The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein subunit vaccine is one of the mainstream technology platforms for the development of COVID-19 vaccines, and most R&D units use the receptor-binding domain (RBD) or spike (S) protein as the main target antigen. The complexity of vaccine design, sequence, and expression systems makes it urgent to establish common antigen assays to facilitate vaccine development. In this study, we report the development of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the antigen content of SARS-CoV-2 protein subunit vaccines based on the United States Pharmacopeia and ICH (international conference on harmonization) Q14 and Q2 (R2) requirements. A monoclonal antibody (mAb), 20D8, was identified as the detection antibody based on its high RBD binding activity (EC50 = 8.4 ng/mL), broad-spectrum anti-variant neutralizing activity (EC50: 2.7–9.8 ng/mL for pseudovirus and EC50: 9.6–127 ng/mL for authentic virus), good in vivo protection, and a recognized linear RBD epitope (369–379 aa). A porcine anti-RBD polyclonal antibody was selected as the coating antibody. Assay performance met the requirements of the analytical target profile with an accuracy and precision of ≥90% and adequate specificity. Within the specification range of 70–143%, the method capability index was >0.96; the misjudgment probability was <0.39%. The method successfully detected SARS-CoV-2 protein subunit vaccine antigens (RBD or S protein sequences in Alpha, Beta, Gamma, or Delta variants) obtained from five different manufacturers. Thus, we present a new robust, reliable, and general method for measuring the antigenic content of SARS-CoV-2 protein subunit vaccines. In addition to currently marketed and emergency vaccines, it is suitable for vaccines in development containing antigens derived from pre-Omicron mutant strains.

Small molecular nucleic acid drugs produce antiviral effects by activating pattern recognition receptors (PRRs). In this study, a small molecular nucleotide containing 5′triphosphoric acid (5′PPP) and possessing a double-stranded structure was designed and named nCoV-L. nCoV-L was found to specifically activate RIG-I, induce interferon responses, and inhibit duplication of four RNA viruses (Human enterovirus 71, Human poliovirus 1, Human coxsackievirus B5 and Influenza A virus) in cells. In vivo, nCoV-L quickly induced interferon responses and protected BALB/c suckling mice from a lethal dose of the enterovirus 71. Additionally, prophylactic administration of nCoV-L was found to reduce mouse death and relieve morbidity symptoms in a K18-hACE2 mouse lethal model of SARS-CoV-2. In summary, these findings indicate that nCoV-L activates RIG-I and quickly induces effective antiviral signals. Thus, it has potential as a broad-spectrum antiviral drug.

The research and development (R&D) of novel adjuvants is an effective measure for improving the immunogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant protein vaccine. Toward this end, we designed a novel single-stranded RNA-based adjuvant, L2, from the SARS-CoV-2 prototype genome. L2 could initiate retinoic acid-inducible gene-I signaling pathways to effectively activate the innate immunity. ZF2001, an aluminum hydroxide (Al) adjuvanted SARS-CoV-2 recombinant receptor binding domain (RBD) subunit vaccine with emergency use authorization in China, was used for comparison. L2, with adjuvant compatibility with RBD, elevated the antibody response to a level more than that achieved with Al, CpG 7909, or poly(I:C) as adjuvants in mice. L2 plus Al with composite adjuvant compatibility with RBD markedly improved the immunogenicity of ZF2001; in particular, neutralizing antibody titers increased by about 44-fold for Omicron, and the combination also induced higher levels of antibodies than CpG 7909/poly(I:C) plus Al in mice. Moreover, L2 and L2 plus Al effectively improved the Th1 immune response, rather than the Th2 immune response. Taken together, L2, used as an adjuvant, enhanced the immune response of the SARS-CoV-2 recombinant RBD protein vaccine in mice. These findings should provide a basis for the R&D of novel RNA-based adjuvants.

Mumps, a disease caused by the mumps virus (MuV), has been spread widely across the world, especially among children and adolescents. Recent frequent local mumps outbreaks were reported worldwide, which may be caused by the decline in the neutralization ability of the existing attenuated live mumps vaccines against circulating MuV strains which were different from the genotype A or B vaccine strains. There is an urgent need to understand the genotypes of MuV strains currently circulated globally and in China. The gene sequences of MuV strains circulated globally were collected and phylogenetic trees were constructed using different strategies. The results showed that the MuV strains previously circulated globally were predominantly genotype G, while genotype F was predominantly circulated in China, followed by genotype G. The molecular evolution of genotype F MuV strains circulated in China is at a low genetic mutation rate, and the analysis of population dynamics pattern indicates that the incidence of genotype F mumps in China showed a rebound trend. These findings provide a basis for the selection or design of vaccine strains, and the decision of the evaluation strains for immunogenicity and protective efficacy, which laid the foundation for the research and development, as well as the application of next-generation MuV vaccines.

Enterovirus A71 (EVA71) is a major pathogen that causes hand, foot, and mouth disease (HFMD). Although the symptoms of HFMD can be self-limiting, severe meningitis, encephalitis, myocarditis, and acute flaccid paralysis may occur. Upon EVA71 infection, the host cells deploy an intricate network of factors to orchestrate cellular responses and maintain cellular homeostasis. However, the virus has evolved various strategies to avoid unfavorable host restrictions and to establish a productive infection process. As response regimens are correlated with disease symptoms, exploring the interactions between the virus and host contributes to understanding the pathogenesis and underlying mechanisms of infection. In this review, we summarized the recent research progress related to pro-viral factors during EVA71 infection and discussed the underlying mechanisms employed by EVA71 to facilitate virion production. These insights may help identify antiviral therapeutic candidates and support vaccine development.

Background: Self-amplifying mRNA (saRNA) holds promising application prospects. However, due to the inclusion of a replicase sequence, its extended length leads to premature termination during in vitro transcription (IVT), resulting in poor product integrity. This study aims to optimize the IVT process for saRNA vaccines to enhance integrity, thereby addressing the key challenge in saRNA vaccine manufacturing. Method: Guided by the Quality by Design (QbD) framework, Design of Experiment (DoE) methodology was employed to design diverse combinations of process parameters for IVT reactions. Predictive models were established to identify critical process parameters (CPPs) influencing integrity and yield. An optimized parameter set and process design space, meeting predefined yield and integrity standards, were developed. The impact of integrity on the immunogenicity of saRNA vaccines was further investigated. Results: Mg²⁺ concentration exerted the most pronounced effect on saRNA integrity. Under optimized IVT conditions, integrity exceeded 85%. Mathematical modeling simulations defined the IVT design space, meeting the preset criteria of ≥80% integrity and ≥600 μg/100 μL yield while accommodating longer saRNA constructs. Notably, murine model data revealed that higher saRNA integrity significantly enhanced antigen-specific antibody and T-cell responses. Conclusion: This study successfully established a multivariate IVT design space fulfilling preset integrity and yield criteria, providing critical data references for the industrialization and quality specification development of saRNA vaccines.