Tarte, P. | bioRxiv Science

bioRxiv2026

Comprehensive Profiling of Monkeypox Virus Antigens Identifies Potent Targets for Next-Generation mRNA Vaccine Development

The 2022 Monkeypox virus (MPXV) outbreak renewed interest in vaccines for orthopoxviruses. Initial development efforts focused on well-established antigen targets, especially A35, B6, and M1. However, orthopoxvirus surfaces are complex, displaying many antigens across two infectious forms, mature virions (MV) and extracellular virions (EV) and targets relevant to protection remain to be comprehensively defined. We leveraged advances in orthopoxvirus protein biology and mRNA vaccine technology to compare immunity to all feasible targets. Mice were immunized with mRNAs encoding each antigen, or antigen complex, and neutralizing antibody responses were measured prior to heterologous challenge with vaccinia virus. Among MV antigens, A28 induced potent complement-mediated neutralizing antibodies, and the A17:G10 complex induced neutralizing antibodies and protected from challenge. For EV antigens, A36 induced neutralizing antibodies and protected from challenge. Our results affirm the consensus strategy focusing on key antigens while highlighting additional targets that could enhance updated MPXV mRNA vaccines. SIGNIFICANCEMonkeypox virus, a member of the Orthopoxvirus genus along with variola virus, has been associated with two recent outbreaks of mpox disease leading to a renewed focus on orthopoxvirus vaccine development. We report an agnostic screen of all monkeypox virus surface antigens where we combined recent advances in structural biology and mRNA technology to evaluate these potential new vaccine targets. We confirmed that historically prioritized antigens M1, A35 and B6 were protective but also discovered new antigens of interest including A28, the A17:G10 complex and A36 that can be the targets of protective immune responses. These findings are critical to inform next-generation vaccine designs should novel orthopoxviruses emerge as human pathogens.

microbiology↗

bioRxiv2024

Improvement of the potency of a N1-methylpseudouridine-modified self-amplifying RNA through mutations in the RNA-dependent-RNA-polymerase

RNA vaccines are sensed as non-self molecules by the innate immune system, and balancing control of the immune activation and vaccine safety and efficacy has remained a challenge, especially for self-amplifying RNAs (saRNAs). Incorporation of modified nucleotides has been widely used to temper immune activation of RNA vaccines. However, it was previously reported that incorporation of modified nucleotides to saRNAs impeded antigen expression. Here, we used a reporter replicon of the attenuated TC-83 strain of Venezuelan equine encephalitis virus (VEEV) to investigate the impact of modified nucleotide incorporation on the replication capacity of the saRNA in transfected cells. {psi} and {psi}-modified molecules showed a profound defect in RNA synthesis compared to unmodified saRNA. Interestingly, the levels of RNA synthesis of m5C-modified RNAs were similar to unmodified molecules, positioning m5C as a promising candidate for saRNA modification. To overcome the impact of {psi} or m1{psi}-modified nucleotide incorporation in RNA synthesis, we explored two alternative approaches: engineering the UTR sequences and tuning polymerase fidelity. Our results uncover a previously unappreciated link between polymerase fidelity and saRNA amplification. Overall, we provide new insights for the design of saRNAs with high levels of heterologous protein expression and potential vaccine applications.

molecular biology↗