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Viral escape from the HLA-A2 restricted cytotoxic T lymphocyte response is not common during chronic hepatitis C virus infection
HEPATOLOGY 905
Vol.
22,
No.
4, Pt.
2, 1995
IN
AASLD
VITRO EFFECTS OF INTERFERON ON HEPATITIS C VIRUS REPLICATION LYMPHOCYTES. X Wang, and AH Sherker. Institute for Medical Research, SMBD-Jewish McGill University, Mont&I, Canada.
ALPHA-ZB IN
906
Lady Davis General Hospital,
THE PERIPHERAL BLOOD CYTOTOXIC T LYMPHOCYTE RESPONSE TO HCV DOES NOT REFLECT DISEASE ACTIVITY DURING CHRONIC HCV INFECTION. B. Rehermann, K.M. Chane. J. 0. MCK.@kka*. M. Houehton* and F.V. Chisnri. Tbe Scripps Research Institute, La Jolla, CA, USA and Chlron Corporation, Emeryville, CA, USA Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been isolated from the peripheral blood and liver of infected patients. The objective of this study was to determine whether the peripheral blood CTL response correlates with disease activity and can be modulated by interferon-therapy. Using a synthetic peptide stimulation strategy, the frequency of peripheral blood CTL precursors (CTLpn to 4 previously defined HCV core and NS3 epitopes and to an immunodominant influenza matrix epitope was determined in 12 HCV-infected patients before, during and after interferon therapy, and in 9 healthy, uninfected conlrols. Brietly, activated B cells pulsed with the individual peptides were used fu stimulate serially diluted PBMC in 24 replicate cultures per PBMC dilufion. On day 19 the cultures were tested for CTL activity in a standard chromium-release assay, and the CTL precursor frequency was calculated. Prior to interferon therapy, chronically infected patients displayed a polyclonal and multispecitic CTL response to HCV with an average HCV-specific CTLpf of approximately 3 CTL per IO‘ PBMC which was 4-12 times lower than the CTLc[!fto the inlluenza matrix peptide. In the uninfected control group, the CTLpf 10 HCV was too low to be detected with any reliability, while the intluenza-specific CTLpf was similar to that observed in the patients. Thus these pafienfs are less strongly primed by their ongoing HCV infection than they are to the unrelated influenza virus, suggesting that the CTL response to HCV may be tou weak to clear the infection. Furthermore, the peripheral GIL response to HCV does not correlate well with disease activity or viral load: although a llare in serum alanine aminotransferase (ALT) (-3 fold) that was preceded by a -4 fold increase in CTLpjwas observed in 4 cases, in 2 other instances there was no change in serum ALT despite a sharp increase (-8 fold) in CTLpf for 3 of the HCV epitopes. HCV-RNA, as determined by bDNA analysis, decreased in patients whose ALT normalized in response to IFN. In conclusion, there was no difference in the CTLpfbetween those patients who responded to IFN and lhose who did not. Whether these results extend to other patient populations and To all HCV genotypes remains to be determined. Since the peripheral CTL response to HCV is very weak in these patients, its specitic enhancement may have therapeutic value.
MOLECULAR MAPPING OF AN lNTR4HEPATlC HEPATITIS C VIRUSSPECIFIC CROSS-GENOTYPE CYl.OTOXIC T-LYMPHOCYTE (HCV-CTL) EPITOPE Marousia CG. Nelson DR. Lau JYN. Section of Hepatobiiary Diseases, University
of Florida, Gainesville, FL.
