Use of equine H3N8 hemagglutinin as a broadly protective influenza vaccine immunogen

Abstract

Development of an efficacious universal influenza vaccines remains a long-sought goal. Current vaccines have shortfalls such as mid/low efficacy and needing yearly strain revisions to account for viral drift/shift. Horses undergo bi-annual vaccines for the H3N8 equine influenza virus, and surveillance of sera from vaccinees demonstrated very broad reactivity and neutralization to many influenza strains. Subsequently, vaccinating mice using the equine A/Kentucky/1/1991 strain or recombinant hemagglutinin (HA) induced similar broadly reactive and neutralizing antibodies to seasonal and high pathogenicity avian influenza strains. Challenge of vaccinated mice protected from lethal virus challenges across H1N1 and H3N2 strains. This protection correlated with neutralizing antibodies to the HA head, esterase, and stem regions. Vaccinated ferrets were also protected after challenge with H1N1 influenza A/07/2009 virus using whole viral or HA. These data suggest that equine H3N8 induces broad protection against multiple influenzas using a unique antigen that diverges from other universal vaccine approaches.

Fig. 1. H3N8 induces bnAbs to human influenza strains.

Sera from vaccinated horses were heat inactivated and RDE treated. A HAI titers were assessed across multiple strains of influenza (n = 15). Line represents a protective reciprocal titer of 40. We do not show baseline pre-vaccination titers here since the horses are a mixed age population. B However, in a limited number of foals that we vaccinated for the first time, we did not find antibodies to any influenza antigen prior to vaccination and found equivalent HAI titers as their peers after vaccination (n = 7, as finding vaccine-free horses is difficult). p < 0.001 against unvaccinated foal sera. Mice received two doses of C H3N8 or D H3N2(A/Texas/50/12) in alum, and HAI serum titers were tested across many strains of influenza. Line indicates a minimum protective reciprocal titer of 40.Similar influenza strains were at least p < 0.001 between H3N8 and H3N2 vaccinees (n = 5 for two reps).

Fig. 2. H3N8 vaccination leads to cross-reactive antibodies and heterosubtypic immunity.

A, B Antibodies from horses were tested against a panel of rHAs (IRR Resources) from seasonal and avian flu strains by ELISA (n = 15). C We vaccinated mice with two doses of our LAIV H3N8 without adjuvant and tested reactivity by ELISA again for IgM and IgG. D, E We then tested for IgM and IgG again after inclusion of alum in the immunization (C–E: n = 5 for two reps each).

Fig. 3. Serum antibodies from vaccinated mice showed binding to HA stem and head domains.

A We performed ELISA for endpoint titers against headless HAs (sHA2) or recombinant HA1 portions of various rHAs (n = 6 mice, 2 reps). HA-reactive antibodies were not detected in sham-vaccinated mice (not shown). p < 0.05 or less for all comparisons to controls. B We performed ELISA competition-binding assays for antigens with pooled vaccinated mice (2x) or horse (1x) sera against panels of known bnAbs. Our sera competed for binding of antibodies specific for the esterase region (mAb H3v-47), head region (mAb H3v-95), or stem region (a recombinant mAb-based on the sequence of CR9114) for binding to HA from H3N2 A/Texas/50/2012. Pools of n = 5 mice or pools of n = 10 horses were used. Antibodies used for this assay are published prior^–.

Fig. 4. Protection against infection after vaccination.

A We vaccinated mice as stated prior in the results twice using rHAs from either H3N8 and tested for HAI responses across many seasonal strains of influenza (n = 5 for two reps). B Next, we tested for survival after H1N1 A/California/07/2009 challenge (4 × LD₅₀). PR8 HA was used here instead of our H1/H3 controls since they contained the H1N1 A/California/07/2009 virus (n = 5 for two reps). C–F Next, we tested for survival after the challenge with 2 × LD₅₀ of viruses stated in the figures. Here, we compared our H3N8 whole vaccine against our H3N8 rHA and a sham or control vaccine containing H1 and H3 viruses (n = 5 for two reps for all animals). Per our animal protocol, we needed to use a 25% weight cutoff before euthanizing animals after the challenge. * p < 0.01 for each survival curve between equine vaccinees (LAIV or rHA) and both controls. The xPR8 in each challenge virus indicates these were viral crosses with A/PR/8/34 to facilitate infection in mice given its mouse adaptation.

Fig. 5. H3N8 protects from heterosubtypic challenge.

A Again, we vaccinated mice with either H3N8 whole virus, rHA from H3N8, sham, or our mix of H1/H3 Cali viruses. We assessed the collected lungs after 5 DP. and stained them with hematoxylin and eosin. (n = 5 with two reps, but representative photos are shown. Shown are A sham control, B H1/H3 Cali, C equine rHA, and D H3N8 LAIV. E Viral lung burdens were determined in 1 mg of the upper right lobe after 6 DPIs from the same mice as (A) by TCID₅₀ assay. 20× magnification was used. The inset for 5C is also 20× magnification but the image size was reduced to show more of the lung histology.

Fig. 6. Vaccination in ferrets leads to cross-reactive HAI antibodies like that in other animal vaccinees.

Ferrets received 3 doses of immunogens with alum and were then challenged 4 weeks after the third vaccination. A HAI responses to the H3N8 virus after each vaccination. * p < 0.001 between equine and control groups. B HAI responses to H1N1 A/California/07/2009 strain after each vaccination. Notice the third vaccine axis is different from subsequent vaccinations. p < 0.001 between equine and control groups. Lines represent HAI titer of 40. A/Texas/50/12 is from H3N2 A/2012.

Fig. 7. Reduced viral shedding in H3N8 vaccinees after challenge with H1N1 A/California/07/09.

Ferrets rested 3 weeks post their third vaccine and were then challenged with 10⁷ TCID₅₀ of H1N1/Cali/09. A Ferrets were euthanized after 6 days, and lungs were stained with anti-influenza A antibody. A representative stain is shown for all groups. Brightfield images were merged with FITC images by the camera software (4× magnification). Green indicates the presence of the influenza virus. B We assessed the viral burdens in nasal swabs 3 days post-infection from the same ferrets using one-step qRT-PCR for a flu-conserved M2 region. C 1 mg of lung tissue was homogenized and filtered, and then virus titer was determined by TCID₅₀ assay. 10× magnification was used in the images.