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Hepatitis C virus-specific CTL responses in PBMC from chimpanzees with chronic hepatitis C: determination of CTL and CTL precursor frequencies using a recombinant canarypox virus (ALVAC)
Journal of Immunological Methods 214 Ž1998. 121–129
Hepatitis C virus-specific CTL responses in PBMC from chimpanzees with chronic hepatitis C: determination of CTL and CTL precursor frequencies using a recombinant canarypox virus ž ALVAC/ Marielle Cohard a,1, Qingyan Liu a , Marion Perkus b, Ethel Gordon a , Betsy Brotman a , Alfred M. Prince a,) a
Lindsley F. Kimball Research Institute, The New York Blood Center, 310 East, 67th Street, New York, NY 10021, USA b Virogenetics, 465 Jordan Road, Rensselaer Technology Park, Troy, NY 12180, USA Received 29 October 1997; revised 26 February 1998; accepted 27 February 1998
Abstract The aim of this study was to evaluate HCV-cytotoxic T lymphocyte response from PBMC in bulk CTL assays and in CTL precursor analyses using in vitro stimulation with canarypox virus ŽALVAC. expressing HCVcapsidrE1rE2rNS2rNS3 antigens. Canarypox virus is naturally host-range restricted and does not replicate or cause cytopathology on mammalian cells. PBMC were obtained from four chimpanzees with chronic hepatitis C infection and one uninfected chimpanzee. CTL from bulk culture of PBMC and CTL precursor frequencies were found in three of the four chronically infected chimpanzees using ALVAC in vitro stimulation. No CTL response was detected in PBMC from the uninfected chimpanzee. The precursor frequencies of CTL specific for capsid, NS2 and NS3 proteins ranged between 1r2663 and 1r27202. No correlation was observed between percent cytolysis in bulk culture and CTL precursor frequencies. This method may prove useful in assessing the correlation between HCV–CTL response and virological or histological status. q 1998 Elsevier Science B.V. All rights reserved. Keywords: HCV–CTL response; CTL precursor frequency; Canarypox virus
1. Introduction Hepatitis C virus ŽHCV. is the major cause of non-A, non-B viral hepatitis. Infection with HCV
Abbreviations: CTL, Cytotoxic T lymphocyte; HCV, Hepatitis C virus; PBMC, Peripheral blood mononuclear cells ) Corresponding author. Tel.: q1-212-570-3279; fax: q1-212570-3180; e-mail: [email protected]. 1 Current address: Schering-Plough Research Institute, 2000 Galloping Hill Road, Kenilworth, NJ 07033.
leads to chronic hepatitis in more than 50% of infected individuals. Ten to 50% of these individuals eventually develop cirrhosis or liver cancer ŽCuthbert, 1994.. Persistent viremia and liver damage have been identified in chronically infected individuals despite the presence of both humoral ŽAlter et al., 1992; Shimizu et al., 1994. and cellular immune response ŽMinutello et al., 1993; Botarelli et al., 1993; Koziel et al., 1993; Koziel et al., 1992.. The mechanism of chronic infection despite vigorous immune responses is unknown. CTL-mediated lysis of
0022-1759r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 8 . 0 0 0 5 4 - 4
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virus infected cells is thought to lead to clearance of the virus or, if incomplete, to viral persistence and chronic tissue injury ŽMondelli et al., 1988.. Class I HLA-restricted CD8 q CTL that recognize viral proteins have been described in the liver of patients with chronic hepatitis ŽKoziel et al., 1992. and in the liver of chimpanzees with acute and chronic hepatitis C ŽErickson et al., 1993.. The role of peripheral blood CTL in HCV infection is unclear. Expansion of virus-specific CTL from PBMC has been accomplished by culturing the cells with epitopic HCV peptides ŽKoziel et al., 1992, 1993; Kita et al., 1993; Shirai et al., 1992, 1994; Cerny et al., 1995; Battegay et al., 1996.. However, the detection of cytolytic activity requires specific experimental conditions with multiple in vitro stimulations with class I HLA restricted peptides. Therefore, it has been suggested that CTL are present at a low frequency in the peripheral blood of some chronically infected subjects. HCV-specific CTL also can be expanded from peripheral blood of individuals with no apparent exposure to the virus ŽCerny et al., 1995.. The purpose of this study was to determine whether canarypox virus vector ŽALVAC. engineered to express CaprE1rE2rNS2r NS3 antigens could be used for specific in vitro stimulation, instead of using synthetic peptides which are potentially responsible for in vitro priming.
2. Materials and methods 2.1. Animals Five chimpanzees Ž Pan troglodytes.: 4 chimpanzees with chronic hepatitis C and one healthy non-infected chimpanzee were studied. They were maintained at Vilab II, New York Blood Center’s primate laboratory in Liberia ŽBrotman et al., 1985.. Chimpanzees 157, 208 and 328 were infected intravenously with 1 ml inoculum containing 5 = 10 4 CID50 of the HCV-H virus stock ŽPrince et al., 1985.. Chimpanzee 159 was infected with a commercial unsterilized factor VIII preparation. The diagnosis of HCV infection was based on standard serological assays using successively first and second-generation Ortho HCV ELISA test system ŽOr-
tho Diagnostic Systems, Raritan, NJ.. PCR was performed as previously described ŽLai et al., 1994. and the results after inoculation were positive for chimpanzees 157, 159, 208 and 328. All four of these animals remain PCR positive to date; they are thus chronically infected. In chimpanzees 157, 159, 208 and 328, liver biopsies revealed ultrastructural features characteristic of HCV infection in chimpanzees up to 10 years after inoculation. One chimpanzee ŽCh. 214. was not inoculated and was used as a negative control in this study. Table 1 summarizes animal characteristics and history of HCV challenge. 2.2. Generation of poxÕirus recombinants expressing HCV proteins Hepatitis C genes from the genotype 1a ŽHutchinson strain. were inserted into two poxvirus: ALVAC, a strain of canarypox ŽTartaglia et al., 1992. and the L variant of WR, a laboratory strain of vaccinia virus ŽPanicali et al., 1981.. Poxvirus recombinants were generated as described previously ŽPerkus et al., 1993.. ALVAC-based recombinant: HCV sequences encompassing capsid through NS3 ŽcapsidrE1rE2r NS2rNS3. were placed under the control of the synthetic vaccinia earlyrlate H6 promoter ŽPerkus et al., 1989.. This expression cassette was inserted into a canarypox donor plasmid flanked by genomic sequences from which a nonessential gene was specifically deleted. Recombination between the donor plasmid and Alvac virus resulted in the recombinant ALVAC-caprE1rE2rNS2rNS3. Three WR vaccinia-based recombinants, cap-vaccinia, NS2-vaccinia and NS3-vaccinia, were generated. The capsid gene was expressed under the control of the vaccinia H6 promoter. NS2 and NS3 genes were expressed under the control of an early promotor. Expression cassettes were inserted immediately downstream from the K1L host range gene ŽGillard et al., 1986. in the WR strain of vaccinia virus. 2.3. Anti-HCV antibodies Monoclonal anti-HCV capsid antibody C7-50 Žkindly provided by Dr. Jack R. Wands. and rabbit anti-HCV NS3 Žkindly provided by Dr. Ramesh A.
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Table 1 Characteristics of chimpanzees Chimpanzee No.
