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Applied Viral Immunology
Symposium on Practical Immunology
Applied Viral Immunology David E. Kahn, D.V.M., Ph.D.*
Viruses interact with both the humoral and cell-mediated components of the immune system. Although it appears that protection against several viral pathogens is afforded primarily by one component or the other (humoral immunity in yellow fever; cell-mediated immunity in herpesvirus infection), the available data suggest that immunologic control of most viral diseases is dependent upon elements of both working in concert. Viral immunoprophylaxis also is affected by the way the pathogen interacts with the host animal (see article on Immunodeficiency Diseases by Cockerell in this symposium). The better-elucidated antiviral immune mechanisms and patterns of virushost interactions will be reviewed. This information will be applied to the immunoprophylactic control of viral diseases of the dog and cat.
HUMORAL ANTIVIRAL IMMUNITY ANTIVIRAL IMMUNOGLOBULINS
The appearance of humoral antibody during convalescence from viral infection was one of the first manifestations of antiviral immunity to be recognized. Three classes of immunoglobulins (IgM, IgG, and IgA) possess antiviral activity. The concentrations of IgM and IgG are greater in plasma than in secretory fluids, while the reverse is true for IgA. 3 • 9 This differential distribution of immunoglobulins suggests that each type is to some extent specialized. 13 Immunoglobulins M and G, considered to be systemic humoral immune factors, respond to parenterally administered antigens, while IgA, considered to be a local humoral immune factor, responds to antigens that contact the mucosa directly. 13 *Head, Department of Virological Research, Pitman-Moore, Inc., Washington Crossing, New Jersey Veterinary Clinics
of North America- Vol. 8,
No.4, November 1978
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Immunoglobulin M, G, and A Specific antibody of the lgM class usually is the first type to be detected in serum after primary exposure of an animal to a virus. Because of its large molecular size, lgM cannot diffuse through endothelial linings. The small quantities found extravascularly are produced locally. Serum IgM concentrations are maximal within several days after the initiation of a viral infection and decrease rapidly in the weeks that follow. After primary viral exposure, the serum concentration of IgG increases more slowly than that of lgM, but persists for a much longer period of time. The molecular size of IgG is small enough to permit its diffusion across capillary membranes; thus, this antibody is found both intravascularly and in the extravascular tissue fluids. The diffusion process accelerates when capillary permeability increases, as during acute inflammation. Re-exposure of the convalescent animal to the same virus stimulates an anamnestic IgG antibody response. A third immunoglobulin class, IgA, occurs in great quantities in lymphoid tissues associated with the digestive, respiratory, and urogenital tracts. 9• 13 Immunoglobulin A is the principal antibody class found in secretions and generally is thought to play a crucial role in protecting mucosal linings of the body from microbial invasion. Studies of several different viral infections indicate that protection from respiratory disease is more highly correlated with the quantity of antibody (principally IgA) present in respiratory tract secretions than with serum antibody titers. 2• 3 Immunoglobulin A is the third most abundant class of immunoglobulin in canine serum. The IgA molecules in secretions are complexed with a protein, the secretory component. The complexed lgA is less susceptible to enzymatic proteolysis than uncomplexed IgA molecules are. Antiviral antibodies of immunoglobulin classes M, G, and A are produced by plasma cells.
MECHANISMS OF HUMORAL ANTIVIRAL IMMUNITY
Viral Neutralization The three immunoglobulins identified in the preceding section share the biological property of specifically neutralizing the infectivity of extracellular viral particles. Maternally derived antibody, obtained primarily by neonatal ingestion of colostrum, may persist in puppies and kittens until they are three to four months old. Maternal antibody affords protection against virulent field strains of viruses, but it also may interfere with vaccination. The phenomenon of specific viral neutralization can be demonstrated in the virology laboratory, and this technique is used common-
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ly for the sero-diagnosis of viral infection (see article on Immunologic Methods by Schultz and Adams in this symposium). Both IgM and IgG also have the property of being able to fix complement. The presence of complement on the surface of virusantibody complexes causes their adherence to platelets and erythrocytes. The resultant aggregates are removed by the reticuloendothelial system more efficiently than are individual virions coated in the antibody, or small complexes of virus particles and antibody molecules. 2 IgM is more efficient than IgG with respect to complement fixation.
Antibody Complement-Mediated Cytotoxicity In addition to neutralization and immune clearance of extracellular virions from blood, extravascular body fluids, and secretions, antibody can play a role in the destruction of virus-infected cells. 2 This occurs when viral antigens are incorporated in the cytoplasmic membrane of cells during viral replication. Antiviral antibody combines with these antigens, fixes complement, and results in cytolysis. Death of the infected host cell aborts the production of new virions.
