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. 2022 Apr 13;96(7):e0165221.
doi: 10.1128/jvi.01652-21. Epub 2022 Mar 15.

Bivalent H1 and H3 COBRA Recombinant Hemagglutinin Vaccines Elicit Seroprotective Antibodies against H1N1 and H3N2 Influenza Viruses from 2009 to 2019

Affiliations

Bivalent H1 and H3 COBRA Recombinant Hemagglutinin Vaccines Elicit Seroprotective Antibodies against H1N1 and H3N2 Influenza Viruses from 2009 to 2019

James D Allen et al. J Virol. .

Abstract

Commercial influenza virus vaccines often elicit strain-specific immune responses and have difficulties preventing illness caused by antigenically drifted viral variants. In the last 20 years, the H3N2 component of the annual vaccine has been updated nearly twice as often as the H1N1 component, and in 2019, a mismatch between the wild-type (WT) H3N2 vaccine strain and circulating H3N2 influenza strains led to a vaccine efficacy of ∼9%. Modern methods of developing computationally optimized broadly reactive antigens (COBRAs) for H3N2 influenza viruses utilize current viral surveillance information to design more broadly reactive vaccine antigens. Here, 7 new recombinant hemagglutinin (rHA) H3 COBRA hemagglutinin (HA) antigens were evaluated in mice. Subsequently, two candidates, J4 and NG2, were selected for further testing in influenza-preimmune animals based on their ability to elicit broadly reactive antibodies against antigenically drifted H3N2 viral isolates. In the preimmune model, monovalent formulations of J4 and NG2 elicited broadly reactive antibodies against recently circulating H3N2 influenza viruses from 2019. Bivalent mixtures of COBRA H1 and H3 rHA, Y2 + J4, and Y2 + NG2 outperformed multiple WT H1+H3 bivalent rHA mixtures by eliciting seroprotective antibodies against H1N1 and H3N2 isolates from 2009 to 2019. Overall, the newly generated COBRA HA antigens, namely, Y2, J4, and NG2, had the ability to induce broadly reactive antibodies in influenza-naive and preimmune animals in both monovalent and bivalent formulations, and these antigens outperformed H1 and H3 WT rHA vaccine antigens by eliciting seroprotective antibodies against panels of antigenically drifted historical H1N1 and H3N2 vaccine strains from 2009 to 2019. IMPORTANCE Standard-of-care influenza virus vaccines are composed of a mixture of antigens from different influenza viral subtypes. For the first time, lead COBRA H1 and H3 HA antigens, formulated as a bivalent vaccine, have been investigated in animals with preexisting immunity to influenza viruses. The cocktail of COBRA HA antigens elicited more broadly reactive anti-HA antibodies than those elicited by a comparator bivalent wild-type HA vaccine against H1 and H3 influenza viruses isolated between 2009 and 2019.

Keywords: COBRA; H1N1; H3N2; bivalent; bivalent vaccine; broadly reactive antibody; cocktail; hemagglutinin; hemagglutinin vaccine; imprinting; influenza; preimmunity; universal influenza.

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Conflict of interest statement

The authors declare no conflict of interest.

We declare no competing interests.

