We previously reported the neutralization activities of 32 HIV-1-positive sera against a large panel of virus isolates from clade A (n
= 11), clade B (n
= 24), clade C (n
= 11), and clade E (n
= 1). Five sera, designated as sera 1, 18, 20, 30, and 45 neutralized most viruses tested and were designated as broadly neutralizing. Two sera (sera 1 and 45) were particularly potent and were previously studied (27
). Six sera with weak neutralization and limited breadth were also identified. The potency of the broadly neutralizing sera was confirmed by determining the serum ID50
titer against a selected panel of virus isolates (see Fig. S2A and B in the supplemental material). We subsequently identified three additional broadly neutralizing sera (B7B5, US12, and US21), all derived from SP individuals. These three sera, along with sera 18 and 20, were analyzed with an expanded panel of adsorption and peptide competition reagents. This included single amino acid mutant gp120 reagents to identify antibodies directed against the CD4bs and the coreceptor binding region, as well as peptides designed to detect antibodies with specificities similar to MAbs 2F5 and 4E10. In these analyses, we occasionally utilized the previously characterized sera 1 and 45 as positive controls. Where appropriate, selected sera that were relatively limited in neutralization breadth were used to compare and contrast the antibody specificities that mediated virus neutralization relative to the more broadly neutralizing sera.
Cross-clade neutralization is gp120 directed.
We initially analyzed whether the neutralizing activity of the broadly neutralizing sera was directed against known determinants present on native gp120 (see Fig. S1 in the supplemental material). This analysis involves adsorption of the sera with native, solid-phase-coupled gp120, followed by a comparison of the binding and neutralizing properties of the adsorbed sera, as well as analysis of the gp120-eluted antibodies (27
) (Fig. ). The coupling efficiency of gp120 to the beads was determined to be between 70 and 100% by SDS-PAGE analysis (see Fig. S3A in the supplemental material). We next confirmed the integrity of the gp120WT coupled to the beads by a FACS-based binding analysis using a panel of ligands. The panel of ligands included the conformational, anti-gp120 monoclonal antibodies b12, 2G12, and 447-52D, the CD4-induced antibody 17b, and the conformational ligand CD4 (see Fig. S3B in the supplemental material) (not shown). We further validated the specificity of the gp120WT beads by demonstrating their ability to adsorb the binding activity of the 2G12 and 447-52D antibodies and to adsorb the neutralizing capacity of the CD4bs antibody (b12) (see Fig. S3C in the supplemental material).
We performed adsorptions of sera using the gp120-coupled beads and verified that the flowthrough contained nearly undetectable levels of gp120-specific antibodies by ELISA, indicating that the absorbed sera were fully depleted of gp120-binding antibodies (see Fig. S4B in the supplemental material). For specificity controls, adsorption of BSA-conjugated beads and blank beads was performed in parallel, and the beads showed only minor effects on serum neutralization capacity compared to untreated serum (Table ) (see Fig. S3D in the supplemental material). In contrast, we observed a significant decrease in the neutralizing activity of the gp120 flowthrough (gp120ft) fraction for several of the broadly neutralizing sera. Table shows serum neutralization ID50 titers after adsorption with gp120WT-coupled beads or negative-control beads, including BSA-coupled beads or blank beads. The proportion of gp120-specific neutralization against a particular virus isolate was determined by the percent reduction in ID50 by gp120WT relative to blank beads (when available) or BSA-coupled beads. For instance, the flowthrough antibody fraction for serum B7B5 had reciprocal ID50 titers against JRFL of 2,281 (blank beads) and 240 (gp120WT beads), respectively. Thus, 89% [(2,281 − 240)/2,281] of JRFL neutralization was reduced by gp120WT bead adsorption and can be termed gp120-directed neutralization. For all seven broadly neutralizing sera, the majority of neutralization of most viruses tested was gp120-directed neutralization. Furthermore, since a single clade B monomeric gp120 protein (derived from YU2) removed the neutralizing activity against representative clade A and C isolates (RW020 and ZA12), the data suggested that the gp120-specific activity was directed against well-conserved surfaces of the clade B HIV-1 gp120 protein. Accordingly, we then turned to mapping of the neutralizing specificity to the most conserved and exposed regions on gp120, either the CD4 binding site or the coreceptor binding region.
