Protection against lethal simian immunodeficiency virus SIVsmmPBj14 disease by a recombinant Semliki Forest virus gp160 vaccine and by a gp120 subunit vaccine.

Infection of pigtail macaques with SIVsmmPBj14, biological clone 3 (SIV-PBj14-bc13), produces an acute and usually fatal shock-like syndrome 7 to 14 days after infection. We used this simian immunodeficiency virus (SIV) model as a rapid and rigorous challenge to evaluate the efficacy of two SIV Env vaccine strategies. Groups of four pigtail macaques were immunized four times over a 25-week span with either a recombinant Semliki Forest virus expressing the SIV-PBj14 Env gp160 (SFV-SIVgp160) or purified recombinant SIV-PBj14 gp120 (rgp120) in SBN-1 adjuvant. Antibody titers to SIV Env developed in all immunized animals (mean peak titers prior to challenge, 1:1,700 for SFV-SIV gp 160 and 1:10,500 for rgp120), but neither neutralizing antibodies nor SIV-specific T-cell proliferative responses were detectable in any of the vaccinees. All macaques were challenged with a 100% infectious, 75% fatal dose of SIV-PBj14-bc13 at week 26. Three of four control animals died of acute SIV-PBj14 syndrome on days 12 and 13. By contrast, all four SFV-SIVgp160-immunized animals and three of the four rgp120-immunized animals were protected from lethal disease. While all virus-challenged animals became infected, symptoms of the SIV-PBj14 syndrome were more severe in controls than in vaccinees. Mean virus titers in plasma at 13 days postchallenge were approximately 10-fold lower in vaccinated than control animals. However, there was no apparent correlation between survival and levels of peripheral blood mononuclear cell-associated culturable virus, provirus load, or any antiviral immunologic parameter examined. The results indicate that while immunization with SFV-SIVgp160 and rgp120 did not protect against virus infection, these Env vaccines did lower the virus load in plasma and protect against the lethal SIV-PBj14 challenge.     

Recombinant modified vaccinia virus ankara expressing the surface gp120 of simian immunodeficiency virus (SIV) primes for a rapid neutralizing antibody response to SIV infection in macaques

Neutralizing antibodies were assessed before and after intravenous challenge with pathogenic SIVsmE660 in rhesus macaques that had been immunized with recombinant modified vaccinia virus Ankara expressing one or more simian immunodeficiency virus gene products (MVA-SIV). Animals received either MVA-gag-pol, MVA-env, MVA-gag-pol-env, or nonrecombinant MVA. Although no animals were completely protected from infection with SIV, animals immunized with recombinant MVA-SIV vaccines had lower virus loads and prolonged survival relative to control animals that received nonrecombinant MVA (I. Ourmanov et al., J. Virol. 74:2740-2751, 2000). Titers of neutralizing antibodies measured with the vaccine strain SIVsmH-4 were low in the MVA-env and MVA-gag-pol-env groups of animals and were undetectable in the MVA-gag-pol and nonrecombinant MVA groups of animals on the day of challenge (4 weeks after final immunization). Titers of SIVsmH-4-neutralizing antibodies remained unchanged 1 week later but increased approximately 100-fold 2 weeks postchallenge in the MVA-env and MVA-gag-pol-env groups while the titers remained low or undetectable in the MVA-gag-pol and nonrecombinant MVA groups. This anamnestic neutralizing antibody response was also detected with T-cell-line-adapted stocks of SIVmac251 and SIV/DeltaB670 but not with SIVmac239, as this latter virus resisted neutralization. Most animals in each group had high titers of SIVsmH-4-neutralizing antibodies 8 weeks postchallenge. Titers of neutralizing antibodies were low or undetectable until about 12 weeks of infection in all groups of animals and showed little or no evidence of an anamnestic response when measured with SIVsmE660. The results indicate that recombinant MVA is a promising vector to use to prime for an anamnestic neutralizing antibody response following infection with primate lentiviruses that cause AIDS. However, the Env component of the present vaccine needs improvement in order to target a broad spectrum of viral variants, including those that resemble primary isolates.     

