Structure-Guided Molecular Grafting of a Complex Broadly Neutralizing Viral Epitope

Abstract

Antigenic variation and viral evolution have thwarted traditional influenza vaccination strategies. The broad protection afforded by a "universal" influenza vaccine may come from immunogens that elicit humoral immune responses targeting conserved epitopes on the viral hemagglutinin (HA), such as the receptor-binding site (RBS). Here, we engineered candidate immunogens that use noncirculating, avian influenza HAs as molecular scaffolds to present the broadly neutralizing RBS epitope from historical, circulating H1 influenzas. These "resurfaced" HAs (rsHAs) remove epitopes potentially targeted by strain-specific responses in immune-experienced individuals. Through structure-guided optimization, we improved two antigenically different scaffolds to bind a diverse panel of pan-H1 and H1/H3 cross-reactive bnAbs with high affinity. Subsequent serological and single germinal center B cell analyses from murine prime-boost immunizations show that the rsHAs are both immunogenic and can augment the quality of elicited RBS-directed antibodies. Our structure-guided, RBS grafting approach provides candidate immunogens for selectively presenting a conserved viral epitope.

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

Figure 1:. RBS grafts and sequence alignments.

A, Phylogeny of influenza subtypes. Group 1 and 2 influenzas are annotated. Highlighted in pink are the seasonal subtypes currently circulating in the human population. B, Representative H1 antigenic clusters: H1 Massachusetts/1/1990 (MA-90), H1 Solomon Islands/03/2006 (SI-06) and H1 California/07/2009 (CA-09) are listed. Sequence alignment is in reference to SI-06 and conserved residues are marked as (.); segments defining the H1 RBS graft, S1–4, are colored. C, Residues comprising S1–4 of the acceptor scaffolds (H3 numbering) from non-circulating influenzas H4 New Brunswick/00464/2010 (H4 NB-10), H6 Wisconsin/617/1983 (H6 WI-83), H14 Wisconsin/10OS3941/2006 (H14 WI-06) and H16 Delaware Bay/296/1998 (H16 DB-98). D, Influenza HA trimer (PDB 5UGY) in surface representation. HA1 is in silver, HA2 in dark gray and S1–4 are colored. e, LSTc (sialylneolacto-N-tetraose c, stick representation) modeled in complex with HA. S1–4 is colored and HA is in silver.

Figure 2:. Structure of K03.12 in complex with rsH4NBv1 and scaffold improvement.

A, Antibody K03.12 Fab (heavy and light chains are colored blue and green, respectively) in complex with rsH4NBv1 HA1 “head” (silver). The CDR H3 (magenta) is marked. B, Close-up of the antigen-binding site. The CDR H3 (magenta) is shown in sticks with key interacting HA residues (silver). Hydrogen bonds are denoted in yellow, dashed-lines. C, Comparison of the RBS of H1 SI-06, yellow, (PDB 4YJZ), H4 A/duck/Czechoslovakia/1956 (H4 CZ-56), black (PDB 5XL3) and rsH4NBv1, silver. The segments of the grafts are labeled and colored. D, rsH4NBv2 and e, rsH4NBv3 with residues changed for each construct represented by a colored sphere. The original, unchanged segments from rsH4NBv1 are labeled and colored.

Figure 3:. Reactivity of RBS-directed IgGs for rsHAs.

CH67 (blue), H2526 (red), H2227 (green), 641 I-9 (violet) and K03.12 (orange) IgGs were titrated against A, wildtype H4 NB-10, B, rsH4NBv1, C, rsH4NBv2, D, rsH4NBv3, E, K_Ds obtained from curves in A-D for H4 constructs displayed as a “heatmap”: warm colors are high affinity and cool colors low affinity. The legend is in M. IgG titrations for F, wildtype H14 WI-10, G, rsH14WIv1, H, rsH14WIv2 and I, control H1 SI-06 HA constructs. J, K_Ds obtained from curves in F-I for H14 constructs. ELISA measurements were done in duplicate over the concentration range except for A, and F, where only 1μM final concentration of IgG was tested. The wildtype H1 SI-06 values in E and J are both derived from I. Curve fitting was done with a nonlinear regression model and K_Ds were determined using GraphPad Prism version 8.0.

Figure 4:. in vivo assessment of rsHA immunogenicity.

A, schematic representation of the immunization strategy B, CH67 competition to H1 SI-06 FLsE HA against the sera from wild-type non-immunized B6 mice (black), J_H6 mice primed and boosted with H1 SI-06 (blue) and mice primed with H1 SI-06, then boosted with rsH4NBv3 (pink), C, total GC B cell counts in the animal cohorts. n=4 mice for B6 and n=4 mice for J_H6. *p < 0.05, Welch’s t test. Error bars represent SD, D, serum reactivity against H1 SI-06, e, serum reactivity against rsH4NBv3. The mean values are plotted for d and e with error bars indicating SD.

Figure 5:. Identification and biochemical characterization of RBS-directed antibodies.

A, CH67 competition experiment against the single B cell culture supernatants from homologous (H1 SI-06; blue) and heterologous (H1 SI-06, rsH4NBv3; pink) immunizations. The competition was performed on Luminex beads coated with either H1 SI-06 or with rsH4 NB-10, antibody competition is expressed as % inhibition (x-axis); antibodies with >75% inhibition are colored. Representative monoclonal antibodies Ab18, Ab133 and Ab144 were selected for further characterization. B, Binding to a panel of historical H1 HAs and the resurfaced rsH4 NB-10 was assayed with recombinant Fabs using bio-layer interferometry. Affinity constants (K_Ds, in μM) were obtained by applying a 1:1 binding isotherm using vendor-supplied software. The “heatmap” color scheme is a visualization aid: warm colors are high affinity and cool colors low affinity. H1 SI-06 – A/Solomon Islands/03/2006, H1 USSR-77 – A/USSR/90/1977, H1 MA-90 – A/Massachusetts/1/1990, H1 FL-93 – A/Florida/2/1993, H1 BE-95 – A/Beijing/262/1995, H1 NC-03 – A/North Carolina/3/2003, rsH4 NB-10 - resurfaced duck/New Brunswick/00464/2010; N.D. – non-detectable. C, Visualization of the HA-Ab133 complex by negative stain electron microscopy. The top panels are of HA alone with and without crosslinking to Ab133 with glutaraldehyde. Ab133 is colored green in the bottom right panel and compared to an RBS antibody bound to HA (PDB ID: 5UG0). One copy of the RBS antibody is not shown for clarity.