Viral Immunology and Immunopathology

america

Authors and Participants

Lewellys. F Barker*,     Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland

Joseph. A Bellanti*,     Georgetown University School of Medicine, Washington, D.C.

R.V. Blanden*,     John Curtin School of Medical Research, Australian National University, Canberra, Australia

Barry. R Bloom*,     Albert Einstein College of Medicine, Bronx, New York

WilIiam. H Burns*,     National Institutes of Health, Bethesda, Maryland

Robert. M Chanock*,     National Institutes of Health, Bethesda, Maryland

Gerald. A Cole*,     School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland

Frank. M Collins*,     Trudeau Institute, Saranac Lake, New York

Charles. A Daniels*,     Duke University Hospital, Durham, North Carolina

Jules. L Dienstag,     National Institutes of Health, Bethesda, Maryland

Frank. J Dixon,     Scripps Clinic and Research Foundation, La Jolla, California

Lila. R Elveback,     Mayo School of Medicine, Mayo Clinic, Rochester, Minnesota

Frank Fenner,     Centre for Resource and Environmental Studies, Australian National University, Canberra, Australia

John. P Fox*,     School of Public Health and Community Medicine, University of Washington, Seattle, Washington

W. Gerhard*,     The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania

Harold. S Ginsberg*,     College of Physicians and Surgeons, Columbia University, New York, New York

Gertrude. Henle*,     Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania

Werner. Henle*,     Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania

Martin. S Hirsch*,     Harvard Medical School, Boston, Massachusetts

Eugene. D Johnson,     School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland

Richard. T Johnson,     School of Medicine, The Johns Hopkins University, Baltimore, Maryland

Elvin. A Kabat*,     College of Physicians and Surgeons, Columbia University, New York, New York

Edwin. D Kilbourne*,     Mt. Sinai School of Medicine, New York, New York

H. Koprowski*,     The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania

Henry. F McFarland*,     School of Medicine, The Johns Hopkins University, Baltimore, Maryland

Andrew. A Monjan,     School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland

Abraham Morag,     School of Medicine, State University of New York at Buffalo, Buffalo, New York

Brian. R Murphy,     National Institutes of Health, Bethesda, Maryland

Neal. Nathanson*,     School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland

Abner. Louis Notkins*,     National Institutes of Health, Bethesda, Maryland

Robert. C Nowinski*,     McArdle Laboratory for Cancer Research, University of Wisconsin—Madison, Madison, Wisconsin

Pearay. L Ogra*,     School of Medicine, State University of New York at Buffalo, Buffalo, New York

Michael. B.A. Oldstone*,     Scripps Clinic and Research Foundation, La Jolla, California

Hillel. S Panitch,     School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland

John. M Peterson*,     National Institutes of Health, Bethesda, Maryland

Gudmundur Petursson,     The Institute for Experimental Pathology, The University of Iceland, Reykjavik, Iceland

Theodore. Pincus*,     Stanford University School of Medicine, Stanford, California

David. D Porter*,     University of California School of Medicine, Los Angeles, California

Allen. Portner*,     St. Jude Children’s Research Hospital, Memphis, Tennessee

Carroll Pridgen,     St. Jude Children’s Research Hospital, Memphis, Tennessee

Max. R Proffitt,     Harvard Medical School, Boston, Massachusetts

Robert. H Purcell*,     National Institutes of Health, Bethesda, Maryland

Bracha Rager-Zisman,     Albert Einstein College of Medicine, Bronx, New York

William. E Rawls*,     McMaster University, Hamilton, Ontario, Canada

Linda. S Richardson,     National Institutes of Health, Bethesda, Maryland

Douglas. D Richman,     National Institutes of Health, Bethesda, Maryland

Wallace. P Rowe*,     National Institutes of Health, Bethesda, Maryland

Albert. B Sabin*,     Medical University of South Carolina, Charleston, South Carolina

Thomas. J Schnitzer,     National Institutes of Health, Bethesda, Maryland

Jerome. Schulman*,     Mt. Sinai School of Medicine, New York, New York

Alexis. Shelokov*,     University of Texas Health Science Center, San Antonio, Texas

