Fig. 3.

Long-term spike-specific cellular and humoral immunity is induced by ISR52. A) Female C57BL/6J mice were immunised twice with Spike S1 alpha variant vaccine by the indicated routes of administration. Two weeks after the final immunisation, mice were euthanized and blood, BAL and spleens were harvested. Anti-alpha variant Spike RBD IgG responses in serum were assessed by ELISA, and the average optical density (OD) for each vaccinated group is plotted for each dilution of serum. B) BAL samples from mice from (A) were taken, diluted, and analysed for anti-alpha variant Spike RBD IgA by ELISA. The average optical density (OD) for each vaccinated group is plotted for each dilution. C) ELISpot analysis of T cells from spleens harvested two weeks after the second immunisation described in (A) for IFN-γ producing T cells stimulated either with medium only or by a peptide pool covering the S1 domain of the alpha variant Spike protein. Results and median for individual mice are plotted. Statistical testing by ANOVA with Sidak’s multiple comparison test, where *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates P > 0.05 in a comparison with the corresponding medium-stimulated samples. D) T cells from vaccinated mice as per (C) were stimulated twice with S1 peptide pool or left untreated, followed by T cell marker and intracellular IFN-γ staining and flow cytometry. The proportion of IFN-γ positive CD4+ T cells is indicated. Statistical testing by ANOVA with Kruskal- Wallis multiple comparison test, where ** indicates P < 0.01. All other comparison were not significant. E) As (D), but showing the proportion of CD8+ T cells. F) Female C57BL/6J mice were immunised twice with Spike S1 alpha variant vaccine by the indicated routes of administration. At day 182 post initial vaccination (6 months), all animals ere euthanized, and T cells from spleens were analysed for response to S1 peptide pool as described in (D). Statistical testing by ANOVA with Sidak’s multiple comparison test, where ** indicates P < 0.01and ns indicates P > 0.05. G) Serum samples from mice in (F) were analysed at the indicated times post immunisation for anti-Alpha variant Spike RBD2 IgG responses by ELISA. The average optical density (OD) for each vaccinated group is plotted for the 1:100 dilution of serum.