Vaccine-elicited memory cytotoxic T lymphocytes contribute to Mamu-A*01-associated control of simian/human immunodeficiency virus 89.6P replication in rhesus monkeys

The expression of particular major histocompatibility complex (MHC) class I alleles can influence the rate of disease progression following lentiviral infections. This effect is a presumed consequence of potent cytotoxic T-lymphocyte (CTL) responses that are restricted by these MHC class I molecules. The present studies have examined the impact of the MHC class I allele Mamu-A*01 on simian/human immunodeficiency virus 89.6P (SHIV-89.6P) infection in unvaccinated and vaccinated rhesus monkeys by exploring the contribution of dominant-epitope specific CTL in this setting. Expression of Mamu-A*01 in immunologically naive monkeys was not associated with improved control of viral replication, CD4+ T-lymphocyte loss, or survival. In contrast, Mamu-A*01+ monkeys that had received heterologous prime/boost immunizations prior to challenge maintained higher CD4+ T-lymphocyte levels and better control of SHIV-89.6P replication than Mamu-A*01- monkeys. This protection was associated with the evolution of high-frequency anamnestic CTL responses specific for a dominant Mamu-A*01-restricted Gag epitope following infection. These data indicate that specific MHC class I alleles can confer protection in the setting of a pathogenic SHIV infection by their ability to elicit memory CTL following vaccination.     

Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag

The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(-) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(-) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.     

Cytotoxic T-lymphocyte escape does not always explain the transient control of simian immunodeficiency virus SIVmac239 viremia in adenovirus-boosted and DNA-primed Mamu-A*01-positive rhesus macaques

Adenovirus 5 (Ad5) vectors show promise as human immunodeficiency virus vaccine candidates. Indian rhesus macaques vaccinated with Ad5-gag controlled simian-human immunodeficiency virus SHIV89.6P viral replication in the absence of Env immunogens that might elicit humoral immunity. Here we immunized 15 macaques using either a homologous Ad5-gag/Ad5-gag (Ad5/Ad5) or a heterologous DNA-gag/Ad5-gag (DNA/Ad5) prime-boost regimen and challenged them with a high dose of simian immunodeficiency virus SIVmac239. Macaques vaccinated with the DNA/Ad5 regimen experienced a brief viral load nadir of less than 10,000 viral copies per ml blood plasma that was not seen in Mamu-A*01-negative DNA/Ad5 vaccinees, Mamu-A*01-positive Ad5/Ad5 vaccinees, or vaccine-naive controls. Interestingly, most of these animals were not durably protected from disease progression when challenged with SIVmac239. To investigate the reasons underlying this short-lived vaccine effect, we investigated breadth of the T-cell response, immunogenetic background, and viral escape from CD8+ lymphocytes that recognize immunodominant T-cell epitopes. We show that these animals do not mount unusually broad cellular immune response, nor do they express unusual major histocompatibility complex class I alleles. Viral recrudescence occurred in four of the five Mamu-A*01-positive vaccinated macaques. However, only a single animal in this group demonstrated viral escape in the immunodominant Gag181-189 CM9 response. These results suggest that viral "breakthrough" in vaccinated animals and viral escape are not inextricably linked and underscore the need for additional research into the mechanisms of vaccine failure.     

Multispecific vaccine-induced mucosal cytotoxic T lymphocytes reduce acute-phase viral replication but fail in long-term control of simian immunodeficiency virus SIVmac239

