Oral Administration of Bacillus subtilis Endospores Displaying Influenza Virus Matrix Protein 1 Elicit Cellular Immune Response in Mice

Abstract

Open Access Short communicationimmune response targets conserved antigens and can provide cross-immunity against different subtypes [2]. Influenza virusspecific Cytotoxic T Lymphocytes (CTLs) have been shown in animal studies to limit influenza A virus replication and to protect against lethal influenza A virus challenge [3]. In last year's a new live antigen carrier system emerged [4]. Recent studies show that recombinant endospores of Bacillus subtilis can serve as antigen carriers by fusing peptide of interest to spore coat proteins [5]. In our study we constructed Bacillus subtilis strain producing spores presenting influenza M1 protein on their surface. M1 is most abundant protein in influenza virons and it is conserved among various strains. Moreover it has been shown that vaccine based on M1 protein can elicit significant cellular response in vaccinated individuals [6]. Recombinant spores were orally administrated to mice to evaluate the immunogenic properties of constructs. This work indicates that spores can serve as influenza antigen carriers and induce cell-mediated immune response. Moreover co-administration of constructed spores with spores presenting interleukin 2 significantly boosted observed cellmediated response. Materials and Methods Bacterial Strains and TransformationAll strains used in this study are listed in Table 1. All cloning experiments were done using Escherichia coli DH5α [7]. Bacteria were transformed according to the previously described methods: CaCl 2 -induced competence of E. coli cells [8] and transformation of B. subtilis [9]. Construction of Recombinant Bacillus subtilis StrainTo generate genetic fusion, gene cotZ coding for the coat protein was PCR-amplified together with its natural promoter using the B. subtilis 168 chromosomal DNA as a template and AbstractBacterium Bacillus subtilis is a gram-positive bacilli which produce endospores. Being metabolically dormant, spores are resistant to many environmental stressors such as UV radiation, desiccation, heat or freezing. There are evidence that oral or intranasal administration of spores presenting antigens induces a specific, both cellular and humoral immune response which can protect animals from infection. In our study, using a genetic approach we constructed Bacillus subtilis strains producing spores presenting influenza A virus matrix protein 1 (M1) on their surface. M1 protein was fused to spore coat CotZ protein and was stably exposed on the spore surface as demonstrated by the immunostaining of intact, recombinant spores. The immunogenicity of recombinant spores was tested by oral administration in mice. As proved by an IFN-γ ELISPOT assay, constructed spores elicited significant cell-mediated immune response.