Birthdate
Sex
Inoculation date
Inoculuma
Re-challengeb date
157 159 208 328 214
1977 1975 1978 1982 1979
M M M M M
1982 1981 1985 1988 y
Hutch Ž1a. Factor VIII c Hutch Ž1a. Hutch Ž1a. y
y 1994 y 1994 y
a
Chimpanzees 157, 208 and 328 were infected intravenously with 1 ml inoculum containing 5 = 10 E4 CID50 of the HCV-Hrvirus stock ŽPrince et al., 1985.. Chimpanzee 214 was not inoculated with HCV and was considered as negative control. b Animals were re-challenged with the same inoculum. c The inoculum was 10 ml of a commercial unsterilized factor VIII preparation.
Bhat. were used to identify the expression of HCV protein. 2.4. Detection of HCV protein expression by the recombinant Õiruses COS-1 cells were grown in Dulbecco’s modified Eagle’s medium ŽDMEM. containing 4.5 mgrml glucose and 10% fetal bovine serum ŽFBS. at 378C in 5% CO 2 rair. The cells were seeded into 35 mm petri dishes and cultured overnight or until the cells became monolayers. The following day, the cells were infected by recombinant pox viruses or their parents with a MOI of 10 pfurcell in DMEM containing 2% FBS. After 1 h incubation, the virus containing supernatant was removed and replaced with 2 ml DMEM and 2% FBS, culture was continued for 24 h. The cells were harvested by adding 1 = SDS loading buffer containing 50 mM Tris hydrochloride, pH 6.8, 100 mM dithiothreitol, 2% Žwrv. sodium dodecyl sulfate, 10% glycerol and 0.2% bromphenol blue. The mixture was then heated at 1008C for 5 min, sheared by sonication and centrifuged for 5 min at room temperature at 14,000 rpm in a microcentrifuge. The samples were separated by 12% SDS-PAGE and electrotransferred to nitrocellulose membrane with a Bio-Rad blotting apparatus ŽBio-Rad Laboratories.. The membranes were blocked in 5% non-fat dry milk in PBS at room temperature for 1 h. Monoclonal anti-HCV capsid Ž1:1000 dilution. and rabbit anti-HCV NS3 Ž1:1000 dilution. antibodies were used as primary antibodies, and HRP-labeled goat anti-mouse or goat anti-rabbit IgG ŽH q L. Ž1:5000 dilution, KPL. were applied as
secondary antibodies to detect the expression of HCV capsid and NS3 proteins. Signals were developed by enhanced chemiluminescence ŽECL; Amersham.. 2.5. Target cells Autologous B-lymphoblastoid cell lines ŽB-LCL. were generated from each animal. Briefly, Ficoll Hypaque-separated PBL were cultured in supernatant from the EBV-producing marmoset cell line B95-8 Žkindly provided by Dr. Maria Proytcheva at the New York Blood Center., in RPMI 1640 containing 20% fetal bovine serum ŽFBS. and 2 m grml cyclosporine A ŽNeitzel, 1986.. Transformed cells were maintained in RPMI 1640 supplemented with Lglutamine Ž2 mM., penicillin Ž100 Urml., streptomycin Ž100 m grml., Hepes Ž20 mM. and 20% heat-inactivated FBS. 2.6. Effector cells Peripheral blood mononuclear cells ŽPBMC. drawn between 1994 and 1996 were purified by Ficoll-Hypaque and then cryopreserved at 18Crmin in the presence of 10% DMSO and 20% FBS. PBMC were thawed and plated in 24-well plates at 6 to 10 = 10 6rwell in 2 to 4 ml of culture medium consisting of RPMI 1640, 10% heat-inactivated FBS, antibiotics and 2 mM L-glutamine. Cultures included stimulator cells which were prepared by infecting autologous PBMC or EBV-transformed B lymphoblastoid cell lines ŽB-LCL. at a MOI of 10 pfurcell with a canarypox virus ŽALVAC. vector expressing capsid, E1 E2, NS2, NS3 proteins or a
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parental-Alvac vector. PBMC-to-stimulator cells ratio of 5:1 and 10:1 were studied. When B-LCL were used as stimulator cells, 1 = 10 6 rml irradiated human PBL Ž3000 Rad. were added to cultures as feeder cells. Bulk CTL assays were performed after 7 days of culture at 378C Ž5% CO 2 . without IL-2.
2.7. Bulk cytotoxic T lymphocyte assays CTL assays using autologous B-LCL infected with recombinant vaccinia virus ŽVV. were performed. All B-LCL were infected with recombinant vaccinia HCV vectors containing the genes of capsid, NS2 or NS3, or control vaccinia virus at a multiplicity of infection ŽMOI. of 2 pfurcell for 1 h at 378C then washed and cultured 16 h prior to the assay. VV-infected autologous B-LCL were labelled with 100 m Ci of 51 Cr for 1 h and washed three times in cold assay medium ŽRPMI1640, 10% FBS, 2 mM Lglutamine, Antibiotics, 25 mM Hepes, 5 = 10y5 MB-mercaptoethanol Ž2 BME.. and used as targets in a standard 51 Cr-release assay in which the effector cells were PBMC. Effector-to-target ŽErT. ratios between 50:1 and 10:1 were assessed. Cytolytic activity was determined in a standard 4–6 h 51 Cr-release assay using U-bottom 96-well plates containing 5000 targets per well. The background lysis by vaccinia and EBV-specific CTL was reduced in most assays by the use of cold target inhibition as previously described ŽLubaki et al., 1994.. Briefly excess non-chromium-labeled control vaccinia-infected autologous B-LCL Žcold targets. were added to prior to the initiation of the CTL assay. All assays were performed in duplicate. Percent cytotoxicity was calculated using the formula: 100 = wŽexperimental release in cpm y spontaneous release in cpm.rŽmaximum release in cpm y spontaneous release in cpm. x. Maximum release was determined by lysis of targets with detergent Ž1% Triton X-100; Sigma Chemical.. Spontaneous release was determined from targets in medium alone. Assays were excluded from analysis if the spontaneous release value was ) 30% of maximum release. Results were reported as the mean of duplicate values. A difference in the specific lysis of recombinant-VV-infected targets and control-VV-infected target cells of
at least 10% seen in at least 2 effectorrtarget ratio was considered to represent a positive CTL response. 2.8. Limiting dilution assays of memory cytotoxic T lymphocytes Precursor frequencies of HCV- capsid, HCV-NS2 and HCV-NS3-specific CTL were estimated by performing limiting dilutions on isolated PBMC followed by in vitro stimulation to allow expansion of CTL. PBMC were diluted to give 315 to 20,000 lymphocytes per well in 24 replicates in 96-U bottomed microtiter plates. Stimulator cells Žautologous PBMC. were infected with recombinant-Alvac expressing CaprE1rE2rNS2rNS3 proteins, then gamma-irradiated Ž3000 rad. and added at 25,000 cells per well in complete medium to the responder cells. Effector and stimulator cells were cultured at 378C, 5% CO 2 in culture medium containing RPMI1640, 10% FBS, Antibiotics, 2 BME and Hepes. On days 3 and 10, half of the culture medium was changed for complete medium containing IL-2 at 50 Urml ŽR & D.. A second round of specific stimulation with the recombinant Alvac vector was performed as above on day 7. At day 14, wells were split and assayed for cytotoxicity on 51 Cr-labeled autologous B-LCL infected with vaccinia-capsid expression vector or vaccinia-NS2 or NS3 vector and control–vaccinia virus in parallel, in a 6 hour 51 Crrelease assay. The fraction of nonresponding wells was taken as the number of wells in which 51 Cr-release did not exceed the mean plus three standard deviations of the spontaneous release of the 24 control wells. 2.9. Statistical analysis The CTL precursor frequencies ŽCTLpf. estimates and confidence intervals were calculated using Miller’s software as described by Carl Taswell ŽTaswell, 1984.. Minimal estimates of CTL frequency were obtained according to the Poisson distribution equation as the slope of a line relating the number of responder cells per microwell Žplotted on a linear axis. and the percentage of microwells that failed to develop cytolytic activity Žplotted on a
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3. Results 3.1. HCV protein expression by the recombinant Õiruses
Fig. 1. COS-1 cells were infected by recombinant pox viruses and their parents Žlane 1 and 5: WR parents, lane 2: WR-HCV capsid, lane 6: WR-HCV NS3, lane 3 and 7: ALVAC parents, lane 4 and 8: ALVAC-HCV capsidrE1rE2rNS2rNS3.. Twenty-four hours later, the infected cells were harvested using 1= SDS loading buffer, boiled, sheared by sonication and centrifuged. The samples were separated by 12% SDS-PAGE and analyzed with immunoblotting. The positions of HCV capsid and NS3 proteins Žarrows., and of the molecular mass standards Žin kilodaltons. are indicated at the sides of the gel.
