Enhancement of pathogen-specific immunity following co-administration of whole inactivated respiratory vaccines

Abstract

Influenza A virus (IAV) and Streptococcus pneumoniae are two of the most prominent respiratory pathogens affecting humans worldwide. While IAV and S. pneumoniae cause considerable morbidity and mortality individually, their synergistic pathogenicity poses the greatest threat to human health. Co-infection is associated with dramatically increased disease severity, particularly during IAV pandemics. The 1918 pandemic remains the most lethal on record, with an estimated 50 million deaths. However, a large portion of fatalities have since been attributed to secondary bacterial infection, with S. pneumoniae being heavily implicated. Given the ongoing risk of a future pandemic with highly pathogenic avian IAV, protective strategies against both IAV and S. pneumoniae represent an urgent and unmet need. Current vaccines against each of the individual pathogens are restricted to the induction of strain- and serotype-specific responses. Thus, our group has been developing novel vaccines that confer broad-spectrum protection after mucosal administration. These vaccines consist of whole IAV and whole un-encapsulated pneumococci that have been sterilised using gamma (γ)-irradiation, to generate γ-Flu and γ-PN. Irradiation effectively sterilises each pathogen by damaging the genomic material, whilst pathogen structure and antigenic proteins are maintained. This study describes the enhancement of safety and immunogenicity of both vaccines to facilitate future clinical advancement. Treatment of γ-Flu with a high radiation dose of 50 kGy was shown to have minimal impact on vaccine efficacy whilst exceeding a Sterility Assurance Level of 10-6. Establishing the efficacy of 50 kGy-treated preparations will aid in the inclusion of highly pathogenic strains in future vaccine formulations, such as avian H5N1 or H7N9. Such strains must be irradiated with a very high dose for sterilisation, and generation of g-Flu based on 50 kGy-treated avian strains would be immensely beneficial in the event of a future pandemic. Utilisation of high radiation dose may also aid in the transfer of this inactivation approach to other highly pathogenic agents for vaccine purposes, particularly when CD8⁺ T-cell responses are needed. The safety profile of our g-PN vaccine was also heightened in the current study. A growth attenuating mutation was introduced (generating γ-PN(∆PsaA)), which is an additional safety parameter to facilitate future clinical use of our vaccine. Interestingly, the supplementation of media with manganese required to restore normal growth in vitro was found to have immunomodulatory effects. Specifically, manganese supplementation was associated with enhanced TLR2 signalling by both live and irradiated samples of the pneumococcal vaccine. This phenomenon was unique to the further attenuated strain and is expected to enhance the magnitude of immune responses induced in vivo. In addition, antibody responses induced by g- PN(DPsaA) were found to react against a wider range of pneumococcal antigens compared to those induced by the original γ-PN. While the adjuvant activity of γ-Flu to co-administered γ-PN has been reported previously, the subsequent combination of the two optimised vaccines revealed direct interaction of γ-Flu and γ-PN(∆PsaA) in suspension, suggesting bi-directional adjuvant activities. Mixing the two vaccines resulted in enhanced uptake of γ-Flu virions by epithelial cells and macrophages in vitro, and co-vaccination with γ-Flu + γ-PN(∆PsaA) was associated with significant enhancement of IAV-specific Tissue Resident Memory cell populations in the lung. Furthermore, co-vaccination enhanced the protection in mice against lethal challenge with both drifted and heterosubtypic IAV strains. My data indicate that our novel approach of mixing whole inactivated viral and bacterial vaccine components could enhance pathogen-specific immunity, and may revolutionise vaccine design to combat infectious diseases.