Antibody response to Mycobacterium tuberculosis p27-PPE36 antigen in sera of pulmonary tuberculosis patients.

We have expressed, characterized and studied the immune response of mice against the Mycobacterium tuberculosis PPE36 protein. This protein encoded by the gene Rv2108 is a 27-kDa cell-wall associated protein. Here, we compare the antibody isotypes distribution of p27-PPE36 in sera of pulmonary tuberculosis patients and in sera from healthy control subjects by enzyme-linked immunosorbent assay (ELISA). We detected in the sera of affected individuals a significant increase of IgA antibody response, a no significant augmentation of IgM response, a complete lack of IgG2, 3, and 4 responses and a weak IgG1 response in a few subjects.

Antibody to heat shock protein 70 (HSP70) inhibits human T-cell lymphoptropic virus type I (HTLV-I) production by transformed rabbit T-cell lines.

Adult T cell leukemia is a fatal malignant transformation caused by the human T-cell lymphoptropic virus type I (HTLV-I). HTLV-I is only associated with the development of this disease in a small percentage of infected individuals. Using two rabbit transformed T-cell lines; RH/K30 (asymptomatic) and RH/K34 (leukemogenic), we have investigated the expression of heat shock proteins (HSP) 90 and 70 and the role of anti-HSPs antibodies on virus production. HSPs surface expression was higher on RH/K34 than RH/K30 cells. Heat treatment of cells increased the expression of HSPs proteins and virus production; HSPs augmentation was stabilized after 12 h and virus production reached a maximum between 8 h-12 h then returned to normal level after 24 h of culture. Incubation of cells only with rabbit anti-HSP 70 antibodies prevented virus production specifically in the leukemogenic cell line. The results indicate a relationship between HSP 70 and virus production.

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.

In a previous study, we used bacterial flagellin to deliver antigens such as p27 of Mycobacterium tuberculosis to a host immune system and obtained a potent Th1 response compared to those obtained with Freund's adjuvant and DNA immunization. In the current study, using a POMP91B antigen of Chlamydophila abortus, a human and animal pathogen, as a model, we found that this antigen is unable to promote Th1 response. However, this antigen, unlike others, was able to induce a good Th2 response and IL-4 production after immunization by recombinant protein in Freund's adjuvant or in phosphate buffered saline. Our results suggest that immune response is not only dependent on the immunization adjuvant, but also dependent on the nature of antigen used.

Flagellin as a good carrier and potent adjuvant for Th1 response: study of mice immune response to the p27 (Rv2108) Mycobacterium tuberculosis antigen.

We have recently expressed and characterized the product of the gene Rv2108 of Mycobacterium tuberculosis (MT), the p27 protein. Here, we investigated the immune responses against the p27 protein in the context of different pathogen associated molecular patterns (PAMPs). Different immunization protocols were used. BALB/c mice were immunized either with the p27 recombinant protein in Freund's adjuvant or in phosphate saline buffer (PBS), with a pcDNA3 plasmid containing the gene encoding the p27 protein, or with the Escherichia coli bacteria expressing the p27 protein genetically fused into the flagellin. We found that p27 expressed into the flagellin led to the strongest cellular responses, where we obtained the highest production of IFN-gamma and cell proliferation, an indication of specific Th1-like orientation of the immune response. We confirmed the role of flagellin in this response by using different immunization combinations.

Constitutive and induced activation of JAK/Stat pathway in leukemogenic and asymptomatic human T-cell lymphoptropic virus type 1 (HTLV-1) transformed rabbit cell lines.

We have shown that Vav and C-cbl are activated in the leukemogenic HTLV-I transformed rabbit T cell line RH/K34 but not in the asymptomatic one RH/K30. We extended these observations and investigated the activation of JAKs (Janus Kinase) and the STATs (signal transducers and activators of transcription) pathway in these cell lines. We found that Tyk2 and Stat3 are constitutively tyrosine phosphorylated in the leukemogenic cell line. Phosphorylation of Tyk2 can be induced in RH/K30 by treatment with IL-10, interferon alpha (INFalpha) and by the supernatant of RH/K34 which contain both these cytokines. Stat3 tyrosine phosphorylation can be induced in RH/K30 by treatment with IL-10. Transfection of RL-5, a rabbit T-cell line, with the RH/K34 viral clone transiently increased the expression of serine/threonine phosphorylated Stat3. Differences were also observed on induced Stat5 phosphorylation. These results highlight the relation between the virulence of HTLV-I and the activation of the Jak/Stat pathway.

Homologous recombination with linear DNA to insert antigenic protein in the flagellin: improvement of the Th1 immune response.

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.