Given the current difficulties generating vaccine-induced neutralizing antibodies to human immunodeficiency virus (HIV), the focus of the vaccine community has shifted toward creating cytotoxic-T-lymphocyte (CTL)-based vaccines. Recent reports of CTL-based vaccine trials in macaques challenged with simian/human immunodeficiency virus SHIV-89.6P have supported the notion that such vaccines can ameliorate the course of disease. However, almost all of these studies included Env as an immunogen and since SHIV-89.6P is sensitive to neutralizing antibodies it is difficult to determine the mechanism(s) of protection. Consequently, SHIV-89.6P challenge of macaques may be a poor model for determining vaccine efficacy in humans. To ascertain the effect of vaccine-induced multispecific mucosal CTL, in the absence of Env-specific antibody, on the control of an immunodeficiency virus challenge, we vaccinated Mamu-A*01(+) macaques with constructs encoding a combination of CTL epitopes and full-length proteins (Tat, Rev, and Nef) by using a DNA prime/recombinant modified vaccinia virus Ankara (rMVA) boost regimen. The vaccination induced virus-specific CTL and CD4(+) helper T lymphocytes with CTL frequencies as high as 20,000/million peripheral blood mononuclear cells. The final rMVA vaccination, delivered intravenously, engendered long-lived mucosal CTL. At 16 weeks after the final rMVA vaccination, the vaccinees and naive, Mamu-A*01(+) controls were challenged intrarectally with SIVmac239. Massive early anamnestic cellular immune responses controlled acute-phase viral replication; however, the three vaccinees were unable to control virus replication in the chronic phase. The present study suggests that multispecific mucosal CTL, in the absence of neutralizing antibodies, can achieve a modicum of control over early viral replication but are unable to control chronic-phase viral replication after a high-dose mucosal challenge with a pathogenic simian immunodeficiency virus.     

Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys.

In vivo passage of a chimeric simian-human immunodeficiency virus (SHIV-89.6) expressing the human immunodeficiency virus type 1 (HIV-1) tat, rev, vpu, and env genes generated pathogenic viruses (SHIV-89.6P) inducing rapid CD4+ lymphocyte depletion and AIDS-like illness in rhesus monkeys (K. Reimann, J. T. Li, R. Veazey, M. Halloran, I.-W. Park, G. B. Karlsson, J. Sodroski, and N. L. Letvin, J. Virol. 70:6922-6928, 1996). To characterize the molecular changes responsible for this increase in virulence, infectious proviral clones of SHIV-89.6P isolates were derived. Viruses generated from some of these clones caused a rapid and profound decline of CD4+ lymphocytes in a high percentage of inoculated monkeys. Nucleotide changes potentially responsible for the increased virulence of SHIV-89.6P were limited to the env, tat, or long terminal repeat sequences, with most of the observed changes in env. Nucleotide changes in env altered 12 amino acids in the gp120 and gp41 exterior domains, and a 140-bp deletion in env resulted in the substitution of the carboxyl terminus of the SIVmac gp41 glycoprotein for that of the HIV-1 gp41 glycoprotein. The availability of pathogenic proviral clones should facilitate dissection of the molecular determinants of SHIV-89.6P virulence.     

A chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys.

The utility of the simian immunodeficiency virus of macaques (SIVmac) model of AIDS has been limited by the genetic divergence of the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and the SIVs. To develop a better AIDS animal model, we have been exploring the infection of rhesus monkeys with chimeric simian/human immunodeficiency viruses (SHIVs) composed of SIVmac239 expressing HIV-1 env and the associated auxiliary HIV-1 genes tat, vpu, and rev. SHIV-89.6, constructed with the HIV-1 env of a cytopathic, macrophage-tropic clone of a patient isolate of HIV-1 (89.6), was previously shown to replicate to a high degree in monkeys during primary infection. However, pathogenic consequences of chronic infection were not evident. We now show that after two serial in vivo passages by intravenous blood inoculation of naive rhesus monkeys, this SHIV (SHIV-89.6P) induced CD4 lymphopenia and an AIDS-like disease with wasting and opportunistic infections. Genetic and serologic evaluation indicated that the reisolated SHIV-89.6P expressed envelope glycoproteins that resembled those of HIV-1. When inoculated into naive rhesus monkeys, SHIV-89.6P caused persistent infection and CD4 lymphopenia. This chimeric virus expressing patient isolate HIV-1 envelope glycoproteins will be valuable as a challenge virus for evaluating HIV-1 envelope-based vaccines and for exploring the genetic determinants of HIV-1 pathogenicity.     