ADCC Develops Over Time during Persistent Infection with Live-Attenuated SIV and Is Associated with Complete Protection against SIVmac251 Challenge

Live-attenuated strains of simian immunodeficiency virus (SIV) routinely confer apparent sterilizing immunity against pathogenic SIV challenge in rhesus macaques. Understanding the mechanisms of protection by live-attenuated SIV may provide important insights into the immune responses needed for protection against HIV-1. Here we investigated the development of antibodies that are functional against neutralization-resistant SIV challenge strains, and tested the hypothesis that these antibodies are associated with protection. In the absence of detectable neutralizing antibodies, Env-specific antibody-dependent cell-mediated cytotoxicity (ADCC) emerged by three weeks after inoculation with SIVΔnef, increased progressively over time, and was proportional to SIVΔnef replication. Persistent infection with SIVΔnef elicited significantly higher ADCC titers than immunization with a non-persistent SIV strain that is limited to a single cycle of infection. ADCC titers were higher against viruses matched to the vaccine strain in Env, but were measurable against viruses expressing heterologous Env proteins. In two separate experiments, which took advantage of either the strain-specificity or the time-dependent maturation of immunity to overcome complete protection against SIV_mac251 challenge, measures of ADCC activity were higher among the SIVΔnef-inoculated macaques that remained uninfected than among those that became infected. These observations show that features of the antibody response elicited by SIVΔnef are consistent with hallmarks of protection by live-attenuated SIV, and reveal an association between Env-specific antibodies that direct ADCC and apparent sterilizing protection by SIVΔnef.     

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.     

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.     

Vaccine-induced virus-neutralizing antibodies and cytotoxic T cells do not protect macaques from experimental infection with simian immunodeficiency virus SIVmac32H (J5).

To gain further insight into the ability of subunit vaccines to protect monkeys from experimental infection with simian immunodeficiency virus (SIV), two groups of cynomolgus macaques were immunized with either recombinant SIVmac32H-derived envelope glycoproteins (Env) incorporated into immune-stimulating complexes (iscoms) (group A) or with these SIV Env iscoms in combination with p27gag iscoms and three Nef lipopeptides (group B). Four monkeys immunized with recombinant feline immunodeficiency virus Env iscoms served as controls (group C). Animals were immunized intramuscularly at weeks 0, 4, 10, and 16. Two weeks after the last immunization, monkeys were challenged intravenously with 50 monkey 50% infectious doses of virus derived from the J5 molecular clone of SIVmac32H propagated in monkey peripheral blood mononuclear cells. High titers of SIV-neutralizing antibodies were induced in the monkeys of groups A and B. In addition, p27gag-specific antibodies were detected in the monkeys of group B. Vaccine-induced cytotoxic-T-lymphocyte precursors against Env, Gag, and Nef were detected on the day of challenge in the monkeys of group B. Env-specific cytotoxic-T-lymphocyte precursors were detected in one monkey from group A. In spite of the observed antibody and T-cell responses, none of the monkeys was protected from experimental infection. In addition, longitudinal determination of cell-associated virus loads at weeks 2, 4, 6, 9, and 12 postchallenge revealed no significant differences between vaccinated and control monkeys. These findings illustrate the need to clarify the roles of the different arms of the immune system in conferring protection against primate lentivirus infections.     

A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge.

