Incorporation of 5 methylcytidine alleviates innate immune response to self-amplifying RNA vaccine

Abstract

In order to improve vaccine effectiveness and safety profile of existing synthetic RNA-based vaccines, we have developed a self-amplifying RNA (saRNA)-based vaccine expressing membrane-anchored receptor binding domain (RBD) of SARS-CoV-2 S protein (S-RBD) and have demonstrated that a minimal dose of this saRNA vaccine elicits robust immune responses. Results from a recent clinical trial with 5-methylcytidine (5mC) incorporating saRNA vaccine demonstrated reduced vaccine-induced adverse effects while maintaining robust humoral responses. In this study, we investigate the mechanisms accounting for induction of efficient innate and adaptive immune responses and attenuated adverse effects induced by the 5mC-incorporated saRNA. We show that the 5mC-incorporating saRNA platform leads to prolonged and robust expression of antigen, while induction of type-I interferon (IFN-I), a key driver of reactogenicity, is attenuated in peripheral blood mononuclear cells (PBMCs), but not in macrophages and dendritic cells. Interestingly, we find that the major cellular source of IFN-I production in PBMCs is plasmacytoid dendritic cells (pDCs), which is attenuated upon 5mC incorporation in saRNA. In addition, we demonstrate that monocytes also play an important role in amplifying proinflammatory responses. Furthermore, we show that the detection of saRNA is mediated by a host cytosolic RNA sensor, RIG-I. Importantly, 5mC-incorporating saRNA vaccine candidate produced robust IgG responses against S-RBD upon injection in mice, thus providing strong support for the potential clinical use of 5mC-incorporating saRNA vaccines.

Self-Amplifying RNA: A Second Revolution of mRNA Vaccines against COVID-19

“…But this was an assumption that had not been tested due to the fact that saRNA does not tolerate the incorporation of the most commonly modified nucleotides used in conventional mRNAs, such as pseudouridine. However, this scenario has recently changed with the discovery that saRNA can be modified with certain nucleotides, like 5-mC, and that these modifications improve the performance of these vectors [ 93 , 94 ]. Remarkably, one of these modified saRNAs has already been tested in a phase I clinical trial in Japan with promising results [ 95 ].…”

“…Until now, it was believed that this was not feasible, since many groups had failed to modify saRNA with pseudouridine, the most common modification used in conventional mRNA vaccines. However, two recent reports showed that saRNA can be modified with other nucleotides, such as 5-hydroxymethylcytidine, 5-methylcytidine (5-mC), and 5-methyluridine [ 93 , 94 ]. These modifications led to a considerable reduction in IFN-I induction both in vitro and in vivo, resulting in a higher and more robust expression of transgenes compared to unmodified saRNA.…”

“…These modifications led to a considerable reduction in IFN-I induction both in vitro and in vivo, resulting in a higher and more robust expression of transgenes compared to unmodified saRNA. The reduction of IFN-I production mainly affected plasmacytoid dendritic cells (pDCs), which seemed to be the main source of this cytokine in response to saRNA [ 93 ]. In fact, one of these studies showed that a fully 5-mC modified saRNA vaccine led to significant protection in mice against a lethal challenge with a mouse-adapted SARS-CoV-2 strain using a 100-fold lower dose than a modified mRNA vaccine [ 94 ].…”

Trans-Amplifying RNA: A Journey from Alphavirus Research to Future Vaccines

“…However, its low dose of only 5 µg probably reduced reactogenicity to such an extent that it became tolerable [ 4 , 5 ]. Encouragingly, a recent comprehensive test of a variety of modified nucleotides has shown that 5-methyl cytidine (5 mC) reduces the immunogenicity of saRNA without affecting its function [ 82 , 83 ], raising hopes for even less reactogenic saRNA in the future.…”

Safety and immunogenicity of VLPCOV-02, a SARS-CoV-2 self-amplifying RNA vaccine with a modified base, 5-methylcytosine

“…Therefore, innate responses were minimized in VLPCOV-02 by incorporation of 5-MeC, an approach supported by both preclinical in vitro and animal model observations. 19 The resulting modified product, VLPCOV-02, is currently being studied as a booster vaccine in a clinical trial setting in Japan.…”