A Chimeric B6M1 circRNA Vaccine Confers Robust and Complete Protection Against Monkeypox Virus

The 2025 Cell Reports paper by Wu et al. presents a rationally designed circular RNA (circRNA) vaccine encoding a bivalent chimeric immunogen (B6M1) for protection against the monkeypox virus (MPXV).

Introduction:

The global outbreak of mpox (caused by the monkeypox virus, MPXV) has been declared a Public Health Emergency of International Concern twice, underscoring the urgent need for effective, specific vaccines. Current vaccines, like ACAM2000 and JYNNEOs, are based on the live vaccinia virus (VACV) and offer cross-protection due to the high genetic similarity between VACV and MPXV. However, as live virus vaccines, they carry risks for immunocompromised individuals and cannot completely prevent infection. This highlights the necessity for safer, next-generation vaccines with defined antigens.

MPXV produces two infectious forms: intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs), which are responsible for different stages of infection. Key surface proteins from these virions, such as B6 (from EEVs) and M1 (from IMVs), are prime targets for neutralizing antibodies. While mRNA vaccine technology proved successful during the COVID-19 pandemic, it faces challenges related to stability and immunogenicity. Circular RNA (circRNA) presents a promising alternative due to its covalently closed structure, which confers superior stability and lower innate immunogenicity, even without nucleotide modification.

Finding Highlights:

• A novel circRNA-based vaccine encoding a chimeric immunogen (B6M1) was developed.
• The vaccine elicits potent and balanced neutralizing antibodies and T-cell responses against both B6 and M1 antigens.
• It provides complete cross-protection in mice against a lethal challenge with the vaccinia virus (VACV).
• The single-chain chimeric antigen design offers a cost-effective and industrially scalable strategy for multivalent vaccine development.

Method Description

The researchers employed a multi-step process to develop and test their vaccine:

1. Antigen Selection & circRNA Synthesis: They first screened four MPXV antigens (B6, M1, A35, A29) as monovalent circRNA vaccines. The circRNAs were synthesized using an optimized permuted intron-exon (PIE) system, which allows for precise circularization via self-splicing. The successful synthesis and circularity of the RNAs were confirmed through RT-PCR, sequencing, urea-PAGE, and resistance to RNase R digestion.
2. Chimeric Immunogen Design: Based on the initial results, the two most promising antigens, B6 and M1, were tandemly fused to create two chimeric immunogens: B6M1 and M1B6. Their structural integrity was predicted using AlphaFold and experimentally validated using Surface Plasmon Resonance (SPR) to ensure key neutralizing epitopes were preserved.
3. Vaccine Formulation: The circRNAs encoding these immunogens were purified via High-Performance Liquid Chromatography (HPLC) and encapsulated into Lipid Nanoparticles (LNPs) to form the final LNP-circRNA vaccine.
4. Immunological and Efficacy Testing: BALB/c mice were immunized with the vaccines. Immune responses were evaluated by measuring antigen-specific antibodies (ELISA), neutralizing antibodies against both MPXV and VACV, and T-cell responses (ELISpot). The ultimate test of efficacy was a lethal intranasal challenge with VACV, monitoring survival and weight loss.
5. Safety Evaluation: The safety of the lead vaccine candidate was assessed by monitoring post-vaccination weight, analyzing blood biochemical parameters, and conducting histopathological examinations of major organs.

Key Results and Figure Interpretation

1. Construction and Verification of Monovalent circRNA Vaccines

The study began by evaluating four circRNAs—CircRNAB6, CircRNAM1, CircRNAA35, and CircRNAA29—encoding distinct antigens from Monkeypox virus (MPXV). The vaccine templates were constructed using the PIE self-splicing system, incorporating a Coxsackievirus B3 IRES element to ensure robust translation (Figure 1A). Circularization was confirmed by junction-specific RT‑PCR and Sanger sequencing; urea–PAGE and RNase R digestion further validated the closed circular form. Upon transfection into Expi293F cells, Western blots confirmed antigen expression (Figure 1B). The circRNAs were encapsulated into spherical LNPs (~100 nm diameter) with a mild negative zeta potential (Figures 1C–1E), confirming proper formulation for immunization.

Figure 1. Design, characterization, and encapsulation with LNP of the circRNAs encoding single MPXV antigens

2. Identifying Promising Monovalent Antigen Candidates
   To identify the best antigens for a chimeric vaccine, the team first created and tested four separate monovalent circRNA vaccines (CircRNA^B6, CircRNA^M1, CircRNA^A35, CircRNA^A29). Mice were immunized with these, and their immune responses and protective efficacy against a lethal VACV challenge were compared. Figures 2B-E showed that CircRNA^B6 and CircRNA^M1 induced high titers of antigen-specific antibodies, while CircRNA^A35 did not. Most critically, in the challenge study (Figures 2F-G), only CircRNA^B6 (87.5% survival) and CircRNA^M1 (50% survival) provided significant protection. The other groups, including A35 and A29, offered almost no protection. This pivotal experiment identified B6 and M1 as the most promising protective antigens, justifying their selection for the chimeric immunogen.

