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. 2019 Jan 17;93(3):e00946-18.
doi: 10.1128/JVI.00946-18. Print 2019 Feb 1.

Elicitation of Protective Antibodies against 20 Years of Future H3N2 Cocirculating Influenza Virus Variants in Ferrets Preimmune to Historical H3N2 Influenza Viruses

James D Allen  1 , Hyesun Jang  1 , Joshua DiNapoli  2 , Harold Kleanthous  2 , Ted M Ross  3   4
Affiliations

Elicitation of Protective Antibodies against 20 Years of Future H3N2 Cocirculating Influenza Virus Variants in Ferrets Preimmune to Historical H3N2 Influenza Viruses

James D Allen et al. J Virol. .

Abstract

The vast majority of people already have preexisting immune responses to influenza viruses from one or more subtypes. However, almost all preclinical studies evaluate new influenza vaccine candidates in immunologically naive animals. Recently, our group demonstrated that priming naive ferrets with broadly reactive H1 COBRA HA-based vaccines boosted preexisting antibodies induced by wild-type H1N1 virus infections. These H1 COBRA hemagglutinin (HA) antigens induced antibodies with HAI activity against multiple antigenically different H1N1 viral variants. In this study, ferrets, preimmune to historical H3N2 viruses, were vaccinated with virus-like particle (VLP) vaccines expressing either an HA from a wild-type H3 influenza virus or a COBRA H3 HA antigen (T6, T7, T10, or T11). The elicited antisera had the ability to neutralize virus infection against either a panel of viruses representing vaccine strains selected by the World Health Organization or a set of viral variants that cocirculated during the same time period. Preimmune animals vaccinated with H3 COBRA T10 HA antigen elicited sera with higher hemagglutination inhibition (HAI) antibody titers than antisera elicited by VLP vaccines with wild-type HA VLPs in preimmune ferrets. However, while the T11 COBRA vaccine did not elicit HAI activity, the elicited antibodies did neutralize antigenically distinct H3N2 influenza viruses. Overall, H3 COBRA-based HA vaccines were able to neutralize both historical H3 and contemporary, as well as future, H3N2 viruses with higher titers than vaccines with wild-type H3 HA antigens. This is the first report demonstrating the effectiveness of a broadly reactive H3N3 vaccine in a preimmune ferret model.IMPORTANCE After exposure to influenza virus, the host generates neutralizing anti-hemagglutinin (anti-HA) antibodies against that specific infecting influenza strain. These antibodies can also neutralize some, but not all, cocirculating strains. The goal of next-generation influenza vaccines, such as HA head-based COBRA, is to stimulate broadly protective neutralizing antibodies against all strains circulating within a subtype, in particular those that persist over multiple influenza seasons, without requiring an update to the vaccine. To mimic the human condition, COBRA HA virus-like particle vaccines were tested in ferrets that were previously exposed to historical H3N2 influenza viruses. In this model, these vaccines elicited broadly protective antibodies that neutralized cocirculating H3N2 influenza viruses isolated over a 20-year period. This is the first study to show the effectiveness of H3N3 COBRA HA vaccines in a host with preexisting immunity to influenza.

Keywords: COBRA; H3N2; broadly protective vaccine; ferrets; hemagglutination inhibition; influenza; neutralization.

