Computationally Optimized Broadly Reactive Hemagglutinin Elicits Hemagglutination Inhibition Antibodies against a Panel of H3N2 Influenza Virus Cocirculating Variants

Abstract

Each influenza season, a set of wild-type viruses, representing one H1N1, one H3N2, and one to two influenza B isolates, are selected for inclusion in the annual seasonal influenza vaccine. In order to develop broadly reactive subtype-specific influenza vaccines, a methodology called computationally optimized broadly reactive antigens (COBRA) was used to design novel hemagglutinin (HA) vaccine immunogens. COBRA technology was effectively used to design HA immunogens that elicited antibodies that neutralized H5N1 and H1N1 isolates. In this report, the development and characterization of 17 prototype H3N2 COBRA HA proteins were screened in mice and ferrets for the elicitation of antibodies with HA inhibition (HAI) activity against human seasonal H3N2 viruses that were isolated over the last 48 years. The most effective COBRA HA vaccine regimens elicited antibodies with broader HAI activity against a panel of H3N2 viruses than wild-type H3 HA vaccines. The top leading COBRA HA candidates were tested against cocirculating variants. These variants were not efficiently detected by antibodies elicited by the wild-type HA from viruses selected as the vaccine candidates. The T-11 COBRA HA vaccine elicited antibodies with HAI and neutralization activity against all cocirculating variants from 2004 to 2007. This is the first report demonstrating broader breadth of vaccine-induced antibodies against cocirculating H3N2 strains compared to the wild-type HA antigens that were represented in commercial influenza vaccines.IMPORTANCE There is a need for an improved influenza vaccine that elicits immune responses that recognize a broader number of influenza virus strains to prevent infection and transmission. Using the COBRA approach, a set of vaccines against influenza viruses in the H3N2 subtype was tested for the ability to elicit antibodies that neutralize virus infection against not only historical vaccine strains of H3N2 but also a set of cocirculating variants that circulated between 2004 and 2007. Three of the H3N2 COBRA vaccines recognized all of the cocirculating strains during this era, but the chosen wild-type vaccine strains were not able to elicit antibodies with HAI activity against these cocirculating strains. Therefore, the COBRA vaccines have the ability to elicit protective antibodies against not only the dominant vaccine strains but also minor circulating strains that can evolve into the dominant vaccine strains in the future.

Keywords: COBRA; H3N2; hemagglutination-inhibition; influenza; mice.

FIG 1

Characterization of the H3N2 influenza COBRA HA vaccines. (A) The unrooted phylogenetic tree was inferred from HA amino acid sequences derived from 17 representative H3N2 isolates and also the 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 (46). (B) Genetic map of the HA of H3N2 viruses isolated between 1968 and 2015 that was generated from the numbers of amino acid substitutions between strains in the antigenic map. The two principal components, principal component 1 (PC1) and principal component 2 (PC2), are shown on the x and y axes. The clusters are colored according to the era of isolation. The spheres that represent each COBRA HA are shown in gold circles, and wild-type HA proteins are depicted as black dots. (C) Schematic of the timeline of H3N2 influenza infections in humans. Each bubble represents a time period. (Top) The time period that H3N2 HA sequences were used to develop each COBRA HA sequence. (Bottom) Time interval that each H3N2 vaccine component was used in the seasonal influenza inactivated vaccine over the past 48 years, from 1968 to 2016.

FIG 2

Schematics of putative glycosylation sites in selected H3 HA vaccines. (A) Nine wild-type H3 HA and four COBRA HA amino acids are depicted as putative N-linked glycosylation sites based upon the N{P}[ST]{P} search parameters, and these potential N-glycosylation sites were analyzed by using the NetNGlyc 1.0 server (37). Each putative N-glycosylation site is depicted with a purple arrow underneath the Arg amino acid in the HA sequence. (B) The N-glycosylation site predicted with a ++ or +++ score would be identified as a strong potential one with asparagine N-glycosylated. Low or no N-glycosylation sites, with +, −, or −− scores for N-glycosylation results, indicated low likelihood of an N-glycosylation site.

