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Low immunogenicity is one of the major problems limiting the clinical use for DNA vaccines, which makes it impossible to obtain a strong protective immune response after vaccination. In order to explore whether Ag85A DNA vaccine could mount more efficiently protective immune response through new RNA sensor and its signal transduction pathway of antigen presentation we designed and synthesized Ag85A gene fragment containing multiple points mutations and transfected the gene fragment into the dendritic cell line (DC2.4) by CRISPR/Cas9. Subsequently, we focused on the changes of RNA sensors RIG-I, Mda-5, and the downstream adaptors MAVS, IRF3, IRF7 and IFN-β. The results indicated the significant increases in the mRNA and protein expression of RNA sensors RIG-I, Mda-5 and related adaptors MAVS, IRF3, IRF7, and IFN-β in the mutant DC 2.4 cells. The flow cytometry results demonstrated that the expression of MHC II on the surface of DC 2.4 significantly increased when compared with that in control. Therefore, it is suggested that Ag85A mutant DNA could release immunogenic message through RNA sensors and related adaptors via non protein pathway. There is at least one RNA signal transduction pathway of Ag85A DNA in DC2.4 cell. The work provides a new mode of action for nucleic acid vaccine to improve immunogenicity and meaningful data for the better understanding of the mechanisms of DNA vaccine. [ABSTRACT FROM AUTHOR]

Antigen-85A (Ag85A) is one of the major proteins secreted by Mycobacterium tuberculosis. Many studies on animal models have shown that vaccination with the recombinant Ag85A-DNA or Ag85A protein induces powerful immune response. However, these vaccines cannot generate sufficient protective immunity in the systemic compartment. CD226, a member of the immunoglobulin superfamily, is expressed in the majority of NK cells, T cells, monocytes, and platelets, and can be served as a co-stimulator that contributes to multiple innate and adaptive responses. However, there has been no study where either CD226 protein or DNA has been used as an adjuvant for vaccine development. The aim of this study was to develop a novel Ag85A DNA vaccine with CD226 as the genetic adjuvant to increase the immune efficacy induced by Ag85A. Oral vaccination with pcDNA3.1-Ag85A-CD226 DNA induced potent immune responses in mice. CD226 was an effective genetic adjuvant that improved the immune efficacy induced by Ag85A and enhanced the activity of cytotoxic T lymphocytes (CTL) and NK cells in mice. Th1 dominant cytokines (i.e. IL-2, IFN-γ and TNF-α), cellular immunity (i.e. CD4 + IFN-γ + T cells and CD8 + IFN-γ + T cells in splenocytes) and MLNs were also significantly elevated by pcDNA3.1-Ag85A-CD226 DNA vaccination. Our results suggest that CD226 is an effective adjuvant to enhance the immune efficacy induced by Ag85A. Our findings provide a new strategy for the development of a DNA vaccine co-expressing Ag85A and CD226. [ABSTRACT FROM AUTHOR]

Abstract: Interleukin (IL)-17A contributes to the development of asthma, especially in severe asthma which has characteristic neutrophil infiltration in airways. However, IL-17A-blocking antibody could escalate T helper (Th) 2 cytokines, such as IL-13, IL-4 in murine models. We aimed at determining the effect of mycobacterial Ag85A and IL-17A fusion protein—Ag85A-IL-17A on airway inflammation in a murine model of asthma. IL-17A recombinant protein fused mycobacterial immunodominant antigen Ag85A was constructed, expressed and purified. The fusion protein was then administrated into BALB/c mice and its anti-inflammatory effects in the infiltration of inflammatory cells, Th2/Th17 cytokines in BALF, histopathological changes of lung tissues as well as chemokines in lung tissues were evaluated in the murine model of asthma. We found that administration of mycobacterial Ag85A and IL-17A fusion protein induced IL-17A specific immunoglobulin (Ig)G in sera and significantly decreased IL-17A and IL-6 levels in bronchoalveolar lavage fluid (BALF). Ag85A-IL-17A vaccinated mice also showed marked reduction in the infiltration of inflammatory cells in peribronchiolar region and significant decrease in total cells, eosinophil cells and neutrophil cells in BALF. The increased levels of IL-13 and IL-4 in BALF of ovalbumin-sensitized mice were significantly reduced by the administration of Ag85A-IL-17A. Furthermore, CD3+CD4+IL-13+ splenocytes stimulated with OVA and CXCL1 mRNA, CCL2 mRNA and GATA-3 mRNA expressed in lung tissues were decreased markedly in Ag85A-IL-17A vaccinated group. Our results demonstrate remarkable antiallergic effects of Ag85A-IL-17A in a murine model of asthma and it may have protective effects on allergic asthma. [Copyright &y& Elsevier]

Abstract: The ability of dendritic cells to provide all the signals required for T-cell activation makes them an ideal cancer vaccine platform. With the use of established DC2.4 cell line, originated from C57BL/6 mice and developed by superinfecting GM-CSF transduced bone marrow cells with myc and raf oncogenes, we investigated whether the DC 2.4 cell line transfected with Ag85A gene could enhance immunity against bladder cancer. Both phenotypic and functional analyses of Ag85A‐DCs were done with use of FCM and T cell proliferation test. The cytotoxicity of Ag85A-DCs loaded with tumor cell lysate was verified by LDH. Finally, the production of interferon gamma was assayed by both ELISA and FCM. The immunotherapeutic effect of DC vaccine on murine bladder cancer was assessed pharmacologically and pathologically. Our results showed that Ag85A gene transfected DCs expressed high levels of key surface markers such as CD80, CD86 and MHC-II. The CTL primed with MB49 lysate-pulsed Ag85A-DCs elicits higher activity against MB49 tumor cells and upregulated level of IFN-γ production. Furthermore, the significant inhibitive effect on tumor growth in mice was found in the group of Ag85A-DC vaccine. The infiltration of CD4+ or CD8+ T cell within established tumor treated by Ag85A-DC vaccine significantly increased as compared with control groups. It is therefore concluded that DCs engineered by Ag85A gene exerts enhanced anti-tumor immunity against bladder cancer and this study might provide a meaningful mode of action with the use of Ag85A engineered DC vaccination in anti-cancer immunotherapy. [Copyright &y& Elsevier]