al., 1994; Yuki et al., 1997), yet sequential changes in HVR1 were also observed in an asymptomatic patient (Nakazawa et al., 1994) and quasispecies ...
Journal of General Virology (1999), 80, 317–325. Printed in Great Britain ...................................................................................................................................................................................................................................................................................
Ontogeny of hepatitis C virus (HCV) hypervariable region 1 (HVR1) heterogeneity and HVR1 antibody responses over a 3 year period in a patient infected with HCV type 2b Ayaz Majid,1 Peter Jackson,3 Zarah Lawal,1, 2 Gavin M. J. Pearson,3 Hayley Parker,1 Graeme J. M. Alexander,2 Jean-Pierre Allain1 and Juraj Petrik1 1, 2
Division of Transfusion Medicine1 and Department of Medicine2, University of Cambridge School of Clinical Medicine, Cambridge, UK 3 East Anglia Blood Centre, National Blood Service, Cambridge, UK
Hypervariable region 1 (HVR1) sequences of 96 clones at six time-points representing 27 variants in two major and one minor group were identified in a patient with chronic hepatitis C virus (HCV) infection over 3 years. Major and selected minor variants were used to design synthetic peptides corresponding to the HVR1 C terminus. Peptide ELISA reactivity with IgG was plotted against the corresponding clone frequency, and three patterns emerged : (1) three peptides were unreactive ; (2) antibodies against two peptides followed emergence of the corresponding variant, suggesting isolate-specificity ; (3) antibodies against four peptides preceded the appearance of the corresponding variant, indicating cross-reactivity or previous exposure. Cross-reactivity was investigated further : sera from six time-points were tested against 11 unrelated HVR1 peptides, seven of which (63n6 %) showed cross-reactivity at all time-points. Cross-reactivity of nine patientspecific peptides tested against a panel of 45 heterologous sera from chronic HCV carriers ranged between 0 and 20 %. Only three of 27 variants appeared at more than one time-point and in two cases specific and/or cross-reactive HVR1 antibodies coexisted with the corresponding variant, consistent with emergence of escape mutants. In addition, analysis of HVR1 IgG reactivity within a group of closely related patient-specific peptides revealed a loss of reactivity in one peptide attributable to a single amino acid substitution. Interferon-α treatment considerably reduced viral RNA but, paradoxically, heterogeneity increased.
Introduction Most hepatitis C virus (HCV) infections evolve to chronicity and a proportion develop serious liver disease (Colombo et al., 1991 ; DiBisceglie et al., 1991), but mechanisms underlying the development of chronicity or disease progression remain unclear. HCV (in common with other RNA viruses) has a high mutation rate and a virus population consisting of related but not identical genomes termed quasispecies (Martell et al., 1992). It has been proposed that HCV evades immunological surveillance through the emergAuthor for correspondence : Juraj Petrik. Present address : Medical Research Council, LMB, Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, UK. Fax j44 1223 402140. e-mail jp4!mrc-lmb.cam.ac.uk The EMBL accession numbers of the sequences reported in this paper are AJ228353–AJ228446.
0001-5826 # 1999 SGM
ence of escape mutants (Weiner et al., 1992 ; Taniguchi et al., 1993 ; Kato et al., 1993). The role of selective immune pressure in this process (Weiner et al., 1991 ; Higashi et al., 1993) is supported by the decreased complexity of quasispecies in immunosuppressed (Martell et al., 1994 ; Lawal et al., 1997) or agammaglobulinaemic (Kumar et al., 1994) patients. Most mutations are concentrated within a short region of hypervariable region 1 (HVR1) in the envelope E2 gene (Kurosaki et al., 1993 ; Higashi et al., 1993 ; Kato et al., 1994 ; Sakamoto et al., 1994 ; Nakazawa et al., 1994) and some neutralizing antibodies are directed against epitope(s) in HVR1. Antibodies against synthetic peptides corresponding to regions of HVR1 were shown to prevent infection of susceptible cell lines (Shimizu et al., 1996 ; Zibert et al., 1995) or chimpanzees (Farci et al., 1996). Quasispecies from infected cell lines have also shown decreased complexity compared to the inoculating virus (Sugiyama et al., 1997). While some reports described isolate-specific HVR1 antibodies (Kato et al., 1994 ; DBH
A. Majid and others
Allander et al., 1997), others (Lesniewski et al., 1993 ; Scarcelli et al., 1995 ; Jackson et al., 1997) observed cross-reactivity even against distantly related HVR1. Although there are at least six main HCV genotypes and numerous subtypes (Simmonds et al., 1993) most longitudinal studies of HVR1 in patients or chimpanzees focused on the most prevalent, type 1 (Hohne et al., 1994 ; Sugiyama et al., 1997 ; Allander et al., 1997 ; van Doorn et al., 1994). In view of potential type-dependent differences in disease severity and interferon-α (IFN-α) responsiveness (Enomoto et al., 1994, 1995), obtaining comparative data on other subtypes may be important. We therefore studied the specificity and development of HVR1 immune responses longitudinally in a patient infected with HCV genotype 2b. The extent of cross-reactivity of patient sera with unrelated HVR1 peptides or patient-specific HVR1 peptides with unrelated sera was also investigated. In addition, the effect of IFN-α treatment on HVR1 heterogeneity, serum HCV RNA and immune responses was studied.
