeric simian-human immunodeficiency virus (SHIV) which contains the core of SIV mac and the envelope glycoprotein of HIV-1 was created to address this question. Newly created SHIVs were infectious but not pathogenic and thus vaccines could not be evaluated for efficacy in preventing disease. The recent derivation of virulent SHIV (SHIV KU .,) which causes almost total loss of CD4 + T cells and AIDS in macaques in six months after inoculation established a new and reproducible model which accurately reflected HIV-1 disease compressed in a short time frame (Joag, S. V., Li, Z., Foresman, L., et al. J. Virol. 70, 3189-97. (1996); Joag, S. V., Li, Z., Foresman, L., et al. AIDS Res. Hum. Retroviruses 13, 635-645 (1997); Joag, S. V., Adany, I., Li, Z., et al. J. Virol. 71, 4016-4023 (1997)). Moreover, the new virus is virulent after intravenous, oral, or intravaginal infusion. This virus thus proved ideal for evaluating efficacy of vaccines. The choice of a live-virus vaccine was based on observations of genetic changes occurring in the virus during animal passages which yielded SHIV KU _,. The original molecularly cloned SHIV-4 is avirulent and the vpu gene does not encode a functional protein. SHIV KU ., which is extremely virulent, encodes a functional Vpu and has amino acid substitutions in other viral proteins, including gρl60, Nef, Tat, Rev and Vpr (Stephens, E. B., Joag, S. V., Sheffer, D., et al. J. Med. Primatol. 25, 175-185 (1996); Stephens, E. B., Mukherjee, S., Sahni, M., et al. Virology 231, 313-321 (1997)). Assuming that development of virulence of SHIV KL was multifactoral (i.e., associated with amino acid changes in multiple viral proteins) including changes in vpu and nef genes, both genes from original SHIV-4 were deleted to develop a vaccine. Materials and Methods Viruses: SHIV-4 DNA encoding the env, tat, rev, and vpu genes of HIV-1 HXBc2 on a background of SIV mac 239 (Li, J., Lord, C. I., Haseltine, W., Letvin, N. L. & Sodroski, J. J. AIDS 5, 639-646 (1992)) was obtained from Dr. Joseph Sodroski, Harvard University. Viral DNA was transfected into CEMxl74 cells to produce a virus that was used to initiate passage in macaques. Virus from passage 4 which was associated with AIDS and death of macaque PNb at 6 months was amplified in culture of peripheral blood mononuclear cells (PBMC) from a normal macaque, as described previously. Supernatant fluid from this culture (SHIV KlM ) has a titer of 10,000 TCID 50 /ml in PBMC and in C8166 cells. Aliquots of this SHIV KU.| stock were stored in liquid nitrogen. Construction of Avpu nef SHIV-4 . The original SHIV-4 DNA consisted of 2 plasmids with the 5 ' and 3 ' regions, respectively. All manipulations were performed with the p3 ' SHIV-4. In the first step, shown in Figure 6, plasmid pUC19 was digested with Sspl, blunt-ended by Klenow fragment of DNA polymerase and ligated to generate pDS. The Sphl-Kpnl fragment (nt 6450 to 6985) of p3 ' SHIV-4 was subcloned into pDS to generate pDSvpu. Plasmid pDSvpu was digested with Sspl and Bbsl, blunt-ended, and religated to delete a 60 bp Sspl-Bbsl fragment and generate pDSDvpu. For construction of AvpuAnef SHIV-4, the Sphl-Kpnl fragment of 3 ' SHIV-4 (Figure 7A) was replaced with the corresponding fragment of pDSDvpu to yield p3 ' ΔvpwSHIV-4 (Figure 7B). Next, p3 ' Δv. wSHIV-4 was digested with RsrII and Ncol, blunt-ended, and ligated, resulting in the deletion of a 216 bp Rsrll-Ncol fragment (including 205 bp of the nef gene, encoding the first 69 amino acids of Nef). This plasmid was designated as nAvp uΔrce SHIV-4 (figure 7C). The n AvpuAnef SHIV-4 and p5 ' SHIV-4 plasmids were digested with Sphl, ligated with T4 DNA ligase and the ligated DNA used to transfect C8166 cells as described previously (Stephens, E. B., Mukherjee, S., Sahni, M., et al. Virology 231, 313-321 (1997)). Virus stocks were prepared and aliquots were stored at -80°C. The virus stock had a titer of 10,000 TCID 50 /ml in C8166 cells. Vaccination of macaques and challenge with SHIV Klj .,. Eight sexually mature (3 to 10 year old) pig-tailed macaques (Macaca nemestrina) were obtained from the Yerkes Primate Center, Atlanta, Georgia. Six of the eight macaques were inoculated subcutaneously with 1.0 ml AvpuAnef SHIV-4 stock. The two remaining control animals were mock-inoculated with saline. Seven months (29 weeks) after vaccination, the 6 vaccinated and 2 control macaques were inoculated intravaginally with 1.0 ml undiluted SHIV KL stock, representing 30 AID 50 by the intravaginal route (Joag, S. V., Adany, I., Li, Z., et al. J. Virol. 71, 4016-4023 (1997)). A second intravaginal challenge with the virus was given 5 weeks after the first challenge. Cell cultures. Human T cell line C8166 was used as indicator cells to measure virus infectivity. Cells were cultured at a concentration of 1 x 10 6 /ml in RPMI medium (RPMI supplemented with 10 mM Hepes buffer pH 7.3, 50 ug/ml gentamicin, 5 x 10 "5 M 2-mercaptoethanol, and 2 mM glutamine) with 10% fetal bovine serum (FBS). Processing of samples. Heparinized blood obtained from the femoral vein was centrifuged to separate plasma and buffy coats. Plasma was assayed for p27 using a capture ELISA kit (Coulter Laboratories, Hialeah, FL), and for infectivity in C8166 cells. Peripheral blood mononuclear cells (PBMC) were separated from buffy coats by centrifugation through a Ficoll-Paque (Pharmacia) density gradient. Infectious cell frequency was measured by inoculation of serial ten-fold dilutions of PBMC, starting with 1 x 10 6 cells into 24-well tissue culture plates containing 1 x 10 5 indicator C8166 T cells which were observed for development of syncytial cytopathic effects (CPE) during a 7 day period, after which cells and supernatant fluid in 100 μl from each well were transferred to another plate, fresh indicator cells added, and these observed for a further 7 days (Joag, S. V., Stephens, E. B., Adams, R. J., Foresman, L. & Narayan, O. Virology 200, 436-446 (1994)). Results were expressed as the number of infectious cells/10 6 PBMC. Mesenteric lymph nodes were obtained at 19 weeks post-challenge by biopsy. A single cell suspension was prepared and infectious cell frequency assessed as for PBMC. Other portions of the biopsy material were used for analysis of DNA or formalin-fixed for histological examination. FACS analysis. PBMC or lymph node cells were reacted with mAb to CD4 or CD8. After washing, the cells were stained with FITC-conjugated goat anti-mouse IgG , fixed in 1% buffered formalin, and analyzed on a fluorescence-activated cell counter. Neutralizing antibody assays. This test is performed as described previously. Briefly, serial doubling dilutions of plasma in RPMI were prepared in quadruplicate in 96-well plates, 10 to 20 TCID 50 of the virus were added to each well, plates incubated 1 h at 37°C and 1 x 10 4 indicator C8166 cells added to each well. Plates were observed for CPE 7 days later, wells scored individually, and the 50% neutralization end point was calculated using the Karber method (Lennette, E. H. in Diagnostic procedures for viral and rickettsial infections (eds Lennette, E.H. & Schmidt, N.J.) Vol.4th, 1-65 ( American Public Health Association, New York, 1969)). Detection of vaccine and challenge viruses using PCR. Nested PCR was used to detect SIV gag sequences in DNA of PBMC of the vaccinates using gαg-specific oligonucleotides and conditions of amplification as described earlier. PCR of the vpu gene was used distinguish between vaccine and challenge viruses in PBMC and mesenteric lymph nodes of the six vaccinates and the non- vaccinated control animals after challenge. For the first round of PCR amplification of vpu, oligonucleotide primers 5'-CCTAGACTAGAGCCCTGGAAGCATCC-3' (SEQ ID NO:l) and GTACCTCTGTATCATATGCTTTAGCAT-3' (antisense) (SEQ ID NO:2), which are complementary to nucleotides 5845 to 5870 and 6393 to 6420, respectively of the HIV-1 (HXb2) genome were used. One ug of genomic DNA was used in the PCR containing 4.0 mM MgC12, 200 uM each of the four deoxynucleotide triphosphates, 100 pM each oligonucleotide primer and 2.5 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, Conn.). The template was denatured at 95°C for 3 min and PCR amplification performed with an automated DNA Thermal Cycler (Perkin-Elmer Cetus) for 35 cycles using the following profile: denaturation at 92°C for 1 min, annealing at 55°C for 1 min and primer extension at 72°C for 3 min. Amplification was completed by incubation of the PCR for 10 minutes at 72°C. One ul of the PCR product from above was used in a nested PCR using the same reaction conditions described above. The second round of amplification used oligonucleotide primers 5'-TTAGGCATCTCCTATGGCAGGAAGAAG-3' (sense) (SEQ ID NO:3) and 5'-CACAAAATAGAGTGGTGGTTGCTTCCT-3' (SEQ ID NO:4), which are complementary to nucleotides 5956 to 5984 and 6386 to 6413, respectively of the HIV-1 (HXb2) genome. Following the second round of amplification, a 10 ul aliquot was removed and run on a 1.5% agarose gel and bands visualized by staining with ethidium bromide. The result of this PCR was the amplification of a 397 base pair fragment if the deleted vpu was present (i.e., the vaccine virus) or a 457 base pair fragment if the intact vpu was present (i.e., the challenge virus). T cell proliferation assays. PBMC were tested for T cell proliferative responses against equal concentrations of extracts of SHIV-infected and control cells. Additionally, a panel of 6 synthetic peptides was used (peptides 61, 63, 104, 1 1 1, 1 13, 1 16) representing T cell epitopes present in the gpl60 of HIV-1, and against a control peptide. The sequences and synthesis of the gpl60 and control peptides have been previously described (Nehete, P. N., Satterfield, W. C, Matherne, C. M., Arlinghaus, R. B. & Sastry, K. J. AIDS Res. Hum. Retroviruses 9, 235-240 (1993)). Cells were suspended at 2 x lOVml in medium and 0.1 ml aliquots dispensed into 96-well U bottom microtiter plates and incubated with different concentrations of peptides or medium alone. All treatments were done in triplicate and cells were