Harry. W Snyder, Jr.,     Memorial Sloan-Kettering Cancer Center, New York, New York

Susan. B Spring,     National Institutes of Health, Bethesda, Maryland

Moti. L Tiku,     School of Medicine, State University of New York at Buffalo, Buffalo, New York

Wayne. A.F. Tompkins,     McMaster University, Hamilton, Ontario, Canada

E. Frederick Wheelock*,     Jefferson Medical College, Philadelphia, Pennsylvania

Jack. F Woodruff*,     Cornell University Medical College, New York, New York

Judith. J Woodruff*,     State University of New York, Downstate Medical Center, Brooklyn, New York

*An asterisk designates a participant in the workshop

Preface

The purpose of this book is to bring together into a single volume the new information that has emerged over the last several years concerning the role of cellular and humoral immunity in viral infections, factors responsible for the persistence and recurrence of viral infections in the presence of immunity, mechanisms of viral immunopathology and new concepts in the development of vaccines.

The authors, all active investigators in their respective fields, have attempted to write brief but critical reviews with emphasis on concepts and mechanisms.

The 24 chapters in the book are organized systematically so that the book can serve as a text for advanced students and as an up-to-date review for active workers in the field. It begins with a chapter on the history of viral immunology and proceeds to cover the synthesis and properties of viral antigens, the humoral immune response to viruses, mechanisms of viral neutralization, cellular immunity, the role of inflammatory cells and effector molecules in combating viral infections, and genetic control of resistance.

The emphasis of the book is on mechanisms rather than on description of the immune response to a variety of individual viruses. For illustrative purposes, however, three viruses of considerable current interest (hepatitis virus, leukemia virus, and Epstein-Barr virus) were singled out for in-depth analysis. The book then turns to herd immunity and the latest thinking about vaccines. It concludes with five chapters on viral immunopathology and a chapter on the future of viral immunology and immunopathology.

The appendix contains a summary of a 3-day workshop that deals with many of the problems discussed in the book. The workshop on Viral Immunology and Immunopathology was sponsored by The National Institute of Dental Research on April 7–9, 1975, in Airlie House, Airlie, Virginia.

Abner Louis Notkins

CHAPTER 1

HISTORY OF VIRAL IMMUNOLOGY

FRANK FENNER and R.V. BLANDEN,     Centre for Resource and Environmental Studies, Australian National University, Canberra; Department of Microbiology John Curtin School of Medical Research, Australian National University, Canberra

Publisher Summary

This chapter presents the history of viral immunology. The use of viral components for vaccination has been the subject of experimental clinical trials with adenoviruses and influenza subunit vaccines are in current use. The effect of the production of antibodies against viral components during the course of infection is in vivo complement fixation, demonstrable in practice by the developing anti-complementary activity of serum. Other antibody reactions, which may or may not result in neutralization of infectivity, may play an important role in the production of lesions, especially in certain persistent viral infections. Immune complexes in which viral antigens rather than virions are involved may also play an important role in the pathogenesis of some acute infections, such as hemorrhagic dengue and in the glomerulonephritis found in several chronic viral infections. Indeed, antigenemia and the early formation of immune complexes, rather than viremia as such, may provide the pathogenetic basis for the fever and symptoms that mark the end of the incubation period in many generalized viral infections and the prodromal rash may have a similar explanation.

INTRODUCTION

A history of viral immunology is clearly more concerned with tracing the development of immunological concepts than with an analysis of the history of virology, yet the latter is not unimportant. Viruses obviously differ in important respects from non-living antigens, and they differ fundamentally from living cells, including the pathogenic microorganisms. Viral immunology is a transdisciplinary subject, and several of the great figures in its history, Jenner in the eighteenth century, Pasteur in the nineteenth and Burnet in the twentieth century, are honored as pioneers by both virologists and immunologists. Since the immune response is an aspect of resistance to disease that is restricted to vertebrate animals, we shall therefore have to consider only the minority of organisms that are vertebrates, and the minority of viruses that infect vertebrates. Indeed, we are ultimately concerned with the human host and the viruses that infect man, and with model systems that enable us to study problems of human medical relevance experimentally.