Table 2. Patient-specific and unrelated HVR1 peptides Amino acids used for coupling to BSA are underlined. Peptide* 82 83 84 85 86 87 88 89 90 91 92 93 26 27 28 29 30 31 S64 S65 S66 S67 S68
Methods
Patient. A male blood donor, aged 43, positive by IgG EIA (Abbott), indeterminate repeatedly with RIBA 2 confirmatory test (Ortho), but confirmed positive when tested retrospectively using RIBA 3, had detectable HCV RNA in serum (Table 1). HCV genotype was 2b, as determined from the deduced C-terminal sequence of the E1 gene (Bukh et al., 1993) and by InnoLipa (Innogenetics). Further details of the clinical evaluation of the patient are available from the authors.
Variant†
Sequence
1a 1c 2e 2h 3a 3f 4a 5a 4cell 2e 3a 4a
CTGFSNLFNLGSQQKV CGGLTNLFSLGSQQKV CHGFTGLFSLGPSQTI CAALASFLSLGPQQKN CTGFSKLFNMGSQQKV CTGFSKHNNMGSQQKV CAALANFLSLGPQQKI CAALASFLSLGPQQKI CAALANSLSLGPQQKI CWGTYVTGGASGRTVH CATTYTTGAQMGQGIT CATTHITGAQTGRGTA STLTSLFRPGASQNV STLTSLFTPGPAQNV STLTSLFSPQPSQNV STLTSLFTPGASQNV STFTSLLAPGAKQNV SGLTSLFTPGASQNV CSTFTSLLAPGAKQNV CRRVASFFSPGSAQKI CLGIASFLTRGPKQNI CRTLTGMFSLGARQKI CRGFTSLFSAGSAQN
* 82–90, Patient-specific C-terminal HVR1-derived peptides ; 91–93, patient-specific N-terminal HVR1-derived peptides ; 26–31 and S64–68, unrelated C-terminal HVR1-derived peptides. † Time-point and letter code of the variant used for designing the peptide (see Table 4, Fig. 1 and the text).
Sera. Sera were obtained from three blood donations at time-points 1–3 and at three subsequent clinic attendances (time-points 4–6 ; Table 1). All sera were stored at k40 mC. Sera from 45 HCV carriers described previously (Jackson et al., 1997) were used for comparative purposes.
