The endpoint titer was calculated as the reciprocal serum dilution giving O.D 450 nm readings 2 and the background levels which was calculated using pre-bleed serum at the same dilutions. Western Blot Analysis For Western blot analysis, the S and RBD soluble proteins were separated in 12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel and transferred to a polyvinylidene fluoride (PVDF) membrane. RBD, and S2 domains, respectively, by structural and immunoinformatic analysis. We have shown as a proof of principle in the murine model, the potential role of these novel epitopes in-inducing humoral and cellular immune responses. Further analysis has shown that RBD and S2 directed epitopes were able to efficiently inhibit the replication of SARS-CoV-2 wild-type virus suggesting their role as virus entry Acarbose inhibitors. Structural analysis revealed that S2-epitope is a part of the heptad repeat 2 (HR2) domain which might have plausible inhibitory effects on virus fusion. Taken together, this study discovered novel epitopes that might have important implications in the development of potential SARS-CoV-2 spike-based vaccine and therapeutics. and 0.05), where *** 0.001 (one-way ANOVA). Next, we investigated peptide-specific IgG subclass switching in sera from the second boost immunization to determine Th1/Th2 polarization (Supplementary Figure 2). IgG1 subclass was found to be dominated in the sera from the immunized mice with all the three peptides (Supplementary Figure 2A), whereas, no titer was detected against IgG2c subclass (Supplementary Figure 2D). In the S1-pep1 immunized sera, there was no significant titer against IgG2a and 2b (Supplementary Figures 2B,C, left panel). In the RBD-pep2 immunized sera, high IgG2b titers were seen (Supplementary Figure 2C, middle panel), followed by IgG2a responses (Supplementary Figure 2B, middle panel). Immunization with S2-pep3 also induces significantly higher titers to IgG2b (Supplementary Figure 2C, right panel). In contrast, in Rabbit polyclonal to ZNF625 the immunized sera of mixed peptides, the IgG1 titers are found to be the highest against S2-pep3 followed by RBD-pep2 and S1-pep1 (Supplementary Figure 2E). In addition, the mixed immunized sera showed the highest IgG2a and moderate IgG2b titers to S2-pep3 (Supplementary Figure 2F, left and middle panels) followed by reactivity to RBD-pep2 (Supplementary Figure 2G, left and middle panels); however, no reactivity was Acarbose shown to IgG2c (Supplementary Figures 2F,G, right panel). In addition, the mixed immunized mice sera showed a very low titer of IgG2a, 2b, and 2c Acarbose against S1-pep1 (data not shown). Peptide Prime-Boost Immunization Efficiently Mounted Antigen-Specific CD8+ T Cell Responses T-cell responses to S1-pep1, RBD-pep2, and S2-pep3 and mixed peptide preparation were characterized in the mice immunized with Acarbose respective peptides. For this purpose, spleens were isolated from each of the above-immunized groups and stimulated in the presence of PMA + Ionomycin or respective peptide antigen and compared with the mock control group. Characterization of various T-cell populations was then carried out based on the presence of CD4, CD8 surface markers, and cytokines. In peptide stimulated groups, both the T helper as well as T cytotoxic cells showed ~10-fold upregulation in IFN- (Interferon-gamma) production in the mixed peptide immunized mice followed by RBD-pep2 group which showed ~6-fold upregulation as compared to the control group (Figure 3). Similar significant upregulation of total IFN- production was also observed in the presence of mixed peptide and RBD-pep2, indicating a robust IFN- mediated anti-viral response in the presence of these peptides (Figure 3). Consistent with the antigen-specific stimulation, PMA + Ionomycin stimulation also resulted in significant IFN- production in the mixed-peptide group and RBD-pep2 immunized group (Supplementary Figure 3). Interestingly, the upregulated IFN- production was also observed in the case of S1-pep1 group in both antigen-specific and PMA+Ionomycin stimulation. However, no significant changes were observed either in IL2 or IL17A production suggesting that our immunization strategy particularly with mixed peptide, RBD-pep2, and S1-pep1 induces a strong IFN- production which may be driven by Th1 axis. Open in a separate window Figure.