Biological, immunological and functional properties of two novel multi-variant chimeric recombinant proteins of CSP antigens for vaccine development against Plasmodium vivax infection.

The circumsporozoite protein (CSP) of the malaria parasite Plasmodium vivax is a major pre-erythrocyte vaccine candidate. The protein has a central repeat region that belongs to one of repeat families (VK210, VK247, and the P. vivax-like). In the present study, computer modelling was employed to select chimeric proteins, comprising the conserved regions and different arrangements of the repeat elements (VK210 and VK247), whose structure is similar to that of the native counterparts. DNA encoding the selected chimeras (named CS127 and CS712) were synthetically constructed based on E. coli codons, then cloned and expressed. Mouse monoclonal antibodies (mAbs; anti-Pv-210-CDC and -Pv-247-CDC), recognized the chimeric antigens in ELISA, indicating correct conformation and accessibility of the B-cell epitopes. ELISA using IgG from plasma samples collected from 221 Iranian patients with acute P. vivax showed that only 49.32% of the samples reacted to both CS127 and CS712 proteins. The dominant subclass for the two chimeras was IgG1 (48% of the positive responders, OD492=0.777±0.420 for CS127; 48.41% of the positive responders, OD492=0.862±0.423 for CS712, with no statistically significant difference P>0.05; Wilcoxon signed ranks test). Binding assays showed that both chimeric proteins bound to immobilized heparan sulphate and HepG2 hepatocyte cells in a concentration-dependent manner, saturable at 80µg/mL. Additionally, anti-CS127 and -CS712 antibodies raised in mice recognized the native protein on the surface of P. vivax sporozoite with high intensity, confirming the presence of common epitopes between the recombinant forms and the native proteins. In summary, despite structural differences at the molecular level, the expression levels of both chimeras were satisfactory, and their conformational structure retained biological function, thus supporting their potential for use in the development of vivax-based vaccine.

Designing and structure evaluation of multi-epitope vaccine against ETEC and EHEC, an in silico approach.

Diarrheal diseases represent a major health problem in developing countries. Several viruses and bacterial agents, such as Enterotoxigenic Escherichia coli (ETEC) and Enterohemorrhagic Escherichia coli (EHEC) are responsible for human enteric infections. In humans, EHEC infections result in bloody or non-bloody diarrhea, which may be complicated by haemorrhagic colitis and haemolytic uraemic syndrome (HUS). Infection by ETEC is accompanied by a non inflammatory watery diarrhea. E. coli follows a common strategy of infection: colonization on a mucosal site, evasion of host defenses, multiplication, and host damage. Intimin, Stx, Lt and Cfa proteins are the virulence factors expressed by these strains. Antibiotic treatment is generally not recommended for most cases of diarrhea, since antibiotic usage may lead to antibiotic resistance in ETEC and may also change the intestinal flora. We hypothesized that the chimeric forms of these effectors as vaccine candidates would reduce the colonization of bacteria. This study is based on an in silico analysis of chimeric protein structure and its stability and solubility. The secondary and tertiary structures of selected domains were also predicted. Moreover, T and B cell epitopes were mapped. Protein structure Prediction showed that each domain of antigen was separated completely also stable for recombinant expression. We believe that this chimeric vaccine candidate is effective for prevention of bacteria caused diarrheal diseases.

In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications.

BACKGROUND: In silico techniques are highly suited for both the discovery of new and development of existing vaccines. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological lesion (attaching/effacing). The genes encoding the products responsible for this phenotype are clustered on a 35-kb pathogenicity island. Among these proteins, Intimin, Tir, and EspA, which are expressed by attaching-effacing genes, are responsible for the attachment to epithelial cell that leads to lesions. RESULTS: We designed synthetic genes encoding the carboxy-terminal fragment of Intimin, the middle region of Tir and the carboxy-terminal part of EspA. These multi genes were synthesized with codon optimization for a plant host and were fused together by the application of four repeats of five hydrophobic amino acids as linkers. The structure of the synthetic construct gene, its mRNA and deduced protein and their stabilities were analyzed by bioinformatic software. Furthermore, the immunogenicity of this multimeric recombinant protein consisting of three different domains was predicted. CONCLUSION: a structural model for a chimeric gene from LEE antigenic determinants of EHEC is presented. It may define accessibility, solubility and immunogenecity.