HIV-1 Envgp160 based vaccines protect monkeys against a homologous SHIV challenges [18, 19]. The ability to match the gene sequences used in the vaccine to the possible exposure virus in humans is not possible. Therefore, studies that use a matched Envgp160 in the vaccine to the challenge strain is appropriate for proof-of concept studies, but our group set a more challenging goal to protect against a challenge virus with a mismatched vaccine with a limited number of vaccinations. We report here that each consensus sequence representing clade A, B, C, E, in a polyvalent mixture or as a single consensus Envgp160 representing the entire Group M elicited anti-Envgp140 antibodies that bound to a broad panel of HIV-1 Envgp160s. The immunization schedule used was not optimal for antibody affinity maturation; nonetheless, the regimen did induce modest neutralizing antibody titers to the challenge Envgp160. However, the elicited immunity did not prevent infection by SHIVSF162p4.
In previous studies, consensus sequences designed for clades B and C Gag and Envgp140 elicited increased breadth of humoral and cellular immune responses [12, 20–22]. Consensus Envgp160 sequences representing Group M, termed CON-S, elicited antibodies that neutralized multiple Envgp160s, as well as eliciting cross-clade cellular immune responses [13, 14]. However, viral challenges of CON-S vaccinated monkeys were not reported and therefore the efficacy of the induced immune response elicited by these vaccines is unknown.
Compared to Envgp120 monomers, Envgp140 trimers may expose binding and neutralizing epitopes that are present only in Env’s quaternary state [23, 24]. Our consensus Envgp140 trimers have similar antigenic properties as wild-type Envgp160s, as demonstrated by attaching to human CD4 and binding to the monoclonal antibody b12. The b12 antibody recognizes a conserved region on gp120 mapped to a discontinuous epitope overlapping the CD4 binding site .
Following three intramuscular vaccinations, all monkeys seroconverted by day 14 following the final vaccination. Nonetheless, there were differences in the vaccine efficacy following challenge between the two vaccine groups. Both non-neutralizing and neutralizing antibodies have been implicated in reducing rates of infection by HIV-1 [26, 27]. A report based on the analysis of the sera samples of vaccinated volunteers in the RV144 clinical trial stated that the vaccine elicited antibodies against the V2 region of the HIV-1 Envgp160 were correlated with lower rates of HIV infection . Antisera collected from these vaccinated individuals did not neutralize the infection in vitro. Additionally, vaccine induced protection against a neutralization resistant virus in macaques was correlated with antibodies to the V2 region of Envgp160. Whether antibodies that bind to the V2 region are correlated with protection against SHIVSF162p4 infection in this study is unclear. There were no antibodies elicited in monkeys vaccinated with Con M or polyvalent consensus Envgp140 vaccines that recognized SF162 Envgp160 linear peptides, including those specific to V2 (data not shown). Further studies are necessary to determine if the two protected animals in the Con M Envgp140 group elicited antibodies recognizing conformational epitopes, such as the V1/V2 scaffold proteins. The V1/V2 scaffold was used to analyze human sera collected from vaccinated volunteers in the RV144 clinical trial [30, 31]. Determining if antibodies specific to various conformational epitopes on Envgp160 may explain the differences observed in vaccinated animals following SHIV challenge.
Two monkeys that had no detectable viral levels following SHIV infection were M1 and M4. M2 and M3 had detectable viral levels and therefore were not protected against infection. Monkeys vaccinated with either polyvalent consensus or Con M Envgp140 trimers had neutralizing titers to HIV-1SF162. Neutralizing antibodies against Envgp160 can protect monkeys against viral challenge [32, 33]. However, only one monkey (M4) in the present study had high neutralizing antibodies (1:320) against SHIVSF162p4 and had undetectable viral titer 14 days after challenge. However, following CD8+ T cell depletion, virus was detected (<1×105 RNA copies /ml) in the blood indicating that infection was not blocked, but may have been controlled by the vaccine elicited antibodies. T cell responses did not appear to play a role in protecting the monkeys from infection. There was no difference in the number or kinetics in the elicitation of Envgp160 or Gag specific IFNγ producing cells following challenge in any of the vaccinated monkeys compared to mock vaccinated animals.
Upon CD8+ depletion, it was not unexpected that M2 and M3 had a rebound in blood titer virus, but the detection M4 was unexpected. In contrast to monkey M4, no virus was detected in monkey M1 even after depletion of CD8+ T cells. Both IFNγ specific T cells and neutralizing antibodies were detected, but it is unclear which of these immune responses may have contributed to the protection. In addition, the MHC class I haplotype did not appear to correlate with protection. Even though no viremia was ever detected in monkey M1, it is possible that virus could be located in reservoirs, such as the bone marrow or gut mucosa . The M-T807R1 monoclonal antibody used for CD8+ T cell depletion is specific for cells in the serum and lymph nodes , therefore, it may have not depleted cells in reservoirs of hidden virus. In an effort to identify possible reasons for M1 protection the animals’ halotypes were determined. Monkey M1 had a Mamu-B*008 MHC class I haplotype, which has been associated with control of SIVmac239 virus; the parent virus of the challenge SHIVSF162p4. Therefore, a combination of the neutralizing antibodies, non-neutralizing antibodies and the Mamu-B*008 MHC class I haplotype may have resulted in “sterilizing” protection after viral challenge. However, the Mamu-B*008 MHC class I haplotype was also present in monkey M3, which had similar binding and neutralizing antibody titers as monkey M1, but was not protected from SHIV infection. Interestingly, two monkeys vaccinated with the polyvalent consensus vaccine, P2 and P4, had a rebound in viral titers at day 40 post-infection before returning to undetectable levels (Figure 3B). The rebound virus could have been a variant that escaped the vaccine elicited immune response, however, sequencing of the virus in the blood collected at day 40, did not show any significant variation of the viral sequence compared to the input virus on day 0.
While Envgp140 only vaccines have been successful against homologous challenge, both the human RV144 trial and previous monkey studies showed significant protection from heterologous challenge, included other HIV protein components [8, 37, 38]. Including Tat in the vaccine formulation induces strong and persistent CD4+ T cells  and broadens T cell responses directed against Gag and Envgp160[40, 41]. Gag is known for inducing strong cellular responses that may lead to reduced viral loads [42, 43]. Addition of Gag and/or Tat to our Con M vaccine may have prevented infection or controlled undetectable virus in vaccinated animals more effectively than Con M Envgp140 alone. Even though some of these studies use Envgp140 proteins, they are combined with other HIV proteins to elicit a broadly reactive response. In our vaccine presented here, the purified VLPs only have Gag and Envgp140 expressed in different modalities than VLPs and we achieve a broadly reactive anti-Envgp140 response using our consensus Envgp160s. For example, viral vectors are used to express Gag and Envgp160 independently in the RV144 human trial, which really does not allow for comparison with our VLP strategy.