Search

As sexual transmission of human immunodeficiency virus-1 (HIV-1) occurs via the mucosa, an ideal HIV-1 vaccine should induce both mucosal and systemic immunity. We therefore sought to evaluate the induction of mucosal responses using a DNA env prime–gp120 protein boost approach in which sequential nasal and parenteral protein administration was performed with two novel carbohydrate-based adjuvants. These adjuvants, Advax-M and Advax-P, were specifically designed for mucosal and systemic immune enhancement, respectively. Murine intranasal immunization with gp120/Advax-M adjuvant elicited gp120-specific IgA in serum and mucosal secretions that was markedly enhanced by DNA priming. Boosting of DNA-primed mice with gp120/Advax-M and gp120/Advax-P by sequential intranasal and intramuscular immunization, or vice versa, elicited persistent mucosal gp120-specific IgA, systemic IgG and memory T- and B-cell responses. Induction of homologous, but not heterologous, neutralizing activity was noted in the sera of all immunized groups. While confirmation of efficacy is required in challenge studies using non-human primates, these results suggest that the combination of DNA priming with sequential nasal and parenteral protein boosting, with appropriate mucosal and systemic adjuvants, could generate strong mucosal and systemic immunity and may block HIV-1 mucosal transmission and infection.

Nonhuman primates represent a robust model to evaluate preclinical efficacy of HIV-1 vaccine and therapeutic strategies. Plasma and tissue viral RNA as well as tissue proviral DNA load are key parameters in assessing efficacy of vaccines and therapeutics against simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) challenge. To quantitate SIV RNA in plasma and tissues, an isothermal nucleic acid sequence-based amplification (NASBA) method using real-time detection of amplified RNA with molecular beacons was developed. This assay has accuracy and reproducibility over seven orders of magnitude and has advantages over the electrochemiluminescence-based NASBA assay described previously, both in terms of higher throughput and sensitivity. Reproducibility and accuracy were also demonstrated for a TaqMan real-time PCR assay for quantitating proviral DNA load in PBMCs and lymphoid tissues. In infected macaques, the level of plasma viremia correlated with the tissue viral RNA but not always with proviral DNA loads. Further, animals with undetectable levels of viral RNA in plasma and proviral DNA in tissues, showed no sign of seroconversion and activation of Gag-specific CD8+ or CD4+ T cells in peripheral blood. These results suggest that simultaneous application of real-time NASBA and PCR assays provides quantitative evaluation of challenge outcome in macaques.

Simian betaretroviruses (SRV), formerly known as simian type D retroviruses, are endemic in many populations of Asian monkeys of the genus Macaca. Asian monkeys have been used extensively as animal models for preclinical HIV vaccine development, therapeutics, and other biomedical studies. SRV infection can sometimes lead to immune deficiency disease, which complicates such studies; thus, it is important to screen for SRV infection and remove infected animals from test populations. Real-time PCR assays were developed to specifically quantify SRV-1/3, SRV-2, and SRV-5 proviral DNA. The SRV provirus copy numbers were standardized relative to real-time PCR measurements of the rhesus macaque albumin gene. The primers and TaqMan probe sequences for the rhesus macaque (Indian origin) albumin gene also detect cynomolgus macaque and rhesus macaque (Chinese origin) albumin genes. The SRV primers and probes were designed to amplify gag gene sequences of SRV-1/3 (GeneBank accession number M11841), SRV-2 (GeneBank accession number M16605), and SRV-5 (GeneBank accession number AF252389). The optimized reactions for detection of each SRV serotype and the macaque albumin gene had amplification efficiencies of greater than 90% with a linear range spanning 1 x 10(1) to 2.5 x 10(6) copies per reaction. The R(2) values of all standard curves were greater than 0.995. Of 40 animals housed in quarantine, four animals were positive for SRV-1/3 with 28, 5450, 9780, and 14,500 copies of provirus per 10(6) PBMCs, and one animal was positive for SRV-2 with provirus copy number of 7790 per 10(6) PBMCs. All of 40 animals appeared to be seronegative and had normal CD4(+) and CD8(+) T-cell counts. These quantitative real-time PCR assays enhance the detection and quantitation of SRV infection and will facilitate the elimination of this virus from macaque colonies.

This report describes the safety observations following administration of a polyvalent DNA prime–protein boost HIV-1 vaccine formulated with adjuvant QS21. Local injection site reactions were the most common (65% of subjects), and included type IV delayed-type hypersensitivity (DTH) reactions at prior DNA inoculation sites in 12 of 28 (43%) subjects following protein vaccination. Systemic reactions revealed two cases of vasculitis temporally related to inoculation with recombinant Env protein + QS21 adjuvant. Questions remain regarding the cause of the vasculitis, but the unique DTH observation may have contributed to the high level of immune responses previously reported for this vaccine.

Selection of potent yet low reactogenic adjuvants for protein immunization is important for HIV-1 vaccine development. Immunogenicity of electroporated DNA (HIV env) and recombinant gp120, administered with either QS-21 or the orally administered immunomodulator, Talabostat, was evaluated in BALB/c mice. Electroporation of low dose DNA elicited Th1 cytokines and anti-envelope antibodies. Immunization with gp120 protein alone with or without Talabostat elicited lower Th1 and Th2 cytokine levels but comparable anti-gp120 antibodies to QS-21-formulated protein. Boosting of DNA-primed mice with gp120/Talabostat induced similar anti-gp120 antibody titers and slightly higher levels of Th1 and Th2 cytokines relative to QS-21-formulated protein. Induction of CD8(+) and CD4(+) T cells and functional CTL activity was noted. These results highlight the potential use of orally administered Talabostat for efficient protein boosting of antibody and T-cell responses primed by DNA.

Intramuscular needle injection of HIV-1 DNA vaccines typically elicits weak immune responses in immunized individuals. To improve such responses, the immunogenicity of a vaccine consisting of electroporated DNA followed by intramuscular protein boost was evaluated in rabbits and macaques. In macaques, electroporation of low dose DNA encoding HIV-1 env followed by gp120 protein elicited Th1 cytokines and functional CTL that persisted for over 1 year. In both macaques and rabbits, robust anti-envelope antibodies, elicited by electroporated DNA, were augmented by gp120 protein and such responses neutralized sensitive SHIV isolates. These findings highlight efficient priming of immune responses by electroporated DNA that in conjunction with protein boost may give rise to long-term immunity in immunized hosts.

As the diversity of potential immunogens increases within certain classes of vectors, the possibility has arisen of employing heterologous prime/boost immunizations using diverse members of the same family of vectors. The present study was initiated to explore the use of divergent pox vectors in a prime/boost regimen to elicit high-frequency cellular immune responses to human immunodeficiency virus type 1 envelope and simian immunodeficiency virus gag in rhesus monkeys. We demonstrated that monkeys vaccinated with a recombinant modified vaccinia virus Ankara (rMVA) prime/recombinant fowlpox virus (rFPV) boost regimen and monkeys vaccinated with a recombinant vaccinia virus prime/rFPV boost regimen developed comparable cellular immune responses that were greater in magnitude than those elicited by a homologous prime/boost with rMVA. Nevertheless, comparable magnitude recall cellular immune responses were observed in monkeys vaccinated with heterologous and homologous recombinant poxvirus following challenge with the CXCR4-tropic SHIV-89.6P. Consistent with this finding, comparable levels of containment of viral replication and CD4 + T-lymphocyte preservation were seen in these groups of recombinant poxvirus-vaccinated monkeys. This study supports further exploration of combining recombinant vectors of the same family in prime/boost immunization strategies to optimize vaccine-elicited cellular immune responses.

