Homologous Recombination with Linear DNA to Insert Antigenic Protein in the Flagellin: Improvement of the Th1 Immune Response

Abstract

Bacterial flagellin is a surface protein with numerous advantages for the presentation of exogenous peptides. However, the production of recombinant bacteria and the expression of fusion proteins is laborious and time consuming. Here, we present a simple way to produce modified bacteria. Partially deleted, non-functional, chromosomal flagellin gene (fliC ) was changed using homologous recombination by a functional linear fliC gene in which we introduced an exogenous oligonucleotide encoding for the peptide of interest. The modified fliC gene was produced by polymerase chain amplification. Linear amplicons were introduced into the non-motile E. coli by electroporation. The formation of functional flagellar filaments allowed the discrimination of motile transformants from non-motile, non-transformed cells. Thus antibiotic selection and gene expression inductors are not required since transformed bacteria can be easily isolated and used as a vector and adjuvant for immunization. To validate this hypothesis, we studied the immune response against the N-terminal peptide of Clostridium tyrobutyricum flagellin fragment. BALB/c mice were immunized either with the protein displayed as flagellin fusion protein on the surface of E. coli, with the recombinant protein in Freund's adjuvant (FA), or with the pcDNA3 vector bearing the DNA fragment encoding this protein. Immunization with the flagellin recombinant bacteria induced a strong Th1 response as measured by high level of IFN-gamma production and the lack of IL-4 production. The results indicate that the flagellar filament protein carrying a specific epitope can be a potent inducer of the Th1 cellular response.

Antigen-Containing Liposomes Engrafted with Flagellin-Related Peptides Are Effective Vaccines That Can Induce Potent Antitumor Immunity and Immunotherapeutic Effect

“…+ T cells is crucial for the induction of an effective CTL response and protective antitumor immunity (23,47). Further confirmation that pFlg liposomes can be used to elicit Ag-specific responses was provided by our demonstration that vaccination of C57BL/6 mice with 9Flg-and 42Flg-OVA liposome induced Agspecific T cell priming, as assessed by the ability to induce increased Ag-specific cell proliferation (Fig.…”

“…Consistent with this, a number of studies showed that flagellin has adjuvant-like properties (17,18) Vaccination with recombinant flagellin-Ag fusions induced potent T and B cell responses, including Ag-specific CD8 + T cell and Ab responses to model Ags (19,20), as well as to a number of disease-relevant Ags (21)(22)(23), including influenza M2e Ag (24), Plasmodium vivax merozoite surface protein-1 (25), and Mycobacterium tuberculosis Ag p27 (26). The responses induced were often much greater than those induced by vaccination of the Ag emulsified in CFA (20)(21)(22)(23)(24)(25)(26). Also, the linking of flagellin to Ag is essential to induce detectable Ab responses, because a simple mixing of the two is not sufficient (20)(21)(22)(23)(24)(25)(26).…”

“…This approach has distinct advantages over the use of Ag-flagellin fusion proteins to target Ag to TLR5 (19)(20)(21)(22)(23)(24)(25)(26) or single-chain variable fragment-engrafted Ag-containing liposomes to target Ag-uptake receptors to DCs (28). First, the relatively short pFlgs can readily be produced synthetically and do not require the production of recombinant proteins, such as Ag-flagellin fusions and short-chain variable fragments, which can be cumbersome to produce and difficult to use because of poor stability.…”

“…The responses induced were often much greater than those induced by vaccination of the Ag emulsified in CFA (20)(21)(22)(23)(24)(25)(26). Also, the linking of flagellin to Ag is essential to induce detectable Ab responses, because a simple mixing of the two is not sufficient (20)(21)(22)(23)(24)(25)(26). This underscores the notion that TLR activation and Ag delivery to the same APC, or indeed to the same location/compartment at the cell surface or within the cell, may be required to elicit an effective adjuvant response.…”

Immune Response to Chlamydophila abortus POMP91B Protein in the Context of Different Pathogen Associated Molecular Patterns (PAMP); Role of Antigen in the Orientation of Immune Response

“…The antibody response, systemic (IgG) or secreted (IgA), against flagellin was dependent on the route of immunization [17] and finally, immunization of mice with a fusion protein containing a flagellin-enhanced green protein was capable of stimulating antigen presenting cells and thus develop a specific T-cell response [18]. We previously used this strategy and obtained a good Th1 response against two different bacterial antigens [19,20]. …”