Antibody-Dependent Cell-Mediated Cytotoxicity Antiviral antibody also interacts with leukocytes to abort viral infection. Even minute quantities of antibody adsorbed to the surface of infected cells will result in their destruction by lymphocytes, polymorphonuclear leukocytes, and macrophages. It has been suggested that this mechanism may be especially important during the early recovery from viral infections when antibody levels are low. 2
CELL-MEDIATED ANTIVIRAL IMMUNITY After it was observed that individuals suffering from immunoglobulin deficiencies were not defenseless against viral infections, investigators began the search for additional antiviral defense mechanisms. Much of the current research effort in viral immunology is concerned with elucidation of cell-mediated immune mechanisms.1· 2 • 7 • 13 The role of these mechanisms is better documented as responsible for recovery from herpesvirus infections than it is for infections by other viral pathogens. Herpesvirus infections of domestic animals include a neonatal disease of the dog, equine rhinopneumonitis, feline viral rhinotracheitis, and infectious bovine rhinotracheitis. Cell-mediated antiviral immunity requires that specifically sensitized T lymphocytes recognize viral infected target cells and destroy them. The mechanism of this destruction is poorly understood, as is the recognition of antigens (self and viral) on the target cell. The cells involved in cell-mediated immunity are distinct from those concerned with humoral immunity (antibody synthesis).
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EFFECTOR MOLECULES OF CELL-MEDIATED IMMUNITY While in the humoral immune response antigenic stimulation of a sensitized lymphocyte results in proliferation of plasma cells and antibody production, in the cell-mediated immune response contact with the antigen forms sensitized lymphocytes that release a number of biologically active effector molecules called lymphokines.
Interferon For many years interferon has been recognized as important in the defense of a nonimmune individual against viral disease. Recently it has been demonstrated that interferon also is an effector molecule released by sensitized (immune) leukocytes when they are stimulated by re-exposure to viral antigens. Interferon is a nonimmunoglobulin protein that can be produced by a number of different cell types. It can be released by interaction between these cells and any of a rather broad group of both natural and synthetic substances, including viruses, bacteria, nucleic acids, and polynucleotides. Interferon is not antigen-specific and does not neutralize viral infectivity. Instead, it inhibits viral replication in host cells and thereby aborts the infection. Both nonsensitized and sensitized leukocytes can release interferon. Comparative studies conducted in vitro indicate that greater quantities of interferon are released after exposure of the sensitized leukocytes to virus. It is assumed that the same thing occurs in the virus-infected host animal.
Chemotactic Factors When lymphocytes collected from immune animals are stimulated in vitro with virus on other antigens, they release a group of chemical substances (chemotactic factors) that attract polymorphonculear leukocytes, macrophages, and lymphocytes. At present the exact chemical and physical characteristics of all the chemotactic factors are poorly defined, but some have been reported. It has been determined that reaction of a sensitized lymphocyte with antigen induces macrophages to destroy intracellular parasites, including viruses. The effector molecule responsible, termed the macrophage arming factor, is also considered to be a lymphokine.
Migration Inhibitory Factor One of the best studied lymphokines is designated the migration inhibitory factor. In laboratory studies release of migration inhibitory factor from sensitized leukocytes has been demonstrated to prevent the migration of macrophages, monocytes or polymorphonuclear neutrophils. Presumably this factor is important in preventing macrophages from leaving sites of viral infection.
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Lymphotoxins Activated lymphocytes are known to secrete substances that are toxic to virus-infected cells. Lymphotoxins are presumed to be important in ridding virus-infected cells from the immune host.
Lymphoblast Transformation Factor This effector molecule is released by sensitized lymphocytes and results in the recruitment and proliferation of previously uncommitted lymphocytes. In this way exposure of a few immune lymphocytes to viral antigen can trigger the rapid formation of many newly sensitized lymphocytes to ward off a viral infection.
Antigen-Specific Transfer Factor An effector substance of very small molecular weight has been extracted from immune human leukocytes. Its addition to populations of nonimmune lymphocytes causes these cells to participate in various cell-mediated immune responses, such as cutaneous delayed type hypersensitivity reactions and production of migration inhibitory factor, when they are exposed to the same antigen used to immunize the donor leukocytes. This transfer factor is considered to be a critical mediator of antigen-specific cell-mediated immunity. There is hope that methods will be developed for its use as an antiviral immunotherapeutic drug, however much work remains to be done to characterize and identify the importance of this factor in the dog and cat.
MECHANISMS OF ANTIVIRAL CELL-MEDIATED IMMUNITY Evidence that cell-mediated immune responses are important in the defense of animals from viral diseases is largely circumstantial. In most cases the biologically active effector molecules have been studied in tissue culture systems, and it has been hypothesized that similar mechanisms are operational in the intact animal. Primary exposure of an animal to a virus sensitizes a group of lymphocytes to the viral antigens. Re-exposure is believed to activate these sensitized lymphocytes to release the various effector molecules identified in the preceding section. In this way viral replication is interfered with, new lymphocytes are recruited, inflammatory cells attracted and confined to the site of virus infection, and infected cells are destroyed.
