A NEW SEROLOGICAL SYSTEM FOR PREDICTING HCV TREATMENT RESPONSE, PRELIMINARY RESULTS

Tatiana Kuznetsova1, Tatjana Tallo1, Vadim Brjalin2, Irina Reshetnjak1, Maria Smirnova3, Alexei Shevelev3, Valentina Tefanova1

1Department of Virology, NIHD, Tallinn, Estonia2West-Tallinn Central Hospital, Tallinn, Estonia

3Federal State Budgetary Institution «M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides» of RAMS, Moscow, Russia

Previously we analyzed complete NS5A sequences from Estonian patients with chronic HCV-1b infection who had received combination therapy with PegIFNα-2a plus ribavirin. Phylogenetic analysis revealed four subclades tending to confer a different treatment outcome. These subclades were characterized by several steady inherited amino acid substitutions. However, detection of amino acid substitutions in HCV is precluded by irreproducible PCR of relevant NS5A region. Serological methods, e. g. solid-phase immune-assay (ELISA), provides an efficient and inexpensive alternative to PCR for routine assay. However, there are no commercial kits available for assay of antibody response towards NS5A regions. Additionally, NS5A protein is not easily available as an antigen for serological tests.

Background

ResultsMethods

Conclusions and plans

Objectives Our work was focused on designing short derivatives of NS5A gene adapted for efficient production in E. coli and allowing NS5A putative subclades

discrimination within HCV1b genotype by serological testing.The first stage required establishing an innovative screening method for designing truncated antigens. This approach included in vitro constructing libraries of

NS5A fragments and their screening for expression efficiency in bacteria. The second stage included selection of an appropriate clones by serological methods (Western-blot). The third stage involved engineering high-efficient producers of designed NS5A mini-derivatives fused with green fluorescent protein (GFP).

1. Nicking PCR product with DNAse. NS5A PCR products were treated with DNAse in several dilutions to produce nicks in DNA. Probes with mild extent of degradation were used for the next stage.

2. Generation of deletion derivatives. DNA probes purified by phenol-chloroform extraction were treated with DNA polymerase I for extending the gaps in DNA. Then DNA probes were mixed with random primers bearing constant adapter sequence at 5’-end and ligated. The library of deletion derivatives was eventually produced by PCR with a single adapter primer. 3. Cloning library of gene fragments into specially designed LacZ-based vector was performed. Phenotypical selection for brightness of colonies was carried out. Level of protein expression was evaluated by beta-galactosidase activity test.

4. PCR with standard primers for determination of insertion size in the selected clones was performed. Obtained PCR products were sequenced.

5. LacZ-based protein production and Western-blot with HCV-positive sera from patients were performed.

6. Engineering GFP-based producers of selected NS5A derivatives was carried out.

The scheme of producing short derivatives library was designed.One water-soluble immune-positive protein was obtained. Its purification and further serological testing in ELISA with

panels of serum samples is in a progress. Testing alternative enzymes for extending the gaps in DNA, e.g. exonuclease III and lambda phage nuclease, is in course. Obtaining and testing new NS5A-derived proteins is considered.

Delution of DNAse 104 105 106 conc. 1 kb Ladder

Probes: a s n r 100 bp Ladder

beta-galactosidase activity Precipitate

Supernatant

PAGE electrophoresis under denaturing and quasi-native conditions

kDa

Extraction by: Tris SDS Ac.acid Tris SDS Ac.acid

M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides

1. Selection of DNA probes with mild extent of degradation

2. Results of PCR with a single adapter primer

3. Quantification of beta-galactosidase activity in soluble cell fractions

4. Insertion size range: from 50 bp to 700 bp

5. One out of 40 clones was immune-positive

6. Primary purification of a fused protein with GFP