Rhesus macaques chronically infected with highly pathogenic simian immunodeficiency virus (SIV) SIVmac251 were treated with antiretroviral drugs and vaccinated with combinations of DNA vectors expressing SIV antigens. Vaccination during therapy increased cellular immune responses. After the animals were released from therapy, the virus levels of 12 immunized animals were significantly lower ( P = 0.001) compared to those of 11 animals treated with only antiretroviral drugs. Vaccinated animals showed a persistent increase in immune responses, thus indicating both a virological and an immunological benefit following DNA therapeutic vaccination. Several animals show a long-lasting decrease in viremia, suggesting that therapeutic vaccination may provide an additional benefit to antiretroviral therapy.

Although replication-competent adenovirus (Ad) vectors are promising in AIDS vaccine design, their safety in immune compromised hosts is unknown. To initially address this question, enteric-coated tablets containing a replicating Ad vector were orally administered to SHIV- and SIV-infected rhesus macaques with normal, intermediate or low CD4 T cell counts and stable disease. The vector was detected within a week after tablet administration in stools of all animals but not in nasal secretions, indicating no spread of virus to the upper respiratory tract. CD4 T cell counts and viral loads remained stable in all animals and no signs of fever, weight loss, or other clinical symptoms of Ad-induced disease were observed during 10 weeks of follow-up. Oral delivery of the replicating Ad vector was safe and well tolerated by SHIV- and SIV-infected hosts. Oral enteric-coated tablets may prove safe for administering replicating Ad-vectored vaccines in areas with high HIV prevalence.

Due to the alarming spread of human immunodeficiency virus type 1 (HIV-1) infection worldwide, the development of a prophylactic vaccine is critical. Vaccine strategies for AIDS have included the use of structural and nonstructural HIV subunit proteins or peptides, naked DNA, bacterial and viral live vectors, or combinations of the above. Most of the vaccines developed thus far induce T-cell responses but are unable to induce neutralizing antibodies to the Env of primary HIV-1 isolates. The fact that a decrease in plasma viremia (9, 41) is concomitant with the appearance of virus-specific cytotoxic T lymphocytes (CTLs) and depletion of CD8+ T cells in simian immunodeficiency virus (SIV)-infected macaques or HIV-infected chimpanzees suggests that CD8+ T cells are able to partially control HIV-1/SIV replication (15, 36, 43, 52). Indeed, several “T-cell vaccines” based on DNA and live vectors confer protection from high-level replication of challenge viruses in rhesus macaques (2, 4, 8, 30, 31, 48). The extent of the decrease in viral replication induced by these vaccines is variable in nonhuman primates, and protection from disease appears to be dependent on the virulence of the virus used in the challenge experiments (28). The relative efficacy of poxvirus-based vaccine candidates with various degrees of attenuation has been extensively studied in rhesus macaques after challenge with SIV, SHIV, and HIV-2 isolates (1, 3, 8, 24, 25, 29-31, 48). These vaccine modalities elicit variable levels of cell-mediated immune response and prevent infection after challenge exposure to viruses with low virulence, such as HIV-2 (3, 25), and to other somewhat-attenuated SIV isolates (34, 35). Importantly, these vaccine modalities were also able to significantly reduce virus load after challenge with highly pathogenic SIV isolates (8, 29, 32, 48). An ALVAC-SIV vaccine encoding the gag, pol, and env genes of the SIVmac251 isolate was able to reduce plasma virus load during primary infection and conferred protection from CD4+ loss during both acute and chronic phases of infection after rectal exposure to a highly pathogenic SIVmac251 isolate (50). Among the pox vector-based vaccines, several ALVAC-based HIV-1 vaccines have been tested in phase I and II clinical trials and have been shown to be safe and immunogenic in humans (10, 14, 18, 21, 27). Whether the immunogenicity of these vaccine candidates will be sufficient to protect humans from HIV-1 remains unknown (5, 11, 44). The ongoing human phase III trial in Thailand will provide key information in this regard. Here, we designed a study to assess whether systemic immunization with recombinant canarypox expressing either HIV-1 gp120 or gp160 followed by a boost with either purified gp120 or gp140 proteins would confer protection after mucosal challenge with the pathogenic SHIVKU2. We also investigated whether the addition of the Tat protein of HIV-1 as an immunogen could provide better protection, since a few studies have demonstrated that immunization of macaques with Tat protein either alone or as a Nef-Tat fusion protein in a multicomponent subunit vaccine confers an advantage in a SHIV89.6P model (12, 13, 56) but not in other studies (53). Here, we found that immunization of macaques with this type of ALVAC-based vaccine formulation elicited both antibody and cellular responses and significantly decreased plasma viremia and CD4+ T-cell loss after rectal exposure to the SHIVKU2 isolate. Tat protein immunization had no additive effect on the reduction of virus load in vaccinated rhesus macaques, as also observed by others (42, 46, 53).

While DNA vaccines have been shown to prime cellular immune responses, levels are often low in nonhuman primates or humans. Hence, efforts have been directed toward boosting responses by combining DNA with different vaccination modalities. To this end, a polyvalent DNA prime/protein boost vaccine, consisting of codon optimized HIV-1 env (A, B, C, E) and gag (C) and homologous gp120 proteins in QS-21, was evaluated in rhesus macaques and BALB/c mice. Humoral and cellular responses, detected following DNA immunization, were increased following protein boost in macaques and mice. In dissecting cellular immune responses in mice, protein-enhanced responses were found to be mediated by CD4+ and CD8+ T cells with a Th1 cytokine bias. Our study reveals that, in addition to augmenting humoral responses, protein boosting of DNA-primed animals augments cellular immune responses mediated by CD8+ CTL, CD4+ T-helper cells and Th1 cytokines; thus, offering much promise in controlling HIV-1 in vaccinees.

A toxicity and immunogenicity study, evaluating the safety of a polyvalent DNA prime/protein boost HIV-1 vaccine (DP6-001), was examined in rabbits. Animals were primed with a cocktail of six different DNA plasmids expressing five HIV-1 env genes and one gag gene followed by boosting with five gp120 proteins homologous to the DNA vaccines. The vaccine was shown to be immunogenic as evident from the induction of high-titered anti-Env and anti-Gag antibodies. There was an absence of detectable adverse effects on key toxicology parameters. Although plasmids persisted in the injection sites following single administration for 64 days, no evidence of integration into the host genomic DNA was observed. These studies demonstrate that a novel polyvalent DNA prime/protein boost vaccine lacks signs of toxicity and DNA integration in a rabbit model, and immunogenicity and toxicology data support clinical testing of the vaccine in humans.

