T-cell vaccine for hepatitis C: Virus-host interaction in hepatitis C: analysis of the virusspecific T-lymphocyte response as a basis for the development of a T-cell vaccine

• Forschungsstelle: EU FRP
• Projektnummer: 95.0138
• Projekttitel Englisch: T-cell vaccine for hepatitis C: Virus-host interaction in hepatitis C: analysis of the virusspecific T-lymphocyte response as a basis for the development of a T-cell vaccine

Schlüsselwörter (Deutsch)

Hepatitis C-Virus1 Immunantwort; T-ZeII-Impfung; Wirt-Virus-Interaktion

Alternative Projektnummern (Englisch)

EU project number: BMH4CT961064

Forschungsprogramme (Englisch)

EU-programme: 4. Frame Research Programme - 4.2 Agriculture and agroindustry

Kurzbeschreibung (Englisch)

See abstract

Partner und Internationale Organisationen (Englisch)

Klinik Grosshadern München, Universität Bari, Medizinische Fakultät Lissabon, University of London, INSRM Unité 271, Lyon, Swedish Institute tor infectious disease Control, Stockholm

Abstract (Englisch)

The progress report will discuss the work done in the period 1.4.98 through 31.3.99 first, according to the specific aims and second, relative to the time schedule defined in the grant proposal.

Aim 1. Serial analysis of the CD8+ T cell mediated immune response to HCV in the natural and Interferon-alpha treatment-modified course of chronic infection as compared to the acute phase of infection. Two approaches to expand and detect HCV specific CTL will be used:
a) a peptide based stimulation strategy using known HLA class I restricted CTL epitopes,
b) a stable tightly regulated HCV expression system.
CTL lines and clones generated will be characterized according to phenotypic and functional parameters and used for experiments described in specific aim 2.

The studies described under aim 1.a) are reported in the manuscript Cucchiarini M et al. which we have submitted to the European Journal of Immunology. Efforts to obtain more samples of acutely infected patients are ongoing.

As an extension of aim 1.a) we have used HCV-derived peptides enclosed into virosomes. Virosomes are liposomes containing influenza haemagglutinin and neuraminidase. This vaccine delivery system was developed by the Swiss Serum and Vaccine Institute Berna. We have shown that virosomes can be used to selectively target peptides into the endogenous MHC class I pathway. Virosomes can thus be used to restimulate HCV specific CTL as well as for the induction of naive CTL in vitro. This work has been reported in abstract form (Hunziker et al.). A manuscript is in preparation.

As a collaboration with Dr. P. Klenerman (Oxford) and Prof. G. Pape (Munich) we have studied alternative peptide-based techniques for the detection of HCV specific CTL. One technique involves gamma-interferon staining of peptide stimulated cells by ELISPOT, the other MHC class tetramers refolded with HCV peptides (Abstract Lechner F. et al). Both techniques confirm that chronically infected patients have very low levels of CTL. The levels are somewhat higher in acutely infected patients, but they are still far lower than in acute CMV, EBV or HIV infection.

Aim 1b): Studies done in collaboration with Dr. D. Moradpour from Prof. Blum's laboratory in Freiburg have shown that the expression system developed in a human osteosarcoma cell line is suitable to present HCV derived proteins via MHC class I (Abstract Grabscheid B et al.). Cotransfection with B7.1 was successful but we so far failed to induce HCV specific primary CTL in coculture systems.

Characterisation of HCV specific CTL clones from chronically infected patients is ongoing. We have focussed our attention on a HCV core (core 178-186) derived epitope with homology to cytochrome p450 as described under aim 2.a) below.

Aim 2. Elucidation of potential mechanisms of viral persistence and tissue injury. We will focus our studies on
a) the role of viral sequence variation in the course of the acute and chronic infection and determine if it results in viral evasion mechanisms such as immune escape, antagonism or molecular mimicry.
b) the effect of HLA class I subtypes on the antiviral response focusing on the role of HLA-A2 subtypes in the HCV core specific CTL response.
c) interference of viral products with the HLA class I-mediated induction or effector phase of the antiviral immune response.

