Search tips
Search criteria 


Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
J Virol. 2012 July; 86(13): 7052–7059.
PMCID: PMC3416332

Envelope Variable Region 4 Is the First Target of Neutralizing Antibodies in Early Simian Immunodeficiency Virus mac251 Infection of Rhesus Monkeys


A major goal of AIDS vaccine development is to design vaccination strategies that can elicit broad and potent protective antibodies. The initial viral targets of neutralizing antibodies (NAbs) early after human or simian immunodeficiency virus (HIV/SIV) infection are not known. The identification of early NAb epitopes that induce protective immunity or retard the progression of disease is important for AIDS vaccine development. The aim of this study was to determine the Env residues targeted by early SIV NAbs and to assess the influence of prior vaccination on neutralizing antibody kinetics and specificity during early infection. We previously described stereotypic env sequence variations in SIVmac251-infected rhesus monkeys that resulted in viral escape from NAbs. Here, we defined the early viral targets of neutralization and determined whether the ability of serum antibody from infected monkeys to neutralize SIV was altered in the setting of prior vaccination. To localize the viral determinants recognized by early NAbs, a panel of mutant pseudoviruses was assessed in a TZM-bl reporter gene neutralization assay to define the precise changes that eliminate recognition by SIV Env-specific NAbs in 16 rhesus monkeys. Changing R420 to G or R424 to Q in V4 of Env resulted in the loss of recognition by NAbs in vaccinated monkeys. In contrast, mutations in the V1 region of Env did not alter the NAb profile. These findings indicate that early NAbs are directed toward SIVmac251 Env V4 but not the V1 region, and that this env vaccination regimen did not alter the kinetics or the breadth of NAbs during early infection.


Passive immunization studies in nonhuman primates and correlates of protection studies in both nonhuman primates and human vaccinees have demonstrated that antibodies can contribute to preventing the acquisition of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) (1, 10, 1114, 25, 27, 28, 33, 35, 37, 41). Therefore, considerable efforts are being directed toward identifying HIV-1 immunogens that elicit broadly neutralizing antibody (NAb) responses. The identification of regions of HIV-1 Env that are targeted by NAbs and an understanding of the immunogenicity of these regions in the setting of infection may guide the development of vaccine immunogens that elicit a protective humoral immune response.

The SIV-infected rhesus monkey model of AIDS provides an important system for dissecting the targets of NAbs and assessing the evolution of the humoral immune response following vaccination and/or infection. The emergence of variants of SIV that escape recognition by NAbs has been well documented in SIV-infected rhesus monkeys (4, 5, 31, 32, 38, 40, 45). Several NAb epitopes have been previously identified in the V1/V2 and V4 regions of SIV Env (2, 6, 9, 1518, 20, 34, 39, 40). In addition, we have recently demonstrated that mutations in variable regions 1 and 4 of SIVmac251 Env are responsible for the escape from recognition by NAbs that develop following mucosal infection (3, 45). However, the precise early neutralization antibody determinants during acute SIV infection have not been defined. The identification of epitopes that play a role in inducing protective immunity early in infection is crucial for AIDS vaccine development.

The primary objective for the present study was to identify the principal neutralization determinants of SIVmac251 during early infection and assess whether prior vaccination with an Env immunogen altered the kinetics and specificity of the humoral immune response. Based on a previous study of longitudinal env sequence analysis in rhesus monkeys that were mucosally infected with SIVmac251 (45), we hypothesized that the initial neutralizing antibody response against SIV is directed against the variable region 4 of Env. Furthermore, we hypothesized that prior immunization with an SIVmac Env immunogen alters the early neutralizing antibody kinetics and specificities that develop following infection. To test these hypotheses, we utilized a pseudovirion-based, TZM-bl reporter gene neutralization assay to characterize the early neutralizing antibody responses in a cohort of monkeys that were immunized with vaccine regimens that either did or did not include SIVmac env. To elucidate the targets of the early NAb responses, we constructed a panel of SIVmac251 Env pseudoviruses that have specific site-directed mutations in Env, and we used these mutant viruses to probe sera from vaccinated monkeys infected with SIVmac251.



