Exploring the Contribution of CD4+ T Cell Help in Boosting Mucosal Humoral Immunity

Vaccination is a critical public health measure that protects individuals from bacterial and viral infections. However, viruses like influenza can evade vaccine-induced immunity, particularly antibody-mediated protection, reducing vaccine effectiveness. Current influenza vaccines also rely largely on intramuscular injection, which fails to trigger adequate mucosal immunity at sites of infection. This thesis aimed to enhance local mucosal humoral immunity by investigating the role of CD4⁺ T cell help to B cells, elicited by mucosal subunit peptide vaccination. Subunit peptide vaccines are advantageous as they incorporate small, targeted antigenic peptides to defined regions of influenza proteins, such as those that are conserved between virus strains, and can be safely delivered via mucosal routes. However, peptide vaccines often exhibit low immunogenicity. To address this, we created a self-adjuvanted vaccine by conjugating peptides to alpha-galactosylceramide (αGC), which activates Natural Killer T (NKT) cells in the mucosa. In turn these cells can provide immunostimulatory signals that stimulate antigen presenting cells (including both dendritic cells and B cells). The vaccine included either the universal non-virally derived pan DR epitope (PADRE) peptide or the influenza nucleoprotein derived NP₃₁₁₋₃₂₅ CD4⁺ T cell peptide, conjugated to a B cell antigen.

We used this vaccine platform to discern which CD4⁺ T cell peptide best stimulates CD4⁺ T follicular helper cells (Tfh) known to support B cells, and optimal humoral immunity. We found peptide vaccines containing PADRE induced stronger mucosal CD4⁺ Tfh cell responses compared to NP₃₁₁₋₃₂₅ and could elicit robust antibody responses. We also addressed when CD4⁺ T cell help is most important using various recall and non-recall response models. Interestingly, recalling virus specific memory NP₃₁₁₋₃₂₅ did not offer any significant benefits over 𝑑𝑒 𝑛𝑜𝑣𝑜 responses. Based on these findings, we identified PADRE as an effective CD4⁺ T cell peptide and incorporated it into an influenza peptide vaccine with αGC and a conserved influenza B cell peptide. Intranasal administration of this vaccine generated influenza peptide specific antibodies and provided protection against influenza. Notably, protection was also achieved with vaccines lacking the influenza B cell peptide, suggesting that mucosal NKT cells play a key protective role in these vaccine platforms. While the antibody response did not play a key role in protection against the PR8 H1N1 influenza strain, it is not known whether antibodies specific to this minimal-peptide based vaccine can confer protection against other influenza virus strains, highlighting the difficulty of identifying epitopes that confer broad specificity. In summary, this thesis highlights the potential of PADRE peptide vaccines in enhancing mucosal humoral immunity and supports its incorporation into future influenza vaccines.