Jee, Pui Fong (2018) Influenza hemagglutinin 1 (HA1) surface-displayed on lactococcus lactis in the induction of protective mucosal immunity against influenza virus / Jee Pui Fong. PhD thesis, University of Malaya.
Abstract
Influenza virus infection can result in respiratory illnesses and may contribute to high rate of morbidity and mortality. In addition to the pandemic influenza, seasonal influenza remains a major health concern worldwide. To date, vaccination is still the most effective approach for the prevention and control of influenza. Vaccines are usually delivered by intramuscular (IM) injection using needle, which causes pain and possibility of blood transmissible infections. Side effects associated with conventional vaccines, such as local reactions at the injection site, are among the issues necessitating alternative modes of vaccine delivery. Currently, there is great interest in developing mucosal vaccine as an alternative over the conventional IM route. It is suggested that administration of mucosal vaccines will eliminate the need for needles and skilled personnel, and these would be a more favorable approach to mass vaccination, especially of children. One of the current mucosal vaccine strategies is by oral administration of antigen displayed on Lactococcus lactis. In the present study, influenza A (H1N1) hemagglutinin 1 (HA1) was surface-displayed on non-recombinant L. lactis. The objective of the study was to evaluate the non-recombinant L. lactis ability to induce mucosal immune response and to accord protection against influenza virus infection in mice. The HA1/L/AcmA recombinant protein was constructed by fusing HA1 with N-acetylmuramidase (AcmA) binding domain using a single-chain variable fragment (scFv) peptide linker comprising (Gly4Ser)3. The inclusion of this peptide linker in the recombinant protein was investigated for its ability to improve the surface display on L. lactis. Flow cytometry and immunoblotting analysis suggested that the amount of HA1 bound on L. lactis was improved in the presence of this peptide linker. The recombinant protein completely agglutinated red blood cells (RBCs), suggesting that insertion of the scFv peptide linker between HA1 and AcmA binding iv domain did not affect the biological function of HA. The non-recombinant L. lactis displaying HA1/L/AcmA recombinant protein, LL-HA1/L/AcmA, was administered into mice orally to evaluate the host immune responses. Mice immunized with LLHA1/L/AcmA developed detectable specific sIgA in faecal extract, small intestine wash, BAL fluid and nasal fluid. The results obtained suggested that oral immunization of mice with LL-HA1/L/AcmA elicited mucosal immunity in both the gastrointestinal tract and the respiratory tract. The protective efficacy of LL-HA1/L/AcmA in immunized mice against a lethal dose challenge with A/TN/1-560/2009-MA2(H1N1) influenza virus was also assessed. Upon challenge, the PBS-treated group (control) of mice showed total body weight loss up to 20%, suggesting high susceptibility to influenza virus infection. In contrast, 7/8 of mice immunized with LL-HA1/L/AcmA and 6/8 of mice immunized with HA1/L/AcmA recombinant protein survived. The latter group however, had severe sickness and total body weight loss when compared to the former group, suggesting that oral immunization with LL-HA1/L/AcmA resulted in less morbidity and better survival upon challenge with influenza virus. In conclusion, oral administration of LL-HA1/L/AcmA in mice induced mucosal immunity and provided protection against lethal challenge with influenza virus. These results highlight the potential application of L. lactis as a platform for delivery of influenza virus vaccine.
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