Gene-based vaccination with a mismatched envelope protects against simian immunodeficiency virus infection in nonhuman primates

The RV144 trial demonstrated that an experimental AIDS vaccine can prevent human immunodeficiency virus type 1 (HIV-1) infection in humans. Because of its limited efficacy, further understanding of the mechanisms of preventive AIDS vaccines remains a priority, and nonhuman primate (NHP) models of lentiviral infection provide an opportunity to define immunogens, vectors, and correlates of immunity. In this study, we show that prime-boost vaccination with a mismatched SIV envelope (Env) gene, derived from simian immunodeficiency virus SIVmac239, prevents infection by SIVsmE660 intrarectally. Analysis of different gene-based prime-boost immunization regimens revealed that recombinant adenovirus type 5 (rAd5) prime followed by replication-defective lymphocytic choriomeningitis virus (rLCMV) boost elicited robust CD4 and CD8 T-cell and humoral immune responses. This vaccine protected against infection after repetitive mucosal challenge with efficacies of 82% per exposure and 62% cumulatively. No effect was seen on viremia in infected vaccinated monkeys compared to controls. Protection correlated with the presence of neutralizing antibodies to the challenge viruses tested in peripheral blood mononuclear cells. These data indicate that a vaccine expressing a mismatched Env gene alone can prevent SIV infection in NHPs and identifies an immune correlate that may guide immunogen selection and immune monitoring for clinical efficacy trials.     

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.     

Glycosylation of Simian Immunodeficiency Virus Influences Immune-Tissue Targeting during Primary Infection, Leading to Immunodeficiency or Viral Control

Glycans of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) play pivotal roles in modulating virus-target cell interactions. We have previously reported that, whereas SIVmac239 is pathogenic, its deglycosylated essentially nonpathogenic mutant (Δ5G) serves as a live-attenuated vaccine, although both replicate similarly during primary infection. These findings prompted us to determine whether such a polarized clinical outcome was due to differences in the immune tissues targeted by these viruses, where functionally and phenotypically different memory CD4(+) T cells reside. The results showed that Δ5G replicates in secondary lymphoid tissue (SLT) at 1- to 2-log-lower levels than SIVmac239, whereas SIVmac239-infected but not Δ5G-infected animals deplete CXCR3(+) CCR5(+) transitional memory (TrM) CD4(+) T cells. An early robust Δ5G replication was localized to small intestinal tissue, especially the lamina propria (effector site) rather than isolated lymphoid follicles (inductive site) and was associated with the induction and depletion of CCR6(+) CXCR3(-) CCR5(+) effector memory CD4(+) T cells. These results suggest that differential glycosylation of Env dictates the type of tissue-resident CD4(+) T cells that are targeted, which leads to pathogenic infection of TrM-Th1 cells in SLT and nonpathogenic infection of Th17 cells in the small intestine, respectively.     

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.     

Vaccine protection by a triple deletion mutant of simian immunodeficiency virus.

Twelve rhesus monkeys were vaccinated with SIVmac316 delta nef (lacking nef sequences), and 12 were vaccinated with SIVmac239 delta3 (lacking nef, vpr, and upstream sequences in U3). SIVmac316 and SIVmac239 differ by only eight amino acids in the envelope; these changes render SIVmac316 highly competent for replication in macrophages. Seventeen of the animals developed persistent infections with the vaccine viruses. Seven of the 24 vaccinated animals, however, developed infections that were apparently transient in nature. Six of these seven yielded virus from peripheral blood when tested at weeks 2 and/or 3, three of the seven had transient antibody responses, but none of the seven had persisting antibody responses. The 24 monkeys were challenged in groups of four with 10 rhesus monkey infectious doses of wild-type, pathogenic SIVmac251 at weeks 8, 20, and 79 following receipt of vaccine. None of the seven with apparently transient infections with vaccine virus were protected upon subsequent challenge. Analysis of cell-associated viral loads, CD4+ cell counts, and viral gene sequences present in peripheral blood in the remainder of the monkeys following challenge allowed a number of conclusions. (i) There was a trend toward increased protection with length of time of vaccination. (ii) Solid vaccine protection was achieved by 79 weeks with the highly attenuated SIV239 delta3. (iii) Solid long-term protection was achieved in at least two animals in the absence of complete sterilizing immunity. (iv) Genetic backbone appeared to influence protective capacity; animals vaccinated with SIV239 delta3 were better protected than animals receiving SIV316 delta nef. This better protection correlated with increased levels of the replicating vaccine strain. (v) The titer of virus-neutralizing activity in serum on the day of challenge correlated with protection when measured against a primary stock of SIVmac251 but not when measured against a laboratory-passaged stock. The level of binding antibodies to whole virus by enzyme-linked immunosorbent assay also correlated with protection.     

