The World Health Organization (WHO) has recently declared another global health emergency due to the rapidly spreading monkeypox (Mpox) outbreak in numerous African countries. To address the unmet need to contain the outbreak using the existing vaccines, this study developed a lyophilization process for an effective, scalable and affordable Mpox mRNA-LNP vaccine candidate to address the global health crisis. A comprehensive evaluation and optimization of the vaccine formulation (the type/concentration of cryoprotectants, the type/concentration of buffer system, as well as the mRNA concentration and reconstitution solvent) and the freeze-drying process parameters (freezing method, temperature, cooling rate and primary/secondary drying conditions) were conducted. The freeze-dried product exhibits a uniform appearance and a moisture content of less than 1%. Reconstitution of the lyophilized mRNA-LNP resulted in equivalent particle size/polydispersity index, encapsulation efficiency and mRNA integrity compared to that of freshly prepared mRNA-LNP. Furthermore, the lyophilization process can be scaled up 100-fold to 2000 vials/batch. Notably, the lyophilized mRNA-LNP demonstrated a storage stability of at least 12 months at 4°C, and at ambient temperature for a minimum of 8 h post-reconstitution, exhibiting minimal deterioration in product quality. The in vitro biological activity and in vivo immunogenicity of the lyophilized mRNA-LNP was comparable to that of the freshly prepared mRNA-LNP. These results provide a compelling rationale for the utilization of lyophilization technology in enhancing the accessibility of the Mpox mRNA vaccine in developing countries, a strategy that is crucial for containing the global epidemic of Mpox infection.

The global outbreak of the 2022 monkeypox virus infection of humans and the 2023 documentation of a more virulent monkeypox in the Democratic Republic of the Congo raised public health concerns about the threat of human-to-human transmission of zoonotic diseases. Currently available vaccines may not be sufficient to contain outbreaks of a more transmissible and pathogenic orthopoxvirus. Development of a safe, effective, and scalable vaccine against orthopoxviruses to stockpile for future emergencies is imminent. In this study, we have developed an mRNA vaccine candidate, ALAB-LNP, expressing four vaccinia viral antigens A27, L1, A33, and B5 in tandem in one molecule, and evaluated the vaccine immunogenicity in rodent models. Immunization of animals with the candidate mRNA vaccine induced a potent cellular immune response and long-lasting antigen-specific binding antibody and neutralizing antibody responses against vaccinia virus. Strikingly, the sera from the vaccine-immunized mice cross-reacted with all four homologous antigens of multiple orthopoxviruses and neutralized monkeypox virus in vitro, holding promise for this mRNA vaccine candidate to be used for protection of humans from the infection of monkeypox and other orthopoxvirus.

Emulsion-based antigen delivery systems have emerged as a novel approach to enhance the effectiveness of subunit vaccines. This study presents the development of a newly formulated oil-in-water (o/w) nanoemulsion adjuvant (NEA) composed of squalene oil and α-tocopheryl polyethylene glycol 1000 succinate (TPGS), which serves dual roles as an emulsifier and an immunostimulator. In comparison to AS03, an FDA-approved emulsion adjuvant that includes α-tocopherol, squalene, and polysorbate 80, NEA is devoid of α-tocopherol and exhibits comparable physicochemical properties, including particle size, polydispersity index, morphology, pH, zeta potential, and viscosity. Stability assessments conducted using Turbiscan Lab indicated that NEA undergoes an uplift process without experiencing flocculation, agglomeration or delamination. Model subunit antigens of recombinant glycoprotein E (gE) targeting the varicella-zoster virus (VZV) and highly purified hemagglutinin (HA) protein against trivalent seasonal influenza viruses (TIV) were employed to assess the adjuvanticity of NEA. It was revealed that the specific anti-gE IgG titers induced by the gE/NEA were markedly higher than those generated by gE alone, with titers of 13,000 vs 3,000 for the primary vaccination, and 5 × 10⁶ vs 5 × 10⁴ for the booster vaccination. Additionally, the TIV/NEA group exhibited a significantly improved immunogenic response relative to TIV alone across all three HA antigens at six-week after immunization, as evidenced by anti-HA titers of 256 vs 32. Furthermore, the NEA demonstrated no significant difference in efficacy compared to AS03 in both the VZV and TIV vaccines. Consequently, NEA presents a promising alternative to AS03 for the development of effective subunit vaccines.