Novel strategies are constantly being involved to combat viruses and different other pathogens. The search for the perfect adjuvant that has the potential to optimize the immune response of the host is critical in this aspect. In this study, we have shown the mechanistic details of the functional alteration of the key immune cells and molecules in shaping the immune response against influenza by differently charged lipid adjuvants. While both the anionic L3 and cationic N3 adjuvant showed remarkably enhanced immune response as compared to the non-adjuvant control, each one of them has a preferential immune pathway for achieving the same.
Anionic L3 adjuvants have the potential of inducing the higher cDC population with better costimulatory molecules CD80/86 expression (Fig. 1D) with high MHCII (Fig. 1E) and DEC205 levels (Fig. 1F). DEC205 is a critical dendritic cell protein marker that takes the novel pathway of antigen uptake as they are recycled through MHCII-rich late endosomal compartments increasing antigen presentation to CD4 + helper T cells 33,41. An increase in DEC205 expression in the L3 group as compared to N3 (Fig. 1F) justifies the elevated MHCII expression and CD4T cell-mediated B cell activation that we have observed in the L3 groups.
The key difference in antigen processing and its presentation via different MHC molecules (MHCI vs MHC II) leads to the differential activation of T cell subsets (CD8T vs CD4T) and subsequent immune response. It needs to be highlighted here that the treatment with either L3 or N3 do not lead to ‘all or none’ activation of either the Th1/Th17 response or the Th2 humoral response. Both adjuvant subtypes are potent enough to activate both arms. Both L3 and N3 treatments showed enhanced immune response than non-adjuvant antigen control. It is just that L3 preferred the humoral arm while N3 induce a stronger Th1/Th17 immunity arm. Both of them are perfectly capable to activate and enhance their non-preferred arm as well to provide optimum immune protection.
Significantly higher Th17 population (Fig. 3D), higher CD8T population and CD28 expression along with increased Th1 cytokine IFNγ expression in the CD4T population (Fig. 4A, 4B) of the N3 treated group indicated a Th1/Th17 dominant response from the cationic lipid adjuvant. With the higher intracellular level of IFNγ within both the CD4T and CD8T cells in the N3 groups, it is evident that N3 adjuvant promotes the cell-mediated immune response considerably higher than that of L3 treatment (Fig. 4A). This supports our previous finding of the same via the ELISPOT assay against influenza antigen, where the N3 treated group produces more IFNγ spots than the L3 groups 19. It has to be noted that both L3 and N3-treated groups have exhibited a considerable level of IFNγ than the non-adjuvant control group. While assessing the absolute number of T cell populations expressing IFNγ we find a higher number of CD4T than CD8T cells (Fig. 4B). This is also evident from the ELISPOT data reported before 19.
The anti-influenza HA specific serum IgG titer is high in both the adjuvant treatments as compared to the control (Fig. 5A). This along with the significantly low immature B cell and high plasma B cells in the L3 group (Fig. 4C, 4D) indicates a stronger humoral response from the L3 adjuvant. L3 treatment also induce significant enhancement of HA-specific IgA titer as compared to the non-adjuvant control, while no significant increase observed in N3 groups (Fig. 5B). As reported before N3 vaccination failed to stimulate the synthesis of influenza-specific serum IgA 19,42,43, although they have they secrete mucosal IgA specific to the pathogen 19. This difference might originate from the differential source of IgA. While the mucosal IgA is produced from the nasal-associated lymphoid tissue (NALT), the serum IgA originates mainly from the plasma cells of the spleen and the lymph nodes 44. This fact justifies the higher splenic plasma B level in L3 treatment than in N3 treatment (Fig. 4C), as the former produces both anti-influenza IgG and IgA, while the latter focused mostly on IgG production only (Fig. 5A, B).
Serum collected after each dose of immunization before the viral challenge with these adjuvants previously showed a steady increase in the HA-specific antibody level with enhanced neutralization potential 19. The neutralization potential is significantly higher in both the adjuvant-treated groups as compared to the control (Fig. 5C). The neutralization capacity is significantly high in the L3 treatment as compared to the N3 treatment as shown in this (Fig. 5C) and a previous study 19. This along with higher anti-influenza IgA levels in the L3-treated groups demonstrates a stronger humoral immune response as compared to N3 groups.
Another key finding from this study is the lowering of the dose of the antigen in the vaccination formulations. High doses of the antigen have been associated with detrimental side effects. The use of both of these adjuvants showed promising immune responses even with low antigenic doses in some of the parameters studied here. Earlier studies with these adjuvants showed a significant reduction in the influenza virus RNA copy number in the lungs 22 even with a low antigenic dose.
Since we are focused on identifying the immune response exhibited from the adjuvant-antigen vaccine formulation alone and not generated from the whole virus, we did not have the scope to assess the post-challenged status in this study. It has already been reported that these adjuvants proved to be effective in eliciting the immune response post-viral challenge in influenza 19–22. We have limited scope to study one of each anionic and cationic variant of the lipid adjuvants over here. It will be interesting to explore other variants of lipid adjuvants with different charges in future studies.
This study is critically important for demonstrating the differences in the mechanism of action of the differentially charged adjuvants in modulating the immune response elicited by the same vaccine candidate antigen. Fitting our findings with the previously reported mucosal immune response 20, 28–30 from the intra-nasal administration of the lipid adjuvants can lead to better understanding and designing of customized vaccines that can potentially target the population having a deficiency in one or the other arm of immune responses. A vast majority of the population have a deficit or weaker immune response generated from the cell-mediated or humoral immunity depending on the comorbidities, age, gender, etc. It is easier to change the adjuvant accompanied with the same vaccine antigen rather than altering and finding another antigen candidate itself. Especially in the context of the COVID19 vaccine, we have learned that while the humoral response is important for sterilizing immunity, the presence of a sufficient T-cell response is important in protecting against severe disease as antibody levels wane. Thus, the proper use and administration of the different adjuvants may be the key to better protection against the influenza virus.