PATTERNS OF VIRUS-HOST INTERACTION VIRUS-CELL INTERACTION
Viruses can interact with their target cells in the host animal in several different ways. Each has a different outcome for the infected
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cell. These have been designated as the cytolytic, steady-state, and integrated patterns of virus-cell interaction. 2 Cytolytic Infection
In viral infections that run a relatively short clinical course the host cell becomes infected, produces new virus particles, and is destroyed at the time these progeny virions are released. Cell lysis usually occurs a relatively short time after infection. The short replication cycle and the release of virus particles to the extracellular environment make the cytolytic viruses more susceptible to control by humoral than cell-mediated immune mechanisms. Feline panleukopenia and infectious canine hepatitis are two examples of cytolytic infections. Steady-State Infection
Less commonly, the infected cell remains viable and may continue to produce and release progeny virions for long periods of time. This type of infection has a more protracted course than the cytolytic pattern. Immunological control of steady-state viral infections involves neutralization of virus infectivity, antibody-mediated destruction of cells whose cytoplasmic membranes have been modified by the incorporation of viral antigens, and the destruction of infected cells by cellmediated mechanisms. Feline viral rhinotracheitis and canine distemper viruses have the capability of inducing steady-state infections. Integrated Infection
In the last type of virus-cell interaction to be considered, a portion or all of the viral genome, or a DNA copy of an RNA genome, is integrated into that of the host cell. Even when such cells are not replicating virus they are recognizable by the presence of virusspecified transformation antigens on their surface membranes. It is believed that cell-mediated immune mechanisms function most efficiently to eliminate these transformed cells, protecting the body from neoplasia. Feline leukemia virus has the capacity to become an integrated infection and to induce cell transformation.
VIRUS-HOST INTERACTION
The susceptibility of a given viral pathogen to humoral or cellmediated immune mechanisms is dependent upon the pattern of infection it initiates in the host animal, as well as by the way in which the virus interacts with its target cells. If, for example, a virus is restricted in its replication to the skin or mucosal surfaces, it will be more subject to local than to systemic immune mechanisms. On the other hand, when the pathogen initiates a generalized infection by spreading via the hemic-lymphatic system from primary replication site to
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secondary target cells in distant organs of the body, it is very sensitive to systemic immune mechanisms. Highly cell-associated viruses are more readily controlled by cell-mediated than by humoral mechanIsms.
IMMUNOPROPHYLAXIS Approximately one-half of the small animal practitioner's professional activities are concerned with vaccination of animals to prevent infectious diseases. The list of biological products available for use in dogs and cats has increased rapidly in recent years (Table 1). A number of technological changes have been adopted by the biologicals industry to produce vaccines more uniform in potency and less likely to induce untoward reactions than their predecessors. Of current interest to veterinarians is whether inactivated vaccines are as effective as their counterparts that contain modified live viruses. Another topic of debate is whether topical administration of vaccines to the mucosa results in better protection against locally invasive viral pathogens than parenteral vaccination.
Table 1. Canine and Feline Viral Vaccines Licensed by United States Department of Agriculture and Plant Health Inspection Service TYPE
VACCINE
Canine Distemper Vaccine Canine Distemper-Measles Vaccine Canine Hepatitis Vaccine Canine Parainfluenza Vaccine Canine Distemper-Hepatitis-Parainfluenza Vaccine (available in combination with leptospira bacterin) Feline Panleukopenia Vaccine Feline Panleukopenia Vaccine Feline Pneumonitis Vaccine Feline Rhinotracheitis Vaccine Feline Rhinotracheitis-Calicivirus Vaccine Feline RhinotracheitisCalicivirus-Panleukopenia Vaccine Feline RhinotracheitisCalicivirus-Panleukopenia Vaccine Rabies Rabies Rabies Rabies
Vaccine-SAD Stain Vaccine, Flury Stain-Low Egg Passage Vaccine, Goat Brain Origin Vaccine, Mouse Brain Origin
Modified Modified Modified Modified
Live Live Live Live
Virus Virus Virus Virus
Vaccine Vaccine Vaccine Vaccine
Modified Live Virus Vaccine Inactivated Virus Vaccine Modified Live Virus Vaccine Modified Live Chlamydia Vaccine Modified Live Virus Vaccine Modified Live Virus Vaccine Modified Live Virus Vaccine Modified Live and Inactivated Virus Vaccine Modified Live Virus Vaccine Modified Live Virus Vaccine Inactivated Virus Vaccine Inactivated Virus Vaccine
From Veterinary Services Biologics Notice No. 24, United States Department of Agriculture, Animal and Plant Health Inspection Service.
