A global HIV-1 vaccine must elicit effective immune responses to diverse viral isolates. In fact, broadly cross-reactive HIV-1-specific T-cell immune responses have been described. HIV-1-infected individuals develop T-lymphocyte responses that recognize viral sequences from a diversity of HIV-1 clades (3
). Cross-clade-reactive cytotoxic T lymphocytes (CTL) have also been detected in uninfected volunteers who have been vaccinated with recombinant canarypox constructs (8
). However, because these studies employed CTL clones or in vitro-cultured PBL to assess cross-clade T-cell reactivity, the true breadth of these HIV-1-specific immune responses is unknown. In the present study we demonstrate that immunization of rhesus monkeys with a DNA prime-rAd boost vaccine that includes multiple Env immunogens elicits cellular and humoral immune responses that exhibit a greater breadth of Env-specific recognition than that observed in monkeys immunized with single Env immunogens.
PBL from monkeys immunized with single HIV-1 Env immunogens demonstrated a high frequency of cellular immune responses to peptide pools matching the vaccine-encoded Env immunogen, with a lower frequency of responses to peptides of Env proteins not included in the vaccine. These cross-reactive responses may reflect T-lymphocyte recognition of conserved viral epitopes, as well as cross-reactive recognition of variant epitopes that may differ by limited numbers of amino acids (6
). The highest degree of heterologous Env recognition in this study was the reactivity of PBL of monkeys immunized with the clade-B HXBc2/BaL Env immunogen against peptide pools representing 89.6P Env, a heterologous clade-B Env (Fig. ). HXBc2/BaL Env shares 81% amino acid identity with 89.6P Env and only 75 and 72% identity, respectively, with the clade-A and -C Env sequences used in these immunizations. These data suggest that immunizing with single-Env immunogens may elicit the highest frequency of cross-reactive T-cell responses against Envs of viruses of the same clade.
A concern with a vaccine that includes viral proteins from multiple clades of HIV-1 is that interference between these diverse antigens may diminish immune responses. In fact, such antigenic interference has been observed in vaccines that include proteins of multiple pathogens (7
). Moreover, studies have shown that complex mixtures of plasmid DNA vaccines can lead to decreased protein expression and immunogenicity in vivo (13
). The findings in the present study demonstrate that the inclusion of Env immunogens from several clades of HIV-1 in a single vaccine can increase the breadth of vaccine-elicited Env-specific T-cell and antibody responses. Thus, monkeys immunized with the multiclade Env vaccine developed high frequencies of cellular immune responses and antibody responses to all vaccine-encoded Env antigens. The magnitudes of T-lymphocyte responses to the clade-B and clade-C Env peptide pools following the DNA prime and rAd boost with the multiclade Env immunogens were similar to those observed in monkeys receiving the high-dose single clade-B or -C Env vaccine. Furthermore, no deleterious effects on the magnitudes of Gag- or Pol-specific cellular immune responses were detected in the multiclade Env-immunized monkeys. These results support the findings of previous studies in mice demonstrating that multiclade HIV-1 vaccines can elicit robust cellular and humoral immune responses to all vaccine-encoded antigens, with no evidence of antigenic interference (4
While this immunization strategy can elicit high-titer HIV-1-specific antibody responses, both the potency and the breadth of the neutralizing antibodies induced by these Env immunogens were limited. We employed appropriate controls to confirm the presence of antibody-mediated neutralization by the immune plasma. These controls included the use of preimmune plasma from each animal and an irrelevant MuLV control virus. Although only low levels of neutralization were detected, the specificity of the demonstrated neutralization also lends credibility to the results. For example, the clade-A viruses were more strongly neutralized by plasma from animals that received the clade-A, -B, and -C multiclade Env immunogens than by plasma from animals that received only the clade-B immunogen. Several of the isolates in the panel of clade-B viruses that was used to evaluate the breadth of neutralization are known to be particularly neutralization sensitive (e.g., HxB2, SF162, Bx08). The clade-A virus isolates evaluated in this study also appear to be more sensitive to neutralization than the clade-C isolates (Fig. ). The panel of 8 clade-A and 11 clade-C viruses used in this study was composed of viruses readily available to us. No a priori criteria were applied in choosing these specific viruses. In fact, we have little prior experience in evaluating vaccine-elicited antibody neutralization of non-clade-B viruses, and it is possible that the clade-A viruses we used in this panel were inherently more sensitive to neutralization than the clade-C viruses.
We have previously demonstrated that DNA prime-rAd boost immunization of rhesus monkeys with vaccine antigens that include 89.6P Env or a clade-B Env fails to elicit neutralizing antibodies against the SHIV-89.6P challenge virus (15
). Several other vaccine studies have also demonstrated SHIV-89.6P to be a neutralization-resistant isolate (17
). Thus, while we did not evaluate plasma-mediated neutralization of SHIV-89.6P in this study, we are confident that the immunogens that were employed would not elicit neutralizing antibodies to the challenge virus. Neutralizing antibodies to SHIV-89.6P do develop after SHIV challenge, but our prior study of a homologous 89.6P Env immunogen was unable to demonstrate that Env immunization accelerated the development of these antibodies (15
). We therefore believe it is highly unlikely that an anamnestic antibody response played a significant role in the clinical protection observed in this study.
It is encouraging that the combination of the clade-A, -B, and -C Env immunogens elicited neutralizing antibodies to some viral isolates not included in the vaccine. Clearly, this neutralization was low in potency, and further efforts will be required to design more-potent Env immunogens. We have yet to examine in depth whether this multiclade Env immunization elicits T-cell cross-reactivity to diverse clade-A, -B, or -C Envs that are not included in the vaccine or whether it simply elicits immune responses to the vaccine antigens that are additive; however, given our observation that single-clade Env immunization elicits immune responses that are highly cross-reactive with Env sequences from the same clade, it may be expected that T lymphocytes of multiclade Env-immunized monkeys will exhibit a high frequency of cross-reactive cellular immune responses to heterologous clade-A, -B, and -C Envs.
The present data show that multiclade Env immunization does not diminish vaccine-elicited immune protection against SHIV-89.6P infection. Monkeys receiving DNA prime-rAd boost vaccines encoding either a single Env or multiple-clade Env immunogens demonstrated equivalent viral containment during acute and chronic infection, and comparable preservation of CD4+
T lymphocytes. We have previously demonstrated that DNA prime-rAd boost vaccine-elicited protection against SHIV-89.6P infection was associated with an anamnestic antigen-specific cellular rather than a neutralizing-antibody response (15
). It is therefore not surprising that no significant differences in clinical protection were evident between the various groups of vaccinated monkeys; they all demonstrated robust prechallenge cellular immune responses to SIV Gag and Pol, as well as some degree of cellular immune cross-reactivity to 89.6P Env. In fact, the ELISPOT responses to 89.6P Env increased rapidly in the PBL of all groups of Env-vaccinated monkeys following challenge, suggesting that vaccine-elicited T lymphocytes capable of recognizing 89.6P Env epitopes expanded in response to the replicating virus (Fig. ).
The present study demonstrates that the inclusion of viral proteins from multiple clades of HIV-1 is a viable approach for a global HIV-1 vaccine. Whether this strategy proves superior to the use of immunogens created from ancestral or consensus HIV-1 viral sequences for increasing the breadth of immune recognition of HIV-1 isolates remains to be determined (9
). Nevertheless, this study illustrates the feasibility of constructing vaccine immunogens that address the problem of the extreme variability of HIV-1 isolates throughout the world.