Figure 2. Evaluation of the circRNAs encoding single MPXV antigens

3. Rational design and characterization of chimeric antigens (B6M1 and M1B6)

Having selected B6 and M1, the researchers designed two fusion proteins: B6M1 and M1B6. A major concern was whether fusing the antigens would disrupt their three-dimensional structure and hide the critical epitopes targeted by neutralizing antibodies.
AlphaFold modeling indicated preserved tertiary folds (Figures 3C–3D), while gel filtration chromatography confirmed proper folding (Figures 3E–3F). They then quantitatively tested this using SPR with known neutralizing antibodies (hMB668 for B6 and 7D11 for M1). Table 1 and Figure S6 confirmed that while binding affinity was slightly reduced, both chimeras maintained strong, nanomolar-level binding. This indicated that the key neutralizing epitopes were preserved in the fusion proteins, the antigenic integrity of B6M1 and M1B6 as chimeric immunogens.

Figure 3. Design and characterization of B6 and M1 antigen fusions

4. Preparation and optimization of circRNAs encoding chimeric antigens

The team then generated CircRNAB6M1 and CircRNAM1B6 using the same PIE system. Both CircRNAB6M1 and CircRNAM1B6 circRNAs were successfully circularized (Figure 4A), purified by HPLC (Figures 4B–4E), and verified for structural integrity via PAGE. Following Expi293F transfection, Western blotting confirmed antigen production (Figure 4F). Quantitative ELISA (Figure 4G) revealed higher expression from CircRNAB6M1, over twofold that of M1B6. This suggested B6M1’s superior stability, guiding its selection for LNP encapsulation.

Figure 4. Design, synthesis, and characterization of circRNA encoding B6 and M1 antigen fusions

5. Humoral and cellular immune responses induced by CircRNAB6M1

For immunological evaluation, a dose-ranging study (2-16 μg) identified 8 μg as optimal for consistent B6/M1 antibodies, followed by comparative immunization in mice: CircRNAB6M1, monovalents, mixture, LNP, or VACV-TT (Figure 5A). ELISA showed CircRNAB6M1 induced similar prime/boost IgG titers (10^5-10^6) to monovalents/mixture for B6/M1, significantly higher than LNP but comparable to VACV-TT (Figures 5B-5C). Neutralization assays observed superior MPXV titers for CircRNAB6M1 versus VACV-TT or mixture (Figure 5D), and equivalent VACV PRNT50 to mixture but better than VACV-TT (Figure 5E). ELISpots detected comparable IFN-γ/IL-2/IL-4 T cells to mixture, higher than VACV-TT (Figures 5F-5H). This interpreted CircRNAB6M1’s chimeric design as eliciting balanced, potent humoral/cellular responses, with cross-neutralization attributed to preserved epitopes, outperforming live virus in MPXV specificity due to focused antigen presentation.

Figure 5. Immunological evaluation of the CircRNAB6M1 MPXV vaccine

6. Protective efficacy in fatal challenge models

To evaluate functional protection, mice were challenged intranasally with either a low (2×10⁵ PFU) or high (10⁶ PFU) dose of VACV-WR two weeks after boosting. Mice immunized with LNP control rapidly lost body weight and died within five days, while those vaccinated with live VACV-TT showed partial survival (∼50%) at high viral dose. In sharp contrast, CircRNAB6M1 provided complete protection under both challenge conditions, with no measurable weight loss or mortality (Figures 6A–6D). Interestingly, while the co-delivered CircRNAB6 + CircRNAM1 mixture offered partial protection, the single chimeric construct achieved full protection, indicating synergistic immune activity derived from its tandem design.

Figure 6. The CircRNAB6M1 vaccine protects mice from the lethal VACV challenge

Conclusion and Impact

This study presents a major advancement in circRNA vaccine design, demonstrating that a single circRNA encoding a B6–M1 chimeric immunogen can induce potent humoral and cellular immunity against MPXV and provide complete protection from lethal orthopoxvirus challenge in mice. By unifying two key antigens into one open reading frame, the authors achieved strong cross-protective immunity in a cost-effective and manufacturable format.

The findings also highlight the unique advantages of circRNA over traditional mRNA vaccines: enhanced molecular stability, prolonged antigen expression, and reduced innate immunogenicity—all contributing to stronger and more durable immune responses. The single-component multivalent design simplifies production, lowers cost, and accelerates translation for emergency deployment.

Beyond MPXV, this work demonstrates the feasibility of circRNA as a next-generation RNA vaccine platform capable of targeting complex viral pathogens through rational chimeric antigen design. The CircRNAB6M1 vaccine thus serves as a prototype for future RNA-based vaccines against emerging orthopoxviruses and potentially other large DNA viruses that demand broad, durable immunity.

References:

https://www.cell.com/cell-reports/fulltext/S2211-1247(25)01203-3