logarithmic y-axis.. This analysis yields minimal frequency estimate Žrange., and a x 2 estimate of probability Ž p .. In this system, significance is indicated by p greater or equal to 0.05. P values for the differences between the CTLpf were determined by the Mann–Whitney U-test.
As shown in Fig. 1, both 21 kDa and 19 kDa HCV capsid proteins ŽC191 and C174. were detected in the WR-HCV capsid and ALVAC-HCV capsidrE1rE2rNS2rNS3 infected COS-1 cells Žlane 2, 4., not in the parental infected cell lysates Žlane 1, 3.. The expression of 70 kDa HCV-NS3 protein was identified in the WR-HCV NS3 and ALVAC-HCV capsidrE1rE2rNS2rNS3 infected COS-1 cells Žlane 6, 8.. The expression of NS2 in ALVAC-HCV capsidrE1rE2rNS2rNS3 and WRHCV NS2 was not detected, due to lack of a good anti-HCV NS2 antibody. 3.2. Bulk assays: HCV-specific CTL actiÕity Assays were considered positive for CTL if the lysis of targets was greater or equal to 10% higher than the lysis of control vaccinia-infected targets at two different E:T ratios. One of 4 chronically infected chimpanzees had detectable capsid-specific CTL activity. Three of 4 animals had detectable
Fig. 2. HCV-specific CTL response observed from bulk culture after 7 days of in vitro stimulation with recombinant canarypox virus ŽALVAC. expressing CaprE1rE2rNS2rNS3 proteins. Autologous B-LCL were infected overnight with L-Var Žwild type Vaccinia Virus. or with VV-encoding capsid or NS2-vaccinia or NS3-vaccinia, then labeled with 51 Cr and were incubated with effector cells Ž4 to 6 h 51 Cr release assay.. Results with an E:T ratio of 25:1 are shown. Chimpanzee 214 was not inoculated with HCV and was used as a negative control. A difference in the specific lysis of recombinant VV-infected target versus control-VV target cells ŽL-Var. of at least 10% was considered to represent a positive CTL response Žshown as an asterisk..
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Table 2 Frequencies of HCV-specific CTL precursors in chimpanzees with chronic hepatitis C Chimpanzee No.
Targetsa
Frequency Žper thousand cells. 95% CI b
Reciprocal frequency
p value c
157
Cap NS3 Cap NS3 NS2 NS3
0.043 Ž0.012–0.074. 0.250 Ž0.076–0.424. 0.037 Ž0.020–0.054. 0.074 Ž0.042–0.106. 0.375 Ž0.179–0.572. 0.113 Ž0.03–0.224.
23 020 3997 27 202 13 490 2663 8811
0.150 0.501 0.193 0.460 0.191 0.833
208 328 a
CTLpf were determined by adding varying numbers of T cells to 24-replicate wells containing 5000 51 Cr-labeled autologous targets ŽB-LCL. infected with Cap-vaccinia or NS2-vaccinia or NS3-vaccinia or L-Var Žwild type vaccinia.. Positive cultures were defined as those in which 51 Cr release exceeded the mean value in parallel control microculture by at least 3 standard deviations. From wells containing targets infected with L-Var, no precursor frequency was listed because this frequency was not calculated as all wells failed to exhibit significant cytolysis even at the highest effector cell concentration. b Precursor frequencies were determined as described in Section 2. Values in parentheses represent 95% confidence intervals ŽCI. of the calculated frequencies. c In this system, significance is indicated by a p greater or equal to 0.05.
NS3-specific CTL activity and one of 4 had detectable NS2-specific CTL activity ŽFig. 2.. Two chimpanzees reacted to 2 of the targets ŽChimpanzees 157 and 328. and one chimpanzee Ž208. reacted with only one target. There was no detectable HCVspecific CTL activity following stimulation of PBMC with autologous PBMC infected with a canarypox
construct expressing CaprE1rE2rNS2rNS3 genes in a healthy, uninfected chimpanzee Žchimpanzee 214.. The activity in ex vivo expanded CTL was detectable at an E:T ratio of at least 20:1. Attempts to detect HCV-CTL activity from the same animals with chronic hepatitis by non-specific in vitro stimulation of PBMC using anti-CD3 anti-
Fig. 3. Quantification of memory NS3-specific CTL. Limiting dilution was performed on PBMC from HCV-infected chimpanzees followed by in vitro stimulation with ALVAC vectors as described in Section 2. Targets were autologous B-LCL infected with vaccinia virus: L-Var ŽL-Variant WR was the wild type vaccinia., vaccinia-based recombinant cap-vaccinia, NS2-vaccinia and NS3-vaccinia. Results represent precursor frequencies of NS3-specific CTL in three chimpanzees: Chimp 157 Ž1r3997., Chimp 208 Ž1r13490., chimp 328 Ž1r8811.. No precursor frequency is listed for L-Var, because that frequency was not calculable as all wells failed to exhibit significant cytolysis at any effector cell concentration.
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bodies were not successful Ždata not shown.. Additional experiments used stimulator cells prepared by infecting autologous EBV-transformed B lymphoblastoid cells lines ŽB-LCL. with ALVAC expressing CaprE1rE2rNS2rNS3 proteins and then gamma-irradiated. Background lysis of the EBVtransformed B-LCL was enhanced in 50% of the experiments when B-LCLs were used as stimulator cells Ždata not shown.. Excess unlabeled, vacciniainfected, autologous targets included in those assays did not prevent the high background lysis. 3.3. Frequencies of HCV-specific CTL precursors in PBL To determine the frequency of memory HCVspecific CTL precursors in the PBMC of chronically infected chimpanzees, limiting dilution analysis following in vitro stimulation with recombinant Alvac vector expressing CaprE1rE2rNS2rNS3 was performed. The results of precursor frequency determinations on 3 animals are summarized in Table 2. Results representing NS3-specific CTL precursor frequencies are shown in Fig. 3. Using B-LCL infected with vaccinia recombinants, chimpanzees with chronic hepatitis C were shown to have CTL precursors that recognized capsid, NS2 and NS3 proteins. The precursor frequencies of CTL specific for Cap, NS2 and NS3 proteins in general ranged between 1r2663 and 1r27 202. The mean precursor frequency of CTL specific for Capsid protein and for NS3 protein were, respectively, 1r25 111 Žrange 1r23 020–1r27 202. and 1r8766 Žrange 1r3997– 1r13 490.. The difference was not statistically significant Ž p s 0.08.. CTL precursors were detected against capsid in chimpanzee 208, whereas there was no capsidspecific CTL activity detectable in bulk culture. No CTL precursors were detected in one out of four chimpanzees with chronic hepatitis C ŽChimpanzee 159. and in the healthy uninfected control chimpanzee Žchimpanzee 214..
