We have demonstrated that non-denaturing (native) deglycosylation of HIV-1 JRFL and the group M consensus gp140 envelope oligomers with PNGase F resulted in enhanced Env binding to gp41 broadly neutralizing mAbs 4E10 and 2F5, and as well, induced or enhanced binding of gp140s to 2F5 and 4E10 reverted unmutated ancestor antibodies. Moreover, immunization of the JRFL deglycosylated gp140 Env resulted in enhanced antibody induction to the gp41 2F5 peptide epitope.
Treatment of Env with glycosylation inhibitors and neuraminidase has been reported to enhance HIV-1 binding and neutralization by mannose-binding lectin, and to facilitate immunocapture of virions by both mAbs 2G12 and 2F5 
. Thus, the selective removal of complex glycans by non-denaturing PNGase F treatment may similarly expose the 2F5 and 4E10 MPER binding site for enhanced mAbs 2F5 and 4E10 binding. MAb 2G12 has been reported to bind to gp120 N-linked high mannose residues at N295, N332, and N392, as well as with peripheral glycans from 386 and 448 
. Interestingly, our mass spectrometry analysis of deglycosylated JRFL gp140 demonstrated selective loss of N-linked glycans particularly around V1 and V2 with high mannose residues at N295, N332, and N392 remaining intact 
. The non-denatured deglycosylated JRFL gp140 Env bound well to mAb 2G12 and also bound to DC-SIGN. In this regard, the gp120 high mannose residues involved in 2G12-gp120 binding have been reported to be involved in gp120-DC-SIGN binding 
. Thus, selective removal of gp140 glycans may induce conformational changes or unmasking of the gp41 MPER 4E10 epitope that results in enhanced on-rate (ka) of mAbs 4E10 and 2F5 binding.
Walker et al. have recently reported that 25% chronically infected HIV-1 subjects with broad neutralizing antibodies had neutralizing plasma antibody activity targeted at Env glycans at N332, and at N295 in some subjects 
. Although N332 and N295 glycans are involved in mAb 2G12 binding, their N332 and/or N295 activity was found to bind a glycan epitope distinct from that of 2G12 
. That PNGase treatment of HIV-1 Env under non-denaturing condition retained all N-linked glycans involved in 2G12 and in the novel N295/N332 neutralizing epitopes, suggests natively deglycosylated HIV-1 gp120 or gp140 Env may be of use in vaccine formulations targeted at elicitation of anti-glycan gp120 neutralizing antibodies.
Yuste et al.
, has demonstrated that mutations in gp41 N-linked glycosylation sites exposed a neutralization epitope N-terminal to the MPER 
. From Yuste et al.
, we would predict that cleavage of N-linked glycans at 611 616, 625 and/or 637 might also be responsible for directly unmasking the 2F5 and 4E10 gp41 epitopes, or inducing conformational changes of the Env that indirectly expose the gp41 membrane proximal external region, or both. Indeed, three out of four glycosylation sites in gp41 were occupied in recombinant untreated JRFL gp140, and were removed in natively deglycosylated JRFL gp140 (). Additionally, we and others have previously shown that enhanced exposure of the MPER epitope by single amino acid changes can increase the binding and neutralizing activity by broadly neutralizing MPER antibodies 
Since the 4E10 antibody is markedly polyreactive 
, it was important to consider that the 4E10 epitope exposed on PNGase F-treated Env gp140 might be distant from the 672
gp41 4E10 nominal epitope. Hages-Braun et al
. have suggested that two ancillary 4E10 binding sites are located on gp120 at 34
and on gp41 at 512
. However, our studies demonstrated no 4E10 binding to PNGase F-treated gp120, and PNGase F-treated gp140, but not gp120, absorbed the binding of mAb 4E10 to the gp41 peptide (P-4E10, SLWNWFNITNWLWYIK). These data suggested that the reason that mAbs 2F5 and 4E10 RUAs can bind PNGase-treated JRFL gp140 is most likely due to enhanced exposure via glycan unmasking of the gp41 MPER, or an induced gp41 MPER conformational change that allowed for a faster on-rate for mAbs 2F5 and 4E10 to bind to gp41. That most of the deglycosylated forms of JRFL and CON-S gp140 are oligomeric and only a minor species is monomeric for both Envs (Figure S1
), demonstrates that the exposure of the MPER is on oligomeric Env forms.