Immunization of rhesus macaques with a DNA prime/modified vaccinia virus Ankara boost regimen induces broad simian immunodeficiency virus (SIV)-specific T-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic SIVmac239

Producing a prophylactic vaccine for human immunodeficiency virus (HIV) has proven to be a challenge. Most biological isolates of HIV are difficult to neutralize, so that conventional subunit-based antibody-inducing vaccines are unlikely to be very effective. In the rhesus macaque model, some protection was afforded by DNA/recombinant viral vector vaccines. However, these studies used as the challenge virus SHIV-89.6P, which is neutralizable, making it difficult to determine whether the observed protection was due to cellular immunity, humoral immunity, or a combination of both. In this study, we used a DNA prime/modified vaccinia virus Ankara boost regimen to immunize rhesus macaques against nearly all simian immunodeficiency virus (SIV) proteins. These animals were challenged intrarectally with pathogenic molecularly cloned SIVmac239, which is resistant to neutralization. The immunization regimen resulted in the induction of virus-specific CD8(+) and CD4(+) responses in all vaccinees. Although anamnestic neutralizing antibody responses against laboratory-adapted SIVmac251 developed after the challenge, no neutralizing antibodies against SIVmac239 were detectable. Vaccinated animals had significantly reduced peak viremia compared with controls (P < 0.01). However, despite the induction of virus-specific cellular immune responses and reduced peak viral loads, most animals still suffered from gradual CD4 depletion and progressed to disease.     

Significant protection against high-dose simian immunodeficiency virus challenge conferred by a new prime-boost vaccine regimen

We constructed vaccine vectors based on live recombinant vesicular stomatitis virus (VSV) and a Semliki Forest virus (SFV) replicon (SFVG) that propagates through expression of the VSV glycoprotein (G). These vectors expressing simian immunodeficiency virus (SIV) Gag and Env proteins were used to vaccinate rhesus macaques with a new heterologous prime-boost regimen designed to optimize induction of antibody. Six vaccinated animals and six controls were then given a high-dose mucosal challenge with the diverse SIVsmE660 quasispecies. All control animals became infected and had peak viral RNA loads of 10(6) to 10(8) copies/ml. In contrast, four of the vaccinees showed significant (P = 0.03) apparent sterilizing immunity and no detectable viral loads. Subsequent CD8(+) T cell depletion confirmed the absence of SIV infection in these animals. The two other vaccinees had peak viral loads of 7 × 10(5) and 8 × 10(3) copies/ml, levels below those of all of the controls, and showed undetectable virus loads by day 42 postchallenge. The vaccine regimen induced high-titer prechallenge serum neutralizing antibodies (nAbs) to some cloned SIVsmE660 Env proteins, but antibodies able to neutralize the challenge virus swarm were not detected. The cellular immune responses induced by the vaccine were generally weak and did not correlate with protection. Although the immune correlates of protection are not yet clear, the heterologous VSV/SFVG prime-boost is clearly a potent vaccine regimen for inducing virus nAbs and protection against a heterogeneous viral swarm.     

