Persistence of low levels of simian immunodeficiency virus in macaques that were transiently viremic by conventional testing

Transient SIV viremia after experimental SIV challenge has been documented. Whether SIV persists in these transiently viremic macaques remains unclear. In the present study, we applied a sensitive PCR and found persistent low levels of SIVmne infection (LLSI) (range: 0.1-5.3 SIV DNA copies/10(6) PBMC) in seven macaques that were transiently positive by conventional assays, which was 10(2)- to 10(6)-fold less than those of SIVmne infected monkeys with typical disease progression. SIV envelope V1 sequences remained homogeneous in these macaques for the 6-year study period, with a mean evolution rate of 0.005% per site per year, which was not different from zero (P = 0.612) and significantly lower than that (0.56-1.18%) in macaques with progressive infection of SIVmne. LLSI macaques have remained free from SIV-associated illness, and are still alive 10 years after virus inoculation. Understanding the mechanisms underlying this outcome may provide valuable insight into therapy and vaccine development.     

Immune Responses but No Protection against SHIV by Gene-Gun Delivery of HIV-1 DNA Followed by Recombinant Subunit Protein Boosts

The efficacy of combining immunization with human immunodeficiency vitus type 1 (HIV-1) DNA and HIV-1 recombinant proteins to obtain protection from chimeric simian/human immunodeficiency virus (SHIV) was determined. Four cynomolgus monkeys received four gene-gun immunizations intraepidermally of plasmid DNA encoding HIV-1laienv(gp160),gag, tat, nef,andrevproteins. Ten micrograms of DNA was used per immunization. The animals were boosted twice intramuscularly with 50 μg of HIV-1laiEnv (MicroGeneSys), Gag, Tat, Nef, and Rev recombinant proteins mixed in Ribi adjuvant. The antibody responses were amplified following the administration of the recombinant subunit boosts. One month after the final subunit immunization, the vaccinated animals together with four control animals were challenged intravenously with 10 monkey infectious doses of SHIV that expresses theenv, tatandrevgenes of HIV-1 and gag and nef from SIV. However, only low titers of neutralizing antibodies were present at the day of challenge. The consecutive HIV-1 DNA and recombinant protein immunizations induced B- and T-cell responses but not protection against SHIV replication nor reduction of the viral load.     

Novel simian immunodeficiency virus CTL epitopes restricted by MHC class I molecule Mamu-B*01 are highly conserved for long term in DNA/MVA-vaccinated, SHIV-challenged rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques provides an excellent model for investigating the basis of protective immunity against human immunodeficiency virus (HIV). One limitation of this model, however, has been the availability of a small number of known MHC class I-restricted CTL epitopes for investigating virus-specific immune responses. We assessed CTL responses against SIV Gag in a cohort of DNA/modified vaccinia virus Ankara (MVA)-vaccinated/simian-human immunodeficiency virus (SHIV)-challenged rhesus macaques. Here, we report the identification of five novel SIV CTL epitopes in Gag for the first time (Gag(39-46) NELDRFGL, Gag(169-177) EVVPGFQAL, Gag(198-206) AAMQIIRDI, Gag(257-265) IPVGNIYRR and Gag(296-305) SYVDRFYKSL) that are restricted by the common MHC class I molecule Mamu-B*01. CTL responses to these epitopes were readily detected in cryopreserved PBMC in multiple animals up to 62 weeks post-infection, both by IFN-gamma enzyme-linked immunospot assay and intracellular IFN-gamma staining. Importantly, viral sequencing results revealed that these epitopes are highly conserved in the SIV-challenged macaques over a long period of time, indicating functional constraints in these regions. Moreover, the presence of CTL responses targeting these epitopes has been confirmed in two independent cohorts of rhesus macaques that have been challenged by SHIV or SIV. Our findings provide valuable candidates for poly-epitope vaccines and for long-term quantitative monitoring of epitope-specific CD8(+) responses in the context of this common Mamu class I allele. It may thus help increase the supply of rhesus macaques in which epitope-specific immunity can be studied in the context of SIV vaccine design.     

Replication of simian immunodeficiency virus (SIV) in ex vivo lymph nodes as a means to assess susceptibility of macaques in vivo

Six macaques, apparently uninfected, following low-dose exposure to the pathogenic SIV(mac251) and SIV(SME660) by the mucosal route, were used in a pilot study to investigate whether infectability of ex vivo lymph nodes could predict resistance and/or susceptibility to SIV infection in vivo. Of six macaques exposed to the less-pathogenic virus SIV(MNE), four resisted viral infection. Analysis of the susceptibility of the PBMC of these four animals before SIV(MNE) challenge indicated that all of them were resistant to infection by the SIV(BK28) isolate and, in three of them, this resistance was dependent on CD8+ T cells. Blocks of lymph nodes of these four macaques were resistant to SIV(MNE) infection ex vivo following SIV(MNE) viral challenge exposure. However, the same blocks from the same animals were permissive to the more virulent SIV(251(32H)). Accordingly, three of these macaques were readily infected following challenge exposure with SIV(251(32H)). Lymphoproliferative responses in blood or lymph nodes, local C-C chemokine production in the lymph-node explants, and cytotoxic T-cell activity measured throughout the study did not correlate with ex vivo resistance or susceptibility to in vivo infection. In conclusion, PBMC and lymph-node resistance or susceptibility to infection ex vivo appeared to correlate with in vivo infectivity and, thus, these approaches should be further tested for their predictive value for in vivo infection.     

SIV from stump-tailed macaques: molecular characterization of a highly transmissible primate lentivirus.

Over the past 6 years, simian immunodeficiency viruses (SIVs) have been isolated from four distinct species of macaques (Macaca mulatta, M. fascicularis, M. nemestrina, and M. arctoides) in captivity in the United States. However, the epidemiologic and genetic relationships among SIVs from the four species are not well understood. SIV from stump-tailed macaques (M. arctoides) (SIVstm) is unusual in that it has been associated with outbreaks of infection characterized by aggressive spread within stump-tailed macaque colonies at two separate primate centers in the United States. To characterize SIVstm at the molecular level, we have derived six biologically active viral DNA clones by polymerase chain reaction amplification of genomic DNA from infected cells. Nucleotide sequence analyses of one clone (SIVstm/37.16) showed that SIVstm was indeed a member of the previously defined group of simian lentiviruses that are closely related to the human immunodeficiency virus type 2 (HIV-2). However, our data indicate that SIVstm is equidistantly related to the other SIVs from macaques (SIVmac 251/142 and SIVmne) and SIV from African sooty mangabeys (SIVsmm). These findings suggest that SIV from captive macaques may have originated from several cross-species transmissions from imported sooty mangabeys and that additional spread has been fostered by the exchange of macaques among primate centers.     

Immunization of macaques with live simian human immunodeficiency virus (SHIV) vaccines conferred protection against AIDS induced by homologous and heterologous SHIVs and simian immunodeficiency virus

To evaluate the vaccine potential of SHIVs attenuated by deletion of viral accessory genes, seven rhesus macaques were sequentially immunized with Delta vpu Delta nefSHIV-4 (vaccine-I) followed by Delta vpuSHIV(PPC) (vaccine-II). Despite the absence of virological evidence of productive infection with the vaccine strains, based on analysis of infectivity among peripheral blood mononuclear cells (PBMC) of the vaccinated animals, all seven animals developed binding as well as neutralizing antibodies against both vaccine-I and -II. The animals also developed vaccine virus-specific CTLs that recognized homologous as well as heterologous pathogenic SHIVs and SIV, and also soluble inhibitory factors that blocked the in vitro replication of the vaccine strains and different challenge viruses. Virus-specific cellular and humoral responses were sustained throughout a 58-week prechallenge period. To model aspects of natural transmission, the animals received a mucosal (rectal) challenge, with a mixture of three challenge viruses, SHIV(KU), SHIV(89.6)P, and SIV(mac)R71/17E. Two mock-vaccinated control animals inoculated with the same mixture of challenge viruses developed large numbers of infectious PBMC, high plasma viremia, and precipitous loss of CD4(+) T cells. The control animals did not develop any immune responses and succumbed to AIDS between 6 and 7 weeks postchallenge. All seven vaccinated animals became infected with challenge viruses as indicated by the presence of infectious cells in the PBMC and/or viral RNA in plasma. However, peak plasma viremia in vaccinates was two to nearly five logs lower than in the control animals and later plasma viral RNA became undetectable in all vaccinates. Vaccinated animals maintained normal CD4(+) T cell levels throughout the study. Challenge with pathogenic viruses caused massive anamnestic responses as determined by quantitation of virus-specific CD4(+) and CD8(+) T cells by intracellular IFN-gamma staining, and these cells persisted for at least 74 weeks. The study is still in progress and at this time DNA of SIV has become undetectable in lymph nodes of six of the seven vaccinates, SHIV(89.6)P in five of the seven, and SHIV(KU) in three of the seven animals.     

Vaccination of rhesus macaques with a vif-deleted simian immunodeficiency virus proviral DNA vaccine

Studies in non-human primates, with simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) have demonstrated that live-attenuated viral vaccines are highly effective; however these vaccine viruses maintain a low level of pathogenicity. Lentivirus attenuation associated with deletion of the viral vif gene carries a significantly reduced risk for pathogenicity, while retaining the potential for virus replication of low magnitude in the host. This report describes a vif-deleted simian immunodeficiency virus (SIV)mac239 provirus that was tested as an attenuated proviral DNA vaccine by inoculation of female rhesus macaques. SIV-specific interferon-gamma enzyme-linked immunospot responses of low magnitude were observed after immunization with plasmid containing the vif-deleted SIV provirus. However, vaccinated animals displayed strong sustained virus-specific T cell proliferative responses and increasing antiviral antibody titers. These immune responses suggested either persistent vaccine plasmid expression or low level replication of vif-deleted SIV in the host. Immunized and unvaccinated macaques received a single high dose vaginal challenge with pathogenic SIVmac251. A transient suppression of challenge virus load and a greater median survival time was observed for vaccinated animals. However, virus loads for vaccinated and unvaccinated macaques were comparable by twenty weeks after challenge and overall survival curves for the two groups were not significantly different. Thus, a vif-deleted SIVmac239 proviral DNA vaccine is immunogenic and capable of inducing a transient suppression of pathogenic challenge virus, despite severe attenuation of the vaccine virus.     

Protection from immunodeficiency virus challenges in rhesus macaques by multicomponent DNA immunization.

Multicomponent DNA vaccines were used to elicit immune responses, which can impact viral challenge in three separate rhesus macaque models. Eight rhesus macaques were immunized with DNA vaccines for HIV env/rev and SIV gag/pol and were challenged intravenously with 10 animal infective doses (AID(50)) of cell-free SHIV IIIB. Three of eight immunized rhesus macaques were protected, exhibiting no detectable virus. Animals protected from nonpathogenic SHIVIIIB challenge were rested for extended periods of time and were rechallenged first with pathogenic SIV(mac239) and subsequently with pathogenic SHIV89.6P viruses. Following the pathogenic challenges, all three vaccinated animals were negative for viral coculture and antigenemia and were negative by PCR. In contrast, the control animals exhibited antigenemia by 2 weeks postchallenge and exhibited greater than 10 logs of virus/10(6) cells in limiting dilution coculture. The control animals exhibited CD4 cell loss and developed SIV-related wasting with high viral burden and subsequently failed to thrive. Vaccinated animals remained virus-negative and were protected from the viral load, CD4 loss, disease, and death. We observed strong Th1-type cellular immune responses in the protected macaques throughout the study, suggesting their important roles in protection. These studies support the finding that multicomponent DNA vaccines can directly impact viral replication and disease in a highly pathogenic challenge system, thus potentially broadening our strategies against HIV.     

Substantial envelope-specific CD8 T-cell immunity fails to control siv disease

It is unknown which HIV proteins to target by vaccination in order to generate the most effective CD8 T-cell immunity. We recently immunized SIV_mac251-infected pigtail macaques with Gag peptides or a cocktail of peptides spanning all SIV proteins, including SIV Env. High-level SIV Env-specific CD8 T-cell responses were generated and 7 novel Env-specific CD8 T-cell epitopes in 10 animals were mapped. Env-specific CD8 T-cell responses were significantly inferior to Gag-specific responses, and no better than unvaccinated control animals, in the control of SIV replication and prevention of disease. Escape mutations emerged within several Env-specific CTL epitopes, suggesting at least some pressure imparted by the Env CTL responses, but this did not correlate with significantly reduced SIV replication. We conclude Env-specific CTL may not be the most effective response to induce by vaccination.     

Chimeric human papilloma virus-simian/human immunodeficiency virus virus-like-particle vaccines: immunogenicity and protective efficacy in macaques

Vaccines to efficiently block or limit sexual transmission of both HIV and human papilloma virus (HPV) are urgently needed. Chimeric virus-like-particle (VLP) vaccines consisting of both multimerized HPV L1 proteins and fragments of SIV gag p27, HIV-1 tat, and HIV-1 rev proteins (HPV-SHIV VLPs) were constructed and administered to macaques both systemically and mucosally. An additional group of macaques first received a priming vaccination with DNA vaccines expressing the same SIV and HIV-1 antigens prior to chimeric HPV-SHIV VLP boosting vaccinations. Although HPV L1 antibodies were induced in all immunized macaques, weak antibody or T cell responses to the chimeric SHIV antigens were detected only in animals receiving the DNA prime/HPV-SHIV VLP boost vaccine regimen. Significant but partial protection from a virulent mucosal SHIV challenge was also detected only in the prime/boosted macaques and not in animals receiving the HPV-SHIV VLP vaccines alone, with three of five prime/boosted animals retaining some CD4+ T cells following challenge. Thus, although some immunogenicity and partial protection was observed in non-human primates receiving both DNA and chimeric HPV-SHIV VLP vaccines, significant improvements in vaccine design are required before we can confidently proceed with this approach to clinical trials.     

Improved SIV DNA vaccine can be effectively used as a boost for Ad5 in prime-boost immunization strategy

In the study under evaluation, optimized SIV DNA were used to boost T-cell responses induced by a highly immunogenic SIV Ad5-prime in Chinese rhesus macaques. A regular prime-boost regimen (SIV DNA-prime and rAd boost) and naive macaques were used as the control. After vaccination, the animals were challenged intrarectally with SIVmac251, and partial protection was observed in the macaques immunized by the Ad5-prime DNA-boost regimen. SIV-specific T-cell responses in the enzyme-linked immunospot assay were significantly higher in the Ad5-prime DNA-boost, compared with the responses in the control macaques. Viral control correlated with the generation of HLA-DR+ T cells 2 weeks after the viral challenge. Further studies using prime and boost strategies and alternative routes of vaccination (including a simultaneous approach) are warranted to fully explore the potential of prime and boost regimens for HIV-1 vaccine development.     

Direct measurement of CD8+ T cell responses in macaques infected with simian immunodeficiency virus

The simian immunodeficiency virus (SIV) macaque model system has been used extensively to study AIDS pathogenesis and to test candidate vaccines for their ability to protect against homologous or heterologous challenge with pathogenic SIV or SHIV. Recent studies suggest that stimulation of HIV-1-specific CTL responses is important for effective vaccination against HIV-1. While quantitative measurements of SIV-specific cytotoxic T lymphocyte (CTL) responses have been facilitated by the use of tetrameric peptide complexes, this technique is currently limited to the study of Mamu-A*01-positive rhesus macaques. Furthermore, very few SIV-specific CTL epitopes have been identified, and there is limited identification of other MHC alleles in macaques. In this study, cytokine flow cytometry (CFC) was used to quantify SIV-specific CD8+ antigen-reactive T cells in macaques infected with SIV. We found a strong correlation (r = 0.96, P < 0.001) between CD8+ antigen-reactive T cells stained with the Mamu-A*01 p11C, C-M tetramer and production of intracellular TNF-alpha in the CFC assay. Furthermore, the CFC assay was used to identify a novel SIV-specific CTL epitope in Envelope (SIV Env, a.a. 486-494, sequence AEVAELYRL). The use of the CFC assay facilitates the study of antigen-reactive T cell responses in SIV infection and vaccination.     

Functional gene analysis of individual response to challenge of SIVmac239 in M. mulatta PBMC culture

It has previously been shown in macaques that individual animals exhibit varying responses to challenge with the same strain of SIV. We attempted to elucidate these differences using functional genomics and correlate them to biological response. Unfractionated PBMC from three rhesus macaques were isolated, activated, and infected with SIVmac239. Interestingly, one of the three animals used for these experiments exhibited a completely unique response to infection relative to the other two. After repeated attempts to infect the PBMC from this animal, little or no infectivity was seen across the time points considered, and corresponding to this apparent lack of infection, few genes were seen to be differentially expressed when compared to mock-infected cells. For the remaining two animals, gene expression analysis showed that while they exhibited responses for the same groups of pathways, these responses included differences specific to the individual animal at the gene level. In instances where the patterns of differential gene expression differed between these animals, the genes being differentially expressed were associated with the same categories of biological process, mainly immune response and cell signaling. At the pathway level, these animals again exhibited similar responses that could be predicted based on the experimental conditions. Even in these expected results, the degree of response and the specific genes being regulated differed greatly from animal to animal. The differences in gene expression on an individual level have the potential to be used as markers in identification of animals suitable for lentiviral infection experiments. Our results highlight the importance of individual variation in response to viral challenge.     

Ability of herpes simplex virus vectors to boost immune responses to DNA vectors and to protect against challenge by simian immunodeficiency virus

The immunogenicity and protective capacity of replication-defective herpes simplex virus (HSV) vector-based vaccines were examined in rhesus macaques. Three macaques were inoculated with recombinant HSV vectors expressing Gag, Env, and a Tat-Rev-Nef fusion protein of simian immunodeficiency virus (SIV). Three other macaques were primed with recombinant DNA vectors expressing Gag, Env, and a Pol-Tat-Nef-Vif fusion protein prior to boosting with the HSV vectors. Robust anti-Gag and anti-Env cellular responses were detected in all six macaques. Following intravenous challenge with wild-type, cloned SIV239, peak and 12-week plasma viremia levels were significantly lower in vaccinated compared to control macaques. Plasma SIV RNA in vaccinated macaques was inversely correlated with anti-Rev ELISPOT responses on the day of challenge (P value<0.05), anti-Tat ELISPOT responses at 2 weeks post challenge (P value <0.05) and peak neutralizing antibody titers pre-challenge (P value 0.06). These findings support continued study of recombinant herpesviruses as a vaccine approach for AIDS.     

Changes in simian immunodeficiency virus reverse transcriptase alleles that appear during infection of macaques enhance infectivity and replication in CD4+ T cells

We previously showed that a slowly replicating, minimally pathogenic clone of simian immunodeficiency virus (SIV), SIVmneCl8, evolves increased ability to replicate in T cells with the onset of AIDS in pig-tailed macaques. Moreover, molecular clones derived from late stages of infection (SIVmne170 and SIVmne027) replicate to high levels in vivo compared to SIVmneCl8. Here, we investigated the role of rt mutations in SIVmne variant replication. We demonstrate selection for rt alleles that enhance viral infectivity and replication capacity in CD4(+) T cells. Moreover, the ability of SIVmne to be induced from resting CD4(+) T cells by anti-CD3/CD28 stimulation is more strongly influenced by the variant rt alleles than nef alleles. Taken together, our data underscore the importance of RT determinants for pathogenicity of SIV and for the capacity to replicate in CD4(+) T cell populations.     

Detection of macaque perforin expression and release by flow cytometry, immunohistochemistry, ELISA, and ELISpot

Simian immunodeficiency virus (SIV)-infection in macaques provides an important animal model for human immunodeficiency virus-1 (HIV-1) infection. The involvement of perforin (PFN), released by cytotoxic cells to mediate killing of virus-infected cells, has been difficult to assess in this experimental model due to a lack of reagents. We therefore evaluated monoclonal antibodies (mAbs) Pf-80, Pf-164 and Pf-344, previously raised against human PFN, for cross-reactivity with macaque PFN. Mabs Pf-164 and Pf-344 reacted with intracellular PFN in peripheral blood mononuclear cells (PBMC) from cynomolgus and rhesus macaques by flow cytometry and stained PFN in rhesus lymphoid tissue by immunohistochemistry (IHC). Moreover, PFN capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays utilizing mAbs Pf-164/Pf-80 for capture and mAb Pf-344 for detection were used to quantify PFN release by mitogen-stimulated cynomolgus and rhesus PBMC. The PFN ELISpot was further used to quantify antigen-specific CD8+ T cells by ex vivo stimulation of PBMC from cynomolgus macaques immunized against SIV/HIV-1. These macaque PFN-reactive mAbs and immunoassays will be valuable new tools for investigation of cytotoxic T lymphocyte (CTL) responses in non-human primate models of infectious diseases as well as for vaccine development.     