Backmound Iotrahepatic HCV-CTL, mostly against core-E& are important in controlling HCV replication, a virus with marked genetic heterogeneity. In designing CTL-based immunotherapy, conserved CTL epitopes are more useful. & To map and characterize an HLA-A2 restricted cross-genotype CTL epitope within HCV core. Methods HLA-A2 restricted epitopes were studied since (1) HLA-A2 is present in half of the population, and (2) the binding motif is well characterized (x&xxxxxV~. Patients CTLs were cloned from intrahepatic bulk-expanded naive CTLs with HCV-specific CTL activity (towards core or core-E2) from 3 HLAA2( +) (HCV type lb, 2a, 3a) and 3 HLA-AZ(-) patients (HCV types la, la, 3a). E&one Mnmin~ As CTL activity was detected based on HCVl (type la), predicted HLA-A2 epitopes within core/E2 were synthesized. CTL clones were generated by limiting dilution and expanded with IL-2/antXD3/irradiated autologous feeder cells. Multiple clones from each patient were screened for CTL activity against autologous EBV-transformed B-cells (HLA re &ted) sensitized by either HLA-A2 No clone from HCV epitopes, or an unrelated peptide using % Cr release. m HLA-A2(-) and 2/3 HLA-A2( +) patients had CTL activity against HLA-A2 peptides within core-E2. Patient 94-13 (HCV type lb) had 2/3 clones re&ive to peptide 1 (aa 132-140, DLMGYIPLV). For patient 94-21 (type 2a), 5/9 clones were reactive to the same peptide. 2/4 remaining clones were reactive to peptide 6 within E2 (aa49E503, HLHQNIVDV, conserved between la/2a). Chamctekeion of the eoitove aa132-140 This epitope is well conserved across genotypes, with type 2a having XL--W instead of z&xxx&V [V in position 81. Nucleotide sequencing of the patient’s (94-21, with type 2a) HCV shoved the same predicted amino acid sequence in 132-140 as HCV-M, the prototype 2a. Further mapping was performed using peptides with different combiiations of valine and leucine in positions 8,9 and 10 to determine the requirements of the carboxyl terminus. All clones from patients 94-21 (HCV type 2a) and 94-U (HCV type lb) which showed activity against a&32140 xLxxxc&V also showed activity to an 8 amino acid peptide &xxxxx~, but not to other combiiations, indicating that the anchor can be in either position 8 or 9, but not other combiations. Conclusion This cross-genotype CTL epitope within HCV core (aa132-140) is highly conserved functionally. lmolication This epitope is a good candidate for the development of immunotherapy.
There is evidence. that hepatitis C virus (HCV) is present within peripheral blood mononuclear cells (PBMCs) in chronically infected patients. In an attempt to study the in vi&o effects of interferon alpha-2b we have isolated PBMCs from chronically infected patients using tbe Ficoll-Hypaque method. Lymphocytes were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in RPMI 1640/10% fetal calf serum in the presence of recombinant interleukin-2 (IL2). Media was supplemented every other day in the absence or presence of lo-fold increasing concentrations of interferon alpha-2b (lOU/ml-1COOOUlml). Cells harvested at seven days showed no apparent toxicity and were analyzed by strandspecific RT/PCR using primers from the 5’-noncoding region of the virus. Southern transfer of the F’CR products of the viral minus- and plus-strand specific reactions were perfomed and hybridized with a phospholuminescent viraI DNA probe internal to the PCR primers. An inhibitory effect of interferon alpha-2b was seen at the lower doses of lo-lOOU/ml. This inhibitory effect is less marked at the higher doses of 1000 and 10000U/ml. This paradoxical effect of massive doses of intetferon may relate to proliferative effects of type I interferons on PBMCs at high doses through increased production of cytokines including interlenkins 1 and 2. In support of this explanation is the observation we have made that when chronic HCV-infected patients’ PBMCs are isolated and cultured for seven days in the absence of PHA, IL-2 or any other mitogens, there is a reproducible dose-related increase in viral replication at high doses of interferon alpha-2b.
907
333A
ABSTRACTS
908
ESCAPE FROM THE HLA-A2 RESTRICTED CYTOTOXIC T LYMPHOCYTE RESPONSE IS NOT COMMON DURING CHRONIC HEPATITIS C VIRU> INFECTION. J.G. M The Scripps Research Institute7 La Jolla, CA USA.