Accelerated heterologous adenovirus prime-boost SIV vaccine in neonatal rhesus monkeys

A pediatric human immunodeficiency virus type 1 (HIV-1) vaccine would be desirable to protect infants against HIV-1 transmission from breast-feeding. Such a vaccine would need to induce protective immunity at mucosal surfaces in neonates as soon as possible after birth. Recombinant adenovirus (rAd) vectors have been shown to elicit potent systemic and mucosal virus-specific immune responses in adult nonhuman primates and humans, but these vectors have not previously been comprehensively studied in infants. In this study, we demonstrate that a single injection of rAd26 encoding simian immunodeficiency virus mac239 (SIVmac239) Gag on the day of birth elicited detectable Gag-specific cellular immune responses in rhesus monkeys, but these responses were transient and waned quickly. In contrast, an accelerated heterologous prime-boost regimen involving administration of rAd35 at birth and rAd26 at 4 weeks of life elicited potent and durable Gag-specific cellular and humoral immune responses in neonatal rhesus monkeys, including mucosal responses that remained detectable at 1 year of age. These results suggest the potential of an accelerated heterologous rAd prime-boost regimen as a candidate HIV-1 vaccine for newborns.     

Simian immunodeficiency virus SIVmac239Deltanef vaccination elicits different Tat28-35SL8-specific CD8+ T-cell clonotypes compared to a DNA prime/adenovirus type 5 boost regimen in rhesus macaques

Different human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) vaccine vectors expressing the same viral antigens can elicit disparate T-cell responses. Within this spectrum, replicating variable vaccines, like SIVmac239Δnef, appear to generate particularly efficacious CD8(+) T-cell responses. Here, we sequenced T-cell receptor β-chain (TRB) gene rearrangements from immunodominant Mamu-A 01-restricted Tat(28-35)SL8-specific CD8(+) T-cell populations together with the corresponding viral epitope in four rhesus macaques during acute SIVmac239Δnef infection. Ultradeep pyrosequencing showed that viral variants arose with identical kinetics in SIVmac239Δnef and pathogenic SIVmac239 infection. Furthermore, distinct Tat(28-35)SL8-specific T-cell receptor (TCR) repertoires were elicited by SIVmac239Δnef compared to those observed following a DNA/Ad5 prime-boost regimen, likely reflecting differences in antigen sequence stability.     

Protection of Macaques with Diverse MHC Genotypes against a Heterologous SIV by Vaccination with a Deglycosylated Live-Attenuated SIV

HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc), following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a successful HIV vaccine against diverse isolates.     

Early cytokine and chemokine gene expression in lymph nodes of macaques infected with simian immunodeficiency virus is predictive of disease outcome and vaccine efficacy.

Competitive PCR was used to evaluate the expression of cytokine, granzyme B, and chemokine genes in lymph nodes of macaques recently infected with the simian immunodeficiency virus (SIV) pathogenic molecular clone SIVmac239 (n = 16), the nonpathogenic vaccine strain SIVmac239 delta nef (n = 8), and the nonpathogenic molecular clone SIVmac1A11 (n = 8). For both SIVmac239 and its nef-deleted derivative, strong expression was observed as early as 7 days postinfection for interleukin 1beta (IL-1beta), IL-6, tumor necrosis factor alpha, gamma interferon, and IL-13. The levels of gene induction were equally intense for both viruses despite a lower viral load for SIVmac239 deltanef compared with that for SIVmac239. However, the nature of the cytokine network activation varied with the viral inocula. Primary infection with SIVmac239 was characterized by a higher level of IL-4, IL-10, MIP-1alpha, MIP-1beta, MCP-1, and RANTES gene expression and a lower level of IL-12 and granzyme B gene expression compared with infection with SIVmac239 delta nef. Thus, infection with nef-deleted SIV was associated with a preferential Th1 versus Th2 pattern of cytokine production. Infection with SIVmac1A11 was characterized by a delayed immune response for all markers tested. The unique patterns of cytokine and chemokine gene expression in lymph nodes correlated nicely with the pathogenic potential of the SIV strains used as well as with differences in their ability to serve as protective vaccines.     

