Despite multiple attempts, no immunogen has successfully elicited high-titer bNt Abs against the MPER. Importantly, some success has been recently achieved by Ye et al. (60
), who produced a chimeric immunogen comprising the influenza virus HA1 domain fused to the full sequence of HIV-1 gp41, including its TM and CT. Immunization of guinea pigs with this fusion protein, in the form of a virus-like particle (VLP) or expressed by a DNA vaccine, elicited low-titer (1:50) Nt activity against Tier-2 viruses and a simian immunodeficiency virus-HIV-MPER (SIV-HIV-MPER) chimeric virus; importantly, this Nt activity was completely blocked by preincubation with a peptide bearing the MPER sequence. These results strengthen the hypothesis that the MPER must be tethered by the TM and presented in the context of the PM to fully form its NCS. The work of Ye et al. builds on earlier studies which showed the affinities of MAbs 2F5 and 4E10 for their epitopes on a gp41 fragment, comprising the MPER and TM and part of the CT, were increased when associated with lipid (16
). Their results, taken together with the results reported here, suggest that conclusions from NMR studies of synthetic MPER peptides and their interaction with lipid micelles (46
), which showed the residues critical for 4E10 MAb binding to be buried in a lipid micelle, can be explained by the absence of the TM (and possibly the CT) in these peptides. We offer a model based upon biochemical evidence to explain this discrepancy; the NCS of the MPER, and especially its 4E10 epitope region, depends upon both a lipid environment and the HIV-1 gp41 TM, with each contributing to its conformation.
The majority of previous work analyzing the MPER and the bNt MAbs that recognize it was performed with synthetic peptides whose sequence is limited to the MPER or recombinant, soluble proteins (reviewed in reference 33
). We show here that the TM plays an important role in epitope presentation when the MPER is displayed in the context of the PM ( and ) and provide molecular models to explain the difference in exposure of the 4E10 epitope observed between MPER anchored by the PDGFR TM versus the gp41 TM. We speculate that the extended external region of the PDGFR-TM allows the MPER C terminus freedom to move in the PM, resulting in the burial of core residues in the 4E10 epitope (A); this was confirmed by the results obtained using this external region as a linker between the MPER and gp41 TM in the MPER-PS-TM1 construct (A). While it may have affected the structure of the MPER directly, a relatively hydrophilic, flexible linker is not expected to strongly affect the hydrophobic 4E10 epitope. Instead, it is more likely that the conformational constraints imposed by the adjacent gp41 TM directly affect exposure of the 4E10 epitope ().Our analysis of the gp41 TM predicts that it spans residues 676 to 699; thus, the TM begins in what is currently described as the C-terminal region of the MPER (aa 676 to 683). We suggest that this region acts to tether the MPER closely to the PM interface (B), and in doing so, this constraint more fully exposes the 4E10 epitope.
Further, based upon a model initially proposed in studies by Cleveland et al. (11
), the TM of MPER-TM1 construct could span the membrane twice; conflicting views exist regarding the degree of membrane spanning of the gp41 TM (28
). The TM also appears to play a role in the overall exposure of the MPER, as selected amino-acid substitutions in this region of the TM affect binding by all three Nt MAbs (). Though the results did not reach statistical significance, a trend was observed when Ala substitution of hydrophobic amino acids surrounding the N terminus of the TM, Trp678, Ile682 and Ile684 increased the binding of all three Nt MAbs, whereas Ala substitution of a hydrophilic aa in that region, Lys683, decreased MAb binding. Thus, we propose that residues in the N terminus of the TM directly affect positions of the MPER's C-terminal region. Exposure of the MPER, and the 4E10 epitope in particular, may also be affected by the quaternary structure of the TM (monomeric versus trimeric) (B versus 7D); this awaits further study.
The TM of HIV, like that of PIV5, is unusually long compared to other TMs, possibly to stabilize a fusion intermediate (5
). For these long TMs, decreased hydrophobicity at one end can be compensated by sliding the other end of the TM further into the membrane. In this case, replacement of hydrophobic residues near the TM's N terminus would make movement out of the membrane more favorable, thus increasing the accessibility of MPER epitopes. On the other hand, loss of a hydrophilic residue, such as Lys683, may cause deeper burial of this region, which in turn may weaken Ab binding. Others have found that binding of 4E10 and 2F5 is inversely correlated to the extent of immersion of epitope residues (14
), supporting the role of membrane anchoring in exposing the MPER. The recent observation of 4E10-resistant strains that have substituted hydrophilic and aromatic residues in this region (38
) also supports a role for membrane anchoring in 4E10 binding. However, the full effect of increased exposure of epitopes in the MPER on immunogenicity has yet to be evaluated.
