Env-Independent Protection Induced by Live, Attenuated Simian Immunodeficiency Virus Vaccines

Live attenuated simian immunodeficiency viruses (SIV), such as nef deletion mutants, are the most effective vaccines tested in the SIV-macaque model so far. To modulate the antiviral immune response induced by live attenuated SIV vaccines, we had previously infected rhesus monkeys with a nef deletion mutant of SIV expressing interleukin 2 (SIV-IL2) (B. R. Gundlach, H. Linhart, U. Dittmer, S. Sopper, S. Reiprich, D. Fuchs, B. Fleckenstein, G. Hunsmann, S. Stahl-Hennig, and K. Überla, J. Virol. 71:2225–2232, 1997). In the present study, SIV-IL2-infected macaques and macaques infected with the nef deletion mutant SIVΔNU were challenged with pathogenic SIV 9 to 11 months postvaccination. In contrast to the results with naive control monkeys, no challenge virus could be isolated from the SIV-IL2- and SIVΔNU-infected macaques. However, challenge virus sequences could be detected by nested PCR in some of the vaccinated macaques. To determine the role of immune responses directed against Env of SIV, four vaccinated macaques were rechallenged with an SIV-murine leukemia virus (MLV) hybrid in which the env gene of SIV had been functionally replaced by the env gene of amphotropic MLV. All vaccinated macaques were protected from productive infection with the SIV-MLV hybrid in the absence of measurable neutralizing antibodies, while two naive control monkeys were readily infected. Since the SIV-MLV hybrid uses the MLV Env receptor Pit2 and not CD4 and a coreceptor for virus entry, chemokine inhibition and receptor interference phenomena were not involved in protection. These results indicate that the protective responses induced by live attenuated SIV vaccines can be independent of host immune reactions directed against Env.     

Limited Protection from a Pathogenic Chimeric Simian-Human Immunodeficiency Virus Challenge following Immunization with Attenuated Simian Immunodeficiency Virus

Two live attenuated single-deletion mutant simian immunodeficiency virus (SIV) constructs, SIV_239Δnef and SIV_PBj6.6Δnef, were tested for their abilities to stimulate protective immunity in macaques. During the immunization period the animals were examined for specific immune responses and virus growth. Each construct generated high levels of specific immunity in all of the immunized animals. The SIV_239Δnef construct was found to grow to high levels in all immunized animals, with some animals remaining positive for virus isolation and plasma RNA throughout the immunization period. The SIV_PBj6.6Δnef was effectively controlled by all of the immunized animals, with virus mostly isolated only during the first few months following immunization and plasma RNA never detected. Following an extended period of immunization of over 80 weeks, the animals were challenged with a pathogenic simian-human immunodeficiency virus (SHIV) isolate, SIV_89.6PD, by intravenous injection. All of the SIV_239Δnef-immunized animals became infected with the SHIV isolate; two of five animals eventually controlled the challenge and three of five animals, which failed to check the immunizing virus, progressed to disease state before the unvaccinated controls. One of five animals immunized with SIV_PBj6.6Δnef totally resisted infection by the challenge virus, while three others limited its growth and the remaining animal became persistently infected and eventually died of a pulmonary thrombus. These data indicate that vaccination with attenuated SIV can protect macaques from disease and in some cases from infection by a divergent SHIV. However, if animals are unable to control the immunizing virus, potential damage that can accelerate the disease course of a pathogenic challenge virus may occur.     