4. Discussion It has been reported that HCV-specific CTLs are detectable from the liver of patients and chimpanzees
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with chronic hepatitis C. The intrahepatic CTL response in humans and chimpanzees is similar in its broad specificity for multiple HCV epitopes Žboth in structural and nonstructural regions. that are presented primarily by MHC class I A or B alleles. The long-lived and multi-specific nature of the CTL response has been demonstrated in some chimpanzees ŽWalker, 1996.. It has been suggested that HCVspecific CTL might be sequestered at the site of infection. A direct comparison of cytolytic activity in the peripheral blood and liver could determine whether HCV-specific T cells are sequestered at the site of infection ŽWalker, 1996.. Multiple stimulations with the HLA-A2.1 restricted peptides were usually required for detection of cytolytic activity. Furthermore, it has been reported that HCV-specific CTL could also be detected from peripheral blood of individuals with no exposure to the virus ŽCerny et al., 1995. raising the possibility that the response might reflect in vitro priming by the peptides rather than in vivo priming by exposure to virus. In order to better understand the antiviral immune response by CD8 q CTL and to explain why some individuals develop self-limited infections rather than chronic infection, further studies are needed to investigate HCV-specific T lymphocytes in the peripheral blood of acutely and chronically infected chimpanzees and humans. Access to peripheral blood is easy whereas liver tissue biopsy is considered as an invasive technique and is not routinely performed in acute HCV infection in human. It is difficult to isolate and characterize CTLs from peripheral blood following non-specific expansion using CD3 antibodies ŽCerny and Chisari, 1994.. Therefore, there is a need for standardizing and developing new strategies to increase the sensitivity of detection of CTL from peripheral blood without the risk of in vitro priming. Our attention has focused on antigen-specific stimulation using a nonreplicating canarypox virus vector ŽCox et al., 1993.. The canarypox virus is naturally host-range restricted and does not replicate or cause cytopathology, on mammalian cells. The advantage of using canarypox virus ŽALVAC. as a vector infecting cells used as stimulator cells for expansion of HCV-specific CTL is the absence of a need to inactivate Alvac before culturing. Other strategies using recombinant vaccinia virus as a vec-
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tor for infecting B-LCL need to treat the stimulator cells with psoralen and UV light irradiation ŽLubaki et al., 1994; Shankar et al., 1995., prior to their use for in vitro stimulation, to inactivate vaccinia virus and thereby prevent vaccinia infection of the effector T cells. We have been successful in demonstrating CTL response from PBMC in bulk culture assays and in CTL precursor analyses using Alvac stimulation. Our results show that CTL precursors are present in the circulation when CTL are detectable in bulk culture. We did not observe a correlation between percent cytolysis in bulk culture and CTL precursor frequencies. This may reflect differences in the degree of stimulation in the two assays used. The in vitro ALVAC stimulation may have increased the CTL precursor frequency to levels above those seen in unstimulated peripheral blood. Previous authors ŽCerny et al., 1995. observed much lower HCVspecific CTL precursor frequencies in peripheral blood probably because their experimental systems focused on single epitopes. Due to the small number of animals studied, it is not possible to correlate the CTL response to the clinical or virological status of the chimpanzees. However, it is interesting to observe that we could not detect HCV-specific CTL responses in one chimpanzee with chronic hepatitis C and a high viremia Ž15 to 20 times higher than the other animals studied. Ždata not shown.. Further studies are necessary to confirm whether there is a correlation between HCV-specific CTL response and viral load. The methods described in this report are simple and require less effector cells than needed with peptides in vitro stimulation. Therefore, this method could be used to evaluate CTL response from bulk culture and CTL precursor frequencies in blood of patients with acute or chronic hepatitis C in studies aimed to assess correlation between CTL response and virological or histological status. This method could also be valuable in developing vaccine strategies to assess CTL response from peripheral blood after DNA based immunization in animals. Acknowledgements M. Cohard was supported by Grants of Research in Gastroenterology and Hepatology, from Beaufour
Laboratories ŽFrance. and Glaxo-Welcome Laboratories ŽFrance. and by a fellowship of the Institut Electricite´ Sante´ ŽFrance.. Animals were held in pairs or larger groups in cages that exceeded minimal specifications of requirements of the National of Health Guide for Care of Laboratory Animals. Financial support: Pasteur Merieux Serum and Vaccins ŽFrance..
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M. Cohard et al.r Journal of Immunological Methods 214 (1998) 121–129 epitope on hepatitis C virus nucleocapsid protein. Hepatology 18, 1039. Koziel, M.J., Dudley, D., Wong, J., Dienstag, J., Houghton, M., Ralston, R., Walker, B.D., 1992. Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis. J. Immunol. 149, 3339. Koziel, M.J., Dudley, D., Afdhal, N., Choo, Q.L., Houghton, M., Ralston, R., Walker, B., 1993. Hepatitis C virus-specific cytotoxic T lymphocytes recognize epitopes in the core and envelope proteins of HCV. J. Virol. 67, 7522. Lai, J.P., Prince, A.M., Wolfe, L.L., Andrus, L., 1994. A simplified method for PCR detection of hepatitis C virus RNA from human serum. PCR Methods Apll. 3, 308. Lubaki, M.N., Egan, M.A., Silicano, R.F., Weinhold, K.K., Bollinger, R.C., 1994. A novel method for detection and ex vivo expansion of HIV type 1-specific cytotoxic T lymphocytes. AIDS Res. Hum. Retroviruses 11, 1427. Minutello, M.A., Pileri, P., Unutmaz, D., Censini, S., Kuo, G., Houghton, M., Brunetto, M.R., Bonino, F., Abrignani, S., 1993. Compartmentalization of T lymphocytes to the site of disease: intrahepatic CD4qT cells specific for the protein NS4 of hepatitis C virus in patients with chronic hepatitis C. J. Exp. Med. 178, 17. Mondelli, M., Manns, M., Ferrari, C., 1988. Does the immune response play a role in the pathogenesis of chronic liver disease. Arch. Pathol. Lab. Med. 112, 489. Neitzel, H., 1986. A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum. Genet. 73, 320. Panicali, D., Davis, S.W., Mercer, S.R., Paoletti, E., 1981. Two major DNA variants present in serially propagated stocks of the WR strain of vaccinia virus. J. Virol. 37, 1000. Perkus, M.E., Limbach, K., Paoletti, E., 1989. Cloning and expression of foreign genes in vaccinia virus, using a host range selection system. J. Virol. 63, 3829.