Oligonucleotides and peptides. Oligonucleotides were synthesized by the Protein and Nucleic Acid Synthesis Facility, Department of Biochemistry, University of Cambridge. Peptides were synthesized by Severn Biotech (purity 85 %). Patientspecific and unrelated peptides correspond to residues 14–28 of HVR1,
except peptides 91–93, which correspond to amino acids 1–14 (Table 2). We designed five C-terminal HVR1 peptides (82, 84, 86, 88, 89) corresponding to major variants and three (83, 85, 87) corresponding to minor variants. Peptide 90 differed by just one amino acid from peptide
Table 1. Biochemical, serological and virological data RIBA 3 Timepoint TP1 TP2 TP3 TP4 TP5 TP6
Days of follow-up
ALT* (IU/l)
C100
C33c
c22
NS5
Result†
HCV RNA titre (molecules/ml)
0 173 349 610 894 1079
75 87 67 105 119 50
1j 1j 1j j\k j\k j\k
3j 2j 2j 2j 2j 2j
4j 4j 4j 4j 4j 4j
– – – – – –
C C C C C C
8n0i10& 4n4i10& 8n0i10# 2n5i10& 4n0i10' ‡
* Alanine transaminase levels (normal value, 50). † C, Confirmed positive. ‡ Below detection limit in quantitative assay (500–700 copies\ml). DBI
HCV variability and immune responses 88 and was derived from a sequence of one of a few clones amplified and sequenced from the peripheral blood mononuclear cells of the patient. Two N-terminal peptides (91, 92) were derived from major variants at time-points 2 and 3, from which C-terminal peptides 84 and 86 were derived. N-terminal peptide 93 was derived from the same clone as Nterminal peptide 90. In all cases N-terminal and C-terminal peptides (15mers) overlapped by 1 amino acid.
Table 3. Summary of nucleotide and amino acid analysis of HVR1 sequences
HVR1 amplification, cloning and sequencing. HCV RNA was specifically captured from serum using a multisample U-capture method (Petrik et al., 1997). Eluted RNA (12 µl) was used directly in a combined 20 µl cDNA PCR reaction and 1 µl aliquot reamplified in the nested PCR (Lawal et al., 1997). An aliquot of nested PCR product (571 bp) was cloned using a TA cloning kit (Invitrogen) and plasmid DNA was sequenced using standard procedures and a Sequenase II kit (USB). The HVR1 region of 9–21 clones for each time-point was sequenced in both directions using internal sense (nt 1436–1454) and antisense (nt 1706–1689) sequencing primers.
Substitutions No. of Nonper genome site nucleotide synonymous per year changes mutations ( %) (i10−2)
RNA quantification. RNA was extracted and amplified as above, although the number of cycles was reduced to 12 in the first PCR and 27 in nested PCR in order to achieve a linear relationship between the amount of RNA present and the signal obtained after gel electrophoresis. Southern transfer and hybridization to the 5h digoxigenin-labelled probe. The signal was compared to that obtained from various dilutions of control samples quantified by different methods (Petrik et al., 1997).
Peptide immunoassay. Peptides were coupled to BSA and used to coat a 96-well microtitre plate (Nunc-Immuno plate, Maxisorp ; Life Technologies) ; their reactivity was tested against IgG in patient sera by ELISA (Jackson et al., 1997). Wells were blocked with BSA and incubated for 1 h at 40 mC with diluted (1 : 100) plasma samples and subsequently for 1 h at 40 mC with goat anti-human IgG conjugated to peroxidase at 1 : 14 dilution. After each incubation the wells were washed three times with 0n5 % (v\v) Tween 20 (BDH) solution. The peroxidase reaction was visualized using o-phenylenediamine solution in substrate dilution buffer. The cut-off value for each plate was calculated as the mean plus four times the standard deviation for at least eight negative controls included on each plate. Anti-HVR1 IgA and IgM antibodies were measured in the same way except anti-human IgA–peroxidase conjugate (Sigma) or anti-human IgM–peroxidase conjugate (Abbott) were used at 1 : 1000 and 1 : 20 dilution, respectively.
Total anti-HCV IgG and IgM. EIA detection kits (Abbott) were used according to manufacturers ’ instructions.
Phylogenetic analysis. Phylogenetic trees were constructed using a 32 amino acid region at the N terminus of HCV envelope glycoprotein E2 (containing HVR1) and the computer package PAUP3 (Swofford, 1991). Trees were generated from 25 random additional replicates using an unordered matrix.
