SVIBOR - Project code: 3-01-169

MINISTRY OF SCIENCE AND TECHNOLOGY

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Project code: 3-01-169


Mechanisms of control of cytomegalovirus infection


Main researcher: JONJIĆ, STIPAN (95983)


Assistants
Type of research: basic
Duration from: 02/01/91. to 02/01/94.
Papers on project (total): 36
Papers on project quoted in Current Contents: 12
Institution name: Medicinski fakultet, Rijeka (62)
Department/Institute: Department of physiology and immunology
Address: Braće Branchetta 20
City: 51000 - Rijeka, Croatia
Communication
Phone: 385 (0)51 51-32-22
Fax: 385 (0)51 51-49-15
Summary: The experiments planed in this project are aimed to investigate the role of various components of innate and acquired immunity involved in control of acute CMV infection as well as in establishment and maintenance of CMV latency. T lymphocytes involved in CMV control belong to both, CD8+ and CD4+ T cell subset. We have previously shown that CD8+ T cells do not require the cooperation of CD4 helper T cells to function (Jonjić et al. J. Exp. Med. 169:1199 - 1212, 1989). Mice depleted of CD4 subset were capable of controlling and eliminating infectious virus from all tissues with the exception of the salivary gland, where the CD4+ T cells are essential to terminate productive infection. The most compelling evidence for the dominant protective activity of CD8+ T cells is provided by cell transfer studies where murine CMV (MCMV) infected immunodeficient mice were transferred with syngeneic lymphocytes from mice that had recovered from CMV infection. However, despite their prominent role CD8+ T cells are neither indispensable for CMV control nor they can terminate the productive infection (Jonjić et al., J. Virol. 64:5457-5464. 1990.). Namely, CD8-subset-depleted mice clear the virus by CD4-subset-dependent mechanism with clearance kinetics indistinguishable from that of normal animals. Among several possibilities how CD4 cells compensate the lack of CD8 cells the obvious one is that they facilitate the production of neutralizing antibodies. However, this possibility is recently excluded by the experiments with mice carrying deletion of the transmembrane exon of IgM heavy chain and therefore lack B cells and antibodies (Jonjić et al. J.Exp. Med. 179:1713, 1994.). B cell deficient mice depleted of CD8 subset also clear the virus, even in salivary glands, definitely confirming that the lack of antibodies could not be the explanation for CMV persistence in CD4 depleted mice. Yet, although essential for the compensatory antiviral activity in CD8 depleted host, CD4 T lymphocytes are not protective on their own. This findings strongly point to the role of cytokines in control of CMV infection. The experiments using in vivo neutralization of IFN-gamma and TNF-alpha (Lučin et al., J. Virol. 66:1977-1984, 1992., Pavić et al. J. Gen. Virol. 74:2215-2223, 1993.) provided evidence for the role of this cytokine in T-cell mediated virus clearance. In vivo depletion of IFN-gamma abolished the antiviral activity of CD4 T cells in immunocompetent as well as in CD8 subset-depleted mice. The results also imply a role of IFN-gamma in the CD8-subset mediated control of CMV infection. The function of IFN-gamma, in concert with TNF-alpha, is to directly regulate the replication of CMV in cytokine conditioned cells. In addition to direct antiviral activity the role of cytokines may be to counteract the evasion potential of CMV. It is well established that early viral gene products of MCMV block the presentation of immediate early antigens to CTL. Also the reduction of cell-surface MHC class I was observed by MCMV early gene products. However, despite the lack of presentation in vitro and down-regulation of cell surface class I molecules, CD8+ T cells specific for CMV antigens are generated and have protective role in vivo. It appears that cytokines released by cells from infected tissue and by the cells of the immune system counteract the evasion potential of CMV (Hengel et al., J. Virol. 68:289-297, 1993. Also, the preincubation of cells with IFN-gamma prevents the MCMV induced effect on MHC class I expression and rescue the antigen presentation. To asses the compensatory capacity of CD4+ lymphocytes we also used b2-microglobulin-deficient mice, which fail to express MHC class I molecules and lack peripheral CD8+ T lymphocytes. We show that virus organ titers and virus clearance kinetics are not altered in b2-microglobulin mutant mice. There was no indication for the diminished virus propagation. These mice suffered form the lack of CD8+ T lymphocytes that was only partially compensated by CD4+ T lymphocyte function. An organ specific function of NK cells was observed and was not affected by the mutation. The unique immune control of salivary gland infection was maintained in MHC class I deficient mice. Altogether the data confirm the role of MHC class I molecules in immune surveillance but question their biological impact regarding virus cell interaction and virus productivity (Polić et al., J. Gen. Virol., in press).