Outbred, adult male Indian-origin rhesus monkeys were housed at an AAALAC-accredited institution (Bioqual, Rockville, MD). All procedures were performed with full protocol approvals from the Institutional Animal Care and Use Committee for Vaccine Research Center, NIAID, NIH, and in accordance with the Guide for the Care and Use of Laboratory Animals (30). All animal studies were approved by the Vaccine Research Center Animal Care and Use Committees.

Immunization and viral challenge.

Eight of 16 monkeys were part of a cohort that has been previously published (24). Eight monkeys were immunized with a plasmid DNA construct carrying SIVmac239 gag/pol/nef on a schedule of 0, 4, and 8 weeks at a dose of 4 mg/plasmid/inoculation, followed by intramuscular immunization with a recombinant adenovirus 5 (rAd5) vector carrying gag/pol at a dose of 1011 particles at week 40. The 8 monkeys in the env arm of the study received the same plasmid DNA construct carrying SIVmac239 gag/pol/nef and an additional plasmid DNA construct carrying SIVmac239 env gp140ΔCFI (7) on the same 0-, 4-, and 8-week schedule with 4 mg/plasmid/inoculation. At week 40, the latter group was inoculated with one rAd5 vector carrying SIVmac239 gag/pol and another carrying SIVmac239 env pg140 ΔCFI, each at a dose of 1011 particles. Following immunization, monkeys in both arms of the study were challenged intravenously 19 weeks following the rAd immunizations with SIVmac251. This uncloned stock was expanded on human PBMC, and we determined the in vivo titers in rhesus monkeys for use in intravenous challenge studies. This virus stock has been previously characterized (19) and has a concentration of 3 × 108 SIV RNA copies/ml. All monkeys received 1 ml of a 1:3,000 dilution of this stock by the intravenous route.

Env mutations and cloning.

Various Env mutations were introduced into the plasmid containing wild-type (WT) parental SIVmac251.30 Env. ΔV1 deletions from residues 139 to 141, including I129 to T (I129T), N143 to D (N143D), N202 to K (N202K), K254 to E (K254E), M325 to K (M325K), W345 to R (W345R), T368 to N (T368N), R420 to G (R420G), R424 to Q (R424Q), and R426 to Q (R426Q), and ΔV4 deletions from residues 416 to 421 were created by PCR mutagenesis with a QuikChange kit (Stratagene, La Jolla, CA). Oligonucleotide primers used for site-specific mutagenesis are outlined in Table S1 in the supplemental material.

Construction of mutant pseudoviruses.

The env variants were cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA), transformed into TOP10F′ chemically competent cells, and selected with ampicillin. Inserts were confirmed by restriction digestion and direct sequencing. 293T cells were transfected with Lipofectamine (Qiagen, Valencia, CA) or LipoD293 DNA in vitro transfection reagent (SignaGen Laboratories, Rockville, MD) per the manufacturer's instructions. Env-containing plasmids were cotransfected with SIVmac239Δenv backbone. Pseudoviruses were harvested 24 to 48 h later, made cell free by filtration through 0.45-μm filters, and stored at −80°C until use. Pseudovirus stocks were titrated on TZM-bl cells and utilized in neutralization assays to target approximately 100,000 relative light units (RLU).

Neutralizing antibody assay.

Neutralizing antibodies in sera were measured in a luciferase reporter gene assay that utilized TZM-bl cells. The 50% inhibitory dose (ID50) titer was defined as the serum dilution that resulted in a 50% reduction in RLU compared to that of virus control wells after the subtraction of cell control RLUs. Any sample that did not have a detectable ID50 titer at a dilution of 1:10 is reported as <10.

Statistical analyses.