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INACTIVATED AND MoDIFIED LivE VIRus VACCINES
The first veterinary viral vaccines were prepared by harvesting tissues from diseased animals and inactivating the virus, usually by the addition of formalin. Later, the process was refined by the development of methods to grow viruses in tissue culture systems. Inactivated vaccines have proven efficacious only in a limited number of viral diseases. The best examples in veterinary medicine probably are rabies and feline panleukopenia. Humoral immunity appears to be important in the control of both diseases. Inactivated viral vaccines have proven less effective in the prevention of diseases controlled by cell-mediated immune factors. For example, the responses of cattle parenterally vaccinated with inactivated or modified live infectious bovine rhinotracheitis (IBR) virus preparations have been compared. Both immunogens induced similar levels of humoral antibody, but only the modified live virus vaccine stimulated cellmediated immune responses. 11 It is possible that the efficacy of inactivated viral vaccines can be improved by the inclusion of adjuvants to stimulate cell-mediated antiviral immunity. Several years ago a series of new viral vaccines were developed for the prevention of canine and feline respiratory illnesses. 4 • 5 Multivalent vaccines are available that combine canine parainfluenza vaccine with distemper, hepatitis, and leptospirosis vaccines. Similarly, multivalent feline viral rhinotracheitis-calicivirus and feline panleukopenia vaccines are distributed commercially. Both the canine and fe.., line vaccines have been demonstrated to protect against diseases for 12 months, or longer; after parenteral vaccination. The recognition of local humoral and cell-mediated immune factors as being important in the defense of the mucosal surfaces of the body has prompted study of the feasibility of inducing local immunity by the topical application of vaccine viruses. This method has been explored in the quest for effective vaccines for the prevention of influenza and the common cold. In veterinary medicine, intranasal vaccines have been marketed for use in cattle (IBR-parainfluenzavirus-3 vaccines), cats (feline viral rhinotracheitis-calicivirus vaccines), and horses (equine rhinopneumonitis vaccines). Aside from the problems of persistent shed of vaccine virus from intranasally immunized animals and a higher incidence of postvaccinal illness with certain intranasal products than has been associated with similar parenterally administered biological products, the intranasal vaccines have not proven more efficacious because of the fact that the parenteral vaccines also are capable of inducing local immunity in the respiratory tract. Just as respiratory tract infection induces systemic humoral antibody as well as secretory IgA, the parenteral administration of a viral immunogen can result in secretory IgA and local cell-mediated
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immunity, as well as induce systemic immune responses. It was reported that no quantitive differences in local antibody were observed in cattle administered IBR virus vaccines intranasally or intramuscularly.8 Similar findings have been published for parainfluenza virus-3 immunization of lambs. 12 It is now recognized that if sufficient viral antigen to cause systemic infection is administered parenterally, local immunity can be induced. 13 We have compared intranasal and parenteral routes of immunization and found similar results with regard to the degree of protection induced against feline respiratory viral pathogens. 5• 6
CONCLUSION Viruses are capable of inducing a variety of immune responses. Both humoral and cell-mediated immune factors contribute to protection of the host animal from viral diseases. It has been possible to define immune mechanisms that result in the neutralization of extracellular virus, the clearance of virus particles from plasma and extravascular fluids, and the arrest of viral replication in infected cells. Viral vaccines appear to stimulate the same immune responses induced by virulent viruses, while not producing disease. 4 • 10 At the present time, modified live viral vaccines are the more potent stimulators of antiviral cell-mediated immunity. Because of the importance of cell-mediated immune factors in the control of many viral diseases, there are many more efficacious modified live than inactivated vaccines available for viral immunoprophylaxis.
REFERENCES 1. Allison, A. C.: On the role of monouclear phagocytes in im!Il'Unity against viruses. Prog. Med. Virol., 18:15, 1974. 2. Bellanti,]. A.: Immunology. Philadelphia, W. B. Saunders Co., 1971, Chapters 11 and 18. 3. Douglas, R. G., Jr., Rossen, R. D., Butler, W. T., et a!.: Rhinovirus neutralizing antibody in tears, parotid saliva, nasal secretions and serum. J. Immunol., 99:297, 1967. 4. Emery,]. B., House,]. A., Bittle,]. L., et al.: A canine parainfluenza viral vaccine: immunogenicity and safety. Am.]. Vet. Res., 37:1323, 1976. 5. Kahn, D. E., and Hoover, E. A.: Feline caliciviral disease: experimental immunoprophylaxis. Am.]. Vet. Res., 37:279, 1976. 6. Kahn, D. E., Hoover, E. A., and Bittle, ]. L.: Induction of immunity to feline caliciviral disease. Infect. Immunol., 11:1003, 1975. 7. McFarland, H. F.: In vitro studies of cell-mediated immunity in an acute viral infection.]. Immunol., 113:173, 1974. 8. McKercher, D. G., and Crenshaw, G. L.: Comparative efficacy of intranasally and parenterally administered infectious bovine rhinotracheitis vaccines. J .A.V .M.A., 159:1362, 1971.
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9. Molinari, J. A., Ebersole, J. L., and Platt, D.: Investigation of secretory immunoglobulins in saliva from germfree mice. Infect. Immunol., 10:1207, 1974. 10. Schultz, R. D.: Failure of attenuated canine distemper virus (Rockborn Strain) to suppress lymphocyte blastogenesis in dogs. Cornell Vet., 66:27, 1976. 11. Sheffy, B. E., and Rodman, S.: Activation of latent infectious bovine rhinotracheitis infection. J.A.V.M.A., 163:850, 1973. 12. Smith, W. D., Dawson, A.M., Wells, P. W., eta!.: Immunoglobulins in the serum and nasal secretions of lambs following vaccination and aerosol challenge with parainfluenza 3 virus. Res. Vet, Sci., 21:341, 1976. 13. Waldman, R. H., and Ganguly, R.: Immunity to infections on secretory surfaces. J. Infect. Dis., 130:419, 1974. Department of Virological Research Pitman-Moore, Inc. Washington Crossing, New Jersey 08560
Applied Viral Immunology David E. Kahn, D.V.M., Ph.D.*
Viruses interact with both the humoral and cell-mediated components of the immune system. Although it appears that protection against several viral pathogens is afforded primarily by one component or the other (humoral immunity in yellow fever; cell-mediated immunity in herpesvirus infection), the available data suggest that immunologic control of most viral diseases is dependent upon elements of both working in concert. Viral immunoprophylaxis also is affected by the way the pathogen interacts with the host animal (see article on Immunodeficiency Diseases by Cockerell in this symposium). The better-elucidated antiviral immune mechanisms and patterns of virushost interactions will be reviewed. This information will be applied to the immunoprophylactic control of viral diseases of the dog and cat.