Pre- and postadsorption neutralization ID50 values for 10 sera with various neutralization breadths and potencies with wild-type or CD4 binding site mutant D368R gp120-coupled beads
Mapping neutralizing specificity to the CD4 binding site.
We screened for the presence of CD4bs-directed neutralization activity using the well-defined gp120 point mutation D368R (32
). This mutation eliminates the binding of both CD4 and the CD4bs antibodies, such as b12 and F105 (27
). We previously confirmed that the critical gp120D368R
point mutant protein coupled to the Dynabeads was well recognized by the gp120-specific antibodies 2G12 and 447-52D, but not by the CD4bs neutralizing antibody, b12 (27
). To further confirm the specificity of this analysis, we demonstrated that the gp120D368R
bead could adsorb all binding activity of the broadly neutralizing antibody (2G12), the CD4-induced (CD4i) antibody (17b) (see Fig. S4A in the supplemental material), and the V3 antibody (447-52D) (data not shown), but not the b12 antibody (see Fig. S4A in the supplemental material).
We extended the differential solid-phase adsorption to the five broadly neutralizing sera 18, 20, US12, US21, and B7B5 (Fig. ). We also included one weakly neutralizing serum (serum sample 19) in the analysis. After adsorption with gp120WT and gp120D368R beads in parallel, serum antibody binding was tested both for binding to gp120WT and gp120D368R and for virus neutralization capacity. As expected, an ELISA confirmed that binding to the homologous protein, either gp120WT or gp120D368R, was undetectable after adsorption of each of the serum samples. However, for some sera, a significant level of gp120WT binding remained after the gp120D368R bead adsorption (see Fig. S4B in the supplemental material), suggesting that a fraction of the binding antibodies are CD4bs directed. This is consistent with our previous data regarding sera 1 and 45. In fact, such a differential binding assay can be used to detect the presence of CD4bs antibodies in polyclonal sera.
To test whether this subset of antibodies mediates virus neutralization, neutralization assays against clade B and non-clade B viruses were performed. We observed a range of CD4bs-directed neutralization potency profiles (Fig. 2A and summarized in Table ). CD4bs-directed neutralizing activity was assessed by comparing the serum ID50 titer after gp120WT and gp120D368R mutant bead adsorption relative to blank beads or BSA beads, as summarized in Table . Similar to the calculation of the proportion of gp120-directed neutralizing activity, the gp120D368R-directed (non-CD4bs-directed) neutralizing proportion was calculated as percent reduction in ID50 by gp120D368R relative to blank beads (when available) or BSA-coupled beads. Then, the portion of CD4bs-directed neutralizing activity in the total gp120-directed neutralization was calculated as [(percentage of gp120WT-directed neutralizing activity − percentage of gp120D368R-directed neutralizing activity)/percentage of gp120-directed neutralizing activity]. For example, when tested against virus JRFL, serum B7B5 has 89% gp120WT-directed neutralizing activity [(2,281 − 240)/2,281] and 25% gp120D368R-directed neutralizing activity [(2,281 − 1,719)/2,281]. Therefore, approximately 72% of the B7B5 gp120-directed neutralizing activity is focused on CD4bs [89% − 25%)/89%].
The B7B5 serum was the most broadly neutralizing of the newly assessed sera and also displayed the most predominantly CD4bs-focused neutralization. The large majority of B7B5 neutralizing activity was gp120-directed activity, and depending on the virus, most of the gp120 neutralization was specific for the CD4bs (Fig. and Table ). The lower fraction of B7B5 CD4bs-directed neutralization for HXBc2 and SF162 may be related to the fact that these two viruses are sensitive to many other gp120 antibody specificities in sera. Serum 19, US12, and US21 contained CD4bs-directed neutralizing antibodies, but this fraction of antibodies neutralized only the very sensitive HXBc2 virus, suggesting a limited potency for the CD4bs antibody subset against more resistant primary viruses (Table ). Neutralization by serum 20 was largely gp120 directed (except against JRFL), but little of this gp120-directed neutralization was CD4bs directed. Of note, these types of serum fractionation experiments involve several steps, and we consider the values in Table to be general estimates of the proportion of antibody specificities rather than hard and fast numbers. Also, when the proportion of gp120-directed neutralization was less than 50% (such as for serum 20 against JRFL), further calculations of the proportion of gp120 activity are likely to be of limited accuracy. Hence, in this example, serum sample 20 had 34% gp120-directed activity against JRFL; we would not suggest that the value of 24% CD4bs-directed activity is highly accurate. In contrast, serum B7B5 demonstrated 89% gp120-directed neutralization of JRFL, and of this fraction, 72% was CD4bs directed. Here, we can be more confident that 65% (i.e., 72% of 89%) of the total neutralizing activity of this serum is CD4bs directed.
