Protein antigens have drawn a lot of attention from investigators working on tuberculosis vaccines. These proteins can be used to improve the immunogenicity of the new generation BCG vaccines or even replace them completely. Recombinant technology is used to insure the production of pure mycobacterial antigens in high quantities. Mycolyl transferase 85B (Ag85B) is a potent, mycobacterial antigen that significantly stimulates immune responses. Since Ag85B is an apolar protein, production of the water-soluble antigen is of interest. In this work, we report a systematic optimization strategy concerning cloning systems and purification methods, aiming at increasing the yield of recombinant Ag85B. Our optimized method resulted in a yield of 8 mg of recombinant Ag85B from 1 liter of induced culture (400 μg/ml) by using pET32a(+), Escherichia coli Rosseta-gami™(DE3) pLysS and a Ni-NTA agarose-based procedure and on-column re-solubilization. The purified recombinant Ag85B showed strong immunostimulating properties by inducing high levels of TNF-α, IFN-γ, IL-12, and IgG2a in immunized mice, therefore it can effectively be applied in TB vaccine researches.

Control of brucellosis as a worldwide zoonotic disease is based on vaccination of animals and diagnosis of infected cases to be eradicated. Accurate and rapid detection of infected animals is of critical importance for preventing the spread of disease. Current detection of brucellosis is based on whole-cell antigens and investigating serum antibodies against Brucella lipopolysaccharide (LPS). The critical disadvantage is misdiagnosis of vaccinated animals as infected ones and also cross-reactions with other Gramnegative bacteria. Recombinant outer membrane protein 2b (Omp2b) of Brucella abortus was evaluated as a novel serodiagnostic target in comparison to conventional tests which are based on LPS. Recombinant Omp2b (rOmp2b) was expressed in Escherichia coli BL21 and purified by Ni 2+-based chromatography. rOmp2b was evaluated in an indirect enzyme-linked immunosorbent assay (ELISA) system for diagnosis of brucellosis, with sera from Brucella-infected mice along with negative sera and sera from mice which were inoculated with other Gram-negative species for assurance of specificity. Thereafter, cattle sera collected from different regions were assessed along with known negative and known positive serum samples. We found that Omp2b can discriminate between Brucella-infected animals and non-infected ones. Results for assessment of two hundred and fifty cattle sera by Omp2b-based indirect ELISA which were compared to Rose Bengal plate agglutination test (RBPT) and serum tube agglutination test (SAT) showed that our proposed procedure has the sensitivity of 88.5%, specificity of 100%, and accuracy of 90.8%. We suggest that recombinant Omp2b could be used as a protein antigen for diagnosis of brucellosis in domestic animals and can be evaluated for detection of human brucellosis.

Despite a huge number of studies towards vaccine development against human immunodeficiency virus-1, no effective vaccine has been approved yet. Thus, new vaccines should be provided with new formulations. Herein, a new DNA vaccine candidate encoding conserved and immunogenic epitopes from HIV-1 antigens of tat, pol, gag and env is designed and constructed. After bioinformatics analyses to find the best epitopes and their tandem, nucleotide sequence corresponding to the designed multiepitope was synthesized and cloned into pcDNA3.1+ vector. Expression of pcDNA3.1-tat/pol/gag/env plasmid was evaluated in HEK293T cells by RT-PCR and western-blotting. Seven groups of BALB/c mice were intramuscularly immunized three times either with 50, 100, 200 µg of plasmid in 2-week intervals or with similar doses of insert-free plasmid. Two weeks after the last injection, proliferation of T cells and secretion of IL4 and IFN-γ cytokines were evaluated using Brdu and ELISA methods, respectively. Results showed the proper expression of the plasmid in protein and mRNA levels. Moreover, the designed multiepitope plasmid was capable of induction of both proliferation responses as well as IFN-γ and IL-4 cytokine production in a considerable level compared to the control groups. Overall, our primary data warranted further detailed studies on the potency of this vaccine.

Streptococcus pyogenes , a leading human pathogen, is responsible for a wide range of diseases, including skin and soft tissue infections and severe invasive diseases. S. pyogenes produces a large arsenal of virulence factors, including several immune evasion factors. We have identified an open reading frame ( spy0136 ) in the S. pyogenes SF370 genome encoding a protein of unknown function. Using recombinant Spy0136 in a pull-down assay with human plasma and ELISA, we have identified four complement proteins (C1r, C1s, C3, and C5) as binding partners. Treatment of the complement proteins with PNGase F abrogated binding to C1s, C3, and C5, indicating glycan-dependent interactions. rSpy0136 inhibited complement-mediated hemolysis and interfered with all three complement pathways in a Wieslab complement assay. Furthermore, rSpy0136 inhibited deposition of the C3b opsonin and the membrane attack complex (MAC) on the surface of S. pyogenes . We therefore named the previously unknown protein ‘complement evasion factor’ (CEF). An S. pyogenes Δspy0136/cef deletion mutant showed decreased virulence in an in-vitro whole blood killing assay and a Galleria mellonella (wax moth) infection model. Furthermore, an L. lactis spy0136/cef gain-of-function mutant showed increased survival during growth in whole human blood. Analysis of serum samples from patients with invasive S. pyogenes revealed Spy0136/CEF sero-conversion indicating expression during disease. In summary, we have identified a novel S. pyogenes immune evasion factor that binds to several complement proteins to interfere with complement function. This is the first example of a S. pyogenes virulence factor binding to several different target proteins via glycan-dependent interactions.