Glycan repositioning of influenza hemagglutinin stem facilitates the elicitation of protective cross-group antibody responses

The conserved hemagglutinin (HA) stem has been a focus of universal influenza vaccine efforts. Influenza A group 1 HA stem-nanoparticles have been demonstrated to confer heterosubtypic protection in animals; however, the protection does not extend to group 2 viruses, due in part to differences in glycosylation between group 1 and 2 stems. Here, we show that introducing the group 2 glycan at Asn38HA1 to a group 1 stem-nanoparticle (gN38 variant) based on A/New Caledonia/20/99 (H1N1) broadens antibody responses to cross-react with group 2 HAs. Immunoglobulins elicited by the gN38 variant provide complete protection against group 2 H7N9 virus infection, while the variant loses protection against a group 1 H5N1 virus. The N38HA1 glycan thus is pivotal in directing antibody responses by controlling access to group-determining stem epitopes. Precise targeting of stem-directed antibody responses to the site of vulnerability by glycan repositioning may be a step towards achieving cross-group influenza protection.


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The number of animals selected for each study was chosen based on our prior experience with similar vaccine regimens and virus challenge studies. A group size of 10 animals is used. Assuming variance in immune response and/or lethality is proportional to mean for a given group (constant CV of 30%, typical for this type of experiments), a group size of 10 will give 89% power to detect 2-fold differences or 49% power to detect 1.5-fold differences between vaccine groups (set for 4) in the magnitude of the immunological parameters based on a two-tailed test of means with alpha set to 0.05 (calculation was performed by 1-way ANOVA pairwise tools at powerandsamplesize.com). A consistent 1.5-to 2-fold difference in immunological parameters is the minimum amount of difference between vaccine groups that would be relevant for biological significance.
No data have been excluded.
All analyses for antibody binding, specificity, and virus neutralization assays have been performed twice or more with similar results. Most of the experiments including animal studies have been repeated with similar results. All of the data in which we could perform statistical analysis showed that the differences observed were significant and highly consistent across experiments.
All animals were age and gender matched and randomly assigned to experimental groups.
In vivo challenge studies were done in a blinded manner. All of the serological assays including virus neutralization assays, and structural, biochemical characterization of the particles were not performed in a blinded manner.
All the antibodies used were made recombinantly by cloning antibody heavy and light chains into the mammalian expression vectors. Antibodies were produced in mammalian cells (Expi293 cells) by transient transfection of expression vectors and purified by protein A affinity chromatography. Sequences, specificity and function of the antibodies were verified for each antibody. Secondary antibodies used in ELISA were purchased from Southern Biotech.
All the antibodies were tested for their reactivity and specificity by ELISA, BLI or other assays prior to be used in the study.