GraphPad Prism version 5 was used for statistical comparisons. To compare the fold-decrease in neutralizing ID50 titer against V4 mutant pseudovirions relative to that of WT Env in the 2 groups of vaccinated monkeys, we divided the WT ID50 titer of serum from each monkey by the average ID50 titer of the same serum specimen against the 5 V4 mutants. An ID50 of <10 was treated as 10 for the statistical analysis. The fold decreases of the average titer of V4 mutant viruses compared to WT Env in gag/pol/nef groups were compared to those of 8 monkeys in the gag/pol/nef/env group using the Mann-Whitney test. P < 0.05 was considered significant.


Development of neutralizing antibody responses in vaccinated monkeys infected with SIVmac251.

To assess the evolution of SIVmac Env-specific neutralizing antibody responses in rhesus monkeys following infection, we evaluated 8 monkeys that were primed with a plasmid DNA construct carrying SIVmac239 gag/pol/nef and boosted with a recombinant adenovirus 5 (rAd5) vector carrying gag/pol (control group without env vaccination), and we also evaluated an additional 8 monkeys that were primed with the same plasmid DNA construct carrying SIVmac239 gag/pol/nef plus an additional plasmid DNA construct carrying SIVmac239 env gp140ΔCFI and boosted with rAd5 vectors carrying SIVmac239 gag/pol and env gp140 ΔCFI (experimental group with env vaccination). Monkeys in both arms of the study were challenged intravenously with SIVmac251 at 19 weeks following the rAd immunizations. Sera from monkeys that were vaccinated prior to challenge were chosen for this study, because we wanted to determine whether autologous NAbs in these monkeys would be detected earlier than in monkeys immunized with a vaccine regimen lacking an env component. In addition, we were interested in determining whether prior Env vaccination resulted in a greater breadth of Env-specific neutralizing antibody responses.

We assessed the development of neutralizing antibodies in longitudinal serum samples using pseudotyped virions expressing the wild-type (WT) SIVmac251 Env (SIVmac251.30) in a TZM-bl reporter gene assay. This clone represented the predominant Env species in the SIVmac251 quasispecies inoculum that was used to initiate SIV infection in this study and has been shown to be relatively resistant to antibody neutralization (3, 45). Immunization with Env did not induce a detectable neutralizing antibody response prior to infection (Table 1). Neutralization activity against the WT Env pseudovirus was first detected between weeks 12 and 32 postchallenge (p.c.) in both groups of monkeys. The magnitudes of these neutralizing antibody titers increased over time and reached titers of >1,000 in 2 monkeys not vaccinated with env (AX93 at week 32 p.c. and DA1H at week 28 p.c.) and 1 monkey vaccinated with env (13K at week 66 p.c.). There was no difference in time to the development of a NAb response between the 2 groups of vaccinated monkeys (P = 0.59 by Mann-Whitney test), suggesting that the addition of an env immunogen to the vaccination regimen did not contribute to a more rapid emergence of a NAb response following SIV infection.

Table 1
Serum antibody neutralization of WT Env pseudovirus

Neutralizing antibodies target V4 region of Env early following infection.

We previously demonstrated that the emergence of autologous NAbs coincided with the presence of multiple nucleotide substitutions in the variable regions of SIV Env (45). Furthermore, Env variants cloned from these SIV-infected monkeys that contained multiple mutations in the V1 and V4 regions were shown to escape antibody neutralization (3, 45). In the present study, our goal was to identify precisely the determinants in Env that are targeted by NAbs early after SIV infection. To determine which Env amino acid residues were targets of the host humoral immune response, we tested the ability of the earliest sera that recognized SIVmac251 WT Env (SIVmac251.30) to neutralize variant Env pseudovirions with defined mutations. For this study, we considered a minimum of a 3-fold decrease of ID50 titer relative to the ID50 titer against SIV WT Env a true loss of neutralization (full virus escape). A minimum of a 2-fold decrease of ID50 titer relative to the ID50 titer against SIV WT Env was considered a partial loss in neutralization (partial virus escape).

