|
Dr. Xuhuai
Ji Stanford University 3801 Miranda Avenue, 154C Bidg 101, Rm C4-171 Mail Code 154C Palo Alto, CA 94304 Dear Dr. Ji: Thank you for agreeing to present at
DDW® 2001 in Atlanta, GA during the AASLD Topic Forum "Hepatitis C: Treatment- I,
which will be held on Monday, May 21 2001 from 2:15 PM to 3:45 PM in the Georgia World
Congress Center Room 170W-171W Xuhuai Ji, MD & PhD (Refer to this abstract
as # 101269) Stanford University Medicine/Gastroenterolgy & Hepatology 3801 Miranda Avenue, Bidg
101, Rm C4-171 Mail Code 154C Palo Alto, CA 94304 USA Analysis of genomic diversity in the
coding region of HCV NS3 Xuhuai Ji, Xiao-Song He, Harry B Greenberg, Stanford
University, Palo Alto, CA BACKGROUND: The mechanism by which
hepatitis C virus (HCV) persists in infected individuals is thought to
represent a complex interplay between viral and
host factors, and the diversity of the HCV genome may be one of these
factors. To examine the possible role of viral
genomic diversity in HCV persistence, we analyzed the coding sequence for HCV
NS3, one of the HCV proteins that is a target
for HCV-specific CTL response. METHODS: Nine HLA-A2+ patients with chronic
HCV infection were studied. A 293bp
fragment within the NS3 coding region was amplified by RT-PCR, followed by
molecular cloning. Multiple clones of the NS3
fragment from each patient were sequenced. Phylogenetic diversity analysis
was carried out by using the PAUP* software
package. NS3-specific CD8+ T cells in peripheral blood were quantified with
an HLAA2 tetramer loaded with the peptide NS3
1406-1415 (KLVALGINAV). RESULTS: A total of 219 clones from 9 patients were analyzed. All the clones belonged to
known HCV subtypes, while viral sequences from each patient were of distinct
origins as indicated by separate clusters with
strong bootstrap support. Eight patients (89%) were found to harbor
quasi-species within the context of HCV subtypes. Five to 24
variants at the nucleotide acid level were identified in different
individuals. From three patients (33%) clones of both HCV
genotypes I and 2 were isolated, suggesting co-infection or super-infection
of two HCV strains, although standard genotyping
assay (Inno-LiPA assay) at the 5'-noncoding region failed to detect the
mixed-genotype infection. Within the known
HLA-AZ-restricted epitope NS3 1406-1415, a total of 12 variant peptide
sequences were identified. CD8 T cells specific for
this epitope were detected in four (44%) patients by tetramer staining. The
functional response oftetramer-positive cells to
the different variant peptides is currently being studied. CONCLUSIONS: In
chronically infected patients the coding sequence
for the HCV protein NS3 is highly heterogeneous, as indicated by the frequent
detection of quasi-species and different
genotypes. This genomic heterogeneity results in the aminoacid sequence
variation of a potential CTL epitope in NS3. Importantly the
frequency of mixed-genotype infection appears to be higher than previously
thought, probably due to the limitation in
sensitivity and specificity of commonly used assays. Studies on the
interaction between HCV- specific CD8 T cells and variant
peptides may lead to better understanding offactors determining the outcome
of HCV infection. |