An HIV-1 vaccine able to induce broad CD4+ and CD8+ T cell responses may provide long-term control of viral replication. In this study we directly assess the relative benefit of immunization with vaccines expressing three structural Ags (Gag, Pol, and Env), three early regulatory proteins (Rev, Tat, and Nef), or a complex vaccine expressing all six Ags. The simultaneous administration of all six Ags during vaccination resulted in Ag competition manifested by a relative reduction of CD8+ T cell and lymphoproliferative responses to individual Ags. Despite the Ag competition, vaccination with all six Ags resulted in a delay in the onset and a decrease in the extent of acute viremia after mucosal challenge exposure to highly pathogenic SIVmac251. Reduced levels of acute viremia correlated with lower post-set point viremia and long-term control of infection. In immunized animals, virus-specific CD4+ T cell and lymphoproliferative responses were preserved during acute viremia, and the maintenance of these responses predicted the long-term virological outcome. Taken together, these results suggest that the breadth of the immune response is probably more important than high frequency responses to a limited number of epitopes. These data provide the first clear evidence of the importance of nonstructural HIV Ags as components of an HIV-1 vaccine.

HV MNE is an Epstein–Barr virus (EBV)-like lymphocryptovirus (LCV) originally isolated from a Macaca nemestrina with CD8 + T cell mycosis fungoides/cutaneous T cell lymphoma (Blood 98 (2001), 2193). HV MNE transforms rabbit T cells in vitro and causes T cell lymphoma in New Zealand white rabbits. Here we demonstrate that HV MNE also immortalizes T cells from mustached tamarins but not those from owl monkeys, common marmosets, squirrel monkeys, black-capped capuchins, and humans. Cytogenetic and FACS analysis revealed the true origin and T cell lineage of the transformed tamarin T cell lines. Tamarin T cells contained HV MNE DNA sequence and displayed a decreased requirement for the IL-2 cytokine for growth. Thus, this EBV-like virus from M. nemestrina differs from the other EBV-like viruses found in nonhuman primates inasmuch as it appears to preferentially transform T cells.

Despite extensive efforts made to combat human immunodeficiency virus (HIV) infection and AIDS since the discovery of the virus, the number of people infected with HIV and developing the disease worldwide is still increasing rapidly. The need for a vaccine against HIV is now one of the world's greatest public health problems; however, development of a safe and effective HIV vaccine has proved difficult due to several unique challenges presented by the virus. These include difficulty in eliciting broadly reactive neutralizing antibodies, the high variability of the virus, and integration of HIV proviral DNA into the host genome, resulting in latent infection and making achievement of sterilizing immunity nearly impossible (44). Considering recent reports associating either humoral or cellular immune responses with protection against HIV infection or disease progression, it is difficult to define requirements for protective immunity against HIV (1, 4, 9, 23, 24, 32, 38, 40). Accumulating evidence indicates that an ideal HIV vaccine should induce broad humoral, cellular, and mucosal immunity against multiple viral antigens in order to combat infectious viral particles and HIV-infected cells at any point during infection (19, 25, 33, 50). To achieve this goal, many strategies are being investigated, including recombinant viral proteins and peptides, naked DNA, live viral and bacterial vectors, and prime-boost combinations (19). Adenovirus (Ad) is one of the live viral vectors being developed for use as an HIV vaccine. Ad infects a broad spectrum of human cells, including immature dendritic cells, leading to efficient antigen presentation and causing their maturation without polarizing the T-helper response (22, 53, 54). Because AIDS is mainly a sexually transmitted disease, vaccine-elicited mucosal immunity against HIV is critical. Ad vectors are therefore highly attractive, because they target epithelial cells at mucosal surfaces and can be administered orally and intranasally. Both replication-competent and replication-defective Ad recombinants have been investigated as potential AIDS vaccines. Replication-defective Ad vectors, long used in gene therapy applications, have been adapted for use as HIV vaccines (5, 46, 51). Recent studies with an E1- and E3-deleted Ad5-SIVgag recombinant to immunize rhesus macaques elicited high-frequency SIV p11C-tetramer-positive cells. Following challenge with pathogenic SHIV89.6P the monkeys exhibited significantly reduced viral burdens and were protected against SHIV-induced disease (46). We have taken a different approach, using replication-competent Ad recombinants with deletions only in the E3 region. Because of the inability of human Ad to replicate in most mammalian species, our studies initially were carried out with chimpanzees, which are permissive for Ad replication. Replication-competent, E3 region-deleted Ad-HIVenv and -HIVgag/pro recombinants were investigated and shown to elicit cellular immune responses, antibody responses in mucosal secretions, high-titer serum antibodies able to neutralize both T-cell-line-adapted and primary HIV isolates, and significant protective efficacy (20, 21, 30, 31, 43, 55). Chimpanzees immunized with an Ad-HIVenv priming/gp120 boosting regimen were protected against both low- and high-dose HIV challenges, including challenge with a heterologous primary HIV isolate. The protection elicited was shown to be long lasting. To further develop this approach in a macaque model, we took advantage of an Ad5 host range mutant (Ad5hr) (41) and carried out experiments by using an Ad5hr-SIVsmH4env/rev recombinant shown to replicate in monkey cells in vitro (8). Again by using a recombinant priming/gp120-boosting regimen, we demonstrated that the Ad5hr recombinant also replicated in vivo and elicited SIV-specific cellular immunity and humoral immune responses in serum and secretory fluids (6, 7). This solely envelope-based vaccine achieved a reduction in acute-phase viral burden following intravaginal challenge with pathogenic SIVmac251; however, the viral load began increasing by 8 weeks postchallenge. Accumulating data have shown the potential benefit of incorporating additional viral antigens that elicit strong cellular immune responses into vaccine regimens. Therefore, we added an SIV gag component to the vaccination regimen. We have reported that coadministration of the Ad5hr-SIVsmH4env/rev plus Ad5hr-SIVgag recombinant elicits cellular immune responses to each of the encoded SIV gene products, Env, Gag, and Rev, and that the responses are boosted by a second administration of the same recombinants (35, 52). These recombinant immunizations also prime strong neutralizing antibodies and both IgG and IgA SIV-specific antibodies in secretory fluids (J. Pinczewski, J. Zhao, L. J. Patterson, N. Malkevitch, G. Alvord, and M. Robert-Guroff, unpublished results). Here we have investigated the ability of the dual immunization regimen to protect against an intrarectal challenge with pathogenic SIVmac251 and have explored whether particular immune responses are correlated with the protective outcome. We report improved protective efficacy against a mucosal SIVmac251 challenge, with viremia reduced at both the acute phase and set point of infection. Several immune parameters contributed to the protective outcome.

Rhesus macaques were immunized with a replication-deficient vaccinia virus (MVA) expressing human immunodeficiency virus type 1 89.6 envelope (env) and SIV gagpol (MVA/SHIV89.6) with or without a protein boost consisting of soluble 89.6 env (gp140). Immunization with MVA/SHIV89.6 alone elicited binding antibodies in all animals and neutralizing antibodies in 5 of 15 animals. Both types of antibodies were enhanced by protein boosting. In addition, CD8 cells exhibiting CM9 tetramer binding were detected in the subset of animals that were Mamu-A*01 positive. Animals were challenged intravenously with either SHIV-89.6 (Study 1) or the more pathogenic derivative SHIV-89.6P (Study 2). In Study 1, all control and vaccinated animals except one became infected. However, the levels of viremia were as follows: controls > rMVA alone > rMVA + protein. The differences were statistically significant between immunized and control groups but not between the two immunized groups. In Study 2, all animals became infected; however, the vaccinated group exhibited a 5-fold reduction in peak viremia and a 10-fold reduction in the postacute phase viremia in comparison to the controls. All of the controls required euthanasia by 10 months after challenge. A relationship between vaccine-induced antibody titers and reduction in virus burden was observed in both studies. Thus, immunization with MVA/SHIV89.6 alone or with a protein boost stimulated both arms of the immune system and resulted in significant control of viremia and delayed progression to disease after challenge with SHIV-89.6P.