Figures

FIG 1
FIG 1
Influenza-naive mice, day 70 hemagglutination inhibition (HAI) H3N2 2012 to 2019 historical vaccine strain panel. Serum collected from 75 influenza-naive BALB/c mice (n = 5/group) on day 70 post initial vaccination were assessed for HAI activity against a panel of historical H3N2 vaccine strains isolated during 2012 to 2019 (x axis). Log2 HAI titers are reported as absolute mean values ± standard error of the mean (SEM) (y axis). The lower dotted line represents an HAI titer of 1:40, and the upper dotted line represents an HAI titer of 1:80. The mice were vaccinated with 3 μg recombinant hemagglutinin (rHA) as follows: TJ5 (A), J1 (B), J2 (C), J3 (D), J4 (E), NG1 (F), NG2 (G), NG3 (H), Switz/13 (I), HK/14 (J), Sing/16 (K), Kan/17 (L), Switz/17 (M), SA/19 (N), and mock (O). HAI titers were statistically analyzed using nonparametric one-way analysis of variance (ANOVA) by Prism 9 software (GraphPad Software, Inc., San Diego, CA). A P value of less than 0.05 was defined as statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). The H3N2 viruses belong to the following clades: Tx/12 (3c2), Switz/13 (3c3.a), HK/14 (3c.2a), Sing/16 (3c2.a1), Kan/17 (3c3.a), Tx17 (3c3.a), Switz/17 (3c3.a2), SA/19 (3c2.ab/131K), and HK/19 (3c2.a1b/137F).
FIG 2
FIG 2
HAI titers of preimmune mice on day 72: monovalent vaccine groups. Serum collected from 72 H1 + H3 preimmune DBA/2J mice (n = 8/group) that were vaccinated with monovalent formulations of computationally optimized broadly reactive antigens (COBRA) or wild-type (WT) rHA, on day 72 post initial infection were assessed for HAI activity against a panel of historical H1N1 vaccine strains from 2007 to 2019 (green bar) and H3N2 vaccine strains from 2012 to 2019 (blue bar) (separated by the vertical dotted line) (x axis). Log2 HAI titers are reported as absolute mean values ± SEM (y axis). The lower dotted line represents an HAI titer of 1:40, and the upper dotted line represents an HAI titer of 1:80. The mice were vaccinated with 3 μg rHA as follows: Bris/07 (A), Cal/09 (B), Y2 (C), Switz/13 (D), Sing/16 (E), TJ5 (F), J4 (G), NG2 (H), and mock (I). HAI titers were statistically analyzed using nonparametric one-way ANOVA by Prism 9 software (GraphPad Software, Inc., San Diego, CA). A P value of less than 0.05 was defined as statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). The H3N2 viruses belong to the following clades: Tx/12 (3c2), Switz/13 (3c3.a), HK/14 (3c.2a), Sing/16 (3c2.a1), Kan/17 (3c3.a), Tx17 (3c3.a), Switz/17 (3c3.a2), SA/19 (3c2.ab/131K), and HK/19 (3c2.a1b/137F).
FIG 3
FIG 3
HAI titers of preimmune mice on day 72: bivalent cocktail vaccine groups. Serum collected from 64 H1 + H3 preimmune DBA/2J mice (n = 8/group) that were vaccinated with bivalent cocktail formulations of H1 + H3 COBRA or WT rHA, were assessed on day 72 post initial infection for HAI activity against a panel of historical H1N1 vaccine strains from 2007 to 2019 (green bar) and H3N2 vaccine strains from 2012 to 2019 (blue bar) (separated by the vertical dotted line) (x axis). Log2 HAI titers are reported as absolute mean values ± SEM (y axis). The lower dotted line represents an HAI titer of 1:40, and the upper dotted line represents an HAI titer of 1:80. The mice were vaccinated with 3 μg total rHA as follows: Bris/07 + Switz/13 (A), Bris/07 + Sing/16 (B), Cal/09 + Switz/13 (C), Cal/09 + Sing/16 (D), Y2 + TJ5 (E), Y2 + J4 (F), Y2 + NG2 (G), and mock (H). HAI titers were statistically analyzed using nonparametric one-way ANOVA by Prism 9 software (GraphPad Software, Inc., San Diego, CA). A P value of less than 0.05 was defined as statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). The H3N2 viruses belong to the following clades: Tx/12 (3c2), Switz/13 (3c3.a), HK/14 (3c.2a), Sing/16 (3c2.a1), Kan/17 (3c3.a), Tx17 (3c3.a), Switz/17 (3c3.a2), SA/19 (3c2.ab/131K), and HK/19 (3c2.a1b/137F).
FIG 4
FIG 4
FRA titers of preimmune mice on day 72: H1N1 virus panel. On day 72 post initial infection, serum collected from 136 H1 + H3 preimmune DBA/2J mice (n = 8/group) that were vaccinated with monovalent and bivalent formulations of H1+H3 COBRA or WT rHA were pooled for each group and assessed for FRA neutralization against a panel of historical H1N1 vaccine strains from 2009 to 2019. Sera from mice vaccinated with monovalent antigens were tested against the following H1N1 viruses: A/California/07/2009 (A), A/Brisbane/2/2018 (B), and A/Guangdong Maonan/SWL1536/2019 (C). Sera from mice vaccinated with cocktails of bivalent H1+H3 antigens were tested against the following H1N1 viruses: A/California/07/2009 (D), A/Brisbane/2/2018 (E), and A/Guangdong Maonan/SWL1536/2019 (F). The lower dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50), and the upper dotted line represents no neutralization of viral infection.
FIG 5
FIG 5
FRA titers of preimmune mice on day 72: H3N2 virus panel. On day 72 post initial infection, serum collected from 136 H1 + H3 preimmune DBA/2J mice (n = 8/group) that were vaccinated with monovalent and bivalent formulations of H1+H3 COBRA or WT rHA were pooled for each group and assessed for FRA neutralization against a panel of historical H3N2 vaccine strains from 2016 to 2019. Sera from mice vaccinated with monovalent antigens were tested against the following H1N1 viruses: A/Singapore/IFNIMH-16-0019/2016 (A), A/Kansas/14/2017 (B), and A/Hong Kong/2671/2019 (C). Sera from mice vaccinated with cocktails of bivalent H1+H3 antigens were tested against the following H1N1 viruses: A/Singapore/IFNIMH-16-0019/2016 (D), A/Kansas/14/2017 (E), and A/Hong Kong/2671/2019 (F). The lower dotted line represents 80% neutralization (Neut80), the middle dotted line represents 50% neutralization (Neut50), and the upper dotted line represents no neutralization of viral infection.
FIG 6
FIG 6
D86 H3N2 viral challenge weight loss. A total of 136 preimmune DBA/2J mice (n = 8/group) were infected on day 86 with 6.7 × 106 PFU/50 μL of A/Kansas/14/2017 (H3N2) influenza virus. The mice were tracked for 14 consecutive days (D1 to D14) following challenge (x axis), and the percentage of their original body weight lost is reported (y axis). Data are separated by mice vaccinated with monovalent vaccine antigens (A) and those vaccinated with cocktails of bivalent rHA antigens (B). The lower dotted line represents the weight loss associated with the humane endpoint (80% original body weight), and the upper dotted line represents 100% original body weight.
FIG 7
FIG 7
Day 89 lung viral titers. Lungs were collected from n = 3 mice per group on day 89 (3 days post A/Kansas/14/2017 challenge) to assess the viral load present in the lung tissue. Vaccine groups are listed on the x axis, and PFU/g of lung tissue values are reported on the y axis. A nonparametric one-way ANOVA was used to analyze statistical differences between groups using Prism 9 software (GraphPad Software, Inc., San Diego, CA). A P value of less than 0.05 was defined as statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

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