VIRAL
Hepatitis C virus (HCV) infection is usually persistent despite apparent host immune respOnse against the virus. Selection of mutant viruses CaDable of escaping cyiotoxlc T lymphocytes (CTL) recognition has been sugge&ed as a possible mechanism of HCV persistence. The most direct approach to this question in HCV infected patients is to look for mutations in the amino acid sequence of viral epitopes that occur in the context of a CTL response to the epitope and which abrogate or antagonize the CTL recognition of the epitope. We have begun this kind of systematic study to ascertain whether viral escape from the CIZ response is a common event in HCV-infected patients. Thus far, we have determined the nucleotide sequence of 2 known HLA-AZ restricted CTL epitopes located between core residues 35-44 (YLLPRRGPRL) and 131140 (ADLMGYIF’LV) at 2 time wints (l-3 Years anart) in 8 HLA-A2 nositive pati& with chronic kCV infechon. tie s&en&s h&e been cornpa& with the patients’ peripheral blood CTL tesponse to synthetic peptides which correspond to the HCV-1 amino acid sequences at these positions, looking for a correlation between sequence changes and CTL pressure. To simplify interpretation, we restricted our analysis to those instances where the viral sequence at the first time point was identical to the sequence of the synthetic peptide used in the CTL assay.
Our results can be summarized as follows: First, we were able to assess the influence of immune pressure on viral sequence variation in 13 of the 16 paired data points since variant sequences were present in the first sample in 3 cases. Second, CTL response against the epitopes could be detected in 7 of the 13 cases. However, in 6 of these 7 instances, the epitope sequence did not change, suggesting that the CTL response did not exert selection pressure at these epitopes in the time frame studied. Third, in only 1 case, a mutation at core 35
44 (YLLPBRGPBL
-> YLLPSRGPKL) followed a strong CTL response sequence over 11 months. Studies are in progress to determine the effect of this sequence change on CTL recognition. In conclusion, while many more patients and epitopes need to be studied, our
against the HCV-I preliminary
data suggests that viral escape from the HLA-A2
response to these 2 frequently recognized occurrence during chronic HCV infection.
restricted
CTL
core epitopes is not a common
Vol.
22,
No.
4, Pt.
2, 1995
IN
AASLD
VITRO EFFECTS OF INTERFERON ON HEPATITIS C VIRUS REPLICATION LYMPHOCYTES. X Wang, and AH Sherker. Institute for Medical Research, SMBD-Jewish McGill University, Mont&I, Canada.
ALPHA-ZB IN
906
Lady Davis General Hospital,
THE PERIPHERAL BLOOD CYTOTOXIC T LYMPHOCYTE RESPONSE TO HCV DOES NOT REFLECT DISEASE ACTIVITY DURING CHRONIC HCV INFECTION. B. Rehermann, K.M. Chane. J. 0. MCK.@kka*. M. Houehton* and F.V. Chisnri. Tbe Scripps Research Institute, La Jolla, CA, USA and Chlron Corporation, Emeryville, CA, USA Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been isolated from the peripheral blood and liver of infected patients. The objective of this study was to determine whether the peripheral blood CTL response correlates with disease activity and can be modulated by interferon-therapy. Using a synthetic peptide stimulation strategy, the frequency of peripheral blood CTL precursors (CTLpn to 4 previously defined HCV core and NS3 epitopes and to an immunodominant influenza matrix epitope was determined in 12 HCV-infected patients before, during and after interferon therapy, and in 9 healthy, uninfected conlrols. Brietly, activated B cells pulsed with the individual peptides were used fu stimulate serially diluted PBMC in 24 replicate cultures per PBMC dilufion. On day 19 the cultures were tested for CTL activity in a standard chromium-release assay, and the CTL precursor frequency was calculated. Prior to interferon therapy, chronically infected patients displayed a polyclonal and multispecitic CTL response to HCV with an average HCV-specific CTLpf of approximately 3 CTL per IO‘ PBMC which was 4-12 times lower than the CTLc[!fto the inlluenza matrix peptide. In the uninfected control group, the CTLpf 10 HCV was too low to be detected with any reliability, while the intluenza-specific CTLpf was similar to that observed in the patients. Thus these pafienfs are less strongly primed by their ongoing HCV infection than they are to the unrelated influenza virus, suggesting that the CTL response to HCV may be tou weak to clear the infection. Furthermore, the peripheral GIL response to HCV does not correlate well with disease activity or viral load: although a llare in serum alanine aminotransferase (ALT) (-3 fold) that was preceded by a -4 fold increase in CTLpjwas observed in 4 cases, in 2 other instances there was no change in serum ALT despite a sharp increase (-8 fold) in CTLpf for 3 of the HCV epitopes. HCV-RNA, as determined by bDNA analysis, decreased in patients whose ALT normalized in response to IFN. In conclusion, there was no difference in the CTLpfbetween those patients who responded to IFN and lhose who did not. Whether these results extend to other patient populations and To all HCV genotypes remains to be determined. Since the peripheral CTL response to HCV is very weak in these patients, its specitic enhancement may have therapeutic value.