incubated for 96 hr at 37°C. During the final 16 to 18 hr, 1 μCi of [ 3 H]thymidine (6-7 Ci/mmol; ICN Biochemicals, Costa Mesa, CA) added. Cells were harvested on filter strips, the amount of [ 3 H]thymidine incorporated was measured, and specific radioactivity for each addition calculated as described (Nehete, P. N., et al, supra). CTL assays. PBMC were stimulated with Concanavalin A (10 mg/ml) and expanded in RPMI medium containing 10%FCS and 10 U/ml rIL-2 for 7 days and used as effector cells. Autologous B lymphoblastoid cell lines (B-LCL) generated by transformation with Herpesvirus papio were infected with recombinant vaccinia viruses for expressing HIV-Env (vPE16) or SIV-Gag (vgag), SIV-pol (vpol) or with control vaccinia virus (vSC8) and radiolabeled with 51 Cr for use as target cells as described. (Sastry, K. J., Nehete, P. N., Venkatnarayanan, S., Morkowski, J., Platsoucas, C. D. & Arlinghaus, R. B. Virology 188, 502-509 (1992)). The control and recombinant vaccinia vectors were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. CTL activity was detected in standard 4 hour 5l Cr release assays using various effector : target cell ratios. Results Following subcutaneous inoculation of Avpu Anef SHIV-4, the six vaccinated macaques were bled every two weeks for three months and then monthly for another four months. Co-cultivation of 10 6 PBMC with C8166 cells failed to yield infectious virus from any of the six vaccinates. However, PBMC, depleted of CD8 + T cells, and treated with phytohemagglutinin for two days followed by cultivation in medium containing IL-2 for five days, yielded virus, but only at the week four time point and from only three of the six macaques, 42105, PNa and PDj. Nevertheless, PCR analysis of DNA from PBMC of the six vaccinates at six weeks showed that SIV gag sequences were present in all of the animals, confirming that all six had become infected. At week 29, immediately before challenge with SHIV KU .,, only two of the six animals were positive for viral sequences (Table 6), suggesting that only a low level of replication by the vaccine virus had occurred in the vaccinates. Pre- immunization sera and plasma from the two control macaques lacked antibodies to the virus. All of the six vaccinates developed antibodies to the virus. Immunoprecipitation analysis of plasma showed that all six had produced antibodies to SIV mac gag protein and HIV-1 env gene by eight weeks after immunization and by four months, all six had developed neutralizing antibodies to the virus. These antibody titers fluctuated between sampling periods and varied from 1 :30 to 1 :40 against SHIV-4. The same samples had titers of about 2 fold less to SHIV KL (Figure 8). None of the animals developed evidence of cell mediated immune responses to the virus, since PBMC failed to proliferate after exposure to virus antigen or viral peptides or to kill autologous cells in CTL assays. CD4 ^ T cell counts were normal in all animals throughout the 29 week period. TABLE 6 PCR Analysis of PBMC and lymph node tissue DNA following challenge of macaques with SHIV^ . , " DNA was used in nested PCR with oligonucleotide primers that amplified either the vpu or gag gene. b All DNAs analyzed were from PBMC except for week 19, which were from lymph node tissue. C = challenge virus; V = vaccine virus. The six vaccinated and two control animals were challenged intravaginally with SHIV KU ., at 29 weeks and heparininzed blood collected at 2-3 week intervals subsequently for assays on virus burden and immune responses. From this point onwards, PCR was used to detect not only gag but also detect vpu sequences in order to discriminate between the vaccine virus which has a vpu gene containing a 60 bp deletion, and SHIV KL which has non- deleted and a functional vpu. PCR analysis of vpu sequences on DNA from PBMC of the eight animals at three weeks after challenge showed that seven of eight animals had become infected with SHIV KU ., virus, the exception being a control animal, PFy (Table 6). Virus recovery data coming two weeks later after the initial post-challenge blood sample showed that infectious virus was recoverable from only one control animal, PLy, and three vaccinates, 42105, 42107 and PNa (Table 7). The remaining animals were therefore re-challenged intravaginally with SHIV KU ., at week 5. PCR analysis of gag and vpu sequences on DNA from subsequently collected from PBMC demonstrated the presence of SHIV KU _, sequences in all animals. However, the only time point at which SHIV KU „, sequences were detected in the PBMC from macaque PDj was at 29 weeks (Table 6), suggesting that this animal had a low copy number of viral sequences into its PBMC. Since previous studies have shown that the viral genome can persist in lymph node tissue DNA while absent in PBMC DNA (Stephens, E. B., Joag, S. V., Atkinson, B., et al. Virology 234, 328-339 (1997)), the lymph node DNA from the six vaccinates and the surviving control animal was analyzed. PCR analysis of vpu sequences from lymph node DNA at 19 weeks after challenge revealed that the SHIV KU ., vpu was present in five of six vaccinates (the only exception being macaque PDj) and in the control macaques (Table 6). The analysis indicated that the vaccine viral sequences persisted in the lymph node tissue from macaque PNa and PDj. TABLE 7: Infectious cells/10 6 PBMC after challenge. 3 10 100 0 0 0 0 0 10 5 10 10 0 0 1 0 0 10 7 3 2 1 0 2 3 10000 10 9 10 5 0 0 0 0 10 10 11 0 100 0 0 0 0 10 3 13 10 10 0 0 0 0 10 10 15 10 100 0 0 0 1 5 10 19 2 1 0 0 0 0 10 died 21 10 10 0 0 0 0 10 23 10 10 0 0 0 0 10 25 1 died 0 0 0 0 10 Macaques were vaccinated at week -29 and challenged twice with SHIV Kl at weeks 0 and 5. The two control animals, PFy and PLy, developed typical SHIV KU _, infection. Within one month of becoming infected, both animals developed almost total loss of CD4 + T cells to less than 30/μl (Table 8). During this period plasma from both animals had virus antigen (p27) and infectious virus (viremia) (data not shown). Control macaque PLy had become infected at the first challenge and this animal developed AIDS and died at week 15. Control macaque PFy became infected after the second inoculation at week 5. This animal became antigenemic and viremic (data not shown) and is still alive at week 29 with almost no circulating CD4 + T cells (Table 8). Examination of blood from the vaccinates showed that virus was recovered rarely from PBMC from four of the six vaccinates, PDj, PLk, PNa and PPm. None of these four became viremic or antigenemic and they have remained healthy with normal CD4 + T cell counts. In contrast, infectious virus was recovered from PBMC from vaccinates 42105 and 42107 throughout a 23 week observation period (Table 7). Neither of the two animals developed plasma viremia or antigememia during the first six weeks after challenge. Both animals developed partial loss of CD4 * T cells by two months following challenge but the count subsequently returned pre-challenge levels in 42105 whereas CD4 + T cell counts declined constantly in 42107 which died with AIDS at week 23 (Table 8). This animal developed antigenemia but no viremia during the last half of its infection. TABLE 8: CD4 + T cells/ ul in blood. -29 1570 1060 1300 490 2740 820 1100 1570 -13 1421 894 1666 482 1110 900 983 1897 0 1249 1152 1507 630 1217 1197 835 1729 3 538 449 1728 592 792 1254 796 26 5 884 584 1446 548 974 670 866 1 7 554 415 1174 389 1439 1066 106 22 9 464 341 901 356 1046 983 0 0 11 505 360 1158 472 1341 748 0 0 13 515 342 901 356 1046 983 0 0 15 570 344 1176 699 1076 869 0 0 19 531 98 1479 764 1617 961 0 died 21 907 129 1989 448 661 404 72 23 696 226 1216 743 915 513 0 25 514 died 1601 722 960 460 55 27 900 2111 759 1158 876 4 Macaques were vaccinated at week -29 and challenged twice with SHIV KLM at weeks 0 and 5. None of the vaccinated macaques had T cell proliferative responses directed against extracts of cells infected with SHIV KL , or to synthetic peptides representing T-cell epitopes from highly conserved sequences of the HIV-1 envelope protein (data not shown). Testing for cytotoxic T lymphocyte (CTL) responses directed against target cells infected with recombinant vaccinia viruses for expression of HIV-e«v, SYV-gag, or SYV-p ol was also done. No significant levels of CTL activity specific to HIV-e«v and/or SW-pol were observed in the vaccinates prior to challenge. Discussion These results show that a vaccine which caused subclinical and minimally productive infection in macaques, induced protection in 5/6 animals against loss of CD4 + T cells and AIDS after intravaginal infusion of virulent SHIV KLM . This is the first demonstration that sexually transmitted HIV-1 disease caused by a lentivirus bearing the envelope of HIV-1 could be averted by immunization. The success of the vaccine experiment was defined by the virulence of the challenge virus. This is an extremely virulent virus which shares the envelope of HIV-1 and causes HIV-1 disease, including loss of CD4 + T cells and AIDS, in a predictable manner during a six-month course of infection, even after intravaginal infusion of virus. The fact that the vaccinates were protected against this virus shows that not only could HIV-1 disease be prevented by immunization, but that the protection extended to sexually transmitted disease. The two sham-inoculated control animals challenged with SHIV KU _, succumbed predictably to the challenge virus, having developed massive systemic infection and subtotal loss of CD4+ T cells with viremia and viral antigenemia soon after challenge. This sequence of events had been observed in numerous other animals as reported earlier. The fact that all six of the vaccinates developed systemic infection with the challenge virus showed that the virus had breached the vaginal mucosal barrier and had reached the draining lymph nodes, as confirmed by PCR on PBMC and mesenteric lymph nodes. In an earlier study on pathogenesis of SHIV-infection after intravaginal infusion of the virus, it was shown that soon after virus became detectable in mesenteric