Coexpression of 16.8 kDa antigen of Mycobacterium avium paratuberculosis and murine gamma interferon in a bicistronic vector and studies on its potential as DNA vaccine

Abstract

Paratuberculosis or Johne's disease is a chronic gastric disease of ruminants. For this disease there is no effective treatment or preventive measure available. 16.8 kDa protein is an immunogenic protein of Mycobacterium avium paratuberculosis and can be an ideal candidate for developing a DNA vaccine construct. In present study a bicistronic DNA vaccine construct pIR16.8/IFN was developed using eukaryotic vector pIRES 6.1. Two genes MPT (expressing 16.8 kDa protein) and murine IFNgamma were cloned, expressed and immunoreactivity was studied in murine model. Immunoreactivity was also compared with monocistronic construct pIR16.8 expressing 16.8 kDa protein. Both pIR16.8 and pIR16.8/IFN showed eukaryotic expression of respective proteins in BHK21 cells. The expressed proteins also showed immunoreactivity when reacted with hyperimmune sera raised against recombinant 16.8 kDa protein in western blot assay and immunofluorence assay. Both constructs were used as DNA vaccine in murine model and immunogenecity was studied by DTH, lymphocyte proliferation assay and NO determination. DTH reaction was significantly high in pIR16.8/IFN than pIR16.8 group, similarly lymphocyte proliferation and NO release was higher in pIR16.8/IFN group than pIR16.8 group. This indicated T cell epitopic nature of 16.8 kDa protein. The study also showed that co-expression of IFNgamma with mycobacterial gene can enhance immunogenecity of DNA vaccine and can be used as immunoadjuvant.

Screening of Mycobacterium avium subsp. paratuberculosis mutants for attenuation in a bovine monocyte-derived macrophage model

“…It is likely that Johne's disease vaccines will need to utilize similar strategies to elicit immunity. Promising Johne's disease vaccine candidates utilizing unconventional methods include those that present MAP antigens in a Salmonella backbone, recombinant MAP proteins and DNA constructs (Kadam et al, 2009; Santema et al, 2009, 2011; Chandra et al, 2012; Faisal et al, 2013). …”

Cellular and humoral immune responses in sheep vaccinated with candidate antigens MAP2698c and MAP3567 from Mycobacterium avium subspecies paratuberculosis

“…Recombinant MAP vaccines should have various merits over killed or attenuated vaccines in terms of antigen production and human safety. The most commonly evaluated recombinant proteins have been Hsp 70 (Koets et al, 2006), antigen 85, 74F, SOD, 35 kDa (Chen et al, 2008; Kathaperumal et al, 2008; Park et al, 2008), mpt (Heinzmann et al, 2008), 95 kDa (Bull et al, 2007), P22 (Rigden et al, 2006), 65 kDa (Velaz-Faircloth et al, 1999), and 16.8 kDa (Kadam et al, 2009). Many of the recombinant vaccines were reported to induce strong cellular as well as antibody mediated immune responses (Rigden et al, 2006; Kathaperumal et al, 2008; Roupie et al, 2008).…”

Coexpression of PPE 34.9 Antigen ofMycobacterium aviumsubsp.Paratuberculosiswith Murine Interferon Gamma in HeLa Cell Line and Study of Their Immunogenicity in Murine Model

“…The cell-mediated immunity plays a pivotal role to control the spread of organisms within the host body [32]. DNA vaccines may open new horizons for effective vaccination against paratuberculosis as strong CMI responses including CTL and Th1 type cytokines are induced [20]. …”

“…The ligation mixture was transformed in E. coli competent DH5 α cells. Further, to prepare bicistronic construct pIR PPE/IFN, pIR IFN [20] was used and same strategy was adapted to insert the PPE 34.9 in the frame A.…”

“…Recently from our laboratory, Kadam et al [20], have reported that coexpression of IFN γ with a 16.8 kDa gene of MAP can enhance immunogenicity of DNA vaccine using the same protein. In the present study, we have used a similar approach to clone a 34.9 kDa PPE (PPE34.9) antigen of MAP in the A frame of the bicistronic vector pIRES 6.1 having IFN γ gene in the frame B used by Kadam et al [20]. Further, we have studied the coexpression of these two antigens in HeLa cell line.…”