Figure 1.
Recombinant soluble HA constructs.
A. Full length H3N2 A/Aichi/2/68 hemagglutinin precursor (HA0). TM = transmembrane domain, CT = cytosolic domain. Cleavage site at R328 is indicated by arrow. B. Soluble H3N2 A/Aichi/2/68 HA constructs. Soluble H3N2 HA was generated by eliminating the transmembrane (TM) and cytosolic (CT) domain. The modified trimeric soluble H3N2 HA was generated by fusing the GCN4pII trimerization repeat to the C-terminus of HA with a short peptide linker (G6S9).
Figure 2.
Recombinant H3N2 A/Aichi/2/68 is expressed as the hemagglutinin precursor (HA0).
A,B. Western blot analysis of SDS-PAGE separated sHA (Lane 1) and sHA.GCN4pII (Lane 2) using polyclonal sera or anti-histidine tag monoclonal antibody (primary antibody). Protein bands were developed using goat anti-mouse HRP (secondary antibody) and ECL Plus (GE Healthcare). C. Coomassie blue stain of sHA (Lane 1) and sHA.GCN4pII (Lane 3) after nickel-bead column purification. Lane 2 - molecular weight marker. Each lane was loaded with 1 µg of recombinant protein.
Figure 3.
GCN4pII modification stabilizes the trimeric structure of A/Aichi/2/68 soluble HA.
A. BS3 crosslinking was performed as described in Materials and Methods. Lanes 1 and 2 – sHA, Lanes 3 and 4 – sHA.GCN4pII. Lane 1 and 3, no BS3, Lanes 2 and 4, 3 mM BS3. Trimeric HA corresponded to a band of ∼220 kDa, dimeric HA corresponds to a band of ∼140 kDa, and monomeric HA corresponded to a band of ∼75 kDA Western blot primary antibody, anti-histidine monoclonal antibody. B. Sandwich ELISA. 20 µg of sHA (red circle) or sHA.GCN4pII (blue square) were captured using guinea pig anti-A/Aichi/2/68 then binding affinity of HC67, LC89 or polyclonal sera was detected by absorbance at 450 nm. C. Hemagglutination test using 1 µg of recombinant protein (sHA or sHA.GCN4pII) or PBS. Proteins were diluted in PBS and incubated at room temperature for 30 minutes with 0.05% chicken red blood cells (washed).
Figure 4.
sHA.GCN4pII induces more robust humoral responses compared to sHA.
A. Mice were primed and boosted 3 weeks later with 3 µg of sHA (red), sHA.GCN4pII (blue) or PBS (naïve – green). Blood was collected day 21 after priming (prime) and 21 days after boosting (boost). ELISA plates were coated with 4 µg/mL of A/Aichi/2/68 virus and antigen specific serum IgG ELISA was measured for prime and boost. HRP-conjugated goat anti-mouse IgG was used for detection. B. IgG subtype ELISA. Sera collected 21 days after boosting was tested antigen-specific IgG subclass by ELISA as described in Materials and Methods. ELISA plates were coated with 4 µg/mL of A/Aichi/2/68 and antigen specific IgG subclasses were detected using HRP-conjugated goat anti-mouse IgG1 or IgG2a. C. Hemagglutination inhibition test of prime and boost sera was performed as described in Materials and Methods using live MDCK grown A/Aichi/2/68. (n = 6 per group)
Figure 5.
sHA.GCN4pII provides complete protection after two vaccinations unlike sHA.
Mice primed and boosted were challenged with 5 xLD50 of mouse adapted H3N2 A/Aichi/2/68. A, B. Body weights and survival were followed for 14 days post challenge. sHA (circle), sHA.GCN4 (square), PBS (inverted triangle). Open symbols with dashed line indicate % mean initial body weight of surviving mice for each group. Closed symbols with solid line indicate % mean initial body weight of mice showing signs of morbidity. (n = 6 per group)
Figure 6.
sHA.GCN4pII induces stronger protective immune response after a single vaccination compared to sHA.
A. Serum IgG ELISA. Plates were coated with 4 µg/mL of A/Aichi/2/68 and antigen specific IgG was detected in sera collected on day 14 and day 28 post-vaccination using HRP-conjugated goat anti-mouse IgG. Mice vaccinated with recombinant Aichi HA proteins were challenged on day 31 after vaccination with 5 x LD50 of mouse adapted A/Aichi/2/68. B, C. Body weights and survival were followed for 14 days post challenge. sHA (circle), sHA.GCN4 (square), PBS (inverted triangle). Open symbols with dashed lines indicate % mean initial body weight of surviving mice for each group. Closed symbols with solid lines indicate % mean initial body weight of mice showing signs of morbidity. (n = 6 per group)