Figure 6.

Humoral and Cellular Immune Responses against Tag-free CyRPA Vaccine Formulated with Commonly used Adjuvants. (A) BALB/c mice (n = 6 per group) were immunized intramuscularly with tag-free CyRPA formulated with three adjuvants- Alhydrogel, AddaVax and Montanide ISA 720 and Freund’s adjuvant (CFA/IFA) as a control. Enzyme-Linked Immunosorbent Assay (ELISA) was performed to calculate antibody titers in Day 70 sera. CyRPA (AddaVax) and CyRPA (Freund’s Adjuvant) induced high antibody titers (~32,00,000 and ~25,60,000 respectively) followed by CyRPA (Montanide ISA 720) (~6,40,000) and CyRPA (Alhydrogel) (~4,20,000). ELISA cut-offs were calculated as the mean OD₄₉₀ of pre-bleed sera plus three times standard deviation. The bars and error bars show mean and standard error values of each vaccine group respectively. Individual mice titers are also shown. The P values were calculated by two-way ANOVA with Bonferroni post-hoc testing. (B) Levels of T_H1 and T_H2 responses were measured in BALB/c mice serum immunized with CyRPA+adjuvant or adjuvant alone by cytometric bead arrays (CBA) for mouse serum cytokines using flow-cytometry. CyRPA/Alhydrogel vaccine induced IL-4, IL-6, TNF-α when compared to the Alhydrogel only group while no detectable responses were observed for CyRPA/AddaVax and CyRPA/Montanide ISA 720 groups. However the control group- CyRPA/Freund’s Adjuvant observed increased levels of IFN-γ and IL-4. The pre-bleed, adjuvant and CyRPA+adjuvant plots refer to the cytokines detected in sera from groups of 6 mice before immunization, post-immunization of adjuvant and post-immunization of CyRPA formulation respectively. The bars and error bars show mean and standard error values of each group respectively. The P values were calculated by two-way ANOVA with Bonferroni post-hoc testing. * P≤0.05 ** P≤0.01, *** P≤0.001, **** P≤0.0001.