Results HVR1 amino acid sequence heterogeneity
Between 9 and 21 clones (96 in total) were sequenced from each of the six time-points spanning 3 years. Analysis of nucleotide and deduced amino acid sequences (Table 3) revealed a high mutation rate within HVR1, ranging between 0n72 and 7n2i10−# per genome site per year ; replacement mutations represented 75–95 % of the total. Table 4 lists deduced amino acid sequences together with the frequency of
All changes and mutation rates are calculated in relation to the major sequence in time-point 1 (1a in Tables 2 and 4, Figs 1 and 2, and the text)
Timepoint
No. of clones
1 2 3 4 5 6
16 21 16 16 18 9
8 70 12 19 36 56
75 87 91 88 95 81
7n2 0n80 0n72 0n83 2n06
, Not applicable.
variants (quasispecies). The phylogenetic analysis (Fig. 1) revealed two major and one minor amino acid pattern in HVR1. For convenience we refer to these as TGF (including variants 1a–c, 2a, 2c, 3a–f, 5e–f, 6b–c), AAL (variants 2h, 4a–e, 5a–d, 6d) and HGF (variants 2e–g), respectively, according to the first three amino acids (in a majority of the clones within a group) of the C-terminal half of HVR1. A possible fourth pattern (Fig. 1) is represented by a single sequence, 6a, emerging at the final time-point (Table 4). TGF variants dominated at time-points 1, 3 and 6 and were present at five time-points in total. They represented the most prevalent pattern among deduced amino acid sequences of all clones. AAL variants became dominant at time-points 4 and 5 and were present at four time-points in total. HGF variants were present only at the second time-point, when they were dominant. Only three variants were detected at several time-points. A major variant at time-point 1 (1a) was also present as a minor variant at time-point 2 (2a) and then became undetectable. The minor TGF variant at time-point 2 (2c) was a major variant at time-point 3 (3a), was undetected at time-point 4, was a minor variant at time-point 5 (5e) and became a major variant at timepoint 6 (6b). A minor AAL variant at time-point 2 (2h) was present as a minor variant at both time-points 4 (4d) and 6 (6d). HVR1 sequences of selected variants (see Table 4) were used to design peptides as shown in Table 2. HVR1 heterogeneity and HVR1 antibodies
To study the influence of HVR1 antibody responses on the emergence and survival of mutants, we investigated the timecourse of HVR1 IgG reactivity against selected C-terminal HVR1 peptides. Antibodies to nine synthetic peptides corresponding to five major and four minor variants were DBJ
A. Majid and others
Table 4. Deduced HVR1 amino acid sequences and variability of clones analysed Time-point/ variant
No. of clones
Corresponding peptide*
1a 1b 1c 2a 2b 2c 2d 2e 2f 2g 2h 3a 3b 3c 3d 3e 3f 4a 4b 4c 4d 4e 5a 5b 5c 5d 5e 5f 6a 6b 6c 6d
8 5 3 4 2 2 2 8 1 1 1 11 1 1 1 1 1 1 11 2 1 1 12 1 1 2 1 1 1 5 1 2
82 – 83 – – – – 84 (91) – – 85 86 (92) – – – – 87 88 (93) – – – – 89 – – – – – – – – –
HVR1 sequence TTYTTGAQMGRGITGFSNLFNLGSQQKV ----------K-------HN-------------------VG-LT---S---------------------------------------------A--LA-F-S-----NI ----------Q------K---M-----------------VG-LT----------G--V--GAS--TVH--TG--S--PS-TI G--V--GAS--TVH--TG--SV-RA-TI G-----GAS--TVH--TG--SV-PA-TI A--I----T---TAALASFLS--P---N ----------Q------K---M-----I---------Q------K---M-------------T-Q------K---M-------------I-Q------K---M---------------Q----------M---------------Q------KHN-M-------HI----T--TGAALANFLS--P---I --HI----T--TGAALANFLS--P---N --HI----T--TGAALANFLS------I A--I----T---TAALASFLS--P---N A--I----T---TA-LA-FLS--P---I A--I----T---TAALASFLS--P---I --------A-SSTLALAKF-S------I R-------T-N-AARLASFFS------I --------A--STWA-A-F-SP-A---I ----------Q------K---M---------------G-AM------S------E--I--GSA-YNLG-VAG--RM-----L ----------Q------K---M-----------------AG-LVS---------A--I----T---TAALASFLS--P---N
ISV† 5 6
0n165 7 8 5
6
0n634 7
8 5
6
0n395 7
8 5
6
0n272 7
8 5
6
0n325 7
8
6
5
0n902 7 8
* C-terminal (82–89) or N-terminal (91–93, in brackets) synthetic peptides designed on the basis of deduced amino acid sequences of shown variants. † Intrasample variability for each time-point calculated using the ProtDist program of the Phylip package (Felsenstein, 1989).