The outline of this book illustrates the many-facetted nature of viral immunology, and we cannot hope in this introductory chapter to give a satisfactory history of each of the topics represented by a chapter heading. We shall therefore try first to illustrate the discoveries that have led to our present concepts of what vertebrate viruses are and how they behave, and then illustrate the developing concepts of the host’s response to viral infection that have brought biomedical scientists to the stage of writing this book. In this introductory chapter we shall try not just to pay homage to the great historical figures of our subject, but also in a sense to set the stage for the other more technical chapters of the book.

THE CONCEPT OF VIRUS

Animal virology began as a branch of pathology. Virology as a discipline advanced rapidly only when it freed itself from this association, thanks mainly to the leadership of Delbrück and the phage group (1). The concepts developed with phage were extended into animal virology, and it is now possible for virologists to move back into pathology with a greatly enriched understanding.

The brilliant studies of Pasteur, Koch and their followers had by the end of the nineteenth century established the microbial etiology of many of the infectious diseases of man and his domesticated animals (2). However, there were still a number of common infectious diseases for which neither a bacterium nor a protozoan could be incriminated as the causal agent. In 1898, Loeffler and Frosch (3) demonstrated that the economically important disease of cattle, foot-and-mouth-disease, could be transferred from one animal to another by material which could pass through a filter that retained the smallest bacteria. The same technique produced comparable results with other infectious diseases of unknown etiology. Following these discoveries, such diseases were tentatively ascribed to what were first called ultramicroscopic filterable viruses, then ultra-filterable viruses, and ultimately just viruses. We now know that viruses also occur in all other groups of organisms, from trees to mycoplasmas, and in our present state of knowledge it is not unreasonable to postulate that for every species of organism there will be found to exist one or perhaps many more species of virus (4).

From the practical viewpoint of the plant pathologist and the public health worker, it is convenient to regard the viruses that cause disease as pathogenic microorganisms. Indeed, Burnet entitled his Dunham Lectures, delivered at Harvard University in 1944, Virus as Organism (5). However, at about this time the question was repeatedly posed as to whether viruses, whatever their host, might have common properties that distinguished them sharply from microorganisms. There was a great deal of discussion about the nature of the eclipse period (6), and at about this time Hershey’s demonstration in 1952 of the injection of DNA by bacteriophages (7) opened the way for a proper understanding of viral multiplication. The argument about the differences between viruses and microorganisms was sharpened and focussed by Lwoff (8) whose dictum that viruses are viruses are viruses, i.e., that all viruses show some properties that distinguish them from any microorganism, is now universally accepted (Table 1). Exceptions to some of Lwoff’s generalizations have since been discovered, but three are still valid: (a) unlike even the smallest microorganisms (chlamydiae), viruses contain no functional ribosomes or other cellular organelles, although some enveloped viruses, notably in the genus Arenavirus, contain a few ribosomes derived from the host cell, (b) in RNA viruses the whole of the genetic information is encoded in RNA, a situation unique in biology, and (c) viruses lack enzymes for energy metabolism and they lack genes for such enzymes. Other distinctions apply to some but not all viruses, e.g., the isolated nucleic acid of viruses of several genera is infectious, so that the virus can be generated intracellularly from a single molecule of nucleic acid.

TABLE 1

The essential differences between viruses and microorganisms, e.g., bacteria

It is impossible to define viruses satisfactorily in a sentence or even a paragraph, bearing in mind both their intracellular states and the extracellular particles. The virus particle or virion consists of a genome of either DNA or RNA enclosed within a protective coat of protein molecules, some of which may be associated with carbohydrates or lipids, usually of cellular origin. In the vegetative state and as provirus (see Chapter 2), viruses may be reduced to their constituent genomes, and the simplest viruses (the viroids of Diener, ref. 9) may be transmitted from one host to another as naked molecules of nucleic acid, possibly associated with certain cellular components. At the other extreme, the largest animal viruses, e.g., the poxviruses and the retraviruses are relatively complex and contain many different polypeptides including several enzymes.