Efficacy of Multivalent Adenovirus-Based Vaccine against Simian Immunodeficiency Virus Challenge

The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01⁺/B*17⁻ Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01⁺ cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env ≈ Gag/Pol > Gag ≈ Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.There is a significant body of evidence suggesting that anti-human immunodeficiency virus type 1 (HIV-1) cellular immunity plays a prominent role in controlling viral infection and progression to disease (15, 32, 33). This stimulated substantial research into vaccines capable of eliciting this type of immunity, and several vaccine candidates (5, 6, 8-13, 22, 29-31, 35) have reached various stages of clinical development. However, the viability of this general vaccine approach was recently undermined by the findings in a phase II trial (called the Step Study) that immunization with a replication-defective adenovirus serotype 5 (Ad5) vaccine expressing HIV-1 clade B Gag, Pol, and Nef was not effective in either reducing acquisition rates and/or lowering set point viral loads in infected subjects (2, 25). In fact, more infections were originally observed in the vaccine group than in the placebo arm (2).The outcomes of the Step Study led to several important questions. Do the results argue against the concept of a HIV-1 vaccine based on the induction of specific T lymphocytes? On the other hand, if cytotoxic T-lymphocyte (CTL) responses are intrinsically valuable for an effective vaccine, what are the shortcomings in the vaccine-induced immunity that contributed to the lack of efficacy in the Step trial? What is the predictive value of preclinical challenge studies for selection of future clinical vaccine candidates? The potential role of CTL responses in an effective vaccine is also challenged by the recently reported phase III study results for the ALVAC vCP1521 prime-AIDSVAX B/E boost vaccine. The efficacy of this vaccine in a low-risk population was recently shown to trend toward prevention of HIV acquisition and not reduction of viral loads (30). Unlike the Step study vaccine, the ALVAC/AIDSVAX vaccine approach utilized a heterologous prime-boost regimen and contained an Env component that may have contributed to the type of outcome observed here. A better understanding of the immune correlates for this vaccine may be possible following further experimental investigations of the samples collected from the phase III study and earlier-stage trials.Despite the proven efficacy of Ad5 vaccination against simian-human immunodeficiency virus 89.6P (SHIV89.6P) challenge, subsequent primate studies provided equivocal results. In a homologous prime-boost regimen, Ad5 vaccine expressing Gag was ineffective against a high-dose simian immunodeficiency virus SIVmac239 challenge (4, 24). The same study compared this regimen with the DNA prime/Ad5 boost regimen that was found to be efficacious in Mamu-A*01⁺ monkeys; the level of protection in the overall study was correlated with the breadth of epitopes recognized and the frequency of induced antigen-specific CTLs. In this study, we examine whether the expansion of antigens to include Pol, Nef, and Env gp140 using the Ad5/Ad5 regimen would improve the outcome against the same high-dose SIV challenge. Of particular interest is the combination of Gag, Pol, and Nef, for which the homologous human vaccine was utilized in the Step study (29).     

Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.     

Pre-Clinical Development of a Recombinant,Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

Background

There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.

Methods

The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.

Results

Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.

Conclusions

The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials.     

Vaccination of rhesus macaques with recombinant Mycobacterium bovis bacillus Calmette-Guérin Env V3 elicits neutralizing antibody-mediated protection against simian-human immunodeficiency virus with a homologous but not a heterologous V3 motif

Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations.     

Vaccine-elicited V3 loop-specific antibodies in rhesus monkeys and control of a simian-human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate envelope

Vaccine-elicited antibodies specific for the third hypervariable domain of the surface gp120 of human immunodeficiency virus type 1 (HIV-1) (V3 loop) were assessed for their contribution to protection against infection in the simian-human immunodeficiency virus (SHIV)/rhesus monkey model. Peptide vaccine-elicited anti-V3 loop antibody responses were examined for their ability to contain replication of SHIV-89.6, a nonpathogenic SHIV expressing a primary patient isolate HIV-1 envelope, as well as SHIV-89.6P, a pathogenic variant of that virus. Low-titer neutralizing antibodies to SHIV-89.6 that provided partial protection against viremia following SHIV-89.6 infection were generated. A similarly low-titer neutralizing antibody response to SHIV-89.6P that did not contain viremia after infection with SHIV-89.6P was generated, but a trend toward protection against CD4+ T-lymphocyte loss was seen in these infected monkeys. These observations suggest that the V3 loop on some primary patient HIV-1 isolates may be a partially effective target for neutralizing antibodies induced by peptide immunogens.     

