1The Norwegian University of Life Sciences, 2Institut National De La Recherché Agronomique
Immunoglobulin (Ig) isotypes expressing B-cell lineages, IgM and IgT, have been identified in Salmonids. Activated B-cells undergo V(D)J recombination and somatic hypermutation (SHM) which introduces diversity into the Ig repertoire. Nucleotide substitutions in the complementarity determining regions (CDRs) by SHM, is crucial for high-affinity Ig formation by B-cells engaged in response to antigens. Biases towards CDR3 SHM mutation hotspots are therefore useful when performing repertoire analysis. We are aiming to correlate antibody repertoire expression and CDR3 length spectratypes with immune responses to identify the mutation hotspots, and potential public and private responses. Our focus is B-cell response to viral antigens of infectious pancreatic necrosis virus (IPNV, a dsRNA, naked virus) in which amino acids located in the central region of the VP2 capsid influences the immunogenicity. By immunizing Salmonids with two antigenic variants, rNVI-15TA and rNVI-15PT, we found that antibody response mainly involves IgM, and all variable heavy chain µ (VHCµ) profiles are altered. rNVI-15TA is the most immunogenic strain, and an rNVI-15TA-based vaccine confers superior protection over rNVI-15PT. To map CDR3 length and clonotype frequency of Ig transcripts, we are using a barcode system to increase the accuracy of the PCR output, prior to sequencing and bioinformatics analysis. We look for response in a group of fish, in single individuals, and in single cells, aiming to unveil the footprints of the IPNV infection at different levels. If immunization of Salmonids with rNVI-15TA and rNVI-15PT provokes responses with identical CDR3 footprints, it will be reasonable to suggest that the defined VP2 amino acid residues are not part of the protective epitope, but merely involved in host-pathogen interaction, particularly virus-receptor interaction.
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