CD4bs-directed neutralization is mediated by immunoglobulin.
To further characterize the CD4bs-directed neutralizing specificity in sera, we eluted the absorbed gp120-specific binding activity from the gp120WT protein. This gp120-directed IgG was characterized as pure immunoglobulin by SDS-PAGE (data not shown), and the IgG concentration was determined by radial immunodiffusion. The binding and neutralization activity was then studied as depicted schematically in Fig. . We used serum 1 as a positive control and focused on the most potent and broadly neutralizing of the five newly analyzed sera, B7B5, for this analysis.
To further enrich for the CD4bs-directed antibodies, the gp120-eluted IgG from serum B7B5 was subjected to a second adsorption with the gp120D368R-conjugated beads. This removed gp120-directed antibodies, except those directed against the CD4bs region of gp120 which would remain in the column flowthrough (Fig. ). The CD4bs-enriched IgG fraction was tested for its binding specificity by a competition ELISA using gp120 as the capture antigen in the absence and presence of soluble CD4 as a competitor. The CD4bs-enriched IgG fraction of B7B5 serum specifically competed with sCD4 in a manner similar to that of the CD4bs antibody, b12 (Fig. ). For negative controls, the MAbs 2G12 and 447-52D, which are not directed against the CD4 binding region, did not compete with sCD4 for binding to gp120. The CD4bs antibodies fractionated from the B7B5 serum were then tested for neutralization activity against selected HIV-1 primary isolates to confirm that the elute/enriched IgG can recapitulate the potent and broad neutralization of the serum sample. A severalfold increase in neutralizing potency could be observed in stepwise progression from the initial gp120 eluate IgG fraction to the CD4bs-enriched fraction of antibodies (Fig. , bottom right).
The conserved gp120 coreceptor binding site and neutralization specificity.
The chemokine receptor binding site of HIV-1 is highly conserved, but most known antibodies directed against this region are unable to access their epitopes on the HIV-1 functional spike due to steric or conformational constraints (6
). Consequently, the known coreceptor site MAbs do not generally neutralize primary HIV-1 isolates (8
). However, the possibility exists that broadly neutralizing sera contain antibodies against this conserved region that are capable of neutralization. Therefore, we applied the selective adsorption approach using an I-to-R mutation at position 420. This residue is located in the bridging sheet region of the coreceptor binding site (see Fig. S1 in the supplemental material), and the I-to-R nonconservative mutation eliminates the binding of most known antibodies directed at the CD4i coreceptor binding sites without affecting most other binding specificities (37
). We confirmed these previous observations by performing binding analysis of the gp120I420R
by both ELISA and FACS. By ELISA, the I420R mutation in the BaL gp120 context was recognized at levels equivalent to the levels of gp120WT recognized by MAbs b12, 2G12, and 447-52D, whereas coreceptor binding region MAbs, such as 17b, 48d, E51, and 2.8B, did not recognize the gp120I420R
mutant protein (see Fig. S5 in the supplemental material). We observed a similar selective elimination of MAb 17b binding by FACS analysis of the solid-phase paramagnetic beads coupled to either gp120WT or the gp120I420R
mutant proteins. For controls, recognition by the conformational antibodies b12 and 2G12 to both proteins after coupling was unaffected (Fig. ). We confirmed further the selective specificity of the gp120I420R
beads by demonstrating that this solid-phase mutant could adsorb all the gp120-specific binding activity of the broadly neutralizing antibodies, b12 and 2G12, but not the coreceptor binding site antibody, 17b (Fig. ).