lymph nodes, an explosive burst of virus replication ensued in the lymphoid tissues. However, in the vaccinates reported here, virus replication was completely suppressed in three (PDj, PLk and PNa) since no infectious virus could be recovered from lymph node or PBMC cells despite presence of challenge virus DNA. The other two vaccinates, 42105 and PPm that resisted the virus had very low number of infectious cells, 5 and 10 cells/10 6 PBMC, even though these animals had large number of susceptible CD4+ T cells. This indicated failure of the virus to replicate and spread among susceptible cells. Vaccinate 42107 which succumbed to AIDS had an unanticipated high virus burden, similar to the control animals. It is of interest that both of the vaccinates, 42105 and 42107, that became productively infected with SHIV^., developed partial loss of CD4+ T cells shortly after challenge. Infections in these two animals had diametrically opposite outcomes, since macaque 42105 gradually gained control over the virus with restoration of CD4+ T cell values, whereas in 42107 there was a slowly progressive loss of the CD4+ T cells that ended in development of AIDS. Thus, only one of the six animals developed disease following exposure to the challenge virus. Our results are in keeping with the precedent that SIV mac -induced AIDS in macaques could be prevented by prior infection with SIV whose virulence had been attenuated by deletion of accessory genes including nef. This converted virulent SIV to an avirulent virus, which was then used as a vaccine. In this case, the rationale for the vaccine constructs differed from that of SIV. SIV does not have a vpu gene. During the development of virulence in SHIV by animal to animal passage the vpu gene became functional prior to acquisition of most of the mutations in the env and nef genes which are present in virulent SHIV KU .,. This led to the speculation that deletion of the two accessory genes, vpu and nef, would prevent development of virulence. The strategy worked. Whether deletion of the vpu gene alone in this vaccine construction would have sufficed is not known. While the precise mechanisms associated with development of vaccine-induced resistance to replication of SHIV KU _, are not fully understood, it is clear that the vaccine virus had replicated sufficiently in the animals to induce binding antibodies to both the SIV gag and the HIV-1 envelope proteins and neutralizing antibodies to SHIV KU _,. New results showed that passively-immunized macaques having antibodies with similar titers in their plasma as the vaccinates were refractory to infection with virulent SHIV. Thus, presence of neutralizing antibodies alone may have been capable of averting productive infection with SHIV KU _, in the vaccinates. Interestingly, macaque 42107 had developed neutralizing antibodies to SHIV KU ., at 10 and 6 weeks prior to challenge but the neutralizing titer had dropped to an undetectable level at the time of challenge. This macaque went on to develop productive infection with SHIV and development of AIDS even though the neutralizing antibody titer rose again. Presence of these antibodies prior to challenge may be protective but not after infection. The apparent lack of SHIV-specific proliferative responses in and CTL activity PBMC from the vaccinates was surprising. These results are in striking contrast to the high T cell proliferative activity previously documented in HIV- 1 -infected chimpanzees (Nehete, P. N., Murthy, K. K., Satterfield, W. C, Arlinghaus, R. B. & Sastry, K. J. _ 7E>S 9, 567-572 (1995)) and in rhesus macaques immunized with HIV-1 gpl60-derived peptides (Nehete, P. N., Satterfield, W. C, Matherne, C. M., Arlinghaus, R. B. & Sastry, K. J. AIDS Res. Hum. Retroviruses 9, 235-240 (1993)). However, the results are in concordance with the similar low levels of CTL activity directed against SIV-RT and SW-nef reported to contribute to protection of macaques against mucosal challenge with SIV (Putkonen, P., Makitalo, B., Bottiger, D., Biberfeld, G. & Thorstensson, R. J. Virol. 71, 4981-4984 (1997)). Additionally, since proliferative and CTL responses in lymph node cells were not analized, it cannot exclude the presence of SHIV-specific cellular immune mechanisms in the vaccinated monkeys since the immune cells may have been confined to the lymphoid tissues. That CMI responses may have been involved in resistance to SHIV-disease comes from earlier studies in which macaques that had recovered from infection with macrophage-tropic SIV were refractory to superinfection with SHIV (Stephens, E. B., Joag, S. V., Atkinson, B., et al. Virology 234, 328-339 (1997)). The envelope glycoprofiens of these two viruses are distinctly different and, as expected, serum from the SIV-infected animals failed to neutralize SHIV, yet the animals resisted infection by SHIV. Future studies on CMI responses to vaccine virus using lymph node cells will shed new light on this problem. However, although the mechanisms of protection against SHIV KU _, are not understood fully, the results clearly shows that sexually transmitted HIV-1 disease can be averted by vaccination. Example 7 Direct Infection With Viral DNA Methods using the DNA constructs of the present invention to confer protective immunity and/or other therapeutic benefits on non-infected or HIV infected individuals can be accomplished by the direct administration of DNA. For example, when 100 1 of saline containing 100 g ofΔvpz.SHI V-PPc (equivalent to AvpuSHTV-4) was dropped on to the cornea of two macaques, and the animals monitored for infectious virus in the blood over the next four weeks. As seen in table 9 below, no infectious cells were detected in the blood, indicating that there was no infection by this dose of viral DNA. Subsequently, 200 1 of saline containing 200 g ofΔvpwSHIV-PPc was then injected intradermally into each of the two animals and monitoring continued. One week after the inoculation, infectious cells were detected in the blood of the animals at a concentration of 1000 infectious cells per million, and 10 infectious cells per million in week two. This infection has persisted for three weeks, and appears similar to the type of infection caused by infectious virus inoculated intravenously. Table 9: Viral DNA Inoculation Week -7 to -1 were after Cornea inoculation; Week 1 to 4 after Intra-dermal inoculation. Example 8: Drug Therapy Various drug therapy regimens have been used to treat HIV infection, and have yielded mixed results. Most importantly, presently employed drug therapy, (including DDI, DDC, ZDU/AZT), and proposed drug therapies (cyanovirin-N, calanolide-A) do not convey protective immunity on the treated subjects, and thus are vulnerable to desensitizadon and the evolution of resistant strains. The live virus vaccines, and viral DNA vectors of the present invention do not suffer from this flaw, and can be used to convey protective immunity on non-infected individuals, as well as therapeutic and protective immunity on previously infected individuals. The latter therapeutic administration of the virus or DNA vectors of the present invention, preferably being done in conjunction with conventional drug therapy, which will reduce the viral load. To exam the effects of drug therapy in the current animal model, two macaques (45P and 45Q), were inoculated intravenously with SHIV KU . 2 and one week later, put on therapy with the administration of (R) -9- 2-Phosphonylmethoxypropyl Adenine (PMPA) at a concentration of 30 mg/kg given by daily injections. Blood and CSF were monitored every day for the first week of treatment, and at weekly intervals thereafter. As can be seen from Table 10 below, infectious cells were present on the first day of treatment, and continued until week 1 1, when it gradually disappeared with continued treatment. The infectivity of the plasma also lasted from week 1 to the beginning of week 2. Infectious cells were present in the CSF for the same period as it was present in blood. The CD4+ T cell counts declined rapidly during the first week of infection but instead of declining progressively, as in typical SHIV KU _ 2 infection, the CD4+ T cell counts stabilized, and essentially recovered to close to normal levels during treatment. PMPA treatment was stopped after week 11, and the animals monitored for resurgence in virus replication. This resurgence began very slowly, and as can be seen in Table 10 below, by seven weeks after drug treatment was stopped, infectious cells began to reappear in the blood. At seven weeks after drug cessation, the CD4+ T cell counts were close to normal. These results indicate that immunization with the viral DNA constructs of the present invention to convey protective therapeutic effect and/or immunity, should preferably begin within one week after cessation of anti-viral drug treatment, prior to the resurgence of viral load. However, administration of the viral DNA constructs or virus of the invention can be effective where pathogenic virus is still present at detectable levels. Table 10: PMPA Inhibition of SHIV κυ Pathogenesis Samples taken at time points 1 to 1 1 were during active PMPA therapy. Samples marked with an * correspond to the samples tested and illustrated in table 1 1 below which shows the results of a more sensitive assay to detect virus RNA in the blood via QCPCR. The Q-PCR technique is currently performed at the NIH, and can detect as little as 300 copies of RNA ml of blood. Table 11 illustrates the relatively rapid decline of virus RNA in the blood during PMPA treatment. Table 11: QCPCR of Samples during PMPA Inhibition of SHIV KL , Pathogenesis Q-PCR: RNA Copy Equivalents/ml Example 9 Oral Immunization Six adult macaques were immunized subcutaneously with vaccine 1 (ΔvpuΔnef), and six were immunized orally with vaccine 2 (Δvpu). Both viruses caused infection in all inoculated animals, but whereas vaccine 1 virus caused only a nonproductive type of infection, vaccine 2 virus replicated productively but transiently for a 6- to 10-week period. Both groups were challenged 6-7 months later with pathogenic SHIV KU _, by the intravaginal route. All four unvaccinated controls developed low CD4 + T-cell counts (<200/μl) and AIDS. The 12 vaccinated animals all became infected with SHIV KU _,, and two in group 1 developed a persistent productive infection followed by development of AIDS in one. The other 10 have maintained almost complete control over virus replication even though spliced viral RNA was detected in lymph nodes. This suppression of virus replication correlated with robust antiviral cell-mediated immune responses. This is the first demonstration of protection against virulent SHIV administered by the intravaginal route. This study supports the concept that sexually transmitted HIV diseases can be prevented by parental or oral immunization. MATERIALS AND METHODS Vaccination of macaques and challenge with SHIV KlM . Sixteen sexually mature (3- to 15-year old) female pig-tailed macaques (Macaca nemesirina) were obtained from the Yerkes Primate Center, Atlanta, Ga. Six were inoculated subcutaneously, close to the inguinal and auxiliary lymph nodes, with 1.0 ml of Δv >uΔ/.e SHIV-4 stock (vaccinel), while two control animals (Pfy and Ply) were mock inoculated with saline. Seven months (29 weeks) after vaccination, the six vaccinated and two control macaques were challenged with 1.0 ml of undiluted SHIV KU _, stock which was inoculated intravaginally in nontraumatic fashion as described previously (Joag et al., 1997; J. Virol. 