Keywords: T lymphocytes, cytomegalovirus, cytokines, antiviral antibodies, IFN-gamma, TNA-alpha

Research goals: In an immunocompetent host CMV usually does not cause any clinical symptoms, but rather evades into a state of latency that can reactivate into a productive infection whenever the immune control is compromised. The experiments were aimed to study the immune mechanisms involved in control of cytomegalovirus infection. The infection of mice with murine CMV has been used as a suitable animal model for human CMV infection. The relative importance of the different immune effector mechanisms that are generated during the primary infection was tested. The immune mechanisms operative in the salivary gland, a privileged site for CMV replication and a place of productive virus replication even in immunocompetent host, was of particular importance. Here we have characterized the protective mechanisms by CD4 subset with special emphasis on the role of cytokines, IFN-gamma and TNF-alpha. Following mechanisms were proposed to explain how CD4 cells compensate the lack of CD8 cells: 1. Enhancement the specific antibody response, 2. Release of cytokines that have direct and indirect antiviral activity, 3. Direct effect on viral replication by generating MHC class II-restricted lysis of infected cells, and 4. Enhancement of antiviral activity of NK cells. Using the model of B cell deficient mice we asked the question whether antiviral antibodies are essential for resolution of acute infection as well as for the establishment and maintenance of latency.

COOPERATION - PROJECTS


  1. Name of project: SFB 322/B6 Praesentation viraler Proteinantigene durch MHC-Molekuele: Prozessierung und Peptid-Rezeptor-Interaktion (principal investigator Prof. Dr. U. Koszinowski)
    Name of institution: Institut fuer Mikrobiologie, Abteilung Virologie, Universitaet Ulm
    City: 89069 - Ulm, Deutschland

  2. Name of project: DFG Ko 571/8-5 Regulation der Virusvermehrung bei latenter und rekurrierender muriner Cytomegalovirus-Infektion (principal investigator Prof. Dr. U. Koszinowski)
    Name of institution: Institut fuer Mikrobiologie, Abteilung Virologie, Universitaet Ulm
    City: 89069 - Ulm, Deutschland

  3. Name of project: DFG Ko 571/11-2 Cytomegalovirus-Tropismus fuer die Speicheldruese und immunilogische Kontrolle (principal investigator Prof. Dr. U. Koszinowski)
    Name of institution: Institut fuer Mikrobiologie und Immunologie, Abteilung virologie, Universitaet Ulm
    City: 89069 - Ulm, Deutschland

  4. Name of project: SFB, projekt C8 Selective Modulations des Vesikularen Transporters durch Cytomegalovirus (principal investigator Prof. Dr. U. Koszinowski)
    Name of institution: Abteilung Virologie, Universtaet Heidelberg
    City: 69120 - Heidelberg, Deutschland

COOPERATION - INSTITUTIONS


  1. Name of institution: Institut fuer Mikrobiologie und Immunologie, Abteilung Virologie, Universitaet Ulm
    Type of institution: University/Faculty
    Type of cooperation: Joint project
    City: 89069 - Ulm, Deutschland

  2. Name of institution: Zentrum fuer Hygiene, Institut fuer Medizinische Virologie, Universitaet Heidelberg
    Type of institution: University/Faculty
    Type of cooperation: Joint project
    City: 69120 - Heidelberg, Deutschland
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