measured by ELISA in sera from time-points 1 to 6. The timecourse of the frequency of selected variants was plotted against the time-course of antibody responses against corresponding synthetic peptides (Fig. 2). There were three patterns of reactivity : (1) no response against minor or major variants (Fig. 2 a) ; (2) antibodies present prior to detection of the corresponding variant (Fig. 2 b) ; (3) antibody detected after the appearance of the corresponding HVR1 variant (Fig. 2 c). Two variants were present at three and four time-points, respectively. Peptide 86-corresponding variant was dominant at time-point 3 with an IgG response delayed by 1 year until time-point 5, at which point IgG levels decreased and the variant re-emerged as a major variant at time-point 6. IgG DCA
reacting against peptide 85 was present throughout, although it decreased over the last three time-points. Despite this, the corresponding variant was present as a minor variant at timepoints 2, 4 and 6. Peptides within the TGF variants (82, 86, 87) and AAL variants (85, 88, 89, 90) differ by only one\few amino acids and the contribution of particular amino acids to antigenicity or reactivity could be investigated readily. One of four related AAL variant peptides (peptide 90) showed no significant reactivity (Table 5). When aligning their sequences only the replacement of phenylalanine with serine at position 6 could explain the absence of reactivity. In contrast, the overall reactivity of TGF variant peptides was low, and for peptides 82
HCV variability and immune responses 1a 1
2
1 2 1 4 3
2
5e 6b
1b
2a 5f 2b
2
2
3b
2
2
(a)
2c 3a 1 3e 1 3c 1 1 3d 2 3f
(b)
6c
1 1c 2d 2h
2
1 4a 1
3 1 4d 1
3 5b 1
3 4
4
6 2
4 2
4b
4c 6d
4e 5c
5a (c)
5d 2e 2
11 7
1
2f
2g 6a
Fig. 1. Maximum parsimony tree of the 32 amino acid long region of the HCV E2 protein containing HVR1 (sequences under EMBL accession nos AJ228353–AJ228446). Seven equally parsimoneous trees were identified, of which one is shown above. Branch lengths are proportional to the degree of variation between the sequences.
and 87 the absorbance at all time-points was below the cut-off value. When the sequence of peptide 86 was aligned with 82 and 87 no single amino acid change could explain the reactivity of peptide 86 at later stages (Fig. 2). For comparison, we measured serum reactivity of three Nterminal peptides (91, 92 and 93) by ELISA, but despite the identical method of coupling to BSA, identical ELISA protocol, concentrations and peptide length, reactivity was negligible (not shown). There was no or negligible reactivity for either IgA or IgM with HVR1, although the experimental method was similar to that for IgG against peptides 82–90 (data not shown). Cross-reactivity of patient-specific HVR1 peptides with unrelated sera
Patient-specific C-terminal (82–90) and N-terminal (91–93) HVR1 peptides were tested against a panel of sera from 45 (43 for peptides 91–93) chronic HCV carriers. C-terminal peptide reactivity ranged between zero and nine positive sera per peptide (Fig. 3 b). The mean number of reactors per peptide, however, was lower for patient-specific peptides (3n89p3n41) than for other peptides used against the panel of 45 sera in a
Fig. 2. Serum ELISA reactivity (left axis, solid line, filled symbols) of C-terminal HVR1 peptides (82–90 ; Table 2) is shown in context of the frequency of occurrence (right axis, dotted line, open symbols) of the corresponding variants (Tables 2 and 4). Absorbance/cut-off 1 is considered negative. HVR1 IgG were either unreactive (a), present prior to (b) or following (c) the occurrence of the corresponding variant.
previous study (Jackson et al., 1997) (5n50p4n08 ; P, not significant). N-terminal peptides showed negligible reactivity with homologous sera, but peptide 91 reacted with 11\43 (25n6 %) of heterologous sera tested. AAL peptides with good reactivity against homologous sera (85, 88, 89) reacted weakly with a panel of 45 heterologous sera (three, one and one positive sera, respectively) ; in contrast, TGF peptides that reacted weakly with homologous sera (82, 86) reacted with a higher number of heterologous sera (six and nine, respectively). Cross reactivity of sera obtained at time-points 1–6 with unrelated HVR1 peptides
To evaluate the extent of potential cross-reactivity of patient antibodies, 11 synthetic peptides derived from published or database-derived sequences (and thus not related DCB
A. Majid and others
Table 5. Decrease in the ELISA anti-HVR1 IgG reactivity due to a single amino acid substitution Figures represent a mean of absorbance\cut-off values from four replicates.