71 :4016-4023) This inoculum represents 30 50% animal infectious doses determined by titration of the virus in animals via the intravaginal route of inoculation (Joag et al., 1997). A second intravaginal challenge dose of SHIV KU ., was given 5 weeks after the first challenge to ensure exposure to this virus. Six other macaques were vaccinated with 1.0 ml of AvpuSHIV PPc stock (vaccine 2) by the oral route, while two control animals (4267 and PDb) were mock inoculated with culture medium. Six months later, all eight macaques were challenged by intravaginal inoculation twice, 1 day apart, with 1.0 ml of undiluted SH_V KU _, stock. Cell cultures. The human T-cell line C28100 was used as the indicator line to measure virus infectivity. Cells were cultured at a concentration of 10 6 /ml in RPMI medium (RPMI 1640 supplemented with lOmM HEPES buffer [pH 7.3], 50 μg of gentamicin per ml, 50 μM 2-mercaptoethanol, and 2 mM glutamine) with 10% fetal bovine serum (FBS). Processing of samples was conducted as in Example 6. Inguinal lymph nodes were obtained later from animals in both groups (between weeks 54 and 60 for group 1 and weeks 31 and 37 for group 2) and snap-frozen in liquid nitrogen immediately upon removal. They were stored at - 80°C until processed for RNA analysis. Fluorescence-activated cell sorting analysis was conducted as in Example 6. Neutralizing antibody assays were conducted as in Example 6. Detection of viral DNA in tissues. Tissues from inoculated animals were first screened for the presence of the SIV region of SHIV by PCR using oligonucleotide primers specific for both SIV and SHIV. Total cellular genomic DNA was extracted from PBMC and/or lymph nodes of the animals and was used as a template in nested PCR to amplify SIV gag sequences which were common to both viruses. The gαg-specific oligonucleotides and the conditions for amplification were exactly as described previously (Joag et al. 1994. Virology 300:436-446). PCR techniques were also used to distinguish between the presence and absence of vaccine and/or challenge viruses in macaque tissues, using truncated or full-length vpu as markers for both viruses, as described in Example 6. The result of this PCR was the amplification of a 307-bp fragment if the deleted vpu was present (i.e., the vaccine virus) or a 457-bp fragment if the intact vpu was present (i.e., the challenge virus). Detection of viral RNA in lymph nodes. Snap-frozen lymph nodes were homogenized in Trizol reagent (Gibco-BRL, Gaithersburg, M.D.), by using an Omni-mixer homogenizer (Omni International; Waterbury, Connecticut). Total RNA was isolated by standard procedures, and the final RNA was dissolved in 50μl of distilled H 2 O per 100 mg of original tissue. The quality of the RNAs was assessed by reverse transcriptase (RT)-mediated PCR (RT PCR) for the cellular gene GAPDH mRNA as described previously (Smith et al. 1997. J. Infect. Dis. 176:84-93), using the Titan One-tube Rl PCR system (Boehring Mannheim, Indianapolis, IN.). The possibility of contaminating DNA in the RNA preparation was assessed by parallel reactions in which the RT activity in the reaction was first inactivated by 2 minutes at 99°C. If needed, RNase-free DNase (Gibco-BRL) was used to remove residual DNA from the sample, followed by extraction and precipitation. Sample RNAs were amplified by RT-PCR using primers for the SHIV pol gene (corresponding to the parent SIV sequence: SIVpolA (5OAAAAGATGGAAAAGGATGG3') (SEQ ID NO:5) and SIVpolB (5 GGCTTCTAATGGCTTGC3') (SEQ ID NO:6). One microgram of total RNA was used in the one-step reaction containing the manufacturers buffer and enzyme mix, appropriate primers and 1.6 U of Prime Rnase inhibitor (5 Prime— >3 Prime, Inc., Boulder. Colo.). The reactions were performed with a Perkin-Elmer DNA Thermal Cycler 480 with the following thermal profile: 42°C for 30 min. 1 cycle: 94°C for 30 seconds; 55°C for 30 seconds; and 68°C for 45 seconds for 10 cycles: 94°C for 30 seconds, 55°C for 30 seconds, 68°C for 30 seconds with an additional extension cycle for 25 cycles; and then 68°C for 6 min. Nine microliters of the product was loaded onto an agarose-Tris-borate-EDTA gel containing ethidium bromide. DNA was separated by electrophoresis and the gel was photographed. When no visible product was detected, 1 μl of the initial reaction mixture was added to a neated PCR mixture containing primers SIVpolC(5'ACCAATCCATACAACACC3') (SEQ ID NO:7) and SIVpolD (5'CTGCCCAATTTAATACTCC3') (SEQ ID NO:8) 3 mM MgCl 2 and 1.25 U of Taq enzyme (Sigma, St. Louis, MO.), and a further 35 cycles were performed with the following thermal profile: 97°C for 1 minute, 55°C for 2 minutes and 72°C for 5 minutes, 1 cycle 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 45 seconds, with an additional extension/cycle, 33 cycles: 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 6 minutes. The amplified SIVpolAB fragment is 869 bp, whereas the SIVpolCD nested fragment is 645 bp. For the detection of multiply spliced mRNAs, primers spanning the intron of the tat and rev genes were used (Gunthard et al. 1998. J. Virol. 72:2422-2428; Saksela et al. 1993. J. Virol. 67:7423-7427) without the additional restriction sites: the second-round primers are hemi- nested, containing one of the outer primers plus a nested primer. The PCR products of the Mspl AB primer pair range from 131 to 159 nucleotides, and those for Mspl CB primer pair ranged from 1 10 to 138 nucleotides. Second-round reactions included 1 M betaine (Sigma). Quantitation of plasma RNA viral load. Plasma samples collected in acid citrate dextrose (ACD) from animals in group 1 during weeks 52 and 58 and from group 2 during weeks 27 and 35 were analyzed by a real-time RT-PCR assay as previously described (Suryanarayana et al. 1998. AIDS Res. Hum. Retroviruses 14:183-189). Lymphocyte proliferation assay. PBMC from different macaques, collected 49 weeks after virus challenge, were cultured in triplicate as 10 6 cells/well in 96-well tissue culture dishes in 200 μl of RPMI 1640 containing 10% FBS. Stock SHIV KU ., (10 4 2 TCID /ml) was UV irradiated for 30 minutes and heat treated at 56°C for 60 min before use as antigen; 20μl of this material was added to each well. Three wells each containing unstimulated PBMC from different pig-tailed macaques served as negative controls. Cells were cultured for 6 days before the addition to each well of 1 μCi of [ 3 H]thymidine (Specific activity, 247.9 GBq/minol, NEN, Boston, MA). Cultures were harvested onto glass fiber filter mats by using an automated plate harvester (Skatron, Sterling, VA) 24 h after addition of [ 3 H]thymidine, and [ 3 H]thymidine incorporation was determined with a Microbeta liquid scintillation counter (Packard). Stimulation index (SI) was calculated as mean counts per minute in stimulated wells/mean counts per minute in control well, and an SI of greater than 1.5 was considered significant (Buge et al. 1997. J. Virol. 71:8531-8541.). Development of CD4 + T cell clones. CD4 " cells from group 1 macaques PDj and Pna (week 50 post-challenge) were negatively selected from PBMC first by incubating PBMC with mouse anti-human CD8 monoclonal antibody and then with anti-mouse Immunoglobulin G magnetic beads (Dynal, Lake Success, N.Y.). Cells enriched for CD4 + T cells were then inoculated with Herpesvirus saimiri (American Type Culture Collection Manassas, VA) to generate immoralized CD4 * T cell lines (Beisinger et al. 1992. Proc. Natl. Acad. Sci. USA 89:31 16-31 19). CD4 + T cell clones were derived from the limiting-dilution method and the phenotype was confirmed by staining with mouse anti-human CD4" antibody. These CD4 + T cell clones were inoculated with SHIV KU ., at a multiplicity of infection of about 0.1 and used 7 days later as stimulation or targets. Generation of bulk cytotoxic T-lymphocyte (CTL) population and chromium release assay. PDj and PNa PBMC were collected 50 weeks after virus challenge and co- cultured with UV-irradiated SHIV KL -infected autologues CD4 + T cells (effector/stimulator ratio of 10:1) in 24-well tissue culture plates in RPMI 1640 containing 10% FBS. They were restimulated on day 7 and used as effectors on day 14 in a chromium release assay. Target cells (autologues CD4 T cell close) were either infected with SHIV KU. , or sham infected for 3 days and then used in chromium release assays. Cells were labeled with 100 μCi 51 Cr (specific activity, 962 MBq/ml of sodium chromate (Amershan, Cleveland, OH) for 2 h and then washed thrice with Hanks balanced salt solution. Target cells (2,500/well) were dispensed in triplicate and