Role of the type 1 interferon response in humoral immunity elicited by self-replicating RNA vaccines

Introduction:: RNA vaccines have emerged as a breakthrough technology in the COVID-19 pandemic. An advancement on this technology uses self-replicating RNA (repRNA) in lieu of mRNAs. Compared to the traditional mRNA, repRNA has the potential for much more prolonged expression, which could be effective for promoting humoral immunity to vaccine immunogens. Thus, we studied the role of innate immune response in repRNA vaccine-elicited immunity. In particular, type 1 interferon (IFN) is a key downstream product from the numerous innate sensors involved in repRNA-recognition. This recognition machinery is particularly important to study for repRNAs, because the repRNA is inherently based on the alphaviral RNA, in which the non-structural protein (nsP) segments of the back-bone enable replication of the gene of interest (Fig. 1a). The segment also allows the repRNA construct to be recognized as a viral infection via activation of toll-like receptor-7 (TLR7)-induced response. This innate immune recognition may hinder the response by inhibiting RNA replication and translation, or by overstimulating innate immunity (Fig. 1b). We thus aimed to test the role of one key downstream component of innate repRNA recognition, the type I interferon response.

Materials and Methods:: To test the role of IFN signaling in vaccine responses in vivo, we designed repRNA-loaded lipid nanoparticle (LNP) vaccines to express an HIV immunogen (stabilized SOSIP trimer of the HIV envelope glycoprotein spike) and silence IFN-α/β receptor 1 (Ifnar1) to suppress type 1 interferon response. Three lipid nanoparticle (LNP)-formulated repRNA vaccines are compared: (1) a baseline LNP that loaded repRNA encoding for the antigen or a reporter; (2) an LNP that co-loads repRNA encoding for the antigen or a reporter and an siRNA against Ifnar1; and (3) a cocktail mix of LNPs, one loading repRNA encoding either an antigen or reporter, and the other loading the siRNA against Ifnar1. By silencing Ifnar1 to interrupt the type I IFN positive feedback loop, an increase in vaccine response would indicate that type I IFN-mediated recognition hinders vaccine-elicited immunity, while a decrease would suggest that type I IFN stimulates it.

Results, Conclusions, and Discussions:: We found that suppressing Ifnar1 enhanced vaccine effects in mice; by 2-weeks post-vaccination we saw a 4-fold increase in the antibody titer against the immunogen, as well as increases in the total percentage of germinal center B cells by 1.5-fold, antigen-specific B cells by 6.5-fold, and follicular helper T (T_fh) cells in the draining lymph nodes when compared to mice vaccinated without Ifnar1 silencing or with a negative control siRNA. Moreover, we observed significantly stronger antigen-specific T cell response in the splenocytes of mice vaccinated with siRNA against Ifnar1 compared to mice vaccinated without (1.6-fold difference), or with the negative control siRNA (3-fold difference). Altogether, these results indicate a potentially obstructive role in innate immune recognition of repRNAs. We also studied the mechanism of action of the vaccine by observing changes in the injected muscle; knocking down Ifnar1 improved LNP retention and repGFP expression in the muscles as far as 5 days post-injection. This result indicates that Ifnar1 silencing may enable transfected cells to survive longer to extend repRNA amplification. Overall, this research provides new information on immune recognition of repRNAs, and how we may modulate it to develop novel next-generation vaccines.

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