key: cord-022254-8y5sq72c authors: Nathanson, Neal; Gonzalez-Scarano, Francisco title: IMMUNOSUPPRESSION AND VIRUS INFECTION OF RODENTS date: 2012-12-02 journal: Viral and Mycoplasmal of Laboratory Rodents DOI: 10.1016/b978-0-12-095785-9.50036-6 sha: doc_id: 22254 cord_uid: 8y5sq72c nan Immunosuppression is a standard part of the technical armamentarium used in experimental immunobiology and viral pathogenesis. Over the past 20 years, an increasing number of methods have been developed to produce selective suppression of different elements of the host defensive response. Macrophages, B cells, T cells, and their subsets may be deleted; alternatively their effector molecules, such as immunoglobulins or interferons, may be inactivated. One potential consequence of the use of immunosuppression is the activation or potentiation of latent or intercurrent viral infections which may complicate and invalidate experimental studies. On the other hand, the deliberate use W. Ñ. holds a Javits award (NS 20904) and F. G-S. holds a TIDA (NS 00717) and a Harry Weaver Neuroscience Fellowship from the National Multiple Sclerosis Society. of immunosuppression complemented by immunoreplacement has enhanced our understanding of the role of various host defenses in acute and persistent viral infection. These two themes are the subject of this brief overview. More detailed reviews are listed in the bibliography (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) . Unwanted complications of immunosuppression are rarely published, so that the literature represents only the tip of the iceberg. A representative example was provided by an experience in our own laboratory (17) (18) (19) . An investigator at the Johns Hopkins University was developing treatment regimes for the maintenance of longterm immunological tolerance. While attempting to suppress the response of adult rats to sheep erythrocytes with cyclophosphamide, an alkylating agent which has a broad immunosuppressive activity, he noted that a small proportion of the animals developed hindlimb paralysis. Pathologic examination disclosed an unusual hemorrhagic lesion of the spinal cord. When homogenates of affected tissues were injected into suckling rats, a lethal disease was transmitted, with a more severe form of the same hemorrhagic encephalopathy. A parvovirus was isolated and identified as the causal agent; the isolate was called the HER (hemorrhagic encephalopathy of rats) strain of rat virus, a previously described parvovirus of rodents. The HER strain failed to cause disease when adult rats were infected intracerebrally. However, when potentiated by cyclophosphamide-induced immunosuppression, the infection progressed to paralysis in about 10% of the animals. It was never determined whether the original episode represented activation of a persistent latent infection or potentiation of an acute infection which happened to occur concomitantly with the administration of cyclophosphamide. Many Although, from the earliest days of animal virology it had been recognized that immunity protected against reinfection, it has only been 15 years since it was first shown that the immune response also plays a key role in recovery from primary viral infection. The initial experiments utilized methods, such as x-irradiation, cyclophosphamide, or anti-thymocyte serum, which caused temporary abrogation of both humoral and cell-mediated immunity (3-6). The hallmarks of potentiated infection were: 1) no alteration in virus replication during the first week or so (suppression-insensitive phase of infection); 2) a failure to clear virus during the second week (antigen-sensitive phase of infection); 3) replication to higher tissue titers than in immunocompetent animals; 4) enhancement of virusinduced pathology and increased mortality; 5) increased tissue titers of interferon. All of these observations (20-23, 30-35) supported the old concept that there was a race between the immune response and virus replication; anything which retarded immune induction would favor the virus. Furthermore, immunosuppression had no direct effect on the rate of viral replication but only on immune defenses. The major limitation of the early studies of immunosuppression was the lack of specificity of the methods employed (3-6, 36-38). Two complementary approaches have been taken to refine the experimental dissection of various components of the immune response: specific types of suppression (deletion techniques) and reconstitution with specific immune components (addition techniques). Each approach has its advantages and limitations. Deletion methods may be incomplete or only partially specific; addition methods may fail to mimic the physiologic response of intact animals and constitute therapy rather than reconstitution. A. Specific Immunodeletion T cell deletion may be accomplished with neonatal thymectomy; anti-thymocyte serum; adult thymectomy, x-irradiation, and bone marrow reconstitution (ATxBM); or through the use of homozygous nude (nu/nu) mice. However, such animals lack helper T cells and therefore cannot mount a normal B cell response. Furthermore, it is clear that different effector lymphocytes, such as cytotoxic T lymphocytes (39), NK cells (40), and ADCC-mediating K cells (41), can all play roles in anti-viral defense. This underlines the importance of specific deletion methods. The definition of T cell subjects and markers for them should make it possible to