Enhancing cross-protection against influenza by heterologous sequential immunization with mRNA LNP and protein nanoparticle vaccines

Enhancing influenza vaccine cross-protection is imperative to alleviate the significant public health burden of influenza. Heterologous sequential immunization may synergize diverse vaccine formulations and routes to improve vaccine potency and breadth. Here we investigate the effects of immunization strategies on the generation of cross-protective immune responses in female Balb/c mice, utilizing mRNA lipid nanoparticle (LNP) and protein-based PHC nanoparticle vaccines targeting influenza hemagglutinin. Our findings emphasize the crucial role of priming vaccination in shaping Th bias and immunodominance hierarchies. mRNA LNP prime favors Th1-leaning responses, while PHC prime elicits Th2-skewing responses. We demonstrate that cellular and mucosal immune responses are pivotal correlates of cross-protection against influenza. Notably, intranasal PHC immunization outperforms its intramuscular counterpart in inducing mucosal immunity and conferring cross-protection. Sequential mRNA LNP prime and intranasal PHC boost demonstrate optimal cross-protection against antigenically drifted and shifted influenza strains. Our study offers valuable insights into tailoring immunization strategies to optimize influenza vaccine effectiveness.


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All data that support the findings of this study are available with the manuscript, and a source data file is supplied.
The sample size was determined by ethical considerations and availability, and based on existing data sets from previous experiments with similar readouts and expected statistical errors.All sample sizes are stated in the manuscript.n=5 per group for immune serum samples and mouse challenge studies; n=3 for the evaluation of cellular and mucosal responses.
No data was excluded from the reported analyses.
Replication is described in the legends where applicable.
Mice were randomly assigned to different groups.
For all experiments, animal numbers were provided to investigators, and investigators were blinded to grouping names until after the generation of the raw data.
nature portfolio | reporting summary For ELISA assay, the HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, and IgA antibodies were used at a dilution raning from 1:2000 to 1:4000; mouse anti-influenza A virus NP antibody was used at a dilution of 1:2000.For B cell Elispot assays, the HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, and IgA antibodies were used at a dilution of 1:1000.For T cell Elispot assays, the capture antibodies were used at a dilution of 1:250; the biotin-conjugated detection antibodies were used at a dilution of 1:500.For the FACS assays, the antibodies were used at a dilution of 1:150.For the NP-based ELISA assay in microneutralization assays, the anti-NP antibody was used at a dilution of 1:2000.
The axis scales are clearly visible.Include numbers along axes only for bottom left plot of of group (a (a 'group' is is an an analysis of of identical markers).
All plots are contour plots with outliers or or pseudocolor plots.
A numerical value for number of of cells or or percentage (with statistics) is is provided.Abundant immune cells can be be obtained from mouse spleens and lungs.The T cell numbers in in the BALF, particularly in in naïve or or IM-immunized mice, are comparatively low.Therefore, we we utilized pooled cells for specified groups when cell numbers were limited.

Methodology
For Lung/BALF T cell analysis, cells were gated for cells, singlets, live/dead, CD45+, CD4+ or or CD8+, which were further gated for the specific populations.CD44+ cells were defined as as antigen-experienced T cells.CD44+CD69+ T cells were defined as as resident T cells.For spleen T cell analysis, cells were gated for cells, singlets, live/dead, CD3+, CD4+/CD8+, which were further gated for the specific populations.CD127 expression, as as a selective characteristic marker for long-lived memory T cells was evaluated.For lung B cell analysis, cell were gated for cells, singlets, live/dead, CD19+, IgD-CD38+, and CD69+ to to select the Brm cells.
to to confirm that a figure exemplifying the gating strategy is is provided in in the Supplementary Information.
Sample preparation details were described in in the Methods section.BD BD LSRFortessa Cell Analyzer (BD Biosciences) FlowJo v.10 software