Lwoff’s dictum that viruses are generically different from organisms has had important theoretical and practical consequences; on the one hand, it emphasized their similarities irrespective of the nature of the host (animal, plant or bacterium), and, on the other hand, it led to the establishment of an international body that works in parallel with the international committees of botany, zoology and bacteriology and is charged with the responsibility of classifying and naming viruses (10, 11). However, the operational division of viruses made according to type of host continues to be used by the majority of virologists most of the time. For example, all the contributors to this book are concerned only with viruses of vertebrates, on which we shall henceforth concentrate our attention.

THE CLASSIFICATION OF VIRUSES OF VERTEBRATES

Since the rest of this book will be concerned almost exclusively with immunology, albeit with immunological aspects of viral infection, we shall begin by reviewing briefly the range and nature of the viruses that infect vertebrates. Viruses can be subdivided according to the nature and amount of their nucleic acid, and the shape and size of the virion, into some twenty groups, some of which currently have the status of families, others of genera. Virologists have so far refrained from making any taxa higher than family, reflecting the belief that viruses are polyphyletic and that each family or genus now recognized probably arose independently of each other one, and that all were derived ultimately from cells. The size and shape of representative members of the various groups of viruses of vertebrates are illustrated diagrammatically in Figure 1.

Fig. 1 Diagram illustrating the shapes and relative sizes of animal viruses of the major taxonomic groups (bar = 100 nm). (12)

Many of the features of viruses that fascinate virologists, such as the nature of their genomes (whether double or single-stranded, cyclic or linear, single molecules or fragmented, messenger or complementary strand), and the mode of replication of their genome and the translation of its message, are to a large extent irrelevant to viral immunologists. What is important to them is the way that viruses affect cells (Table 2) and by their effects on cells affect organs and the organism (Tables 3 and 4). While we can state the molecular virologist’s interests fairly easily, it is much more difficult to generalize about the effects of viruses of particular families or genera on cells and organs of particular species of host animal, since these interactions depend upon both the cells affected and the virus involved. Nevertheless, some generalizations are possible and may be valuable as a frame of reference. Tables 3 and 4 have been constructed with this in mind.

TABLE 2

Different kinds of virus-cell interaction

aPK negative mutant of rabbitpox virus

bRSV, Rous sarcoma virus – a mutant avian oncornavirus C

TABLE 3

DNA viruses: families and some genera, characters of genome, characteristics of diseases produced (mainly in man)

a)in millions of daltons; in all cases genome is a single molecule

b)D, double-stranded; S, single-stranded; C, cyclic; L, linear

TABLE 4

RNA viruses: Families and some genera, character of genome, characteristics of diseases produced (mainly in man)

a)in millions of daltons

b)S, single-stranded; D, double-stranded; 1, 7, 4, 10 indicates numbers of pieces of RNA in genome; m, sense of RNA is that of messenger RNA; c, sense of RNA is complementary.

PROTECTION AGAINST REINFECTION

The critical observation that links virology and immunology, and from which immunology was both etymologically and scientifically derived, was an observation that goes back to antiquity, namely, that persons who had recovered from smallpox or measles would not get a second attack – they were immune. This observation was put to practical use by the Chinese, and later the Turks, and through Lady Wortley Montagu, the English, by the practice of variolation as a method of preventing smallpox (see Voltaire, 1733; ref. 13). However, variolation, i.e., the deliberate inoculation of virulent smallpox virus, was a hazardous practice, for some subjects so treated died or were permanently disfigured, and such patients also constituted a source of natural virulent smallpox to their contacts. People living in the countryside had long been aware that persons who tended cows (which also suffered from the pox) did not catch smallpox from them, but merely got blisters on the hands. However, these milkmaids did not get smallpox when this disease hit the rest of the community. Edward Jenner took up this piece of folklore and investigated it systematically, culminating with his experiment with James Phipps and his memoir on the subject published in 1798 (see LeFanu, ref. 14). Although subjected to violent abuse and opposition at the time, Jenner is now properly regarded as the father of immunology – and because he was, although unaware of it, working with a virus, he can reasonably be regarded as the father of virology as well.

