Studies that use SHIVs benefit from the combination of an established animal model for HIV infection and access to an extensive array of HIV-1 reagents, including stored plasma samples and genetically modifiable molecular clones. Using two well-characterized SHIV clones and plasma collected from animals infected with the virus matched to each clone, we designed a straightforward panel of variable-loop chimeras in each SHIV background to evaluate the role of each loop sequence in determining the strain specificity of antibody-mediated neutralization. The beauty of these experiments was that, by using two sets of reciprocal chimeric constructs and matched SHIV-positive plasma samples, we were able to measure both gain and loss of sensitivity to strain-specific neutralization based on the presence or absence of a particular variable-loop sequence. All SHIV-derived variable-loop chimeras that included an exchanged V1/V2 variable loop lost sensitivity to positive plasma samples from monkeys infected with the autologous virus and acquired specific sensitivity to positive plasma from monkeys infected with the SHIV strain containing that V1/V2 sequence originally. The plasma dilutions at which the V1/V2 variable-loop chimeras were 50% neutralized were quite similar to the dilutions needed to neutralize the viruses from which the V1/V2 sequences were derived. The presence of these exchanged V1/V2 loop complexes was the dominant factor in establishing a neutralization phenotype for each variable-loop chimera. These results demonstrate unambiguously that the V1/V2 variable-loop complex is principally responsible for the strain-specific neutralizing activity observed in plasma from monkeys infected with these prototypic SHIVs.
The V3 loop of gp120 is often viewed as a major target of anti-HIV antibody responses and an important immunogen to be considered in vaccine design (8
). Arguments for the importance of V3 include the targeting of V3 by antibodies from the majority of HIV-1-infected individuals (15
), the ability of some anti-V3 monoclonal antibodies to potently neutralize HIV-1 infectivity (19
), and V3's structural conservation (21
). Even though much effort has gone into defining the nature and characteristics of anti-V3 antibody responses, others have pointed out that most anti-V3 antibodies are actually quite limited in their abilities to neutralize primary isolates of HIV-1 (7
). Our results indicate that HIV-1 V3 is not a target of the neutralizing-antibody response to any appreciable extent in monkeys infected with the prototypic SHIV strains DH12 and KB9. It is, of course, possible that V3 may be an important target for neutralization in the context of R5-only HIV-1 infection or at later time points in the course of monkey infection with these same SHIV strains (49
). However, the results reported here provide strong evidence for the predominance of the V1/V2 loop complex in determining the strain-specific neutralizing-antibody response that characterizes both HIV-1 and SHIV infections.
Using very different approaches and reagent sets, Pinter et al. and Ching et al. have also recently concluded that the V1/V2 region is the dominant determinant of HIV-1 neutralization sensitivity (16
). In contrast to their studies, our studies employed plasma matched to the cloned virus with which the monkeys were infected, did not use viruses that were globally sensitive to antibody-mediated neutralization, and employed variable-loop swaps in both directions. Nonetheless, all three studies similarly found a dominant role for V1/V2 in determining sensitivity to antibody-mediated neutralization. It will be important in the future to perform analogous experiments with CCR5-using clade B HIV-1 isolates and matched plasma collected from HIV-1-infected individuals.
Others have found more complex determinants for the strain specificity of the neutralizing-antibody response (17
). The study by Moore et al. published in 2008 (68
) is the only study in addition to our own that performed reciprocal exchanges of variable loops in both directions. Although Moore et al. found a substantial role for V1/V2 in determining the strain specificity of the neutralizing-antibody response to clade C HIV-1 infection, the C3-V4 region also contributed importantly (68
). It is possible that clade, tropism, and individual-to-individual variation could contribute to the degree of dominance of the V1/V2 region.
There are multiple mechanisms by which the V1/V2 loop complex may be acting in order to dramatically alter the neutralization sensitivities of the SHIV-derived V1/V2 variable-loop chimeras. Most directly, the V1/V2 loop sequence may contain the epitope targets of neutralizing antibodies in the plasma from monkeys infected with either SHIV DH12 or SHIV KB9. In this scenario, exchanging the V1/V2 loop complex between the two SHIVs will concomitantly switch the targets for antibody recognition and neutralization. Alternatively, the V1/V2 variable-loop complex might shield particular epitopes from antibody recognition while allowing others to be bound and neutralized by circulating antibody. The conformational change in envelope following a V1/V2 loop exchange may shift this shielding to occlude previously exposed epitopes and expose previously shielded epitopes to antibody recognition, leading to reciprocal gains and losses of neutralization sensitivity. Lastly, the V1/V2 loop complex might be critically involved in the formation of complex, conformational neutralizing determinants. Consequently, the V1/V2 loop exchange would disrupt such epitopes and render the resultant virus refractory to neutralization by autologous SHIV-positive plasma while creating a conformational structure that could be recognized and neutralized by heterologous SHIV-positive plasma. Extensive epitope mapping will be necessary to discern which of these potential mechanisms is principally responsible for the V1/V2-dependent determination of the strain-specific neutralizing activity described by this study.
Despite robust replication and infectivity of both parental viruses in C8166-45 LTR-SEAP cells, three of the SHIV-derived variable-loop chimeras (SHIV DKV3, SHIV KDV12, and SHIV KDV124) exhibited poor infectivity and were not included in further studies. Our inability to obtain infectious recombinant viruses with these chimeras strongly suggests that although one variable loop is able to function within the context of its parental envelope spike, this same loop was nonfunctional when introduced into a different envelope context. The reduced infectivity resulting from the variable-loop exchange does not appear to be an inherent characteristic of the amino acid sequence, as infectivity was not impaired in the reciprocal exchange. These differences are most likely based upon inherent differences in the abilities of the envelope complexes to tolerate a heterologous variable-loop exchange. gp120 is thought to be stabilized in its tight, compact conformation within the envelope spike by intratrimeric interactions between monomers and by an extensive glycosylation network, the pattern of which differs between envelope species (80
). Mismatched variable-loop sequences and/or differential N-linked glycosylation patterns of the SHIV-derived variable-loop chimeras may destabilize the trimeric envelope framework such that specific loop exchanges (DKV3, KDV12, and KDV124) result in viruses with severely reduced infectivity.
One of the most daunting challenges for HIV-1 vaccine strategies aimed at eliciting a protective neutralizing antibody response is overcoming the enormous sequence variability that is a hallmark of the envelope protein. Thus far, such attempts have demonstrated little success, as the neutralizing activities elicited by the particular envelope immunogens tested have characteristically displayed low potency and/or high strain-specific neutralizing activity. An ideal immunogen would elicit potent neutralizing antibodies that were capable of neutralizing a broad range of diverse primary HIV-1 isolates and would avoid inducing antibodies that were weak and strain specific. There are at least two distinct approaches to achieve this. The first is to design an envelope-based immunogen that will elicit antibodies focused on conserved elements within gp120 that are able to access these epitopes in the context of the mature trimer spike on the surface of the virion. An alternative approach is to include a mixture of envelope sequences in an immunogen pool that can cover as broad a range of sequence variation as possible, thus inducing antibodies capable of neutralizing a large spectrum of primary isolates. This approach seems daunting if one is trying to adequately represent the sequence diversity of the entire envelope protein. However, if the sequence variation within the V1/V2 variable loop is the principal determinant of antibody-mediated neutralization, as indicated by the present study, this would considerably limit the range of sequences that would need to be included in such an envelope immunogen pool.