Immunogenicity and Efficacy of Flagellin-Fused Vaccine Candidates Targeting 2009 Pandemic H1N1 Influenza in Mice

We have previously demonstrated that the globular head of the hemagglutinin (HA) antigen fused to flagellin of Salmonella typhimurium fljB (STF2, a TLR5 ligand) elicits protective immunity to H1N1 and H5N1 lethal influenza infections in mice (Song et al., 2008, PLoS ONE 3, e2257; Song et al., 2009, Vaccine 27, 5875–5888). These fusion proteins can be efficiently and economically manufactured in E. coli fermentation systems as next generation pandemic and seasonal influenza vaccines. Here we report immunogenicity and efficacy results of three vaccine candidates in which the HA globular head of A/California/07/2009 (H1N1) was fused to STF2 at the C-terminus (STF2.HA1), in replace of domain 3 (STF2R3.HA1), or in both positions (STF2R3.2xHA1). For all three vaccines, two subcutaneous immunizations of BALB/c mice with doses of either 0.3 or 3 µg elicit robust neutralizing (HAI) antibodies, that lead to > = 2 Log10 unit reduction in day 4 lung virus titer and full protection against a lethal A/California/04/2009 challenge. Vaccination with doses as low as 0.03 µg results in partial to full protection. Each candidate, particularly the STF2R3.HA1 and STF2R3.2xHA1 candidates, elicits robust neutralizing antibody responses that last for at least 8 months. The STF2R3.HA1 candidate, which was intermediately protective in the challenge models, is more immunogenic than the H1N1 components of two commercially available trivalent inactivated influenza vaccines (TIVs) in mice. Taken together, the results demonstrate that all three vaccine candidates are highly immunogenic and efficacious in mice, and that the STF2R3.2xHA1 format is the most effective candidate vaccine format

Efficacious Recombinant Influenza Vaccines Produced by High Yield Bacterial Expression: A Solution to Global Pandemic and Seasonal Needs

It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines

Purification step, yield, and flagellin bioactivity^a of candidate vaccines.

a<p>flagellin bioactivity was assessed by measuring secreted IL-8 with a sandwich ELISA (BD Biosciences) as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020928#pone.0020928-Song1" target="_blank">[9]</a>. HEK293 cells in 96-well plates (n = 9) were stimulated with 278 ng/ml of each vaccine candidate for 16–18 hours at 37°C. IL-8 concentrations in medium were calculated from an IL-8 standard curve, and expressed as means ± SDs. IL-8 levels of cell control samples were < = 30 pg/ml.</p

SDS-PAGE and antigenicity analyses of three purified recombinant. vaccine candidates.

<p>(<b>A</b>) Purified recombinant proteins were separated on an 4–12% SDS PAGE (0.5 µg protein/lane) and stained with Coomassie Blue. Lane1: STF2.HA1; lane 2: STF2R3.HA1; lane 3, STF2R3.2x HA1; and lane 4, Protein Marker. (<b>B</b>) Reactivity of ferret post infection serum to various vaccine candidates or reference antigen. ELISA plates were coated with serially diluted various CA07 proteins or HA CA04 (Protein Sciences) starting at 320 nmol/l in in PBS, reacted to ferret anti-CA7 serum, and detected with HRP-conjugated goat anti-ferret IgG. Mean OD₄₅₀ of triplicates was read and graphed.</p

HAI titers and survival rates of immunized mice.

<p>Mice were immunized <i>s.c.</i> on days 0 and 21, bled on day 35, and challenged I.N. with 500 TCID50 of mouse adapted A/California/04/2009 on day 42. Infected mice were monitored daily (n = 10) for mortality for 21 days. SP: seroprotective titer, mice% with ≥40 HAI titers.</p><p>Two-way ANOVA/Boferroni tests for HAI data (n = 15):</p>a<p>, <i>p</i><0.01 (**) vs STF2.HA1;</p>b<p>, <i>p</i><0.001 (***) vs STF2R3.2xHA1;</p>c<p>, <i>p</i><0.001 (***) vs STF2R3.2xHA1;</p>d<p>, <i>p</i><0.05 vs STF2R3.2xHA1.</p><p>Fisher's exact test for survival data (n = 10):</p>*<p>, <i>p</i><0.05;</p>**<p>, <i>p</i><0.01;</p>***<p>, <i>p</i><0.001;</p>e<p>, <i>p</i><0.05 (*) vs STF2.HA1 at the same dose (0.03 µg/mouse).</p

Longevity of neutralizing antibody response.

<p>Mice (n = 5) were immunized <i>s.c.</i> with the indicated candidates (1 µg/mouse), and bled monthly for 10 months. Serum neutralizing antibody titers were measured by HAI assay and expressed as GMTs +95%CI. Immunization time points (days 0, 21 and 266) are given in arrows.</p

STF2.HA1-1, STF2.HA1-2 and STF2.HA1-3 expression, purification and immmunoreactivity.

<p>STF2.HA1-1, STF2.HA1-2 and STF2.HA1-3 proteins were expressed and purified. Refolded proteins were analyzed by SDS-PAGE and Western blot analyses. A) Coomassie stained gel showing the proteins run in the presence (R) or absence (N) of reductant. Bands of the appropriate molecular weight were observed for each construct. B) Western blot analyses using the anti-flagellin monoclonal antibody, 6H11. C) Western blot using PR8-specific immune sera raised following a sub-clinical infection of mice with the PR8 virus.</p

Immunoreactivity of STF2.HA1-1, STF2.HA1-2 and STF2.HA1-3 in ELISA.

<p>ELISA plates were coated with the 0.2 µg/well of indicated STF2 fusion proteins, PR8 virus or the full length PR8 HA0 ectodomain expressed in Hi5 cells. Plates were probed with either naïve or PR8 convalescent sera at indicated dilution. Following incubation with HRP-conjugated goat anti-mouse IgG, plates were developed with UltraTMB substrate. Results reflect the delta value of OD₄₅₀ (Convalescence-Naïve) of samples performed in duplicate. Naïve values (data not shown) were below 0.02.</p