Influence of Mismatch of Env Sequences on Vaccine Protection by Live Attenuated Simian Immunodeficiency Virus

Vaccine/challenge experiments that utilize live attenuated strains of simian immunodeficiency virus (SIV) in monkeys may be useful for elucidating what is needed from a vaccine in order to achieve protective immunity. Derivatives of SIVmac239 and SIVmac239Δnef were constructed in which env sequences were replaced with those of the heterologous strain E543; these were then used in vaccine/challenge experiments. When challenge occurred at 22 weeks, 10 of 12 monkeys exhibited apparent sterilizing immunity despite a mismatch of Env sequences, compared to 12 of 12 monkeys with apparent sterilizing immunity when challenge virus was matched in its Env sequence. However, when challenge occurred at 6 weeks, 6 of 6 SIV239Δnef-immunized monkeys became superinfected by challenge virus mismatched in its Env sequence (SIV239/EnvE543). These results contrast markedly not only with the results of the week 22 challenge but also with the sterilizing immunity observed in 5 of 5 SIV239Δnef-immunized rhesus monkeys challenged at 5 weeks with SIV239, i.e., with no mismatch of Env sequences. We conclude from these studies that a mismatch of Env sequences in the challenge virus can have a dramatic effect on the extent of apparent sterilizing immunity when challenge occurs relatively early, 5 to 6 weeks after the nef-deleted SIV administration. However, by 22 weeks, mismatch of Env sequences has little or no influence on the degree of protection against challenge virus. Our findings suggest that anti-Env immune responses are a key component of the protective immunity elicited by live attenuated, nef-deleted SIV.     

CD4 independence of simian immunodeficiency virus Envs is associated with macrophage tropism, neutralization sensitivity, and attenuated pathogenicity

To investigate the basis for envelope (Env) determinants influencing simian immunodeficiency virus (SIV) tropism, we studied a number of Envs that are closely related to that of SIVmac239, a pathogenic, T-tropic virus that is neutralization resistant. The Envs from macrophage-tropic (M-tropic) virus strains SIVmac316, 1A11, 17E-Fr, and 1100 facilitated infection of CCR5-positive, CD4-negative cells. In contrast, the SIVmac239 Env was strictly dependent upon the presence of CD4 for membrane fusion. We also found that the Envs from M-tropic virus strains, which are less pathogenic in vivo, were very sensitive to antibody-mediated neutralization. Antibodies to the V3-loop, as well as antibodies that block SIV gp120 binding to CCR5, efficiently neutralized CD4-independent, M-tropic Envs but not the 239 Env. However, triggering the 239 Env with soluble CD4, presumably resulting in exposure of the CCR5 binding site, made it as neutralization sensitive as the M-tropic Envs. In addition, mutations of N-linked glycosylation sites in the V1/V2 region, previously shown to enhance antigenicity and immunogenicity, made the 239 Env partially CD4 independent. These findings indicate that Env-based determinants of M tropism of these strains are generally associated with decreased dependence on CD4 for entry into cells. Furthermore, CD4 independence and M tropism are also associated with neutralization sensitivity and reduced pathogenicity, suggesting that the humoral immune response may exert strong selective pressure against CD4-independent M-tropic SIVmac strains. Finally, genetic modification of viral Envs to enhance CD4 independence may also result in improved humoral immune responses.     

