Divergent H7 immunogens offer protection from H7N9 virus challenge

Abstract

The emergence of avian H7N9 viruses in humans in China has renewed concerns about influenza pandemics emerging from Asia. Vaccines are still the best countermeasure against emerging influenza virus infections, but the process from the identification of vaccine seed strains to the distribution of the final product can take several months. In the case of the 2009 H1N1 pandemic, a vaccine was not available before the first pandemic wave hit and therefore came too late to reduce influenza morbidity. H7 vaccines based on divergent isolates of the Eurasian and North American lineages have been tested in clinical trials, and seed strains and reagents are already available and can potentially be used initially to curtail influenza-induced disease until a more appropriately matched H7N9 vaccine is ready. In a challenge experiment in the mouse model, we assessed the efficacy of both inactivated virus and recombinant hemagglutinin vaccines made from seed strains that are divergent from H7N9 from each of the two major H7 lineages. Furthermore, we analyzed the cross-reactive responses of sera from human subjects vaccinated with heterologous North American and Eurasian lineage H7 vaccines to H7N9. Vaccinations with inactivated virus and recombinant hemagglutinin protein preparations from both lineages raised hemagglutination-inhibiting antibodies against H7N9 viruses and protected mice from stringent viral challenges. Similar cross-reactivity was observed in sera of human subjects from a clinical trial with a divergent H7 vaccine. Existing H7 vaccine candidates based on divergent strains could be used as a first line of defense against an H7N9 pandemic. In addition, this also suggests that H7N9 vaccines that are currently under development might be stockpiled and used for divergent avian H7 strains that emerge in the future.

Importance: Sporadic human infections with H7N9 viruses started being reported in China in the early spring of 2013. Despite a significant drop in the number of infections during the summer months of 2013, an increased number of cases has already been reported for the 2013-2014 winter season. The high case fatality rate, the ability to bind to receptors in the human upper respiratory tract in combination with several family clusters, and the emergence of neuraminidase inhibitor-resistant variants that show no loss of pathogenicity and the ability to transmit in animal models have raised concerns about a potential pandemic and have spurred efforts to produce vaccine candidates. Here we show that antigen preparations from divergent H7 strains are able to induce protective immunity against H7N9 infection.

FIG 1

Phylogenetic and antigenic relationship among H3 and H7 strains. (A) Phylogenetic tree based on HA sequences of North American (blue) and Eurasian (red) lineage H7 strains used in this study or in human clinical trials. H7N9 prototype strains are indicated. Drift in closely related H3N2 strains (95.4% amino acid identity between 2003 and 2009 isolates) mediates escape from HI-active antibodies quickly. HI cross-reactivity between distantly related H7 strains (e.g., mallAlb01 and Shanghai13 share only 84.8% amino acid identity) is probably mediated by conserved antigenic sites. The tree was built by using ClustalW and was visualized by using FigTree software. (B and C) Front view (B) and top view (C) of the HA trimer of A/Shanghai/2/13 (PDB accession number 4N5J [11]). Regions conserved among the vaccine strains tested in this study (Eurasian and North American lineages) are shown in dark gray, while nonconserved regions are shown in red. The completely conserved antigenic site A is indicated by black arrows.

FIG 2

Exposure to divergent H7 antigens protects mice against H7N9 challenge. (A) Weight loss of animals vaccinated once with inactivated H7 viruses derived from the North American (mallAlb01 and rheaNC93) or Eurasian (Shanghai13, homologous) lineage (n = 5 to 6 for vaccine groups; n = 17 for naive animals). (B) Weight loss of animals vaccinated twice with inactivated H7 viruses derived from the North American (mallAlb01 and rheaNC93) or Eurasian (Shanghai13, homologous) lineage (n = 5 for vaccine groups; n = 17 for naive animals, which are the same as those shown in panel A). (C) Weight loss of animals vaccinated once with recombinant H7 HA protein (mallNL00, Shanghai13, and Anhui13) (n = 5 for vaccine groups; n = 17 for naive animals, which are identical to the animals shown in panel A). (D) Weight loss of animals vaccinated twice with recombinant H7 HA protein (mallNL00, Shanghai13, and Anhui13) (n = 5 for vaccine groups; n = 17 for naive animals, which are the same as those shown in panel A). (E) Weight loss of animals preinfected with divergent H7 subtype viruses of the North American (rheaNC93 and mallAlb01) or Eurasian (Shanghai13) lineage, an H11N9 virus, or an H3N2 virus (n = 5 to 7 for preinfection groups; n = 17 for naive animals, which are identical to the animals shown in panel A). Survival rates are indicated in the keys as percentages.

FIG 3

ELISA reactivity of vaccinated or infected animals to recombinant H7 hemagglutinin. Shown is ELISA reactivity of sera from animals vaccinated once (A) or twice (B) with inactivated vaccines or vaccinated once (C) or twice (D) with recombinant HA antigen or of sera from animals preinfected with divergent influenza viruses (E). Recombinant H7 HA derived from Shanghai13 was used as the substrate. To rule out a biased readout, the substrate was expressed with a different trimerization domain and purification tag (GCN4pII trimerization domain and Strep-tag II) than the recombinant HA used for vaccination (T4 fibritin trimerization domain and hexahistidine tag). OD, optical density.

FIG 4

Neuraminidase inhibition activity and viral lung titers in mice. (A) ELISA reactivity of selected groups to N9 NA. (B) N9 NI titers of groups shown in panel C. NI was measured by using A/Shanghai/1/13 virus; values are expressed as the reciprocal serum dilution that was able to inhibit NA activity by 50%. (C to E) Lung titers of mice vaccinated with different vaccination regimens on day 3 (black) and day 6 (red) postinfection. Samples with no detectible virus titer were scored as 10. The naive day 3 and day 6 groups were included in all three panels to facilitate comparison with the vaccine groups. ND, not determined.

FIG 5

Characterization of recombinant H7 hemagglutinin proteins. (A) Coomassie staining of an SDS-PAGE gel with the three different H7 HAs used in the vaccination study. (B) ELISA with stalk-reactive antibody CR9114. This monoclonal antibody binds to a conformational epitope in the HA stalk and was used to assess the structural integrity of the proteins used.