Background: Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, seniors, and immunocompromised individuals, contributing substantially to the global disease burden. Given the limited preventive options available, developing an effective and safe vaccine remains a public health priority. Methods: An mRNA vaccine encoding the RSV PreF protein was designed and prepared. Antigen properties were evaluated in silico, and the coding sequence was optimized using NLP algorithms. The stability and translational efficiency of the mRNA constructs were verified through in vitro and in vivo assays, followed by immunogenicity evaluation of the formulated mRNA vaccines in a BALB/c mouse model. Results: The optimized mRNA showed predicted improvements in structural stability and a lower free energy state, which were associated with increased translational efficacy in vitro. Correct antigen conformation and retention of key epitopes were confirmed by intracellular staining followed by flow cytometry. A balanced Th1-biased immune response was induced in mice, characterized by high levels of neutralizing antibodies and antigen-specific T-cell immunity, along with enhanced memory T-cell proliferation and differentiation, indicating long-term immunological memory. Conclusions: A novel RSV PreF mRNA vaccine was successfully developed via optimization of protein structure and mRNA sequence. Superior immunogenicity was demonstrated in the BALB/c mouse model, together with promising potential in terms of vaccine safety and immunological persistence. These findings represent a promising step forward in the pursuit of an effective RSV vaccine and suggest the potential of the developed mRNA vaccine to induce substantial immune responses that may correlate with protection in future challenge studies.

Madin-Darby canine kidney (MDCK) cells are the recognized cell strain for influenza vaccine production. However, the tumorigenic potential of MDCK cells raises concerns about their use in biological product manufacturing. To reduce MDCK cells’ tumorigenicity and ensure the safety of influenza vaccine production, a B-cell lymphoma extra-large (Bcl-xL) gene, which plays a pivotal role in apoptosis regulation, was knocked-out in original MDCK cells by CRISPR-Cas9 gene editing technology, so that a homozygous MDCK-Bcl-xL ^-/- cell strain was acquired and named as BY-02. Compared with original MDCK cells, the proliferation and migration ability of BY-02 were significantly reduced, while apoptosis level was significantly increased, the endogenous mitochondrial apoptotic pathway were also modulated after Bcl-xL knock-out in MDCK cells. For tumor formation assays in nude mouse tests, all ten mice injected with original MDCK cells presented tumors growth in the injection site, in contrast to only one mouse injected with BY-02 cells presented tumors growth. These findings suggest that Bcl-xL knock-down is an effective strategy to inhibit tumor formation in MDCK cells, making BY-02 a promising genetically engineered cell strain for influenza vaccine production.

Objectives: Seasonal influenza leads to substantial global morbidity and mortality, especially in adults aged 65 years and older, who present poor immune responses to standard-dose influenza vaccines. This study aimed to systematically evaluate the preclinical safety and immunogenicity of a high-dose quadrivalent seasonal influenza split vaccine (HD-QIV), providing preclinical evidence for its clinical application in the elderly. Methods: Following GLP guidelines, we performed single-dose and repeated-dose toxicity tests in Sprague–Dawley rats, active systemic anaphylaxis assays in guinea pigs, and immunogenicity assessments in young and aged BALB/c mice. Safety indicators included general clinical signs, hematology, blood biochemistry, histopathology and allergic reactions; immunogenicity was evaluated via hemagglutination inhibition (HI) antibody titers and antigen-specific cellular immune responses. Results: HD-QIV only caused mild and reversible local irritation in rats without obvious systemic toxicity, and no dose-related systemic anaphylaxis was observed in guinea pigs. HD-QIV induced robust and dose-dependent humoral immune responses, and showed significantly higher HI antibody titers, earlier seroconversion and longer antibody persistence than standard quadrivalent influenza vaccine in aged mice. Cellular immunity was slightly enhanced but not the dominant protective response. Conclusions: The HD-QIV demonstrates favorable preclinical safety and superior immunogenicity, supporting its further clinical development and use as a priority vaccine for the elderly population.