Sera from all 16 vaccinated and infected monkeys neutralized the WT Env pseudovirus with a median ID50 titer of 153 and a range from 70 to 381 (Table 2). There was no difference in the median ID50 titers between the 2 groups of monkeys (P = 0.64 by Mann-Whitney test). Consistently with previously reported data (3), the same sera from 14 monkeys did not neutralize a variant Env (SGA Env) containing multiple mutations in gp160 that was cloned after single-genome amplification (SGA) from a vaccinated and infected monkey (ID50 titers of <10 to 12). This SGA Env clone contains V1 deletions from residues 139 to 141, as well as the following single-amino-acid substitutions: I129 to T (I129T), N143 to D (N143D), N202 to K (N202K), K254 to E (K254E), M325 to K (M325K), W345 to R (W345R), T368 to N (T368N), R420 to G (R420G), I699 to V (I699V), H736 to Y (H736Y), and A836 to V (A836V). A schematic of Env mutants is summarized in Fig. 1, and the full amino acid alignment of the Env mutants is outlined in Fig. S1 in the supplemental material. Although serum from monkey AY28 partially neutralized the SGA Env pseudovirus (Table 2), there was a >2-fold decrease in ID50 titer compared to that of WT Env pseudovirus neutralization by this serum, indicating that SGA Env is a partial escape virus. The SGA Env pseudovirus exhibited an increased sensitivity to serum from monkey 01D068 at 36 weeks p.c. This was an unexpected result that could be accounted for by host variability.

Table 2
Neutralizing antibody titers against mutant viruses
Fig 1
Summary of SIV mutant Envs. A schematic of the Env gp160 is shown with WT SIVmac251.30 on top. The WT sequences for specific residues are represented in black, while the site-directed mutations are shown in red. Amino acid deletions in the V1 and V4 regions ...

To narrow the list of potential NAb determinants of SIVmac251, we introduced gp120 mutations into the WT SIV Env backbone that are present in the SGA Env variant using site-directed mutagenesis (ΔV1.KEKRNG). This mutant pseudovirus did not contain the I699V, H736Y, and A836V mutations in the gp41 region. In addition, we constructed a pseudovirus with an Env that contained WT V1 and the gp41 region with the V2 to V5 mutations (WT.KEKRNG). The effects of these amino acid changes on the neutralization sensitivity of pseudoviruses were tested using the earliest sera from all 16 vaccinated and infected monkeys that contained NAbs against WT SIV Env. Similarly to SGA Env, the ΔV1.KEKRNG and WT.KEKRNG mutants were not neutralized by these sera (ID50 titers of <10 to 56). Although sera from monkey 01D068 was less efficient at neutralizing ΔV1.KEKRNG, the ID50 titer did not meet the preestablished minimum of a 2-fold decrease compared to the WT Env to be considered a true escape virus. These data suggest that the determinants recognized by NAbs early following infection were not in gp41 or the V1 region. Rather, they were in the V2 to V4 regions of Env (Table 2).

To further identify the Env mutations responsible for neutralization escape, specific amino acid substitutions in V4 were introduced into the WT SIV env. We were particularly interested in changes to the V4 region of Env, because in a previous study of a cohort of unvaccinated and mucosally infected monkeys, we found that all SIV sequence variations were also confined in the V4 region at 5 to 8 months postinfection (p.i.) when autologous neutralizing antibodies were first detected against the WT SIVmac Env (45). Although the N202K mutation in the V2 region of Env resulted in a loss of N-linked glycosylation, we did not specifically evaluate the effect of this mutation on sensitivity to NAbs in the present study, because it did not appear in the viral sequence evolution until much later in SIV infection.