HUMORAL ANTIVIRAL IMMUNITY ANTIVIRAL IMMUNOGLOBULINS
The appearance of humoral antibody during convalescence from viral infection was one of the first manifestations of antiviral immunity to be recognized. Three classes of immunoglobulins (IgM, IgG, and IgA) possess antiviral activity. The concentrations of IgM and IgG are greater in plasma than in secretory fluids, while the reverse is true for IgA. 3 • 9 This differential distribution of immunoglobulins suggests that each type is to some extent specialized. 13 Immunoglobulins M and G, considered to be systemic humoral immune factors, respond to parenterally administered antigens, while IgA, considered to be a local humoral immune factor, responds to antigens that contact the mucosa directly. 13 *Head, Department of Virological Research, Pitman-Moore, Inc., Washington Crossing, New Jersey Veterinary Clinics
of North America- Vol. 8,
No.4, November 1978
697
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KAHN
Immunoglobulin M, G, and A Specific antibody of the lgM class usually is the first type to be detected in serum after primary exposure of an animal to a virus. Because of its large molecular size, lgM cannot diffuse through endothelial linings. The small quantities found extravascularly are produced locally. Serum IgM concentrations are maximal within several days after the initiation of a viral infection and decrease rapidly in the weeks that follow. After primary viral exposure, the serum concentration of IgG increases more slowly than that of lgM, but persists for a much longer period of time. The molecular size of IgG is small enough to permit its diffusion across capillary membranes; thus, this antibody is found both intravascularly and in the extravascular tissue fluids. The diffusion process accelerates when capillary permeability increases, as during acute inflammation. Re-exposure of the convalescent animal to the same virus stimulates an anamnestic IgG antibody response. A third immunoglobulin class, IgA, occurs in great quantities in lymphoid tissues associated with the digestive, respiratory, and urogenital tracts. 9• 13 Immunoglobulin A is the principal antibody class found in secretions and generally is thought to play a crucial role in protecting mucosal linings of the body from microbial invasion. Studies of several different viral infections indicate that protection from respiratory disease is more highly correlated with the quantity of antibody (principally IgA) present in respiratory tract secretions than with serum antibody titers. 2• 3 Immunoglobulin A is the third most abundant class of immunoglobulin in canine serum. The IgA molecules in secretions are complexed with a protein, the secretory component. The complexed lgA is less susceptible to enzymatic proteolysis than uncomplexed IgA molecules are. Antiviral antibodies of immunoglobulin classes M, G, and A are produced by plasma cells.
MECHANISMS OF HUMORAL ANTIVIRAL IMMUNITY
Viral Neutralization The three immunoglobulins identified in the preceding section share the biological property of specifically neutralizing the infectivity of extracellular viral particles. Maternally derived antibody, obtained primarily by neonatal ingestion of colostrum, may persist in puppies and kittens until they are three to four months old. Maternal antibody affords protection against virulent field strains of viruses, but it also may interfere with vaccination. The phenomenon of specific viral neutralization can be demonstrated in the virology laboratory, and this technique is used common-
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ly for the sero-diagnosis of viral infection (see article on Immunologic Methods by Schultz and Adams in this symposium). Both IgM and IgG also have the property of being able to fix complement. The presence of complement on the surface of virusantibody complexes causes their adherence to platelets and erythrocytes. The resultant aggregates are removed by the reticuloendothelial system more efficiently than are individual virions coated in the antibody, or small complexes of virus particles and antibody molecules. 2 IgM is more efficient than IgG with respect to complement fixation.
Antibody Complement-Mediated Cytotoxicity In addition to neutralization and immune clearance of extracellular virions from blood, extravascular body fluids, and secretions, antibody can play a role in the destruction of virus-infected cells. 2 This occurs when viral antigens are incorporated in the cytoplasmic membrane of cells during viral replication. Antiviral antibody combines with these antigens, fixes complement, and results in cytolysis. Death of the infected host cell aborts the production of new virions.
