Broadly cross-reactive mimotope of hypervariable region 1 of hepatitis C virus derived from DNA shuffling and screened by phage display library.
The hypervariable region 1 (HVR1) is the target of neutralizing antibodies but with isolate specificity. The aim of this study was to obtain immunogenic mimotopes of HVR1, which can react broadly with different HVR1 antibodies and could be one of the candidate immunogens in an effective vaccine against HCV. Thirty-one HVR1 cDNA fragments were digested by DNase I into a pool of random fragments and reassembled by repeated cycles of annealing in the presence of DNA polymerase to their original size. The shuffled HVR1 was then inserted into the gene III phagemid vector pCANTAB-5E and displayed on the surface of the phage. Eight individual phages were selected after four rounds of biopanning against anti-HVR1. ELISA was carried out on immobilized purified phages, respectively, to detect their reactivity with a panel of sera. DNA sequences of the inserts were analyzed and compared with the consensus sequences defined by Puntoriero et al. [(1998) EMBO J 17:3521-3533]. The reactivity of the eight selected clones to the 30 sera were from 53.3 to 80%. Among these, phage 13 (ETYVSGGSAARNAYGLTSLFTVGPAQK, aa 384-410) reacted most broadly. None of the selected sequences encoded for peptides corresponded to known HVR1 from original viral isolates. The two high reactive phages had the similar amino acid sequences with the consensus, which might play a particular role in determining the frequency of reactivity. In conclusion, this study has used effectively DNA shuffling combined with phage display technology to identify broadly cross-reactive mimotopes recognized by human polyclonal antibodies. Mimotope 13 and 23 appeared to be most reactive immunologically and could be candidate immunogens. Efforts are now underway to identify their neutralizing antibodies by immunization of animals.
Zhang
Xin-Xin
XX
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China.
Deng
Qiang
Q
Zhang
Shen-Ying
SY
Liu
Jing
J
Cai
Qian
Q
Lu
Zhi-Meng
ZM
Wang
Yuan
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D003429:Cross Reactions; D004279:DNA, Viral / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics* / Q000276:immunology*; D006526:Hepatitis C / Q000276:immunology / Q000821:virology; D018937:Hepatitis C Antibodies / Q000097:blood; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology*
D004279:DNA, Viral; C400562:HVR1 protein, Hepatitis C virus; D018937:Hepatitis C Antibodies; D010446:Peptide Fragments; D019151:Peptide Library; D014764:Viral Proteins