A number of studies have suggested that deglycosylation can affect HIV-1 envelope antigenicity and immunogenicity 
. A non-glycosylated outer domain of gp120 has been reported to be immunogenic and induce weak neutralizing antibodies in rabbits 
. For example, loss of sialic acids on gp120 has been reported to improve Env immunogenicity 
. Strategies for native deglycosylation of Env have been described that can improve both immunogenicity and sensitivity of resulting virions to neutralization 
Frey et al.
have reported weak or no binding of recombinant HIV-1 Envs to mAbs 2F5 and 4E10, but strong binding of these antibodies to a stabilized gp41 intermediate molecule 
. Whether the epitope on natively deglycosylated recombinant JRFL gp140 is in the intermediate conformation of gp41 seen by mAbs 2F5 and 4E10 during the infection process is not known. However, we have recently shown that glycosylated JRFL gp140 Env can prime for an MPER peptide-liposome boost and induce antibodies focused on the DKW core 2F5 epitope that bind to the gp41 intermediate epitope (Sekaran, M. Alam, SM, Haynes, BF, unpublished). That deglycosylated JRFL gp140 is more immunogenic in rhesus macaques than glycosylated JRFL for 2F5 epitope MPER antibodies suggests that deglycosylated JRFL may have enhanced immunogenicity as a prime in this prime-boost setting.
Other factors in addition to the conformation of the MPER neutralizing epitopes appear to contribute to regulation of anti-MPER broad neutralizing antibodies. For example, we now have experimental data with mAbs 2F5 and 4E10 knock-in mice that B cells expressing these VHs are controlled by both central and peripheral tolerance mechanisms in these animals 
. Thus, immunogen design for optimal neutralizing epitope exposure is only one-component of the problem for inducing anti-MPER antibodies. Nonetheless, the increased affinity of the non-denaturing PNGase F-treated gp140 Env may render it a preferred Env immunogen in the setting of formulation of Envs with potent adjuvants that are designed to circumvent peripheral tolerance 
While the RUAs in this paper are close approximations of the unmutated ancestor B cell receptors on naïve B cells, it should be noted that they are only inferred and may not precisely mimic the binding of membrane bound IgM B cell receptors on naïve B cells. Our data demonstrate that RUAs of both mAbs 2F5 and 4E10 can recognize the MPER well in the context of natively deglycosylated JRFL gp140, but not in fully glycosylated JRFL gp140. Interestingly, the glycosylated group M consensus Env CON-S gp140 constitutively bound to both 2F5 RUAs. This finding is consistent with our previously published site-specific glycan analysis of the CON-S gp140 in which we found, unlike JRFL gp140, that CON-S gp140 when expressed in 293T cells, has no occupied gp41 glycan sites 
. That native deglycosylation of CON-S gp140 still resulted in enhanced binding to unmutated ancestors of 2F5 and 4E10 suggests that removal of gp120 glycans modulates access to gp41 as well. Thus, the inability of mAbs 2F5 and 4E10 reverted unmutated ancestors to react with recombinant HIV-1 gp140 Envs in some instances may be due to glycan masking or glycan modulation of conformations of MPER neutralizing epitopes, or may be due to “holes” in the native B cell repertoire for glycosylated HIV-1 Env. That the MPER 2F5 and 4E10 gp41 epitopes can be exposed with native deglycosylation, to us suggests the importance of glycan masking on glycosylated gp140 to 2F5 and 4E10 unmutated ancestor antibody non-reactivity. In light of the host immunoregulatory controls that also appear to play roles in control of MPER broad neutralizing antibody induction, both structural considerations of immunogen design targeted to optimized binding to reverted unmutated ancestor antibodies, in concert with methods of immunogen formulation to access and drive MPER-specific naïve and memory B cell proliferation likely will be required for the ultimate safe induction of MPER broad neutralizing antibodies.