The most commonly used animal model for the study of HIV-1 infection in humans is the infection of non-human primates by simian immunodeficiency virus (SIV). The animal hosts used most frequently are different species of macaques, which are readily infected with SIV, and can therefore be used to study natural infection, pathogenesis, therapy, and vaccine efficacy. The study of HIV-1 infection in humans relies heavily on the quantification of HIV-1 load (i.e. viral RNA) in patient plasma. Given the importance of HIV-1 RNA levels in humans, it follows that SIV RNA levels in animals are also relevant to the study of infection in this model system. This report describes the development of the isothermal amplification-based NASBA technology for the quantification of SIV RNA load in macaque plasma. Evaluation of the assay using model systems demonstrated that the assay is accurate and reproducible over nearly four orders of magnitude. Viral RNA load data were compared to other infection measurements in the macaque system. Further, the assay was used to provide copy number levels of SIV RNA in macaque plasma samples, permitting characterization of viral load during the course of SIV infection.

Epstein-Barr virus (EBV) infection of humans has been associated with the development of lymphoid malignancies mainly of B-cell lineage, although occasionally T-cell lymphomas have been reported. We describe here the characterization of a novel EBV-like virus (HVMNE) isolated from a simian T-cell lymphotropic virus type I/II (STLV-I/II) seronegative pigtailed macaque (Macaca nemestrina) with a cutaneous T-cell lymphoma. Immunohistochemistry studies on the skin lesions demonstrated that the infiltrating cells were of the CD3+/CD8+ phenotype. Two primary transformed CD8+ T-cell lines were obtained from cultures of peripheral blood mononuclear cells (PBMC) and skin, and, with time, both cell lines became interleukin-2–independent and acquired the constitutive activation of STAT proteins. Polymerase chain reaction analysis of the DNA from the cell lines and tissues from the lymphomatous animal demonstrated the presence of a 536-bp DNA fragment that was 90% identical to EBV polymerase gene sequences, whereas the same DNA was consistently negative for STLV-I/II sequences. Electron microscopy performed on both cell lines, after sodium butyrate treatment, showed the presence of a herpes-like virus that was designated HVMNE according to the existing nomenclature. In situ hybridization studies using EBV Epstein-Barr viral-encoded RNA probes showed viral RNA expression in both CD8+ T-cell lines as well as in the infiltrating CD8+ T cells of skin-tissue biopsies. Phylogenetic analysis of a 465-bp fragment from the polymerase gene of HVMNE placed this virus within theLymphocryptovirus genus and demonstrated that HVMNEis a distinct virus, clearly related to human EBV and other EBV-like herpesviruses found in nonhuman primates.

Vaccine-induced protection of chimpanzees against laboratory-adapted and syncytium-inducing, multiply passaged primary human immunodeficiency virus type 1 (HIV-1) isolates, but not against non-syncytium-inducing, minimally passaged ones, has been demonstrated. Following challenge with such an isolate, HIV-1 5016 , we obtained complete protection in one of three chimpanzees previously protected against low- and high-dose HIV-1 SF2 exposures after immunization with an adenovirus-HIV-1 MN gp160 priming–HIV-1 SF2 gp120 boosting regimen. At challenge, the protected chimpanzee exhibited broad humoral immunity, including neutralizing antibody activity. These results demonstrate the potential of this combination vaccine strategy and suggest that vaccine protection against an HIV isolate relevant to infection of people is feasible.

It has been previously reported that hydroxyurea (HU) displays anti-HIV-1 activity and potentiates the antiviral effects of didanosine (ddI) in vitro. To assess the antiviral efficacy of HU in an animal model, the effects of HU and ddI, either individually or as combination therapy, were tested in a model using infection of pigtail macaque with the acutely fatal variant SIV(smpbj14). At the high dosage used (100 mg/kg/day), HU monotherapy failed to protect the exposed animals from viral infection and death, which occurred within 10 days postinoculation. However, both of the ddI-treated animals (5 mg/kg/day) survived the SIV(smmpbj14) lethal dose and displayed a reduction in viral load (undetectable SIV RNA or p27gag) in the primary phase of infection. Of the animals treated with the combination of drugs, one died at day 18 after infection and failed to seroconvert to viral antigens. These data suggest that a high dose of HU monotherapy does not protect against death induced by SIV(mmpbj14). However, lower doses of HU as monotherapy or combination therapy deserve further evaluation for their therapeutic effects.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

Vaccine protocols involving multiple immunizations with molecularly attenuated vaccinia virus (NYVAC) or naturally attenuated canarypox virus (ALVAC) HIV-2 recombinants and subunit boosts have conferred longlasting protection against HIV-2 infection of macaques. Similar complex protocols using HIV-1 NYVAC and ALVAC recombinants and subunit boosts have provided cross-protection against HIV-2 challenge. Here a simplified three-immunization regimen over 24 weeks was tested in 18 juvenile rhesus macaques. Twelve macaques were immunized twice with NYVAC or ALVAC recombinants carrying HIV-2 env, gag, and pol genes. Subsequently, macaques in groups of three received either an additional recombinant immunization or an HIV-2 gp160 boost. Six control macaques received three immunizations of NYVAC or ALVAC vector alone and additionally alum at the third immunization. Macaques primed with ALVAC recombinant exhibited sporadic T cell proliferative activity, and all but one failed to develop neutralizing antibodies. In contrast, macaques primed with NYVAC recombinants had no T cell proliferative activity but exhibited neutralizing antibody titers (highest in the three recombinant group) that declined by the time of challenge. None of the macaques exhibited significant cytotoxic T lymphocyte activity. Following challenge at 32 weeks with HIV-2SBL6669 all macaques became infected. Thus, the three-immunization regimen is not sufficient to confer protective immunity in the HIV-2 rhesus macaque model. However, delayed infection in macaques immunized with the NYVAC-HIV-2 recombinant may have been associated with the development of memory B cells capable of providing a neutralizing antibody response on challenge.

Rhesus macaques were immunized with attenuated vaccinia or canarypox human immunodeficiency virus type 1 (HIV-1) recombinants and boosted with HIV-1 protein subunits formulated in alum. Following challenge with HIV-2SBL6669, three out of eight immunized macaques resisted infection for six months and another exhibited significantly delayed infection, whereas all three naive controls became infected. Immunizations elicited both humoral and cellular immune responses; however, no clear correlates of protection were discerned. Although more extensive studies are now called for, this first demonstration of cross-protection between HIV-1 and -2 suggests that viral variability may not be an insurmountable problem in the design of a global AIDS vaccine.

