Protection Against IAV/Sp Co-Infection Via Sp-specific CD4 Memory T Cells

BACKGROUND: Influenza A virus(IAV) is highly contagious and is responsible for pandemics and outbreaks of influenza. In the majority of cases, the high mortality associated with these influenza infection is not due to primary viral pneumonia, but is instead caused by secondary bacterial infections attributed to agents like Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus, with Streptococcus pneumoniae(Sp) the leading cause. However there’s limited understanding on the efficient vaccination for lethal secondary bacterial infection. In this study, we used live Sp immunization to investigate its role in the protection against single and co-infection, and further reported the important role of Sp-specific CD4 memory T cells in protection. Our report highlights the importance of memory T cells in the protection against IAV/Sp co-infection model for the first time.Therefore,targeting memory T cells and regulating its functions may reveal potential prophylactic strategy for the vaccine development and better control over lethal IAV/Sp co-infection.METHODS: We used a lethal IAV/Sp co-infection mouse model with IAV(PR8)on Day 0 and Sp(TIGR4 or P1121)on Day 5 to investigate the characteristics of memory T cells; we longitudinally observed CD4 and CD8 T cells expansion, CD44 expression on memory T cells and cytokine secretion during IAV/Sp co-infection by flow cytometery, meanwhile comparing with single Sp infection. In addition, we took advantage of adoptive transfer to examine the effect of sera or T cells from Sp immunized mice on protection against later challenge to the recipient mice. We performed Luminex to detect the level of cytokines from the BALF, measured the Sp bacterial burden, carried out RT-q PCR to evaluate the viral titer of IAV. Via the recombinant Sp strain that has been inserted with a Lm epitope(LLO190), we set up a new method to detect the immune responses from bacterial-specific CD4 memory T cells and identified the critical role of Sp-specific memory CD4 T cells during co-infection.RESULTS: In our study, we use a C57BL/6 mouse model for primary infection with IAV followed by lethal secondary Sp bacterial infection that has been established. 1) We found that live Sp immunization can protect against lethal single Sp infection, and can partially protect against lethal IAV/Sp co-infection. 2) In single Sp infection, both Sp-specific antibodies and memory T cells contribute to the bacterial clearance in the lung, however, only memory T cells rather than antibodies play a role in the partial protection against co-infection. 3) Memory T cells underwent different immune responses between single Sp and IAV/Sp co-infection. Data showed that there’s a robust IL-17 secretion from memory CD4 T cells in single Sp infection after Sp immunization, however, which was inhibited in IAV/Sp co-infection after Sp immunization, while experienced a shift from IL-17 to IFN-γ secretion, most significant in CD4 memory T cells. 4) Immunization with Listeria monocytogenes can protect against lethal single recombinant Sp(with LLO190 epitope) infection, with enhanced immune responses. 5) Immunization with the recombinant Sp can partially protect against IAV/Sp co-infection with enhanced IFN-γ secretion from antigen specific memory CD4 T cells.CONCLUSIONS: We identified a critical role for Sp-specific memory T cells in lethal IAV/Sp co-infection which is distinctly different from its role in single Sp infection. Sp-specific memory CD4 T cells response to Sp or IAV/Sp challenge robustly after Sp immunization. Therefore, targeting Sp-specific memory T cells will be a potential strategy for the vaccine development and further improving the protection against the lethal IAV/Sp co-infections.