In this study, we have clearly demonstrated that anti-MPER antibodies in three broadly cross-neutralizing plasmas were largely responsible for the heterologous neutralization displayed by these samples. For most viruses, the bulk of the neutralizing activity could be attributed to this single antibody specificity. Furthermore, our data suggested that these antibodies were as potent as existing MAbs and defined novel epitopes within the MPER. These data reinforce the potential of the HIV-1 gp41 MPER as a neutralizing-antibody vaccine target.
We previously showed a significant association between the presence of anti-MPER antibodies and neutralization breadth in plasma samples from a cohort of chronically infected blood donors (12
). We can now confirm that, at least in some cases, anti-MPER antibodies are primarily responsible for this neutralizing activity. The levels of breadth displayed by these three HIV-1 subtype C plasma samples varied, with BB34 being the broadest and CAP206 and SAC21 neutralizing about half the viruses tested. Of those viruses neutralized by BB34 and CAP206, approximately 70% were neutralized via anti-MPER antibodies, and in the majority of cases, these antibodies mediated almost all the activity. The anti-MPER antibodies in SAC21 neutralized fewer viruses, and often they only partially contributed to the overall neutralization, probably due to smaller amounts of specific IgG in the sample. For all three plasmas, there were examples where the adsorption of anti-MPER antibodies did not remove all the neutralizing activity or in some cases had no effect. The latter suggests that other specificities distinct from the adsorbed anti-MPER antibodies were also present in these plasmas. The residual neutralization of C1C by depleted CAP206 and SAC21 plasmas suggested that in some cases they may also be MPER antibodies that failed to bind the linear peptide. This is in line with the observations by others that more than one specificity may be involved in the neutralization breadth displayed by plasmas from some HIV-1-infected individuals (3
Testing of the antibodies eluted from the MPER peptide allowed us to conclusively show that these antibodies mediated cross-neutralization. The potency of the eluted antibodies recapitulated the activity in the original plasma samples, although the IC50 and ID50 values did not always correlate. This may be due to other non-MPER neutralizing antibodies present in these samples, as described above, or perhaps loss of activity during the elution process. Eluates are likely to contain mixtures of MPER-specific antibodies that may differ in binding affinity, as well as neutralization capacity, and thus represent considerably more of a technical challenge than testing purified MAbs. Even if the elution data are more qualitative than quantitative, they nevertheless show that the potencies of these antibodies are in the range of the current MAbs. Interestingly, the CAP206 eluate efficiently neutralized the autologous virus, despite the fact that no significant reduction in the ID50 was observed after depletion of anti-MPER antibodies from the plasma sample (Table ). It is possible that other autologous neutralizing-antibody specificities overshadowed the activities of the anti-MPER antibodies in this plasma sample.
The neutralizing anti-MPER antibodies in plasma BB34 were found to be mainly IgG3. It is interesting that the original hybridoma-derived broadly neutralizing anti-MPER MAbs 4E10 and 2F5 were of the IgG3 subclass (14
) and the neutralizing fraction of a polyclonal human HIV immune globulin was also reported to be IgG3 (29
). IgG3s have a highly flexible hinge region that has been proposed to facilitate access to the MPER and that is thought to be partly buried in the viral membrane and enclosed by the gp120 protomers. However, for both MAbs, a change to IgG1 did not affect the neutralization capacity, suggesting that IgG3s are not essential for MPER-mediated neutralization (14
). Indeed, for CAP206, the IgG3-enriched fraction had less activity, and in this case, neutralization was due to either IgG1 or IgG2. While there was an enrichment of IgG3 in SAC21 eluates, the low potency of these antibodies precluded them from being tested further. Both BB34 and SAC21 were from blood donors with an unknown duration of infection, while CAP206 has been followed prospectively for 3 years since seroconversion. Although IgG3 has been reported to appear early in infection, we will continue to monitor the anti-MPER response in CAP206 to see if the IgG subclass profile, antibody specificities, or neutralization titers change over time.