The entire envelope protein of the human T cell leukemia/lymphoma virus type I (HTLV-I)1711, obtained from the DNA of a West African healthy HTLV-I-infected patient, was expressed in the highly attenuated poxvirus vaccine vectors ALVAC and NYVAC. These live recombinant vaccine candidates were used to immunize New Zealand White rabbits. Immunization regimens included inoculation of the poxvirus recombinant alone as well as prime/boost protocols using gp63 HTLV-I envelope precursor protein in Alum as the subunit boost. All animals were exposed to an HTLV-I cell-associated challenge (5 x 10(4) cells) from a primary culture of the HTLV-IBOU isolate. The results indicated that two inoculations of the ALVAC-based HTLV-Ienv vaccine candidate protected animals against viral challenge 5 months following the last immunization. However, a combination protocol with ALVAC-env and two additional boosts of gp63 surprisingly failed to confer protection, suggesting that administration of the subunit preparation might be deleterious. Further, in the case of the NYVAC HTLV-Ienv recombinant, protection was afforded as early as 2 months following the first immunization. Last, all the protected animals in the NYVAC and ALVAC trials were challenged 5 months following the initial challenge exposure with 5 ml of blood from an HTLV-IBOU-infected animal, and subsequently became infected. Protection conferred by the attenuated HTLV-Ienv recombinant poxvirus vaccine in the rabbit model might be instrumental for optimizing the immunogenicity of poxvirus-based vaccine candidates against human immunodeficiency virus (HIV), particularly because of the need to enhance protection against cell-to-cell transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

BackgroundProgression to AIDS is often associated with the evolution of HIV-1 toward increased virulence and/or pathogenicity. Evidence suggests that a virulence factor for HIV-1 is resistance to CCR5-binding chemokines, most notably RANTES, which are believed to play a role in HIV-1 controlin vivo. HIV-1 can achieve RANTES resistance either by phenotypic switching from an exclusive CCR5 usage to an expanded coreceptor specificity, or by the acquisition of alternative modalities of CCR5 usage. An infectious agent that might promote the evolution of HIV-1 toward RANTES resistance is human herpesvirus 6A (HHV-6A), which is frequently reactivated in HIV-1-infected patients and is a potent RANTES inducer in lymphoid tissue.ResultsSIV isolates obtained from pig-tailed macaques (M. nemestrina) after approximately one year of single infection with SIVsmE660or dual infection with SIVsmE660and HHV-6AGSwere characterized for their growth capacity and sensitivity to HHV-6A- and RANTES-mediated inhibition in human or macaque lymphoid tissuesex vivo. Four out of 4 HHV-6A-coinfected macaques, all of which progressed to full-blown AIDS within 2 years of infection, were found to harbor SIV variants with a reduced sensitivity to both HHV-6A and RANTES, despite maintaining an exclusive CCR5 coreceptor specificity; viruses derived from two of these animals replicated even more vigorously in the presence of exogenous HHV-6A or RANTES. The SIV variants that emerged in HHV-6A-coinfected macaques showed an overall reducedex vivoreplication capacity that was partially reversed upon addition of exogenous RANTES, associated with suppressed IL-2 and enhanced IFN-γ production. In contrast, SIV isolates obtained from two singly-infected macaques, none of which progressed to AIDS, maintained HHV-6A/RANTES sensitivity, whereas the only AIDS progressor among singly-infected macaques developed an SIV variant with partial HHV-6A/RANTES resistance and increased replication capacity, associated with expanded coreceptor usage.ConclusionThese results providein vivoevidence of SIV evolution toward RANTES resistance in macaques rapidly progressing to AIDS. RANTES resistance may represent a common virulence factor allowing primate immunodeficiency retroviruses to evade a critical mechanism of host antiviral defense.

Insights into the host factors that contribute to an effective antiviral immune response may be obtained by examining global gene expression in simian human immunodeficiency virus (SHIV)-infected nonhuman primates that exhibit different virological outcomes. Immune responses and gene expression profiles in peripheral blood mononuclear cells (PBMCs) were compared between animals that controlled or did not control viremia after infection. Rectal inoculation of eight rhesus macaques with R5-tropic SHIV(SF162P3) resulted in a high level of plasma viremia during the acute phase of infection. The viremia was controlled to below levels of detection in six of these animals at the set point (controllers), whereas two animals had persistent viremia throughout the 140 wk that the animals were monitored (non-controllers). CD4(+) T-cell counts declined slightly in both controllers and non-controllers in the acute phase of infection, but CD4(+) T-cell counts continued to decline only in the non-controllers. Neutralizing antibodies to the challenge virus were variable and could not account for the control of viremia. However, analysis of the cellular gene expression profiles in the PBMCs from both groups of animals revealed distinctive gene expression patterns between controllers and non-controllers. Using the paired LPE test, 59 genes with p values0.01 were identified and specific differences in the gene expression profiles in PBMCs from controllers versus non-controllers were detected.

We used optimized DNA expression vectors to compare two gene delivery methodologies in rhesus macaques, namely direct DNA injection and in vivo adaptive constant-current electroporation via the intramuscular route. The use of in vivo electroporation increased levels of gene expression and immune responses. We used an optimized HIV gag expression plasmid to show the development of new cellular immune responses in SIV-infected animals controlling viremia. Furthermore, after vaccination with SIV expression plasmids the recall responses to the SIV antigens were very high, indicating that DNA is a strong boost in the presence of antiretroviral treatment in SIV-infected animals. There was substantial animal-to-animal variability in DNA expression, revealed by plasma measurements of IL-15 produced by co-injected IL-15 DNA. IL-15 expression levels correlated with peak immune responses. Electroporation led to an expansion of antigen-specific CD4+ and CD8+ T cells of both central and effector memory phenotype. These results indicate that improved gene delivery and expression by electroporation dramatically increases immunogenicity of DNA vaccines. Electroporation is thus an important method to improve the effectiveness of DNA vaccination.

An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report on the induction of Human Immunodeficiency Virus Type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime-protein boost HIV-1 vaccine formulation, DP6-001, in a Phase I clinical trial conducted in healthy adult volunteers of both genders. Robust cross-subtype HIV-1-specific T cell responses were detected in IFNgamma ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime-protein boost approach is an effective immunization method to elicit both humoral and cell-mediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds.

Although HIV is the necessary and sufficient causative agent of AIDS, genetic and environmental factors markedly influence the pace of disease progression. Clinical and experimental evidence suggests that human herpesvirus 6A (HHV-6A), a cytopathic T-lymphotropic DNA virus, fosters the progression to AIDS in synergy with HIV-1. In this study, we investigated the effect of coinfection with HHV-6A on the progression of simian immunodeficiency virus (SIV) disease in pig-tailed macaques (Macaca nemestrina). Inoculation of HHV-6A resulted in a rapid appearance of plasma viremia associated with transient clinical manifestations and followed by antibody seroconversion, indicating that this primate species is susceptible to HHV-6A infection. Whereas animals infected with HHV-6A alone did not show any long-term clinical and immunological sequelae, a progressive loss of CD4+T cells was observed in all of the macaques inoculated with SIV. However, progression to full-blown AIDS was dramatically accelerated by coinfection with HHV-6A. Rapid disease development in dually infected animals was heralded by an early depletion of both CD4+and CD8+T cells. These results providein vivoevidence that HHV-6A may act as a promoting factor in AIDS progression.

