Catalytic domain of PDC-E2 contains epitopes recognized by antimitochondrial antibodies in primary biliary cirrhosis

AIM:To search for further immunodominant peptides of the pyruvate dehydrogenase complex E2-component (PDC-E2) recognized by antimitochondrial antibodies (AMA) in primary biliary cirrhosis (PBC). METHODS:Sera from 95 patients with PBC were tested by enzyme-linked immunosorbent assay against 33 synthetic overlapping peptides (25 amino acids; aa) covering the entire length of the E2-subunit of PDC-E2. Furthermore,the inner lipoyl peptide 167-184 was used in an unlip oylated and a lipoylated form as well as coupled to ovalbumin. Sera from 11 AMA negative/ANA posit ive PBC patients,63 patients with other liver disorders and 22 healthy blood donors served as controls.RESULTS:Of the 95 PBC-sera,74% reacted with the peptide 475-499 and 58% with the pept ide 407-431 located within the catalytic domain of PDC-E2. Patients with other disorders or healthy controls were positive in only up to 18%. Antibodies to the unlipoylatedand lip oylated pept ide 167-184 within the inner lipoyl domain were found in only 5% and 11% of the PBC sera,respectively; using ovalbumin-coupled peptides,the incidence increased up to 57% (unlipoylated form). CONCLUSION:Peptides within the catalytic site of PDC-E2 rather than the previously reported lipoyl binding peptide 167-184 may represent major immunodomin ant epitopes recognized by AMA in PBC.

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.

Prokaryotical expression of structural and non-structural proteins of hepatitis G virus

AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.

Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the “a” region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the “a” determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.

An overview on application of phage display technique in immunological studies

Phage display is very strong technique in drug discovery and development. Phage display has many applications in improving the immunological studies. Development of monoclonal antibody, peptides, peptidomimetics and epitope mapping are main application of phage display. Selection of monoclonal antibody or peptides that are displayed on the surface of the phages can be occurred through biopanning process. In biopanning process phage library is incubated with antigen and particular phages can be identified and isolated. Increasing the stringency in the biopanning rounds can be help to select phages with high affinity and specificity. Here, we describe an overview of phage display application with focusing on monoclonal antibody production and epitope mapping.

Phage display creates innovative applications to combat hepatitis B virus

Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20th century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases.

Functional and structural characterization of Ebola virus glycoprotein （1976-2015） -- An in silico study

Ebola virus （EBOV） is the causative agent of a severe hemorrhagic fever disease associ- ated with high mortality rates in humans. This virus has five strains of which Zaire Ebola virus （ZEBOV） is the first and most important strain. It can be transmitted through contact with contaminated surfaces and objects. The genome of EBOV codes one non- structural and seven structural proteins consisting of two forms of glycoprotein （GP）： soluble glycoprotein （sGP） and GP （spike）. In this paper, we attempted to characterize and predict physicochemical properties, B-cell epitopes, mutation sites, phosphorylation sites, glycosylation sites, and different protein structures of EBOV GP to provide com- prehensive data about changes of this GP during a 40-years course （1976-2015）. GP sequences were obtained from NCBI gene bank from 1976-2015. Expasy＇sProtParam, PROTSCALE, immuneepitope, Bepipred, BcePred, ABCpred, VaxiJen, DISPHOS, Net- Phos, and numerous programs were used to predict and analyze all sequences. More variety of mutations were found in 2015 sequences and mutations were related to huge changes in B-cell epitopes, phosphorylation and glycosylation sites. In addition, our results determined different sites of disulfide bonds and an important mutation related to IgE epitope as well as four potent B-cell epitopes （380-387, 318-338, 405-438 and 434-475）. In this study, we suggested the effect of mutations on GP properties, six posi- tions for disulfide bonds and four phosphorylation sites for protein kinase C enzyme. Selected sequences were shown different sites for O-link and N-link glycosyl^tion. A mutation that changed GP to an allergen protein and has a significant role in vaccine designing as well as four potent B-cell epitopes in GP protein were found.