Dissecting the polyclonal antibody response of nurse sharks (Ginglymostoma cirratum) to immunization with influenza A virus suggests different roles for IgNAR and IgM, but a common immunodominance hierarchy with hemagglutinin on top

Abstract

Abstract Cartilaginous fishes are the earliest extant phylogenic group to harness germline recombination and somatic hypermutation of immunoglobulins antibodies (Igs) for immunity. In this study we immunized and 1-year-later boosted, two adult nurse sharks (Ginglymostoma cirratum) with inactivated H1 influenza A virus (IAV). After immunization, polyclonal sera from these sharks had strong antiviral properties. Primary immune and recall sera neutralized viral growth in cell culture, caused hemagglutination inhibition, and inhibited neuraminidase (NA) activity. By ELISA and protein microarray, we examined the contribution of two shark Ig isotypes, IgNAR and IgM. Unique to cartilaginous fishes, IgNAR are heavy-chain-only Igs. IgNAR from immunized sharks bound only the immunizing strain of virus. But, flow cytometry of spleen and blood revealed ~1% of IgNAR-positive cells bound both H1 and H5 HA subtypes. While these crossreactive cells exist, any crossreactive antibodies made by cells in these lineages were below our limit of detection. By contrast, both preimmune and immune serum IgM was highly crossreactive, binding H1, H3, H5, and even headless HA. In cartilaginous fish, secreted IgM comes in two independently-produced forms high affinity monomeric (7s) and pentameric (19s). Serum fractionation determined these crossreactive IgM antibodies were 19s. While capable of binding virus, we found 19s alone could not neutralize virus. Despite these differences, regardless of isotype, and in-line with our previous work with mice and lamprey, the immunodominance hierarchy of shark vaccine-induced IgNAR and IgM antibodies to IAV is consistent—HA is dominant, followed by NA, and then core proteins, such as, nucleoprotein and matrix.