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Perkus, M.E., Kauffman, E.B., Taylor, J., Mercer, S., Smith, D., VanderHoeven, J., Paoletti, E., 1993. Methodology of using vaccinia virus to express foreign genes in tissue culture. J. Tis. Cult. Meth. 15, 72. Prince, A.M., Stephan, W., Dichtelmuller, H., Brotman, B., Huima, ¨ T., 1985. Inactivation of the Hutchinson Strain of Non-A, Non-B hepatitis virus by combined use of b-propiolactone and ultraviolet irradiation. J. Med. Virol. 16, 119. Shankar, P., Fabry, J., Lieberman, J., 1995. A simple method to selectively expand HIV-specific cytotoxic T lymphocytes in vitro. Immunol. Invest. 24, 489. Shimizu, Y.K., Hijikata, M., Iwamoto, A., Alter, H.J., Purcell, R.H., Yoshikura, H., 1994. Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses. J. Virol. 68, 1494. Shirai, M., Akatsuka, T., Pendleton, C.D., Houghten, R., Wychowski, C., Mihalik, K., Feinstone, S.M., Berzofsky, J.A., 1992. Induction of cytotoxic T cells to a cross-reactive epitope in the hepatitis C virus nonstructural RNA polymerase-like protein. J. Virol. 66, 4098. Shirai, M., Okada, H., Nishioka, M., Akatsuka, T., Wychowski, C., Houghten, R., Pendleton, C.D., Feinstone, S.M., Berzofsky, J.A., 1994. An epitope in hepatitis C virus core region recognized by cytotoxic T cells in mice and humans. J. Virol. 68, 3334. Tartaglia, J., Perkus, M.E., Taylor, J., Norton, E.K., Audonnet, J.C., Cox, W.I., David, S.W., VanderHoeven, J., Meignier, B., Riviere, M., Languet, B., Paoletti, E., 1992. NYVAC: a highly attenuated strain of vaccinia virus. Virology 188, 217. Taswell, C., 1984. Limiting dilution assays for the determination of immunocompetent cell frequencies. Validity tests for the Single-Hit Poisson Model. J. Immunol. Methods 72, 29. Walker, C.M., 1996. Cytotoxic T-lymphocyte responses to the hepatitis C virus in humans and chimpanzees. Sem. Virology 7, 13.
Hepatitis C virus-specific CTL responses in PBMC from chimpanzees with chronic hepatitis C: determination of CTL and CTL precursor frequencies using a recombinant canarypox virus ž ALVAC/ Marielle Cohard a,1, Qingyan Liu a , Marion Perkus b, Ethel Gordon a , Betsy Brotman a , Alfred M. Prince a,) a
Lindsley F. Kimball Research Institute, The New York Blood Center, 310 East, 67th Street, New York, NY 10021, USA b Virogenetics, 465 Jordan Road, Rensselaer Technology Park, Troy, NY 12180, USA Received 29 October 1997; revised 26 February 1998; accepted 27 February 1998
Abstract The aim of this study was to evaluate HCV-cytotoxic T lymphocyte response from PBMC in bulk CTL assays and in CTL precursor analyses using in vitro stimulation with canarypox virus ŽALVAC. expressing HCVcapsidrE1rE2rNS2rNS3 antigens. Canarypox virus is naturally host-range restricted and does not replicate or cause cytopathology on mammalian cells. PBMC were obtained from four chimpanzees with chronic hepatitis C infection and one uninfected chimpanzee. CTL from bulk culture of PBMC and CTL precursor frequencies were found in three of the four chronically infected chimpanzees using ALVAC in vitro stimulation. No CTL response was detected in PBMC from the uninfected chimpanzee. The precursor frequencies of CTL specific for capsid, NS2 and NS3 proteins ranged between 1r2663 and 1r27202. No correlation was observed between percent cytolysis in bulk culture and CTL precursor frequencies. This method may prove useful in assessing the correlation between HCV–CTL response and virological or histological status. q 1998 Elsevier Science B.V. All rights reserved. Keywords: HCV–CTL response; CTL precursor frequency; Canarypox virus
1. Introduction Hepatitis C virus ŽHCV. is the major cause of non-A, non-B viral hepatitis. Infection with HCV
Abbreviations: CTL, Cytotoxic T lymphocyte; HCV, Hepatitis C virus; PBMC, Peripheral blood mononuclear cells ) Corresponding author. Tel.: q1-212-570-3279; fax: q1-212570-3180; e-mail: [email protected]. 1 Current address: Schering-Plough Research Institute, 2000 Galloping Hill Road, Kenilworth, NJ 07033.
leads to chronic hepatitis in more than 50% of infected individuals. Ten to 50% of these individuals eventually develop cirrhosis or liver cancer ŽCuthbert, 1994.. Persistent viremia and liver damage have been identified in chronically infected individuals despite the presence of both humoral ŽAlter et al., 1992; Shimizu et al., 1994. and cellular immune response ŽMinutello et al., 1993; Botarelli et al., 1993; Koziel et al., 1993; Koziel et al., 1992.. The mechanism of chronic infection despite vigorous immune responses is unknown. CTL-mediated lysis of
0022-1759r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 8 . 0 0 0 5 4 - 4
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virus infected cells is thought to lead to clearance of the virus or, if incomplete, to viral persistence and chronic tissue injury ŽMondelli et al., 1988.. Class I HLA-restricted CD8 q CTL that recognize viral proteins have been described in the liver of patients with chronic hepatitis ŽKoziel et al., 1992. and in the liver of chimpanzees with acute and chronic hepatitis C ŽErickson et al., 1993.. The role of peripheral blood CTL in HCV infection is unclear. Expansion of virus-specific CTL from PBMC has been accomplished by culturing the cells with epitopic HCV peptides ŽKoziel et al., 1992, 1993; Kita et al., 1993; Shirai et al., 1992, 1994; Cerny et al., 1995; Battegay et al., 1996.. However, the detection of cytolytic activity requires specific experimental conditions with multiple in vitro stimulations with class I HLA restricted peptides. Therefore, it has been suggested that CTL are present at a low frequency in the peripheral blood of some chronically infected subjects. HCV-specific CTL also can be expanded from peripheral blood of individuals with no apparent exposure to the virus ŽCerny et al., 1995.. The purpose of this study was to determine whether canarypox virus vector ŽALVAC. engineered to express CaprE1rE2rNS2r NS3 antigens could be used for specific in vitro stimulation, instead of using synthetic peptides which are potentially responsible for in vitro priming.
