We studied 9 subjects who make strong BNAb responses during chronic HIV-1 infection. Evidence from two experimental approaches supports the hypothesis that HM-glycan is involved in the epitope(s) targeted by BNAb responses in 8 of the 9 study subjects. For subject C1-440, no specific glycan deletion mutation was identified that caused neutralization resistance to this antiserum (), but the NAb activity was enhanced by kifunensine treatment (, bottom middle graph). The simplest interpretation is that HM-glycan is not directly involved in this BNAb response, and the outcome could be due to a methodological weakness of kifunensine treatment (see below). It is also possible that this NAb response is very polyclonal, involving a diverse array of N-glycans, and that this diversity is able to overcome the sensitivity of our screening assay using Env point mutants. A third possibility is that the BNAb activity in this subject may target a glycan-dependent epitope centered at glycan 262; however, since mutation of the potential glycosylation site at position 262 significantly disrupted the function of model Envs of both HIV-1YU2
(), the BNAb activity in this subject may elude our mapping with glycan deletion mutants. In summary, our results demonstrate that a vast majority of BNAb responses during chronic HIV-1 infection target HM-glycan-dependent epitopes in our study subjects. For one subject (C1-219), monoclonal antibodies have been isolated, and the neutralization activity of the neutralizing ones is sensitive to changes in glycosylation (8
). That finding provides validation of our conclusion that glycan-dependent BNAbs are present in the 9 study subjects. Significantly, this result is also in full concordance with the recent isolation of a large panel of glycan-dependent NAbs from 4 independent subjects (54
). Our results are able to attribute a unified phenotype to a large panel of BNAb responses in HIV-1 infection. Collectively, these studies establish that glycan-specific NAb responses are produced naturally and successfully in some HIV-1-infected subjects, providing an important basis for vaccine development.
Importantly, our study used a discriminating factor of 2 1:2 dilutions in neutralization curves of wild-type and mutant Envs. Although not conventional in the field, this requirement ensured that only the dominant NAb activity in the study subjects was identified. Thus, our finding does not preclude the existence of subdominant NAb responses in these subjects that are specific for other epitopes, such as the CD4BS and MPER, and this is under continued investigation.
We used two independent methods to demonstrate that the BNAb epitopes targeted in study subjects contain N-glycan, specifically HM-glycan. Epitope mapping using glycan deletion mutations was straightforward, demonstrating that BNAbs in 8 of 9 study subjects target epitopes composed of or at least having a component of N-glycan. Our interpretation is the simplest explanation for data in the form of a phenotypic change, i.e., neutralization resistance, in response to a genetic change, i.e., removal of a potential glycosylation signal. However, it is conceivable that an off-target effect of removal of a specific glycan on local peptic structure or on glycosylation at other potential N-glycan sites may contribute to the phenotypic change. Also, due to the usage of the screening assay and the high discriminating factor of a 4-fold difference between a mutant and its parental Env, our method of epitope mapping theoretically may miss some subjects who make glycan-specific BNAb responses that involve a diverse array of glycans (including HM-glycan and C-glycan). Enrichment of HM-glycan by use of α-mannosidase inhibitors is an established method (53
). Kifunensine treatment has no impact on neutralization of HIV-1YU2
by most known MAbs whose epitopes do not involve N-glycan. (One notable MAb not reported in this publication is MAb 4E10. Kifunensine treatment of HIV-1YU2
had the opposite effect on neutralization by MAb 4E10 from two independent sources. The interpretation for this is not clear, and thus the results were intentionally excluded.) Kifunensine treatment led to enrichment of HM-glycan-dependent epitopes and enhanced neutralization by MAbs such as 2G12, but its effect on complex epitopes with an N-glycan-dependent component, such as PG9 and PG16, was more complicated and cannot be appreciated straightforwardly. Enrichment of HM-glycan by blocking with α-mannosidase inhibitors such as kifunensine may lead to an enhancement of neutralization in different ways. The most obvious and consistent effect of kifunensine treatment is to increase the epitope frequency for HM-glycan-binding NAbs such as 2G12 in a large population of HIV-1 strains with diverse patterns of glycosylation. Enriching HM-glycan can also make low-affinity HM-glycan-specific antibodies gain neutralization potency (14
). In addition, modification of glycosylation may lead to conformational changes in HIV-1 Env, a possibility which can be inferred from our studies of PG9 and PG16 (see Results) (15
). Our data do not allow for discrimination between these various possibilities. Finally, kifunensine treatment can have different influences on different HIV-1 strains under testing conditions (E. Gray and L. Morris, unpublished results). Notwithstanding these caveats, kifunensine treatment of HIV-1YU2
Env studied under the conditions reported here produced interpretable results within our experimental system: whereas kifunensine treatment caused no increase in the neutralization by known NAbs, except for 2G12, it enhanced neutralization of HIV-1YU2
by 8 of 9 subject antisera. These data support the hypothesis that HM-glycan is involved in BNAb epitopes in a majority of our study subjects. Most importantly, consistent results from two complementary approaches synergize to support the observation that BNAb responses in the majority of our study subjects target HM-glycan-dependent epitopes.
It is a striking finding that the majority of our study subjects make BNAb responses to HM-glycan-dependent epitopes. Given its novelty and its enormous implications for the future direction of HIV-1 vaccine development, validating this finding for independent cohorts of HIV-1-infected patients will be critical. Glycosylation patterns are defined primarily by the presence of the NXS/T consensus signal. However, the profile of different “glycoforms” is heavily influenced by the host cell type and even by cell culture conditions (53
). The nature of glycans on HIV-1 can be influenced by the host cells and culture conditions, including methods of activation and differentiation of lymphocytes or macrophages. In most studies investigating neutralization and neutralization escape mechanisms of HIV-1 Env or measuring NAb responses to vaccination, the neutralization target viruses are produced in immortalized lymphocytes or 293T cells and thus are likely to have different N-glycan patterns from HIV-1 strains replicating in human hosts. In light of the findings reported here, necessary caution should be used in interpreting data on NAb responses to HIV-1 infection or to vaccinations, especially those results produced in experiments using pseudotyped reporter viruses. Glycan-specific antibody responses can be an attractive line of vaccine development, since some glycan-specific MAbs demonstrate great neutralizing breadth and potency (51
). It has also been shown that glycan-binding antibodies can be induced by synthetic glycans (2
) or HM-glycan on non-HIV-1 proteins (1
). Unfortunately, a glycan-binding antibody capable of neutralizing diverse strains of primary HIV-1 has yet to be achieved. Thus, it will not be a straightforward task to develop anti-HIV-1 vaccines by eliciting glycan-specific NAbs. However, our finding should add to the thinking in the overall direction of vaccine development against HIV-1 infection.