The next great figure in the history of viral immunology was one of the great men of history, Louis Pasteur. Trained as a chemist, and responsible for many important discoveries in chemistry, Pasteur’s interest turned to the infectious diseases of man, his domesticated animals (including the silkworm) and his industrial processes, especially the production of wines and beer (2). His important contributions to immunology (15) included the development of live attenuated vaccine, in 1880 with a bacterium, the chicken cholera bacillus (16), and in 1885 with the virus of rabies (17). Recognizing the similarity of his observations to Jenner’s use of cowpox virus to prevent smallpox, Pasteur called his treatment vaccination – a term which has since been extended to cover the injection of bacteria, viruses, and their products as a method of producing future immunity to disease.

THE DISCOVERY OF ANTIBODIES

Pasteur was the first experimental microbiologist and he was a great practical immunologist, but he had no idea how protection was produced by vaccination. This information derived not from a study of viral immunity, but from the discovery in 1888 of diphtheria toxin by Roux and Yersin (18). It was readily shown that immunity can be developed against such a toxin, and the important observation was then made by Behring and Kitasato in 1890 that this immunity could be passively transferred by inoculating serum from an immunized animal into an untreated recipient (19), i.e., there was some specific antibody in the serum.

For the next sixty years immunology in general, and viral immunology in particular, was dominated by studies of antibodies. This is not the place to record details of the development of our understanding of the physico-chemical structures of antibodies, although it is useful to recall that antibodies were not known to be γ-globulins until the late 1930’s (20). This discovery reached its full development with the elucidation of the molecular structure of IgG in 1969 (21).

The elucidation of the structures and properties of the five different immunoglobulins that act as antibodies (Table 5) is part of the history of immunology. Here we shall only summarize briefly our present understanding of the role of these immunoglobulins in viral infections.

TABLE 5

The three immunoglobulin classes important in viral infections

a)Secretory IgA; data for serum IgA in parentheses

IMMUNOGLOBULINS IN THE SERUM AND TISSUE FLUIDS

Table 5 sets out the properties of the three principal classes of immunoglobulin: IgA, IgG and IgM. IgD is omitted, since it has no known significance in viral infections. IgE, which includes the reaginic antibodies that are involved in anaphylactic reactions, is probably of little importance in viral infections.

Although largely neglected, because its study poses no great intellectual challenges, natural passive immunization, i.e., transfer of antibodies from mother to newborn, is of great evolutionary significance, since it protects the highly vulnerable newborn and infant animal from otherwise lethal infections. Brambell (22) has been responsible for our present understanding of the complexities of congenital immunity, which are summarized in Table 6. In mammals, the placenta is permeable to IgG, but not IgM or IgA. This fact is exploited in the diagnosis of congenital rubella, where IgM in the cord blood represents foetal synthesis of antibody, whereas IgG is maternal antibody transferred across the placenta. IgM and IgG dominate the plasma and extracellular fluids, and thus constitute the most important antibodies in systemic viral infections.

TABLE 6

Routes of passive transfer of immunoglobulins from the mother to the extracellular fluids of the fetus or newborn animal

aN.R., not relevant

bImmunoglobulins from milk and colostrum are present and may be active against viruses within the lumen of the gut of the newborn animal, although they may not pass into the circulation

The dynamics of antibody production in mature animals in response to viral antigens resembles the standard response to other non-living antigens – a short inductive phase, the initial appearance of IgM antibodies, and the later appearance, and persistence of IgG (inactivated poliovirus: refs. 23 and 24; inactivated influenza virus: refs. 25, 26 and 27). In viral infections, of course, the initial antigenic dose is minute, and subsequently there is a dynamic interaction between the multiplying virus and viral components and the developing immune response, which is of considerable significance in pathogenesis (28).

THE DURATION OF IMMUNITY AND ORIGINAL ANTIGENIC SIN

The lifelong immunity found after attacks of most generalized viral diseases is proverbial, and in favorable subjects immunity to reinfection and/or persistent circulating antibodies have been demonstrated over very long periods (measles: ref. 29; yellow fever: ref. 30 and poliomyelitis: ref. 31). In several other viral infections, notably those of the upper respiratory tract, immunity is much less durable, partly because strictly homologous immunity that involves adequate production of IgA on mucous surfaces is less efficient than systemic immunity (28) and partly because most of these infections are characterized by a mutliplicity of serotypes.

