RESUMO
In recent years, recombinant lactic acid bacteria (LAB) have been successfully used as safe mucosal delivery vectors. Herein, we report on the development of a Stress-Inducible Controlled Expression (SICE) system in L. lactis for the production and delivery of proteins of health interest (both therapeutic and vaccine related) at mucosal surfaces. This system is episomal in nature and is composed of a vector carrying an expression cassette under the transcriptional control of a stress-inducible promoter. The functionality of the SICE system was validated in vivo using two different routes of administration: oral and intranasal, and in two different murine models of human pathologies: (i) a model of therapy against inflammatory bowel diseases (IBD) and (ii) a model of vaccination against human papillomavirus type-16 (HPV-16).
Assuntos
Vetores Genéticos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Interleucina-10/administração & dosagem , Interleucina-10/metabolismo , Lactococcus lactis/genética , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/administração & dosagem , Administração Oral , Administração Retal , Animais , Sequência de Bases , Modelos Animais de Doenças , Feminino , Papillomavirus Humano 16/imunologia , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Lactococcus lactis/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mucosa/metabolismo , Especificidade de Órgãos , Vacinas contra Papillomavirus/genética , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Reprodutibilidade dos Testes , Estresse Fisiológico , Vacinas SintéticasRESUMO
Human papillomavirus (HPV) is the causative agent of cervical cancer (CxCa) and the most commonly sexually transmitted pathogen worldwide. HPV type 16 (HPV-16) E7 oncoprotein is constitutively produced in CxCa and considered as a good antigen candidate for the development of new therapeutic CxCa vaccines. Here, we report the use of non-genetically modified, E7-expressing lactic acid bacteria (LAB) by using the cell-binding domain from Lactobacillus casei A2 phage lysin as a cell wall anchor. The versatility of this system was validated by investigating E7 stability at the surface of Lactococcus lactis and L. casei, two major species of LAB. Moreover, we demonstrated the successful use of these LAB displaying E7 antigen as a mucosal live vaccine in mice. Altogether, these results show the feasibility of using non-genetically modified LAB for low-cost mucosal immunotherapy against HPV-related CxCa in humans.
Assuntos
Antígenos Virais/imunologia , Técnicas de Visualização da Superfície Celular , Lactobacillus/imunologia , Neoplasias/prevenção & controle , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Administração através da Mucosa , Animais , Antígenos Virais/genética , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Portadores de Fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/administração & dosagem , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
The lysin gene (lysA2) of the Lactobacillus casei bacteriophage A2 was cloned and expressed in Escherichia coli. LysA2 is an endopeptidase that hydrolyzes the bond between the terminal D: -alanine of the peptidoglycan tetrapeptide and the aspartic acid residue that forms the bridge with the L: -lysine of a neighboring peptidoglycan chain, characteristic of Gram-positive bacteria included into the A4 peptidoglycan subgroup. This includes most lactobacilli, Lactococcus lactis, Pediococcus acidilactici, and Pediococcus pentosaceus, the walls of all of which were substrates for the enzyme. Specific binding of LysA2 to the wall of these bacteria is mediated by its C-terminal moiety, does not need the N-terminal catalytic domain for recognition, and is stable: at least 88% of the molecules were still bound to L. casei after 3 days in phosphate buffer at 4°C. The enzyme acts as a monomer, is active at pH values between 4 and 6, and at temperatures ranging between 18°C and 50°C while being independent of divalent cation addition. The enzyme showed strong resistance to incubation at high and low pH values but became progressively inactivated at 50°C and above. LysA2 is bactericidal, the viability of L. casei cultures dropping to 1% in 10 min, under the standard conditions used for the enzymatic assay.