Peptide
Sequence (C terminus)*
TP1
TP2
TP3
TP4
TP5
TP6
85 88 89 90
AALASFLSLGPQQKN AALANFLSLGPQQKI AALASFLSLGPQQKI AALANSLSLGPQQKI
2n28 4n13 3n20 0n43
1n85 2n75 1n56 0n92
2n15 3n80 2n35 0n96
1n71 3n68 2n43 0n66
1n45 2n45 1n48 0n91
1n16 1n38 0n90 0n58
* Variable amino acids are shown in bold. A single amino acid difference between peptide 90 and the other three peptides is underlined.
(a)
biochemical and virological parameters. Cryoglobulins became undetectable and the serum ALT normalized (less than 50 IU\l), together with a decrease of viral RNA titre below the level detectable in our quantitative assay (Table 1). However, quasispecies heterogeneity increased. Relapse on completion of IFN-α therapy was immediate (not shown).
Discussion
(b)
Fig. 3. (a) Cross-reactivity of patienths sera was measured by ELISA against 11 unrelated HVR1-derived peptides as described in Methods. Absorbance to cut-off ratios are shown ; a positive result is defined as 1. (b) Cross-reactivity of patient-specific C-terminal (82–90) or N-terminal (91–93) peptides was measured against 45 unrelated sera from chronic HCV carriers. The number of sera reactivity with each peptide is shown ( %). For the sequences of peptides used in (a) and (b) see Table 2.
closely to patient-derived peptides) were tested against sera obtained at time-points 1–6. A large proportion of these peptides (seven of 11 peptides ; 63n6 %) reacted with patientspecific sera at all time-points (Fig. 3 a). Effect of IFN-α
There was considerable clinical improvement during IFN-α therapy, consistent with objective changes in immunological, DCC
It has been postulated that continuous production and selection of HCV mutants, which cannot be contained by antibodies, might explain some aspects of the chronic character of HCV infection (Taniguchi et al., 1993 ; Shimizu et al., 1994 ; Weiner et al., 1992, 1995), although the fine details of this process are poorly defined. There are conflicting results on the degree of HVR1 antibody specificity. Both isolate-specific (Kato et al., 1993 ; Shimizu et al., 1994 ; Allander et al., 1997 ; Zibert et al., 1997) and cross-reacting (isolate-independent) (Scarcelli et al., 1995 ; Jackson et al., 1997 ; Yoshioka et al., 1997 ; Zibert et al., 1997) antibodies have been described. The situation is further complicated by an apparent existence of other neutralizing epitope(s) within a more conserved region of E2, as indicated by chimpanzee vaccination experiments (Rosa et al., 1996). In this study we observed both patterns ; the first corresponding to isolate-specific antibodies when serum reactivity against a particular synthetic peptide followed the emergence of the corresponding variant ; the second, which suggests isolate-independent antibody cross-reactivity, present prior to detection of the corresponding variant. In the latter case it is also possible that a particular variant may have appeared previously during the prolonged infection or that a variant with a closely related sequence had induced crossreactive antibody previously. Zibert et al. (1997) came to a similar conclusion when following a group of individuals infected with an identical HCV isolate at a defined time. Crossreactivity was unexpectedly high even when patient-specific peptides were reacted with unrelated sera and vice-versa, extending our previous observations (Jackson et al., 1997).
