Administration of multivalent influenza virus recombinant hemagglutinin vaccine in combination-adjuvant elicits broad reactivity beyond the vaccine components

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants. A combination adjuvant comprising MPLA, CpG ODN1018, and AddaVax™ (a squalene-in-water emulsion) was used to formulate recombinant trimers of influenza virus hemagglutinins H1, H5 or H7 into monovalent or trivalent vaccines, which were then evaluated for the breadth of antibody response induced after single dose or prime-boost administration into C57Bl/6 mice. The breadth of response was measured using a custom protein microarray comprising multiple HA variants spanning HA subtypes H1 to H18, represented as full-length (HA0) proteins and HA1 domains.