Based on these data, we hypothesized that amino acid residues in V4 are the first targets of NAbs following SIV infection. To test this hypothesis, we constructed a panel of V4 Env mutants: R420G, R424Q, R420G+R424Q, R426Q, and ΔV4 (Fig. 1; also see Fig. S1 in the supplemental material). The ΔV4 Env mutant with amino acid deletions from positions 416 to 421 (VTTQRP) and R424Q and R426Q mutants were constructed, because these amino acid deletions/mutations were stereotypic in viral sequence evolution in several monkeys in this cohort of vaccinated monkeys (3) and in a separate cohort of unvaccinated monkeys that were mucosally infected with SIV (45). We predicted that if V4 mutations arose as a result of viral escape from neutralizing antibodies, sera from SIV-infected monkeys early following infection that were capable of neutralizing the WT Env pseudovirus would lack the ability to neutralize Env pseudoviruses encoding Envs with various V4 mutations.

Consistently with our prediction, changing R420 to G, R424 to Q, and R426 to Q each independently resulted in complete or partial escape from neutralization by the majority of sera that recognized WT Env (Table 2). Importantly, these mutations did not change the N-linked glycosylation sites in the V4 region. Although the differences in the neutralizing antibody ID50 titers between the WT and the R424Q and R426Q pseudoviruses were small due to the low titers of neutralizing antibodies in sera of monkeys AX93 and 05K, the decrease in ID50 titers suggests that these residues in Env were targeted early following SIV infection in these 2 monkeys. The R420G+R424Q and ΔV4 variant pseudoviruses behaved like NAb escape variants against half of the serum samples tested while retaining sensitivity to neutralization by sera from 2 monkeys that were not immunized with env (DA1H and AX93) and 3 monkeys that were immunized with env (AY24, AY74, and 05K) (Table 2). These data suggest that the mutation and deletion of multiple amino acid residues in V4 alter the sensitivities of these Env variants to NAbs. Overall, the data suggest that amino acid residues 420 and 424 in the SIV V4 region are determinants of early NAb recognition in SIV-infected monkeys.

NAbs detected early following SIV infection are not directed against the V1 region of Env.

In addition to changes in the V4 region of SIV Env at 5 to 8 months after mucosal infection, we previously observed that mutations in the V1 region of SIV Env are hotspots for SIV evolution and thus may represent sites of viral escape from antibody neutralization during chronic SIV infection (3, 45). Because V1 changes emerged around 16 months following infection, we hypothesized that mutations in V1 did not change the neutralization profile of serum samples obtained from monkeys at early time points following SIV infection, even if they represent escape viruses in later stages of infection. To test this hypothesis, we generated pseudoviruses with V1 env mutations, including deletions in residues 139 to 141 (ΔV1), ΔV1 with I129T (ΔV1.I129T), ΔV1 with N143D (ΔV1.I143D), and ΔV1 with I129T and N143D (ΔV1.I129T.I143D), all of which we have previously observed in cohorts of infected monkeys at approximately 16 months p.i. (3). As predicted, the mutations in V1 did not detectably alter recognition by the majority of serum NAb at early infection relative to the parental WT Env virus (ID50 titers of 67 to 500) (Table 2). Of the 16 serum samples examined for the presence of SIV Env-specific NAbs, all samples contained antibodies capable of neutralizing the various V1 mutants. An exception to this general finding was the ΔV1.I143D pseudovirus that was resistant or partially resistant to neutralization by sera from monkeys CF55, AD26, 26J, AY28, and AY24, suggesting the possible presence of NAbs that target these residues in this group of monkeys. Similarly, the ΔV1.I129T pseudovirus was resistant to neutralization by serum from monkey 26J, while the ΔV1.I129T.I143D pseudovirus was resistant to neutralization by serum from monkey AY28. These results indicate that early NAbs in the vaccinated and infected monkeys were, for the most part, not directed against the V1 region. However, it is likely that there was some variability in NAb production by different monkeys.

