Macaques infected with a CCR5-tropic simian/human immunodeficiency virus (SHIV) develop broadly reactive anti-HIV neutralizing antibodies

The development of anti-human immunodeficiency virus (anti-HIV) neutralizing antibodies and the evolution of the viral envelope glycoprotein were monitored in rhesus macaques infected with a CCR5-tropic simian/human immunodeficiency virus (SHIV), SHIVSF162P4. Homologous neutralizing antibodies developed within the first month of infection in the majority of animals, and their titers were independent of the extent and duration of viral replication during chronic infection. The appearance of homologous neutralizing antibody responses was preceded by the appearance of amino acid changes in specific variable and conserved regions of gp120. Amino acid changes first appeared in the V1, V2, C2, and V3 regions and subsequently in the C3, V4, and V5 regions. Heterologous neutralizing antibody responses developed over time only in animals with sustained plasma viremia. Within 2 years postinfection the breadth of these responses was as broad as that observed in certain patients infected with HIV type 1 (HIV-1) for over a decade. Despite the development of broad anti-HIV-1 neutralizing antibody responses, viral replication persisted in these animals due to viral escape. Our studies indicate that cross-reactive neutralizing antibodies are elicited in a subset of SHIVSF162P4 infected macaques and that their development requires continuous viral replication for extended periods of time. More importantly, their late appearance does not prevent progression to disease. The availability of an animal model where cross-reactive anti-HIV neutralizing antibodies are developed may facilitate the identification of virologic and immunologic factors conducive to the development of such antibodies.     

Cellular immunity elicited by human immunodeficiency virus type 1/ simian immunodeficiency virus DNA vaccination does not augment the sterile protection afforded by passive infusion of neutralizing antibodies

High levels of infused anti-human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibodies (MAbs) can completely protect macaque monkeys against mucosal chimeric simian-human immunodeficiency virus (SHIV) infection. Antibody levels below the protective threshold do not prevent infection but can substantially reduce plasma viremia. To assess if HIV-1/SIV-specific cellular immunity could combine with antibodies to produce sterile protection, we studied the effect of a suboptimal infusion of anti-HIV-1 neutralizing antibodies in macaques with active cellular immunity induced by interleukin-2 (IL-2)-adjuvanted DNA immunization. Twenty female macaques were divided into four groups: (i). DNA immunization plus irrelevant antibody, (ii). DNA immunization plus infusion of neutralizing MAbs 2F5 and 2G12, (iii). sham DNA plus 2F5 and 2G12, and (iv). sham DNA plus irrelevant antibody. DNA-immunized monkeys developed CD4 and CD8 T-cell responses as measured by epitope-specific tetramer staining and by pooled peptide ELISPOT assays for gamma interferon-secreting cells. After vaginal challenge, DNA-immunized animals that received irrelevant antibody became SHIV infected but displayed lower plasma viremia than control animals. Complete protection against SHIV challenge occurred in three animals that received sham DNA plus MAbs 2F5 and 2G12 and in two animals that received the DNA vaccine plus MAbs 2F5 and 2G12. Thus, although DNA immunization produced robust HIV-specific T-cell responses, we were unable to demonstrate that these responses contributed to the sterile protection mediated by passive infusion of neutralizing antibodies. These data suggest that although effector T cells can limit viral replication, they are not able to assist humoral immunity to prevent the establishment of initial infection.     

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.     

Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies

The role of antibody in protection against human immunodeficiency virus (HIV-1) has been difficult to study in animal models because most primary HIV-1 strains do not infect nonhuman primates. Using a chimeric simian/human immunodeficiency virus (SHIV) based on the envelope of a primary isolate (HIV-89.6), we performed passive-transfer experiments in rhesus macaques to study the role of anti-envelope antibodies in protection. Based on prior in vitro data showing neutralization synergy by antibody combinations, we evaluated HIV immune globulin (HIVIG), and human monoclonal antibodies (MAbs) 2F5 and 2G12 given alone, compared with the double combination 2F5/2G12 and the triple combination HIVIG/2F5/2G12. Antibodies were administered 24 h prior to intravenous challenge with the pathogenic SHIV-89.6PD. Six control monkeys displayed high plasma viremia, rapid CD4(+)-cell decline, and clinical AIDS within 14 weeks. Of six animals given HIVIG/2F5/2G12, three were completely protected; the remaining three animals became SHIV infected but displayed reduced plasma viremia and near normal CD4(+)-cell counts. One of three monkeys given 2F5/2G12 exhibited only transient evidence of infection; the other two had marked reductions in viral load. All monkeys that received HIVIG, 2F5, or 2G12 alone became infected and developed high-level plasma viremia. However, compared to controls, monkeys that received HIVIG or MAb 2G12 displayed a less profound drop in CD4(+) T cells and a more benign clinical course. These data indicate a general correlation between in vitro neutralization and protection and suggest that a vaccine that elicits neutralizing antibody should have a protective effect against HIV-1 infection or disease.     

Antibody responses elicited in macaques immunized with human immunodeficiency virus type 1 (HIV-1) SF162-derived gp140 envelope immunogens: comparison with those elicited during homologous simian/human immunodeficiency virus SHIVSF162P4 and heterologous HIV-1 infection

The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIV(SF162P4), and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, DeltaV2gp140 (lacking the crown of the V2 loop), DeltaV3gp140 (lacking the crown of the V3 loop), and DeltaV2DeltaV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and DeltaV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and DeltaV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by DeltaV3gp140 or DeltaV2DeltaV3gp140. In contrast, the SHIV(SF162P4)-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.     

Protective immunity of gene-deleted SHIVs having an HIV-1 Env against challenge infection with a gene-intact SHIV

We constructed three simian-human immunodeficiency viruses (SHIVs) lacking regulatory gene(s) and analyzed their induction of protective immunity against challenge infection with gene-intact SHIV in rhesus macaques. Inoculation of SHIV-dn lacking nef and SHIV-drn lacking nef and vpr induced transient viremia, while that of SHIV-dxrn lacking nef, vpr, and vpx induced no viremia. The SHIVs with fewer deletions were more effective in inducing neutralizing antibodies and cytotoxic T lymphocyte responses. When these macaques were challenged with parental gene-intact SHIV-NM-3rN, all the SHIV-dn-vaccinated macaques and two of the four SHIV-drn-vaccinated macaques showed complete resistance. The other two SHIV-drn-vaccinated macaques and all SHIV-dxrn-vaccinated macaques did not show complete resistance, but they did show suppression of replication of the challenge virus. These results suggested that as more genes were deleted, protective immunity was decreased.     

Sequential evolution and escape from neutralization of simian immunodeficiency virus SIVsmE660 clones in rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques has become an important surrogate model for evaluating HIV vaccine strategies. The extreme resistance to neutralizing antibody (NAb) of many commonly used strains, such as SIVmac251/239 and SIVsmE543-3, limits their potential relevance for evaluating the role of NAb in vaccine protection. In contrast, SIVsmE660 is an uncloned virus that appears to be more sensitive to neutralizing antibody. To evaluate the role of NAb in this model, we generated full-length neutralization-sensitive molecular clones of SIVsmE660 and evaluated two of these by intravenous inoculation of rhesus macaques. All animals became infected and maintained persistent viremia that was accompanied by a decline in memory CD4(+) T cells in blood and bronchoalveolar lavage fluid. High titers of autologous NAb developed by 4 weeks postinoculation but were not associated with control of viremia, and neutralization escape variants were detected concurrently with the generation of NAb. Neutralization escape was associated with substitutions and insertion/deletion polymorphisms in the V1 and V4 domains of envelope. Analysis of representative variants revealed that escape variants also induced NAbs within a few weeks of their appearance in plasma, in a pattern that is reminiscent of the escape of human immunodeficiency virus type 1 (HIV-1) isolates in humans. Although early variants maintained a neutralization-sensitive phenotype, viruses obtained later in infection were significantly less sensitive to neutralization than the parental viruses. These results indicate that NAbs exert selective pressure that drives the evolution of the SIV envelope and that this model will be useful for evaluating the role of NAb in vaccine-mediated protection.     

Determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high-titered neutralizing antibodies

We previously reported that high-titered neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope can block the establishment of a simian immunodeficiency virus (SIV)/HIV chimeric virus (SHIV) infection in two monkeys following passive transfer (R. Shibata et al., Nat. Med. 5:204-210, 1999). In the present study, increasing amounts of neutralizing immunoglobulin G (IgG) were administered to 15 pig-tailed macaques in order to obtain a statistically valid protective neutralization endpoint titer in plasma. Using an in vitro assay which measures complete neutralization of the challenge SHIV, we correlated the titers of neutralizing antibodies in plasma at the time of virus inoculation (which ranged from 1:3 to 1:123) with the establishment of infection in virus-challenged animals. Ten of 15 monkeys in the present experiment were virus free as a result of neutralizing IgG administration as monitored by DNA PCR (peripheral blood mononuclear cells and lymph node cells), RNA PCR (plasma), virus isolation, and the transfer of lymph node cell suspensions (10(8) cells) plus 8 ml of whole blood from protected animals to na?ve macaques. The titer of neutralizing antibodies in the plasma calculated to protect 99% of virus-challenged monkeys was 1:38.     

Control of simian/human immunodeficiency virus viremia and disease progression after IL-2-augmented DNA-modified vaccinia virus Ankara nasal vaccination in nonhuman primates

A successful HIV vaccine may need to stimulate antiviral immunity in mucosal and systemic immune compartments, because HIV transmission occurs predominantly at mucosal sites. We report here the results of a combined DNA-modified vaccinia virus Ankara (MVA) vaccine approach that stimulated simian/human immunodeficiency virus (SHIV)-specific immune responses by vaccination at the nasal mucosa. Fifteen male rhesus macaques, divided into three groups, received three nasal vaccinations on day 1, wk 9, and wk 25 with a SHIV DNA plasmid producing noninfectious viral particles (group 1), or SHIV DNA plus IL-2/Ig DNA (group 2), or SHIV DNA plus IL-12 DNA (group 3). On wk 33, all macaques were boosted with rMVA expressing SIV Gag-Pol and HIV Env 89.6P, administered nasally. Humoral responses were evaluated by measuring SHIV-specific IgG and neutralizing Abs in plasma, and SHIV-specific IgA in rectal secretions. Cellular responses were monitored by evaluating blood-derived virus-specific IFN-gamma-secreting cells and TNF-alpha-expressing CD8+ T cells, and blood- and rectally derived p11C tetramer-positive T cells. Many of the vaccinated animals developed both mucosal and systemic humoral and cell-mediated anti-SHIV immune responses, although the responses were not homogenous among animals in the different groups. After rectal challenge of vaccinated and naive animals with SHIV89.6P, all animals became infected. However a subset, including all group 2 animals, were protected from CD4+ T cell loss and AIDS development. Taken together, these data indicate that nasal vaccination with SHIV-DNA plus IL-2/Ig DNA and rMVA can provide significant protection from disease progression.     

CD8+ and CD20+ lymphocytes cooperate to control acute simian immunodeficiency virus/human immunodeficiency virus chimeric virus infections in rhesus monkeys: modulation by major histocompatibility complex genotype

