Immunopotentiating substances with antiviral activity

Pharmac. Ther. VoL 6, pp. 235-273, 1979. Pergamon Press Ltd.

Printed in Great Britain

Specialist Subject Editor: DAVID SHUGAR

I M M U N O P O T E N T I A T I N G SUBSTANCES WITH ANTIVIRAL ACTIVITY GEORGES H. WERNER l~panement de Virologie et d'lmmunologie, Centre Nicolas Griilet, Rh~ne-Poulenc Recherche et 13~velopperaent, 94400 Vitry-sur-Seine, France

1. INTRODUCTION A purposeful struggle against virus diseases of man and domestic animals began almost two centuries ago with Jenner's discovery that inoculation of cowpox to humans made them immune to smallpox. Since that time, through the combined efforts of virologists and immunologists--two scientific disciplines which, until recently, represented a common field of endeavor--effective vaccines have been found against yellow fever, poliomyelitis, influenza, measles, mumps, rubella and rabies, while vaccination against hepatitis B, adenovirus, respiratory syncytial virus, cytomegalovirus and varicella virus infections will probably become a reality before long. In the veterinary field, effective vaccines are used against a number of economically important diseases of cattle, swine and poultry. By comparison with these achievements of specific vaccination, those of antiviral chemotherapy--an area in which intensive work started about 30 years ago--are quite modest indeed, since only a handful of drugs have been shown to exert prophylactic and/or therapeutic activity on poxvirus, herpes virus and influenza virus infections (Table 1). At the present stage, neither vaccines nor antiviral agents show broad spectrum efficacy: in the case of vaccines, their high specificity was to be expected from their very design; it was less obvious for antiviral chemotherapy although it was reasonable to infer from the diverse mechanisms of viral replication that it would be hard to find inhibitors which might, at the same time be highly active on many possible viral processes and nontoxic to the host cells at effective antiviral doses. At that point, one may ask whether interferon does not provide an example of a broad spectrum antiviral substance and indeed, in spite of uncertainties about the future of its therapeutic applications, this natural inhibitor appears much less limited in its scope than the synthetic antiviral substances. One must recall that the discovery of interferon (Isaacs and Lindenmann, 1957) was the outcome of investigations on the phenomenon of interference between viruses, which is readily demonstrable in experimental systems but also certainly takes place in nature. One may thus wonder whether one could not fruitfully exploit toward nonspecific prophylactic and/or therapeutic applications the various mechanisms which underlie natural resistance to and recovery from virus ipfections (Lagrange, 1977): such an attempt, which was systematically initiated about a decade ago, is the subject of the present review. It will first be necessary to summarize our present knowledge about immunity in viral infections, especially with respect to the immunological mechanisms of recovery from such infections; we shall then review the available evidence according to which one can experimentally enhance the host's resistance against viral infections in a nonspecific manner through the use of various so-called immunopotentiating or immunomodulating substances and, finally, we shall consider possible applications of such manipulations to human or veterinary medicine, with due regard to what is known about the immunopathology of virus infections and against the background of what has already been achieved through specific vaccinations. 235

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TABLE I. Year first described 1798 1885 1937 1940 1954 1957 1959 1960 1966 1970 1971 1978

Viral Vaccines vs Antiviral Chemotherapy

Vaccines in present use Smallpox (vaccinia) Rabies Yellow fever Influenza Poliomyelitis (inactivated) Poliomyelitis (live) Mumps Measles Rubella, adenoviruses Japanese encephalitis Hepatitis B Respiratory syncytial virus

Antiviral drugs in present use

1960 Methisazone* 1961 Idoxuridinet 1963 Vidarabine (ara-A)t 1964 Amantadine~t 1972 Ribavirin§

*Poxvirus infections. tHerpesviruses. $Influenza A. §Relatively broad-spectrum.

2. I M M U N I T Y I N V I R A L I N F E C T I O N S For the purpose of this review, it is important to distinguish b e t w e e n natural and acquired resistance to virus infections and, also, b e t w e e n resistance to and r e c o v e r y f r o m such infections: using i m m u n o m o d u l a t i n g agents, one m a y wish to increase the host's relative resistance, b y lifting the threshold of infection n e c e s s a r y to cause o v e r t disease; but one m a y also attempt to improve the acquisition of specific resistance by supplementing vaccines with suitable adjuvants; finally, the m o s t important objective m a y well be to find w a y s of improving on those nonspecific and specific m e c h a n i s m s which, under natural circumstances, result finally in the r e c o v e r y of the host, after a more or less severe and prolonged illness. The m e c h a n i s m s of natural and acquired resistance, and those of s p o n t a n e o u s r e c o v e r y , have m u c h in c o m m o n and the drugs capable of exerting such activities m a y finally turn out to be similar, but the experimental a p p r o a c h e s to their d i s c o v e r y and the conditions for their uses will be different. Natural resistance of animals to a given virus infection, in the absence of any previous experience with this virus or with an antigenically related agent, m a y be absolute or relative. I m m u n i t y has nothing to do with the fact that it is not possible to infect c h i c k e n s with poliomyelitis viruses or mice with h u m a n rhinoviruses, and this species resistance manifests itself at the cellular level (except when infectious nucleic acids can be artificially introduced into the cells of the resistant species). A p a r t f r o m classes of viruses capable of infecting several animal species, there are a n u m b e r of others which are strictly species-specific in their infectivity, although they m a y be closely related in other respects, a fact which can be best explained b y evolutionary m e c h a n i s m s (e.g. measles virus in humans, canine distemper in dogs and rinderpest in cattle). Mechanical or physical barriers also play an important role: a given species will not be infected by a virus if its physiological b o d y t e m p e r a t u r e lies well below or a b o v e the t e m p e r a t u r e range at which this virus can replicate. Much more important for our purpose is the relative resistance to viral infection which, within the same animal species or population, varies from one individual or subgroup to another and will manifest itself in significant differences with respect to f r e q u e n c y or severity of illness. The m e c h a n i s m s of specific acquired resistance to virus infections will be discussed later; we are dealing here with nonspecific resistance, independent of previous experience with a given virus and, inasmuch as this relative resistance will enable the host to go through a particular virus infection without apparent illness or

