Identification and Characterization of Paramyosin from Cyst Wall of Metacercariae Implicated Protective Efficacy against Clonorchis sinensis Infection

Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-γ and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization

Single Dose Novel Salmonella Vaccine Enhances Resistance against Visceralizing L. major and L. donovani Infection in Susceptible BALB/c Mice

Visceral leishmaniasis is a major neglected tropical disease, with an estimated 500,000 new cases and more than 50,000 deaths attributable to this disease every year. Drug therapy is available but costly and resistance against several drug classes has evolved. Despite all efforts, no commercial, let alone affordable, vaccine is available to date. Thus, the development of cost effective, needle-independent vaccines is a high priority. Here, we have continued efforts to develop live vaccine carriers based on recombinant Salmonella. We used an in silico approach to select novel Leishmania parasite antigens from proteomic data sets, with selection criteria based on protein abundance, conservation across Leishmania species and low homology to host species. Five chosen antigens were differentially expressed on the surface or in the cytosol of Salmonella typhimurium SL3261. A two-step procedure was developed to select optimal Salmonella vaccine strains for each antigen, based on bacterial fitness and antigen expression levels. We show that vaccine strains of Salmonella expressing the novel Leishmania antigens LinJ08.1190 and LinJ23.0410 significantly reduced visceralisation of L. major and enhanced systemic resistance against L. donovani in susceptible BALB/c mice. The results show that Salmonella are valid vaccine carriers for inducing resistance against visceral leishmaniasis but that their use may not be suitable for all antigens

Heterologous expression of antigenic peptides in Bacillus subtilis biofilms

Background Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. Results We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. Conclusions In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.ISSN:1475-285