Viruses use antigenic evolution to introduce glycans on their surface glycoproteins to evade immune surveillance by the host adaptive response; HIV and its envelope (Env) glycoprotein is one notable example (1). Indeed, for HIV Env, glycans account for approximately half the mass of the protein, and glycan density and heterogeneity can affect induction of broadly neutralizing antibodies (bnAbs) (1–3). Influenza (4, 5), ebola (6, 7), hepatitis C (8), Lassa (9–11), and coronavirus (12) are other examples of viruses that vary their glycosylation to “shield” conserved epitopes. Influenza has varied its hemagglutinin (HA) glycosylation profiles across subtypes since its introduction into the human population. Circulating H1 HAs have maintained approximately 8 glycans since their re-introduction in 1977 until the introduction of the pandemic H1N1 in 2009 which lost 4 glycans relative to previous historical H1s. H3 HAs, however, have increased overall glycans since the 1968 pandemic from 7 to 13.

Conserved epitopes on influenza HA are targets for broadly protective humoral immunity. These epitopes include the receptor binding site (RBS), stem, and “interface” regions (13). Antibodies that engage the RBS neutralize by impeding viral entry through sterically blocking attachment to its receptor, sialic acid. However, cross-reactive RBS-directed antibodies are subdominant, and their breadth is often dependent on having conserved features in their complementary determining region 3 (HCDR3) to present sialic acid-like contacts (14–20). The stem region of HA is similarly conserved, likely due to functional constraints necessary for undergoing conformational changes required for membrane fusion (21). Antibodies targeting the stem show breadth across nearly all HA subtypes (22–25). However, these antibodies targeting the stem are encoded by a relatively restricted set of variable genes and are difficult to elicit (21). More recently, we and others identified an additional conserved epitope at the HA trimer interface (26–30). This epitope lies at an otherwise occluded site between HA protomers in the native trimeric structure present on the virion surface. Nonetheless, antibodies targeting this epitope have been isolated from both human and murine sources and confer protection via an Fc-dependent mechanism (26–28). This implies that this occluded epitope is transiently exposed enough on the virion surface or the infected cell to be targeted by such antibodies (31).

Current immunogen design efforts for next generation influenza vaccines are focused on eliciting protective antibodies targeting these conserved epitopes (32). Here, we analyzed whether glycan engineering along with cysteine stabilizing mutations could modulate elicited humoral immune responses. We used the H3 A/Hong Kong/1/1968 HA as a model antigen to develop glycan shielded HA immunogens that block variable surface-exposed epitopes; we selectively exposed or concealed the RBS epitope with glycans and further stabilized the trimeric HA by introducing cysteine residues at both the head and stem interfaces. We immunized mice with stabilized or non-stabilized, glycan-modified immunogens and characterized elicited immune responses. We observed a reduction in serum responses to the stabilized immunogens, indicating that blocking surface epitopes with glycans or disulfide stabilization can significantly alter their overall immunogenicity. These data show how different protein engineering strategies can be combined to influence humoral responses to conserved epitopes on the influenza HA.

Based on our previous work, we designed second-generation glycan-modified hemagglutinin (gHA) immunogens based on the historical H3A/Hong Kong/1/1968 HA (HK-68). Our first-generation gHAs included three immunogens with varying degrees of glycosylation: (1) an “RBS-exposed” immunogen (gHA^RBS), in which the entire surface is hyperglycosylated but leaves the RBS exposed, (2) an “RBS-concealed” (gHA^cRBS) immunogen with a single glycan at position 133 to abrogate binding of RBS-directed antibodies, and (3) a “full-shield” immunogen (gHA^shield) which obscures all surface-exposed epitopes. For this study, we modified our initial gHA^RBS immunogen to include two additional predicted N-linked glycans (PNGs) at residues 122 and 126 to create gHA^RBSv2.0; this construct differs from gHA^shield by two PNGs at residues 133 and 158, both in the RBS pocket ( Figure 1 ). We used these two immunogens, gHA^RBSv2.0 and gHA^shield, to discern RBS- from non-RBS directed responses and as templates for further modifications.

Antibodies elicited by our previous immunization with first-generation gHAs focused predominantly to the trimer interface; therefore, we hypothesized that occluding the interface epitope, could re-focus the immune response to the RBS. This may have been a consequence of the engineered glycans destabilizing the HA trimer resulting in mixed trimer and monomeric HAs; the latter readily exposing the interface epitope ( Supplemental Figure 1 ). To that end, we stabilized the HA trimer by engineering cysteines at residues 212 and 216 (33) (H3 numbering) on the HA head and at residues 30 and 376 (34) on the HA stem, in order to occlude immune responses directed to the HA interface ( Figure 1A ). These stabilizing mutations were engineered onto both gHA^RBSv2.0 and gHA^shield, to yield their cysteine-stabilized counterparts, referred to as gHA_cys ^RBSv2.0 and gHA_cys ^shield ( Figures 1B – D ). We confirmed disulfide presence using SDS-PAGE under reducing and non-reducing conditions ( Figure 1E ). The cysteine modified hyperglycosylated HA proteins showed predominantly trimeric bands that collapsed into a monomeric species upon reduction. They also are stable trimers under native conditions, as confirmed by size-exclusion chromatography ( Supplemental Figure 1 ).

