An In Vitro Screen Identifies H1 Influenza Hemagglutinin Substitutions That Alter mRNA-LNP Vaccine Responses Against the Stalk Domain

Background: Conformational stability has been shown to modulate the immunogenicity of structural class 1 viral fusion glycoproteins, yet the relationship between influenza A hemagglutinin (HA) stability and antigenicity remains poorly understood. Methods: Here, we screened a panel of 21 A/Hawaii/70/2019 (H1N1) HA mutants for expression, cleavage, and acid and conformational stability in cells. Twelve mutant HA proteins expressed from transfected plasmid constructs that showed relatively robust expression compared to WT also exhibited either altered stability or glycosylation deletion. mRNA-LNPs were generated containing these 12 HAs, along with the wild-type (WT) HA, to investigate the mutational effects on mRNA-LNP vaccine immunogenicity and protection in mice. Hemagglutination inhibition, microneutralization, and total serum IgG ELISAs were performed using a panel of H1N1 antigens to evaluate humoral immune responses at 28 days post vaccination. The vaccinated mice were then subjected to lethal challenge with a forward-drifted H1N1 virus. Results: Most mutant HA candidates elicited WT-like serological responses and provided protection against challenge, although noticeable decreases in serological responses and, to a lesser extent, protection were observed, especially among G72K- and R109E-vaccinated mice. Of note, substitutions at position E107 enhanced total IgG responses against the HA stalk. Modest, but detectable, increases in antibody-dependent cellular cytotoxicity (ADCC) were also observed, particularly with E107R. Although substitution-specific differences in weight loss were found in E107-vaccinated mice challenged with a mouse-adapted Cal/09 virus, these differences were minor, and protection against the forward-drifted challenge virus and a heterologous PR8 challenge virus was no different from that in mice vaccinated with WT HA. Conclusions: These findings demonstrate the ability of specific substitutions to alter components of humoral immunity by shifting HA domain-specific antibody responses, which could prove useful in the design and development of HA vaccine antigens possessing optimized immunogenicity.