Immune responses and protection following conjugation of rare occurring peptides to the H5N1-HA antigen C-terminus.

Individual 5-mer, 9-mer, or 13-mer peptides consisting of amino acids that occur rarely or never in sequence were added to the end of the H5N1-HA antigen by PCR. (a) Pooled T-cell immune responses generated by rare, semi-common, and common peptides. Groups of 3–4 BALB/c mice were immunized with pools of 10 peptides representing 200 rare, 200 semi-common, and 200 common peptides and cellular immune responses were detected 10 days post-vaccination by visualization of IFN-γ secretion by spot forming cells (SFC). The data, representing the average SFC in each category of peptide, is representative of 3 independent experiments and is normalized based on comparison with the HA DNA vaccine alone to distinguish changes with respect to peptide frequency. The average SFC baseline from unstimulated control splenocytes is (40±8). (b) T-cell responses following vaccination. Groups of 4 BALB/c mice were immunized with 50 µg of each HA-peptide construct and cellular immune responses were detected 10 days post-immunization. (c) Hemagglutination inhibition and neutralizing antibody responses at day 25 post-vaccination. Serum was individually evaluated from 8–10 BALB/c mice immunised with HA-5mer4 (1 µg), HA-5mer6 (1 µg), or HA alone (1 µg). HI antibody titres were detected using horse red blood cells. NAB titres were evaluated by monitoring MDCK cells for the presence of CPE. The dotted line represents the limit of detection of the HI and NAB assays. (*p<0.0001, **p<0.0001) (d) Survival against lethal H5N1-H05 challenge. Groups of 10 BALB/c mice were immunized with control PBS ▪, HA-5mer4 (1 µg) ♦, HA-5mer6 (1 µg) ▴, or H5N1-HA (1□, 5 ▾, or 10 µg •) and then challenged with 100LD50 of homologous H5N1-H05 virus. Animals were monitored over a period of 15 days. (*p<0.01) Error bars represent the standard error of the mean (SEM).