Fig. 2.

LAH-KLH vaccine acts as a robust immunogen, and the elicited serum antibody reacts with multiple hemagglutinin subtypes. (A and B) Sera from individual mice were taken 10 d after primary and secondary immunization and were tested for binding to H3 or H1 hemagglutinin by ELISA. (C) Pooled sera from 20 mice taken after secondary immunization with LAH-KLH were tested by Western blot for reactivity with purified influenza virus subtypes A/Hong Kong/1/1968 (HK68; H3), A/New York/1/2008 (NY08; H3) or A/USSR/90/1977 (USSR77; H1), purified A/California/04/09 (transmembrane domain absent) (Cal09 HA; H1); or Newcastle disease virus (NVD). (D) Pooled sera were tested for binding activity against H2, H5, or H7 hemagglutinin by ELISA. LAH-KLH antiserum has considerable binding activity against all three hemagglutinin subtypes. Positive sera used were from mice infected with either a group 1 influenza virus [A/Singapore/1/57 (SG57); H2 ELISA and A/Viet Nam/1203/2004 (VN04); H5 ELISA] or a group 2 virus [A/Netherlands/219/2003 (Neth03); H7 ELISA] (2). (E) LAH amino acids 76–130 from the HA2 of different hemagglutinin subtypes. White letters on a black background indicate a residue conserved in all five HAs. Conserved residues fall into one of four groups: (i) D/N/E/Q, (ii) I/L/V/M, (iii) K/R, or (iv) S/T. White letters on a gray background indicate a residue conserved in four of five HAs or less stringent conservation (R vs. H near middle conserves charge, but there is a change in size). Bold text on a white background indicates partial conservation (three of five HAs) or less stringent (L vs. A at middle or F vs. M toward left). (F) Isotype profile of hemagglutinin-specific antibody in serum pools from normal (Naïve) mice, mice infected with A/Hong Kong/1/1968 (H3), or mice immunized with LAH-KLH. Recombinant hemagglutinin from A/Hong Kong/1/1968 was used to coat plates for ELISA.