HCV variability and immune responses
We observed that a single amino acid change (phenylalanine to serine, position 401) in one of four closely related AAL peptides studied led to a complete loss of reactivity with our patient sera, although it reacted with some of the unrelated sera. Amino acid 401 is part of two overlapping B-cell epitopes described by Kato et al. (1994). The importance of this region for IgG reactivity was noted in our previous study (Jackson et al., 1997), where the reactivity of two peptides differing only in position 403 differed by more than 50 %. This confirms a similar observation involving an identical position in one of the patients described by Yoshioka et al. (1997). Clearly, for some positions there are more stringent restrictions for replacement mutations, perhaps due to the secondary structure of HVR1. This could explain the strict isolate-specific character of HVR1 antibodies described in some studies (Kato et al., 1993 ; Shimizu et al., 1994 ; Allander et al., 1997 ; Zibert et al., 1997). The V3 loop of human immunodeficiency virus type 1 gp120 may be similar to HCV HVR1 in hypervariability and in containing a major neutralizing epitope. A single amino acid change was shown to result in a conformational alteration with a loss of the neutralizing V3 epitope (Di Marzo Veronese et al., 1993). In this work even the continuous presence of ELISAreactive IgG (whether isolate-specific or cross-reactive) against particular HVR1 peptides did not prevent re-appearance of some of variants at two, three or four time-points, with one of these (no. 86) becoming dominant for the second time, suggesting the emergence of escape mutant(s). There are limitations of HVR1 sequence analysis which need to be considered. It is possible that some minor variants not detected at a particular time-point would be detected if a larger number of clones was analysed (Farci et al., 1996), although it is unlikely to be true for major variants. Length and localization of HVR1-derived peptides may also be important as HVR1 antibodies directed against N-terminal rather than Cterminal HVR1 sequences may have played a role in selflimited infection (Zibert et al., 1997). Several attempts have been made to correlate quasispecies heterogeneity to biochemical and virological markers such as ALT, HCV RNA levels or histological markers of disease progression. Some authors observed increased HVR1 diversity with progressive liver disease (Honda et al., 1994 ; Enomoto et al., 1994 ; Yuki et al., 1997), yet sequential changes in HVR1 were also observed in an asymptomatic patient (Nakazawa et al., 1994) and quasispecies complexity was found to be independent of the stage of chronic HCV infection (Naito et al., 1995). In a similar fashion, attempts to correlate heterogeneity and the outcome of IFN-α treatment have been inconclusive. Okada et al. (1992) reported that patients with more heterogeneous virus populations were less responsive to IFN-α treatment, but other studies reported no difference in HVR1 heterogeneity between responders and non-responders to IFN-α (Nakazawa et al., 1994) or a weak correlation at low level
replication (Shindo et al., 1996). In our study, a clinical response was associated paradoxically with increased heterogeneity. This supports a recent quantitative study showing that IFN-α increased the rate of HVR1 mutations (Polyak et al., 1998). Differential sensitivity of various quasispecies to IFN-α may explain this finding, perhaps mediated by changes in the interferon sensitivity-determining region located within NS5a (Enomoto et al., 1994, 1995), which represses IFN-α-induced protein kinase by direct physical binding (Gale et al., 1997). Generally, there are only limited data on HCV type 2infected patients. In the paper describing type 2b variability, Sakamoto et al. (1994) used direct PCR product sequencing, which is difficult to compare with our study. However, the average heterogeneity of variants from a type 2a-infected patient was higher than in two type 1b-infected patients (Manzin et al., 1998). This agrees with our observation on HVR1 heterogeneity in this patient compared to earlier characterized type 1-infected patients (Lawal et al., 1997). One possible reason may be a higher incidence and activity of HVR1 antibody for genotype 2, as shown for patients with genotype 2a compared to patients with genotype 1b (Yoshioka et al., 1997). The potential for vaccine development using HVR1 peptides to induce neutralizing antibodies has been discussed before (Taniguchi et al., 1993 ; Shimizu et al., 1994 ; Jackson et al., 1997). Cross-reactivity between unrelated HVR1 peptides suggests that a limited set of peptides could induce a wide variety of neutralizing antibodies. In some HVR1 positions the changes observed even in a large set of sequences are very limited (Nakamoto et al., 1996) and the dramatic change in peptide reactivity attributed to a single amino acid change suggests stringent restrictions at particular positions, perhaps due to structural restraints. In order to identify the rules governing HVR1 diversity and its correlation with serological and virological markers, more detailed studies in well-defined but diverse patient groups will be necessary. The authors thank Dr M. Tristem for help with phylogenetic analysis and I. Reeves for titrating anti-HCV IgG.
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