Split inactivated COBRA vaccine elicits protective antibodies against H1N1 and H3N2 influenza viruses

Abstract

Development of broadly reactive or universal influenza vaccines will be a paradigm shifting event for the influenza vaccine field. These next generation vaccines could replace the current standard of care with vaccines that elicit broadly cross-protective immune responses. However, a variety of in vitro and in vivo models are necessary to make the best assessments of these vaccine formulations to determine their mechanisms of action, and allow for downselection of candidates prior to human clinical trials. Our group has developed the computationally optimized broadly reactive antigen (COBRA) technology to develop HA head-based strategies to elicit antibodies against H1, H3, and H5 influenza strains. These vaccines elicit broadly reactive antibody responses that neutralize not only historical and contemporary vaccine strains, but also co-circulating variants in mice. In this study, we used H1 and H3 HA antigens in a split, inactivated vaccine (IIV) formulation in combination with the AF03 squalene-in-water emulsion adjuvant in ferrets immunologically naïve to influenza virus. The H3 COBRA IIV vaccine T11 elicited antibodies with HAI activity against more H3N2 influenza strains compared to IIV expressing wild-type H3 HA antigens, except for IIV vaccines expressing the HA from A/Texas/50/2012 (Tx/12) virus. H1 COBRA IIV vaccines, P1 and X6, elicited antibodies that recognized a similar number of H1N1 viruses as those antibodies elicited by IIV expressing the A/California/07/2009 (CA/09) HA. Ferrets vaccinated with the P1 or X6 COBRA IIV were protected against CA/09 challege and cleared virus from the lungs of the ferrets, similar to ferrets vaccinated with the CA/09 IIV.

Fig 1. Schematic of IIV vaccination of naive ferrets.

Ferrets (n = 8) were vaccinated with IIV vaccine at day 0 and again at day 28. Blood was collected prior to initial infection and at days 28 and 56 post-vaccination. At day 56, the ferrets were challenged with either A/California/07/2009 (H1N1) virus (5x10⁴ PFU) or A/Wisconsin/67/2005 (H3N2) virus (1x10⁷ PFU) and monitored for morbidity and mortality for 14 days post-infection. Nasal washes were collected at days 0, 1, 3, 5, and 7 to assess viral titers.

Fig 2. Hemagglutination inhibition serum antibody titers induced by vaccination of ferrets with H3N2 IIV vaccines expressing COBRA HA antigens or HA proteins from wild-type viruses.

HAI titers were determined for each group of immunologically naïve ferrets (n = 8) vaccinated two times (days 0 and 28) with 1 of the 2 COBRA H3N2 IIV vaccines (T7 or T11) or H3N2 VLP vaccines expressing wild-type HA proteins from Wisc/05, Uru/07, TX/12, Sz/13, HK/14 against a panel of 13 H3N2 influenza viruses. Values are the individual animal HAI titers from antisera collected on day 56. The two dotted lines indicates the 1:40–1:80 HAI titer range. (A) T6 IIV; (B) T11 IIV; (C) Wisc/05 IIV; (D) Uru/07 IIV; (E) TX/12 IIV; (F) Sz/13 IIV; (G) HK/14 IIV; (H) Mock.

Fig 3. Hemagglutination inhibition serum antibody titers induced by vaccination of ferrets with H1N1 IIV vaccine expressing COBRA HA antigens or HA proteins from wild-type viruses.

HAI titers were determined for each group of immunologically naïve ferrets (n = 8) vaccinated two times (days 0 and 28) with 1 of the 3 COBRA H1N1 IIV vaccines (P1, X3 or X6) or H1N1 IIV vaccines expressing wild-type HA proteins from CA/09 against a panel of 8 H1N1 influenza viruses. Values are the individual animal HAI titers from antisera collected on day 56. The two dotted lines indicates the 1:40–1:80 HAI titer range. (A) CA/09 IIV; (B) P1 IIV; (C) X3 IIV; (D) X6 IIV.

Fig 4. Challenge of vaccinated ferrets with influenza viruses.

Vaccinated or unvaccinated ferrets were infected with A/Wisconsin/67/2005 (A and B) or A/California/07/2009 (C) influenza viruses. (A-C) Ferrets were monitored daily for weight loss over an 8-day observation period. Values are average percentages of original weight plus the SEM (error bars). (D) Viral titers were determined from nasal washes infected with A/California/07/2009 and collected at days 1, 3, and 5 post-challenge. Each ferret is represented with a symbol. Lines indicate mean virus titers with the standard deviations (SD). The values for CA/09, P1, and X6 are all significant (p>0.01) compared to the values at each timepoint for X3 and Mock. CA/09 was significant compared to P1 and X6 at day 3 post-infection (p>0.05). Significance was determined using paired or unpaired t tests (*p>0.05; **p >0.01).

Fig 5. Phylogenetic tree of H1 HA sequences.

The unrooted phylogenetic tree was inferred from HA amino acid sequences derived from 11 H1 HA isolates and COBRA HA using the maximum likelihood method. Sequences were aligned with MUSCLE 3.7 software, and the alignment was refined by Gblocks 0.91b software. Phylogeny was determined using the maximum likelihood method with PhyML software. Trees were rendered using TreeDyn 198.3 software [32].

Fig 6. Phylogenetic tree of H3 HA sequences.

The unrooted phylogenetic tree was inferred from HA amino acid sequences derived from 30 H3 HA and COBRA HA sequences using the maximum likelihood method. Sequences were aligned with MUSCLE 3.7 software, and the alignment was refined by Gblocks 0.91b software. Phylogeny was determined using the maximum likelihood method with PhyML software. Trees were rendered using TreeDyn 198.3 software [32].