Aim 2.a) We have focussed our attention on a HCV core (core 178-186) derived epitope with homology for cytochrome p450 and could show that the CTL repertoire of HLA A*0201 positive individuals contains crossreactive cells able to react with self. (Kammer et al. Journal of Experimental Medicine: in press). Molecular mimicry such as this may explain how a virus can initiate a mechanism by which selfreactive cells can mediate bystander tissue injury. Similarity with self may also help the virus survive under condition that the host is tolerant to the self antigen in question.
Studies performed with self-peptides eluted from HLA-A*0201 have led to the discovery that a self-peptide derived from BTG-2 is capable of inducing CTL recognizing endogenously synthesized protein. This work was presented at the 1999 meeting of the Swiss Society of Haematology (Abstract Kremer-Hovinga et al) an won the poster price of the Swiss Society of Haematology. A manuscript is in preparation.

Aim 2.b) These studies are ongoing.

Aim 2.c) In collaboration with M. Groetrupp (St. Gallen) and D. Moradpour (Freiburg) we have studied a panel of HCV expressing cell lines and have so far shown that HCV NS3-4A expression had no effect on the MHC class I expression levels, the IFN induced upregulation of MHC class I, the recognition and lysis of target cells by specific CTL and the processing and presentation of HCV epitopes. ( Abstracts Grabscheid et al., and Moradpour et al.). Experiments using cells expressing other HCV proteins and cells cotransfected with influenza A matrix protein are planned to see if HCV expresssion affects an independent MHC class I response.

We have been given the opportunity to review the literature on mechanisms of viral persistence and hepatic injury (Cerny A , Chisari FV Hepatolgy in press)

Adherence to the time schedule proposed in the grant:

Serial and prospective analysis of the CTL response using HCV derived peptides as well as the HCV expression system (task 1a. and 1b.) will be continued during the whole grant period (36 months). This is ongoing. This task has been extended to include the study of virosome encapsulated HCV derived peptides, as well as ELISPOT and MHC class I tetramer technology (see above).
Methods for the determination of viral sequence variation (quasispecies analysis) of selected CTL epitopes will have to be established in our lab which will take 6 months. Quasispecies analyses will then be done during the whole grant period. The study of consequences of viral sequence variation (task 2a.) will be done during the entire period of this grant. Prof. P. Simmonds, Edinburgh, who is an expert in the field of HCV virology accepted to study our serum samples. A first shipment of samples unfortunately perished due to a power failure in his lab.
Studies on the role of molecular mimicry between HCV core and cytochrome p450 on the clonal level will be completed as outlined (task 2a.) within the first 12 months. A first publication including a larger number of chronic HCV patients is in the press (Kammer et al.).
The search and analysis of HCV infected patients with autoimmune hepatitis (task 2a.) will be initiated in approx. 6 months and should be completed after another 12 months. We were able to find one HLA A*0201 positive patient with autoimmune hepatitis type 2 among the patients seen at the outpatient clinic of the Institute for Clinical Pharmacology. We found no crossreactive CTL however, possibly due the fact that he had undergone immunosuppressive treatment.
The analysis of further self-protein sequences involved in a possible molecular mimicry will be initiated in 12 months and should be completed after another 12 months (task 2a.). The molecular mimicry discovered between HCV core and cytochrome p450 is the only one we found so far.
The effect of HLA class I subtypes on the antiviral response focusing on the role of HLA-A2 subtypes in the HCV core-specific CTL response (task 2b.) will be initiated in approx. 6 months and will be completed in 12 months. These studies have been delayed to perform the additional studies under 4.
Interference of viral products with the HLA class I-mediated induction or effector phase of the antiviral immune response (task 2c.) will be initiated within the next month and are pursued during the whole grant period. These studies are ongoing and described above.

Datenbankreferenzen (Englisch)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
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Swiss Project-Number: 95.0138