Since V1 mutations have previously been observed in combination with V4 mutations in SIV env sequences from monkeys 16 months p.i., we also constructed additional Env mutant pseudoviruses that contained both V1 and V4 mutations (ΔV1.ΔV4 with deletions in residues 139 to 141 and 416 to 421; ΔV1.R420G with deletions in residues 139 to 141 and R420G). Interestingly, V4 substitutions in combination with ΔV1 conferred more neutralization resistance to sera than was observed in pseudoviruses that contained only a ΔV1 mutation. Conversely, the addition of V1 mutations to R420G and ΔV4 mutations also altered the neutralization profiles compared to variants with only the V4 mutations. For example, the addition of ΔV1 to the R420G mutation rendered the pseudovirus more sensitive to neutralization than the R420G-only pseudovirus that was uniformly resistant to antibody neutralization. In contrast, the addition of the ΔV1 to ΔV4 mutations resulted in an increase in neutralization resistance compared to ΔV4 alone. These data suggest that the mutation and deletion of multiple amino acid residues in two separate variable regions of Env altered the sensitivities of these mutant pseudoviruses to NAbs, highlighting the potential importance of interactions between noncontiguous regions of the virus in the variable loop domains.

Env immunization did not alter the activity of early Env-specific antibody response.

To determine whether prior env vaccination affected the breadth of the neutralizing antibodies that developed following SIVmac infection, we determined the neutralization antibody titers against the viral mutants in sera of monkeys that were vaccinated with env or left unvaccinated. In the gag/pol/nef-vaccinated monkeys, sera from 6 monkeys that neutralized WT Env did not neutralize any of the V4 mutant pseudovirions. Sera from 3 gag/pol/nef/env-vaccinated monkeys did not recognize any of the V4 mutant pseudoviruses. There was no difference in the fold decrease in the average neutralizing ID50 titers of the 5 V4 mutant pseudoviruses relative to that of WT Env between the 2 groups of vaccinated monkeys (Table 3) (P = 0.38 by two-sided Mann-Whitney test). Similarly, all sera had detectable NAb titers against all V1 mutant pseudoviruses, and there was no difference in neutralization between the 2 groups. These data suggest that this env vaccination did not significantly alter the breadth of the anti-Env NAbs during early SIV infection.

Table 3
Fold decrease in neutralizing antibody titer of mutant viruses compared to the wild type


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, 4, 9, 20, 42). 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, 32, 45). 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, 22). Recent studies have demonstrated that glycan-dependent epitopes are frequently targeted by broad neutralizing antibody responses following HIV-1 infection (23, 43). 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, 44). 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.

Supplementary Material

Supplemental material:


We thank Rebecca Gelman (supported by Harvard CFAR P30 060354-07) for helpful discussions on statistics.

This work was supported by NIAID K08 AI069995 (W.W.Y.), the Shore Fellowship from Harvard Medical School (W.W.Y.), and the Pfizer Young Investigator Award in Vaccine Development from Infectious Disease Society of America (W.W.Y.); the intramural research program of the Vaccine Research Center, NIAID, NIH; NIH NIAID Center for HIV/AIDS Vaccine Immunology AI067854 (N.L.L.); and the Bill and Melinda Gates Collaboration for AIDS Vaccine Discovery Vaccine Immune Monitoring Consortium Grant 38619 (M.S.S.).

We declare no competing financial interests.