In vivo attenuation of simian immunodeficiency virus by disruption of a tyrosine-dependent sorting signal in the envelope glycoprotein cytoplasmic tail

Attenuated simian immunodeficiency viruses (SIVs) have been described that produce low levels of plasma virion RNA and exhibit a reduced capacity to cause disease. These viruses are particularly useful in identifying viral determinants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxxphi) in the envelope glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This domain constitutes both a potent endocytosis signal that reduces Env expression on infected cells and a sorting signal that directs Env expression to the basolateral surface of polarized cells. Rhesus macaques were inoculated with SIVmac239 control or SIVmac239 containing either a Tyr-721-to-Ile mutation (SIVmac239Y/I) or a deletion of Tyr-721 and the preceding glycine (DeltaGY). To assess the in vivo replication competence, all viruses contained a stop codon in nef that has been shown to revert during in vivo but not in vitro replication. All three control animals developed high viral loads and disease. One of two animals that received SIVmac239Y/I and two of three animals that received SIVmac239DeltaGY remained healthy for up to 140 weeks with low to undetectable plasma viral RNA levels and normal CD4(+) T-cell percentages. These animals exhibited ongoing viral replication as determined by detection of viral sequences and culturing of mutant viruses from peripheral blood mononuclear cells and persistent anti-SIV antibody titers. In one animal that received SIVmac239Y/I, the Ile reverted to a Tyr and was associated with a high plasma RNA level and disease, while one animal that received SIVmac239DeltaGY also developed a high viral load that was associated with novel and possibly compensatory mutations in the TM cytoplasmic domain. In all control and experimental animals, the nef stop codon reverted to an open reading frame within the first 2 months of inoculation, indicating that the mutant viruses had replicated well enough to repair this mutation. These findings indicate that the Yxxphi signal plays an important role in SIV pathogenesis. Moreover, because mutations in this motif may attenuate SIV through mechanisms that are distinct from those caused by mutations in nef, this Tyr-based sorting signal represents a novel target for future models of SIV and human immunodeficiency virus attenuation that could be useful in new vaccine strategies.     

Depo-Provera abrogates attenuated lentivirus-induced protection in male rhesus macaques challenged intravenously with pathogenic SIVmac239

BACKGROUND: Progesterone administration prior to intravaginal challenge with pathogenic SIVmac239 decreases the protective efficacy of live attenuated vaccines in rhesus macaques. METHODS: To determine if progesterone alters the efficacy of live attenuated vaccines through local or systemic effects, seven male rhesus macaques were immunized with SHIV89.6 and then challenged intravenously with SIVmac239. Three of these animals were treated with Depo-Provera 30 days prior to the SIV challenge. RESULTS: The SHIV animals had significantly lower plasma viral RNA levels than the unimmunized control monkeys, but the Depo-Provera treated, SHIV-immunized animals did not. Despite the lack of protection, the Depo-Provera SHIV animals had strong SIV specific T-cell responses. However, altered patterns of NK frequency and CD38 T-cell expression prior to SIV challenge were observed in Depo-Provera SHIV animals. CONCLUSIONS: Depo-Provera eliminates live-attenuated lentivirus vaccine efficacy in male rhesus monkeys through systemic effects on antiviral immunity and/or viral replication.     

Live,attenuated simian immunodeficiency virus SIVmac-M4, with point mutations in the Env transmembrane protein intracytoplasmic domain,provides partial protection from mucosal challenge with pathogenic SIVmac251

