Alternative conformations of a major antigenic site on RSV F

Jones, Harrison G.; Battles, Michael B.; Lin, Chun-Chi; Bianchi, Siro; Corti, Davide; McLellan, Jason S.

Previous studies have demonstrated that AM22 and RSD5 potently neutralize RSV and preferentially bind to the prefusion RSV F conformation, similar to the previously characterized site Ø antibodies D25 and 5C4 [8, 35, 38, 42] . However, differences in antibody kinetics and subtype specificities have not been fully explored. Therefore, we used surface plasmon resonance (SPR) to determine the binding affinity and kinetics of the interaction between three site Ø antibodies (AM22, D25, and RSD5) and prefusion RSV F derived from each subtype (strains A2 and B9320) (Fig 1) . For these studies, we worked with a germline-reverted version of RSD5 Kinetic parameters of AM22, D25, and RSD5-GL antibodies binding to prefusion RSV F. Surface plasmon resonance (SPR) sensorgrams of AM22, D25, and RSD5-GL Fabs binding to prefusion RSV F from (A) subtype A (strain A2) or (B) subtype B (strain B9320). The raw data is plotted as a black line and the fitted curve used to calculate the binding kinetics is plotted as a thicker red line. Each binding curve has a 180 second association phase, followed by a 500 second dissociation phase. The equilibrium dissociation constant (K D ) is displayed immediately above the SPR curve for each Fab. (C) A table of the association rate constants (k a ) and dissociation rate constants (k d ) determined for the binding kinetics of each Fab. A value of <1 x 10 −5 was used as the lower limit for the dissociation rate constant (k d ) that can be accurately measured by a Biacore X100. (RSD5-GL), which had 20 somatic mutations in the framework regions reverted to germline residues to minimize immunogenicity (S1 Fig). Of note, RSD5-GL showed similar neutralization potency and binding kinetics for prefusion RSV F as compared to the parental RSD5 antibody (RSD5-WT) (S2 Fig). Despite similar neutralization potencies, AM22, D25, and RSD5 displayed distinct affinities and binding kinetics when compared to each other as well as when compared individually across the two RSV subtypes. The AM22 antigen-binding fragment (Fab) has an~50-fold higher affinity for subtype A with an equilibrium dissociation constant (K D ) of 0.12 nM, whereas its affinity for subtype B is 6.1 nM. Similarly, D25 Fab binds tightly to prefusion RSV F with a slight preference for subtype A, consistent with previously published data [43] , having a K D of <0.064 nM and 0.33 nM for subtype A and B, respectively. In contrast, RSD5-GL Fab has substantial subtype specificity with a >2,000-fold stronger affinity for subtype B compared to subtype A, with a K D of <0.016 nM and 34 nM, respectively. Preferential binding of RSD5-GL for subtype B is primarily due to the off-rate, which is fast for the subtype A interaction and slow for the subtype B interaction.