delete effector subsets of T cells, such as cytolytic T cells, with consequently improved specificity (42). One example of specific deletion is the use of anti-mu antiserum to delete IgM bearing B cells from neonatal animals, thus blocking the B cell arm of the immune response (43-46). When applied to a model of experimental rabies in the mouse, there was a failure to generate circulating antibody, with definite potentiation of infection; T cell populations and responses were intact (43). Administration of anti-interferon antiserum is another method · of. considerable specificity. There is a large literature (1, 2, 8, 9) documenting the efficacy of interferon as therapy in ongoing acute experimental virus infection in normal animals. However, it was never clear from such therapeutic trials whether interferon played a vital role in the recovery from infection in infected but otherwise unmanipulated animals. The application of anti-interferon antiserum has now provided convincing evidence that, at least in some experimental infections, neutralization of circulating interferon potentiates the virus (47-53). One action of interferon is to increase the activity of natural killer (NK) lymphocytes, which in turn can provide a nonspecific antiviral defense by attacking virus-infected targets (40, 54-57). Antiserum against asíalo GM1, a neutral glycosphingolipid present on the surface of NK cells, can specifically deplete this lymphocyte population. Treatment with anti-asialo GM1 antiserum has been shown to potentiate viral infection (58,59). Complement plays an important ancillary role as a host defense, since in conjunction with specific antiviral antibody, it can lyse either virions or virus-infected cells (60). Decomplementation with cobra venom transiently lowers complement levels and partially potentiates viral infection (43,61). Other methods for specific deletion include reduction in macrophages with silica, anti-macrophage antiserum, or other treatments (62) (63) (64) ; or the use of cyclosporin which appears to retard T cell activation (65) (66) (67) (68) . Each of these methods has certain drawbacks such as partial efficacy, short duration, unwanted side effects, or inadequate specificity. Originally, reconstitution was accomplished with relatively primitive additions, such as polyclonal antiserum or unfractionated spleen, lymph node, or thymus cells. Those early experiments bolstered the conclusions from the suppression experiments and added conviction to hypotheses regarding the role of both B and T cells in the host defense against infectious agents (3-5, 69-74). The advent of monoclonal antibodies (11,75) has made it possible to refine this approach and examine the role of antibodies directed against individual virus proteins. From these more recent experiments (76-85) have come a number of significant conclusions: 1) antibodies against individual epitopes on viral proteins confer surprisingly effective protection against infection, in comparison to polyclonal antisera. This finding alone has major implications for the future development of vaccines and suggests that it may be realistic to develop oligopeptide vaccines directed against immunodominant epitopes. 2) As expected, there is a general correlation between neutralizing activity determined in cell culture assays and the protective efficacy of monoclonal antibodies. However, in some systems selected non-neutralizing monoclonal antibodies are also protective (86) . This may relate to the expression on virus-infected cells of viral epitopes which are not exposed on mature virions. 3) Monoclonal antibodies also permit the examination of the effect of other variables (87) , such as the immunoglobulin isotope, on protection. The recent development of methods for the cloning of T cells and the culture of T cell lines (88) (89) (90) (91) (92) , has made it possible to study the effect of specific T cell subsets upon virus infection (93) (94) (95) (96) . Again, although the data are still limited, it is clear that certain effector subsets, such as cytolytic T cells, are capable of clearing virus or (in the case of virus-induced immunopathology) of mediating disease. It is now well established that certain viral diseases are mediated, not by a direct cytolytic effect of the agent, but by the immune response to the viral antigens. Among such diseases, lymphocytic choriomeningitis (LCM) of mice is the classical prototype, while acute hepatitis B is the most familiar human example (97). The salient aspects of such syndromes are that: 1) immunosuppression prevents disease but does not reduce virus tissue titers; 2) the virus is capable of replication and release from host cells without cytolysis (there are exceptions to this feature); 3) under certain conditions the virus can persist in infected cells or animals, often with little cellular or tissue destruction. The essential features of LCM are worth recapitulating, since they illustrate the role of experimental suppression and immune reconstitution in documenting the ability of a virus to initiate lesions indirectly (12-15, 98-101). In adult mice, LCM virus, injected by the intracerebral route, causes an acute choriomeningitis with convulsions, resulting in death about seven days after infection. Nonspecific immunosuppressive treatments, such as x-irradiation or cyclophosphamide, will prevent this disease and also prevent the underlying lesions. Nude mice and ATxBM mice