Three infectious, attenuated molecular clones of simian immunodeficiency virus (SIVmac) were tested for viral and host determinants of protective immunity. The viruses differed in degree of virulence from highly attenuated to moderately attenuated to partially attenuated. Levels of immune stimulation and antiviral immunity were measured in rhesus macaques inoculated 2 years previously with these viruses. Monkeys infected with the highly attenuated or moderately attenuated viruses had minimal lymphoid hyperplasia, normal CD4/CD8 ratios, low levels of SIV-specific antibodies, and cytotoxic T-lymphocyte activity against p55gag (Gag) or gp160env (Env). Monkeys infected with the partially attenuated virus had moderate to marked lymphoid hyperplasia, normal CD4/CD8 ratios, high levels of SIV-specific antibodies, and cytotoxic T-lymphocyte activity against both Gag and Env. After pathogenic virus challenge, monkeys immunized with the partially attenuated virus had 100- to 1,000-fold-lower viral load in peripheral blood mononuclear cells and lymph node mononuclear cells than naive control animals. One of four monkeys immunized with the highly attenuated virus and two of four monkeys immunized with the moderately attenuated virus developed similarly low viral loads after challenge. These three attenuated strains of SIV induced a spectrum of antiviral immunity that was inversely associated with their degree of attenuation. Only the least attenuated virus induced resistance to challenge infection in all immunized monkeys.     

Comparative efficacy of recombinant modified vaccinia virus Ankara expressing simian immunodeficiency virus (SIV) Gag-Pol and/or Env in macaques challenged with pathogenic SIV

Prior studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high levels of viremia and AIDS following challenge with a pathogenic strain of SIV (V. M. Hirsch et al., J. Virol. 70:3741-3752, 1996). This MVA-SIV recombinant expressed relatively low levels of the Gag-Pol portion of the vaccine. To optimize protection, second-generation recombinant MVAs that expressed high levels of either Gag-Pol (MVA-gag-pol) or Env (MVA-env), alone or in combination (MVA-gag-pol-env), were generated. A cohort of 24 macaques was immunized with recombinant or nonrecombinant MVA (four groups of six animals) and was challenged with 50 times the dose at which 50% of macaques are infected with uncloned pathogenic SIVsmE660. Although all animals became infected postchallenge, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines as compared with animals that received nonrecombinant MVA (P = 0.0011 by repeated-measures analysis of variance). The differences in the degree of virus suppression achieved by the three MVA-SIV vaccines were not significant. Most importantly, the reduction in levels of viremia resulted in a significant increase in median (P < 0.05 by Student's t test) and cumulative (P = 0.010 by log rank test) survival. These results suggest that recombinant MVA has considerable potential as a vaccine vector for human AIDS.     

Immune response of rhesus macaques to recombinant simian immunodeficiency virus gp130 does not protect from challenge infection.

Simian immunodeficiency virus (SIV) infection of rhesus macaques is a model for human immunodeficiency virus (HIV) infection in humans. Inactivated and modified live whole-virus vaccines have provided limited protective immunity against SIV in rhesus macaques. Because of safety concerns in the use of inactivated and live whole-virus vaccines, we evaluated the protective immunity of vaccinia virus recombinants expressing the surface glycoprotein (gp130) of SIVmac and subunit preparations of gp130 expressed in mammalian cells (CHO). Three groups of animals were immunized with recombinant SIV gp130. The first group received SIV gp130 purified from genetically engineered CHO cells (cSIVgp130), the second group was vaccinated with recombinant vaccinia virus expressing SIVmac gp130 (vSIVgp130), and the third group was first primed with vSIVgp130 and then given a booster immunization with cSIVgp130. Although anti-gp130 binding antibodies were elicited in all three groups, neutralizing antibodies were transient or undetectable. None of the immunized animals resisted intravenous challenge with a low dose of cell-free virus. However, the group primed with vSIVgp130 and then boosted with cSIVgp130 had the lowest antigen load (p27) compared with the other groups. The results of these studies suggest that immunization of humans with HIV type 1 surface glycoprotein may not provide protective immunity against virus infection.     

Long-term control of simian immunodeficiency virus mac251 viremia to undetectable levels in half of infected female rhesus macaques nasally vaccinated with simian immunodeficiency virus DNA/recombinant modified vaccinia virus Ankara

The efficacy of two SIV DNA plus recombinant modified vaccinia virus Ankara nasal vaccine regimens, one combined with plasmids expressing IL-2 and IL-15, the other with plasmids expressing GM-CSF, IL-12, and TNF-α, which may better stimulate humoral responses, was evaluated in two female rhesus macaque groups. Vaccination stimulated significant SIV-specific mucosal and systemic cell-mediated immunity in both groups, whereas SIV-specific IgA titers were sporadic and IgG titers negative. All vaccinated animals, except one, became infected after intravaginal SIV(mac251) low-dose challenge. Half of the vaccinated, infected animals (7/13) promptly controlled virus replication to undetectable viremia for the duration of the trial (130 wk) and displayed virological and immunological phenotypes similar to those of exposed, uninfected individuals. When all vaccinated animals were considered, a 3-log viremia reduction was observed, compared with controls. The excellent viral replication containment achieved in vaccinated animals translated into significant preservation of circulating α4β7(high+)/CD4(+) T cells and of circulating and mucosal CD4(+)/C(M) T cells and in reduced immune activation. A more significant long-term survival was also observed in these animals. Median survival was 72 wk for the control group, whereas >50% of the vaccinated animals were still disease free 130 wk postchallenge, when the trial was closed. There was a statistically significant correlation between levels of CD4(+)/IFN-γ(+) and CD8(+)/IFN-γ(+) T cell percentages on the day of challenge and the control of viremia at week 60 postchallenge or survival. Postchallenge immunological correlates of protection were systemic anti-SIV Gag + Env CD4(+)/IL-2(+), CD4(+)/IFN-γ(+), and CD8(+)/TNF-α(+) T cells and vaginal anti-SIV Gag + Env CD8(+) T cell total monofunctional responses.     

A replication-competent adenovirus-human immunodeficiency virus (Ad-HIV) tat and Ad-HIV env priming/Tat and envelope protein boosting regimen elicits enhanced protective efficacy against simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques

We previously demonstrated that replication-competent adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinant prime/protein boost regimens elicit potent immunogenicity and strong, durable protection of rhesus macaques against SIV(mac251). Additionally, native Tat vaccines have conferred strong protection against simian/human immunodeficiency virus SHIV(89.6P) challenge of cynomolgus monkeys, while native, inactivated, or vectored Tat vaccines have failed to elicit similar protective efficacy in rhesus macaques. Here we asked if priming rhesus macaques with replicating Ad-human immunodeficiency virus (HIV) tat and boosting with the Tat protein would elicit protection against SHIV(89.6P). We also evaluated a Tat/Env regimen, adding an Ad-HIV env recombinant and envelope protein boost to test whether envelope antibodies would augment acute-phase protection. Further, expecting cellular immunity to enhance chronic viremia control, we tested a multigenic group: Ad-HIV tat, -HIV env, -SIV gag, and -SIV nef recombinants and Tat, Env, and Nef proteins. All regimens were immunogenic. A hierarchy was observed in enzyme-linked immunospot responses (with the strongest response for Env, followed by Gag, followed by Nef, followed by Tat) and antibody titers (with the highest titer for Env, followed by Tat, followed by Nef, followed by Gag). Following intravenous SHIV(89.6P) challenge, all macaques became infected. Compared to controls, no protection was seen in the Tat-only group, confirming previous reports for rhesus macaques. However, the multigenic group blunted acute viremia by approximately 1 log (P = 0.017), and both the multigenic and Tat/Env groups reduced chronic viremia by 3 and 4 logs, respectively, compared to controls (multigenic, P = 0.0003; Tat/Env, P < 0.0001). The strikingly greater reduction in the Tat/Env group than in the multigenic group (P = 0.014) was correlated with Tat and Env binding antibodies. Since prechallenge anti-Env antibodies lacked SHIV(89.6P)-neutralizing activity, other functional anti-Env and anti-Tat activities are under investigation, as is a possible synergy between the Tat and Env immunogens.     

Protection against High-Dose Highly Pathogenic Mucosal SIV Challenge at Very Low Serum Neutralizing Titers of the Antibody-Like Molecule CD4-IgG2

Passive transfer studies using monoclonal or polyclonal antibodies in the macaque model have been valuable for determining conditions for antibody protection against immunodeficiency virus challenge. Most studies have employed hybrid simian/human immunodeficiency virus (SHIV) challenge in conjunction with neutralizing human monoclonal antibodies. Passive protection against SIV, particularly the pathogenic prototype virus SIVmac239, has been little studied because of the paucity of neutralizing antibodies to this virus. Here, we show that the antibody-like molecule CD4-IgG2 potently neutralizes SIVmac239 in vitro. When administered by an osmotic pump to maintain concentrations given the short half-life of CD4-IgG2 in macaques, the molecule provided sterilizing immunity/protection against high-dose mucosal viral challenge to a high proportion of animals (5/7 at a 200 mg dose CD4-IgG2 and 3/6 at a 20 mg dose) at serum concentrations below 1.5 μg/ml. The neutralizing titers of such sera were predicted to be very low and indeed sera at a 1∶4 dilution produced no neutralization in a pseudovirus assay. Macaque anti-human CD4 titers did develop weakly at later time points in some animals but were not associated with the level of protection against viral challenge. The results show that, although SIVmac239 is considered a highly pathogenic virus for which vaccine-induced T cell responses in particular have provided limited benefit against high dose challenge, the antibody-like CD4-IgG2 molecule at surprisingly low serum concentration affords sterilizing immunity/protection to a majority of animals.     

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.     

Improved protection of rhesus macaques against intrarectal simian immunodeficiency virus SIV(mac251) challenge by a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen

In this study we investigated the ability of a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen to induce protective immunity in rhesus macaques against pathogenic simian immunodeficiency virus(mac251). Immunization of macaques by two sequential administrations of the same recombinants by the same route resulted in boosting and persistence of SIV-specific cellular immune responses for 42 weeks past the initial immunization. Anti-SIV gp120 immunoglobulin G (IgG) and IgA antibodies were induced in secretory fluids, and all macaques exhibited serum neutralizing antibody activity. After intrarectal SIV(mac251) challenge, all of the macaques became infected. However, relative protection, as assessed by statistically significant lower SIV viral loads in plasma at both acute infection and set point, was observed in 8 out of 12 immunized non-Mamu-A(*)01 animals. Elevated mean cellular immune responses to Gag and Env, neutralizing antibody activity, and IgG and IgA binding antibody levels were observed in the eight protected macaques. Statistically significant correlations with protective outcome were observed for cellular immune responses to SIV Env and Gag and for SIV gp120-specific IgG antibodies in nasal and vaginal fluids. Two macaques that exhibited the greatest and most persistent viremia control also exhibited strong CD8(+) T-cell antiviral activity. The results suggest that a spectrum of immune responses may be necessary for adequate control of viral replication and disease progression and highlight a potential role for nonneutralizing antibodies at mucosal sites.     

Immunization with a live, attenuated simian immunodeficiency virus (SIV) prevents early disease but not infection in rhesus macaques challenged with pathogenic SIV.

An infectious, virulence-attenuated molecular clone of simian immunodeficiency virus (SIV), SIVMAC-1A11, was derived from an SIV isolate that causes fatal immunodeficiency in rhesus macaques. When inoculated intravenously in rhesus macaques, SIVMAC-1A11 induced transient viremia (1 to 6 weeks) without clinical disease and a persistent humoral antibody response. The antibodies were directed mainly against the viral envelope glycoproteins, as determined by immunoblots and virus neutralization. The potential of this virulence-attenuated virus to protect against intravenous challenge with a pathogenic SIVMAC strain was assessed. Five rhesus macaques were each given two intravenous inoculations with SIVMAC-1A11 7 months apart. Three of the five immunized monkeys and four naive control animals were then challenged with 100 to 1,000 100% animal infectious doses of pathogenic SIVMAC. All seven animals became persistently viremic following the challenge. Four of four unimmunized animals developed severe clinical signs of simian acquired immunodeficiency syndrome by 38 to 227 days after challenge and were euthanatized 91 to 260 days postchallenge. However, no signs of illness were seen in immunized monkeys until 267 to 304 days postchallenge, when two of three immunized animals developed mild thrombocytopenia and lymphopenia; one of these animals died with clinical signs of simian immunodeficiency disease at 445 days after challenge. The two SIVMAC-1A11-immunized monkeys that were not challenged were healthy and antibody positive 22 months after the initial immunization. Thus, although live SIVMAC-1A11 was immunogenic and did not induce any disease, it failed to protect rhesus macaques against infection with a moderately high dose of pathogenic virus. However, immunization prevented severe, early disease and prolonged the lives of monkeys subsequently infected with pathogenic SIV.     

Complement-mediated, infection-enhancing antibodies in plasma from vaccinated macaques before and after inoculation with live simian immunodeficiency virus.

Rhesus monkeys vaccinated against infection with simian immunodeficiency virus (SIV) were examined, in retrospect, for the presence of infection-enhancing antibodies and possible consequences associated with the presence of these antibodies. At the time of experimental inoculation with live virus, complement-mediated, infection-enhancing antibodies were detected in plasma specimens from six of six animals vaccinated with detergent-inactivated whole virus, from nine of nine animals vaccinated with Formalin-inactivated whole virus, and from seven of eight animals immunized with two SIV subunit preparations. The presence of infection-enhancing antibodies at the time of viral challenge gave no indication of predicting vaccine success or failure. After live-virus challenge, titers of infection-enhancing antibodies, like enzyme-linked immunosorbent assay titers, increased in unprotected animals and decreased or became undetectable in animals protected by vaccination. Thus, vaccine protection against SIV infection can still be achieved in the presence of detectable complement-mediated, infection-enhancing antibodies.     

Sequential priming with simian immunodeficiency virus (SIV) DNA vaccines, with or without encoded cytokines, and a replicating adenovirus-SIV recombinant followed by protein boosting does not control a pathogenic SIVmac251 mucosal challenge

Previously, combination DNA/nonreplicating adenovirus (Ad)- or poxvirus-vectored vaccines have strongly protected against SHIV(89.6P), DNAs expressing cytokines have modulated immunity elicited by DNA vaccines, and replication-competent Ad-recombinant priming and protein boosting has strongly protected against simian immunodeficiency virus (SIV) challenge. Here we evaluated a vaccine strategy composed of these promising components. Seven rhesus macaques per group were primed twice with multigenic SIV plasmid DNA with or without interleukin-12 (IL-12) DNA or IL-15 DNA. After a multigenic replicating Ad-SIV immunization, all groups received two booster immunizations with SIV gp140 and SIV Nef protein. Four control macaques received control DNA plasmids, empty Ad vector, and adjuvant. All vaccine components were immunogenic, but the cytokine DNAs had little effect. Macaques that received IL-15-DNA exhibited higher peak anti-Nef titers, a more rapid anti-Nef anamnestic response postchallenge, and expanded CD8(CM) T cells 2 weeks postchallenge compared to the DNA-only group. Other immune responses were indistinguishable between groups. Overall, no protection against intrarectal challenge with SIV(mac251) was observed, although immunized non-Mamu-A*01 macaques as a group exhibited a statistically significant 1-log decline in acute viremia compared to non-Mamu-A*01 controls. Possible factors contributing to the poor outcome include administration of cytokine DNAs to sites different from the Ad recombinants (intramuscular and intratracheal, respectively), too few DNA priming immunizations, a suboptimal DNA delivery method, failure to ensure delivery of SIV and cytokine plasmids to the same cell, and instability and short half-life of the IL-15 component. Future experiments should address these issues to determine if this combination approach is able to control a virulent SIV challenge.     

Effect of CD8+ lymphocyte depletion on virus containment after simian immunodeficiency virus SIVmac251 challenge of live attenuated SIVmac239delta3-vaccinated rhesus macaques

Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8+ lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Delta3 of CD8+ lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals. The depletion of CD8+ lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8+ cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8+ T-cell responses and lower neutralizing antibody titers than those in Mamu-A*01- animals. Consistent with these findings, the depletion of CD8+ lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in Mamu-A*01+ than in Mamu-A*01- animals. The partial control of postchallenge viremia after CD8+ lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.     

Rhesus macaques previously infected with simian/human immunodeficiency virus are protected from vaginal challenge with pathogenic SIVmac239.

Nontraumatic vaginal inoculation of rhesus macaques with a simian/human immunodeficiency virus (SIV/HIV) chimera containing the envelope gene from HIV-1 89.6 (SHIV 89.6) results in systemic infection (Y. Lu, B. Brosio, M. Lafaile, J. Li, R. G. Collman, J. Sodroski, and C. J. Miller, J. Virol. 70:3045-3050, 1996). A total of five rhesus macaques have each been infected by exposure to at least three intravaginal inoculations of SHIV 89.6. The SHIV 89.6 infection is characterized by a transient viremia that evokes humoral and cellular immune responses to HIV and SIV antigens, but disease does not develop in animals infected with SHIV 89.6. To determine if a previous infection with SHIV 89.6 by vaginal inoculation could protect animals from vaginal challenge with pathogenic SIV, all five animals were intravaginally inoculated twice with pathogenic SIV-mac239. After challenge, all of the SHIV-immunized animals had low or undetectable viral RNA levels in plasma compared to control animals. Three of the five of the SHIV-immunized animals remained virus isolation negative for more than 8 months, while two became virus isolation positive. The presence of SIV Gag-specific cytotoxic T lymphocytes in peripheral blood mononuclear cells and SIV-specific antibodies in cervicovaginal secretions at the time of challenge was associated with resistance to pathogenic SIV infection after vaginal challenge. These results suggest that protection from sexual transmission of HIV may be possible by effectively stimulating both humoral and cellular antiviral immunity in the systemic and genital mucosal immune compartments.     

Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus.

The simian immunodeficiency virus (SIV) macaque model of AIDS has provided a valuable system with which to investigate vaccine approaches for protection against human immunodeficiency virus type 1 (HIV-1) infection. In particular, the ability of macaques persistently infected with attenuated infectious molecular clones of SIV to resist challenge with the pathogenic parental swarm has conclusively demonstrated that protective immunity can be achieved by immunization prior to exposure. The breadth of these protective responses and the immunological correlates of protection, however, have not been identified. In addition, vaccine studies have mainly employed lymphocyte-tropic strains of HIV-1 and SIV. Recent studies have implicated macrophage-tropic strains in the transmission of HIV-1 and have suggested that these virus strains should be examined in vaccine strategies. Macrophage-tropic viruses may confer additional advantages in the induction of protective immunity by replication in antigen-presenting cells. In this study, the immune response of rhesus macaques inoculated with an attenuated macrophage-tropic recombinant of SIVmac239 (SIV/17E-Cl) was evaluated with respect to protective immunity by heterologous challenge at various times after infection. Vigorous type-specific neutralizing-antibody responses restricted to SIV/17E-Cl were evident by 2 weeks postinfection. By 7 months, however, cross-reactive neutralizing antibodies emerged which neutralized not only SIV/17E-Cl but also the heterologous primary isolate SIV/DeltaB670. Challenge of SIV/17E-Cl-infected monkeys with SIV/DeltaB670 at various times postinfection demonstrated that protective responses were associated with the appearance of cross-reactive neutralizing antibodies. Furthermore, passive transfer of sera from SIV/17E-Cl-infected animals passively protected two of four naive recipients.