The binding of all three anti-MPER plasma antibodies depended on the residue at position 674 in the MPER, which has been shown to be the most critical for Z13e1 recognition (21
). The immunogenicity of this residue may be related to its location in the hinge region of the MPER (21
). However, the high level of polymorphism at this position is considered to be one of the main reasons why the Z13e1 MAb neutralizes a narrower set of viruses than the 4E10 MAb. In contrast to MAb 2F5, which seldom neutralizes subtype C viruses due to a subtype-associated polymorphism at position 665 (4
), the residue at position 674 is not associated with a particular subtype. This is consistent with our finding that subtype B and C viruses were equally neutralized by MPER antibodies present in all three plasmas. In addition to this common residue, BB34 and SAC21 also depended on W670, which is not implicated in either 4E10 or Z13e1 recognition. SAC21 showed some overlap with the 4E10 MAb, since it was affected by the F673A mutation. However, the identities of the precise residues required by these antibodies indicated that they are distinct from 4E10 and Z13e1. Furthermore, analysis of the MPER sequences of the viruses neutralized by these plasmas suggested that the residue at position 674 affects their sensitivity, with the majority of viruses harboring a serine showing resistance (see Fig. S1 in the supplemental material). However, not all viruses with an aspartic or asparagine residue at position 674 and, even more, with the same MPER sequence were neutralized equally, suggesting that features outside this region may modulate the presentation of this epitope, as suggested by previous studies (4
The presence of anti-MPER antibodies in broadly cross-neutralizing subtype B plasmas has been reported recently by others. Li and colleagues found that neutralization of the JR-FL virus by plasma no. 20 was outcompeted by a peptide covering the 4E10 epitope, although the extent of the contribution of this specificity to breadth was not determined (16
). Sather and coworkers found 4E10-like activity in plasma VC10008 (28
); however, this sample did not neutralize some 4E10-sensitive viruses, suggesting differences in their specificities. Neither of these studies investigated the precise epitopes recognized by these potentially novel antibodies, so it is not possible to determine if they differ from the ones identified here. A third study described an individual who developed antibodies that recognized a region overlapping the 2F5 epitope (30
). Anti-MPER affinity-purified antibodies from this individual, SC44, displayed broad neutralizing activity. Similar to our study, which identified three samples from among 156 chronically infected individuals, the 2F5-like antibody found by Shen and colleagues was 1 of 311 plasmas analyzed (30
The scarcity of these samples supports the notion that broadly neutralizing anti-MPER antibodies are seldom developed by HIV-1-infected individuals. Haynes et al. proposed that such antibodies are autoreactive and therefore eliminated through B-cell tolerance mechanisms (13
). While CAP206 did not have detectable levels of autoreactive antibodies, BB34 was positive for anti-double-stranded DNA antibodies and rheumatoid factor (12
). Another explanation for the paucity of such antibodies may be the short exposure time of this epitope during the formation of the fusion intermediate (8
). Consistent with this, MAbs 2F5, 4E10, and Z13e1, as well as plasma BB34, neutralize JR-FL after CD4 and CCR5 attachment, when this occluded epitope is exposed (2
). Furthermore, BB34 neutralization was potentiated by coexpression of FcγRI on JC53bl-13 cells, also a feature of 2F5 and 4E10, possibly by providing a kinetic advantage through prepositioning of these antibodies close to the MPER (22
). However, it remains unclear how these antibodies are induced in the context of natural infection despite the exposure constraints of this epitope. Perhaps these antibodies are elicited by more open conformations of the envelope glycoprotein that expose the MPER. Analysis of the autologous viruses that induce such responses may help to answer these questions.
It is noteworthy that the three cross-neutralizing antibodies identified here, while sharing some common residues, had distinct fine specificities. This suggests that the MPER can be recognized in a variety of conformations by the human immune system. It is therefore critical to isolate MAbs that define these novel epitopes within the MPER in order to facilitate a better understanding of the immunogenic structure of this region of gp41 and to identify new targets for HIV vaccine design.