Published ahead of print 24 April 2012

Supplemental material for this article may be found at


1. Baba TW, et al. 2000. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat. Med. 6:200–206 [PubMed]
2. Babas T, et al. 1995. Specificity and neutralizing capacity of three monoclonal antibodies produced against the envelope glycoprotein of simian immunodeficiency virus isolate 251. Virology 211:339–344 [PubMed]
3. Basavapathruni A, et al. 2010. Envelope vaccination shapes viral envelope evolution following simian immunodeficiency virus infection in rhesus monkeys. J. Virol. 84:953–963 [PMC free article] [PubMed]
4. Burns DP, Collignon C, Desrosiers RC. 1993. Simian immunodeficiency virus mutants resistant to serum neutralization arise during persistent infection of rhesus monkeys. J. Virol. 67:4104–4113 [PMC free article] [PubMed]
5. Burns DP, Desrosiers RC. 1991. Selection of genetic variants of simian immunodeficiency virus in persistently infected rhesus monkeys. J. Virol. 65:1843–1854 [PMC free article] [PubMed]
6. Chackerian B, Rudensey LM, Overbaugh J. 1997. Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies. J. Virol. 71:7719–7727 [PMC free article] [PubMed]
7. Chakrabarti BK, et al. 2002. Modifications of the human immunodeficiency virus envelope glycoprotein enhance immunogenicity for genetic immunization. J. Virol. 76:5357–5368 [PMC free article] [PubMed]
8. Chen B, et al. 2005. Structure of an unliganded simian immunodeficiency virus gp120 core. Nature 433:834–841 [PubMed]
9. Choi WS, et al. 1994. Effects of natural sequence variation on recognition by monoclonal antibodies neutralize simian immunodeficiency virus infectivity. J. Virol. 68:5395–5402 [PMC free article] [PubMed]
10. Conley AJ, et al. 1996. The consequence of passive administration of an anti-human immunodeficiency virus type 1 neutralizing monoclonal antibody before challenge of chimpanzees with a primary virus isolate. J. Virol. 70:6751–6758 [PMC free article] [PubMed]
11. Emini EA, et al. 1992. Prevention of HIV-1 infection in chimpanzees by gp120 V3 domain-specific monoclonal antibody. Nature 355:728–730 [PubMed]
12. Hessell AJ, et al. 2009. Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques. Nat. Med. 15:951–954 [PubMed]
13. Hessell AJ, et al. 2009. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS Pathog. 5:e1000433 doi:10.1371/journal.ppat.1000433 [PMC free article] [PubMed]
14. Hessell AJ, et al. 2010. Broadly neutralizing monoclonal antibodies 2F5 and 4E10 directed against the human immunodeficiency virus type 1 gp41 membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus SHIVBa-L. J. Virol. 84:1302–1313 [PMC free article] [PubMed]
15. Javaherian K, et al. 1992. The principal neutralization determinant of simian immunodeficiency virus differs from that of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. U. S. A. 89:1418–1422 [PubMed]
16. Johnson WE, et al. 2002. A replication-competent, neutralization-sensitive variant of simian immunodeficiency virus lacking 100 amino acids of envelope. J. Virol. 76:2075–2086 [PMC free article] [PubMed]
17. Johnson WE, et al. 2003. Assorted mutations in the envelope gene of simian immunodeficiency virus lead to loss of neutralization resistance against antibodies representing a broad spectrum of specificities. J. Virol. 77:9993–10003 [PMC free article] [PubMed]
18. Jurkiewicz E, et al. 1997. Identification of the V1 region as a linear neutralizing epitope of the simian immunodeficiency virus SIVmac envelope glycoprotein. J. Virol. 71:9475–9481 [PMC free article] [PubMed]
19. Keele BF, et al. 2009. Low-dose rectal inoculation of rhesus macaques by SIVsmE660 or SIVmac251 recapitulates human mucosal infection by HIV-1. J. Exp. Med. 206:1117–1134 [PMC free article] [PubMed]
20. Kinsey NE, et al. 1996. Antigenic variation of SIV: mutations in V4 alter the neutralization profile. Virology 221:14–21 [PubMed]
21. Kwong PD, et al. 2000. Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates. Structure 8:1329–1339 [PubMed]
22. Kwong PD, et al. 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648–659 [PubMed]
23. Lavine CL, et al. 2011. High-mannose glycan-dependent epitopes are frequently targeted in broad neutralizing antibody responses during infection of human immunodeficiency virus type 1. J. Virol. 86:2153–2164 [PMC free article] [PubMed]