We next characterized all four gHA immunogens by ELISA using an assembled panel of structurally characterized, conformation-specific antibodies binding unique antigenic sites on HA, including HC19 (35), HC45 (36), FI6 (22), and 8H10 (27), which bind the RBS, vestigial esterase, stem, and interface epitopes, respectively ( Figure 2A ). All four gHA immunogens abrogated binding of HC45 and FI6. Both gHA^RBSv2.0 immunogens engaged HC19 ( Figures 2C, D ), whereas gHA^shield immunogens abrogated binding by HC19 ( Figures 2E, F ), indicating that these immunogens selectively conceal or expose the RBS epitope, respectively. Additionally, both cysteine-stabilized immunogens, gHA_cys ^RBSv2.0 and gHA_cys ^shield, selectively abrogated binding of the interface-directed antibody, 8H10, characterized in our previous study (27); this confirms that the stabilized trimers effectively mask the interface epitope ( Figures 2D, F ). All immunogens engage the conformation-specific MEDI8852 antibody (37), that binds the stem in a different orientation relative to FI6, and confirms structural integrity of the stem epitope ( Supplemental Figure 2 ).

We then assessed the immunogenicity and antigenicity of gHA^RBSv2.0, gHA_cys ^RBSv2.0, gHA^shield, and gHA_cys ^shield. We immunized mice and collected serum at day 0, 7, 14, 21, and 28. Serum IgG responses were assessed via multiplex bead array (Luminex platform) for binding to each of the four immunogens ( Figure 3 and Supplemental Figure 3 ). While the gHA^RBSv2.0 and gHA^shield immunogens elicited a robust self-directed serum response, there was a comparatively weak reactivity to the cysteine modified counterparts indicating a predominantly interface-directed response ( Figures 3A, B ). This pattern persisted throughout the course of the experiment ( Supplemental Figure 3 ). These data are consistent with our previous observations of the role of glycan shielding and immunodominance of the interface epitope (27). While immunizations with cysteine gHA_cys ^RBSv2.0 and gHA_cys ^shield elicited a nearly undetectable response at day 7, by day 28 we observe a similar overall magnitude of response comparable to the non-cysteine stabilized immunogens ( Figures 3C, D and Supplemental Figure 3 ). However, unlike gHA^RBSv2.0 and gHA^Shield, this response was comparable across all four immunogens for both gHA_cys ^RBSv2.0 and gHA_cys ^shield. This potentially indicates that serum responses elicited by disulfide-stabilized gHAs were directed towards a common epitope exposed across all four immunogens. Notably, we did not observe differential reactivity to our “RBS-exposed” and “shield” immunogens at any timepoint, indicating that there was likely not a robust RBS-directed humoral response elicited by our exposed RBS immunogens. Overall, these data indicate that the combination of hyperglycosylation and disulfide stabilization alters the immunogenicity kinetics of gHA immunogens but cannot significantly redirect humoral responses to the exposed RBS epitope.

Next, we performed competition ELISAs with a panel of conformational specific antibodies with d28 serum to epitope map the elicited responses to our immunogens ( Figure 4 ). Our antibody panel included the FI6 (stem), HC45 (vestigial esterase), HC19 (RBS), and 8H10 (interface) epitopes ( Figure 2 ). The H1 RBS-directed 5J8 (38), which does not bind to H3 HK-68, was included as a negative control. Serum from gHA^RBSv2.0, gHA_cys ^RBSv2.0, and gHA_cys ^shield showed significant competition with HC45 ( Figures 4A – C ), that was not present in gHA_cys ^shield ( Figure 4D ). While not as significant as the HC45-like response, both gHA_cys ^RBSv2.0, and gHA_cys ^shield immunogens apparently elicited a HC19-like and 8H10-like response, despite the presence of glycans and inter-protomer disulfides. There was no FI6-like response detected in sera from any of the immunogens; this potentially indicates that despite fewer glycans present to obscure responses targeting the stem, this region remains subdominant.

Finally, we analyzed day 28 serum for breadth and influenza virus neutralization activity. We assessed H3 breadth using a panel of antigenically distinct, historical H3s spanning 1968-2012 ( Figure 5 and Supplemental Figure 4 . Serum IgG from all four immunogens bound to H3 from A/Hong Kong/1/1968, A/Victoria/3/1975, A/Bangkok/1/1979, and A/Leningrad/360/1986, with little to no binding beyond that point ( Figure 5A ). Additionally, serum elicited by all four immunogens had weak neutralization activity, with no appreciable difference between “RBS-exposed” and “shield” immunogens ( Figure 5B ). This is consistent with the above data that these immunogens do not elicit robust RBS-directed serum responses that would contribute to neutralization. Furthermore, the lack of neutralization by serum from gHA^RBSv2.0 and gHA^shield immunogens is likely a consequence of a focused response to HC45-like or interface epitopes that do not contribute to neutralization.

In this work, we determined whether modifying HA using hyperglycosylation and disulfide stabilization could focus antibody responses to the subdominant RBS epitope. We hypothesized that by both shielding surface-exposed epitopes and by restricting access to the immunodominant interface epitope by glycans, theoretically the only accessible epitope is the RBS, and thus should become immunodominant, or at minimum, be able to elicit a detectable RBS-directed response. This epitope masking approach has been effective in focusing responses to the CD4 binding site on HIV-1 Env (39). Additionally, inter-protomer disulfide stabilization has been an effective method for improving or focusing immune responses to RSV (40, 41), norovirus (42), parainfluenza virus (43), and HIV (44). To our knowledge, however, this is the first example to study the effect of inter-protomer disulfide stabilization of full-length influenza A HA in combination with hyperglycosylation on immunogenicity. However, in this study we showed that obscuring all surface-exposed epitopes except the RBS did not robustly direct responses to the RBS.

The observed lack of an RBS-directed serum response could be due to a variety of factors. Human-derived bnAbs that engage the RBS of group 1 and group 2 HAs have been isolated and appear to be relatively abundant (14–17, 19, 20). These RBS-directed antibodies require long HCDR3s (~19 amino acid in length) to engage the recessed RBS. Because the C57BL/6 mice used in this study tend to have shorter HCDR3s, it is likely that this restricts humoral responses targeting the RBS epitope. Additionally, these antibodies could be rare in the mouse antibody repertoire, and thus may not be readily detected in serum. Alternative murine models with human variable heavy genes knocked-in [e.g., JH6 (45)] may be better suited for characterizing immunogens designed to elicit RBS-directed responses.

Despite being nearly “immune silent” at early time points, the disulfide-stabilized immunogens eventually elicited a weak, but detectable, antibody response at days 21 and 28. This may indicate that the engineered glycans do not completely shield surface-exposed epitopes. In other words, it is possible that antibodies target “holes” in the engineered glycan shield that may ultimately contribute to the humoral response. From competition ELISA data, this could be an epitope that partially overlaps with HC45 within the vestigial interface region but is not obscured by glycans on gHA constructs, or another epitope(s) not covered by our antibody panel. A similar phenomenon has been observed in HIV-1, where antibodies targeting the CD4 binding site on gp120 such as VRC01-like antibody, VRC08 (46), as well as N6 (47), could evolve around a glycan at position N276, present on ~95% of circulating HIV-1 strains. Future immunogen design iterations may vary the pattern of engineered glycans to optimize placement and shielding.

Overall, these data show how hyperglycosylation and disulfide stabilization can effectively modulate humoral responses. There appears to be a “limit” on available surface-exposed epitopes on our immunogens that can be effectively shielded by glycans to dampen overall humoral response. This parallels how viruses use glycosylation of their envelope proteins to evade host adaptive immune responses [e.g., HIV (1–3), ebola (6), hepatitis C (8), influenza (4, 5)]. It remains to be seen whether hyperglycosylation alone or in combination with disulfide stabilization can be leveraged to elevate a subdominant epitope to dominance, in particular the RBS. While in wildtype mice this was not achieved, the immunogens designed and characterized here may indeed immune focus to the RBS in humans who can more readily elicit RBS-directed responses.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Duke University Institutional Animal Care and Use Committee.

DT, AS, GS, and AK designed research. DT, AM, ES performed research. DT and AS analyzed data. DT and AS wrote the paper. DT, AS, GS, AM, and AK edited and commented on the paper. All authors contributed to the article and approved the submitted version.

We acknowledge support from R01 AI146779 and P01 AI089618 (to AS). This research has been funded in whole or part with federal funds under a contract from the National Institute of Allergy and Infectious Diseases, NIH contract 75N93019C00050. Multiplex binding and microneutralization assays were performed in the Immunology and Virology Units, respectively, of the Duke Regional Biocontainment Laboratory, which received partial support for construction from the NIH/NIAID (UC6 AI058607; to GS).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor declared a past co-authorship with one of the authors AS.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.