FIG. 3. The coreceptor binding region neutralizing antibody specificity of serum analyzed by differential adsorption. (A) Flow cytometric analysis of gp120WT, coreceptor binding region mutant gp120I420R, and CD4bs mutant gp120D368R proteins following the coupling (more ...)
We then performed differential adsorption of the B7B5 serum with either gp120WT or gp120I420R
and observed that the gp120WT adsorption eliminated virtually all binding to both the gp120WT and I420R mutant protein (Fig. , left). However, gp120I420R
adsorption eliminated binding to the I420R mutant protein, but substantial binding to the gp120WT protein was retained. This indicated that a fraction of the gp120-specific antibodies in this serum was directed against the coreceptor binding region (Fig. , left). To confirm this finding of antibodies directed against the coreceptor binding site, we performed neutralization assays with an HIV-2 strain (7312A_V434M), which is an assay specific for the detection of HIV-1 coreceptor binding site-directed antibodies (8
). Serum B7B5 was absorbed with gp120WT and gp120I420R
, and the flowthrough fractions were tested for neutralization of HIV-2. As expected, gp120WT, but not gp120I420R
, could effectively adsorb the HIV-2 neutralization (Fig. , right).
By this selective adsorption analysis, we found two sera that appear to contain coreceptor binding site-directed neutralization activity (sera 45 and B7B5). In both cases, the gp120WT protein more efficiently adsorbed serum neutralization than the gp120I420R mutant did (Fig. ). The relative fraction of neutralizing antibodies potentially directed against the coreceptor site was calculated as described above for the CD4bs-directed activity, and values for sera 45 and B7B5 against seven isolates are shown (Table ). The potential existence of coreceptor-directed antibodies is an intriguing result that has implications for immunogen design and awaits confirmation by the isolation of neutralizing monoclonal antibodies displaying such specificity. A caveat to this analysis is that a novel neutralizing antibody that is sensitive to both of the D368R and I420R mutations in gp120 could exist. Such an antibody would be neither a classical CD4bs or coreceptor region-specific neutralizing antibody as currently defined.
Pre- and postadsorption neutralization ID50 values for two broadly neutralizing sera with wild-type (WT) or coreceptor binding region (CD4i) mutant gp120 I420R-coupled beads
This possibility is highlighted by inspection of the percentage of gp120-directed neutralization for both the CD4bs and the coreceptor region from serum 45 for the RW020 isolate and for serum B7B5 for the PVO and ZA012 isolates. In both cases, the percentage of gp120-directed neutralization greatly exceeds 100%, which could be explained by a single neutralization specificity sensitive to both mutations (see Table compared to Table for these isolates). Nevertheless, our results highlight the possibility that novel neutralizing specificities directed toward HIV-1 Env may exist in some sera.
gp120 peptide inhibition of neutralization assays specific for the major variable loops.
We further sought to determine whether the neutralizing specificity in the sera might be directed toward the major variable regions of gp120. Due to the immunodominant nature of the V3 region and because of the large variety of available V3-specific antibodies to use as controls, we initially validated and optimized our ability to detect V3-directed neutralization. Although V3-specific antibodies generally do not neutralize the majority of primary isolates, some viruses that are sensitive to V3-directed antibodies exist. Accordingly, peptides derived from the V3 region of BaL.01 were synthesized, and their ability to inhibit neutralization mediated by a panel of seven V3-directed monoclonal antibodies was analyzed. The neutralization of the V3-directed MAbs 2182, 2191, 2219, 2442, 2456, and 1.3D and the conformational V3 antibody 447-52D against primary isolate BaL.01 was efficiently and specifically inhibited by incubation with the V3 peptide prior to incubation of each antibody with virus (data not shown). V3 peptide inhibition of the V3-directed neutralizing monoclonal antibody panel was confirmed using two other primary isolates, SF162 and 6535.3, in similar experiments (data not shown).
We next tested whether there was V3-directed neutralizing activity in selected patient sera. We found no significant effect of V3 peptides for any of the potent and broadly neutralizing sera tested (Table ). However, the neutralization activity of several weakly/narrowly neutralizing sera was appreciably inhibited by preincubation with the V3 peptide, demonstrating V3-directed neutralization in this type of sera (Table ).
