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

Comparative recognition of STF2.HA1-1, STF2.HA1-2, STF2.HA1-3 and PR8 virus by neutralizing monoclonal antibodies.

<p>A) Ribbon diagram depicting of the known antigenic regions of the HA globular head. B–E) ELISA plates were coated with either PR8 virus or the STF2 fusion proteins and probed with a mAb specific for the Sb, Sa, Ca1, Ca2 and Cb region. All STF2 fusion proteins are produced from E coli. F–I) ELISA plates were coated with STF2.HA1-2 and competed against soluble form of HA1-1, HA1-2, STF2.HA1-2, STF2.HA1-3 in binding of the panel of monoclonal antibodies (H37-64, 18 ng/ml; H37-77, 8 ng/ml; H36-11, 188 ng/ml and H163, 500 ng/ml). Bound antibodies were detected by 450 nm absorption. HA1-1 and STF2.HA1-1 were produced in insect cell culture. Both proteins have C-terminal 6His tag. HA1-2, STF2.HA1-2 and STF2.HA1-3 were produced in E. coli. HA1-2 has 6His tag at C-terminus.</p

STF2.HA1-2 mediated protective immunity against lethal challenge <i>in vivo</i>.

<p>BALB/c mice (10/group) were immunized on day 0 and 14 with 3, 0.3 or 0.03 µg of STF2.HA1-2. A group receiving formulation buffer alone was included as a negative control. A) Sera were harvested on day 21 and evaluated for HA-specific antibody responses by ELISA. B–C) On day 28, mice were challenged <i>i.n.</i> with 1×LD₉₀ of influenza PR8. Survival (B) and weight loss (C) of individual mice were monitored for 21 days post-challenge.</p

Depiction of the HA1-1, HA1-2 and HA1-3 globular head subunits.

<p>A) Ribbon diagram of the trimeric PR8 HA0 ectodomain with a monomeric subunit of the HA trimer circled. B) Ribbon diagram of a monomer with the globular head circled. C) Ribbon diagram of the globular head with the boundaries of the HA1-1, HA1-2 and HA1-3 constructs indicated by crosses. Each construct is presented in detail to the right. The beginning and ending residue numbers, in PR8, for the three constructs are labeled. The important secondary structure elements, such as β-sandwich in HA1-1 and the closing β-sheet in HA1-2 are also marked.</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

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