Protection against Simian Immunodeficiency Virus Vaginal Challenge by Using Sabin Poliovirus Vectors

Here we provide the first report of protection against a vaginal challenge with a highly virulent simian immunodeficiency virus (SIV) by using a vaccine vector. New poliovirus vectors based on Sabin 1 and 2 vaccine strain viruses were constructed, and these vectors were used to generate a series of new viruses containing SIV gag, pol, env, nef, and tat in overlapping fragments. Two cocktails of 20 transgenic polioviruses (SabRV1-SIV and SabRV2-SIV) were inoculated into seven cynomolgus macaques. All monkeys produced substantial anti-SIV serum and mucosal antibody responses. SIV-specific cytotoxic T-lymphocyte responses were detected in three of seven monkeys after vaccination. All 7 vaccinated macaques, as well as 12 control macaques, were challenged vaginally with pathogenic SIVmac251. Strikingly, four of the seven vaccinated animals exhibited substantial protection against the vaginal SIV challenge. All 12 control monkeys became SIV positive. In two of the seven SabRV-SIV-vaccinated monkeys we found no virological evidence of infection following challenge, indicating that these two monkeys were completely protected. Two additional SabRV-SIV-vaccinated monkeys exhibited a pronounced reduction in postacute viremia to <10(3) copies/ml, suggesting that the vaccine elicited an effective cellular immune response. Three of six control animals developed clinical AIDS by 48 weeks postchallenge. In contrast, all seven vaccinated monkeys remained healthy as judged by all clinical parameters. These results demonstrate the efficacy of SabRV as a potential human vaccine vector, and they show that the use of a vaccine vector cocktail expressing an array of defined antigenic sequences can be an effective vaccination strategy in an outbred population.     

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.     

Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge

All human immunodeficiency virus (HIV) vaccine efficacy trials to date have ended in failure. Structural features of the Env glycoprotein and its enormous variability have frustrated efforts to induce broadly reactive neutralizing antibodies. To explore the extent to which vaccine-induced cellular immune responses, in the absence of neutralizing antibodies, can control replication of a heterologous, mucosal viral challenge, we vaccinated eight macaques with a DNA/Ad5 regimen expressing all of the proteins of SIVmac239 except Env. Vaccinees mounted high-frequency T-cell responses against 11 to 34 epitopes. We challenged the vaccinees and eight naïve animals with the heterologous biological isolate SIVsmE660, using a regimen intended to mimic typical HIV exposures resulting in infection. Viral loads in the vaccinees were significantly less at both the peak (1.9-log reduction; P < 0.03) and at the set point (2.6-log reduction; P < 0.006) than those in control naïve animals. Five of eight vaccinated macaques controlled acute peak viral replication to less than 80,000 viral RNA (vRNA) copy eq/ml and to less than 100 vRNA copy eq/ml in the chronic phase. Our results demonstrate that broad vaccine-induced cellular immune responses can effectively control replication of a pathogenic, heterologous AIDS virus, suggesting that T-cell-based vaccines may have greater potential than previously appreciated.It has been impossible thus far for vaccines to engender broadly reactive neutralizing antibodies against human immunodeficiency virus (HIV) (12, 54). Investigators have therefore focused their attention on T-cell-based vaccines (9, 18, 26, 30, 34, 39, 48, 55). Previous preclinical studies in nonhuman primates have shown that vaccine-induced T-cell responses can partially control replication of homologous challenge viruses in the chronic phase (34, 56). Unfortunately, however, simian immunodeficiency virus (SIV) loads exceeded 1 million copies in almost every vaccinated animal during the acute phase. Given the high levels of viral replication observed in these vaccinated macaques, it is possible that such T-cell-based vaccines might not be able to reduce transmission during the acute phase of infection in humans. These high levels of replication during the acute phase likely resulted in the generation of diverse viral quasispecies, providing the substrate for immune selection and eventual escape. Furthermore, in these studies, vaccinated animals were challenged with viruses that were similar to the SIV sequences in the vaccine constructs. Given the diversity of HIV, human vaccinees will never be exposed to viruses with a comparable level of sequence similarity to the vaccine constructs.An HIV-1 vaccine that induced T-cell responses exclusively has recently failed to show efficacy against the incidence of HIV infection and viremia in clinical testing. The STEP trial of a recombinant adenovirus 5 (Ad5)-vectored vaccine designed to induce HIV-specific T-cell responses in humans was widely seen as an important test of the T-cell vaccine concept (http://www.hvtn.org/media/pr/step111307.html) (11, 42). The lack of vaccine efficacy in the STEP trial has led some to conclude that T-cell-based vaccines may not be a viable approach to solving the AIDS epidemic (6, 49, 59). However, STEP trial vaccinees that became infected recognized a median of only five epitopes, mostly in the conserved proteins Gag and Pol. Given the sequence diversity of HIV (19), several of these vaccine-elicited T-cell responses may not have recognized epitopes in the infecting virus and, therefore, not constituted an adequate test of the T-cell vaccine concept.We therefore sought to test whether high-frequency vaccine-induced T-cell responses against multiple T-cell epitopes in one of the simian AIDS viruses, SIVmac239, could effectively impact viral replication after a physiologically relevant heterologous mucosal challenge with SIVsmE660. The majority of virus challenges in macaques have been carried out with high doses of homologous viruses. We used a repeated low-dose mucosal challenge with a heterologous SIV strain. We also used a challenge dose intended to mimic HIV mucosal exposures that lead to infection. Here we show that vaccine-induced T-cell responses can reduce heterologous virus replication during both the acute and chronic phases after a relevant viral challenge.     

