Protein & Peptide Letters, 2007, 14, 865-870 865 0929-8665/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd. Application of a Chimeric Synthetic Peptide in the Development of a Sero- logic Method for the Diagnosis of Hepatitis G Virus Infection M. Fernández-Vidal 1 , M.D. Cubero 1 , G. Ercilla 2 , M.J. Gómara 1 and I. Haro 1, * 1 Department of Peptide and Protein Chemistry, IIQAB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain; 2 Immunology Service, ICII-IDIBAPS, Hospital Clínic, Barcelona, Spain Abstract: New putative antigenic peptides corresponding to the N- and C-terminal of the E2 envelope protein of GBV- C/HGV were synthesized using solid-phase chemistry. The antigens were obtained in linear and chimeric forms with the main aim of improving the sensitivity of the enzyme immunoassays. Furthermore, CD and FTIR have been used in con- junction to characterize their conformational changes showing that the chimeric peptide presents a more ordered secon- dary structure than its parent peptides. Keywords: Hepatitis G virus / GB virus C, solid-phase peptide synthesis, chimeric peptides, ELISA, circular dichroism, Fou- rier transform infrared spectroscopy. INTRODUCTION The hepatitis G virus (HGV/GBV-C) is the most closely related human virus to the hepatitis C virus (HCV) both be- longing to the small enveloped viruses of the Flaviviridae family, which is transmitted by contaminated blood and/or blood products, intravenous drug use, from mother to child and by sexual intercourse. The natural history of GBV- C/HGV infection is at present not fully understood and its potential to cause hepatitis in humans is questionable [1,2]. Epidemiological studies have indicated that it does not cause acute or chronic hepatitis. However, recent reports raised interest in this apparently non-pathogenic virus because co- infection with GBV-C/HGV and the human immunodefi- ciency virus (HIV) have been associated with slower pro- gression of the illness and a higher survival rate of patients once AIDS has developed [3-5]. GBV-C/HGV active viremia can be detected by a reverse transcription-polymerase chain reaction (RT-PCR) [6]. Al- though past infection could be detected through the presence of anti-E2 antibodies using an ELISA assay involving the E2 recombinant protein [7], at present, no commercial tests to detect past infection do exist. In this sense, synthetic peptides have been shown to be valuable tools for viral laboratory diagnosis. They can provide a uniform, chemically well- defined antigen for antibody analysis, reducing inter- and intra-assay variation. In recent years, synthetic peptides that mimic specific epitopes of infectious agents proteins have been used in diagnostic systems for various diseases [8-11]. In the context of using synthetic peptides or peptide deriva- tives for the diagnosis of GBV-C/HGV infections, Toniutto et al. [11] studied putative B-cell epitopes in the non- structural regions 4 (NS4) and 5 (NS5) of the GBV-C/HGV polyprotein. More recently, McLinden et al. [12] have sug- *Address correspondence to this author at the Department of Peptide and Protein Chemistry, IIQAB-CSIC, Jordi Girona Salgado 18-26, 08034 Barce- lona, Spain; Tel. + 34-93-400-61-09; Fax: + 34-93-204-59-04; E-mail: ihvqpp@iiaqb.csic.es gested that the E2 protein contains a single immunodominant antigenic site that includes a complex epitope involved in specific cellular binding. In our group, new epitopes located in E2 and NS3 pro- teins of GBV-C/HGV were also identified and synthesized for their use in immunoassays [13]. However, short peptides representing topographic B-cell epitopes are generally poorly antigenic. Although physical mixtures of antigens can be used in the assays, the sensitivity and specificity can be af- fected by the competition for binding on the solid phase and for changes in the spatial distribution of antigenic determi- nants of bound peptides. To overcome these drawbacks it has been described the use of chimeric peptides in order to im- prove the sensitivity and specificity of the assays [8,14,15]. In this context, our group synthesized linear, chimeric and cyclic forms of new putative epitopes located in non- structural proteins of GBV-C/HGV to use in immunoassays [16]. The results showed, on one hand, that the combination of different antigens seems to be necessary to ensure good sensitivity and more specificity and, on the other hand, that cyclic compounds show higher ability to recognize anti- GBV-C/HGV antibodies than its parent peptide. In the present work, new putative antigenic peptides cor- responding to the amino and carboxy terminal of the enve- lope protein E2 of GBV-C/HGV were identified by com- puter-aided prediction of antigenicity. They were synthesized using solid-phase peptide chemistry. The antigens were ob- tained in linear and chimeric forms with the main aim of improving the sensitivity of the enzyme immunoassays. All these synthetic constructs were evaluated by ELISA to estab- lish whether the epitopes in chimeric peptides are more effi- ciently recognized by the specific antibodies compared to the monomeric linear sequences. The conformational analysis of the parent linear sequences and the chimeric peptide by cir- cular dichroism (CD) and Fourier transform infrared spec- troscopy (FTIR) is also described.