In the present study, we evaluated two infectious molecular clones from the SIVsmE660 virus stock and studied their pathogenesis in rhesus macaques. We focused on characterization of the neutralizing antibody response and virus evolution to escape neutralization, providing fundamental information for future studies. Despite their sensitivity to neutralizing antibodies and a rapid autologous neutralizing antibody response, these two clones were able to maintain persistent plasma viremia when inoculated intravenously into rhesus macaques. We restricted our studies to macaques with a moderately sensitive TRIM5 genotype, and therefore, this study may not reflect the full variability among macaques with restrictive or permissive genotypes. However, virus loads were comparable to those in macaques infected with the uncloned SIVsmE660 stock virus (40
). The continual decline of CD4+
T cells in the infected macaques is consistent with disease progression, and one animal was recently euthanized due to opportunistic infections (data not shown). Although limited by the small study size, the pathogenicity appears to be less than that observed in macaques inoculated with neutralization-resistant molecular clones, such as SIVmac239 and SIVsmE543-3. These neutralization-sensitive clones will be useful for investigating the role of NAbs in disease progression and vaccine protection. A number of recent studies have suggested that envelope is a critical component of vaccines in the SIV/macaque model (2
), and envelope antibodies correlate with protection in the recent RV144 trial (20
). However, vaccine studies with SIVmac239 are limited for use in the evaluation of neutralizing antibody response in protection since they are so resistant to neutralization. In addition to the utility of these NAb-sensitive E660 clones, viruses with a neutralizing phenotype intermediate between SIVmac251/239 and SIVsmE660-FL6/FL14, such as these later-stage SIVsmE660 env
clones, are ideal for evaluating potential vaccine strategies since they are more representative of the phenotype of primary HIV-1 isolates.
There is still controversy regarding the role of autologous NAb responses in the initial downregulation of primary viremia in HIV-1 infection (41
). Since we observed high titers of autologous NAb by 4 weeks, it is possible that these responses might have developed earlier. However, NAb was not detected in plasma samples collected at 2 and 3 weeks (data not shown); this result will need confirmation using serum samples since anticoagulants are known to interfere with neutralizing assays. The appearance of autologous NAbs in macaques in the present study was much earlier than that in SIVmac251-infected macaques (66
). Autologous NAbs against parental SIVsmE660 clones were detected by 4 weeks postinfection, more similar to the kinetics in HIV-infected patients (51
). The temporal association of neutralization escape mutants and evolution of an autologous NAb response in this study, as well as in SIVmac251-infected animals (66
), is entirely consistent with a role of NAbs in the pathogenesis of SIV infection of macaques.
Despite the similarities in kinetics of autologous neutralization, NAb titers in SIVsmE660-infected macaques were higher by orders of magnitude than what have been observed in studies on HIV-1-infected patients (3
). This difference may be due to the sensitivity of the E660 clones compared to that of the transmitted/founder virus HIV-1 Envs used for assays to evaluate HIV-1 antibody responses. Measuring NAbs against the inoculated SIV clone should be equivalent to measuring NAbs against the transmitted/founder Env since most of the Env sequences cloned from plasma at the peak of infection are similar to Env sequences of the inoculated SIV clone. But this is also is an explanation for the very high titers in these animals, since the transmitted/founder viruses used for equivalent HIV-1 studies would be significantly more resistant to neutralization, and thus, the titers would be much lower and more difficult to measure. While titers against subtype B HIV-1 Envs tend to remain at moderate levels, there are subtype differences, since early autologous neutralizing antibody responses are quite high in subtype C HIV-1 infection (37
). Interestingly, several studies have recently demonstrated that NAb responses are very potent in HIV-2-infected cohorts, with NAb titers ranging from 104
per ml (13
), levels which are similar to titers in the SIVsmE660-infected macaques in this study. While some studies suggest that HIV-2 does not escape NAb as effectively as HIV-1, a study by de Silva et al. demonstrated a similar pattern of envelope evolution and the development of neutralization resistance (13
). Compared with HIV-1-infected cohorts, disease progression during HIV-2 infection is usually slower and plasma viral loads are lower, suggesting that the potent NAb response may contribute to the delayed disease progression. The SIVsmE660-infected macaques in the present study should provide a good model to test this hypothesis in vivo
. Future studies will compare the disease progressions of macaques infected with SIVsmE660 clones and their more-resistant derivatives that arose during in vivo
passage to further understand the relationship between NAb response and disease progression.