Vaccination with Attenuated Simian Immunodeficiency Virus by DNA Inoculation

Delivering attenuated lentivirus vaccines as proviral DNA would be simple and inexpensive. Inoculation of macaques with wild-type simian immunodeficiency virus strain mac239 (SIV(mac239)) DNA or SIV(mac239) DNA containing a single deletion in the 3' nef-long terminal repeat overlap region (nef/LTR) led to sustained SIV infections and AIDS. Injection of SIV(mac239) DNA containing identical deletions in both the 5' LTR and 3' nef/LTR resulted in attenuated SIV infections and substantial protection against subsequent mucosal SIV(mac251) challenge.     

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

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

Vaccine Protection against a Heterologous, Non-Syncytium-Inducing, Primary Human Immunodeficiency Virus

Vaccine-induced protection of chimpanzees against laboratory-adapted and syncytium-inducing, multiply passaged primary human immunodeficiency virus type 1 (HIV-1) isolates, but not against non-syncytium-inducing, minimally passaged ones, has been demonstrated. Following challenge with such an isolate, HIV-1₅₀₁₆, we obtained complete protection in one of three chimpanzees previously protected against low- and high-dose HIV-1_SF2 exposures after immunization with an adenovirus-HIV-1_MN gp160 priming–HIV-1_SF2 gp120 boosting regimen. At challenge, the protected chimpanzee exhibited broad humoral immunity, including neutralizing antibody activity. These results demonstrate the potential of this combination vaccine strategy and suggest that vaccine protection against an HIV isolate relevant to infection of people is feasible.     

Identification of Highly Attenuated Mutants of Simian Immunodeficiency Virus

Deletion mutants of the pathogenic clone of simian immunodeficiency virus isolate 239 (SIVmac239) were derived that are missing nef, vpr, and upstream sequences (US) in the U3 region of the LTR (SIVmac239Δ3), nef, vpx, and US (SIVmac239Δ3x), and nef, vpr, vpx, and US (SIVmac239Δ4). These multiply deleted derivatives replicated well in the continuously growing CEMx174 cell line and were infectious for rhesus monkeys. However, on the basis of virus load measurements, strength of antibody responses, and lack of disease progression, these mutants were highly attenuated. Measurements of cell-associated viral load agreed well with assays of plasma viral RNA load and with the strengths of the antibody responses; thus, these measurements likely reflected the extent of viral replication in vivo. A derivative of SIVmac239 lacking vif sequences (SIVmac239Δvif) could be consistently grown only in a vif-complementing cell line. This Δvif virus appeared to be very weakly infectious for rhesus monkeys on the basis of sensitive antibody tests only. The weak antibody responses elicited by SIVmac239Δvif were apparently in response to low levels of replicating virus since they were not elicited by heat-inactivated virus and the anti-SIV antibody responses persisted for greater than 1 year. These results, and the results of previous studies, allow a rank ordering of the relative virulence of nine mutant strains of SIVmac according to the following order: Δvpr > Δvpx > ΔvprΔvpx Δnef > Δ3 > Δ3x ≥ Δ4 > Δvif > Δ5. The results also demonstrate that almost any desired level of attenuation can be achieved, ranging from still pathogenic in a significant proportion of animals (Δvpr and Δvpx) to not detectably infectious (Δ5), simply by varying the number and location of deletions in these five loci.     

Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge

Neutralizing antibodies may have critical importance in immunity against human immunodeficiency virus type 1 (HIV-1) infection. However, the amount of protective antibody needed at mucosal surfaces has not been fully established. Here, we evaluated systemic and mucosal pharmacokinetics (PK) and pharmacodynamics (PD) of 2F5 IgG and 2F5 Fab fragments with respect to protection against vaginal challenge with simian-human immunodeficiency virus-BaL in macaques. Antibody assessment demonstrated that 2F5 IgG was more potent than polymeric forms (IgM and IgA) across a range of cellular and tissue models. Vaginal challenge studies demonstrated a dose-dependent protection for 2F5 IgG and no protection with 2F5 Fab despite higher vaginal Fab levels at the time of challenge. Animals receiving 50 or 25 mg/kg of body weight 2F5 IgG were completely protected, while 3/5 animals receiving 5 mg/kg were protected. In the control animals, infection was established by a minimum of 1 to 4 transmitted/founder (T/F) variants, similar to natural human infection by this mucosal route; in the two infected animals that had received 5 mg 2F5 IgG, infection was established by a single T/F variant. Serum levels of 2F5 IgG were more predictive of sterilizing protection than measured vaginal levels. Fc-mediated antiviral activity did not appear to influence infection of primary target cells in cervical explants. However, PK studies highlighted the importance of the Fc portion in tissue biodistribution. Data presented in this study may be important in modeling serum levels of neutralizing antibodies that need to be achieved by either vaccination or passive infusion to prevent mucosal acquisition of HIV-1 infection in humans.     

Temporal Analyses of Virus Replication, Immune Responses, and Efficacy in Rhesus Macaques Immunized with a Live, Attenuated Simian Immunodeficiency Virus Vaccine

Despite evidence that live, attenuated simian immunodeficiency virus (SIV) vaccines can elicit potent protection against pathogenic SIV infection, detailed information on the replication kinetics of attenuated SIV in vivo is lacking. In this study, we measured SIV RNA in the plasma of 16 adult rhesus macaques immunized with a live, attenuated strain of SIV (SIVmac239Δnef). To evaluate the relationship between replication of the vaccine virus and the onset of protection, four animals per group were challenged with pathogenic SIVmac251 at either 5, 10, 15, or 25 weeks after immunization. SIVmac239Δnef replicated efficiently in the immunized macaques in the first few weeks after inoculation. SIV RNA was detected in the plasma of all animals by day 7 after inoculation, and peak levels of viremia (10⁵ to 10⁷ RNA copies/ml) occurred by 7 to 12 days. Following challenge, SIVmac251 was detected in all of the four animals challenged at 5 weeks, in two of four challenged at 10 weeks, in none of four challenged at 15 weeks, and one of four challenged at 25 weeks. One animal immunized with SIVmac239Δnef and challenged at 10 weeks had evidence of disease progression in the absence of detectable SIVmac251. Although complete protection was not achieved at 5 weeks, a transient reduction in viremia (approximately 100-fold) occurred in the immunized macaques early after challenge compared to the nonimmunized controls. Two weeks after challenge, SIV RNA was also reduced in the lymph nodes of all immunized macaques compared with control animals. Taken together, these results indicate that host responses capable of reducing the viral load in plasma and lymph nodes were induced as early as 5 weeks after immunization with SIVmac239Δnef, while more potent protection developed between 10 and 15 weeks. In further experiments, we found that resistance to SIVmac251 infection did not correlate with the presence of antibodies to SIV gp130 and p27 antigens and was achieved in the absence of significant neutralizing activity against the primary SIVmac251 challenge stock.     