2. Materials and methods 2.1. Animals Five chimpanzees Ž Pan troglodytes.: 4 chimpanzees with chronic hepatitis C and one healthy non-infected chimpanzee were studied. They were maintained at Vilab II, New York Blood Center’s primate laboratory in Liberia ŽBrotman et al., 1985.. Chimpanzees 157, 208 and 328 were infected intravenously with 1 ml inoculum containing 5 = 10 4 CID50 of the HCV-H virus stock ŽPrince et al., 1985.. Chimpanzee 159 was infected with a commercial unsterilized factor VIII preparation. The diagnosis of HCV infection was based on standard serological assays using successively first and second-generation Ortho HCV ELISA test system ŽOr-
tho Diagnostic Systems, Raritan, NJ.. PCR was performed as previously described ŽLai et al., 1994. and the results after inoculation were positive for chimpanzees 157, 159, 208 and 328. All four of these animals remain PCR positive to date; they are thus chronically infected. In chimpanzees 157, 159, 208 and 328, liver biopsies revealed ultrastructural features characteristic of HCV infection in chimpanzees up to 10 years after inoculation. One chimpanzee ŽCh. 214. was not inoculated and was used as a negative control in this study. Table 1 summarizes animal characteristics and history of HCV challenge. 2.2. Generation of poxÕirus recombinants expressing HCV proteins Hepatitis C genes from the genotype 1a ŽHutchinson strain. were inserted into two poxvirus: ALVAC, a strain of canarypox ŽTartaglia et al., 1992. and the L variant of WR, a laboratory strain of vaccinia virus ŽPanicali et al., 1981.. Poxvirus recombinants were generated as described previously ŽPerkus et al., 1993.. ALVAC-based recombinant: HCV sequences encompassing capsid through NS3 ŽcapsidrE1rE2r NS2rNS3. were placed under the control of the synthetic vaccinia earlyrlate H6 promoter ŽPerkus et al., 1989.. This expression cassette was inserted into a canarypox donor plasmid flanked by genomic sequences from which a nonessential gene was specifically deleted. Recombination between the donor plasmid and Alvac virus resulted in the recombinant ALVAC-caprE1rE2rNS2rNS3. Three WR vaccinia-based recombinants, cap-vaccinia, NS2-vaccinia and NS3-vaccinia, were generated. The capsid gene was expressed under the control of the vaccinia H6 promoter. NS2 and NS3 genes were expressed under the control of an early promotor. Expression cassettes were inserted immediately downstream from the K1L host range gene ŽGillard et al., 1986. in the WR strain of vaccinia virus. 2.3. Anti-HCV antibodies Monoclonal anti-HCV capsid antibody C7-50 Žkindly provided by Dr. Jack R. Wands. and rabbit anti-HCV NS3 Žkindly provided by Dr. Ramesh A.
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Table 1 Characteristics of chimpanzees Chimpanzee No.
Birthdate
Sex
Inoculation date
Inoculuma
Re-challengeb date
157 159 208 328 214
1977 1975 1978 1982 1979
M M M M M
1982 1981 1985 1988 y
Hutch Ž1a. Factor VIII c Hutch Ž1a. Hutch Ž1a. y
y 1994 y 1994 y
a
Chimpanzees 157, 208 and 328 were infected intravenously with 1 ml inoculum containing 5 = 10 E4 CID50 of the HCV-Hrvirus stock ŽPrince et al., 1985.. Chimpanzee 214 was not inoculated with HCV and was considered as negative control. b Animals were re-challenged with the same inoculum. c The inoculum was 10 ml of a commercial unsterilized factor VIII preparation.
Bhat. were used to identify the expression of HCV protein. 2.4. Detection of HCV protein expression by the recombinant Õiruses COS-1 cells were grown in Dulbecco’s modified Eagle’s medium ŽDMEM. containing 4.5 mgrml glucose and 10% fetal bovine serum ŽFBS. at 378C in 5% CO 2 rair. The cells were seeded into 35 mm petri dishes and cultured overnight or until the cells became monolayers. The following day, the cells were infected by recombinant pox viruses or their parents with a MOI of 10 pfurcell in DMEM containing 2% FBS. After 1 h incubation, the virus containing supernatant was removed and replaced with 2 ml DMEM and 2% FBS, culture was continued for 24 h. The cells were harvested by adding 1 = SDS loading buffer containing 50 mM Tris hydrochloride, pH 6.8, 100 mM dithiothreitol, 2% Žwrv. sodium dodecyl sulfate, 10% glycerol and 0.2% bromphenol blue. The mixture was then heated at 1008C for 5 min, sheared by sonication and centrifuged for 5 min at room temperature at 14,000 rpm in a microcentrifuge. The samples were separated by 12% SDS-PAGE and electrotransferred to nitrocellulose membrane with a Bio-Rad blotting apparatus ŽBio-Rad Laboratories.. The membranes were blocked in 5% non-fat dry milk in PBS at room temperature for 1 h. Monoclonal anti-HCV capsid Ž1:1000 dilution. and rabbit anti-HCV NS3 Ž1:1000 dilution. antibodies were used as primary antibodies, and HRP-labeled goat anti-mouse or goat anti-rabbit IgG ŽH q L. Ž1:5000 dilution, KPL. were applied as
secondary antibodies to detect the expression of HCV capsid and NS3 proteins. Signals were developed by enhanced chemiluminescence ŽECL; Amersham.. 2.5. Target cells Autologous B-lymphoblastoid cell lines ŽB-LCL. were generated from each animal. Briefly, Ficoll Hypaque-separated PBL were cultured in supernatant from the EBV-producing marmoset cell line B95-8 Žkindly provided by Dr. Maria Proytcheva at the New York Blood Center., in RPMI 1640 containing 20% fetal bovine serum ŽFBS. and 2 m grml cyclosporine A ŽNeitzel, 1986.. Transformed cells were maintained in RPMI 1640 supplemented with Lglutamine Ž2 mM., penicillin Ž100 Urml., streptomycin Ž100 m grml., Hepes Ž20 mM. and 20% heat-inactivated FBS. 2.6. Effector cells Peripheral blood mononuclear cells ŽPBMC. drawn between 1994 and 1996 were purified by Ficoll-Hypaque and then cryopreserved at 18Crmin in the presence of 10% DMSO and 20% FBS. PBMC were thawed and plated in 24-well plates at 6 to 10 = 10 6rwell in 2 to 4 ml of culture medium consisting of RPMI 1640, 10% heat-inactivated FBS, antibiotics and 2 mM L-glutamine. Cultures included stimulator cells which were prepared by infecting autologous PBMC or EBV-transformed B lymphoblastoid cell lines ŽB-LCL. at a MOI of 10 pfurcell with a canarypox virus ŽALVAC. vector expressing capsid, E1 E2, NS2, NS3 proteins or a
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parental-Alvac vector. PBMC-to-stimulator cells ratio of 5:1 and 10:1 were studied. When B-LCL were used as stimulator cells, 1 = 10 6 rml irradiated human PBL Ž3000 Rad. were added to cultures as feeder cells. Bulk CTL assays were performed after 7 days of culture at 378C Ž5% CO 2 . without IL-2.