In this study, we found that the neutralization-sensitive SIVsmE660 clones maintained persistent infection in the presence of high titers of NAbs by rapidly escaping from neutralizing antibody responses, as previously described in HIV-1 studies (51
). Sequence analysis of Env sequences from Rh805 and Rh807 showed that substitutions and deletions in the V1 and V4 domains accumulated over time, suggesting that these domains may be the target of NAbs or that changes in these regions influence the overall conformation of the envelope glycoprotein. Most Env clones from 4 and 8 w.p.i. had mutations only in either the V1 or V4 domain, whereas clones from later time points had mutations in both domains. Neutralization assays also showed that early clones from Rh807 escaped neutralization of concurrent serum samples but were still neutralized by late-phase serum samples (). Env variants from 16 w.p.i. and 24 w.p.i. completely escaped neutralization of serum samples collected before 24 w.p.i. These results revealed that escape mutants gradually acquired neutralization resistance and that the resistant Env variants are selected by NAbs.
In contrast with studies in HIV-infected patients (52
), we did not observe consistent changes in glycosylation sites in E660 envelopes during neutralizing antibody escape. Although uncommon in escape variants, we did observe mutations in some Env variants from 24-w.p.i plasma of Rh807 and Rh808 that resulted in the addition of a glycosylation site in the V4 domain. We observed a common S70N mutation, which results in the addition of a glycosylation site in the C1 domain, in all the clones from chronic-stage plasma of Rh807 and Rh808. Interestingly, this glycosylation site is very conserved among SIVmac and SIVsm viruses (27
). Whether this glycosylation in the C1 domain contributes to neutralization escape will be investigated in the future.
antibody responses developed against SIVsmE660 escape variants, a result which has not been observed in SIVmac239-infected macaques (55
). NAbs against the escape variants from 24-w.p.i. plasma can be detected a few weeks later in all the macaques. These results also suggest that the escape variants are capable of being neutralized. Future studies will be required to identify the neutralizing epitopes recognized by the de novo
antibodies. The NAb responses against the escape Env variants were much weaker than those against the parental clones. In Rh805 and Rh808, NAbs against the escape variants were not detected until 6 to 7 months later (48 to 52 w.p.i.). The weak and delayed NAb responses against escape variants may explain why virus replication is not controlled by the potent neutralizing antibody response. The sensitivity to neutralization of these escape variants was also tested with the panel of sera from macaques infected with uncloned SIVsmE660 stock virus or SIVsmE543-3. The escape mutants are more resistant to both homologous and heterologous NAbs than the parental SIVsmE660-FL6 and -FL14 clones, while they are more sensitive to neutralization than SIVsmE543-3. Such variants, with intermediate sensitivity to neutralization, may be a useful addition to challenge virus stocks for vaccine evaluation in the SIV macaque model, used either alone or in combination with their neutralization-sensitive parents.
In summary, we generated two pathogenic SIVsmE660 molecular clones in the present study. These clones are sensitive to neutralization and induce a more potent NAb response in rhesus macaques than neutralization-resistant SIVmac and SIVsmE543-3 viruses and thus can be used as a model to study the role of NAb in disease progression. We also observed virus evolution to escape from neutralization and obtained several escape mutants with intermediate sensitivity to neutralization compared to that of SIVsmE660-FL6, SIVsmE660-FL14, and SIVsmE543-3 clones. SIV clones with intermediate sensitivity to neutralization will be a useful adjunct to vaccine and challenge strains for vaccine development using the SIV model.