ALVAC-SIV-gag-pol-env-based vaccination and macaque major histocompatibility complex class I (A*01) delay simian immunodeficiency virus SIVmac-induced immunodeficiency.

T-cell-mediated immune effector mechanisms play an important role in the containment of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication after infection. Both vaccination- and infection-induced T-cell responses are dependent on the host major histocompatibility complex classes I and II (MHC-I and MHC-II) antigens. Here we report that both inherent, host-dependent immune responses to SIVmac251 infection and vaccination-induced immune responses to viral antigens were able to reduce virus replication and/or CD4+ T-cell loss. Both the presence of the MHC-I Mamu-A*01 genotype and vaccination of rhesus macaques with ALVAC-SIV-gag-pol-env (ALVAC-SIV-gpe) contributed to the restriction of SIVmac251 replication during primary infection, preservation of CD4+ T cells, and delayed disease progression following intrarectal challenge exposure of the animals to SIV(mac251 (561)). ALVAC-SIV-gpe immunization induced cytotoxic T-lymphocyte (CTL) responses cumulatively in 67% of the immunized animals. Following viral challenge, a significant secondary virus-specific CD8+ T-cell response was observed in the vaccinated macaques. In the same immunized macaques, a decrease in virus load during primary infection (P = 0.0078) and protection from CD4 loss during both acute and chronic phases of infection (P = 0.0099 and P = 0.03, respectively) were observed. A trend for enhanced survival of the vaccinated macaques was also observed. Neither boosting the ALVAC-SIV-gpe with gp120 immunizations nor administering the vaccine by the combination of mucosal and systemic immunization routes increased significantly the protective effect of the ALVAC-SIV-gpe vaccine. While assessing the role of MHC-I Mamu-A*01 alone in the restriction of viremia following challenge of nonvaccinated animals with other SIV isolates, we observed that the virus load was not significantly lower in Mamu-A*01-positive macaques following intravenous challenge with either SIV(mac251 (561)) or SIV(SME660). However, a significant delay in CD4+ T-cell loss was observed in Mamu-A*01-positive macaques in each group. Of interest, in the case of intravenous or intrarectal challenge with the chimeric SIV/HIV strains SHIV(89.6P) or SHIV(KU2), respectively, MHC-I Mamu-A*01-positive macaques did not significantly restrict primary viremia. The finding of the protective effect of the Mamu-A*01 molecule parallels the protective effect of the B*5701 HLA allele in HIV-1-infected humans and needs to be accounted for in the evaluation of vaccine efficacy against SIV challenge models.     

Use of Major Histocompatibility Complex Class I/Peptide/β2M Tetramers To Quantitate CD8+ Cytotoxic T Lymphocytes Specific for Dominant and Nondominant Viral Epitopes in Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys

To evaluate the impact of the diversity of antigen recognition by T lymphocytes on disease pathogenesis, we must be able to identify and analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific for multiple viral epitopes. Many of the studies of the role of CD8(+) CTLs in AIDS pathogenesis have been done with simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies have frequently made use of the well-defined SIV Gag CTL epitope p11C,C-M presented to CTL by the HLA-A homologue molecule Mamu-A*01. In the present study we identified and fine mapped two novel Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived epitope p68A (STPPLVRLV) and the human immunodeficiency virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI). The frequency of CD8(+) CTLs specific for the p11C,C-M, p68A, and p41A epitopes was quantitated in the same animals with a panel of tetrameric Mamu-A*01/peptide/beta2m complexes. All SHIV-infected Mamu-A*01(+) rhesus monkeys tested had a high frequency of SIVmac Gag-specific CTLs to the p11C,C-M epitope. In contrast, only a fraction of the monkeys tested had detectable CTLs specific for the SIVmac Pol p68A and HIV-1 Env p41A epitopes, and these responses were detected at very low frequencies. Thus, the p11C,C-M-specific CD8(+) CTL response is dominant and the p41A- and p68A-specific CD8(+) CTL responses are nondominant. These results indicate that CD8(+) CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8(+) CTL responses to nondominant epitopes, due to the low frequency of these epitope-specific cells, may be difficult to detect and quantitate by this approach.     