Antibody-Dependent Cell-Mediated Cytotoxicity Antiviral antibody also interacts with leukocytes to abort viral infection. Even minute quantities of antibody adsorbed to the surface of infected cells will result in their destruction by lymphocytes, polymorphonuclear leukocytes, and macrophages. It has been suggested that this mechanism may be especially important during the early recovery from viral infections when antibody levels are low. 2
CELL-MEDIATED ANTIVIRAL IMMUNITY After it was observed that individuals suffering from immunoglobulin deficiencies were not defenseless against viral infections, investigators began the search for additional antiviral defense mechanisms. Much of the current research effort in viral immunology is concerned with elucidation of cell-mediated immune mechanisms.1· 2 • 7 • 13 The role of these mechanisms is better documented as responsible for recovery from herpesvirus infections than it is for infections by other viral pathogens. Herpesvirus infections of domestic animals include a neonatal disease of the dog, equine rhinopneumonitis, feline viral rhinotracheitis, and infectious bovine rhinotracheitis. Cell-mediated antiviral immunity requires that specifically sensitized T lymphocytes recognize viral infected target cells and destroy them. The mechanism of this destruction is poorly understood, as is the recognition of antigens (self and viral) on the target cell. The cells involved in cell-mediated immunity are distinct from those concerned with humoral immunity (antibody synthesis).
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EFFECTOR MOLECULES OF CELL-MEDIATED IMMUNITY While in the humoral immune response antigenic stimulation of a sensitized lymphocyte results in proliferation of plasma cells and antibody production, in the cell-mediated immune response contact with the antigen forms sensitized lymphocytes that release a number of biologically active effector molecules called lymphokines.
Interferon For many years interferon has been recognized as important in the defense of a nonimmune individual against viral disease. Recently it has been demonstrated that interferon also is an effector molecule released by sensitized (immune) leukocytes when they are stimulated by re-exposure to viral antigens. Interferon is a nonimmunoglobulin protein that can be produced by a number of different cell types. It can be released by interaction between these cells and any of a rather broad group of both natural and synthetic substances, including viruses, bacteria, nucleic acids, and polynucleotides. Interferon is not antigen-specific and does not neutralize viral infectivity. Instead, it inhibits viral replication in host cells and thereby aborts the infection. Both nonsensitized and sensitized leukocytes can release interferon. Comparative studies conducted in vitro indicate that greater quantities of interferon are released after exposure of the sensitized leukocytes to virus. It is assumed that the same thing occurs in the virus-infected host animal.
Chemotactic Factors When lymphocytes collected from immune animals are stimulated in vitro with virus on other antigens, they release a group of chemical substances (chemotactic factors) that attract polymorphonculear leukocytes, macrophages, and lymphocytes. At present the exact chemical and physical characteristics of all the chemotactic factors are poorly defined, but some have been reported. It has been determined that reaction of a sensitized lymphocyte with antigen induces macrophages to destroy intracellular parasites, including viruses. The effector molecule responsible, termed the macrophage arming factor, is also considered to be a lymphokine.
Migration Inhibitory Factor One of the best studied lymphokines is designated the migration inhibitory factor. In laboratory studies release of migration inhibitory factor from sensitized leukocytes has been demonstrated to prevent the migration of macrophages, monocytes or polymorphonuclear neutrophils. Presumably this factor is important in preventing macrophages from leaving sites of viral infection.
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Lymphotoxins Activated lymphocytes are known to secrete substances that are toxic to virus-infected cells. Lymphotoxins are presumed to be important in ridding virus-infected cells from the immune host.
Lymphoblast Transformation Factor This effector molecule is released by sensitized lymphocytes and results in the recruitment and proliferation of previously uncommitted lymphocytes. In this way exposure of a few immune lymphocytes to viral antigen can trigger the rapid formation of many newly sensitized lymphocytes to ward off a viral infection.
Antigen-Specific Transfer Factor An effector substance of very small molecular weight has been extracted from immune human leukocytes. Its addition to populations of nonimmune lymphocytes causes these cells to participate in various cell-mediated immune responses, such as cutaneous delayed type hypersensitivity reactions and production of migration inhibitory factor, when they are exposed to the same antigen used to immunize the donor leukocytes. This transfer factor is considered to be a critical mediator of antigen-specific cell-mediated immunity. There is hope that methods will be developed for its use as an antiviral immunotherapeutic drug, however much work remains to be done to characterize and identify the importance of this factor in the dog and cat.
MECHANISMS OF ANTIVIRAL CELL-MEDIATED IMMUNITY Evidence that cell-mediated immune responses are important in the defense of animals from viral diseases is largely circumstantial. In most cases the biologically active effector molecules have been studied in tissue culture systems, and it has been hypothesized that similar mechanisms are operational in the intact animal. Primary exposure of an animal to a virus sensitizes a group of lymphocytes to the viral antigens. Re-exposure is believed to activate these sensitized lymphocytes to release the various effector molecules identified in the preceding section. In this way viral replication is interfered with, new lymphocytes are recruited, inflammatory cells attracted and confined to the site of virus infection, and infected cells are destroyed.
