1 Biomedical Research and Study Centre of Latvia, Riga, Latvia

1. Presentation of hepatitis C virus hypervariable region 1 from individual patient isolates on HBc virus-like particles Sudmale G.1, Sominskaya I.1, Petrovskis I.1., Ose V.1.,Skrastina D.1, Arsha F.2, Sondore V.2, Walker A.3, Lange M.3,Pumpens P.1 1Biomedical Research and Study Centre of Latvia, Riga, Latvia 2 Infectology Center of Latvia, Riga, Latvia 3 Institute of Virology, Hospital of Essen, University of Diusburg-Essen, Essen, Germany gunita@biomed.lu.lv Background Hepatitis C virus (HCV) remains one of the most important human pathogens, but no HCV vaccine is available, yet. Globally, hepatitis C virus has infected an estimated 170 million people, most of whom are chronically infected and are at risk for developing of chronic liver disease, cirrhosis, and primary hepatocellular carcinoma. HCV variability is one of the reasons that prevent creation of anti-HCV vaccine. Despite high variability, the most promising vaccine candidates are the envelope proteins E1 andE2 especially the first 27 amino acids of E2 are the most variable region of HCV and are referred ashypervariable region 1 (HVRI)(Fig.1.). It contains an in principal antibobody neutralizing and CTL epitopes. Results All recombinant plasmids led to relatively high levels of expression of chimeric proteins in E.coli, which in case of seven constructions resulted in the formation of complete “mature” VLPs(Fig.4.). Chimeric HBc/HVR1 VLPswere purified by gel filtration. Fig.8. Neutralization of HCVpp H77 (1a). HCVpp H77 were incubated with different concentration of anti-HVR antibodies: anti-G44(1a), anti-G47(1a), anti-G53(1a), anti-G55(3a) prior to infection of Huh7.5 and after tree days neutralization was analyzed by luciferase activity. Fig.4. Electronmicroscopy of chimeric HBc/HCV HVR1 VLPs: (A) HBc_modif/HVR1 (1a), (B)HBc_modif/HVR1 (1b)(C) HBc_modif/HVR1 (3a) (D)HBc145/HVR (1a) (E) HBc145/HVR (3a)(F) HBc183/HVR1 (1a) (G) HBc183/HVR1 (3a) VLPs (magnification x 200 000) Fig.1. HCV genome organization, expressed proteins and proteolytic cleaving (Lloyd A R, et all). Fig.9. Neutralization of HCVpp GT 1a. HCVpp GT1a bearing specific HVR 1a sequence were incubated with different concentration of anti-HVR antibodies: anti-G44(1a), anti-G47(1a), anti-G53(1a), anti-G55(3a) prior to infection of Huh7.5 and after tree days neutralization was analyzed by luciferase activity. VLPs are self-assembled from the proteins that make up a virus' outer coat, and are often used as a component of vaccines.Recombinanthepatitis B virus core protein (HBc) is one of the most promising carriers for foreign epitopes because of the HBc high immunogenicity andpossibility to make well-organized particles(Fig.2.). Groups of 4-6 Balb/c mice were immunised with 25 mg recombinant protein and 125 mg adjuvant- alhydrogel and bosted in day 14 and 28 (Fig.5). At day 42 mice were bleeded and tested for specific antibodies. Serial dilutions were made and dilutionsyielded tree times the optical density atl495 obtained with intact mice serum were scored positive (Fig.6) Fig. 2. Model of Hepatitis B core protein viruse-like particle . Fig.5. Immunization sheme of experiment The major aim of the project was development of virus-like particles (VLP) displaying hipervariable region 1 (HVR1) individual isolates on hepatitis B core (HBc) carrier and analysis of anti-HVR antibodies neutralizing effect. Fig.10. Neutralization of HCVpp GT 3a. HCVpps GT3a bearing specific HVR 3a isolate sequence from patient were incubated with different concentration of anti-HVR antibodies: anti-G44(1a), anti-G47(1a), anti-G53(1a), anti-G55(3a) prior to infection of Huh7.5 and after tree days neutralization was analyzed by luciferase activity. Materials and methods HCV HVR1 and core sequences were amplified from ten chronically infected viral hepatitis C patient blood serum samples (Infectology Center of Latvia). To define the HCV genotype core region was sequenced and determined that HCV in researh group represents genotype 1b and 3a. HCV HVR1 region was cloned into pJET1.2 vector and ten clones from each sample were sequenced. Two different HVR1 isolates 1b and 3a from patients and additional HVR1 1a were selected.Further HVR1 isolates were fused to C terminusof full lenght HBc with modified C terminus, non-modifid C terminus and in MIR region of HBc145. Recombinant plasmids weresequenced and expressed as fusion proteinsin BL21 and K802 cell lines (Fig.3.). • Conclusions • Rrecombinant plasmids led to relatively high levels of expression of chimeric proteins in E.coli. • Seven of the nine constructions are able to form “mature” recombinant viruse-like particles. • Results of the current study have demonstrated the principal possibility of using HVR1 for creation of chimeric VLPs on the basis of HBcAg . • HBc/HVR1 VLP arehighlyimmunogenic in mice. • The induced antibodies are able to neutralize also unrelated HCVpp’s • Anti-HVR1 antibodies neutralizespecificHCVpp’sinalldilutions. • Anti-HVR1antibodieshave a virusneutralizingeffect. Fig.6. Specific titer of anti-HBc and anti-HVR antibodies after immunization with HBc/HVR1 VLPs. G44(1a) , G47(1a), G53(1a) and G55(3a) mice sera samples were selected for viruse neutralization test. HCV pseodo-particles (HCVpp) were generated by contrainfection of 293-T cells with equal amonts of expresion plasmids expresing viral gps, E1E2 containing diferent HVR1 sequences and NL4.3.Luc.R-E-. HCVpp’s were incubated with different concentrations of anti-HVR antibodies prior to infection of naive Huh 7.5. – cells. At day 3 post-infection neutralization was analyzed by luciferase activity (Fig.7,8,9,10). G47 G48 G50 G44 G51 G52 G53 G54 G55 Acknovledgements I would like to thank my colleaugues: Dr.biol. I.Petrovskis and his group (Dz.Dreilina, J.Zakova, I.Lieknina) for protein purification and analysis. J.Bogans for protein purification. I.Akopjana for competent cells. G.Zarins for imunobloting. Dr.chem. L.Ignatovica for sequencing analysis; This work was supported by ERAF project2010/0224/2DP/2.1.1.1.0/10/APIA/VIAA/164, ESF project 2009/0204/1DP/1.1.1.2.0/09/APIA/VIAA/150. Fig.7. Neutralization of HCVpp bearing unrelated HVR1 variants. Anti-HVR antibody neutralizing effect was tested with four different HCVpp: YK5829 (1b subtype), G31 consensus sequence (Puntoriero et all, 1998), YK5807 (1b subtype), H77 (1a subtype). Fig.3. HBc/HVR1 chimeric constructs used in the study