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with minimal symptoms, it is clear that the mechanisms of such resistance may be in many ways similar to those which operate in spontaneous recovery from a viral disease. Genetic factors play a role in resistance to virus infections as do other factors such as age, nutrition, sex, hormonal influences. In man, it is difficult to determine the relative importance of these factors: for instance, the fact that in black Africa measles is a highly lethal disease (mortality up to 5%) may be due not only to racial factors, but also to severe malnutrition (protein deficiency). At any rate, the various factors influencing resistance to viral (and other) infections cannot be dismissed as being entirely non-immunological in nature: very young animals are more susceptible, but this parallels their immunological immaturity, and gross protein deficiency is known to exert a suppressive effect on some immune reactions. But even genetic differences in resistance to virus infections are expressed at the level of cells which are part of the immune system; for instance, genetic susceptibility to mouse hepatitis virus and its phenotypic alteration (induced by drugs and immune responses) respond in a parallel manner in the intact mouse and in in vitro cultures of its macrophages, which mirror the susceptibility of the host (Weiser and Bang, 1977): in vitro, genetically susceptible macrophages are converted into resistant cells by administration of concanavalin A and, in vivo, this lectin can prevent mortality in susceptible mice. Similarly, a strain of avian influenza A virus grows in vitro in the peritoneal macrophages of mouse lines which are susceptible in vivo to the lethal effect of a human influenza A virus, and does not grow in the macrophages of mouse lines which naturally survive the latter infection (Lindenmann et al., 1978). In vitro replication of herpes simplex virus (HSV) in murine spleen cells requires simultaneous stimulation with a B cell mitogen like lipopolysaccharide (LPS); spleen cells from C3H/HeJ mice that do not respond to stimulation by LPS do not either support replication of HSV and, in addition, mice of that line are intrinsically resistant to HSV infection in vivo (Kirchner et al., 1978). Mouse lines have been selected according to the intensity of their antibody responses to sheep red blood cells (Biozzi et al., 1975); the low responders possess more 'active' macrophages (in terms of antigen processing) than do high responders. When these two lines were compared with respect to their relative resistance to a number of murine or mouse-adapted human viruses, it was found that the high responders were more resistant than the low responders to murine hepatitis, to herpes simplex and to A0 influenza viruses, while the reverse was true with respect to encephalomyocarditis virus (Floc'h and Werner, 1978a): the genetic factors influencing the macrophage directly influence resistance or susceptibility to virus infections. In strictly immunological terms, resistance against and recovery from virus infections is dependent, in vertebrates, on the three major types of immune response which have probably appeared in the following chronological order, in the course of their evolution: (a) a system based on the cells which mediate largely nonspecific effects of immune reactions, such cells comprising what was previously called the reticulo-endothelial system and is now more adequately designated as the mononuclear phagocyte system, which includes the marrow promonocytes, the blood monocytes and the macrophages (subcutaneous, alveolar, splenic and synovial macrophages, Kupffer cells) and to which should be added granulocytes and mast cells; (b) a system based on specialized sets of T lymphocytes, which, in a more or less broadly specific way, are capable among other functions of lysing virus-infected target cells by direct interaction with the latter; (c) a highly specific and refined system, based on B lymphocytes which produce equally specific antibodies, capable of neutralizing only those viruses against the antigens of which they have been sensitized (either in the course of infection, as a result of past experience or following active immunization). This is, of course, a grossly simplified picture of the situation, to which should be added, in order to visualize more completely the immunological orchestra which may be called to perform in the course of a virus infection, helper and suppressor T cells, delayed type hypersensitivity (DTH) reactions, K and NK cells, complement and polymorphonuclear cells (for reviews, see White, 1977; Burns, 1977). J P T Vol. 6, No. 2 - - B

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The role that the cells of the mononuclear phagocyte system, or, more briefly, the macrophages, play in the pathogenesis of virus infections and in protection against them is a very complex one, inasmuch as it. is nonspecific. Macrophages are generally considered to be the first line of defense against viruses: they play indeed, an important role in clearing viruses from the blood stream and preventing the infection of susceptible cells in target organs. Many viruses, however, far from being digested by the macrophages which ingest them, do multiply in these cells and infected monocytes may actually transport viruses around the body. It may be that the ability to grow in macrophages is a vital factor in the virulence of a given virus since, by infecting the macrophages, the virus produces a break in the body's major nonspecific defense mechanism. Roughly speaking, the macrophage can be pictured as an advanced fortress but also, at times, as a Trojan horse. The first feature epitomizes what happens in viral infections of the respiratory tract and of the alimentary tract, as well as in viral infections of the skin, while obviously the macrophage barrier is at least partly ineffective in the infections usually marked by a viremic phase (such as generalized infections with rash, those involving the central nervous system, and congenital infections). In fact, within the same species and in the face of the same virus infection, macrophages may be permissive or defensive: herpes simplex virus grows readily in the macrophages of newborn mice and causes their rapid death, while, in adult mice, the same peritoneal macrophages certainly act as barrier, as evidenced by the fact that specific killing of these cells with silica greatly increases the susceptibility of the adult animal to this infection, while transfer of adult macrophages to newborn mice enhances their resistance (Zisman et al., 1970; Hirsch et al., 1970). Actually, and as will be discussed later, the activity of immunopotentiating substances may well be, in many cases, to convert a permissive or passive macrophage into a defensive one (Morahan et al., 1977). Besides the multifaceted and nonspecific functions of macrophages, it must also be mentioned that these cells can be 'activated' by lymphocytes or 'armed' with specific antibody, although the role of such mechanisms in viral infections is still speculative; finally, the macrophages are among the cells which can produce interferon, as will be discussed later. It is only recently that the role of cell-mediated immunity (CMI) in the pathogenesis of virus infections (and in resistance against, or recovery from, them) has been precisely analyzed. That viruses elicit CMI reactions could be inferred from the delayed type hypersensitivity (DTH) reactions which follow intradermal injection of various viral antigens (vaccinia, mumps, for instance) into a previously infected host. T cell-mediated immunity can be abrogated in mice by neonatal thymectomy or treatment with antithymocytic globulin: such manipulations aggravate infections of mice with pox- and herpes viruses but show little effect on entero- or togavirus infections and are even possibly beneficial in some cases. In man, some virus infections are particularly severe and frequent in congenital or acquired immunodeficiencies affecting T cells (see Table 2): vaccinia, measles, herpes simplex, varicella-zoster, cytomegalovirus, while congenital or acquired immunodeficiencies affecting antibody production aggravate infections with poliovirus (attenuated strains, nonvirulent for immunologically normal subjects) or hepatitis B. T cell response to virus infection manifests itself not only by DTH reactions, but also by the appearance of sensitized lymphocytes which are capable of selectively lysing cells bearing the viral antigens on their surface (Blanden, 1974). Available evidence suggests that, at least in the mouse, the class of T cells which is responsible for initiating mechanisms of viral clearance in vivo is the same as that which is directly cytotoxic for virus-infected target cells in vitro. Furthermore, it was shown that cytotoxic T cells generated in response to lymphocytic choriomeningitis virus (LCM) infection in the mouse can efficiently lyse LCM-infected target cells only when the latter share the H-2 K or H-2 D region determinants (of the murine histocompatibility complex) with the donors of these cytotoxic cells (Doherty et al., 1974; Zinkernagel and Welsh, 1976). One explanation for this important restriction phenomenon would be that cytotoxic T cells express two distinct receptors, one specific for self (H-2), the other

Immunopotentiating substances with antiviral activity

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TABLE2. Viral Diseases A~ecting (with Unusual Frequency and/or Severity) Patients Presenting Congenital or Acquired lmmunodeficiencies Syndrome or disease Congenital Severe combined immunodeficiency

Defect in HI* CMIi" +

+

Wiskott-Aldrich

+

+

Thymic dysplasia (Swiss type) (Di George) Hypogammaglobulinemia

+ _

+/ +

+

-

+

+

+ +

+ + +

Acquired Leukemia Hodgkin Non-Hodgkin lymphomas Immunosuppressive chemotherapy

Viral infections Vaccinia, measles, V-Z& adenovirus Measles, V-Z, herpes simplex, cytomegalovirus Vaccinia, measles, cytomegalovirus Paralytic poliomyelitis,persistent enterovirus infection Measles,cytomegalovirus, hepatitis B V-Z, herpes simplex, cytomegalovirus V-Z, vaccinia MeaslesV-Z, herpes simplex, cytomegalovirus

*HI: Humorai immunity.

~CMI: Cell-mediated immunity. ~V-Z: Varicella-Zoster. for non-self (virus), antigen, but it could also be that these cells possess receptors which are specific for the 'altered self' antigen produced by the interaction of virus and H-2 molecules. Cytotoxic T cell responses have been shown to occur in the following acute viral infections of mice: Sindbis and Semliki Forest alphaviruses, ectromelia and vaccinia (poxviruses), Sendai paramyxovirus, influenza (orthomyxovirus), rabies (rhabdovirus), Coxsackie (enterovirus), adenoviruses, and, of course, LCM (see review by Blanden, 1977); in all these cases, the peak cytotoxic T cell response was observed from 5 to 9 days after infection, i.e. before production of circulating neutralizing antibody and shortly before activation of macrophages by products from sensitized lymphocytes. Until recently, it was accepted that mice infected with herpes simplex virus showed little or no cytotoxic T lymphocyte (CTL) response, in contrast to the infections listed above: it has been shown, however (Pfizenmaier et al., 1977), that in lymph nodes draining a local site infected with this virus, CTL precursors are actually sensitized and that, upon transfer to in vitro culture conditions, they develop within 3 days into effective CTL. It is reasonable to assume that the CTL generated during the 5-9 days which follow virus inoculation in a mouse exert a favorable influence on recovery by the fact that they destroy virus-infected cells before infectious virus progeny is actually assembled (Zinkernagel and Althage, 1977), thereby preparing their elimination by macrophages; indeed, peak activity of CTL in the spleen coincides in time with a rapid decline of infectious virus titer in the same organ (Blanden and Gardner, 1976) and this was also shown to occur with respect to respiratory infection of mice with the Sendai strain of paramyxovirus (Anderson et al., 1977). More direct evidence on the role of CTL in natural recovery has been adduced by the demonstration (Yap et al., 1978) that transfer of specific cytotoxic T lymphocytes protects mice from death following intranasal inoculation of a virulent strain of influenza A virus: there was a striking correlation between the level of cytotoxic activity of injected immune spleen cells and the capacity of the latter to protect the recipient mice from death. Spleen cell suspensions with the highest cytotoxic activity actually gave complete protection. Indirect evidence also favors an important role of CT L in resistance to, and recovery from, influenza A virus infection in the mouse: T cells cytotoxic to influenza virus-infected cells occur in the course of a primary immune response to influenza virus and, while exhibiting H-2 restriction, these cells have a broad specificity for viral

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determinants, since T cells generated against one strain of influenza virus can lyse cells infected with another A strain containing serologically distinct surface antigens (hemagglutinin and neuraminidase) (Effros. et al., 1977; Zweerink et al., 1977). It so happens that mice inoculated with live A influenza viruses by a nonlethal route exhibit subsequently a marked resistance against respiratory challenge with a normally lethal dose of another A strain, differing from the first one by both surface antigens and that this state of heterotypic immunity begins 5 days after parenteral inoculation of the first virus, i.e. at the time when it is known that broadly specific CTL appear in the spleen (Floc'h and Werner, 1978b). It must also be stressed that, in this model, delayed type hypersensitivity reactions to the whole virions were equally crossreactive. It is important to note that after recovery from a virus infection, the cytotoxic T cells which survive, after having disseminated through loci of infection, become part of the small T lymphocyte pool which carries immunological memory; the importance of CTL memory in resisting secondary infection is still speculative but it would seem to be a significant factor in all cases where viruses escape neutralization by antibody en r o u t e from the portal of entry to target organs (Blanden, 1977). On the other hand, in several virus infections it is clear that the immune response itself is a major factor in causing some pathological changes. A pertinent example is provided by lymphocytic choriomeningitis (LCM) in adult mice: the cell-mediated response, and particularly the CTL, is both beneficial, by clearing virus from lung, liver and spleen, and detrimental in causing lethal inflammatory reactions in the brain. CTL can be isolated from the spinal fluid of moribund mice and transfer of such cells can induce lesions in the choroid plexus and meninges. In man, one may suspect that encephalitis caused by arenaviruses results largely from the destruction of infected cells by CTL. On the other hand, it is reasonable to assume that in measles and other exanthematous diseases of childhood, CTL mechanisms are responsible for the recovery from the infection and also for the rash, which is one of the main symptoms of disease. Subjects with congenital or acquired immunodeficiencies at the T celt level may present atypical cases of measles, without rash but with growth of the virus in the lungs. The CTL response, though probably quite important both in the recovery from and resistance to virus infections, and in some aspects of the immunopathology of such infections, is certainly not the only compartment of cell mediated immunity which comes into play. Sensitized T lymphocytes can liberate various lymphokines and thereby induce migration and specific activation of macrophages: such a mechanism has been clearly demonstrated in infections with nonviral intracellular organisms and deserves further study in the case of virus infections. Delayed-type hypersensitivity reactions have actually been detected following infection with practically all membrane-associated viruses so far studied (poxviruses, myxoviruses, herpesviruses, arenaviruses) and are seldom seen with non-membrane-associated viruses (i.e. picornaviruses). The last, but certainly not the least, important immune mechanism of defense against virus infections is represented by the production of specific antibodies. These antibodies are capable of neutralizing free virions and, in addition, they can in association with complement, lyse virus-infected cells; we are therefore in the presence of two distinct processes which play a vital role in limiting viral infections: (a) virus neutralization by serum IgG or by secretory IgA at the mucosal surfaces, (b) immune cytolysis, through the interaction of antibody, complement, K cells, polymorphs and macrophages. The essential role of circulating IgG antibody and of mucosal IgA antibody in resistance to reinfection by a virus, following recovery from a viral infection or vaccination with a killed or attenuated virus, need not be emphasized. Antibodies, being specific, play of course no role in natural resistance to primary infection and it appears that, in many viral illnesses, termination of primary infection and recovery are largely mediated by the cellular mechanisms which have been discussed above. Antibodies, however, may exert a limiting effect even during a

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primary infection, especially when infectious virus happens to be present in the blood stream: for instance, the importance of circulating antibody in preventing infection of the central nervous system accounts for the efficacy of inactivated poliovirus vaccines and of passive administration of gammaglobulins and is exemplified by the fact that patients with B cell deficiencies, but intact T cell functions, are more prone to paralytic poliomyelitis. Prolonged presence of an enterovirus (echovirus) in the central nervous system of patients with agammaglobulinemia (such a persistence being associated with progressive symptomatic illness) has been recently reported (Wilfert et al., 1977). On the other hand, progressive vaccinia gangrenosa may be observed in patients with T-cell deficiencies, even in the presence of adequate circulating antibody levels: depending on the nature of the virus, the limiting effects of antibody on viremia may not suffice to stop the progression of the disease. Similarly, recurrences of herpes simplex skin lesions can occur in patients with high titers of neutralizing antibody in their serum. Antibody-dependent cell cytotoxicity, effected through K cells, may play a more important role than circulating neutralizing antibody in the actual process of natural recovery from virus infections; and immune cytolysis, which occurs later than the production of cytotoxic T lymphocytes, probably proceeds concurrently with the latter in terminating many viral infections. The importance of antibody-dependent cell-mediated cytotoxicity in the immunity conferred by smallpox vaccination in humans has recently been demonstrated (Perrin et al., 1977; M¢ller-Larsen and Haahr, 1978). As in the case of cell-mediated immunity, antibody production can also exert deleterious effects and contribute to the pathology of some viral infections. A typical example is provided by LCM virus infection in newborn mice: mice infected neonatally exhibit partial immunological tolerance with lifelong persistence of virus in their tissues, but discrete amounts of antibody are produced, which react with virus in the blood to form immune complexes; the latter deposit in the kidney glomeruli, causing glomerulonephritis. Immune complexes certainly play a role in the pathogenesis of persistent viral infections, such as in human chronic hepatitis B, dengue hemorrhagic fever and subacute sclerosing panencephalitis (SSPE). A similar mechanism may account for the severe pattern of disease shown to occur in individuals vaccinated with formalin-inactivated measles virus when they were exposed to an outbreak of natural measles or revaccinated with live attenuated measles virus. It may also explain in part the severe bronchiolitis observed in children immunized with inactivated respiratory syncytial virus (RSV) when they were exposed later to wild RSV. Interferon and interferon inducers are discussed in detail in another review. Although interferon is produced by lymphoid cells and by macrophages, it can also be produced in vitro or in vivo by almost any type Of cell or tissue as a consequence of viral infection and cannot therefore be strictly regarded as an immunological reaction. The role of interferon in host resistance to, and recovery from, viral infections is largely based on indirect evidence: (a) there is a temporal association between the presence of virus and interferon in tissues (and, in many cases, the amount of interferon appears to be mainly a reflection of the extent of virus multiplication); (b) in some situations, such as herpes-zoster-varicella virus infection in immunosuppressed patients, there is a good association between the appearance of high titers of interferon in the vesicle fluid and the commencement of healing (Stevens et al., 1973); (c) passive administration of large amounts of exogenous interferon has been shown, by many investigators, to confer resistance to, or to hasten recovery from, viral infections in laboratory animals and, in some cases, in man. As there are no known instances of an intrinsic inability to produce interferon, 'experiments of nature' cannot be used to establish the role of interferon in natural mechanisms of resistance and recovery, in a way similar to selective B or T cell deficiencies in proving the role of antibodies and cell-mediated immunity. Following experiments showing increased susceptibility to Semliki Forest virus infection in mice treated with sheep anti-mouse interferon serum (Fauconnier, 1970), recent investigations (Gresser et al., 1976a) have

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convincingly demonstrated that neutralization of interferon production can markedly aggravate several virus infections in the mouse. For instance, in the case of infection with encephalomyocarditis (EMC) virus, the course of the disease in mice inoculated with a potent sheep anti-mouse interferon globulin was entirely different from that affecting control mice similarly infected but treated with a control globulin preparation: the latter died toward the fourth or fifth day after infection, with signs of central nervous system involvement; by contrast, the anti-interferon-serum treated mice died within 24-48 hr of an overwhelming systemic infection before virus had multiplied to high titer in the brain. The same authors (Gresser et al., 1976b) also studied the effect of treatment with anti-interferon globulin on the course of infection of mice with herpes simplex, vesicular stomatitis, influenza A and Moloney sarcoma viruses. With the exception of influenza, such a treatment resulted in an accelerated appearance of signs of infection and in a marked aggravation of the disease. It was shown, in addition, that anti-interferon globulin did not block the synthesis of interferon by virus-infected cells but rather neutralized extracellularly its activity. The unavoidable conclusion from these data is that early interferon in situ production must play a significant role in limiting viral multiplication and spread and thereby contribute to recovery. Summarizing this long, still very sketchy, introduction, one may state that vertebrates possess several mechanisms which enable them to survive through virus infections (Table 3): first, a nonspecific, and not always very efficient, barrier, represented by cells of the mononuclear phagocyte system; then, the various functions exerted by specifically sensitized T lymphocytes followed later by humoral events, either narrowly specific (antibodies) or nonspecific (interferon). There are, of course, multiple interactions between these various systems such as the effect on macrophages of the products from sensitized T lymphocytes, the role of T helper cells in antibody production by B cells and the recently demonstrated inhibition by interferon of some T and B cell reactions. Furthermore, as already referred to above, some of these immune reactions actually contribute to the pathogenesis of the viral disease. While specific vaccination with inactivated or live attenuated viruses does nothing more, immunologically speaking, than mimic the natural disease, the mechanisms and possible uses of 'immunopotentiating' drugs--which may be specific with respect to some aspects of the immune response but, by definition, are not specific with respect to the infecting virusmappear extremely complex.

TABLE3. Immunological Mechanisms of Resistance to and Recovery from Virus Infections (Summary) 1. M a c r o p h a g e S ~productionPhag°cyt°sis n

2. T lymphocyt~es [ ~ ~ t a r g e t interfero~ I NK cells*'f J t ~U~PPerre ~ ~ s ceIIs }

3. B lymphocytes

cells)

antibody

f serum IgG ].mucus IgA

: neutralization

+ complement

~ K cells t macrophages

immune : cytolysis

*According to recent data, NK cells (natural killer cells) may not belong to T lymphocytes but might represent a distinct population of B cells (like the K cells).

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3. I M M U N O P O T E N T I A T I N G SUBSTANCES OF N A T U R A L ORIGIN 3.1. MISCELLANEOUS MICROBIAL INFECTIONS AND PRODUCTS Remarkable resistance against challenge with normally lethal doses of the Mengo strain of encephalomyocarditis virus (EMC) was observed in mice which had been infected with obligate intracellular protozoa Toxoplasma gondii and Besnoitia jeUisoni and with the facultative intracellular bacterium Listeria monocytogenes (Remington and Merigan, 1969). In contrast to the prolonged resistance (1 yr or more) demonstrable in the animals chronically infected with protozoa, the resistance to viral challenge of listeria-infected mice lasted only 1-3 months and was less striking. Sustained 'activation' of the macrophages in mice chronically infected with protozoa was considered as the most likely explanation for this enhanced resistance to viruses. Repeated administration to mice of those combined bacterial vaccines which are employed by some physicians for the treatment of bronchial asthma and of recurrent chronic respiratory infections was shown (Degr6 and Dahl, 1973) to afford protection against intranasal infection with an A2 influenza virus. The combined bacterial vaccine gave some protection when it was administered only once a few hours before virus inoculation but the effect was greater when the mice received a first intraperitoneal dose of vaccine 14 days, and a second dose 4hr, before virus inoculation. The increased protection produced by this immunizing schedule was paralleled by an increased serum interferon production in the mice upon i.p. inoculation of Newcastle Disease Virus. The importance of eliciting delayed type hypersensitivity reactions to tile bacterial product before viral challenge in order to obtain optimal nonspecific protection has been clearly demonstrated, (as described in Section 3.3 devoted to mycobacterial products) in the case of Mycobacterium tuberculosis and old tuberculin; similarly, it was shown (Allen and Mudd, 1973) that infection of mice with a non-lethal strain of Straphylococcus aureus did not significantly alter their sensitivity to tail vein inoculation with vaccinia virus, but that specific elicitation of the Staphylococcus-sensitized animals with a staphylococcal phage lysate a few hours before viral challenge resulted in decreased lesions in the tail. 3.2. LIPOPOLYSACCHARIDES(ENOOTOXINS) The presence of biologically active substances in gram-negative bacteria was recognized during the previous century; it was later found that these were constituents of the cell wall and, because of their toxicity, they were named endotoxins. These endotoxins are lipopolysaccharides (LPS), i.e. heteropolymers containing polysaccharide covalently bound to a phospholipid, termed lipid A. LPS exert a wide variety of biological effects and it is now clear that lipid A is the active component in most such effects (Galanos et al., 19:/7). Nonspecific effects of LPS upon resistance have been demonstrated in experimen, tal infections by gram-negative and gram-positive bacteria, mycobacteria, fungi, parasites and viruses (Cluff, 1970). Relevant to the enhancement by LPS of the resistance of mice to virus infections, first demonstrated in the case of ectromelia (mouse pox) (Gledhill, 1959), is the fact that these substances are highly pyrogenic, at minute doses, and that they cause the release of interferon in the circulation of tile animals injected with them. In vivo and in vitro activation of macrophages by LPS is readily demonstrable by various tests, and it is interesting to note that, while viruses can induce interferon in many types of cells in tissue culture, LPS are only effective in cultures of cells of the reticulo-endothelial system. It is therefore likely that interferon production (or, probably, rather release) as well as hyperthermia are the consequences of a direct effect of LPS on such cells. The effects of administration of a purified preparation of LPS on infection of mice with influenza A0, A 2 and B viruses (inoculated intranasally), with EMC virus (s.c. or

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i.m.), with vesicular stomatitis (i.c.), herpes simplex type 1 (i.p.) and vaccinia (i.p.) viruses have been studied (Rolly et al., 1974). LPS was administered intranasally, i.v., i.m., i.p. or i.c. Although, at appropriate doses and times of administration of LPS, an enhancing effect on resistance was seen in all these systems, the most striking activity was observed when the substance was given intranasally, at 0.3-10/~g per mouse, 24 hr before influenza virus inoculation by the same route. In this system, protection was still significant when LPS was administered as early as 120 hr, and as late as 3 hr, before infection, but treatment after infection was ineffective. From the respective kinetics of protection against mortality and of release of circulating interferon by LPS in the mouse, the authors conclude that the latter effect cannot by itself account for the antiviral activity of LPS (which is inactive in vitro). We have studied the effects of LPS preparations from Salmonella typhimurium and Escherichia coil on infections of mice with EMC, murine hepatitis, influenza, Semliki Forest (arbovirus), herpes simplex type 1 and mouse-adapted foot-and-mouth disease viruses and reached essentially similar conclusions (Floc'h and Werner, unpublished): at minute doses and by various routes, these preparations exerted significant protective effects, and the activity observed when infection was performed at the time of peak interferon levels in the blood (i.e. 2-6 hr after LPS injection) was not higher than when it took place after a considerable drop in the titer of circulating interferon (i.e. 24 hr after LPS injection). It is noteworthy that, in these experimental systems, the transient increase of sensitivity to infections by LPS, which is readily demonstrable in many bacterial infections a few hours after the injectio n of the substance and which precedes enhancement of resistance, was not seen on virus infections. One should also note that the various virus infections tested did not respond equally well to LPS, the least sensitive being herpes simplex. Even in those systems which respond well to enhancement of resistance by LPS, dose-effect relationships are not linear: curves are often bell-shaped, with peak activity at an intermediary dose, or they may present M, V or W shapes, as observed in other circumstances (Bliznakov and Adler, 1972). The toxicity of LPS makes it somewhat doubtful that such substances could be used in nonspecific protection of man and domestic animals against virus infections; attempts at detoxification of LPS by various chemical procedures have generally resulted in loss of only some of their interesting biological activities, but enhancement of resistance against infections and interferon release were among the effects most severely modified by such manipulations, together with the least desirable effects, such as pyrogenicity (Chedid et al., 1975; McIntire et al., 1976). Deleterious effects of LPS in experimental virus infections have also been seen: Newcastle disease virus (an avian virus) produces no signs of infection of the central nervous system in normal mice but causes meningo-encephalitis in mice treated with LPS 24 hr previously; this enhancement of infection was ascribed to transudation of the virus resulting from injury to cerebral blood vessels by LPS (Rahman and Luttrell, 1963). One should finally mention that crude extracts from some gram-positive bacteria have also been shown to enhance resistance of mice against several virus infections: it appears that, in the main, their other biological activities, namely interferon release, macrophage activation and pyrogenicity are qualitatively similar to those of LPS. For instance, it was recently shown (Nozaki-Renard, 1978) that extraction of Bacillus subtilis by methods used to obtain LPS from gram-negative organisms, yields substances which cause the release of interferon in mice and exhibit mitogenic as well as pyrogenic activities, while they appear to be of a different chemical nature from that of endotoxins. 3.3. MYCOBACTERIA AND MYCOBACTERIAL PRODUCTS It was shown, more than 20 years ago, that inoculation of live bacillus CalmetteGu6rin (BCG) induces in the mouse a sustained hyperactivity of the phagocytic cells (Biozzi et al., 1954). BCG-inoculated mice exhibit enhanced resistance to infection

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with virulent staphylococci (Dubos and Schaedler, 1957) or to challenge with several transplanted tumors (Biozzi et al., 1959). Following the pioneer investigations of Math6 and his group (Math6 et al., 1969), BCG is, at present, the most widely used agent in immunotherapy trials against malignant diseases of man. Enhanced resistance of BCG-infected mice (1 mg/mouse i.v.) to the Mengo strain of encephalomyocarditis virus was reported several years ago (Old et al., 1963). A 1.0 to 2.5 log10 reduction in the virus LDs0 by the i.v., i.p. or s.c. routes was observed in comparison with control mice, in animals previously infected with BCG; increased resistance was evident by the eighth day following BCG inoculation and persisted for at least 3.5 months; BCG infection did not protect mice against intracerebral virus challenge. Additional interesting observations were made: serum from BCG-inoculated mice did not neutralize Mengo virus nor did it confer passive protection against this challenge; splenectomy of BCG-inoculated mice did not abolish their resistance to the viral challenge (while it is known to inhibit enhanced antibody production against bacterial antigens); in vitro the extent of replication of Mengo virus in peritoneal macrophages from BCG-infected mice was not different from that in cells from control mice, but, as the authors rightly stressed, the behavior of the peritoneal cells did not necessarily reflect that of spleen and liver macrophages. BCG infection by the i.v. route 10-7 days prior to intravaginal inoculation of herpes simplex type 2 virus (HSV-2) in adult female mice did not alter the subsequent disease, characterized by vaginitis, posterior paralysis, encephalitis and death (Baker et al., 1974): it even appeared, in some cases, to enhance the severity of the disease, while passive immunization of the mice with specific HSV-2 antiserum 4 hr before virus inoculation provided significant protection. It is interesting to note however that the combination of BCG inoculation (7 days before infection) and antiserum treatment (4 hr before) produced the greatest degree of protection. Different results were obtained when HSV-2 was inoculated i.p. into newborn mice (Starr et al., 1976): in this situation, BCG administered i.p. or intradermally 6 days before virus challenge increased survival rate, while the other immunopotentiating substances studied (levamisole, typhoid or brucella vaccines) were ineffective. Clearly, in the newborn mouse infected i.p., stimulation of the peritoneal macrophages by BCG is sufficient to enhance resistance against HSV-2, while the mechanisms of resistance of adult mice against intravaginal herpes infection must be less simple. The effect of BCG infection (1 mg/mouse, i.e. about 106 live bacilli, intravenously) on the resistance of adult mice to virus infections was investigated using EMC virus (inoculated s.c.), murine hepatitis (i.p.), herpes simplex types 1 and 2 (i.p.), foot-andmouth disease virus (s.c.), and A0 and A2 influenza viruses (aerosol) (Floc'h and Werner, 1976). In most cases, BCG-inoculated mice exhibited a significantly higher resistance to these lethal infections than controlmice: the overall survival rate in the latter was 18 per cent vs 41 per cent in the BCG-inoculated animals. Enhanced resistance following BCG inoculation (performed from 31 to 15 days before virus challenge) was especially marked in infections with EMC, HSV-1 and influenza A2 viruses; intercurrent infection ofBCG-inoculated mice with non-lethal doses of viruses did not abolish their resistance towards subsequent challenge with lethal doses of an unrelated virus. It is noteworthy that, in the case of A2 influenza virus infection, the BCG-inoculated mice did not show earlier or higher serum antibody production against the virus than the control mice and that their lung lesions were not markedly diminished: the only difference between the two groups which might explain the higher survival rate of the BCG-inoculated mice was a more rapid virus clearance from their lungs. On the other hand, while BCG-inoculated mice were more resistant than controls to HSV-1, they did not exhibit enhanced resistance to a strain of H s v - 2 which required immunosuppression with cyclophosphamide to cause a lethal infection. Nonspecific protection of mice against A0 influenza virus infection following systemic immunization with BCG was recently confirmed (Spencer et al., 1977); it was found, in addition, that greater protection was afforded by intranasal BCG than by

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intraperitoneal BCG immunization against this intranasal virus challenge. This 'compartmentalization' of the response was maximal 4 weeks after BCG inoculation, decreased at 6 weeks and disappeared at 8 weeks, in agreement with what was seen with respect to specific cell mediated immunity following BCG inoculation to guinea pigs by systemic or intranasal route. The same authors reported that in vitro peritoneal exudate cells from BCG-sensitized guinea pigs showed a more rapid fall in the intracellular titer of influenza virus than did macrophages from unimmunized animals. BCG has also been reported to modify tumor development in mice following inoculation of oncornaviruses such as Moloney sarcoma virus: mice injected with BCG 28 days before virus inoculation had shorter latent periods for tumor development but longer survivals than control animals; when BCG was mixed with the virus at the time of inoculation, there was a decrease in mortality, tumor incidence and tumor size compared with controls inoculated with the virus alone; inoculation of mice with BCG 28 days before attempted induction of tumors with the BCG-virus mixture completely inhibited tumor development and protected the animals against death (Schwartz et al., 1971). Thus far, all the experiments reported have been performed in mice, but it has also been shown that BCG immunization of ,abbits enhanced their resistance to infection with type 2 herpes simplex virus (Larson et al., 1972). While control rabbits died from encephalitis subsequent to vaginal or corneal inoculation of the virus, animals which had been inoculated i.v. with 4 × 107 viable units of BCG 4 weeks before viral challenge also developed encephalitis but, in most cases, did not die. On the clinical scene, attempts have been made to treat recurrent herpes genitalis in men and women by nonspecific stimulation of their immunity with BCG. In one study, (Anderson et al., 1974), fifteen patients with frequent recurring genital herpes manifestations were injected intradermally with BCG: all of them experienced a decrease in the frequency and severity of their recurrences and the best responses were obtained in those patients who became, and remained, tuberculin-positive following BCG administration. These encouraging results have not been confirmed in a recent and more extensive study carried out on 100 patients (Corey et al., 1976): patients with previously documented recurrent genital herpes received BCG or placebo (candida antigen) intradermally and were followed at monthly intervals during 6 months. During this study period, there were on the average 2.30 recurrences/100 days in the BCG recipients compared with 2.18 recurrences/100 days in placebo recipients; the mean duration of lesions during recurrences of genital herpes was similar between the BCG and the placebo group, but, among women, the mean duration of pain was shorter (average: 3.5 days) in BCG recipients than in placebo recipients (6.0 days). One may also mention, in view of the probable viral etiology of this condition, that immunostimulation with BCG failed to affect the clinical course of Burkitt's lymphoma, in a randomized trial carried out on forty patients (twenty-one treated with BCG, nineteen with placebo): no significant differences in the length of remission or the site of relapse were observed that could be attributed to the BCG treatment, in spite of the fact that antibody titers to the membrane antigen associated with Epstein-Barr virus increased greatly in the BCG-injected patients but not in control patients and that BCG treatment increased the rate of recovery from the tumorinduced state of immunosuppression (Magrath and Ziegler, 1976). An avirulent strain of Mycobacterium tuberculosis hominis (H 37 Ra) has also been used to induce resistance of mice against viral challenge, in this case intravenous inoculation of vaccinia virus (Allen and Mudd, 1973): infection with this agent did afford some protection against tail lesions resulting from this challenge, but the enhancement of resistance was definitely greater when the H 37 Ra-sensitized mice received an injection of old tuberculin shortly before virus challenge. All the experiments reported thus far have been performed with live Mycobacteria, but Freund's complete adjuvant (FCA) in which the vacilli are killed was shown to cause inhibition of multiplication of foot-and-mouth disease virus (FMDV) in adult mice (Gorhe, 1967; Gorhe et al., 1968). Mice received a first intradermal injection of