PATTERNS OF VIRUS-HOST INTERACTION VIRUS-CELL INTERACTION
Viruses can interact with their target cells in the host animal in several different ways. Each has a different outcome for the infected
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cell. These have been designated as the cytolytic, steady-state, and integrated patterns of virus-cell interaction. 2 Cytolytic Infection
In viral infections that run a relatively short clinical course the host cell becomes infected, produces new virus particles, and is destroyed at the time these progeny virions are released. Cell lysis usually occurs a relatively short time after infection. The short replication cycle and the release of virus particles to the extracellular environment make the cytolytic viruses more susceptible to control by humoral than cell-mediated immune mechanisms. Feline panleukopenia and infectious canine hepatitis are two examples of cytolytic infections. Steady-State Infection
Less commonly, the infected cell remains viable and may continue to produce and release progeny virions for long periods of time. This type of infection has a more protracted course than the cytolytic pattern. Immunological control of steady-state viral infections involves neutralization of virus infectivity, antibody-mediated destruction of cells whose cytoplasmic membranes have been modified by the incorporation of viral antigens, and the destruction of infected cells by cellmediated mechanisms. Feline viral rhinotracheitis and canine distemper viruses have the capability of inducing steady-state infections. Integrated Infection
In the last type of virus-cell interaction to be considered, a portion or all of the viral genome, or a DNA copy of an RNA genome, is integrated into that of the host cell. Even when such cells are not replicating virus they are recognizable by the presence of virusspecified transformation antigens on their surface membranes. It is believed that cell-mediated immune mechanisms function most efficiently to eliminate these transformed cells, protecting the body from neoplasia. Feline leukemia virus has the capacity to become an integrated infection and to induce cell transformation.
VIRUS-HOST INTERACTION
The susceptibility of a given viral pathogen to humoral or cellmediated immune mechanisms is dependent upon the pattern of infection it initiates in the host animal, as well as by the way in which the virus interacts with its target cells. If, for example, a virus is restricted in its replication to the skin or mucosal surfaces, it will be more subject to local than to systemic immune mechanisms. On the other hand, when the pathogen initiates a generalized infection by spreading via the hemic-lymphatic system from primary replication site to
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secondary target cells in distant organs of the body, it is very sensitive to systemic immune mechanisms. Highly cell-associated viruses are more readily controlled by cell-mediated than by humoral mechanIsms.
IMMUNOPROPHYLAXIS Approximately one-half of the small animal practitioner's professional activities are concerned with vaccination of animals to prevent infectious diseases. The list of biological products available for use in dogs and cats has increased rapidly in recent years (Table 1). A number of technological changes have been adopted by the biologicals industry to produce vaccines more uniform in potency and less likely to induce untoward reactions than their predecessors. Of current interest to veterinarians is whether inactivated vaccines are as effective as their counterparts that contain modified live viruses. Another topic of debate is whether topical administration of vaccines to the mucosa results in better protection against locally invasive viral pathogens than parenteral vaccination.
Table 1. Canine and Feline Viral Vaccines Licensed by United States Department of Agriculture and Plant Health Inspection Service TYPE
VACCINE
Canine Distemper Vaccine Canine Distemper-Measles Vaccine Canine Hepatitis Vaccine Canine Parainfluenza Vaccine Canine Distemper-Hepatitis-Parainfluenza Vaccine (available in combination with leptospira bacterin) Feline Panleukopenia Vaccine Feline Panleukopenia Vaccine Feline Pneumonitis Vaccine Feline Rhinotracheitis Vaccine Feline Rhinotracheitis-Calicivirus Vaccine Feline RhinotracheitisCalicivirus-Panleukopenia Vaccine Feline RhinotracheitisCalicivirus-Panleukopenia Vaccine Rabies Rabies Rabies Rabies
Vaccine-SAD Stain Vaccine, Flury Stain-Low Egg Passage Vaccine, Goat Brain Origin Vaccine, Mouse Brain Origin
Modified Modified Modified Modified
Live Live Live Live
Virus Virus Virus Virus
Vaccine Vaccine Vaccine Vaccine
Modified Live Virus Vaccine Inactivated Virus Vaccine Modified Live Virus Vaccine Modified Live Chlamydia Vaccine Modified Live Virus Vaccine Modified Live Virus Vaccine Modified Live Virus Vaccine Modified Live and Inactivated Virus Vaccine Modified Live Virus Vaccine Modified Live Virus Vaccine Inactivated Virus Vaccine Inactivated Virus Vaccine
From Veterinary Services Biologics Notice No. 24, United States Department of Agriculture, Animal and Plant Health Inspection Service.