Recombinant Vaccine-Induced Protection against the Highly Pathogenic Simian Immunodeficiency Virus SIVmac251: Dependence on Route of Challenge Exposure

Vaccine protection from infection and/or disease induced by highly pathogenic simian immunodeficiency virus (SIV) strain SIV_mac251 in the rhesus macaque model is a challenging task. Thus far, the only approach that has been reported to protect a fraction of macaques from infection following intravenous challenge with SIV_mac251 was the use of a live attenuated SIV vaccine. In the present study, the gag, pol, and env genes of SIV_K6W were expressed in the NYVAC vector, a genetically engineered derivative of the vaccinia virus Copenhagen strain that displays a highly attenuated phenotype in humans. In addition, the genes for the α and β chains of interleukin-12 (IL-12), as well as the IL-2 gene, were expressed in separate NYVAC vectors and inoculated intramuscularly, in conjunction with or separate from the NYVAC-SIV vaccine, in 40 macaques. The overall cytotoxic T-lymphocyte (CTL) response was greater, at the expense of proliferative and humoral responses, in animals immunized with NYVAC-SIV and NYVAC–IL-12 than in animals immunized with the NYVAC-SIV vaccine alone. At the end of the immunization regimen, half of the animals were challenged with SIV_mac251 by the intravenous route and the other half were exposed to SIV_mac251 intrarectally. Significantly, five of the eleven vaccinees exposed mucosally to SIV_mac251 showed a transient peak of viremia 1 week after viral challenge and subsequently appeared to clear viral infection. In contrast, all 12 animals inoculated intravenously became infected, but 5 to 6 months after viral challenge, 4 animals were able to control viral expression and appeared to progress to disease more slowly than control animals. Protection did not appear to be associated with any of the measured immunological parameters. Further modulation of immune responses by coadministration of NYVAC-cytokine recombinants did not appear to influence the outcome of viral challenge. The fact that the NYVAC-SIV recombinant vaccine appears to be effective per se in the animal model that best mirrors human AIDS supports the idea that the development of a highly attenuated poxvirus-based vaccine candidate can be a valuable approach to significantly decrease the spread of human immunodeficiency virus (HIV) infection by the mucosal route.     

Neutralizing Antibodies in Sera from Macaques Immunized with Attenuated Simian Immunodeficiency Virus

Infection with attenuated simian immunodeficiency virus (SIV) in rhesus macaques has been shown to raise antibodies capable of neutralizing an animal challenge stock of primary SIVmac251 in CEMx174 cells that correlate with resistance to infection after experimental challenge with this virulent virus (M. S. Wyand, K. H. Manson, M. Garcia-Moll, D. C. Montefiori, and R. C. Desrosiers, J. Virol. 70:3724–3733, 1996). Here we show that these neutralizing antibodies are not detected in human and rhesus peripheral blood mononuclear cells (PBMC). In addition, neutralization of primary SIVmac251 in human and rhesus PBMC was rarely detected with plasma samples from a similar group of animals that had been infected either with SIVmac239Δnef for 1.5 years or with SIVmac239Δ3 for 3.2 years, although low-level neutralization was detected in CEMx174 cells. Potent neutralization was detected in CEMx174 cells when the latter plasma samples were assessed with laboratory-adapted SIVmac251. In contrast to primary SIVmac251, laboratory-adapted SIVmac251 did not replicate in human and rhesus PBMC despite its ability to utilize CCR5, Bonzo/STRL33, and BOB/gpr15 as coreceptors for virus entry. These results illustrate the importance of virus passage history and the choice of indicator cells for making assessments of neutralizing antibodies to lentiviruses such as SIV. They also demonstrate that primary SIVmac251 is less sensitive to neutralization in human and rhesus PBMC than it is in established cell lines. Results obtained in PBMC did not support a role for neutralizing antibodies as a mechanism of protection in animals immunized with attenuated SIV and challenged with primary SIVmac251.     