2.7. Bulk cytotoxic T lymphocyte assays CTL assays using autologous B-LCL infected with recombinant vaccinia virus ŽVV. were performed. All B-LCL were infected with recombinant vaccinia HCV vectors containing the genes of capsid, NS2 or NS3, or control vaccinia virus at a multiplicity of infection ŽMOI. of 2 pfurcell for 1 h at 378C then washed and cultured 16 h prior to the assay. VV-infected autologous B-LCL were labelled with 100 m Ci of 51 Cr for 1 h and washed three times in cold assay medium ŽRPMI1640, 10% FBS, 2 mM Lglutamine, Antibiotics, 25 mM Hepes, 5 = 10y5 MB-mercaptoethanol Ž2 BME.. and used as targets in a standard 51 Cr-release assay in which the effector cells were PBMC. Effector-to-target ŽErT. ratios between 50:1 and 10:1 were assessed. Cytolytic activity was determined in a standard 4–6 h 51 Cr-release assay using U-bottom 96-well plates containing 5000 targets per well. The background lysis by vaccinia and EBV-specific CTL was reduced in most assays by the use of cold target inhibition as previously described ŽLubaki et al., 1994.. Briefly excess non-chromium-labeled control vaccinia-infected autologous B-LCL Žcold targets. were added to prior to the initiation of the CTL assay. All assays were performed in duplicate. Percent cytotoxicity was calculated using the formula: 100 = wŽexperimental release in cpm y spontaneous release in cpm.rŽmaximum release in cpm y spontaneous release in cpm. x. Maximum release was determined by lysis of targets with detergent Ž1% Triton X-100; Sigma Chemical.. Spontaneous release was determined from targets in medium alone. Assays were excluded from analysis if the spontaneous release value was ) 30% of maximum release. Results were reported as the mean of duplicate values. A difference in the specific lysis of recombinant-VV-infected targets and control-VV-infected target cells of
at least 10% seen in at least 2 effectorrtarget ratio was considered to represent a positive CTL response. 2.8. Limiting dilution assays of memory cytotoxic T lymphocytes Precursor frequencies of HCV- capsid, HCV-NS2 and HCV-NS3-specific CTL were estimated by performing limiting dilutions on isolated PBMC followed by in vitro stimulation to allow expansion of CTL. PBMC were diluted to give 315 to 20,000 lymphocytes per well in 24 replicates in 96-U bottomed microtiter plates. Stimulator cells Žautologous PBMC. were infected with recombinant-Alvac expressing CaprE1rE2rNS2rNS3 proteins, then gamma-irradiated Ž3000 rad. and added at 25,000 cells per well in complete medium to the responder cells. Effector and stimulator cells were cultured at 378C, 5% CO 2 in culture medium containing RPMI1640, 10% FBS, Antibiotics, 2 BME and Hepes. On days 3 and 10, half of the culture medium was changed for complete medium containing IL-2 at 50 Urml ŽR & D.. A second round of specific stimulation with the recombinant Alvac vector was performed as above on day 7. At day 14, wells were split and assayed for cytotoxicity on 51 Cr-labeled autologous B-LCL infected with vaccinia-capsid expression vector or vaccinia-NS2 or NS3 vector and control–vaccinia virus in parallel, in a 6 hour 51 Crrelease assay. The fraction of nonresponding wells was taken as the number of wells in which 51 Cr-release did not exceed the mean plus three standard deviations of the spontaneous release of the 24 control wells. 2.9. Statistical analysis The CTL precursor frequencies ŽCTLpf. estimates and confidence intervals were calculated using Miller’s software as described by Carl Taswell ŽTaswell, 1984.. Minimal estimates of CTL frequency were obtained according to the Poisson distribution equation as the slope of a line relating the number of responder cells per microwell Žplotted on a linear axis. and the percentage of microwells that failed to develop cytolytic activity Žplotted on a
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3. Results 3.1. HCV protein expression by the recombinant Õiruses
Fig. 1. COS-1 cells were infected by recombinant pox viruses and their parents Žlane 1 and 5: WR parents, lane 2: WR-HCV capsid, lane 6: WR-HCV NS3, lane 3 and 7: ALVAC parents, lane 4 and 8: ALVAC-HCV capsidrE1rE2rNS2rNS3.. Twenty-four hours later, the infected cells were harvested using 1= SDS loading buffer, boiled, sheared by sonication and centrifuged. The samples were separated by 12% SDS-PAGE and analyzed with immunoblotting. The positions of HCV capsid and NS3 proteins Žarrows., and of the molecular mass standards Žin kilodaltons. are indicated at the sides of the gel.
logarithmic y-axis.. This analysis yields minimal frequency estimate Žrange., and a x 2 estimate of probability Ž p .. In this system, significance is indicated by p greater or equal to 0.05. P values for the differences between the CTLpf were determined by the Mann–Whitney U-test.
As shown in Fig. 1, both 21 kDa and 19 kDa HCV capsid proteins ŽC191 and C174. were detected in the WR-HCV capsid and ALVAC-HCV capsidrE1rE2rNS2rNS3 infected COS-1 cells Žlane 2, 4., not in the parental infected cell lysates Žlane 1, 3.. The expression of 70 kDa HCV-NS3 protein was identified in the WR-HCV NS3 and ALVAC-HCV capsidrE1rE2rNS2rNS3 infected COS-1 cells Žlane 6, 8.. The expression of NS2 in ALVAC-HCV capsidrE1rE2rNS2rNS3 and WRHCV NS2 was not detected, due to lack of a good anti-HCV NS2 antibody. 3.2. Bulk assays: HCV-specific CTL actiÕity Assays were considered positive for CTL if the lysis of targets was greater or equal to 10% higher than the lysis of control vaccinia-infected targets at two different E:T ratios. One of 4 chronically infected chimpanzees had detectable capsid-specific CTL activity. Three of 4 animals had detectable
Fig. 2. HCV-specific CTL response observed from bulk culture after 7 days of in vitro stimulation with recombinant canarypox virus ŽALVAC. expressing CaprE1rE2rNS2rNS3 proteins. Autologous B-LCL were infected overnight with L-Var Žwild type Vaccinia Virus. or with VV-encoding capsid or NS2-vaccinia or NS3-vaccinia, then labeled with 51 Cr and were incubated with effector cells Ž4 to 6 h 51 Cr release assay.. Results with an E:T ratio of 25:1 are shown. Chimpanzee 214 was not inoculated with HCV and was used as a negative control. A difference in the specific lysis of recombinant VV-infected target versus control-VV target cells ŽL-Var. of at least 10% was considered to represent a positive CTL response Žshown as an asterisk..
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Table 2 Frequencies of HCV-specific CTL precursors in chimpanzees with chronic hepatitis C Chimpanzee No.
Targetsa
Frequency Žper thousand cells. 95% CI b
Reciprocal frequency
p value c
157
Cap NS3 Cap NS3 NS2 NS3
0.043 Ž0.012–0.074. 0.250 Ž0.076–0.424. 0.037 Ž0.020–0.054. 0.074 Ž0.042–0.106. 0.375 Ž0.179–0.572. 0.113 Ž0.03–0.224.
23 020 3997 27 202 13 490 2663 8811
0.150 0.501 0.193 0.460 0.191 0.833
208 328 a
CTLpf were determined by adding varying numbers of T cells to 24-replicate wells containing 5000 51 Cr-labeled autologous targets ŽB-LCL. infected with Cap-vaccinia or NS2-vaccinia or NS3-vaccinia or L-Var Žwild type vaccinia.. Positive cultures were defined as those in which 51 Cr release exceeded the mean value in parallel control microculture by at least 3 standard deviations. From wells containing targets infected with L-Var, no precursor frequency was listed because this frequency was not calculated as all wells failed to exhibit significant cytolysis even at the highest effector cell concentration. b Precursor frequencies were determined as described in Section 2. Values in parentheses represent 95% confidence intervals ŽCI. of the calculated frequencies. c In this system, significance is indicated by a p greater or equal to 0.05.
