RESUMO
Bacillus Calmette-Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT-ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT-ESAT-6-CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis.
Assuntos
Antígenos de Bactérias/biossíntese , Proteínas de Bactérias/biossíntese , Calreticulina/biossíntese , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Calreticulina/genética , Calreticulina/imunologia , Contagem de Colônia Microbiana , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Interferon gama/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Organismos Livres de Patógenos Específicos , Baço/microbiologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
AIMS: To create and provide a strain of the food-grade bacterium Lactococcus lactis able to efficiently secrete a modified form of the E7 protein from the human papilloma virus (HPV) type-16. METHODS AND RESULTS: We cloned the coding sequence of a modified E7 (E7m) from the HPV-16 in a plasmid regulated by the strong expression promoter p59. Secretion of the E7m was made by the signal peptide of the usp45 gene. The E7m was detected by Western blot in the cell-free-medium fraction, showing no degradation or aberrant forms. CONCLUSIONS: We constructed a strain of L. lactis able to secrete efficiently a HPV-16 E7 modified protein with diminished transforming activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Human papilloma virus infection is associated with more than 99% of cervical cancers. Immunotherapy targeting E7 to treat HPV-associated cervical malignancies has been demonstrated to be highly efficient. However, native E7 maintains transforming activity. We present this new strain of a food-grade bacterium able to efficiently secrete a HPV-16 E7-modified protein with diminished transforming activity. This new strain could be used as a live vaccine to deliver E7 at a mucosal level and generate antitumour immune responses against HPV-associated tumours.
Assuntos
Antineoplásicos/metabolismo , Papillomavirus Humano 16/metabolismo , Lactococcus lactis/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Western Blotting , Feminino , Regulação da Expressão Gênica , Papillomavirus Humano 16/genética , Humanos , Lactococcus lactis/genética , Proteínas E7 de Papillomavirus/genética , Plasmídeos/genética , Regiões Promotoras Genéticas , Sinais Direcionadores de ProteínasRESUMO
The endoplasmic reticulum (ER) is where the major histocompatibility complex (MHC) class I molecules are loaded with epitopes to cause an immune cellular response. Most of the protein antigens are degraded in the cytoplasm to amino acids and few epitopes reach the ER. Antigen targeting of this organelle by Calreticulin (CRT) fusion avoids this degradation and enhances the immune response. We constructed a recombinant adenovirus to express the E7 antigen with an ER-targeting signal peptide (SP) plus an ER retention signal (KDEL sequence). In cell-culture experiments we demonstrated that this new E7 antigen, SP-E7-KDEL, targeted the ER. Infection of mice with this recombinant adenovirus that expresses SP-E7-KDEL showed interferon induction and tumour-protection response, similar to that provided by an adenovirus expressing the E7 antigen fused to CRT. This work demonstrated that just by adding a SP and the KDEL sequence, antigens can be targeted and retained in the ER with a consequent enhancement of immune response and tumour protection. These results will have significant clinical applications.