Safety and efficacy of an intramuscular bivalent vaccine against influenza and Streptococcus pneumoniae infections in mice.

Influenza virus and Streptococcus pneumoniae are major respiratory pathogens responsible for significant global mortality. While influenza causes seasonal flu, pneumococcus is associated with pneumonia, meningitis, sepsis, and otitis, often worsening influenza cases through secondary infections. Aiming to develop a bivalent vaccine against these two pathogens, we used reverse genetics to construct a recombinant influenza virus that carries the gene of the pneumococcal PspA protein (Flu-PspA). This study assessed the safety and efficacy of a heterologous prime-boost vaccine protocol consisting of Flu-PspA prime and followed by a boost with recombinant PspA plus alum (Flu-PspA/PspA4 + Alum), administered intramuscularly in C57BL/6 mice. Following immunization, anti-PspA and anti-influenza antibody titers in serum and bronchoalveolar lavage fluid (BALF) were quantified by ELISA. The breadth of the immune response was evaluated by measuring complement deposition across multiple pneumococcal strains. Vaccine protection and efficacy were evaluated 21 days after final immunization by challenging mice with a lethal dose of 7 × LD50 of S. pneumoniae (strain ATCC6303) or 100 × LD50 of influenza A/PR8/34 virus. Mid-term pneumococcal protection was assessed similarly 90 days post-boost. Three days post-pneumococcal challenge, bacterial loads in BALF and blood were quantified. Additionally, bacterial colonization and secondary infection dynamics were evaluated and quantified after nasal colonization challenge (strain EF3030), infection with influenza H3N2 virus and secondary infection with pneumococcus and after nasal colonization with strain EF3030 and H3N2 virus infection. The results demonstrated that the vaccination regimen elicited a robust humoral immune response, conferring broad protection against diverse pneumococcal strains. Following lethal challenge with either pathogen, the vaccine provided 100% survival, by significantly reducing bacterial burden in blood and lungs. Notably, even 90 days post-boost, mice retained partial protection with minimal weight loss. Furthermore, the protocol reduced bacteremia following secondary infections and mitigated weight loss in H3N2-infected colonized mice. These findings demonstrate that the vaccine confers robust protection with broad-spectrum efficacy when administered intramuscularly, highlighting its potential as a viable preventive strategy against both influenza and pneumococcal infections, including co-infection scenarios.