We also performed a similar analysis using peptides derived from the V1 and V2 regions of the YU2 isolate (28
). When V1 and V2 peptides were tested in the peptide inhibition of neutralization assay, none of the patient sera were affected by any of the V1- or V2-derived peptides, despite the fact that selected sera have positive binding to pools of the V1 and V2 peptides (data not shown). The caveat here is that we lacked positive-control V1 or V2 neutralizing antibodies to YU2, and it is possible that linear peptides may not inhibit neutralization by an antibody to a nonlinear epitope on V1 or V2.
Analysis of potential gp41-directed neutralization.
The gp41 MPER contains two well-characterized linear, conserved epitope clusters recognized by the broadly neutralizing MAbs 2F5 and 4E10 (see Fig. S1 in the supplemental material). Because the MPER is the only major neutralizing epitope defined on gp41 and because the structure of the linear epitopes and their cognate antibody for MAbs 2F5 and 4E10 are well defined, we focused on analyzing the MPER to determine whether antibodies to this region were mediating broad neutralization. Many of the sera tested had detectable levels of antibodies binding to MPER-containing peptides (see Fig. S6 in the supplemental material) or to the MPER arrayed on hepatitis B virus surface antigen particles (data not shown).
To assess whether the gp41 and MPER-specific binding antibodies were neutralizing, we used a chimeric HIV-2 virus that contains the complete 25-amino-acid sequence of the HIV-1 YU2 gp41 MPER (15
) (Fig. ). We verified that this chimeric HIV-2/MPER virus is highly sensitive to neutralization by both MAbs 2F5 and 4E10 (IC50
of <0.2 μg/ml for each). No neutralization of the HIV-2 chimeric virus was accomplished by the two most broadly neutralizing control sera, serum 1 or 45 (Fig. ), consistent with the relatively low levels of MPER-specific binding activity detected in these sera (see Fig. S6 in the supplemental material). Interestingly, sera derived from patients 6, 15, 19, 20, 30, and B7B5 demonstrated a range of neutralizing titers against the HIV-2/MPER chimeric virus (Fig. and Table ). We then sought to confirm whether antibodies to the MPER were mediating neutralization of HIV-1.
FIG. 4. Neutralization assays to detect antibodies specific for the membrane-proximal region. (A) The MPER sequence of HIV-2 and HIV-2/HIV-1 MPER chimeric virus are shown as well as peptides derived from the HIV-1 MPER that overlap with the 2F5 (purple) and 4E10 (more ...)
Serum ID50 titers in the absence and presence of MPER-derived peptides
We generated soluble MPER-derived peptides overlapping the core epitopes of MAbs 2F5 and 4E10 and that specifically inhibited the virus neutralization (Fig. ). Of note, the 4E10 peptide is hydrophobic and can affect entry of some viruses. Hence, the peptide alone was tested in all assays and used to control for any effect on viral entry. Sera were tested against HXBc2, SF162, and JRFL, using the 2F5 and 4E10 peptides, along with mock and irrelevant peptide controls. For serum sample 20 neutralization of HXBc2, we observed an approximately twofold decrease of the serum ID50 titer in the presence of the 4E10 peptide. Since gp120-directed neutralization may mask the contribution of MPER neutralizing antibodies, selected serum samples were adsorbed with gp120WT protein to remove gp120-directed neutralizing antibodies. We first confirmed that the gp120ft fraction from two sera, 19 and 20, could still neutralize the sensitive HIV-1 isolate HXBc2, whereas the gp120ft fraction from two other sera, 6 and 30, completely lost the capacity to neutralize HXBc2 (Table ).
We then used the MPER-containing peptides to test whether the remaining non-gp120 was targeting the gp41 MPER region. Neutralization of HXBc2 by the gp120ft fractions of sera 19 and 20 was completely eliminated in the presence of 4E10 epitope peptide but was not affected by 2F5 epitope peptide (Fig. ). Similar results were observed for these two serum samples when tested with virus isolate SF162 (Table ). Furthermore, we demonstrated that the 4E10 epitope-directed antibodies in the potent serum 20 were contributing to neutralization of the relatively resistant primary virus isolate JRFL (Fig. ). Since we estimated that about half (45%) of the serum sample 20 neutralization of JRFL was gp120 directed (Table ), the data here suggest that the remaining 50% of JRFL neutralization is directed against the MPER and specifically to the 4E10 epitope.