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Figures

FIG 1
FIG 1
HAI serum antibody titers induced by vaccination of ferrets with wild-type H3N3 VLP vaccines. HAI titers were determined for each group of immunologically naive ferrets (n = 4) vaccinated two times (days 84 and 168) with one of the four COBRA H3N3 VLP vaccines (T6, T7, T10, or T11) or H3N3 VLP vaccines expressing wild-type HA proteins from Wisc/05 or TX/12 against a panel of 13 H3N2 influenza viruses. Values are the log2 HAI titers of each individual animal from antisera collected on day 182. The two dotted lines indicate the 1:40 to 1:80 HAI titer range. (A) T6 VLP; (B) T7 VLP; (C) T10 VLP; (D) T11 VLP; (E) Wisc/05 VLP; (F) TX/12 VLP.
FIG 2
FIG 2
HAI serum antibody titers induced by H3N2 viral infections. HAI serum antibody titers induced by H3N2 influenza virus infection of immunologically naive ferrets were determined. Ferrets were infected with Pan/99 (A), Wisc/05 (B), or TX/12 (C) H3N2 influenza viruses. At day 84, serum samples were collected, and HAI titers were determined for each group of ferrets against a panel of 13 H3N2 influenza viruses. Values are the log2 HAI titers of each individual animal. Dotted lines indicate the 1:40 to 1:80 HAI titer range.
FIG 3
FIG 3
HAI serum antibody titers induced by COBRA HA and wild-type HA H3N3 VLP vaccination in Pan/99 preimmune ferrets. Immunologically naive ferrets were infected with the Pan/99 influenza virus. At days 84 and 168 postinfection these preimmune ferrets were vaccinated with H3N3 VLP vaccines expressing different HA proteins. (A) T6 VLP; (B) T7 VLP; (C) T10 VLP; (D) T11 VLP; (E) Wisc/05 VLP; (F) TX/12 VLP. HAI titers were determined for each group of ferrets at day 182 against a panel of 13 H3N2 influenza viruses isolated between 1995 and 2016. Values are the log2 HAI titers of each individual animal from antisera collected on day 182. Dotted lines indicate the 1:40 to 1:80 HAI titer range.
FIG 4
FIG 4
FRA. FRA neutralizing titers were determined for each group of immunologically naive ferrets (n = 4) vaccinated two times (days 84 and 168) with one of the four COBRA H3N3 VLP vaccines (T6, T7, T10, or T11) or H3N3 VLP vaccines expressing wild-type HA proteins from Wisc/05 or TX/12. At day 182, sera were collected and tested in an FRA against four H3N2 viruses isolated between 2012 and 2016 representing clades 3c.1 (TX/12) (A and I), 3c.3a (Switz/13) (B and J), 3c.2a (HK/14) (C and K), and 3c.2a1 (Sing/16) (D and L). Alternatively, ferrets were infected with wild-type H3N2 influenza viruses (Pan/99, Wisc/05, or TX/12), and sera collected at day 182 postinfection were also tested in an FRA assay against the four H3N2 viruses (A to D). Ferrets infected with Pan/99 were also subsequently vaccinated with H3N3 VLPs expressing one of four COBRA H3 HA antigens or the Wisc/05 or TX/12 wild-type HA proteins. Collected sera was assayed against the four H3N2 viruses (I to L). For each virus, the virus concentration was standardized to 1.2 × 104 FFU/ml. The dotted lines represent the 50% inhibition and the 80% inhibition of viral infection by antisera compared to virus-only control wells (A to D and I to L). A heat map of the log2 serum dilution titer for the 50 and 80% inhibition per virus for each group of ferrets (E to H and M to P) was also generated. Colors range from white (lowest level of inhibition) to dark blue (highest level of inhibition).
FIG 5
FIG 5
Comparison of log2-fold change in HAI GMT, and 50 and 80% FRA neutralization titers between VLP-vaccinated and preimmune vaccinated ferrets. Fold changes in HAI and FRA at day 182 were compared between immunologically naive ferrets (n = 4) vaccinated two times (days 84 and 168) with one of the four COBRA H3N3 VLP vaccines (T6, T7, T10, or T11) or H3N3 VLP vaccines expressing wild-type HA proteins from Wisc/05 or TX/12 and preimmune ferrets that were vaccinated two times (days 84 and 168) with H3N3 VLP vaccines expressing the same HA proteins used to immunize the naive ferrets. Fold change differences were compared against four H3N2 viruses isolated between 2012 and 2015 representing clades 3c.1 (TX/12) (A), 3c.3a (Sz/13) (B), 3c.2a (HK/14) (C), and 3c.2a1 (Sing/16) (D). Statistical analyses were performed using unpaired parametric t tests to determine significance of vaccination in the preimmune setting compared to naive ferrets. P values of <0.05 are considered significant and are colored blue.
FIG 6
FIG 6
Frequencies of influenza HA clusters in consecutive seasons between 2007 and 2016. Influenza HA sequences posted in GISAID databases were aligned and clustered into families per Northern and Southern Hemisphere seasons. The clusters in each pie chart that differ by 5% in amino acids in the HA sequence are depicted by a different color and slice of the pie chart. Each influenza season for the Northern and Southern Hemispheres from 2007 to 2017 to 2018 is depicted. Representative influenza viruses from each cluster in the pie charts are listed and match the color of the pie charts.
FIG 7
FIG 7
Phylogenetic tree of H3 HA sequences. The rooted (A/Nangchang/933/1995) phylogenetic tree was inferred from COBRA HA and wild-type HA amino acid sequences derived from the representative H3N2 variant viruses from 1995 to 2015 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 (45).
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