effector cells added in triplicate in effector/target (E/T) ratios of 80, 40, 20, 10, and 5 into each well of 96-well U-bottom plates. Chromium release was determined after 4 hours of incubation at 37°C in a 5% CO 2 incubator. Plates were spun at 1 ,200 rpm for 5 minutes, and 100 μl of supernatant from each well was counted in a gamma counter (Packard, Meridian, Con.). Each experiment had three wells containing only targets and medium that provided the data for spontaneous release, while three wells containing targets and 5% sodium dodecyl sulfate (SDS) served as wells for maximal release of chromium. The percent specific cytotoxity was calculated as (test release — spontaneous release) divided by (maximum release — spontaneous release) x 100. Spontaneous lysis of relevant control target was always less than 25% of maximum release. RESULTS Uncoagulated blood samples were collected weekly for the first month, at 2-week intervals for the next month, and monthly thereafter from all inoculated animals. Fresh plasma was tested for ineffectivity, content of p27, viral RNA, and antiviral antibodies. PBMC were examined for virus content (infectious center assays) and for CD4 " and CD8 T T cell markers and were used for cellular immunity studies. Table 12. Viral burden in macaques whose CD4 + T counts are shown in Table 2 Infectious cells/ 10'' PBMC Wk Contro I Vaccine Group i 1 Vaccine Group 2 PFy PLy 4267 PDb 42105 42107 PDj PLk PNa PPm 42106 7024 8124 PEy PWi PWv -22 to -20 0 0 0 0 0 0 0 0 0 0 10 10 10 10 10 10 -19 to-17 0 0 0 0 0 0 0 0 0 0 10 10 10 100 1000 100 - 15 to - 1 1 0 0 0 0 0 0 0 0 0 0 0 1 1 0 100 0 -9 to -3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 to 2 10,000 ND 100 1,000 0 0 0 0 0 0 0 5 0 0 0 3 to 4 10 10 10,000 10,000 10 100 0 0 0 0 2 0 100 2 0 0 5 to 6 10 10 10 10 10 10 0 0 0 0 2 0 0 0 0 4 7 to 8 10 10 4 10 4 2 1 0 2 4 4 0 0 0 0 0 9 to 1 1 5 10 10 100 0 0 0 12 to 13 10 10 10 10 10 0 0 0 0 0 0 0 0 0 0 14 to 17 10 10 1 ,000 10 100 0 0 0 0 0 0 1 1 0 0 18 to 20 10 2 10 0 0 0 0 0 21 to 23 10 10 10 10 0 0 0 0 0 0 1 0 0 0 24 to 26 2 10 1 100 0 0 0 0 0 0 0 0 0 0 27 to 30 10 10 0 0 0 0 0 0 0 0 0 0 0 31 to 34 0 5 0 0 0 0 0 0 0 0 0 0 0 35 to 38 100 5 0 0 0 0 0 0 0 0 0 0 0 39 to 42 10 10 0 0 0 0 0 0 0 0 0 0 0 43 to 46 5 0 0 0 0 0 47 to 50 10 0 0 0 0 0 51 to 54 100 10 0 0 10 5 55 to 58 5 0 0 0 0 59 to 62 10 0 0 0 0 * ND, not determined Vaccine Group 1. Six Macaques were inoculated subcutaneously with 1 ml of tissue culture fluid containing 10 4 TCID 50 of ΔvpuΔnef SHIV-4, and heparinized blood was collected from all six according to the schedule outlined above to assess virus replication and host responses. Cocultivation of 10 6 PBMC of each animal with C8166 cells failed to yield infectious virus from any of the vaccinated animals at any time point (Table 12), even after depletion of CD8 + T cells. However, PCR analysis of PBMC from all six revealed SIV mac gag DNA sequences, showing that all six had become infected with the vaccine virus. The infection was substantiated by immunoprecipitation analysis of plasma obtained 16 weeks after vaccination. This assay showed that all six had developed binding antibodies to SIV mac gag and HIV-1 env proteins. By 19 weeks, all six had developed neutralizing antibodies at titers of 1 : 10 and 1 :20 tp SHIV KLM ; CD4 + T cell counts remained normal in all of the six vaccinated macaques. Thus, although the vaccine virus caused a persistent infection there was little evidence of productive replication of the virus. At 7 months following vaccination, the six vaccinated macaques and two unvaccinated controls (PFy and PLy) were inoculated intravaginally with 1 ml of undiluted SHIV KU ., containing 10 4 TCID 50 . The inoculation was repeated 5 weeks later to ensure adequate exposure of the animals to the challenge virus. Following challenge, the two unvaccinated controls developed the same massive systemic infection typically caused by SH_V KLM in pig-tailed macaques (Joag et al. 1997. J. Virol. 71:4016-4023.; Joag et al. 1997. AIDS Res. Hum. Retroviruses 13:635-645). These control animals developed infectious viremia of 10 3 to 10 4 TCID 50 /ml and plasma antigenemia with p27 levels of between 1 ,000 and 2,000 pg/ml during the first 3 weeks post-challenge, accompanied by high numbers of circulating infectious PBMC. The productive infection was followed by rapid loss of more than 90%> of CD4 + T cells within 3 weeks following exposure to the virus (Table 12). Macaque PLy developed AIDS approximately 12 weeks after challenge. The other animal (PFy) developed an acute infection similar to that in PLy but developed a more chronic disease course. It became progressively cachectic, and at 58 weeks, when it was euthanized in extremis its viral RNA burden in plasma was 2.9 x 10 6 copies/ml (Table 13). The six vaccinated macaques all became infected with SHIV KU _ ! after challenge, as indicated by PCR analysis of DNA obtained from lymph node biopsies. PCR analysis showed evidence of an open vpu gene, consistent with that of SHIV KL (Fig. 4; Table 15). However, none of the six developed the highly productive infection seen in the two unvaccinated controls and as is typical of the infection caused by the SHIV KU _,. Examination of PBMC from PDj, PLk, PNa, and PPm showed that nearly all of the sequentially collected blood samples lacked infectious cells. Rarely, low numbers of infectious PBMC appeared, but these occurrences were transient. At 52 weeks, the viral RNA level in PPm, PDj, and PLk was less that 600 copies/ml (Table 14). One blood sample from PNA at week 52 showed infection in 10 cells per 10 6 PBMC and this was accompanied by a plasma RNA level of approximately 6,000 copies/ml. Two weeks later, however, infected cells had again disappeared from blood sample, and at 58 weeks its viral RNA level in plasma was back to <300 copies/ml. Macaques 42105 and 42107 developed a more productive type of infection characterized by the persistent presence of small numbers of infectious cells in PBMC (Table 12), although neither animal developed infectious viremia or plasma antigenemia. Both animals developed approximately 50% loss of CD4 T cells by 3 weeks following virus challenge, and this cell count stabilized at this level in macaque 42105 (Table 13). However, in 42107, the cell count underwent a second period of decline at approximately 15 weeks, and the animal developed AIDS at 24 weeks. Macaque 42105 developed better control over virus replication, with no evidence of infectious cells in blood between weeks 26 and 50. However, infectious PBMC reappeared after this time period. At 52 weeks post-challenge, when the blood sample had 10 infectious cells per 10 6 PBMC, the corresponding plasma sample had approximately 5,000 copies/ml of viral RNA. At 58 weeks, the plasma had 70,000 copies/ml (Table 14). Thus, of the six animals that were vaccinated and challenged, all became infected with the challenge virus. One developed a productive infection and succumbed to AIDS, and another remains at risk for developing late-onset disease. Except for transient sporadic appearances of infectious cells in blood, the other four macaques developed total control over replication of the challenge virus and have resisted disease. Vaccine Group 2 Since the death of macaque 42107 and the low-grade persistent infection in macaque 42105 may have been the result of inadequate immunity resulting from poor replication of vaccine virus 1, we sought to use another vaccine virus that had the potential for more robust replication and also one that could be administered orally. Table 13. CD4 * counts in blood of macaques following intravaginal inoculation with SHIV k KU-1 No. of CD4 * Cells/ml Wk Controls Vaccine Group 1 Vaccine Group 2 PFy PLy 4267 PDb 42105 42107 PDj PLk PNa PPm 42106 7024 8124 PEy PW1 PWv 0 866 1,729 467 1,394 1,249 1,152 1,507 630 1,217 1,197 1,056 1,636 676 1,149 1.053 1.137 1-2 106 ND" 9 290 ND ND ND ND ND ND 974 1.355 666 1,721 909 1,104 3-4 0 26 1 51 538 449 1,728 592 792 1,254 1,151 1,033 734 1,531 995 1,304 5-6 0 1 1 99 884 584 1,446 548 974 670 940 968 523 1,588 1,008 1,235 7-8 0 22 0 127 554 415 1,174 389 1,439 1,066 972 1,372 616 1,557 1,008 1,092 9-11 0 0 ND ND 505 360 1,158 472 1,341 748 ND ND ND ND ND ND 12-13 ND 0 6 68 515 342 1,243 521 1,046 774 1,176 684 674 1,608 1,251 1,062 14-17 72 0 86 570 344 1,176 699 1,076 869 745 1,209 436 1,352 975 918 18-20 55 0 531 98 1,479 764 1,617 961 236 430 113 812 616 573 21-23 0 54 696 226 1,216 743 915 513 575 825 86 694 324 646 24-26 160 0 514 139 1,601 722 960 460 857 1,466 442 1,088 886 1,331 27-30 141 163 283 1,527 592 706 508 255 359 121 561 319 544 31-34 9 65 818 1,434 693 1,071 544 255 916 72 689 624 290 35-38 15 145 515 566 522 392 480 255 779 156 291 724 702 39-42 22 5 569 749 439 1,056 501 386 858 195 1,421 734 900 43-46 0 573 497 272 182 69 47-50 68 656 528 271 511 277 51-54 0 639 1,003 763 668 446 55-58 0 280 516 169 258 214 59-62 833 947 693 895 534 α ND, not deteπnined Six macaques were inoculated orally with 10 4 TCID 50 of vaccine 2 in 1 ml of tissue culture medium. The inoculum was deposited on both sides of the base of the tongue, aimed at the pharyngeal tonsils. As in tests on animals inoculated with ΔvpuΔnef SHIV-4, we bled the second vaccine group at indicated intervals and tested for evidence of virus replication. As shown in Table 12, all six animals developed a productive infection in PBMC by week 2 after oral inoculation. This was characterized by the appearance of infectious PBMC, although none of the vaccinated animals developed infectious viremia or plasma antigenemia. Infection in the PBMC lasted 6 to 10 weeks, after which productive replication was brought under control and virus could no longer be isolated from PBMC of the animals, even after depletion of CD8 + T cells. All six animals developed immunoprecipitating antibodies to the gag of SIV mac and env of HIV-1, and also neutralizing antibodies at titers of 1 :10 to 1 :80 to SHIV, K..U-1 - Table 14. Detection of viral load in plasma and viral RNA in lymph nodes of study animals Detection of lv ph node RT-PCR Animal Plasma viral RNA at indicated pol Multiply spliced wk postinlection GAPDH Outer pπmei Inner Primei Outer pπmei Inner pπmer Vaccine group 1 52 58 PFy" 380,000 2,900,000 ++ +++ + ++ p L αb ND C ND ND ND ND ND ND 42105 5,200 70,000 - + 42107 B ND ND ND ND ND ND ND PDj <600 <600 + +++ PLk <600 <600 + + PNa 6,100 <300 +++ +++ PPm <600 <300 ++ +++ Vaccine group 2 27 35 PDb" 440,000 490,000 ++ + +++ + ++ 4267"" ND ND ND ND ND ND ND 702 <600 <300 + + 8124 <600 <300 + + + 42106 <600 <300 ++ +++ PEy <600 <300 + PW1 <600 <300 ++ PWv <600 <300 + " Mock Vaccinated h Dead horn AIDS at tune of test c ND, not determined At 24 weeks following immunization, the six vaccinated macaques and two unvaccinated controls were inoculated intravaginally with SHIV KU .