Compared to other vaccines, the inherent properties of messenger RNA (mRNA) vaccines and their interaction with lipid nanoparticles make them considerably unstable throughout their life cycles, impacting their effectiveness and global accessibility. It is imperative to improve mRNA vaccine stability and investigate the factors influencing stability. Since mRNA structure, excipients, lipid nanoparticle (LNP) delivery systems, and manufacturing processes are the primary factors affecting mRNA vaccine stability, optimizing mRNA structure and screening excipients can effectively improve mRNA vaccine stability. Moreover, improving manufacturing processes could also prepare thermally stable mRNA vaccines with safety and efficacy. Here, we review the regulatory guidance associated with mRNA vaccine stability, summarize key factors affecting mRNA vaccine stability, and propose a possible research path to improve mRNA vaccine stability.

Neutralizing antibody (NtAb) levels are key indicators in the development and evaluation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines. Establishing a unified and reliable WHO International Standard (IS) for NtAb is crucial for the calibration and harmonization of NtAb detection assays. National and other WHO secondary standards are key links in the transfer of IS to working standards but are often overlooked. The Chinese National Standard (NS) and WHO IS were developed by China and WHO in September and December 2020, respectively, the application of which prompted and coordinated sero-detection of vaccine and therapy globally. Currently, a second-generation Chinese NS is urgently required owing to the depletion of stocks and need for calibration to the WHO IS. The Chinese National Institutes for Food and Drug Control (NIFDC) developed two candidate NSs (samples 33 and 66–99) traced to the IS according to the WHO manual for the establishment of national secondary standards through a collaborative study of nine experienced labs. Either NS candidate can reduce the systematic error among different laboratories and the difference between the live virus neutralization (Neut) and pseudovirus neutralization (PsN) methods, ensuring the accuracy and comparability of NtAb test results among multiple labs and methods, especially for samples 66–99. At present, samples 66–99 have been approved as the second-generation NS, which is the first NS calibrated tracing to the IS with 580 (460–740) International Units (IU)/mL and 580 (520–640) IU/mL by Neut and PsN, respectively. The use of standards improves the reliability and comparability of NtAb detection, ensuring the continuity of the use of the IS unitage, which effectively promotes the development and application of SARS-CoV-2 vaccines in China.

The rapid mutation and spread of SARS-CoV-2 variants recently, especially through the emerging variants Omicron BA5, BF7, XBB and BQ1, necessitate the development of universal vaccines to provide broad spectrum protection against variants. For the SARS-CoV-2 universal recombinant protein vaccines, an effective approach is necessary to design broad-spectrum antigens and combine them with novel adjuvants that can induce high immunogenicity. In this study, we designed a novel targeted retinoic acid-inducible gene-I (RIG-I) receptor 5′triphosphate double strain RNA (5′PPP dsRNA)-based vaccine adjuvant (named AT149) and combined it with the SARS-CoV-2 Delta and Omicron chimeric RBD-dimer recombinant protein (D-O RBD) to immunize mice. The results showed that AT149 activated the P65 NF-κB signaling pathway, which subsequently activated the interferon signal pathway by targeting the RIG-I receptor. The D-O RBD + AT149 and D-O RBD + aluminum hydroxide adjuvant (Al) + AT149 groups showed elevated levels of neutralizing antibodies against the authentic Delta variant, and Omicron subvariants, BA1, BA5, and BF7, pseudovirus BQ1.1, and XBB compared with D-O RBD + Al and D-O RBD + Al + CpG7909/Poly (I:C) groups at 14 d after the second immunization, respectively. In addition, D-O RBD + AT149 and D-O RBD + Al + AT149 groups presented higher levels of the T-cell-secreted IFN-γ immune response. Overall, we designed a novel targeted RIG-I receptor 5′PPP dsRNA-based vaccine adjuvant to significantly improve the immunogenicity and broad spectrum of the SARS-CoV-2 recombinant protein vaccine.