Improved childhood vaccination and preparedness against seasonal and pandemic influenza: prospects for cellular immunity

"Immune responses against influenza infection are mediated by influenza specific antibodies (measured as HI titres), while increasing evidence points to an important role of T cell mediated immunity (CMI: CD4; T helper cells and CD8; T cytotoxic cells) in reducing disease severity. CMI targets conserved influenza virus antigens inducing immunity against multiple strains, whereas antibodies, which are directed against the variable antigens of the virus, are only effective against one specific strain. Defining new immune responses that correlate to protection is crucial for universal vaccine development. Using bioinformatic tools, we have characterized unique human biobank material to identify conserved and cross-reactive T cell epitopes (universal biomarkers), including material from: 1) vaccine trials of seasonal or experimental vaccines, 2) pregnant women and their offspring during the 2009 pandemic, and 3) unvaccinated Ethiopian children. 1) Universal biomarkers for measuring cross-protective CD4 and CD8 T cell responses We have retrieved optimal epitopes from the Immune Epitope Database. Libraries of CD4 and CD8 T cell epitopes were selected from influenza antigens commonly present in seasonal and pandemic influenza strains from 1934-2009. Stimulating cells with these peptides induced significant CD4 and CD8 T cell responses in healthy donors. This suggests that these peptides are able to induce universal cross-reactive immune responses and are thereby candidates for universal influenza vaccines. 2) Seasonal, pandemic, and experimental vaccines a) Impact of repeated versus single vaccination with the trivalent inactivated vaccine (TIV) in healthcare workers (HCW): Specific antibody and T-cell responses persisted up to 4 years after pandemic vaccination in healthy adults. Repeated annual influenza vaccination maintained the cross-reactive IFN-g+CD4+ and multi-cytokine-secreting responses. The cross-reactive IFN-g+CD8+T cells remained stable despite being vaccinated multiple times. b) Pandemic H5N1, H7N1 vaccines: Pandemic H7 or H5 vaccination induced cross-reactive stalk specific antibodies after the first dose of vaccine and increased CD4+ T helper cytokine responses, whereas no increase was observed after the second, suggesting that one dose of these pandemic vaccines are promising candidates for priming for universal vaccines. c) Universal NA-vaccine: The cross-protective potential of neuraminidase (NA), a second influenza surface antigen, showed homologous and heterologous protective capacity. This suggests that inclusion of NA in a universal vaccine may offer increased protection against influenza infection. d) Live attenuated influenza vaccine trial (LAIV): 151 healthy patients (2-59 yrs) were recruited. LAIV elicits mucosal IgA and memory B cells in young children that have not previously experienced influenza infection. LAIV induced long-lived memory T and B cell responses in young children persisting for at least one year after vaccination. Furthermore, LAIV induces cross protective T cells in young children, which cross react with both old and new strains. 3) Protective immune responses in pregnant women during the 2009 pandemic The Norwegian Influenza Cohort (NorFlu), established during the 2009 pandemic, includes 2600 mother and child pairs with biological samples, questionnaire data and linked registry data for exposures and outcomes. By studying the association between CMI and symptomatic influenza-like illness (ILI), we found that ILI-symptoms correlated inversely with higher frequencies of CD8+ memory T cells and multi-cytokine-secreting CD4+T cells, and increased frequencies of NK cells compared to asymptomatic cases and controls. Virus specific and functionally distinct T and NK cell populations may thus serve as cellular immune correlates of clinical outcomes of pandemic influenza disease in pregnant women, information important for future universal influenza vaccine design. 4) Ethiopian children 103 healthy children (2-5 yrs) recruited from Addis Ababa, Ethiopia, were followed over a 1-year period. Blood samples were collected at two time points and clinical data were recorded. Ninety-six percent of the children were seropositive to influenza at the start of the study, and CMI against cross-subtypic conserved viral antigens increased with repeated exposures. Of the 70 follow-up participants, 40 achieved a new influenza infection during the one-year observational period. Of these, 20 became seriously ill, whereof 9 with bacterial infections. On the other hand, 36 of the 70 became seriously ill during the observation year, whereof 15 with bacterial infections. The sick children had significantly lower vitamin D levels than the healthy, and viral infections may predispose for seriousness of bacterial disease. Taken together, the results from these studies represent important contributions for development of a future universal cross-protective influenza vaccine."

Seasonal influenza is a major contributor to childhood pneumonia in low-income countries, possibly by paving the way for bacterial pathogens. A cheap and broadly protective influenza vaccine might therefore be a better choice than the expensive conjugated pneumococcal vaccines to protect children against lethal pneumonia. While antibody-mediated immunity against influenza is primarily restricted to protect against the same viral strain as used for vaccination, T cell responses are directed against conserved antigens common to a wide range of both seasonal and pandemic influenza strains. This cross-protective immunity provides the basis for development of universal influenza vaccines. We will develop bio-informatic tools for prediction and use of synthetic peptides representing conserved T cell epitopes, allowing efficient measures of cross-reactivity against a broad panel of relevant influenza strains after vaccination without handling pathogenic viruses. In addition, this tool will be used to evaluate preparedness against new and emerging strains in children from a low income country (Ethiopia). High throughput assays for measuring cross-protective cellular immunity (granzyme B assay, multi parameter flow cytomety, and multiplex cytokine detection) have recently been introduced by partners of the consortium, and provides the methodological basis. We will analyse cross-reactive cellular immunity induced by live attenuated and inactivated whole virus vaccines given by the intranasal route compared with parenteral split and virosomal vaccines. Moreover, cellular immunity induced after pandemic vaccination (pH1N1) in pregnancy will be compared to non-pregnant controls with respect to the same immune parameters. The strength of this project is related to the introduction of novel methods for evaluating cellular immunity, and the utilization of clinical material from several vaccine trials, in addition to biological material from clinically ill and healthy individuals. This enables us to define correlates of cross-protective immunity (universal) that is highly relevant for improved preparedness against seasonal and pandemic influenza.