are likewise protected, while "B less" mice treated with anti-mu antiserum are susceptible. Virus titers in suppressed and protected animals are high and, in fact, these mice become longterm virus carriers. Reconstitution experiments (98, 99, 102) show that antiviral antiserum will not reconstitute disease, while immune T cells (102), or cytolytic T cell clones (94,95), will induce acute disease when transferred into syngeneic recipients. Studies of the arenaviruses (of which LCM is the prototype) have extended these classical studies in several directions (15). Arenaviruses, in addition to causing inflammatory lesions, can also cause acute tissue destruction of specific organs, such as the cerebellum or liver (103, 105). Persistent viremia is usually accompanied by continuous synthesis of antiviral antibodies and the resulting immune complexes, over a period of months to years, produce chronic and eventually fatal glomerulonephritis (106, 107) . Persistently infected animals can be cleared of their virus by adoptive immunization with virus-specific immune T cells (108) , and this can be accomplished by cloned (110); the post-infectious demyelination caused by canine distemper virus (CDV); rabies early death syndrome (111) (112) (113) ; dengue hemorrhagic fever (101); respiratory syncytial virus pneumonia; possibly some of the hemorrhagic fever-shock syndromes caused by arenaviruses (Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever); and possibly hemorrhagic fever renal syndrome (HFRS) caused by hantaviruses, such as Korean hemorrhagic fever virus (114) . One important concept that has emerged from the experimental studies described above is the dual role of the immune response. The immune response can both act as a host defense and cause disease, and the mechanisms involved are identical. Thus, the attack on virus-infected target cells, by either antibody and complement or by cytolytic T cells, simultaneously destroys host tissue and removes the sites of viral replication. Likewise, the binding of antibody to free infectious virions, both neutralizes and forms immune complexes which potentially can cause vasculitis and nephritis. The kinetics of virus replication and immune induction may together determine which of these effects predominates. Thus, if the immune response is sufficiently brisk, the number of infected cells available as targets is limited and the destruction of host tissue is minimal. Conversely, if many cells are infected, their removal by immune attack can produce severe or lethal lesions. These concepts have been amply documented in studies of LCMV infection of mice. For instance, it is possible to immunize against LCMV infection and thereby protect against an immunopathology (6,115,116 ) . However, if mice are inadequtely immunized, instead of protection there occurs an accelerated form of the immunopathology. Likewise, incomplete post-exposure (post-infection) immunization in experimental rabies can cause "early death" in some animals, which die before any of their infected but unimmunized counterparts (111) (112) (113) . The dual role of the immune response is not merely an immunological curiosity, since it is seen very clearly in human hepatitis B (97). Here, some infected subjects develop a brisk immune response and clear their infection rapidly, with minimal or no clinical disease; others fail to develop an adequate response against the HBs antigen, become persistent virus carriers, but are clinically healthy; only those persons who have extensive infection prior to an immune response to HBs antigen, develop severe hepatitis in the process of clearing the infection. The proposal that virus infection could initiate an auto-immune response has been suggested repeatedly in the past. Only recently, however, have experimental data evolved to support this hypothesis (16). The advent of B cell hybridoma technology has made it possible to determine whether specific antibodies to viral proteins might also recognize epitopes on self proteins (sometimes called molecular mimicry). It now appears that, if a sufficient number of antibodies against any virus are examined, a small but finite number will also react with epitopes present in one or multiple organs (117) (118) (119) (120) (121) (122) (123) (124) . Furthermore, infections of mice with certain viruses, such as reovirus, result in an autoimmune disease which can be prevented by immunosuppression (118, 125) . Results of this type make it plausible that an acute or persistent virus infection could give rise to an autoimmune process. Recently, a possible example of virus-initiated autoimmunity has been reported in a murine model of virusinduced demyelination (126) . Rats infected with mouse hepatitis virus (MHV) developed demyelinating lesions about one month later. Some of these animals had spinal cord lesions resembling experimental allergic encephalitis (EAE). When spleen or lymph node cells from such rats were cultured in vitro with myelin basic protein (BP), they proliferated as measured by thymidine incorporation. These antigendriven cells, when transferred into a syngeneic (but not an allogeneic) recipient, induced EAE, in about one week. Several controls indicated that MHV was not being transferred with the cells, and the one week interval was too short for virus-induced demyelination. An inescapable conclusion is that every attempt must be made to utilize animals obtained from sources