Although HIV-1 is the necessary and sufficient causative agent of AIDS, genetic and environmental factors markedly influence the pace of disease progression. Clinical and experimental evidence suggests that human herpesvirus 6 (HHV-6), a cytopathic T-lymphotropic agent, may act as an accelerating factor in the progression of HIV disease, although conclusive in vivo evidence has yet to be attained. To evaluate the effect of HHV-6A on the course of AIDS in a relevant model system, we infected pig-tailed macaques (M. nemestrina) either with HHV-6A (strain GS) or with a pathogenic SIV strain (smE660), or with both viruses. Extensive longitudinal virologic, immunologic and clinical follow-up demonstrated that HHV-6A coinfection dramatically accelerated the progression toward full-blown AIDS. Rapid disease development in coinfected animals was associated with an early depletion of both CD4+ and CD8+ T cells. Simultaneous replication of both viruses was documented in coinfected lymph node tissue. These data establish a new animal model for the study of HHV-6 infection and provide the first conclusive in vivo evidence that HHV-6A acts as a cofactor in the progression of primate immunodeficiency virus disease.

A cohort of 22 rhesus macaques of Indian origin infected as neonates, juveniles, or adults by Rev-independent strains of SIV was monitored over several years. After the initial acute phase, virus replication was controlled and plasma virus loads were persistently below the threshold of the assay. The animals were monitored for up to 7.6 years after infection for viral loads, cellular and humoral immune responses, hematological changes, and overall health and no signs of immune dysfunction or AIDS were observed. This study represents several years of additional observation compared to the previously published results, and indicates that the Rev-independent SIV clones tested do not cause AIDS-like progressive disease within 7.6 years from infection. All the animals showed persistent humoral and cellular SIV-specific immune responses, consistent with chronic infection. Different Rev-independent SIV strains showed similar properties and lack of pathogenicity. Multicolor flow cytometric analysis demonstrated preservation of the Central Memory subset of T cells in the attenuated SIV-infected animals. This study demonstrates a potent, long-lasting control of the Rev-independent attenuated SIV in macaques independent of the age at virus exposure.

Natural HIV transmission occurs through mucosa, but it is debated whether mucosal cytotoxic T lymphocytes (CTLs) can prevent or reduce dissemination from the initial mucosal site to the systemic circulation. Also, the role of CTL avidity in mucosal AIDS viral transmission is unknown. To address these questions, we used delay in acute-phase peak viremia after intrarectal challenge as an indicator of systemic dissemination. We found that a peptide-prime/poxviral boost vaccine inducing high levels of high-avidity mucosal CTLs can have an impact on dissemination of intrarectally administered pathogenic SHIV-ku2 in macaques and that such protection correlates better with mucosal than with systemic CTLs and particularly with levels of high-avidity mucosal CTLs.

Address: 1Vaccine Branch, NCI, NIAID, NIH, Bethesda, MD 20892, USA, 2Division of Information and Mathematical Sciences, Genome Institute of Singapore, 138672, Singapore, 3Laboratory Of Clinical Investigation, NIAID, NIH, Bethesda, MD 20892, USA, 4Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, 20892, USA, 5Advanced BioScience Laboratories, Inc., Kensington, MD 20895, USA, 6Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, LA 70112, USA and 7Southern Research Institute, Frederick, MD, USA

Immunization of macaques with multivalent DNA encoding gp120 genes from HIV-1 subtypes A, B, C and E and a gag gene followed by boosting with homologous gp120 proteins elicited strong anti-gp120 antibodies capable of neutralizing homologous and to a lesser degree heterologous HIV-1 isolates. Both Env- and Gag-specific cell mediated immune (CMI) responses were detected in the immunized animals. Following rectal challenge with an SHIV isolate encoding HIV-1(Ba-L)env, plasma viremia in the infected immunized animals was significantly lower than that observed in the naïve animals. Further, one of six immunized animals was completely protected whereas all six naïve animals were infected. These results demonstrate that a vaccine based on priming with a polyvalent DNA vaccine from multiple HIV-1 subtypes followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of controlling rectal transmission of SHIV(Ba-L).

The immunogenicity of a polyvalent HIV-1 vaccine comprised of Env antigens from primary R5 isolates was evaluated in rhesus macaques. DNA vaccines encoding four Env antigens from multiple HIV-1 subtypes and HIV-1 Gag antigen from a single subtype elicited a persistent level of binding antibodies to gp120 from multiple HIV-1 isolates that were markedly enhanced following boosting with homologous gp120 proteins in QS-21 adjuvant irrespective of the route of DNA immunization. These sera neutralized homologous and, to a lesser degree, heterologous HIV-1 isolates. Four of the six immunized animals were completely protected following rectal challenge with a SHIV encoding Env from HIV-1Ba-L, whereas the virus load was reduced in the remaining animals compared to naïve controls. Hence priming with DNA encoding Env antigens from multiple HIV-1 clades followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of protecting macaques against mucosal transmission of R5 tropic SHIV isolate.

Although a consensus has emerged that an HIV vaccine should elicit a cytotoxic T lymphocyte (CTL) response, the characteristics of an effective vaccine-induced T lymphocyte response remain unclear. We explored this issue in the simian human immunodeficiency virus/rhesus monkey model in the course of assessing the relative immunogenicity of vaccine regimens that included a cytokine-augmented plasmid DNA prime and a boost with DNA or recombinant pox vectors. Recombinant vaccinia virus, recombinant modified vaccinia Ankara (MVA), and recombinant fowlpox were comparable in their immunogenicity. Moreover, whereas the magnitude of the peak vaccine-elicited T lymphocyte responses in the recombinant pox virus-boosted monkeys was substantially greater than that seen in the monkeys immunized with plasmid DNA alone, the magnitudes of recombinant pox boosted CTL responses decayed rapidly and were comparable to those of the DNA-alone-vaccinated monkeys by the time of viral challenge. Consistent with these comparable memory T cell responses, the clinical protection seen in all groups of experimentally vaccinated monkeys was similar. This study, therefore, indicates that the steady-state memory, rather than the peak effector vaccine-elicited T lymphocyte responses, may be the critical immune correlate of protection for a CTL-based HIV vaccine.

Whereas several recent AIDS vaccine strategies have protected rhesus macaques against a pathogenic simian/human immunodeficiency virus (SHIV) 89.6P challenge, similar approaches have provided only modest, transient reductions in viral burden after challenge with virulent, pathogenic SIV, which is more representative of HIV infection of people. We show here that priming with replicating adenovirus recombinants encoding SIV env/rev , gag , and/or nef genes, followed by boosting with SIV gp120 or an SIV polypeptide mimicking the CD4 binding region of the envelope, protects rhesus macaques from intrarectal infection with the highly pathogenic SIV mac251 . Using trend analysis, significant reductions in acute-phase and set point viremia were correlated with anti-gp120 antibody and cellular immune responses, respectively. Within immunization groups exhibiting significant protection, a subset (39%) of macaques have exhibited either no viremia, cleared viremia, or controlled viremia at the threshold of detection, now more than 40 weeks postchallenge. This combination prime-boost strategy, utilizing replication competent adenovirus, is a promising alternative for HIV vaccine development.

Nucleotide sequences were obtained for portions of the envelope and gag genes of 4 HIV-1 isolates from Nigeria. The gag gene sequences clustered with previously described gag sequences from Gabon, Zaire, and Taiwan, which form the G clade of HIV-1. This documented for the first time the presence of subgroup G viruses in Nigeria. The env gene sequences were most closely related to env sequences reported from 2 isolates previously classified as gag subgroup G and, with those 2 env sequences, defined the subgroup G env genotype. Virus isolates were obtained by cocultivation of peripheral blood mononuclear cells (PBMCs) from Nigerian AIDS patients (isolates JV1018 and JP88) or healthy prostitutes (G3 and G9) with normal uninfected PBMCs (JV1083, JP88, and G3) or GEM-SS, a T cell line (G9). Clones containing env and gag gene sequences were obtained by polymerase chain reaction amplification, using DNA from these cultures. The region of gag amplified by primers SK37 and SK39 containing BamHI and KpnI sites, respectively, in 5' tails, was cloned into the KpnI-BamHI site of pKSBluescript and sequenced. When the gag sequences from 2 of the Nigerian viruses were subtyped by weighted parsimony, both viruses grouped with others of the gag G subgroup. A similar analysis of env sequences from all 4 Nigerian isolates showed that they were most closely related to 2 env sequences that had been assigned to env subgroup G. The Nigerian env sequences clustered more closely with each other than they did with the other 2 subgroup G viruses. Consensus of the inferred amino acid sequences of the 4 env genes showed that the V3 loop closely resembled the A to F consensus. 8 positions in the Nigerian consensus sequence appeared to be unique. However, whether these are signature residues for Nigerian subgroup G isolates or for subgroup G isolates will require the characterization of more isolates.

Elucidation of host factors that modulate susceptibility to infection with human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) and influence disease outcome will not only broaden our understanding of virus-cell interactions but will also have important practical implications. Knowledge of natural host defense mechanisms may lead to their exploitation for therapeutic or prophylactic purposes. Identification of host susceptibility factors may influence treatment decisions and further define risk factors for HIV acquisition. Clarification of host susceptibility and resistance factors in nonhuman primates should limit the variability in experimental groups and lead to improved design of preclinical studies. A few immunologic and genetic host factors have been identified which influence HIV or SIV infection and viral replication (11). The former includes acquired immunity resulting from viral infection, as well as innate immunity involving many inducible cytokines and chemokines. Genetic factors, such as HLA haplotypes, can influence the host's immune response. Two haplotypes have been associated with rapid disease progression after HIV infection (10), whereas another has been linked to long-term nonprogression (39). A similar protective effect of the rhesus macaque Mamu A*01 genotype has been recently demonstrated (43). The most dramatic genetic influence on HIV transmission and disease progression involves a 32-bp deletion (Δ32) in the CCR5 gene, the major coreceptor for macrophage-tropic, non-syncytium-inducing HIV isolates. This deletion results in a truncated, nonfunctional gene product and is associated with protection against HIV infection in individuals homozygous for the CCR5 Δ32 allele (20, 30, 49) and with delayed disease progression and decreased CCR5 expression on T cells among heterozygous individuals (13, 45, 59). Additional CCR5 polymorphisms exist but have not been shown to modulate HIV infection (37). Polymorphisms in the CCR5 promoter have been associated with accelerated disease progression (34, 36), but the mechanisms for this are unclear. A minor coreceptor mutation, CCR2-64I, with strong linkage disequilibrium with a CCR5 promoter region mutation (25), was initially associated with delayed disease progression (53), although subsequent studies have yielded conflicting results (33, 37, 38). Alterations in chemokine or cytokine genes or their promoters can also affect the course of HIV disease. Delayed disease progression has been attributed to a polymorphism in the RANTES promoter (29) and to an SDF-1 gene mutation, although the latter finding is controversial (19, 41, 57, 58). Polymorphisms in the interleukin-10 (IL-10) promoter have been linked to AIDS progression (52). Identification of host factors which contribute to susceptible or resistant phenotypes is difficult in humans. Our observation, summarized below, of unusually strong resistance to SIV infection in a rhesus macaque, presented a unique opportunity for investigating novel host resistance mechanisms. In general, intravaginal infection of rhesus macaques can result in either transient or persistent viremia (40), as well as in occult systemic infection in some cases (35). In earlier preclinical vaccine studies in which rhesus macaques were challenged with infectious, pathogenic SIVmac251, we observed such variable outcomes in both controls and immunized monkeys with regard to infection and disease progression (7, 8). One naive control macaque, 359, resisted two intravaginal exposures with escalating dosages of SIVmac251. Here we report that, after an additional intrarectal challenge with SIVmac32H, macaque 359 became only transiently viremic and cleared virus from the peripheral blood. In vitro studies confirmed that the animal's peripheral blood mononuclear cells (PBMCs) were highly resistant to SIV infection, even though adequate levels of CCR5 were expressed on the surface of the cells. In order to elucidate the basis for the unusually strong resistance of macaque 359 to in vivo and in vitro SIV infection, early events in the viral infection and replication process were examined. We examined several coreceptor genes for mutational changes. Genetic polymorphisms were detected in the CCR5 gene; however, none of the changes led to amino acid substitutions. Investigation of reverse transcription events revealed significant inhibition in the accumulation of early DNA replication intermediates. However, macaque 359 cells were able to fuse with cells expressing a SIVmac251 envelope or a CCR5-tropic HIV envelope as readily as cells from macaques highly susceptible to SIV infection. Taken together, our results indicate that the resistance of macaque 359 to SIV infection is due to postentry inhibition of viral replication and implicate a host cell mechanism in this process.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.

The conserved, immunogenic CD4 binding site on the viral envelope is an attractive HIV or SIV vaccine candidate. Polymerization of an 18 amino acid segment derived from the C4 domain of SIV gp120 produced a peptide polymer or "peptomer," having an alpha-helical conformation possibly mimicking a proposed structure of the C4 domain in the unbound native protein. The SIV peptomer and native gp120 were compared as subunit boosts following two adenovirus type 5 host range (Ad5hr)-SIVenv recombinant priming immunizations. Both vaccine regimens successfully elicited SIV-specific CTL responses in five of six immunized macaques. Peptomer-boosted macaques exhibited significantly higher envelope-specific T cell proliferative responses than either the gp120-boosted macaques or controls. Peptomer immunization also elicited peptomer and SIV gp120-specific binding antibodies, but only native gp120 boosting elicited SIV neutralizing antibodies. Upon intrarectal challenge with SIVmac32H, all nine macaques became infected. The solely envelope-based vaccine conferred no protection. However, changing the boosting immunogen to the C4 peptomer did not improve protective efficacy in spite of its elicitation of humoral and cellular immune responses, including robust T-helper activity. In spite of the peptomer's strong immunogenicity and potential for induction of broadly protective immune responses, it was not effective as a subunit vaccine.

Prolonged antiretroviral therapy (ART) is not likely to eradicate human immunodeficiency virus type I (HIV-I) infection. Here we explore the effect of therapeutic immunization in the context of ART during primary infection using the simian immunodeficiency virus (SIV251) macaque model. Vaccination of rhesus macaques with the highly attenuated poxvirus-based NYVAC-SIV vaccine expressing structural genes elicited vigorous virus-specific CD4 + and CD8+ T cell responses in macaques that responded effectively to ART. Following discontinuation of a six-month ART regimen, viral rebound occurred in most animals, but was transient in six of eight vaccinated animals. Viral rebound was also transient in four of seven mock-vaccinated control animals. These data establish the importance of antiretroviral treatment during primary infection and demonstrate that virus-specific immune responses in the infected host can be expanded by therapeutic immunization.