Multigene DNA priming-boosting vaccines protect macaques from acute CD4+-T-cell depletion after simian-human immunodeficiency virus SHIV89.6P mucosal challenge

We evaluated four priming-boosting vaccine regimens for the highly pathogenic simian human immunodeficiency virus SHIV89.6P in Macaca nemestrina. Each regimen included gene gun delivery of a DNA vaccine expressing all SHIV89.6 genes plus Env gp160 of SHIV89.6P. Additional components were two recombinant vaccinia viruses, expressing SHIV89.6 Gag-Pol or Env gp160, and inactivated SHIV89.6 virus. We compared (i) DNA priming/DNA boosting, (ii) DNA priming/inactivated virus boosting, (iii) DNA priming/vaccinia virus boosting, and (iv) vaccinia virus priming/DNA boosting versus sham vaccines in groups of 6 macaques. Prechallenge antibody responses to Env and Gag were strongest in the groups that received vaccinia virus priming or boosting. Cellular immunity to SHIV89.6 peptides was measured by enzyme-linked immunospot assay; strong responses to Gag and Env were found in 9 of 12 vaccinia virus vaccinees and 1 of 6 DNA-primed/inactivated-virus-boosted animals. Vaccinated macaques were challenged intrarectally with 50 50% animal infectious doses of SHIV89.6P 3 weeks after the last immunization. All animals became infected. Five of six DNA-vaccinated and 5 of 6 DNA-primed/particle-boosted animals, as well as all 6 controls, experienced severe CD4(+)-T-cell loss in the first 3 weeks after infection. In contrast, DNA priming/vaccinia virus boosting and vaccinia virus priming/DNA boosting vaccines both protected animals from disease: 11 of 12 macaques had no loss of CD4(+) T cells or moderate declines. Virus loads in plasma at the set point were significantly lower in vaccinia virus-primed/DNA-boosted animals versus controls (P = 0.03). We conclude that multigene vaccines delivered by a combination of vaccinia virus and gene gun-delivered DNA were effective against SHIV89.6P viral challenge in M. nemestrina.     

Immunogenicity study of glycoprotein-deficient rabies virus expressing simian/human immunodeficiency virus SHIV89.6P envelope in a rhesus macaque

Rabies virus (RV) has recently been developed as a novel vaccine candidate for human immunodeficiency virus type 1 (HIV-1). The RV glycoprotein (G) can be functionally replaced by HIV-1 envelope glycoprotein (Env) if the gp160 cytoplasmic domain (CD) of HIV-1 Env is replaced by that of RV G. Here, we describe a pilot study of the in vivo replication and immunogenicity of an RV with a deletion of G (DeltaG) expressing a simian/human immunodeficiency virus SHIV(89.6P) Env ectodomain and transmembrane domain fused to the RV G CD (DeltaG-89.6P-RVG) in a rhesus macaque. An animal vaccinated with DeltaG-89.6P-RVG developed SHIV(89.6P) virus-neutralizing antibodies and SHIV(89.6P)-specific cellular immune responses after challenge with SHIV(89.6P). There was no evidence of CD4(+) T-cell loss, and plasma viremia was controlled to undetectable levels by 6 weeks postchallenge and has remained suppressed out to 22 weeks postchallenge.     