which monitor for viruses and which are relatively virus-free· Perhaps a greater problem is the need to isolate such animals from enzootic agents in the experimenters 1 own facility. Our recent experience in a university research facility is that these are very real problems. A barrier facility is probably necessary in most institutions to protect newly introduced animals from rapidly acquiring infections. In addition, minibarriers provided by the use of isolators and filter top cages are important adjuncts. Also, the institution must monitor constantly to determine whether enzootic agents can be excluded from clean rodents imported into the facility. Finally, it is likely that certain institutions fail to meet these criteria; such institutions are probably inappropriate settings for many immunological and viral studies conducted in animals. Adverse effects of interferon in virus infection, autoimmune diseases and acquired immunodeficiency Selective effects of antimacrophage serum, silica, and antilymphocyte serum on pathogenesis of herpes virus infection of young adult mice Modifications by sodium aurothio-malate of the expression of virulence by defined strains of Semliki Forest virus Use of silica to identify host mechanisms involved in suppression of established Friend virus leukemia Beneficial effect of cyclosporin A on the lymphocytic choriomengitis virus infection in mice Modulation by cyclosporin A of murine natural resistance against herpes simplex virus infection. I. Interference with the susceptibility to herpes simplex virus infection Cyclosporin A-usefulness, risks, and mechanism of action Cyclophilin: a specific cytosolic binding protein for cyclosphorin A Suppressor T cells for delayed-type hypersensitivity to Japanese encephalitis virus Recovery from lethal herpes simplex virus type 1 infection is mediated by cytotoxic T lymphocytes. Infect, and Immun Mechanism of recovery from systemic herpes simplex virus infection. I. Comparative effectiveness of antibody and reconstitution of immune spleen cells on immunosuppressed mice An adoptive cell transfer system for the evaluation of immunity to herpes simplex virus in mice E2 glycoprotein of semliki Forest virus can protect mice from lethal encephalitis Protection against lethal challenge of BALB/c mice by passive transfer of monoclonal antibodies to five glycoproteins of herpes simplex virus type 2 Use of monoclonal antibodies for analysis of antibodydependent immunity to ocular herpes simplex virus type 1 Non-neutralizing monoclonal antibodies can prevent lethal alpha virus encephalitis Inductin of subacute murine measles encephalitis by monoclonal antibody to virus haemagglutinin Cloning of functional T lymphocytes l!n_: Concepts in viral pathogenesis Specificity of in vitro cytotoxic clones directed againt vesicular stomatitis virus Biology of clone cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. I. Generation and recognition of virus strains and H-2b mutants Characterization of the murine Th response to influenza virus hemagglutinin: evidence for three major specificities Production and characterization of T cell clones specific for mouse hepatitis virus stain JHM: in vivo and in vitro analysis Protection of mice from fatal herpes simplex virus type 1 infection by adoptive transfer of cloned virusspecific and H-2-restricted cytotoxic T lymphocytes neonatal rats: protective effect of immunosuppression with anti-lymphoid serum The effect of neonatal thymectomy on Tamiami, virus-induced central nervous system disease Virus-induced immune complex formation and disease: definition, regulation Effect of immunosuppression on chronic LCM virus Virus elimination in acute lymphocytic chorimeningitis virus infection. Correlation with virus-specific delayed-type hypersensitivity rather than cytotoxicity Virus-induced demyelination In: Concepts in viral pathogenesis Pathogenesis of borna disease in rats: Immune-mediated viral ophthalmoencephalopathy causing blindness and behavioral abnormalities Acute rabies death mediated by antibody Immunopathologic aspects of infection with Lagos bat virus of the rabies serogroup Dual role of the immune response in street rabies virus infection of mice Hemorrhagic fever viruses The timing of the immune response in relation to virus growth determines the outcome of the LCM infection Immune responses to LCM virus infection ±η_ vivo and in vitro. Mechanisms of immune-mediated disease Infection with vaccinia favors the selection of hybridomas synthesizing autoantibodies against intermediate filaments, one of them cross-reacting with virus hemagglutinin Virus-induced diabetes mellitus: autoimmunity and polyendocrine disease prevented by immunosuppression Autoimmunity induced by syngeneic membranes carrying irreversibly adsorbed paramyxovirus. Infect, and Immun Molecular mimicry in virus infection: cross reaction of measles virus phosphoprotein or of herpes simplex virus protein with human intermediate filaments Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen Virus-induced autoimmunity: monoclonal antibodies that react with endocrine tissues Three monoclonal antibodies against measles virus F protein cross-react with cellular stress proteins Virus-induced autoimmunity: Monoclonal antibodies that react with endocrine tissues Virus-induced diabetes mellitus. XX. Polyendocrinopathy and autoimmunity Adoptive transfer of EAE-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis