Influenza Antigen Engineering Focuses Immune Responses to a Subdominant but Broadly Protective Viral Epitope

Abstract

Viral glycoproteins are under constant immune surveillance by a host's adaptive immune responses. Antigenic variation including glycan introduction or removal is among the mechanisms viruses have evolved to escape host immunity. Understanding how glycosylation affects immunodominance on complex protein antigens may help decipher underlying B cell biology. To determine how B cell responses can be altered by such modifications, we engineered glycans onto the influenza virus hemagglutinin (HA) and characterized the molecular features of the elicited humoral immunity in mice. We found that glycan addition changed the initially diverse antibody repertoire into an epitope-focused, genetically restricted response. Structural analyses showed that one antibody gene family targeted a previously subdominant, occluded epitope at the head interface. Passive transfer of this antibody conferred Fc-dependent protection to influenza virus-challenged mice. These results have potential implications for next-generation viral vaccines aimed at directing B cell responses to preferred epitope(s).

Keywords: broadly neutralizing antibodies; immunogen design; influenza hemagglutinin; protein engineering.

Figure 1.. HA glycan evolution and construct design.

(A) Number of putative N-linked glycans (PNGs) of historical H1 (gray, dashed line) and H3 (solid, black line) HAs beginning from 1977 and 1968, respectively. The template immunogen, H3 Hong Kong/1/1968 (HK-68) and the natively most glycosylated, H3 Victoria/361/2011 (VC-11), are noted. (B) The wildtype H3 HK-68 with its native glycans are marked with additional glycans marked (C) in red for gHA^RBS, (D) in green for gHA^cRBS and (E) in purple for gHA^shield. The total number of glycans for each construct is noted. See also Figure S1.

Figure 2.. Amplitude of antibody serum responses is unaffected by HA glycosylation.

C57BL/6J (B6) mice were immunized with wt H3 HK-68 (n=7), gHA^RBS (n=6), gHA^cRBS (n=6) or gHA^shield (n=4) and bled 16 days post-immunization. The sera were screened for binding to (A) wt H3 HK-68, (B) gHA^RBS, (C) gHA^cRBS, and (D) gHA^shield in a Luminex-based assay. Mean fluorescence intensity (MFI) signal ± SD is reported; n= number of mice used. See also Figure S2.

Figure 3.. Single-cell cultures reveal differences in antibody epitopes elicited by gHAs.

Single GC B cells were sorted from the popliteal lymph nodes of immunized B6 mice and cultured with NB-21.2D9 feeder cells. Individual single-cell culture supernatants from mice immunized with (A) wt H3 HK-68 (blue, n=150), (B) gHA^RBS (red, n=268 ), (C) gHA^cRBS (green, n= 77), and (D) gHA^shield (purple, n=245) were harvested and screened for binding in a Luminex-based assay. The binding signal to the immunizing antigen (x axis) was compared to the other three HA immunogen constructs (y axis). The coefficient of determination (R²) of a linear regression fit is reported. n = number of monoclonal antibodies from supernatants used in screening. See also Figure S3.

Figure 4.. Adaptive immune responses to gHAs are genetically restricted, in contrast to the wt HA.

V_H gene usage frequencies in B6 mice immunized with (A) wt H3 HK-68, (B) gHA^RBS, (C) gHA^cRBS, and (D) gHA^Shield shown as pie charts with the total number of GC B cells marked in the center of the charts. The three major V_H gene [IGHV1-9 (purple), 1-69 (pink) and 5-9-1 (grey)] family members are highlighted and reported in a table summarizing their clonotypes (as defined by their CDR H3 loops) and the number (#) of members in each lineage. See also Figure S4.

Figure 5.. X-ray crystal structures of V_H 5-9-1 antibodies show convergence towards a common epitope.

(A) Structure of 8H10 Fab bound with HA head of vH4 NB-10; (B-C) structures of FL-1056 and FL-1066 Fabs bound with HA head of H3 HK-68. The top panels show the overall structures and indicate the immune focusing to a common epitope. The middle panels highlight the Fab footprints on HA and antibody CDR loops involved in binding. Contacts made by the CDR loops are detailed in the bottom panels. Essential HA residues are underlined. See also Figure S5 and Table S1.

Figure 6.. V_H 5-9-1 antibodies are non-neutralizing but protect against a viral challenge through an Fc-dependent mechanism.

Mouse challenge experiments were performed with intranasal viral infection (5LD₅₀) with X31-68 (H3N2) of mice injected with 100 μg of recombinant 8H10 as IgG2c or IgG1 isotype. (A total of 10 mice were used per group, including the PBS control.) The (A) body weight loss was monitored and the (B) humane endpoint was set at 25% relative to the initial body weight beyond which point the mice were euthanized. (C) Microneutralization titers of 8H10 antibody are reported.