Rhesus macaques were immunized with a combination vaccine regimen consisting of adenovirus type 5 host range mutant-simian immunodeficiency virus envelope (Ad5hr-SIVenv) recombinant priming and boosting with native SIV gp120. Upon intravaginal challenge with SIVmac251, both persistently and transiently viremic animals were observed (S. L. Buge, E. Richardson, S. Alipanah, P. Markham, S. Cheng, N. Kalyan, C. J. Miller, M. Lubeck, S. Udem, J. Eldridge, and M. Robert-Guroff, J. Virol. 71:8531–8541, 1997). Long-term follow-up of the persistently viremic immunized macaques, which displayed significantly reduced viral burdens during the first 18 weeks postchallenge compared to controls, has now shown that one of four became a slow progressor, clearing virus from plasma and remaining asymptomatic with stable CD4 counts for 134 weeks postchallenge. Reboosting of the transiently viremic macaques did not reactivate latent virus. Rechallenge with two sequential SIVmac251 intravaginal exposures again resulted in partial protection of one of two immunized macaques, manifested by viral clearance and stable CD4 counts. No single immune parameter was associated with partial protection. Development of a strong antibody response capable of neutralizing a primary SIVmac251 isolate together with SIV-specific cytotoxic T lymphocytes were implicated, while CD8 + T-cell antiviral activity and mucosal immune responses were not associated with delayed disease progression. Our data show that even a third immunization with the same Ad5hr-SIVenv recombinant can elicit significant immune responses to the inserted gene product, suggesting that preexisting Ad antibodies may not preclude effective immunization. Further, the partial protection against a virulent, pathogenic SIV challenge observed in two of six macaques immunized with a vaccine regimen based solely on the viral envelope indicates that this vectored-vaccine approach has promise and that multicomponent vaccines based in the same system merit further investigation.

Protection against intravenous simian immunodeficiency virus (SIV) challenge was assessed in rhesus macaques after immunization with a highly attenuated vaccinia (NYVAC)-SIV recombinant. One-third of vaccinated animals controlled viral infection and progressed to disease more slowly than control animals (Benson J, et al.: J Virol 1998;72:4170). However, this protection was not associated with neutralizing antibodies, cytotoxic T lymphocytes, or helper T cell responses. To explore other potential correlates of protection, we examined CD8+ T cell antiviral activity in macaques vaccinated with NYVAC-SIV, with or without added cytokine adjuvants, and in controls receiving only IL-12 or IL-12 plus IL-2. Before immunization, naive macaques exhibited a broad range of CD8+ T cell antiviral activity. Nevertheless, in the course of immunization, the vaccinated macaques as a group developed increased CD8+ T cell antiviral activity while the controls remained stable. Infectious SIV exposure also increased antiviral activity. Prechallenge antiviral activity levels of vaccinated macaques were not sufficient to prevent SIV transmission or control viral replication during acute infection. However, vaccinated animals consistently exhibited reduced viral loads postchallenge compared with controls. Moreover, high suppressive activity 8 weeks postchallenge, at which time the viremia set point was established, was significantly correlated with reduced viral load and slow disease progression. Prechallenge antiviral activity influenced this result, as decreased viremia and slow progressor status were more apparent in macaques with high suppressive activity both pre- and postchallenge. Our data demonstrate the impact of CD8+ antiviral activity on viral replication and disease progression, and suggest that vaccine designs able to elicit high levels of this activity will contribute significantly to protective efficacy.

Immunization schemes employing priming with vector-based vaccine candidates followed by subunit booster administrations have been explored and shown to have merit in the human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus systems. In this study, we have assessed the priming capacity of highly attenuated poxvirus vector (NYVAC and ALVAC)-based HIV-2 recombinants, as well as Salmonella typhimurium HIV-2 recombinants in rhesus macaques. ALVAC- and NYVAC-based vaccine candidates expressing the HIV-2 gag, pol, and env genes or NYVAC-based recombinants expressing either gp160 or gp120 were used to immunize rhesus macaques in combination protocols with alum-adjuvanted HIV-2 rgp160. Following intravenous challenge exposure with 100 infectious doses of the HIV-2SBL6669 parental virus genotype mixture, seven of eight animals were protected from infection. The seven protected animals were rechallenged 6 months postprimary challenge, without additional booster inoculations, with the same dose of the HIV-2SBL6669 parental virus. Five of the seven animals remained protected against HIV-2 infection at 6 months following the second challenge. In contrast, oral immunization with recombinant Salmonella expressing the HIV-2 gag and the gp120 portion of the envelope either alone or in combination with alum-adjuvanted rgp160 failed to confer protection. These results suggest that the NYVAC- and ALVAC-based recombinants may confer long-lasting protection and that these two highly attenuated poxvirus vaccine vectors may represent promising candidates for developing an acquired immunodeficiency syndrome vaccine.

Kaposi's sarcoma (KS) is a proliferative disease of vascular origin particularly frequent in HIV-1-infected homosexual men (AIDS-KS) and characterized by proliferating spindle-shaped cells, angiogenesis, and inflammatory cell infiltration. Previous work has suggested that KS spindle cells are of endothelial cell origin and that chronic immune activation via the release of inflammatory cytokines may cooperate with basic fibroblast growth factor (bFGF) and the HIV-1 Tat protein in the induction and progression of AIDS-KS. Here we show that KS spindle cells have features of activated endothelial cells, and that conditioned media from activated T cells, rich in the same inflammatory cytokines increased in HIV-1-infected individuals, induce normal endothelial cells to acquire the phenotypic and functional features of KS cells. These include (a) acquisition of a similar pattern of cell surface antigen expression; (b) similar proliferative response to bFGF; (c) induction of the responsiveness to the mitogenic effect of extracellular HIV-1 Tat protein that is now able to promote the G1-S transition of endothelial cell cycle; and (d) induction in nude mice of vascular lesions closely resembling early KS as well as the lesions induced by inoculation of KS cells. These results suggest that chronic immune activation, via release of inflammatory cytokines, may play a role in the induction of KS.

Eighteen rhesus macaques were inoculated with either an infectious molecularly cloned human immunodeficiency virus type 2 (HIV-2)SBL/ISY, or with one of eight mutants defective in one or more accessory genes. The immune responses generated by the macaques were monitored for up to 2 years postinfection. All the macaques except those that received mutants lacking the vpr or vif genes demonstrated low to moderate antibody titers. Macaques inoculated with vpx- mutants exhibited a persistent serological response, suggesting continuous virus expression even in the absence of detectable virus in the peripheral blood mononuclear cells (PBMCs). Neutralizing antibodies developed in only four macaques. In general, low-level cytotoxic T lymphocyte (CTL) activity, not clearly HIV-2 specific, was detected in PBMCs. However, one virus-negative macaque exhibited significant HIV-2-specific CTL activity in an enriched CD8+ cell population from PBMCs, suggesting clearance of the viral infection. In addition, CTL activity against the Env and Gag/Pol epitopes of HIV-2 by CD8+ lymphocytes from the spleens and lymph nodes of two infected macaques, in one case requiring CD8+ T cell enrichment and in the other clearly evident in unfractionated tissue lymphocytes, was demonstrated for the first time. This sequestration of tissue CTLs occurred in the absence of significant levels of circulating CTLs in the blood. Our results suggest that routine monitoring of PBMCs may sometimes be inadequate for detecting cell-mediated immune responses. Elucidation of immune correlates of vaccine protection may therefore require sampling of lymphoid tissues and assessment of enriched CD8+ populations.