Attenuated molecular clones of simian immunodeficiency virus (SIVmac) are important tools for studying the correlates of protective immunity to lentivirus infection in nonhuman primates. The most highly attenuated SIVmac mutants fail to induce disease but also fail to induce immune responses capable of protecting macaques from challenge with pathogenic virus. We recently described a novel attenuated virus, SIVmac-M4, containing multiple mutations in the transmembrane protein (TM) intracytoplasmic domain. This domain has been implicated in viral assembly, infectivity, and cytopathogenicity. Whereas parental SIVmac239-Nef(+) induced persistent viremia and simian AIDS in rhesus macaques, SIVmac-M4 induced transient viremia in juvenile and neonatal macaques, with no disease for at least 1 year postinfection. In this vaccine study, 8 macaques that were infected as juveniles (n = 4) or neonates (n = 4) with SIVmac-M4 were challenged with pathogenic SIVmac251 administered through oral mucosa. At 1 year postchallenge, six of the eight macaques had low to undetectable plasma viremia levels. Assays of cell-mediated immune responses to SIVmac Gag, Pol, Env, and Nef revealed that all animals developed strong CD8(+) T-cell responses to Gag after challenge but not before. Unvaccinated control animals challenged with SIVmac251 developed persistent viremia, had significantly weaker SIV-specific T-cell responses, and developed AIDS-related symptoms. These findings demonstrate that SIVmac-M4, which contains a full-length Nef coding region and multiple point mutations in the TM, can provide substantial protection from mucosal challenge with pathogenic SIVmac251.     

Systemic immunization with an ALVAC-HIV-1/protein boost vaccine strategy protects rhesus macaques from CD4+ T-cell loss and reduces both systemic and mucosal simian-human immunodeficiency virus SHIVKU2 RNA levels

Transmission of human immunodeficiency virus type 1 (HIV-1) occurs primarily via the mucosal route, suggesting that HIV-1 vaccines may need to elicit mucosal immune responses. Here, we investigated the immunogenicity and relative efficacy of systemic immunization with two human ALVAC-HIV-1 recombinant vaccines expressing Gag, Pol, and gp120 (vCP250) or Gag, Pol, and gp160 (vCP1420) in a prime-boost protocol with their homologous vaccine native Env proteins. The relative efficacy was measured against a high-dose mucosal exposure to the pathogenic neutralization-resistant variant SHIV(KU2) (simian-human immunodeficiency virus). Systemic immunization with both vaccine regimens decreased viral load levels not only in blood but unexpectedly also in mucosal sites and protected macaques from peripheral CD4+ T-cell loss. This protective effect was stronger when the gp120 antigen was included in the vaccine. Inclusion of recombinant Tat protein in the boosting phase along with the Env protein did not contribute further to the preservation of CD4+ T cells. Thus, systemic immunization with ALVAC-HIV-1 vaccine candidates elicits anti-HIV-1 immune responses able to contain virus replication also at mucosal sites in macaques.     

Preexisting vaccinia virus immunity decreases SIV-specific cellular immunity but does not diminish humoral immunity and efficacy of a DNA/MVA vaccine

The influence of preexisting immunity to viral vectors is a major issue for the development of viral-vectored vaccines. In this study, we investigate the effect of preexisting vaccinia virus immunity on the immunogenicity and efficacy of a DNA/modified vaccinia Ankara (MVA) SIV vaccine in rhesus macaques using a pathogenic intrarectal SIV251 challenge. Preexisting immunity decreased SIV-specific CD8 and CD4 T cell responses but preserved the SIV-specific humoral immunity. In addition, preexisting immunity did not diminish the control of an SIV challenge mediated by the DNA/MVA vaccine. The peak and set point viremia was 150- and 17-fold lower, respectively, in preimmune animals compared with those of control animals. The peak and set point viremia correlated directly with colorectal virus at 2 wk postchallenge suggesting that early control of virus replication at the site of viral challenge was critical for viral control. Factors that correlated with early colorectal viral control included 1) the presence of anti-SIV IgA in rectal secretions, 2) high-avidity binding Ab for the native form of Env, and 3) low magnitude of vaccine-elicited SIV-specific CD4 T cells displaying the CCR5 viral coreceptor. The frequency of SIV-specific CD8 T cells in blood and colorectal tissue at 2 wk postchallenge did not correlate with early colorectal viral control. These results suggest that preexisting vaccinia virus immunity may not limit the potential of recombinant MVA vaccines to elicit humoral immunity and highlight the importance of immunodeficiency virus vaccines achieving early control at the mucosal sites of challenge.     

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.