Attenuated poxvirus-based simian immunodeficiency virus (SIV) vaccines given in infancy partially protect infant and juvenile macaques against repeated oral challenge with virulent SIV

An infant macaque model was developed to test pediatric vaccine candidates aimed at reducing HIV transmission through breast-feeding. Infant macaques were given multiple immunizations during the first 3 weeks of life with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins Gag, Pol, and Env (ALVAC-SIV or modified vaccinia virus Ankara [MVA]-SIV). After repeated daily oral inoculations with virulent SIVmac251 at 4 weeks of age, significantly fewer ALVAC-SIV-immunized infants were infected compared with unimmunized infants. Monkeys not infected after oral challenge in infancy were rechallenged at 16 months of age or older by repeated weekly oral SIV exposure; unimmunized animals were infected after fewer SIV exposures than were animals vaccinated with ALVAC-SIV or MVA-SIV. When infected, ALVAC-SIV- and MVA-SIV-vaccinated animals also had reduced viremia compared with unimmunized animals. The results of these investigations suggest that immunization of human infants with poxvirus-based HIV vaccine candidates may offer protection against early and late HIV infection through breastfeeding.     

Detection of simian immunodeficiency virus RNA from infected rhesus macaques by the polymerase chain reaction.

A rapid method for the detection of simian immunodeficiency virus (SIV) RNA from peripheral blood mononuclear cells (PBMC) of experimentally infected rhesus macaques by the polymerase chain reaction (PCR) is reported. The PCR was carried out with a complementary DNA (cDNA) template using 3 pairs of primers that were designed to anneal to homologous sequences in conserved regions of 3 molecular clones of SIVmac. The specificity of the primers was confirmed by performing the PCR with template DNA from the 3 molecular clones. SIV-specific RNA was detected from 30 and 50 infected PBMC/6.25 x 10(5) PBMC of two animals.     

Immunogenicity in pig-tailed macaques of poliovirus replicons expressing HIV-1 and SIV antigens and protection against SHIV-89.6P disease

In the search for an effective vaccine against the human immunodeficiency virus (HIV), novel ways to deliver viral antigens are being evaluated. One such approach is the use of nonreplicating viral vectors encoding HIV and/or SIV genes that are expressed after infection of host cells. Nonreplicating poliovirus vectors, termed replicons, that expressed HIV-1/HXB2 and SIVmac239 gag and various HIV-1 env genes from different clades were tested for immunogenicity and protective efficacy against intravenous challenge of pig-tailed macaques with SHIV-89.6P. To maximize both cellular and humoral immune responses, a prime-boost regimen was used. Initially, macaques were immunized four times over 35 weeks by either the intranasal and intrarectal or the intramuscular (im) route with mixtures of poliovirus replicons expressing HIV-1 gag and multiple env genes. Immunization with replicons alone induced both serum antibodies and lymphocyte proliferative responses. After boosting with purified Env protein, neutralizing antibodies to SHIV-89.6P were induced in four of five immunized animals. In a second experiment, four macaques were immunized im three times over 27 weeks with replicons expressing the SIVmac239 gag and HIV-1/HXB2 env genes. All immunized animals were then boosted twice with purified HIV-1-89.6 rgp140-Env and SIVmac239 p55-Gag proteins. Four control animals received only the two protein inoculations. Immunized and control animals were then challenged intravenously with the pathogenic SHIV-89.6P. After challenge the animals were monitored for virus isolation from peripheral blood mononuclear cells and plasma viremia and for changes in virus-specific antibody titers. Na?ve pig-tailed macaques experienced rapid loss of CD4(+) T cells and died between 38 and 62 weeks after infection. In contrast, macaques immunized with replicons and proteins rapidly cleared plasma virus and did not experience sustained loss of CD4(+) lymphocytes. Furthermore, two of the four macaques that were immunized only with purified proteins maintained high viral burdens and lost greater than 95% of their CD4(+) lymphocytes within 2 to 4 weeks after challenge. Thus, poliovirus replicons expressing HIV-1 and SIV antigens were immunogenic in pig-tailed macaques and appeared to enhance the protective effects observed after administration of purified proteins alone.