We have elucidated the specific viral determinants recognized by NAbs that were responsible for antibody-mediated neutralization during early SIVmac251 infection by screening a panel of mutant pseudoviruses for their sensitivity to neutralization by serum antibodies from vaccinated and SIVmac251-infected rhesus monkeys. By introducing amino acid substitutions in Env, we determined that neutralization resistance in early SIVmac251 infection was conferred by specific amino acid changes in V4. Changing R420 to G, R424 to Q, and R426 to Q in the V4 region significantly altered the sensitivity of these pseudovirions to early NAbs, indicating that the SIVmac251 Env V4 region contains immunodominant epitope(s) and represents the earliest target for NAb. These results are consistent with previous reports demonstrating the importance of the V4 region for recognition by SIVmac neutralizing antibodies (2
). Early NAbs were not directed to the SIVmac251 V1 region, since V1 mutations did not alter the sensitivity of pseudoviruses to neutralizing antibodies in acute infection. In addition, prior vaccination with this Env immunogen did not alter the kinetics or the specificities of early NAbs in this cohort of monkeys.
A limitation of these studies is that the mutations in the V4 region of Env may have affected the folding of SIVmac251 Env. Therefore, any change in antibody neutralization profile of particular pseudoviruses in the present study may be a reflection of the structural integrity of Env rather than a consequence of the specificity of antibody response. It is also likely that the neutralizing activity of sera from monkeys following immunization is not exclusively directed to epitopes in V4; other regions of the Env were also targeted. The mutant Env pseudoviruses used to probe the NAb specificities in the present study were constructed based on sequencing data obtained from a subset of vaccinated monkeys in this cohort (27I, AD26, 26-J, 13K, 63-K, and 05K) (3
) and other unvaccinated/SIVmac251-infected monkeys (45
). These pseudoviruses were derived from the predominant env
quasispecies that were circulating in immunized monkeys that served as the source of 6 of 16 sera evaluated in the present study. However, since we did not examine env
sequences from every monkey in this cohort and did not clone all circulating env
genes, it is possible that potential Env mismatches between the Env pseudoviruses used in the present study and the specific viral sequences that were targeted by NAbs in some monkeys contributed to the variability in the sensitivity of these pseudoviruses to serum NAbs. In addition, the variability in NAb specificities in genetically diverse and outbred rhesus monkeys also may have contributed to the lack of statistical significance when we compared the kinetics and breadth of Env-specific NAbs between the 2 groups of vaccinated monkeys. It is also possible that the association between env
vaccination and a broader NAb response will be more apparent if autologous pseudoviruses were used to probe sera from a larger cohort of monkeys.
The NAb determinants of SIVmac251 Env demonstrated in the present study are consistent with viral sequence evolution data described by our group and others (3
). Our previous studies demonstrated that viral variants with nonsynonymous nucleotide substitutions in the V4 region of Env emerged at 5 to 8 months p.i. as a result of substantial selection pressure exerted on the replicating virus population in vivo
by the autologous NAb response. Although NAbs directed against the V4 region of Env appear not to be protective and do not prevent viral escape from immune pressure, it will be important to examine this region of Env closely to understand the properties that allow it to be uniformly targeted by NAbs. Glycosylation patterns and the exposure of the V4 region on the outer domain of Env and the neighboring residues in gp120 may be playing a role in eliciting the initial humoral immune response following SIVmac251 infection (21
). Recent studies have demonstrated that glycan-dependent epitopes are frequently targeted by broad neutralizing antibody responses following HIV-1 infection (23
). It has also been shown that changes in V4 glycosylation can affect the sensitivity of an HIV-1 isolate to strain-specific neutralizing antibodies directed against noncontiguous sites in Env (44
). These studies suggest that glycans are important targets on HIV-1 glycoproteins for broadly neutralizing antibody responses in vivo
. Although the R420 to G, R424 to Q, and R426 to Q changes did not directly alter the sequences of the predicted glycosylation sites, it is possible that these amino acid changes resulted in Env structure changes that shifted the glycan shield. It is also possible that the SIVmac251 V4 region is immunogenic, because it is not highly glycosylated. Previous studies have shown that the removal of N-linked glycans in HIV-1 V1 to V3 results in an enhanced ability of these constructs to induce NAb responses (26
We did not model the amino acid residues that were shown to be involved in the recognition of SIVmac251 Env by early NAbs, because the V4 region was deleted from the SIVmac239 gp120 crystal structure reported by Chen et al. (8
). Studies have shown that the V4 region of HIV-1 Env does not contribute to receptor binding, virus entry, or viral replication (36
). The HIV-1 Env V4 region appears to tolerate substantial changes in amino acid composition and glycosylation without compromising Env function. However, a study by Moore et al. showed a substantial role for the C3-V4 region of Env in determining the specificity of the early autologous neutralizing antibody response to HIV-1 subtype C infection (29
). The significance of neutralization by antibodies that target functionally unimportant regions of the Env glycoproteins in HIV-1 infection is unclear. Regardless, the identification of early autologous NAb epitopes, such as those in SIVmac251 V4, and the elucidation of the properties that render certain epitopes in the transmitted early virus particularly immunogenic may provide insights into strategies for engineering Env immunogens that are effective in inducing potent and broadly protective NAbs.