NS3-specific CTL activity and one of 4 had detectable NS2-specific CTL activity ŽFig. 2.. Two chimpanzees reacted to 2 of the targets ŽChimpanzees 157 and 328. and one chimpanzee Ž208. reacted with only one target. There was no detectable HCVspecific CTL activity following stimulation of PBMC with autologous PBMC infected with a canarypox
construct expressing CaprE1rE2rNS2rNS3 genes in a healthy, uninfected chimpanzee Žchimpanzee 214.. The activity in ex vivo expanded CTL was detectable at an E:T ratio of at least 20:1. Attempts to detect HCV-CTL activity from the same animals with chronic hepatitis by non-specific in vitro stimulation of PBMC using anti-CD3 anti-
Fig. 3. Quantification of memory NS3-specific CTL. Limiting dilution was performed on PBMC from HCV-infected chimpanzees followed by in vitro stimulation with ALVAC vectors as described in Section 2. Targets were autologous B-LCL infected with vaccinia virus: L-Var ŽL-Variant WR was the wild type vaccinia., vaccinia-based recombinant cap-vaccinia, NS2-vaccinia and NS3-vaccinia. Results represent precursor frequencies of NS3-specific CTL in three chimpanzees: Chimp 157 Ž1r3997., Chimp 208 Ž1r13490., chimp 328 Ž1r8811.. No precursor frequency is listed for L-Var, because that frequency was not calculable as all wells failed to exhibit significant cytolysis at any effector cell concentration.
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bodies were not successful Ždata not shown.. Additional experiments used stimulator cells prepared by infecting autologous EBV-transformed B lymphoblastoid cells lines ŽB-LCL. with ALVAC expressing CaprE1rE2rNS2rNS3 proteins and then gamma-irradiated. Background lysis of the EBVtransformed B-LCL was enhanced in 50% of the experiments when B-LCLs were used as stimulator cells Ždata not shown.. Excess unlabeled, vacciniainfected, autologous targets included in those assays did not prevent the high background lysis. 3.3. Frequencies of HCV-specific CTL precursors in PBL To determine the frequency of memory HCVspecific CTL precursors in the PBMC of chronically infected chimpanzees, limiting dilution analysis following in vitro stimulation with recombinant Alvac vector expressing CaprE1rE2rNS2rNS3 was performed. The results of precursor frequency determinations on 3 animals are summarized in Table 2. Results representing NS3-specific CTL precursor frequencies are shown in Fig. 3. Using B-LCL infected with vaccinia recombinants, chimpanzees with chronic hepatitis C were shown to have CTL precursors that recognized capsid, NS2 and NS3 proteins. The precursor frequencies of CTL specific for Cap, NS2 and NS3 proteins in general ranged between 1r2663 and 1r27 202. The mean precursor frequency of CTL specific for Capsid protein and for NS3 protein were, respectively, 1r25 111 Žrange 1r23 020–1r27 202. and 1r8766 Žrange 1r3997– 1r13 490.. The difference was not statistically significant Ž p s 0.08.. CTL precursors were detected against capsid in chimpanzee 208, whereas there was no capsidspecific CTL activity detectable in bulk culture. No CTL precursors were detected in one out of four chimpanzees with chronic hepatitis C ŽChimpanzee 159. and in the healthy uninfected control chimpanzee Žchimpanzee 214..
4. Discussion It has been reported that HCV-specific CTLs are detectable from the liver of patients and chimpanzees
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with chronic hepatitis C. The intrahepatic CTL response in humans and chimpanzees is similar in its broad specificity for multiple HCV epitopes Žboth in structural and nonstructural regions. that are presented primarily by MHC class I A or B alleles. The long-lived and multi-specific nature of the CTL response has been demonstrated in some chimpanzees ŽWalker, 1996.. It has been suggested that HCVspecific CTL might be sequestered at the site of infection. A direct comparison of cytolytic activity in the peripheral blood and liver could determine whether HCV-specific T cells are sequestered at the site of infection ŽWalker, 1996.. Multiple stimulations with the HLA-A2.1 restricted peptides were usually required for detection of cytolytic activity. Furthermore, it has been reported that HCV-specific CTL could also be detected from peripheral blood of individuals with no exposure to the virus ŽCerny et al., 1995. raising the possibility that the response might reflect in vitro priming by the peptides rather than in vivo priming by exposure to virus. In order to better understand the antiviral immune response by CD8 q CTL and to explain why some individuals develop self-limited infections rather than chronic infection, further studies are needed to investigate HCV-specific T lymphocytes in the peripheral blood of acutely and chronically infected chimpanzees and humans. Access to peripheral blood is easy whereas liver tissue biopsy is considered as an invasive technique and is not routinely performed in acute HCV infection in human. It is difficult to isolate and characterize CTLs from peripheral blood following non-specific expansion using CD3 antibodies ŽCerny and Chisari, 1994.. Therefore, there is a need for standardizing and developing new strategies to increase the sensitivity of detection of CTL from peripheral blood without the risk of in vitro priming. Our attention has focused on antigen-specific stimulation using a nonreplicating canarypox virus vector ŽCox et al., 1993.. The canarypox virus is naturally host-range restricted and does not replicate or cause cytopathology, on mammalian cells. The advantage of using canarypox virus ŽALVAC. as a vector infecting cells used as stimulator cells for expansion of HCV-specific CTL is the absence of a need to inactivate Alvac before culturing. Other strategies using recombinant vaccinia virus as a vec-
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M. Cohard et al.r Journal of Immunological Methods 214 (1998) 121–129
tor for infecting B-LCL need to treat the stimulator cells with psoralen and UV light irradiation ŽLubaki et al., 1994; Shankar et al., 1995., prior to their use for in vitro stimulation, to inactivate vaccinia virus and thereby prevent vaccinia infection of the effector T cells. We have been successful in demonstrating CTL response from PBMC in bulk culture assays and in CTL precursor analyses using Alvac stimulation. Our results show that CTL precursors are present in the circulation when CTL are detectable in bulk culture. We did not observe a correlation between percent cytolysis in bulk culture and CTL precursor frequencies. This may reflect differences in the degree of stimulation in the two assays used. The in vitro ALVAC stimulation may have increased the CTL precursor frequency to levels above those seen in unstimulated peripheral blood. Previous authors ŽCerny et al., 1995. observed much lower HCVspecific CTL precursor frequencies in peripheral blood probably because their experimental systems focused on single epitopes. Due to the small number of animals studied, it is not possible to correlate the CTL response to the clinical or virological status of the chimpanzees. However, it is interesting to observe that we could not detect HCV-specific CTL responses in one chimpanzee with chronic hepatitis C and a high viremia Ž15 to 20 times higher than the other animals studied. Ždata not shown.. Further studies are necessary to confirm whether there is a correlation between HCV-specific CTL response and viral load. The methods described in this report are simple and require less effector cells than needed with peptides in vitro stimulation. Therefore, this method could be used to evaluate CTL response from bulk culture and CTL precursor frequencies in blood of patients with acute or chronic hepatitis C in studies aimed to assess correlation between CTL response and virological or histological status. This method could also be valuable in developing vaccine strategies to assess CTL response from peripheral blood after DNA based immunization in animals. Acknowledgements M. Cohard was supported by Grants of Research in Gastroenterology and Hepatology, from Beaufour
Laboratories ŽFrance. and Glaxo-Welcome Laboratories ŽFrance. and by a fellowship of the Institut Electricite´ Sante´ ŽFrance.. Animals were held in pairs or larger groups in cages that exceeded minimal specifications of requirements of the National of Health Guide for Care of Laboratory Animals. Financial support: Pasteur Merieux Serum and Vaccins ŽFrance..
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