Innate immunity is the first line of host defense against pathogens. Many innate immune cells can recognize molecular patterns (e.g., PAMPs and damage-associated molecular patterns(DAMPs)) through Toll-like receptors. Recognition of viral PAMPs by pattern recognition receptors of host immune cells ultimately leads to producing type I IFNs and proinflammatory cytokines (TNF-α, IL-1β, and IFN-β) through a signal transduction system [2,13]. TNF-α, an inflammatory cytokine mainly produced by macrophages and Th1 cells, can activate macrophages to recruit other immune cells and initiate adaptive immune responses [14]. As previously reported, two vita-PAMPs have been characterized: prokaryotic mRNA and c-di-AMP [5]. Sander et al. found that adding total bacterial RNA to an inactivated vaccine significantly increased secretion of IL-1β and IFN-β compared with the inactivated vaccine alone.
Moreover, c-di-AMP could quickly mobilize cell-autonomous responses to activate type I IFN production, including activation of endoplasmic reticulum stress and reticular phagocytosis and inactivation of the mechanistic target of rapamycin [15,16]. Here, we found that serum TNF-α, IL-1β, and IFN-β levels of mice immunized with inactivated PRV vaccine supplemented with c-di-AMP were significantly higher than those immunized with inactivated PRV vaccine alone. Moreover, the numbers of DCs recruited to superficial cervical lymph nodes were significantly higher in mice immunized with Inactivated vaccine-c-di-AMP. These results are consistent with a previous study showing significantly increased levels of TNF-α and type I IFN in HCV rE1E2-immunized mice when c-di-AMP was added as an adjuvant, T cell activation, and cellular immune responses were increased [17]. In conclusion, c-di-AMP, a representative vita-PAMPs, could activate an inactivated PRV vaccine's early, efficient, and specific immune responses.
In recent years, innate detection of bacterial RNA, a signature of microbial viability, directly enhanced Tfh cell responses [18]. In addition, DCs were found to play important roles in the activation of CD4+ T cells, humoral immune responses, and antibody production [13]. Hence, this study evaluated the numbers of DCs in draining lymph nodes and percentages of Tfh cells induced by c-di-AMP. Our results show that co-administration of c-di-AMP with Inactivated vaccine induced significantly higher percentages of DCs and Tfh cells than Inactivated vaccine alone, not significantly different from those elicited by LV.
As the primary helper cell for B cells to participate in immune responses, Tfh cells promote GC formation and induce the production of high-affinity antibodies, thereby playing an important role in memory B cell production [19]. Sander et al. found that prokaryotic mRNA induced specific cytokine profiles in human APCs that promoted Tfh cell differentiation, ultimately, efficient and lasting humoral immune responses [18,20]. Therefore, we detected activation levels of B cells in the GC (CD38− GL-7+B220+) and production levels of longevity PCs (CD138+B220-). The results show that the addition of c-di-AMP to the inactivated PRV vaccine significantly increased GC formation and B cell activation in draining lymph nodes.
GC formation can induce the production of high-affinity antibodies and plays an important role in the production of memory B cells [21]. Previous studies found that administration of inactivated bacteria with vita-PAMPs induced increased IFN-β and IL-1β secretion, as well as serum antibody titers, in a vaccination model [9,22,23]. In the present study, we also detected serum levels of IgG antibody in mice at 7 dpi and evaluated antibody duration at 3 months post-immunization. The results show that c-di-AMP acted as an immunopotentiator for the PRV vaccine to promote high-level antibody production and persistent antibody responses more effectively.
Our study demonstrated that as an immunopotentiator of the inactivated PRV vaccine, c-di-AMP could activate more comprehensive and efficient immune responses, including promoting cytokine secretion, APC recruitment, and high-efficiency antibody production. Thus, c-di-AMP is a promising immunopotentiator for developing inactivated PRV vaccines.
As we all know c-di-AMP is one of vita-PAMPs, and another vita-PAMPs which has been best studied is cyclic di-GMP (c-di-GMP)[24].In the recent reported, Ogunniyi AD et al. found that Intraperitoneal coadministration of c-di-GMP with PdB or PspA before challenge resulted in significantly higher antigen-specific antibody titers and increased survival of mice, compared to that obtained with alum adjuvant[25]. Karaolis DK et al. found that treatment with exogenous c-di-GMP inhibits Staphylococcus aureus infection in a mouse model[26].Hou L et al. found that c-di-GMP improved the humoral and cellular immune responses induced by PRV inactivated vaccine and its effects on immunity reached the level comparable to that of a live attenuated vaccine[27]. Their studies all indicated that cyclic dinucleotides like c-di-GMP can be used clinically in humans and animals as an immunopotentiator, immunotherapeutic, immunoprophylactic, or vaccine adjuvant. Although c-di-AMP was discovered more recently, the field is rapidly advancing and exposing a physiological complexity that is comparable to the c-di-GMP network. Such as STING has been shown to bind to c-di-GMP and c-di-AMP, similar approaches could be assessed as strategies for the treatment of bacterial infections[28]. Although the specific mechanism by which c-di-AMP elicits such needs futher research, we could truly believe that it could be a forceful immunopotentiator in the future.