Envelope-Modified Single-Cycle Simian Immunodeficiency Virus Selectively Enhances Antibody Responses and Partially Protects against Repeated,Low-Dose Vaginal Challenge

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV_mac239 or SIV_mac251_UCD, neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV_mac251_UCD, six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.Development of a safe and effective vaccine for human immunodeficiency virus type 1 (HIV-1) is an urgent public health priority, but remains a formidable scientific challenge. Passive transfer experiments in macaques demonstrate neutralizing antibodies can prevent infection by laboratory-engineered simian-human immunodeficiency virus (SHIV) strains (6, 33, 34, 53, 59). However, no current vaccine approach is capable of eliciting antibodies that neutralize primary isolates with neutralization-resistant envelope glycoproteins. Virus-specific T-cell responses can be elicited by prime-boost strategies utilizing recombinant DNA and/or viral vectors (3, 10, 11, 16, 36, 73, 77, 78), which confer containment of viral loads following challenge with SHIV_89.6P (3, 13, 66, 68). Unfortunately, similar vaccine regimens are much less effective against SIV_mac239 and SIV_mac251 (12, 16, 31, 36, 73), which bear closer resemblance to most transmitted HIV-1 isolates in their inability to utilize CXCR4 as a coreceptor (18, 23, 24, 88) and inherent high degree of resistance to neutralization by antibodies or soluble CD4 (43, 55, 56). Live, attenuated SIV can provide apparent sterile protection against challenge with SIV_mac239 and SIV_mac251 or at least contain viral replication below the limit of detection (20, 22, 80). Due to the potential of the attenuated viruses themselves to cause disease in neonatal rhesus macaques (5, 7, 81) and to revert to a pathogenic phenotype through the accumulation of mutations over prolonged periods of replication in adult animals (2, 35, 76), attenuated HIV-1 is not under consideration for use in humans.As an experimental vaccine approach designed to retain many of the features of live, attenuated SIV, without the risk of reversion to a pathogenic phenotype, we and others devised genetic approaches for producing strains of SIV that are limited to a single cycle of infection (27, 28, 30, 38, 39, 45). In a previous study, immunization of rhesus macaques with single-cycle SIV (scSIV) trans-complemented with vesicular stomatitis virus (VSV) G elicited potent virus-specific T-cell responses (39), which were comparable in magnitude to T-cell responses elicited by optimized prime-boost regimens based on recombinant DNA and viral vectors (3, 16, 36, 68, 73, 78). Antibodies were elicited that neutralized lab-adapted SIV_mac251_LA (39). However, despite the presentation of the native, trimeric SIV envelope glycoprotein (Env) on the surface of infected cells and virions, none of the scSIV-immunized macaques developed antibody responses that neutralized SIV_mac239 (39). Therefore, we have now introduced Env modifications into scSIV that facilitate the development of neutralizing antibodies.Most primate lentiviral envelope glycoproteins are inherently resistant to neutralizing antibodies due to structural and thermodynamic properties that have evolved to enable persistent replication in the face of vigorous antibody responses (17, 46, 47, 64, 71, 75, 79, 83, 85). Among these, extensive N-linked glycosylation renders much of the Env surface inaccessible to antibodies (17, 48, 60, 63, 75). Removal of N-linked glycans from gp120 or gp41 by mutagenesis facilitates the induction of antibodies to epitopes that are occluded by these carbohydrates in the wild-type virus (64, 85). Consequently, antibodies from animals infected with glycan-deficient strains neutralize these strains better than antibodies from animals infected with the fully glycosylated SIV_mac239 parental strain (64, 85). Most importantly with regard to immunogen design, animals infected with the glycan-deficient strains developed higher neutralizing antibody titers against wild-type SIV_mac239 (64, 85). Additionally, the removal of a single N-linked glycan in gp120 enhanced the induction of neutralizing antibodies against SHIV_89.6P and SHIV_SF162 in a prime-boost strategy by 20-fold (50). These observations suggest that potential neutralization determinants accessible in the wild-type Env are poorly immunogenic unless specific N-linked glycans in gp120 and gp41 are eliminated by mutagenesis.The variable loop regions 1 and 2 (V1V2) of HIV-1 and SIV gp120 may also interfere with the development of neutralizing antibodies. Deletion of V1V2 from HIV-1 gp120 permitted neutralizing monoclonal antibodies to CD4-inducible epitopes to bind to gp120 in the absence of CD4, suggesting that V1V2 occludes potential neutralization determinants prior to the engagement of CD4 (82). A deletion in V2 of HIV-1 Env-exposed epitopes was conserved between clades (69), improved the ability of a secreted Env trimer to elicit neutralizing antibodies (9), and was present in a vaccine that conferred complete protection against SHIV_SF162P4 (8). A deletion of 100 amino acids in V1V2 of SIV_mac239 rendered the virus sensitive to monoclonal antibodies with various specificities (41). Furthermore, three of five macaques experimentally infected with SIV_mac239 with V1V2 deleted resisted superinfection with wild-type SIV_mac239 (51). Thus, occlusion of potential neutralization determinants by the V1V2 loop structure may contribute to the poor immunogenicity of the wild-type envelope glycoprotein.Here we tested the hypothesis that antibody responses to scSIV could be improved by immunizing macaques with strains of scSIV engineered to eliminate structural features that interfere with the development of neutralizing antibodies. Antibodies to Env-modified strains were selectively enhanced, but these did not neutralize the wild-type SIV strains. We then tested the hypothesis that immunization might prevent infection in a repeated, low-dose vaginal challenge model of heterosexual HIV-1 transmission. Indeed, while all six naïve control animals became infected, two of eight immunized animals remained uninfected after 20 weeks of repeated vaginal challenge. Relative to the naïve control group, reductions in peak and set point viral loads were statistically significant in the immunized animals that became infected.     