„ and the inoculation was repeated 24 hours later. The two unvaccinated controls developed the typical massive infection caused by SHIV KU .,. Viremia, high numbers of infectious PBMC and more that 90% loss of CD4 + T cells characterized the early phase of these infections (Tables 12 and 13). One was euthanized with AIDS at 12 weeks and the other PDb, is still alive in a cachectic state. Biopsy and examination of axillary lymph nodes from five of the six vaccinated macaques at 18 weeks showed an open vpu gene in three (Table 15), demonstrating that the animals had become infected with SHIV KU _,. However, as for the four macaques in group 1, virus was recovered only rarely from PBMC of these vaccinated macaques at any time point. The animals have remained healthy and failed to develop virus- associated loss of CD4 + T cells during more than 40 weeks of examination. Considerable menstrual blood loss among these retired breeders, especially 8124, appeared to be associated with the wide fluctuations of CD4 + T-cell counts in individuals. Whatever the reason for loss of these cells, it was not associated with virus replication (Tables 12 and 15). Thus, although all six had become infected with virus crossing the vaginal mucosa and entering the regional lymph nodes, no systemic productive viral replication ensued in any of them. At 27 and 35 weeks post-challenge, plasma RNA levels in the six vaccinated macaques were less that 600 and 300 copies/ml, respectively, whereas the surviving unvaccinated challenge control animal had 490,000 copies/ml (Table 14). Since nearly all of the animals receiving either vaccine had undetectable levels of virus in the plasma, we sought to determine the status of virus replication in lymph nodes. Studies of HIV-infected people have shown that replication-competent virus can be rescued from lymph nodes (Chun et al. 1997. Proc. Natl. Acad. Sci. USA 94: 13193-13197; Finzi et al. 1997. Science 278: 1295-1300; Wong et al. 1997. Science 278: 1291-1295), and active virus replication can be demonstrated in lymph nodes with the use of primers for spliced mRNAs, even while the plasma has less than 400 copies of viral RNA/ml (Gunthard et al. 1998. J. Virol. 72:2422-2428). Since lymph nodes of vaccinated macaques contained pro viral DNA of the challenge virus, SHIV KU _,, we considered it important to determine whether the virus was inactive (trapped in follieular dendritic cells) or was replicating. Lymph node RNA was prepared from biopsies obtained from all surviving 11 of the 12 vaccinated macaques at weeks 54 and 60 (group 1) and weeks 31 and 37 (group 2). RT-PCR amplification of lymph node RNA from the five vaccinated macaques of group 1, using primers to the SIV pol gene, did not reveal detectable SHIV after 35 cycles despite obvious low-grade infection in PBMC of 42105 (Table 14). However, the RNA from the lymph node of 42105 was not of good quality, as shown by the lack of GAPDH signal. The one remaining unvaccinated challenged animal, Pfy, showed a strong signal in the initial reaction with the outer primers. However, lymph nodes of four of the five vaccinated macaques in group 1 did show a specific signal upon nested set amplification of 34 further cycles. In group 2, again, only the unvaccinated animal showed a signal upon the initial RT-PCR (not shown), but five of the six vaccinated animals (the exception being 42106) showed a signal upon nested set amplification. These assays detected full-length mRNAs. Since these could have been viral RNA in virion particles trapped in the lymph nodes, we used a different set of primers spanning the introns of the tat and rev genes to detect multiply spliced viral mRNAs, indicative of replication. The two remaining unvaccinated challenge animals (Pfy and PDb) and 1 of the 1 1 vaccinated macaques (macaque 42105 in group 1) had a detectable signal with these primers. However, nested PCR showed that two other group 1 animals (PD and PPm) and 3 group animals (8124, 42106, and PWv;) had positive signals (summarized in Table 14). Thus, virus replication, albeit at an extremely low level, was still in progress in three of the five vaccinated macaques in group 1 and three of the six in group 2. Table 15. Infectious cell frequency and PCR analysis of mesenteric lymph nodes at week 19 post-challenge from vaccine group 1 and Pfy and of axillary lymph nodes at week 18 post- challenge from PDj and vaccine group 2 Animal Infected cells/ PCR 10 h LNC gag V Controls Pfy 100 + +c PDb 10 + +c Vaccine Group 1 42105 5 + +c 42107 1,000 + +c PDj 0 - +v PLk 0 + +c PNa 0 + +c PPm 10 + +c Vaccine Group 2 7024 0 + +v 8124 0 + +c PEy 0 + +V PWv 0 + +c 42106 0 + +c The vpu that was amplified is indicated as C (challenge vims [SH1V KLM ] or V (vaccine vims). Both challenge and vaccine vims sequences were amplified from Pna Since virus replication was in progress in the lymphoid tissues but had been kept in check in 10 of the 12 vaccinated macaques, we sought to determine whether the vaccinated macaques had developed anti-viral cell-mediated immune (CMI) responses. We determined first whether the animals had acquired virus-specific T-helper (Th) cells by using assays for lymphocyte proliferation after exposure of PBMC to virus. Four of the five macaques in group 1 and all six macaques in group 2 displayed moderate to excellent responses when stimulated in vitro with inactivated challenge virus (Table 16), suggesting that major hisotcompatibility complex class Il-restricted antiviral Th cells may have been expanded in these vaccinated macaques. The single surviving unvaccinated challenge control animal, PDb, failed to display any significant proliferation of T cells in vitro. It is thus of interest that this animal, which had DNA of both vaccine and challenge viruses in it lymph node and had controlled virus replication completely, showed no significant proliferation of T cells. In contrast, macaque 42105, the only member of the group that had an ongoing productive infection, had the highest SI value. Primed relevant Th cells were detected in all of the vaccinated macaques in group 2. While SI values may not be markers of resistance to virus replication, they may contribute to the development of such resistance. In addition to performing lymphoproliferation assays, we are also deterring whether the resistant animals had developed antiviral CTL and report here on preliminary studies on two vaccinated macaques. We took advantage of the fact that not only did the disease- resistant, challenged vaccinated macaques have CD4 T T cells in peripheral blood that were uninfected with SHIV KU . | , but these cells were fully susceptible to infection with this virus I culture. We therefore immortalized CD4 + T cells by infection with Herpesvirus saimiri and obtained clones which were then expanded, inoculated with SHIV KL , and used in autologous reactions as stimulators and targets. Two animals, PDj and PNa, from which we had derived our first T-cell clones, were selected for study. PBMC collected at 50 weeks post-challenge from these two animals were stimulated with SH_V KU _, -infected autologous CD4" T cells at an effector/stimulator ratio of 10: 1 and resfimulated again in the same fashion on day 7. These bulk T-cell lines were used as effector cells for detecting CTL activity 2 weeks after the first stimulation. Both cell populations displayed excellent cytotoxic activity while showing no (PNa) or very little (PDj) lysis against control targets. sponses to SHIV KU. ,in Vaccinated Animals Immunogen Monkey SI Vaccine 1 42105 9.10 PLk 8.19 PPm 7.87 PDj 2.29 PNa 1.31 Vaccine 2 42106 3.78 7024 7.17 8124 12.39 PEy 3.62 Pwl 2.78 PWv 4.94 Virus Control PDb 0.91 DISCUSSION Six sexually mature macaques inoculated orally on a single occasion with ΔvpuSHIVpp. (group 2) developed an uneventful, transiently productive infection that conferred protection against AIDS caused by intravaginally inoculated, highly virulent SHIV KU _, (χ 2 test, P = 0.005. All four of the unvaccinated control macaques inoculated intravaginally with this virus developed highly productive infection, subtotal loss of CD4 + T cells, and AIDS, confirming our finding (Joag et al. 1997. J. Virol. 