MOLECULAR MAPPING OF AN lNTR4HEPATlC HEPATITIS C VIRUSSPECIFIC CROSS-GENOTYPE CYl.OTOXIC T-LYMPHOCYTE (HCV-CTL) EPITOPE Marousia CG. Nelson DR. Lau JYN. Section of Hepatobiiary Diseases, University
of Florida, Gainesville, FL.
Backmound Iotrahepatic HCV-CTL, mostly against core-E& are important in controlling HCV replication, a virus with marked genetic heterogeneity. In designing CTL-based immunotherapy, conserved CTL epitopes are more useful. & To map and characterize an HLA-A2 restricted cross-genotype CTL epitope within HCV core. Methods HLA-A2 restricted epitopes were studied since (1) HLA-A2 is present in half of the population, and (2) the binding motif is well characterized (x&xxxxxV~. Patients CTLs were cloned from intrahepatic bulk-expanded naive CTLs with HCV-specific CTL activity (towards core or core-E2) from 3 HLAA2( +) (HCV type lb, 2a, 3a) and 3 HLA-AZ(-) patients (HCV types la, la, 3a). E&one Mnmin~ As CTL activity was detected based on HCVl (type la), predicted HLA-A2 epitopes within core/E2 were synthesized. CTL clones were generated by limiting dilution and expanded with IL-2/antXD3/irradiated autologous feeder cells. Multiple clones from each patient were screened for CTL activity against autologous EBV-transformed B-cells (HLA re &ted) sensitized by either HLA-A2 No clone from HCV epitopes, or an unrelated peptide using % Cr release. m HLA-A2(-) and 2/3 HLA-A2( +) patients had CTL activity against HLA-A2 peptides within core-E2. Patient 94-13 (HCV type lb) had 2/3 clones re&ive to peptide 1 (aa 132-140, DLMGYIPLV). For patient 94-21 (type 2a), 5/9 clones were reactive to the same peptide. 2/4 remaining clones were reactive to peptide 6 within E2 (aa49E503, HLHQNIVDV, conserved between la/2a). Chamctekeion of the eoitove aa132-140 This epitope is well conserved across genotypes, with type 2a having XL--W instead of z&xxx&V [V in position 81. Nucleotide sequencing of the patient’s (94-21, with type 2a) HCV shoved the same predicted amino acid sequence in 132-140 as HCV-M, the prototype 2a. Further mapping was performed using peptides with different combiiations of valine and leucine in positions 8,9 and 10 to determine the requirements of the carboxyl terminus. All clones from patients 94-21 (HCV type 2a) and 94-U (HCV type lb) which showed activity against a&32140 xLxxxc&V also showed activity to an 8 amino acid peptide &xxxxx~, but not to other combiiations, indicating that the anchor can be in either position 8 or 9, but not other combiations. Conclusion This cross-genotype CTL epitope within HCV core (aa132-140) is highly conserved functionally. lmolication This epitope is a good candidate for the development of immunotherapy.
There is evidence. that hepatitis C virus (HCV) is present within peripheral blood mononuclear cells (PBMCs) in chronically infected patients. In an attempt to study the in vi&o effects of interferon alpha-2b we have isolated PBMCs from chronically infected patients using tbe Ficoll-Hypaque method. Lymphocytes were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in RPMI 1640/10% fetal calf serum in the presence of recombinant interleukin-2 (IL2). Media was supplemented every other day in the absence or presence of lo-fold increasing concentrations of interferon alpha-2b (lOU/ml-1COOOUlml). Cells harvested at seven days showed no apparent toxicity and were analyzed by strandspecific RT/PCR using primers from the 5’-noncoding region of the virus. Southern transfer of the F’CR products of the viral minus- and plus-strand specific reactions were perfomed and hybridized with a phospholuminescent viraI DNA probe internal to the PCR primers. An inhibitory effect of interferon alpha-2b was seen at the lower doses of lo-lOOU/ml. This inhibitory effect is less marked at the higher doses of 1000 and 10000U/ml. This paradoxical effect of massive doses of intetferon may relate to proliferative effects of type I interferons on PBMCs at high doses through increased production of cytokines including interlenkins 1 and 2. In support of this explanation is the observation we have made that when chronic HCV-infected patients’ PBMCs are isolated and cultured for seven days in the absence of PHA, IL-2 or any other mitogens, there is a reproducible dose-related increase in viral replication at high doses of interferon alpha-2b.
907
333A
ABSTRACTS
908
ESCAPE FROM THE HLA-A2 RESTRICTED CYTOTOXIC T LYMPHOCYTE RESPONSE IS NOT COMMON DURING CHRONIC HEPATITIS C VIRU> INFECTION. J.G. M The Scripps Research Institute7 La Jolla, CA USA.
VIRAL
Hepatitis C virus (HCV) infection is usually persistent despite apparent host immune respOnse against the virus. Selection of mutant viruses CaDable of escaping cyiotoxlc T lymphocytes (CTL) recognition has been sugge&ed as a possible mechanism of HCV persistence. The most direct approach to this question in HCV infected patients is to look for mutations in the amino acid sequence of viral epitopes that occur in the context of a CTL response to the epitope and which abrogate or antagonize the CTL recognition of the epitope. We have begun this kind of systematic study to ascertain whether viral escape from the CIZ response is a common event in HCV-infected patients. Thus far, we have determined the nucleotide sequence of 2 known HLA-AZ restricted CTL epitopes located between core residues 35-44 (YLLPRRGPRL) and 131140 (ADLMGYIF’LV) at 2 time wints (l-3 Years anart) in 8 HLA-A2 nositive pati& with chronic kCV infechon. tie s&en&s h&e been cornpa& with the patients’ peripheral blood CTL tesponse to synthetic peptides which correspond to the HCV-1 amino acid sequences at these positions, looking for a correlation between sequence changes and CTL pressure. To simplify interpretation, we restricted our analysis to those instances where the viral sequence at the first time point was identical to the sequence of the synthetic peptide used in the CTL assay.
Our results can be summarized as follows: First, we were able to assess the influence of immune pressure on viral sequence variation in 13 of the 16 paired data points since variant sequences were present in the first sample in 3 cases. Second, CTL response against the epitopes could be detected in 7 of the 13 cases. However, in 6 of these 7 instances, the epitope sequence did not change, suggesting that the CTL response did not exert selection pressure at these epitopes in the time frame studied. Third, in only 1 case, a mutation at core 35
44 (YLLPBRGPBL
-> YLLPSRGPKL) followed a strong CTL response sequence over 11 months. Studies are in progress to determine the effect of this sequence change on CTL recognition. In conclusion, while many more patients and epitopes need to be studied, our
against the HCV-I preliminary
data suggests that viral escape from the HLA-A2
response to these 2 frequently recognized occurrence during chronic HCV infection.
restricted
CTL
core epitopes is not a common