Background/Objectives: Influenza viruses are highly transmissible and mutable, posing a significant burden on public health. This study aimed to design a recombinant multi-epitope vaccine with broad protective potential. Methods: Immunoinformatic approaches were employed to predict epitopes from over 30,000 protein sequences retrieved from protein databases. Epitopes were filtered using four key indicators: antigenicity, allergenicity, toxicity, and conservancy. Population coverage analysis was conducted to estimate the proportion of the global population that could potentially benefit from the vaccine. Secondary and tertiary structures of the recombinant vaccine were predicted using the PSIPRED server and AlphaFold2. The vaccine efficacy was validated through an immune simulation, molecular docking, and molecular dynamics simulation. Results: A recombinant multi-epitope vaccine demonstrating strong antigenicity, no allergenicity or toxicity, and high conservation across different subtypes was successfully constructed. Population coverage analysis indicated that the vaccine could elicit an immune response in 90.14% of the global population. Both the secondary and tertiary structures of the vaccine were accurately predicted. Molecular dynamics simulations further validated the structural stability and interactions of the vaccine components with TRL4. Molecular docking confirmed the robust binding affinity of T-cell epitopes to MHC molecules. Simulated immunity studies showed that the vaccine induced the proliferation of memory B cells and T cells, enabling rapid antibody production during viral challenges. Conclusions: This study provides a promising basis for the development of a broadly protective influenza vaccine, leveraging cutting-edge immunoinformatics and molecular dynamics simulations to address the global challenge posed by influenza virus variability.

Norovirus is a major etiological agent of nonbacterial gastroenteritis around the world. Due to its in vitro culture complexity, high genome diversity, and the lack of cross-reactive immunity between genogroups, there is an unmet urgent need for polyvalent norovirus vaccines that provide broad-spectrum protection, and no vaccine has gained global approval to date. In this study, we constructed a bivalent norovirus vaccine, based on the highly attenuated poxvirus [strain VG9] vector, expressing the major capsid protein VP1 from genotypes GII.4 and GII.17. VG9-NOR exhibited a comparable replication ability to the authentic virus while preserving good safety. After the intramuscular and intranasal immunization of mice, VG9-NOR induced high IgG- and IgA-binding antibody (Ab) titers against GII.4 and GII.17, increased the secretion of GII.4 and GII.17-specific HGBA-blocking antibodies, and enhanced GII.17-specific mucosal immunity. Furthermore, VG9-NOR also induced a Th1-mediated cellular response. These results demonstrate that the polyvalent poxvirus vector vaccine expressing VP1 variants from different subtypes is able to elicit effective protection. Our study highlights the VG9 vector as a highly promising candidate for the development of polyvalent norovirus vaccines.

Influenza (flu) is a highly prevalent respiratory illness caused by influenza viruses, representing a significant global health burden due to its substantial morbidity and mortality rate. Vaccination remains the most effective strategy for influenza prevention, and well-characterized animal models of influenza infection serve as essential tools for evaluating vaccine protective efficacy. However, animal models utilizing live influenza virus strains pose significant biosafety concerns, and many such strains are not readily available for research. To address these challenges, we established a novel visual mouse infection model using an HIV-based vector system. This model employs influenza pseudoviruses carrying a luciferase reporter gene, enabling real-time monitoring of viral load and in vivo tracking of viral distribution during infection. Using this infection model, we assessed the in vivo protective efficacy of an influenza vaccine and cross-validated the pseudovirus-based evaluation results against a live virus-infected mouse model. Our study thus establishes a safer and more convenient platform for evaluating influenza vaccine efficacy, including the assessment of broad-spectrum neutralization capacity.

Background/Objectives: The development of human papillomavirus (HPV) vaccines is increasingly trending toward multivalency, and accurate quantification of HPV virus-like particles (VLPs) is critical for vaccine quality control. All marketed products currently rely on ELISA-based antigen quantification, which is complex and time-consuming. In accordance with the 3Rs principles, in vitro potency testing is expected to progressively replace in vivo testing. Therefore, efficient and convenient antigen quantification methods for multivalent HPV vaccines are essential to improve testing efficiency. Methods: In this study, photosensitive substances were loaded into beads of different diameters using a swelling method, and carboxyl groups were introduced onto the bead surfaces. By coupling HPV monoclonal antibodies via carboxyl groups, the use of high-risk coupling reagents was avoided, and a one-step light-initiated chemiluminescence assay (LiCA) was established for the in vitro relative potency testing of HPV vaccines. Results: The LiCA method established using 200 nm carboxyl-coupled beads for the HPV16/18 bivalent vaccine exhibited excellent linear range, specificity, accuracy, and precision. The LiCA results were comparable to those of ELISA; moreover, compared with ELISA, LiCA showed a lower detection limit and a wider dynamic range for both HPV16 and HPV18 VLP antigens, and was more convenient and time-saving to operate. Conclusions: A one-step LiCA was established using carboxyl-coupled beads with optimized diameters, providing an efficient and convenient approach for the in vitro relative potency testing of multivalent HPV vaccines based on large-particle VLP systems, which contributes to improved vaccine quality control and testing efficiency.