Induction of antibodies in guinea pigs and rhesus monkeys against the human immunodeficiency virus type 1 envelope: neutralization of nonpathogenic and pathogenic primary isolate simian/human immunodeficiency virus strains

We have compared the abilities of human immunodeficiency virus type 1 (HIV-1) envelope V3 peptides and recombinant gp120 to induce antibodies that neutralize simian/human immunodeficiency viruses (SHIVs). SHIV-89.6 is a nonpathogenic SHIV that expresses the envelope protein of primary HIV-1 isolate 89.6. SHIV-89.6P, clone KB9, is a pathogenic SHIV variant derived from SHIV-89.6. Infection of rhesus monkeys with these SHIVs rarely induces anti-V3 region antibodies. To determine the availability of the gp120 V3 loop for neutralizing antibody binding on SHIV-89.6 and KB9 virions, we have constructed immunogenic C4-V3 peptides from these SHIVs and induced anti-V3 antibodies in guinea pigs and rhesus monkeys. We found that both SHIV-89.6 and KB9 C4-V3 peptides induced antibodies that neutralized SHIV-89.6 but that only SHIV-KB9 C4-V3 peptide induced antibodies that neutralized SHIV-KB9. Immunoprecipitation assays demonstrated that SHIV-KB9 C4-V3 peptide-induced antibodies had a greater ability to bind SHIV-KB9 envelope proteins than did antibodies raised against SHIV-89.6 C4-V3 peptide. We have used a series of mutant HIV-1 envelope constructs to map the gp120 determinants that affect neutralization by anti-V3 antibodies. The residue change at position 305 of arginine (in SHIV-89.6) to glutamic acid (in SHIV-KB9) played a central role in determining the ability of peptide-induced anti-V3 antiserum to neutralize primary isolate SHIVs. Moreover, residue changes in the SHIV-89.6 V1/V2 loops also played roles in regulating the availability of the V3 neutralizing epitope on SHIV-89.6 and -KB9. Thus, SHIV-89.6 and -KB9 V3 region peptides are capable of inducing neutralizing antibodies against these primary isolate SHIVs, although the pathogenic SHIV-KB9 is less easily neutralized than its nonpathogenic variant SHIV-89.6. In contrast to natural infection with SHIV-89.6, in which few animals make anti-V3 antibodies, C4-V3 peptides frequently induced anti-V3 antibodies that neutralized primary isolate SHIV strains.     

Immunodomination in the evolution of dominant epitope-specific CD8+ T lymphocyte responses in simian immunodeficiency virus-infected rhesus monkeys

Because the control of HIV-1 replication is largely dependent on CD8+ T lymphocyte responses specific for immunodominant viral epitopes, vaccine strategies that increase the breadth of dominant epitope-specific responses should contribute to containing HIV-1 spread. Developing strategies to elicit such broad immune responses will require an understanding of the mechanisms responsible for focusing CD8+ T lymphocyte recognition on a limited number of epitopes. To explore this biology, we identified cohorts of rhesus monkeys that expressed the MHC class I molecules Mamu-A*01, Mamu-A*02, or both, and assessed the evolution of their dominant epitope-specific CD8+ T lymphocyte responses (Gag p11C- and Tat TL8-specific in the Mamu-A*01+ and Nef p199RY-specific in the Mamu-A*02+ monkeys) following acute SIV infection. The Mamu-A*02+ monkeys that also expressed Mamu-A*01 exhibited a significant delay in the evolution of the CD8+ T lymphocyte responses specific for the dominant Mamu-A*02-restricted SIV epitope, Nef p199RY. This delay in kinetics was not due to differences in viral load kinetics or magnitude or in viral escape mutations, but was associated with the evolution of the Mamu-A*01-restricted CD8+ T lymphocyte responses to the highly dominant SIV epitopes Gag p11C and Tat TL8. Thus, the evolution of dominant epitope-specific CD8+ T lymphocyte responses can be suppressed by other dominant epitope-specific responses, and this immunodomination is important in determining the kinetics of dominant epitope-specific responses.     