24. Letvin NL, et al. 2006. Preserved CD4+ central memory T cells and survival in vaccinated SIV-challenged monkeys. Science 312:1530–1533 [PMC free article] [PubMed]
25. Letvin NL, et al. 2011. Immune and genetic correlates of vaccine protection against mucosal infection by SIV in monkeys. Sci. Transl. Med. 3:81ra36 [PubMed]
26. Li Y, et al. 2008. Removal of a single N-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses. J. Virol. 82:638–651 [PMC free article] [PubMed]
27. Mascola JR, et al. 1999. Protection of macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J. Virol. 73:4009–4018 [PMC free article] [PubMed]
28. Mascola JR, et al. 2000. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat. Med. 6:207–210 [PubMed]
29. Moore PL, et al. 2008. The c3-v4 region is a major target of autologous neutralizing antibodies in human immunodeficiency virus type 1 subtype C infection. J. Virol. 82:1860–1869 [PMC free article] [PubMed]
30. National Research Council 1996. Guide for the care and use of laboratory animals. National Academy Press, Washington, DC
31. Overbaugh J, Rudensey LM. 1992. Alterations in potential sites for glycosylation predominate during evolution of the simian immunodeficiency virus envelope gene in macaques. J. Virol. 66:5937–5948 [PMC free article] [PubMed]
32. Overbaugh J, Rudensey LM, Papenhausen MD, Benveniste RE, Morton WR. 1991. Variation in simian immunodeficiency virus env is confined to V1 and V4 during progression to simian AIDS. J. Virol. 65:7025–7031 [PMC free article] [PubMed]
33. Parren PW, et al. 2001. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. J. Virol. 75:8340–8347 [PMC free article] [PubMed]
34. Petry H, Pekrun K, Hunsmann G, Jurkiewicz E, Luke W. 2000. Naturally occurring V1-env region variants mediate simian immunodeficiency virus SIVmac escape from high-titer neutralizing antibodies induced by a protective subunit vaccine. J. Virol. 74:11145–11152 [PMC free article] [PubMed]
35. Prince AM, et al. 1991. Prevention of HIV infection by passive immunization with HIV immunoglobulin. AIDS Res. Hum. Retrovir. 7:971–973 [PubMed]
36. Ren X, Sodroski J, Yang X. 2005. An unrelated monoclonal antibody neutralizes human immunodeficiency virus type 1 by binding to an artificial epitope engineered in a functionally neutral region of the viral envelope glycoproteins. J. Virol. 79:5616–5624 [PMC free article] [PubMed]
37. Rerks-Ngarm S, et al. 2009. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N. Engl. J. Med. 361:2209–2220 [PubMed]
38. Rudensey LM, Kimata JT, Long EM, Chackerian B, Overbaugh J. 1998. Changes in the extracellular envelope glycoprotein of variants that evolve during the course of simian immunodeficiency virus SIVMne infection affect neutralizing antibody recognition, syncytium formation, and macrophage tropism but not replication, cytopathicity, or CCR-5 coreceptor recognition. J. Virol. 72:209–217 [PMC free article] [PubMed]
39. Rybarczyk BJ, et al. 2004. Correlation between env V1/V2 region diversification and neutralizing antibodies during primary infection by simian immunodeficiency virus sm in rhesus macaques. J. Virol. 78:3561–3571 [PMC free article] [PubMed]
40. Sato S, et al. 2008. Potent antibody-mediated neutralization and evolution of antigenic escape variants of simian immunodeficiency virus strain SIVmac239 in vivo. J. Virol. 82:9739–9752 [PMC free article] [PubMed]
41. Shibata R, et al. 1999. Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys. Nat. Med. 5:204–210 [PubMed]
42. Torres JV, et al. 1993. An epitope on the surface envelope glycoprotein (gp130) of simian immunodeficiency virus (SIVmac) involved in viral neutralization and T cell activation. AIDS Res. Hum. Retrovir. 9:423–430 [PubMed]
43. Walker LM, et al. 2011. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477:466–470 [PMC free article] [PubMed]
44. Wei X, et al. 2003. Antibody neutralization and escape by HIV-1. Nature 422:307–312 [PubMed]
45. Yeh WW, et al. 2010. Autologous neutralizing antibodies to the transmitted/founder viruses emerge late after simian immunodeficiency virus SIVmac251 infection of rhesus monkeys. J. Virol. 84:6018–6032 [PMC free article] [PubMed]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)