We have previously described two isogenic molecularly cloned simian immunodeficiency virus/human immunodeficiency virus chimeric viruses (SHIVs) that differ from one another by 9 amino acids and direct distinct clinical outcomes in inoculated rhesus monkeys. SHIV(DH12R-Clone 7), like other highly pathogenic CXCR4-tropic SHIVs, induces rapid and complete depletions of CD4+ T lymphocytes and immunodeficiency in infected animals. In contrast, macaques inoculated with SHIV(DH12R-Clone 8) experience only partial and transient losses of CD4+ T cells, show prompt control of their viremia, and remain healthy for periods of time extending for up to 4 years. The contributions of CD8+ and CD20+ lymphocytes in suppressing the replication of the attenuated SHIV(DH12R-Clone 8) and maintaining a prolonged asymptomatic clinical course was assessed by treating animals with monoclonal antibodies that deplete each lymphocyte subset at the time of virus inoculation. The absence of either CD8+ or CD20+ cells during the SHIV(DH12R-Clone 8) acute infection resulted in the rapid, complete, and irreversible loss of CD4+ T cells; sustained high levels of postpeak plasma viremia; and symptomatic disease in Mamu-A*01-negative Indian rhesus monkeys. In Mamu-A*01-positive animals, however, the aggressive, highly pathogenic phenotype was observed only in macaques depleted of CD8+ cells; SHIV(DH12R-Clone 8) was effectively controlled in Mamu-A*01-positive monkeys in the absence of B lymphocytes. Taken together, these results indicate that both CD8+ and CD20+ B cells contribute to the control of primate lentiviral infection in Mamu-A*01-negative macaques. Furthermore, the major histocompatibility complex genotype of an infected animal, as exemplified by the Mamu-A*01 allele in this study, has the additional capacity to shift the balance of the composite immune response.     

Pathogenic Consequences of Vaginal Infection with CCR5-Tropic Simian-Human Immunodeficiency Virus SHIVSF162P3N

We previously reported efficient transmission of the pathogenic R5 simian-human immunodeficiency virus SHIV(SF162P3N) isolate in Indian rhesus macaques by intravenous and intrarectal inoculations, with a switch to CXCR4 coreceptor usage in ～50% of infected animals that progressed rapidly to disease. Since women continue to be disproportionately affected by HIV, we developed an animal model based on the intravaginal challenge of female rhesus monkeys with SHIV(SF162P3N) and sought to validate the utility of this model to study relevant aspects of HIV transmission and pathogenesis. The effect of viral dose on infection outcome was evaluated to determine the optimal conditions for the evaluation of HIV-1 preventive and therapeutic strategies. We found that the virus can successfully cross the vaginal mucosal surface to establish infection and induce disease with coreceptor switch, but with lower efficiencies compared to intravenous and rectal transmissions. In contrast to intrarectal infection, peak and cumulative viral load over a 1 year-infection period were significantly greater in macaques exposed intravaginally to lower rather than higher inoculum doses. Moreover, low and transient viremia was observed only in macaques that were challenged intravaginally twice within the same day with a high dose of virus, which can be seen as doubling the dose. Taken together, these results show that SHIV(SF162P3N) can successfully transmit across the genital mucosa, undergo coreceptor switch, and induce disease. However, the administered dose appears to impact SHIV(SF162P3N) vaginal infection outcome in an unexpected manner.     

Immune responses following simian/human immunodeficiency virus (SHIV) challenge of rhesus macaques after human immunodeficiency virus (HIV)-1 third variable domain (V3) loop-based genetic immunization

Following DNA immunization of rhesus macaques with a plasmid encoding the human immunodeficiency virus (HIV)-1 third variable domain (V3) loop, presented by pseudo-viral envelope particles of hepatitis B virus, specific immune responses were induced. The primates were then inoculated with a chimeric simian/human immunodeficiency virus (SHIV). All the animals were infected, but the V3-specific immunization provided a relative attenuation of the acute phase of infection in the absence of neutralizing antibody. In all animals, SHIV-specific cytotoxic T-lymphocyte precursors (CTLp) were detected early in peripheral blood and lymph nodes. The viremia peak correlated significantly with the decrease in CD4+ T cells and with a transient increase in the percentage of natural killer cells. The infection induced an oligoclonalization of the CD8+ T-cell variable beta chain repertoire in the blood. Surprisingly, HIV envelope-specific CTLp generated by genetic immunization may be governed by distinct circulation rules compared to SHIV-specific CTLp induced by infection.     