71 :4016-4023) on development of disease in six of six animals that became infected after nontraumatic intravaginal infection of this virus. The first vaccine virus, pvpupnefSHI V-4, was incapable of causing infection after mucosal inoculation (and therefore was injected subcutaneously) and replicated poorly in all six macaques, but nevertheless induced binding and neutralizing antibodies to the virus, proving infection. Four of these six vaccinated macaques resisted productive infection and disease following intravaginal challenge with SHIV KLM (χ 2 test, P = 0.102). Of the two animals that developed productive infection, one developed AIDS and another remains at risk of developing AIDS. Given this prognosis, ony 2 of the 12 vaccinated macaques, compared to four of four of the control animals, developed disease ( 2 test, P = 0.003). These results demonstrated that sexually transmitted disease caused by HIV-1 in human beings can probably be prevented by prophylactic immunization. Both vaccine viruses caused persistent infection irrespective of whether they replicated productively during the first few weeks following inoculation. Six months following immunization, even though infectious virus could no longer be isolated from PBMC from any of the 12 vaccinated macaques, vaccine virus DNA was still detectable. All 12 animals had developed binding and very low neutralizing antibody titers to SHIV KU _, in plasma by the time of virus challenge (approximately 6 months following vaccination). As shown, all 12 became infected with the challenge virus, since its DNA was clearly detectable in PBMC or mesenteric lymph node a few months following challenge. Two of the six vaccinated macaques in group 1 developed a persistent productive infection in PBMC and one, 42107, succumbed to AIDS approximately 6 months following challenge. The other macaque, 42105, had a phase of low-grade productive infection for 30 weeks followed by a 12-week period when it had no circulating infections PBMC in blood. However, from week 50 onward, infectious PBMC, accompanied by relatively low concentrations of viral RNA (5,000 to 70,000, versus 4 X 10 5 to 3 X 10 6 in controls) have reappeared in blood. At 60 weeks post-challenge, this animal still has large numbers of uninfected CD4 + T cells (which are highly susceptible to infection), in contrast to control animals, nearly all of whose CD4 + T cells became infected and eliminated during the first 3 weeks of infection. Nevertheless, the resurgence of virus in macaque 42105 suggests that this animal may be at risk for developing late-onset AIDS. The other 10 vaccinated macaques, 4 in group 1 and all six in orally immunized group 2, developed a unique type of control over replication of SHIV KU .,. Rarely and transiently, small numbers of infectious cells have appeared in blood, but at nearly all time points, replication-competent virus could not be isolated from PBMC or lymph (sampled two to three times during this period of observations) by any procedure. Attempts have included three successive cocultivations of CD8-depleted PBMC with mitogen-activated normal PBMC or T cell lines. Nevertheless, both viral DNA and viral RNA have persited in lymph node tissue. Further, the detection of spliced viral RNA in lymph nodes of few animals in both groups suggested that an extremely low level of replication of SHIV KU _, has probably been in progress continuously in all of the vaccinated macaques. This type of replication of SHIV KU _, has probably been in progress continuously in all of the vaccinated macaques. This type of replication of this virus had never been observed before. We have previously demonstrat4ed (see above) that viral DNA became detectable by day 2 after inoculation of mesenteric lymph nodes and that explosive replication of the agent occurred during the following week. In this study, although the intravaginal route of inoculation was shown to be about 30-fold less sensitive than the intravenous route, all animals that became infected after intravaginal inoculation nevertheless developed disease. Extrapolating these data to our vaccinated macaques, since all had developed infection in lymph nodes, we surmised that systemic rather than mucosal factors were responsible for curtailment of virus replication. It is doubtful that neutralizing antibodies had a significant role in curtailment of virus replication in the vaccinated macaques. In an earlier report (Foresman et al. AIDS Res. Hum. Retroviruses 14:1035-1043), we showed that passively administered immune serum was effective only when given before parental inoculation of the virus. This serum had no therapeutic effect when given 2 h following virus inoculation. Worse still, unlike the success at preventing infection after parental inoculation of the virus, we were unable to protect macaques from the dire effects of infection by infusing the serum 24 h before mucosal inoculation of the agent. Immune serum therefore had no apparent effect on an already established infection, a well-known phenomenon in lentivirus infections, nor apparently could the previously administered serum prevent infection across mucosal surfaces. Restriction of virus replication in our 10 vaccinated macaques must therefore have been mediated by factors other than neutralizing antibodies. Studies on CMI responses of the vaccinated macaques have only recently begun. That this type of immunity is important came from an earlier finding that macaques that had "recovered" from infection with virulent SIV mac were protected from effects of infection with SHIV KU _, (Stephens et al. 1997. Virology 234:328-339). Since these two viruses have distinctly different envelopes, the protection was probably mediated by CMI responses to viral core proteins shared by the two viruses. In the present study, our findings that CD4 + T cells in the vaccinated macaques at week 50 in group 1 and in week 30 in group 2 proliferated when exposed to antigens of SHIV Klw , showed that the animals had developed anti-SHIV CMI responses that have persisted along with the infection. The further preliminary finding that 2 of the 10 vaccinated macaques that have controlled virus replication have developed CTL to the virus has strengthened the premise that CMI responses are responsible for control and curtailment of replication of the virulent virus. Possibly, a dynamic interaction between virus replication and CTL curtailment responses is continuously at play. A recent study of CTL responses in HIV-infected humans showed an inverse correlation between the levels of CTL and plasma viral RNA loads (Musey et al. 1997. N. Engl. J. Med. 337:1267-1274), implicating a role for CTL control of viral replication and delay of disease progression. Example 10. Reduction in challenge virus DNA burden HIV-1 causes both highly productive and clinically latent phases of infection, but vaccination approaches have focused primarily on prevention of the former. We used the SHIV KU _, macaque model of HIV infection/disease to investigate the feasibility of vaccine prevention of both parameters of infection. Using an oral vaccine, Δ-vpuSHIV PPc (V-virus), we show that vaccinated animals became infected in lymph nodes with challenge virus SHIV KL (C-virus), but productive C-virus replication was suppressed. DNA of both V- and C-viruses persisted in lymph nodes of all six animals, but during the second year after challenge, four of the six animals lost the C-virus DNA signal. This was associated with persistence of V-virus and prominent CTL responses. This is the first demonstration that vaccination against a pathogenic primate lentivirus can cause reduction of challenge virus burden, including its DNA intermediate phase in lymph nodes. This suggests that a reduction in the level of challenge virus DNA could be included among the efficacy requirements of an HIV vaccine. The ultimate goal of all viral vaccines is to emulate the specific immunity that heralds the end of infection by pathogenic viruses (Murphy BR & Chanock RM. Immunization against virus disease. In Fields Virology (Ed. Fields, B.N.et al.) 467-97 (Lippincott-Raven, New York, 1996). Application of this concept to HIV-1 creates a theoretical stumbling block for vaccine development because, in most cases of HIV infection, specific immunity to the virus causes reduction in viral replication, but it does not eliminate viral DNA from cellular reservoirs in lymph nodes where it is thought to cause latent infection. (Chun, T.W. et al. Nature 387, 183-8 (1997)) A large number of vaccines have been tested in the SIV-macaque model of HIV infection and pathogenesis, and claims of success have been based on prevention or delay in disease onset, or reduction of productive virus replication, but none has yet approached the question of elimination of DNA of the challenge virus from lymph nodes. To explore the question of limits of protection against replication of challenge virus, we inoculated two groups of six pig-tailed macaques with two live vaccines, ΔvpuΔnefSHIV- 4 (vaccine 1 , or V-I) and ΔvpuSHIV PPc (vaccine 2, or V-II), respectively, and challenged them by vaginal inoculation with SHIV KU _, (C-virus) six months later. MATERIALS AND METHODS Identification of vaccine virus and challenge virus DNA sequences by PCR amplification of the vpu region. DNA was isolated from lymph node tissue or PBMC by standard procedures The vpu/gpl20 regions of viral genomes were amplified by nested PCR that produces fragments diagnostic for either vaccine virus DNA or challenge virus DNA as described above. After the second round of amplification, challenge virus DNA, which has an intact vpu gene, yields a fragment of 431 bp while vaccine virus DNA, with a 60 base pair deletion in the vpu gene, yields a fragment of 371 bp. Amplification of each sample was repeated 4 times in separate reactions to ensure accuracy. Virus recovery from lymph nodes. Lymph node cells were obtained from mesenteric lymph node tissue and CD4 * cells were negatively selected using immunomagnetic beads as described above. The CD4 * cells were counted, stimulated with PHA (lμg/ml) for 48 hr, and 2 xlO 6 cells were co-cultured with 1 x 10 6 C8166 indicator cells. After co-cultivation for 7 days, the cultures were examined for CPE. Lymphocyte proliferation assay. PBMC from different macaques, collected at week 98 or 99 post-challenge, were cultured in triplicate in the presence or absence of UV irradiated and heat inactivated SHIV KU _, for 4 days. Cells were then pulsed with lμCi [3H] thymidine for 18 hours before being subjected to scintillation spectroscopy. Stimulation indices were calculated as mean counts per minute (CPM) in stimulated wells/mean CPM in control wells. Generation of bulk CTL population and chromium release assay. CTL activity was determined as described elsewhere 12 . Briefly, CTL effectors were prepared by stimulating PBMCs from different macaques with UV irradiated autologous CD4 + T cells infected with SHIV KL . Autologous CD4 + T cells infected with SHIV KU _, or autologous B cells infected with recombinant vaccinia virus expressing HIVenv or SIVgag or SIVpol were used as targets in 4 hr. chromium