FIG 3

HAI serum antibody titers induced by vaccination of mice with wild-type H3N2 VLP vaccines. HAI titers were determined for each group of mice (n = 5) vaccinated three times (days 0, 28, and 56) with 1 of the 10 H3N2 VLP vaccines expressing surface wild-type HA proteins against a panel of 14 H3N2 influenza viruses. Values are the geometric mean titers plus standard errors of the means (SEM) (error bars) from antisera collected on day 70. The gray bar indicates the 1:40 to 1:80 HAI titer range. The strains are grouped by antigenic eras as depicted by colored bars on the x axis.

FIG 4

HAI serum antibody titers induced by vaccination of mice with COBRA H3N2 VLP vaccines. HAI titers were determined for each group of mice (n = 5) vaccinated three times (days 0, 28, and 56) with 1 of the 17 H3N2 COBRA VLP vaccines against a panel of 14 H3N2 influenza viruses. Values are the geometric mean titers plus standard errors of the means (SEM) (error bars) from antisera collected on day 70. The gray bar indicates the 1:40 to 1:80 HAI titer range. The strains are grouped by antigenic eras as depicted by colored bars on the x axis.

FIG 5

Total number of strains and antigenic eras from vaccine panel recognized by the vaccine-elicited antibodies. Antisera collected from mice vaccinated with wild-type or COBRA VLP vaccines were tested for HAI activity. (A and B) Viral strain (A) or antigenic era of strains (B) detected by the antisera from each mouse in a vaccine group was considered positive only if all mice had antisera with a GMT greater than 1:40 or 1:80 and, for the antigenic era, a titer of 1:80. The strains detected were assessed as positive, and the number of strains in the panel inhibited at the indicated titers was recorded.

FIG 6

Frequencies of influenza HA clusters in consecutive seasons between 2004 and 2007. Influenza HA sequences posted in GISAID databases were aligned and clustered into family per season. Three Northern Hemisphere seasons (A, C, and E) and two Southern Hemisphere seasons (B, D, and F) that differ by 2% in amino acids in the HA sequence are depicted in clusters in each pie chart. (G) Representative influenza viruses from each cluster in the 5 pie charts are listed and match the color of the pie charts. (H) The unrooted phylogenetic tree was inferred from COBRA HA and HA amino acid sequences derived from the representative H3N2 variant viruses from seasonal clusters between 2004 and 2007 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 Geneious software (46).

FIG 7

H3N2 influenza drift variants circulating between 2004 and 2007. Pooled mouse sera from mice vaccinated with one of four wild-type HA VLP vaccines (Fuj/02, Wisc/05, Bris/07, and Per/09) and four COBRA HA VLP vaccines (T-7, T-8, T-10, and T-11) were tested for HAI activity against variant viruses representing different clusters of influenza H3N2 isolates, as depicted in Fig. 6. Values are the average titers from antisera collected on day 70 against the panel of 15 vaccine and variant H3N2 viruses, representing four vaccine and 11 variant strains isolated from 2006 and 2007.

FIG 8

Focal reduction assay (FRA). BALB/c mice were vaccinated at days 0, 28, and 56 with VLPs expressing WT HA antigens from Fuj/02, Wisc/05, Bris/07, or Per/09 or COBRA HA T-7, T-8, T-10, and T-11 antigens. At day 70, sera were collected and tested in an HAI assay and the FRA. (A to E) For each virus, the virus concentration was standardized to 1.2 × 10⁴ FFU/ml (corresponding to 600 FFU/50 μl, which is the volume of virus added to each plate). A monolayer of MDCK SIAT cells (2.5 × 10⁵ to 3 × 10⁵ cells/ml) (100 μl/well in a 96-well plate) was added the day before the assay is run. Cells were 95 to 100% confluent at the time of the assay to determine the number of foci detected as percent infected cells normalized to 100%. Pooled sera from each group of mice were tested against two vaccine strains, Wisc/05 (A) and Uru/07 (B), and three deep-dive viruses, Sant/06 (C), Henan/06 (D), and TX/06 (E). The dotted lines represent 50% and 80% inhibition by sera compared to virus-only control wells. (F) A heat map of the log₂ serum dilution titer for 50% inhibition per virus for each vaccine. (G to K) The GMT HAI and FRA (50% inhibition) titers for each vaccine listed for Wisc/05 (G), Uru/07 (H), Sant/06 (I), Henan/06 (J), and TX/06 (K) viruses.