An interesting immunological feature of repeated infections by related serotypes is what Francis in 1955 called original antigenic sin (32). He found, and others have confirmed, that the serological response of an individual to influenza type A, for example, was dominated throughout life by antibody specific to the first type A strain that he was exposed to. This response is not a peculiar feature of influenza, but is characteristic of infections of long-lived animals with any virus of which there are several cross-reacting antigenic types. It can be reproduced experimentally with inactivated influenza virus vaccines (33). It is probably explained by the fact that the influenza hemagglutinin subunits possess at least two different antigenic determinants; one common to many different strains and the other strain-specific (34). Immunological memory in thymus-independent (B) lymphocytes (see below) to the common determinant results in a powerful secondary response to this determinant upon challenge with any strain of influenza virus that possesses it; the primary response to the unique determinant is comparatively weaker (35).

IMMUNOGLOBULINS ON MUCOSAL SURFACES

Until relatively recently, serum antibodies were the only ones that attracted the attention of immunologists interested in immunity to disease. Besredka’s (36) pioneering concepts of local immunity in 1927, Burrows’ (37) publications in 1947 on coproantibodies in cholera, and the elegant experiments of Fazekas de St. Groth (38) on experimental influenza failed to make much impression on immunologists who were preoccupied with the spillover concept of antibody found on mucosal surfaces. It was only with the demonstration in the early 1960’s by Hanson (39) and Tomasi and Zigelbaum (40) that IgA, a minor immunoglobulin in serum, was the predominant immunoglobulin class on mucosal surfaces such as those of the respiratory and gastrointestinal tracts that led to acceptance of its major role in the prevention of respiratory and enteric infections (41, 42).

NEUTRALIZATION OF VIRUSES BY ANTIBODY

For a long period, from the early 1930’s (43) to the early 1960’s, viral immunology was dominated by studies of the neutralization reaction, essentially as a study of the in vitro reaction between virus and antibody, as measured by subsequent tests for viral infectivity. Burnet’s first review article on an immunological topic (44) was concerned very largely with observations and speculations about neutralization, based mainly on his own studies using pock-counting on the chorioallantoic membrane. Dulbecco studied the subject soon after he had established the plaque method for animal virus (45), and subsequently Fazekas de St. Groth (46) wrote extensively about neutralization of the persistent fraction, largely in an adversary posture to Dulbecco.

This subject is still a topic of interest, and will figure at some length in Chapter 5 of this book. However, we wonder how relevant much of this earlier work was to the problem of the intact animal, since it was carried out with inactivated serum and sought to minimize, where possible, the role of cells other than those subject to infection with the viral inoculum. Neutralization of infectivity by antibodies, nevertheless, remains a matter of vital interest to virologists. It is of major importance in controlling reinfections; the whole vast structure of immunization as a public health measure rests upon the production of antibodies. It is also important in controlling the spread of viruses between cells when the virions are free in the extracellular fluids or plasma. Even though it may not reflect what happens in infected animals, the neutralization reaction as tested in animals or in cell cultures is still a valuable method for epidemiological studies.

VIRAL VACCINES

The outstanding practical aspect of viral immunology is unquestionably the use of viruses or viral components for vaccination (Chapters 17–19). The prototype is, of course, Jennerian vaccination against smallpox. Then, with the cultivation of influenza virus in the chick embryo in 1940 (47) and the fear of a repetition of the World War I experiences of influenza, a major effort was made to produce vaccines against influenza. Although a rather unsatisfactory product, largely because of the predictable (antigenic drift) and unpredictable (antigenic shift) changes in its surface antigens (48), killed influenza virus vaccine, first shown to be moderately effective in 1941 by Horsfall et al. (49), remains the only vaccine of clinical significance against this important disease, and the only inactivated vaccine still in medical use.

The principle of attenuation, first developed in 1884 by Pasteur for rabies (17), has resulted in the production of effective vaccines for yellow fever (50), poliomyelitis (51), measles (52), and rubella (53), as well as numerous veterinary vaccines. There is now renewed interest in live virus vaccines for influenza (54), using modern genetic principles for building the required properties of avirulence, growth capacity, and antigenicity into the vaccine strain. Further, in view of the demonstrated importance of IgA in infections of the respiratory tract, there is now a renewed emphasis on administration of some attenuated virus vaccines by this route. It is interesting to recall that Burnet (55) used both these principles, an attenuated virus given normally, in his wartime studies on vaccination against influenza.