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INACTIVATED AND MoDIFIED LivE VIRus VACCINES
The first veterinary viral vaccines were prepared by harvesting tissues from diseased animals and inactivating the virus, usually by the addition of formalin. Later, the process was refined by the development of methods to grow viruses in tissue culture systems. Inactivated vaccines have proven efficacious only in a limited number of viral diseases. The best examples in veterinary medicine probably are rabies and feline panleukopenia. Humoral immunity appears to be important in the control of both diseases. Inactivated viral vaccines have proven less effective in the prevention of diseases controlled by cell-mediated immune factors. For example, the responses of cattle parenterally vaccinated with inactivated or modified live infectious bovine rhinotracheitis (IBR) virus preparations have been compared. Both immunogens induced similar levels of humoral antibody, but only the modified live virus vaccine stimulated cellmediated immune responses. 11 It is possible that the efficacy of inactivated viral vaccines can be improved by the inclusion of adjuvants to stimulate cell-mediated antiviral immunity. Several years ago a series of new viral vaccines were developed for the prevention of canine and feline respiratory illnesses. 4 • 5 Multivalent vaccines are available that combine canine parainfluenza vaccine with distemper, hepatitis, and leptospirosis vaccines. Similarly, multivalent feline viral rhinotracheitis-calicivirus and feline panleukopenia vaccines are distributed commercially. Both the canine and fe.., line vaccines have been demonstrated to protect against diseases for 12 months, or longer; after parenteral vaccination. The recognition of local humoral and cell-mediated immune factors as being important in the defense of the mucosal surfaces of the body has prompted study of the feasibility of inducing local immunity by the topical application of vaccine viruses. This method has been explored in the quest for effective vaccines for the prevention of influenza and the common cold. In veterinary medicine, intranasal vaccines have been marketed for use in cattle (IBR-parainfluenzavirus-3 vaccines), cats (feline viral rhinotracheitis-calicivirus vaccines), and horses (equine rhinopneumonitis vaccines). Aside from the problems of persistent shed of vaccine virus from intranasally immunized animals and a higher incidence of postvaccinal illness with certain intranasal products than has been associated with similar parenterally administered biological products, the intranasal vaccines have not proven more efficacious because of the fact that the parenteral vaccines also are capable of inducing local immunity in the respiratory tract. Just as respiratory tract infection induces systemic humoral antibody as well as secretory IgA, the parenteral administration of a viral immunogen can result in secretory IgA and local cell-mediated
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immunity, as well as induce systemic immune responses. It was reported that no quantitive differences in local antibody were observed in cattle administered IBR virus vaccines intranasally or intramuscularly.8 Similar findings have been published for parainfluenza virus-3 immunization of lambs. 12 It is now recognized that if sufficient viral antigen to cause systemic infection is administered parenterally, local immunity can be induced. 13 We have compared intranasal and parenteral routes of immunization and found similar results with regard to the degree of protection induced against feline respiratory viral pathogens. 5• 6
CONCLUSION Viruses are capable of inducing a variety of immune responses. Both humoral and cell-mediated immune factors contribute to protection of the host animal from viral diseases. It has been possible to define immune mechanisms that result in the neutralization of extracellular virus, the clearance of virus particles from plasma and extravascular fluids, and the arrest of viral replication in infected cells. Viral vaccines appear to stimulate the same immune responses induced by virulent viruses, while not producing disease. 4 • 10 At the present time, modified live viral vaccines are the more potent stimulators of antiviral cell-mediated immunity. Because of the importance of cell-mediated immune factors in the control of many viral diseases, there are many more efficacious modified live than inactivated vaccines available for viral immunoprophylaxis.
REFERENCES 1. Allison, A. C.: On the role of monouclear phagocytes in im!Il'Unity against viruses. Prog. Med. Virol., 18:15, 1974. 2. Bellanti,]. A.: Immunology. Philadelphia, W. B. Saunders Co., 1971, Chapters 11 and 18. 3. Douglas, R. G., Jr., Rossen, R. D., Butler, W. T., et a!.: Rhinovirus neutralizing antibody in tears, parotid saliva, nasal secretions and serum. J. Immunol., 99:297, 1967. 4. Emery,]. B., House,]. A., Bittle,]. L., et al.: A canine parainfluenza viral vaccine: immunogenicity and safety. Am.]. Vet. Res., 37:1323, 1976. 5. Kahn, D. E., and Hoover, E. A.: Feline caliciviral disease: experimental immunoprophylaxis. Am.]. Vet. Res., 37:279, 1976. 6. Kahn, D. E., Hoover, E. A., and Bittle, ]. L.: Induction of immunity to feline caliciviral disease. Infect. Immunol., 11:1003, 1975. 7. McFarland, H. F.: In vitro studies of cell-mediated immunity in an acute viral infection.]. Immunol., 113:173, 1974. 8. McKercher, D. G., and Crenshaw, G. L.: Comparative efficacy of intranasally and parenterally administered infectious bovine rhinotracheitis vaccines. J .A.V .M.A., 159:1362, 1971.
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9. Molinari, J. A., Ebersole, J. L., and Platt, D.: Investigation of secretory immunoglobulins in saliva from germfree mice. Infect. Immunol., 10:1207, 1974. 10. Schultz, R. D.: Failure of attenuated canine distemper virus (Rockborn Strain) to suppress lymphocyte blastogenesis in dogs. Cornell Vet., 66:27, 1976. 11. Sheffy, B. E., and Rodman, S.: Activation of latent infectious bovine rhinotracheitis infection. J.A.V.M.A., 163:850, 1973. 12. Smith, W. D., Dawson, A.M., Wells, P. W., eta!.: Immunoglobulins in the serum and nasal secretions of lambs following vaccination and aerosol challenge with parainfluenza 3 virus. Res. Vet, Sci., 21:341, 1976. 13. Waldman, R. H., and Ganguly, R.: Immunity to infections on secretory surfaces. J. Infect. Dis., 130:419, 1974. Department of Virological Research Pitman-Moore, Inc. Washington Crossing, New Jersey 08560