Enhanced infectivity of an R5-tropic simian/human immunodeficiency virus carrying human immunodeficiency virus type 1 subtype C envelope after serial passages in pig-tailed macaques (Macaca nemestrina)

The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating strategies against the transmission of the virus. A chimeric simian/human immunodeficiency virus (SHIV), SHIV(CHN19), was generated with a primary, non-syncytium-inducing HIV-1 subtype C envelope from a Chinese strain in the background of SHIV(33). Unlike R5-tropic SHIV(162), SHIV(CHN19) was not found to replicate in rhesus CD4(+) T lymphocytes. SHIV(CHN19) does, however, replicate in CD4(+) T lymphocytes of pig-tailed macaques (Macaca nemestrina). The observed replication competence of SHIV(CHN19) requires the full tat/rev genes and partial gp41 region derived from SHIV(33). To evaluate in vivo infectivity, SHIV(CHN19) was intravenously inoculated, at first, into two pig-tailed and two rhesus macaques. Although all four animals became infected, the virus replicated preferentially in pig-tailed macaques with an earlier plasma viral peak and a faster seroconversion. To determine whether in vivo adaptation would enhance the infectivity of SHIV(CHN19), passages were carried out serially in three groups of two pig-tailed macaques each, via intravenous blood-bone marrow transfusion. The passages greatly enhanced the infectivity of the virus as shown by the increasingly elevated viral loads during acute infection in animals with each passage. Moreover, the doubling time of plasma virus during acute infection became much shorter in passage 4 (P4) animals (0.2 day) in comparison to P1 animals (1 to 2 days). P2 to P4 animals all became seropositive around 2 to 3 weeks postinoculation and had a decline in CD4/CD8 T-cell ratio during the early phase of infection. In P4 animals, a profound depletion of CD4 T cells in the lamina propria of the jejunum was observed. Persistent plasma viremia has been found in most of the infected animals with sustained viral loads ranging from 10(3) to 10(5) per ml up to 6 months postinfection. Serial passages did not change the viral phenotype as confirmed by the persistence of the R5 tropism of SHIV(CHN19) isolated from P4 animals. In addition, the infectivity of SHIV(CHN19) in rhesus peripheral blood mononuclear cells was also increased after in vivo passages. Our data indicate that SHIV(CHN19) has adapted well to grow in macaque cells. This established R5-tropic SHIV(CHN19)/macaque model would be very useful for HIV-1 subtype C vaccine and pathogenesis studies.     

Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV(162P4). Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.     

Neutralizing Antibodies in Sera from Macaques Infected with Chimeric Simian-Human Immunodeficiency Virus Containing the Envelope Glycoproteins of either a Laboratory-Adapted Variant or a Primary Isolate of Human Immunodeficiency Virus Type 1

The magnitude and breadth of neutralizing antibodies raised in response to infection with chimeric simian-human immunodeficiency virus (SHIV) in rhesus macaques were evaluated. Infection with either SHIV-HXB2, SHIV-89.6, or SHIV-89.6PD raised high-titer neutralizing antibodies to the homologous SHIV (SHIV-89.6P in the case of SHIV-89.6PD-infected animals) and significant titers of neutralizing antibodies to human immunodeficiency virus type 1 (HIV-1) strains MN and SF-2. With few exceptions, however, titers of neutralizing antibodies to heterologous SHIV were low or undetectable. The antibodies occasionally neutralized heterologous primary isolates of HIV-1; these antibodies required >40 weeks of infection to reach detectable levels. Notable was the potent neutralization of the HIV-1 89.6 primary isolate by serum samples from SHIV-89.6-infected macaques. These results demonstrate that SHIV-HXB2, SHIV-89.6, and SHIV-89.6P possess highly divergent, strain-specific neutralization epitopes. The results also provide insights into the requirements for raising neutralizing antibodies to primary isolates of HIV-1.     

Envelope variable region 4 is the first target of neutralizing antibodies in early simian immunodeficiency virus mac251 infection of rhesus monkeys

A major goal of AIDS vaccine development is to design vaccination strategies that can elicit broad and potent protective antibodies. The initial viral targets of neutralizing antibodies (NAbs) early after human or simian immunodeficiency virus (HIV/SIV) infection are not known. The identification of early NAb epitopes that induce protective immunity or retard the progression of disease is important for AIDS vaccine development. The aim of this study was to determine the Env residues targeted by early SIV NAbs and to assess the influence of prior vaccination on neutralizing antibody kinetics and specificity during early infection. We previously described stereotypic env sequence variations in SIVmac251-infected rhesus monkeys that resulted in viral escape from NAbs. Here, we defined the early viral targets of neutralization and determined whether the ability of serum antibody from infected monkeys to neutralize SIV was altered in the setting of prior vaccination. To localize the viral determinants recognized by early NAbs, a panel of mutant pseudoviruses was assessed in a TZM-bl reporter gene neutralization assay to define the precise changes that eliminate recognition by SIV Env-specific NAbs in 16 rhesus monkeys. Changing R420 to G or R424 to Q in V4 of Env resulted in the loss of recognition by NAbs in vaccinated monkeys. In contrast, mutations in the V1 region of Env did not alter the NAb profile. These findings indicate that early NAbs are directed toward SIVmac251 Env V4 but not the V1 region, and that this env vaccination regimen did not alter the kinetics or the breadth of NAbs during early infection.     

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.     

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.     

Immunization with recombinant HLA classes I and II, HIV-1 gp140, and SIV p27 elicits protection against heterologous SHIV infection in rhesus macaques

Major histocompatibility complex (MHC) molecules expressed on the surface of human immunodeficiency virus (HIV) are potential targets for neutralizing antibodies. Since MHC molecules are polymorphic, nonself MHC can also be immunogenic. We have used combinations of novel recombinant HLA class I and II and HIV/simian immunodeficiency virus (SIV) antigens, all linked to dextran, to investigate whether they can elicit protective immunity against heterologous simian/human immunodeficiency virus (SHIV) challenge in rhesus macaques. Three groups of animals were immunized with HLA (group 1, n = 8), trimeric YU2 HIV type 1 (HIV-1) gp140 and SIV p27 (HIV/SIV antigens; group 2, n = 8), or HLA plus HIV/SIV antigens (group 3, n = 8), all with Hsp70 and TiterMax Gold adjuvant. Another group (group 4, n = 6) received the same vaccine as group 3 without TiterMax Gold. Two of eight macaques in group 3 were completely protected against intravenous challenge with 18 50% animal infective doses (AID₅₀) of SHIV-SF162P4/C grown in human cells expressing HLA class I and II lineages represented in the vaccine, while the remaining six macaques showed decreased viral loads compared to those in unimmunized animals. Complement-dependent neutralizing activity in serum and high levels of anti-HLA antibodies were elicited in groups 1 and 3, and both were inversely correlated with the plasma viral load at 2 weeks postchallenge. Antibody-mediated protection was strongly supported by the fact that transfer of pooled serum from the two challenged but uninfected animals protected two naïve animals against repeated low-dose challenge with the same SHIV stock. This study demonstrates that immunization with recombinant HLA in combination with HIV-1 antigens might be developed into an alternative strategy for a future AIDS vaccine.     

Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus.

An animal model for the heterosexual transmission of human immunodeficiency virus (HIV) was developed by the application of simian immunodeficiency virus (SIV) onto the genital mucosas of both mature and immature, male and female rhesus macaques. Virus preparations were infused into the vaginal vaults or the urethras (males) of the animals through a soft plastic pediatric nasogastric feeding tube. The macaques that were infected by this route (six males and nine females) developed SIV-specific antibodies, and SIV was isolated from peripheral mononuclear cells of all seropositive animals. One male and one female infected by this route developed severe acquired immunodeficiency syndrome-like disease with retroviral giant-cell pneumonia. As few as two inoculations of cell-free SIV containing 50 50% tissue culture infective doses induced persistent viremia. Cell-free virus preparations were capable of producing infection by the genital route. Much higher doses of virus were required to transmit SIV by this route than are required for transmission by intravenous inoculation. Thus, it appears that the mucous membranes of the genital tract act as a barrier to SIV infection. Spermatozoa and seminal plasma were not required for the genital transmission of SIV. Rarely, SIV was recovered from mononuclear cells in semen and vaginal secretions. The SIV-rhesus macaque model is suitable for assessing the role of cofactors in heterosexual transmission of HIV and will be useful for testing the effectiveness of spermicides, pharmacologic agents, and vaccines in preventing the heterosexual transmission of HIV.