Mamu-A*01 allele-mediated attenuation of disease progression in simian-human immunodeficiency virus infection

Expression of several major histocompatibility complex (MHC) class I alleles is associated with a protective effect against disease progression in both human immunodeficiency virus type 1 and simian immunodeficiency virus infection. To understand the mechanism underlying this effect, we investigated the expression of the MHC class I allele Mamu-A*01 in simian-human immunodeficiency virus (SHIV) infection, one of the major models for evaluation of AIDS vaccine candidates. We found that disease progression was significantly delayed in Mamu-A*01-positive rhesus monkeys infected with the highly pathogenic SHIV 89.6P. The delay corresponded not only to a noted Mamu-A*01-restricted dominant cytotoxic T-lymphocyte (CTL) response but also to a lower viral load in lymph nodes (LN) and, importantly, to minimal destruction of LN structure during early infection. In contrast, Mamu-A*01-negative monkeys exhibited massive destruction of LN structure with accompanying rapid disease progression. These data indicate that MHC class I allele-restricted CTL responses may play an important role in preservation of lymphoid tissue structure, thereby resulting in attenuation of disease progression in immunodeficiency virus infection.     

Removal of a single N-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses

Glycans on human immunodeficiency virus (HIV) envelope protein play an important role in infection and evasion from host immune responses. To examine the role of specific glycans, we introduced single or multiple mutations into potential N-linked glycosylation sites in hypervariable regions (V1 to V3) of the env gene of HIV type 1 (HIV-1) 89.6. Three mutants tested showed enhanced sensitivity to soluble CD4. Mutant N7 (N197Q) in the carboxy-terminal stem of the V2 loop showed the most pronounced increase in sensitivity to broadly neutralizing antibodies (NtAbs), including those targeting the CD4-binding site (IgG1b12) and the V3 loop (447-52D). This mutant is also sensitive to CD4-induced NtAb 17b in the absence of CD4. Unlike the wild-type (WT) Env, mutant N7 mediates CD4-independent infection in U87-CXCR4 cells. To study the immunogenicity of mutant Env, we immunized pig-tailed macaques with recombinant vaccinia viruses, one expressing SIVmac239 Gag-Pol and the other expressing HIV-1 89.6 Env gp160 in WT or mutant forms. Animals were boosted 14 to 16 months later with simian immunodeficiency virus gag DNA and the cognate gp140 protein before intrarectal challenge with SHIV89.6P-MN. Day-of-challenge sera from animals immunized with mutant N7 Env had significantly higher and broader neutralizing activities than sera from WT Env-immunized animals. Neutralizing activity was observed against SHIV89.6, SHIV89.6P-MN, HIV-1 SF162, and a panel of subtype B primary isolates. Compared to control animals, immunized animals showed significant reduction of plasma viral load and increased survival after challenge, which correlated with prechallenge NtAb titers. These results indicate the potential advantages for glycan modification in vaccine design, although the role of specific glycans requires further examination.     

The simian immunodeficiency virus envelope glycoprotein contains two epitopes presented by the Mamu-A*01 class I molecule.

Cytotoxic T lymphocyte (CTL) responses against the simian immunodeficiency virus (SIV) envelope and Gag proteins were monitored in a Mamu-A*01-positive rhesus macaque infected with SIVsmE660. Peripheral blood mononuclear cells (PBMC) cultured with synthetic peptides spanning the entire gp160 and Gag coding region recognized a total of three epitopes. One located in Gag was identified as the previously described Mamu-A*01-restricted p11cC-->M epitope (CTPYDINQM). The other two epitopes, designated p15m and p54m, were located in the gp160 envelope protein. Both were nine amino acids in length and were predicted to bind Mamu-A*01 because they contained proline and leucine residues at positions 3 and 9, respectively. Indeed, expression of this class I major histocompatibility complex molecule was required for target cell recognition by envelope-specific CD8(+) T cells directed against both epitopes. These Mamu-A*01-restricted epitopes in the SIV envelope will be useful for monitoring immune responses in vaccinated or infected animals.