The use of viral components for vaccination (56) has been the subject of experimental clinical trials with adenoviruses (57) and influenza subunit vaccines (58) are in current use (59). Further purification of the influenza virus hemaglutinin is now feasible. The principal difficulty is that purified subunits are not sufficiently immunogenic; current work on this topic will be explored in Chapter 18.

IMMUNE COMPLEXES IN VIVO

One early effect of the production of antibodies against viral components during the course of infection is in vivo complement fixation, demonstrable in practice by the developing anti-complementary activity of serum (60, 61). Other antibody reactions, which may or may not result in neutralization of infectivity, may play an important role in the production of lesions, especially in certain persistent viral infections. Immune complexes in which viral antigens rather than virions are involved may also play an important role in the pathogenesis of some acute infections, such as hemorrhagic dengue (62) and in the glomerulonephritis found in several chronic viral infections (Table 7; Chapter 20). Indeed, antigenemia and the early formation of immune complexes, rather than viremia as such, may provide the pathogenetic basis for the fever and symptoms that mark the end of the incubation period in many generalized viral infections, and the prodromal rash may have a similar explanation. Complement may be important in these reactions (see Chapter 6), as well as in neutralization in vivo (63).

TABLE 7

Chronic infections in which virus is always demonstrable; late immunopathological or neoplastic disease, nonneutralizing antibodies

IMMUNOLOGICAL TOLERANCE

Immunological tolerance still looms large on the horizon of the viral immunologist, especially because of its bearing on autoimmunity (Chapter 23). It was contemplation of Traub’s (64) results on lymphocytic choriomeningitis (LCM) virus infection in mice and those of Owen (65) on erythrocyte mosaicism in cattle, that led to the postulation of immunological tolerance by Burnet and Fenner in 1949 (66). Initial experimental tests of the hypothesis with influenza in eggs (67) were negative, and it remained for Medawar and his colleagues (68) to establish experimental proof of tolerance by skin graft experiments in mice.

Although tolerance has added a new dimension to immunology, the situation is much more complex than originally supposed. In viral infections contracted in utero there is often only partial tolerance, in which non-neutralizing antibody circulates combined with virus, whereas in other cases, like rubella, tolerance fails to become manifest; the fetus develops IgM and also receives maternal IgG via the placenta.

PHAGOCYTOSIS IN VIRAL INFECTIONS

Bacteriologists have long been conscious of the role of phagocytic cells in the control of bacterial infections (e.g. 69, 70). The controversies of the latter nineteenth century between Metchnikoff’s school, which championed phagocytic cells as the major factor in resistance to bacterial infections, and von Behring and Pfeiffer, who claimed that humoral factors (antibodies) were all-important, has been resolved in favor of both sides; effective host resistance depends upon both opsonins and phagocytes.

The microphages (polymorphonuclear leukocytes) and macrophages (including blood monocytes) have very different roles. Clinical experience and experimental studies show that agranulocytosis (deficiency of polymorphonuclear leukocytes) greatly enhances susceptibility to bacterial infections, but hardly affects the course of viral infections, and much additional evidence shows that these cells do not play an important role in defense against viral infections. However, macrophages, both in the tissues and those circulating in the blood as monocytes, affect the course of viral infections in a number of ways. The fixed tissue macrophages, especially the littoral cells of the liver and spleen, clear viruses from the circulation and may then destroy them, or alternatively act as foci for their multiplication (71). Several of the viruses that cause persistent infections multiply preferentially in macrophages (Table 7). Blood monocytes are also important as effector cells in antiviral reactions. They are produced by dividing precursors located mainly in bone marrow, and spend a relatively short time in blood (of the order of one day) before migrating into tissues (72, 73). In this way, fixed tissue macrophages are constantly replenished by new cells from blood, and there is a blood-borne supply of mobile phagocytic cells available to enter sites of infection or other inflammatory areas.

RECENT LINKS BETWEEN VIROLOGY AND IMMUNOLOGY

We have seen that immunology and virology can be traced to a common origin in Jenner, and that Pasteur was unaware of a distinction between these two sciences. More recently, the fundamental immunological conception of tolerance came from Burnet, via Traub’s studies on lymphocytic choriomeningitis virus infection of mice. Another major theoretical advance in immunology was the enunciation of the clonal selection theory of antibody production by Burnet in 1957 (74, 75). As Burnet (55) recounts, this theory, which has had a major impact on modern immunology, arose from his contemplation of Jerne’s (76) natural selection hypothesis for antibody production. Jerne also produced a technical method that has had a major impact in modern experimental immunology, the plaque assay for antibody-forming cells (77). It is no accident that both Burnet and Jerne, who have contributed so significantly to immunological theory and practice, spent many years thinking in population terms about viral genetics and the origin and selection of mutant viruses. Jerne applied the population biological approach to populations of natural antibody molecules, Burnet to populations of somatic (immunocompetent) cells.

However, more recent studies on the cellular basis of immune phenomena, which now constitutes a major part of modern immunology (cellular immunology, see Burnet, ref. 78, of which the study of cell-mediated immunity is a part) have not been influenced directly by virology, but they greatly influence current work in viral immunology. In the following sections, we shall briefly trace the history of cellular immunology as a background for modern studies on this subject that will be described elsewhere in the book.

THE ROLE OF LYMPHOCYTES IN IMMUNOLOGICAL REACTIVITY

The major innovative discovery that opened the way for the study of cellular aspects of immunology was not exploited for many years. It was the demonstration of Landsteiner and Chase in 1942 (79) and Chase (80) that specific immune reactivity (as reflected by delayed hypersensitivity reactions) can be transferred by living lymphoid cells from immunized donors, but not by their serum. This negative definition of cell-mediated immunity, i.e. immunity in which free antibodies are not involved, is still sound and useful.

The next major advance was the elucidation of the immunological significance of the lymphocyte, and the physiological aspects of lymphocyte traffic. These topics were elegantly defined by Gowans in 1959 (81, 82) who showed that lymphocytes constantly recirculate from blood to lymph nodes, and then via efferent lymphatics and the major lymphatic ducts back to the blood. The fundamental work of Miller in 1961 (83) then firmly established that the thymus played a key role in the development of the capacity of the lymphocytes of the mouse to mount an immune response to skin grafts. In the present context it is interesting to recall that at the time of this major immunological discovery, Miller was carrying out thymectomy on mice to determine the role of the thymus in viral leukemogenesis (84).

It is now known that in mature mammals the bone marrow and the thymus constitute the primary sources of new lymphocytes, which migrate via the blood to secondary lymphoid organs such as lymph nodes and spleen. Following the observations of Claman et al. in 1966 (85) that thymus and bone marrow cells acted synergistically in antibody formation, the elegant experiments of Mitchell and Miller (86), Miller and Mitchell (87), Davies (88) and Nossal et al. (89) unequivocally established that antibody-forming lymphocytes were derived from bone marrow, and that thymus-derived lymphocytes played a helper role in this response.

Following Bruton’s (90) pioneering demonstration in 1952 that some children had congenital deficiencies in gammaglobulin synthesis, a wide array of congenital deficiency diseases of the immune system has been recognized in man. Some involve one or several of the immunoglobulins; in others there appears to be thymic dysplasia and abnormalities of cell-mediated immunity. Bacterial and fungal infections are potentiated in both types of deficiency; viral infections, in general, are unaffected by immunoglobulin deficiencies but are potentiated in thymic dysplasias, pointing to the importance of cell-mediated immunity in these diseases (91, 92).

Out of these and other observations grew the concept that there are two broad classes of lymphocytes, thymus-derived (T) cells responsible for the helper function (see above) and for cell-mediated immunity, including delayed hypersensitivity and homograft rejection, and thymus-independent (B) cells responsible for antibody production. The letter B was derived from the avian Bursa of Fabricius which is essential for B cell ontogeny in birds (93). In mammals bone marrow is certainly one source of B cells, but other analogs of the avian bursa have also been