Acute-Phase CD8 T Cell Responses That Select for Escape Variants Are Needed to Control Live Attenuated Simian Immunodeficiency Virus

The overall CD8 T cell response to human/simian immunodeficiency virus (HIV/SIV) targets a collection of discrete epitope specificities. Some of these epitope-specific CD8 T cells emerge in the weeks and months following infection and rapidly select for sequence variants, whereas other CD8 T cell responses develop during the chronic infection phase and rarely select for sequence variants. In this study, we tested the hypothesis that acute-phase CD8 T cell responses that do not rapidly select for escape variants are unable to control viral replication in vivo as well as those that do rapidly select for escape variants. We created a derivative of live attenuated SIV (SIVmac239Δnef) in which we ablated five epitopes that elicit early CD8 T cell responses and rapidly accumulate sequence variants in SIVmac239-infected Mauritian cynomolgus macaques (MCMs) that are homozygous for the M3 major histocompatibility complex (MHC) haplotype. This live attenuated SIV variant was called m3KOΔnef. Viremia was significantly higher in M3 homozygous MCMs infected with m3KOΔnef than in either MHC-mismatched MCMs infected with m3KOΔnef or MCMs infected with SIVmac239Δnef. Three CD8 T cell responses, including two that do not rapidly select for escape variants, predominated during early m3KOΔnef infection in the M3 homozygous MCMs, but these animals were unable to control viral replication. These results provide evidence that acute-phase CD8 T cell responses that have the potential to rapidly select for escape variants in the early phase of infection are needed to establish viral control in vivo.