Intranasal M2e Peptide Immunogenicity

Six to 10 week old female BALB/c mice were immunized by intramuscular injection (i.m.) twice, 3 weeks apart. Each group contained five mice. Group 1 received control buffer (50 μL of 10 mM Tris–HCl, 150 mM NaCl pH 8.0); Group 2 received PapMV-C and free M2e peptide; Group 3 received PapMV-C coupled to M2e; Group 4 received PapMV-N and free M2e peptide; Group 5 received PapMV-N coupled to M2e. To assess the humoral response to the M2e peptide, blood samples were collected before each boost-immunization on days 20 and 42. Serum was separated from the blood by centrifugation in BD Microtainer SST blood collection tubes (BD, East Rutherford, NJ, USA) for 2 min at 10,000× g. The immunoglobulin G (IgG) 2a endpoint titer to M2e peptides in the sera of immunized mice was determined by enzyme linked immunosorbent assay (ELISA) as described previously [33 (link)]. Briefly, 96-well flat-bottom nunc^TM MaxiSorp plates (VWR, Radnor, PA, USA) were coated overnight at 4 °C with the M2e peptide at 1 μg/mL. Two-fold step serial dilutions of mice sera, starting at 1 in 50, were prepared. Primary antibodies were revealed using peroxidase-conjugated goat anti-mouse IgG2a (Jackson Immunoresearch, West Grove, PA, USA). Results are expressed as antibody endpoint titers greater than threefold OD_450nm of the background value consisting of a pool of pre-immune sera.