Our results also suggest that the presence of gp41 domains N-terminal to the MPER does not add to bNt MAb binding, as it was not enhanced for constructs comprising the CHR and NHR domains in addition to the MPER (B). One caveat regarding our experiments is that they involved recombinant, transiently expressed MPER constructs on the surface of a primate kidney cell line instead of the native viral membrane. Structural differences may exist between the viral and cellular forms of the MPER, as the CT has been shown to produce antigenic differences between the Env on viral particles versus the same Env on the cell surface (52
). For native gp41, which exists in numerous prefusogenic and fusogenic states, kinetic restrictions may limit bNt Ab binding to the MPER, whereas the recombinant constructs could allow better exposure of the MPER to bNt Ab binding; however, better exposure may not translate into better immunogens if obstruction by the domains upstream from the MPER must be overcome by a Nt Ab. Nevertheless, many of the amino acid substitutions we found to affect Ab binding have also been shown to affect neutralization (39
), as is consistent with these constructs acting as functional mimics of the NCS. Our results also agree with those of Maeso et al. (30
) who showed differential binding between 2F5 Fab and the Fab mutants of Julien et al. (20
) to MPER peptides in the context of the lipid bilayer; the differences in binding mirrored their Nt activity.
We envision the NCS of the MPER to be structurally akin to a “speed bump” on the surface of the viral membrane that is constrained by a TM tether; as our results suggest, a highly flexible Ab paratope is required for binding to this structure and subsequent viral neutralization. Crystal structures of 2F5 (9
) and 4E10 (10
) Fabs bound to synthetic MPER peptides show that the CDR-H3 apex of these Fabs extends well beyond the peptide, and yet amino acid substitutions or deletions at the CDR-H3 apex of 2F5 (20
) or 4E10 (2
) decrease or ablate neutralization, respectively. While binding to peptides or gp41 in solution is not affected by alterations in the CDR-H3 apex, binding to the MPER in our cell lysate ELISA was reduced by half for the 2F5 Fab mutant, F100B(H)A, and completely ablated for the 2F5 Fab mutant in which the CDR H3 loop was truncated; these losses in MPER reactivity correspond to the partial and full loss of Nt activity for these Fabs, respectively, compared to WT 2F5 Fab, and similar observations were found for 4E10 and its mutant Abs. Yet these mutant Abs still retained the ability to bind to peptides in solution and, in our study, to peptides bearing their respective epitopes and recombinant-gp41 adsorbed to polystyrene. Thus, it appears that MPER-TM1 antigenicity closely follows the neutralization activities of the bNt MAbs and their mutants. These results provide further support for the continued development of the MPER-TM1 construct as an immunogen.
Notably, this same effect, albeit present to a lesser degree, has been shown for peptides that are directly adsorbed to ELISA wells both here and by others (20
). Taken together, the results suggest a mechanism in which a long CDR-H3 loop enhances paratope flexibility, allowing it to open up to engage the speed bump conformation of the MPER in the context of the PM; this very likely also involves direct interaction of the CDR-H3 with the PM. Interestingly, in separate work, we obtained similar results with two other HIV Nt MAbs, m66 and m66.6, which bind an epitope that mostly overlaps with the 2F5 epitope (64
). The m66 and m66.6 Abs are identical, except m66 has 6 fewer amino acid replacements than m66.6 and the neutralization breadth of m66 is more limited than that of m66.6, and yet they bind MPER synthetic peptide with similar affinities. In similarity to the other mutant Abs with lower Nt activity, the m66 Fab bound the MPER-TM1 more weakly than the m66.6 Fab, and yet both Fabs were affected by the same amino-acid substitutions in the MPER-TM1, indicating that their overall binding preferences were identical. Thus, in this case, a difference in Nt activity, caused by differences in the variable domain of the immunoglobulin light chain (VL), not the heavy chain (VH) or the CDR-H3, mirrored binding to the MPER-TM1 and is consistent with our hypothesis that Ab paratope flexibility is required in binding the NCS of the MPER. Future work with a recently discovered MPER-specific Nt MAb, CAP206-CH12, possessing overlapping epitopes with the 4E10 and Z13e1 MAbs, sharing the same germ line heavy and variable kappa light chains as the 4E10 MAb, and exhibiting CDR-H3 sequence similarities to Z13e1 MAb (35
), should further clarify the role of Ab paratope flexibility in neutralization.
As mentioned above, Ye et al. reported that guinea pigs immunized with an influenza virus HA/HIV-1 gp41 chimeric protein elicited low-titer, MPER-specific Nt Abs (60
). Low-titer, MPER-binding Abs that lacked Nt activity were elicited in preliminary rabbit-immunization studies using the DNA constructs described here (unpublished data); if this was not merely an issue of low-titer Ab, then we must conclude that we had not fully replicated the NCS of the MPER. However, both studies were impeded by a major hurdle that must be overcome in designing an MPER-targeting vaccine: elicitation of low-titer anti-MPER Abs. We speculate that titers are low because of low expression or copy numbers of MPER-TM-CT fragments presented in the context of the PM, for current DNA and VLP vaccines, respectively. It may be possible to produce synthetic, liposome- or nanoparticle-based vaccines that present the MPER-TM in the context of a virus-like lipid bilayer, recapitulating the viral NCS. If that can be achieved, we suggest a strategy for producing high-titer Nt Ab responses based on DNA or VLP priming immunizations followed by boosts comprising liposome or nanoparticles bearing high-copy-number MPER. The recent success of Ye et al. (60
) indicates that a strategy focused on improving Nt Ab titers is promising. Coupled with this, further work in designing immunogens that more faithfully mimic the NCS of the MPER in vivo
should further inform vaccine design and improve bNt Ab responses.