Pam3CSK4 enhances adaptive immune responses to recombinant Mycobacterium bovis bacille Calmette-Guérin expressing Plasmodium falciparum C-terminus merozoite surface protein-1

Objective: To determine the effects of toll-like receptor 2 (TLR-2) agonist, Pam3CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum. Methods: Six groups of mice (n=6 per group) received intraperitoneal phosphate buffered saline T80 (PBS-T80), BCG or rBCG in the presence or absence of Pam3CSK4. Enzyme-linked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2a, and IgG2b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN-γ via enzyme-linked immunosorbent assay. Results: The production of total IgG and the isotype IgG1, IgG2a and IgG2b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3CSK4 further enhanced their productions. A similar pattern was also observed in IFN-γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3CSK4. Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2a, IgG2b, as well as IFN-γ in response to rBCG. However, the presence or absence of Pam3CSK4 had no effect on IL-4 production.

Role of toll-like receptor 4 in eliciting adaptive immune responses against recombinant BCG expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum

Objective:To determine the role of toll-like receptor 4 (TLR-4) in eliciting cellular and humoral immune responses against recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum.Methods:Six groups of mice (n=6 per group) were injected with phosphate buffered saline T80,BCG or rBCG intraperitoneally,in the presence or absence of a TLR-4 inhibitor;TAK-242.Enzyme-linked immunosorbent assay was carried out for serum total IgG,IgG1,IgG2a and IgG2b determination.Spleens were also harvested and splenocytes cultured for determination of intracellular cytokines;IL-4 and IFN-γ via enzyme-linked immunosorbent assay.Results:The production of total IgG,and the subclasses IgG 1,IgG2a and IgG2b was significantly higher in rBCG-immunised mice than BCG and phosphate buffered saline immunised mice in the absence of TAK-242.A significant rise in total IgG occurred with more booster immunisations.The level of IgG2a was highest,followed by IgG2b,then IgG1.The production of both IL-4 and IFN-γ was also highest in the rBCG immunised groups.These significant rises were inhibited in the presence of TAK-242.Conclusions:We present evidence of the role of TLR-4 in the increased production of total IgG,IgG1,IgG2a and IgG2b,as well as IL-4 and IFN-γ in response to our rBCG construct.

Role of toll like-receptor 2 in inflammatory activity of macrophage infected with a recombinant BCG expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum

Objective: To investigate the role of toll-like receptor 2 (TLR2) in inflammatory activity of macrophage infected with the recombinant Mycobacterium bovis bacillus Calmette-Guerin (rBCG). Methods: Mouse macrophage cell line J774A.1 was infected with Mycobacterium bovis bacillus Calmette-Guerin (BCG) and rBCG cultures for 48 h in the presence or absence of 10 μg/mL of TLR2 inhibitor. Untreated macrophages were used as a negative control while lipopolysaccharide-stimulated macrophages were used as a positive control. The ability of the macrophage to engulf the BCG and rBCG in the absence or presence of TLR2 inhibitor was assessed using a phagocytic assay, while the production of inflammatory cytokines and nitric oxide by the infected macrophages was evaluated using ELISA and Griess reagent method, while the expression of the inducible nitric oxide synthase was determined using Western blot analysis. Results: The results showed that blocking TLR2 function reduced the phagocytic activity, nitric oxide production and proinflammatory cytokine secretion such as TNF-α, IL-1β and IL-12p40 as well as inducible nitric oxide synthase expression in the infected macrophages. These data showed the importance of TLR2 in the activation of macrophages following BCG and rBCG infections. Conclusions: Through exploring the immunological mechanism which underlies the protection conferred by the candidate vaccine, this study will improve our understanding of the vaccine candidate's mechanism to protect the host from malaria infection.