release assay. The percent specific cytotoxicity was calculated as (test release- spontaneous release)/(maximum release-spontaneous release) X 100. Initially, four unvaccinated control macaques developed typical acute infection with subtotal loss of CD4 + T cells within one month, and also subsequently developed AIDS. All twelve vaccinates became infected in lymph nodes with C-virus and infection spread to peripheral blood mononuclear cells (PBMC) in some of the animals during the early weeks following challenge. Two animals in the V-I group developed a slowly progressive infection that culminated in AIDS. The other four in the group eventually curtailed replication of the virus in PBMC and remained healthy with normal levels of CD4 * T cells. However, infectious C-virus was rescued from lymph node cells from three of these animals two years later. This indicated that the infection had regressed to a persistent, clinically latent stage in the lymph nodes. Among the animals in the V-II group, all six resisted productive replication of the virus and all have also remained healthy for more than two years. Furthermore, infectious SHIV KU. , (C-virus) was rescueable from lymph node cells from only one of the six. Using primers that distinguish between the truncated vpu of the V-viruses and the full length vpu of C-virus, we monitored the animals for evidence of persistence of DNA of either or both viruses in PBMC and in lymph nodes, and compared these results with results regarding recovery of infectious virus from lymph node tissue from these animals. These data are summarized in Table 17. Animals were also monitored for humoral and cellular immune responses to C-virus. C-virus-antigen-specific CD4 + T cells were detected in all ten animals throughout the post-challenge period between weeks 40 and 110. All ten surviving animals also had CTLs that recognized HIV env, and SIV gag and pol epitopes expressed by H. papio transformed B cells infected with recombinant vaccinia viruses. They also developed long- lasting CTLs directed against C-virus. Table 17. Virus infection after challenge with SHIV κυ + indicates presence of infectious PBMC D = dead/week; * = virus isolated at time of death Virus isolation from LN: C = challenge virus V = vaccine virus Viral DNA in LN: C, V, or C+V indicates which virus DNA is represented All four surviving animals in the V-I group had DNA of V- and C-viruses, and as mentioned above, C-virus was isolated from CD8 " -depleted lymph node cells from three of the four at week 87. Examination of the V-II animals showed surprising results; V-virus was detected in all six animals throughout the post-challenge period up to the last timepoint at 103 weeks. Whereas all six animals in the VII group had C-virus DNA at week 18 post-challenge, the DNA signal for this virus became undetectable in one (PWv) at week 55, and in three others (PEy, PW1, and 7024) by week 63. The lack of C-virus DNA signal in lymph node and PBMC of the four animals was confirmed in tissue samples obtained at weeks 91 and 103. Thus, at the last time point sampled, 103 weeks post-challenge, four of the six animals showed only V-virus DNA. V-II-like viruses were isolated from CD8 * depleted lymph node cells from two of these animals. Of the remaining two that had DNA of both viruses (42106 and 8124), lymph node cells from one (8124) yielded C-virus. Isolated viruses were identified by the presence or absence of vpu, and by nucleotide sequence analysis of gp\2Q and nef. Comparison of these sequences with those of V-II virus DNA and C-virus DNA showed that the newly isolated viruses have scattered mutations in gp\2Q and nef. The viruses isolated from PEy and PW1 in the V-II group are variants of V-II virus, and the agent isolated from 8124 is a variant of C-virus (data not shown). There was no evidence of recombination between V- and C-virus DNAs in any of the three newly derived agents. The validity of the claim for reduction in C-virus DNA burden in the V-II animals is supported by the fact that a single set of primers was used to amplify both species of DNA. Because the primers bind to identical sites in both V-virus DNA and C-virus DNA, and the sequences of the amplified fragments differ only by a deletion of 60 bp, preferential amplification of one species was extremely unlikely. Effectively, the amplification of one species served as an internal control for the amplification of the other species. Thus, the detection of V-virus DNA and C-virus DNA in PBMC at week 55, and detection of only V- virus DNA in the four animals at 3 later time points, reflected an actual decline in the level of C-virus DNA in these animals. This finding is further supported by the failure to recover C- virus from any of these four animals. The results from this study show that ten of the vaccinates not only resisted C-virus- mediated disease, but appeared to have gained control over productive replication by the virus, since CD4+ T cell counts in the animals were within normal ranges, their PBMC were devoid of infectious virus and viral DNA, and all ten had equivalent levels of cell-mediated immune responses to the virus. Antiviral immunological responses of animals in the two groups provided no indication of the differences in effectiveness between the two vaccines. CD4 + T cells were equally responsive to viral antigens and CTLs recognized epitopes in all three of the structural proteins of the virus and in autologous CD4 + T cells infected with C-virus (Table 18). Although the study on CMI responses was not initiated until approximately 40 weeks post-challenge, this immune response was already in place in animals in both groups when, in the V-II group, not only had infectious virus been eliminated, but viral DNA was in the process of being eliminated in most of the animals. This contrasted with the V-I group in which infectious C-virus persisted. Thus, effector CTLs to the structural proteins of the virus did not determine whether or not infectious virus persisted in lymph nodes. It is possible that the sudden decline in the C viral DNA signal was associated with activation of replication of the vaccine virus since antigenically distinct vaccine viruses were obtained from 2 of the 4 vaccinates at 63 weeks. The success of the vaccine may have been due to its ability to replicate and concomitantly induce CTLs to both structural and non-structural proteins of the virus. This constellation of immune responses may be required for complete elimination of pathogenic virus. Table 18- Cellular immunological profile in macaques immunized with Vaccine I or Vaccine II and challenged with SHIV KU .,. Vaccine Macaque Stimulation' Percent specific lysis against b index SHIV KU ., , c HIVenv d SI VI PDj 23.1 54.2 43.2 29.7 32.1 PNa 4.3 48.6 39.5 28.4 21.7 PPm e 6.3 46.0 29.5 21.1 20.5 PLk 6.0 51.2 45.1 32.3 25.2 42105 3.9 14.1 0.0 18.5 12.2 VII 42106 45 471 276 376 321 7024 35 377 273 501 366 8124 302 627 275 386 270 PEy 45 430 363 443 360 PW1 46 514 385 523 405 PWv 44 469 309 439 362 a Macaques inoculated with either VI or VII were tested for SHIV,. υ , specific T-helper cells at week 98 and 99 post-challenge, respectively b Macaques inoculated with either VI or VII were tested for CTL activity at week 106 and 96, post-challenge respectively Specific lysis shown here are at Effector/Target ratio of 40 1 c SHIV ku , specific lysis was determined by infecting Con-A stimulated CD8 + depleted PBMC with SHIV KL , and using them as targets in a 4 hr chromium release assay d Envelope, gag, and pol specific lysis was determined by infecting H papio transformed B-LCL with recombinant vaccinia virus expiessmg either protein, and using them as targets m a 4 hr chromium release assay 'Macaque PPm's Effectors were tested for CTL activity at the time of necropsy (Week 92) The invention now being fully descπbed, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spiπt or scope of the invention

Claims: 1. A method for inoculating a subject to inhibit virus infection by sexually transmitted HIV, comprising orally administering to a subject to be inoculated, a vaccine comprising a virus wherein the DNA of said virus comprises DNA consisting of HIV-2 LTR, gag, pol, and nef genes and HIV-1 env, tat, rev, and vpu genes, wherein said vpu gene is rendered nonfunctional by full or partial deletion, or by insertion of DNA into the vpu gene. 2. The method of claim 1 wherein said vpu gene is rendered non- functional by partial deletion. 3. The method of claim 1 wherein said vpu gene is absent. 4. The method of claim 1 wherein said nef gene is rendered non-functional or is deleted. 5. A method for inoculating a subject to inhibit virus infection by sexually transmitted HIV, comprising orally administering to a subject to be inoculated, a vaccine comprising a virus wherein the DNA of said virus comprises HIV-1 DNA wherein the vpu gene of HIV-1 has been rendered non-functional by full or partial deletion, or by insertion of DNA into the vpu gene. 6. The method of claim 5 wherein said vpu gene is rendered non-functional by partial deletion. 7. The method of claim 5 wherein said vpu gene is rendered non-functional by full deletion. 8. A method for inoculating a primate to inhibit virus infection by SIV or HIV, comprising orally administering to a subject to be inoculated, a vaccine comprising a virus wherein the DNA of said virus comprises DNA consisting of SIV LTR, gag, pol, and nef genes and HIV-1 env, tat. rev, and vpu genes, wherein said vpu gene is rendered nonfunctional by full or partial deletion, or by insertion of DNA into the vpu gene. 9. The method of claim 8 wherein said vpu gene is rendered non-functional by partial deletion. 10. The method of claim 8 wherein said vpu gene is absent. 1 1. The method of claim 8 wherein said we/gene is rendered non-functional or is deleted.

Inventors: NARAYAN OPENDRA, NARAYAN, OPENDRA
Applicants: NARAYAN OPENDRA, UNIV KANSAS MEDICAL CENTER, NARAYAN, OPENDRA, UNIVERSITY OF KANSAS MEDICAL CENTER
Application year: 1999
Main classification: A61K39
IPC codes: A61K39, A61P31