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FCA 10 days before intraperitoneal challenge with FMDV and a second one 3 days before challenge: only 33 per cent of the animals treated in this way showed evidence of viremia, vs 100 per cent in the control mice, and the extent of virus multiplication in their pancreas was greatly diminished. Increased resistance was also shown by the fact that it took about 100 times more virus in FCA-treated mice than in controls to kill 50 per cent of the animals. An important observation was that, in FCA-treated mice, the titer of interferon in the spleen rose rapidly after virus inoculation to a significant level and actually preceded the peak of viral replication, while it followed the latter in control animals. A wax D preparation from M. tuberculosis, containing lipid, polysaccharide and peptide moieties exerted essentially the same activities as FCA. It is important to note that in these positive experiments, the killed bacilli or their wax fraction were injected as an emulsion in mineral oil and that two injections were performed 1 week apart. We have found (unpublished observations) that a single treatment of mice with delipidated Mycobacterium phlei, in aqueous suspension, shortly (48 hr to 2 hr) before viral challenge did not significantly protect the animals against infection with encephalomyocarditis (EMC), routine hepatitis, herpes simplex or influenza viruses. Relatively little information is available, on the other hand, concerning the activity on virus infections of the various fractions which have recently been isolated from Mycobacteria by several groups. A water soluble fraction consisting of a polysaccharide bound to a peptidoglycan, with a molecular weight of 15,000 to 30,000, extracted from M. tuberculosis hominis, was shown to protect mice against EMC virus infection when administered i.v. (at doses ranging from 0.5 to 4.5 mg/kg) 2 hr before viral challenge by the s.c. route (Werner et al., 1975); this same substance was without effect against respiratory infection with influenza virus when administered intranasally 2-5 hr before challenge. Only indirect evidence is available concerning an antiviral activity of the MER (methanol extraction residue) fraction of Mycobacteria, a powerful immunostimulating agent widely used in tumor immunotherapy trials in man: mice that had been injected with MER remained free of symptoms during outbreaks of a naturally occuring pneumonitis, of presumed viral origin, which affected animal quarters in which they were housed (Weiss et al., 1964). Similarly there does not seem to exist any information about possible antiviral activities of cord factor (trehalose-6,6'dimycolate), a glycopeptide produced by mycobacteria which, in addition to exerting antitumor immunopotentiation, has been shown, together with other synthetic trehalose-6,6'-diesters, to enhance resistance of mice to infection with Klebsiella pneumoniae and with Listeria monocytogenes when administered in oily emulsion 14 days before challenge (Parant et al., 1978). The minimal structure required for the adjuvant activity of mycobacterial cell wall preparations has recently been shown to consist of a muramyldipeptide, viz. N acetylmuramyl-L-alanyl-D-isoglutamine (MDP), of which various analogs have been synthesized (Adam et al., 1976). While MDP exhibits, in addition to its adjuvant effect on antibody production and delayed type hypersensitivity, an enhancing effect on resistance of mice to bacterial infections (Chedid et al., 1976), even when administered orally, its activity on viral infections has apparently not been investigated. It is known, on the other hand, that MDP, injected together with a vaccine consisting of sub-units of influenza virus, exerts, in the mouse and in the hamster, a definite adjuvant effect on the production of antibodies to the virus hemagglutinin (Audibert et al., 1977; Webster et al., 1977). In view of the remarkable effects of infection with a live attenuated Mycobacterium strain like BCG on non-specific resistance to virus infections (at least in laboratory models), it is hoped that more information will soon become available on the activity of the mycobacterial products which exhibit many of the immunopotentiating effects of the whole bacilli. Direct stimulation of the mononuclear phagocyte system is the most likely mechanism accounting for the antiviral activity of BCG; in addition, in

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animals inoculated with this agent, there appear after some time specifically sensitized T lymphocytes which, through interaction with the microbial antigen persisting i n their organism, are induced to release factors capable of activating macrophages. Such a phenomenon may be difficult to reproduce with a nonliving fraction, unless the substance can actually persist in the tissues or be administered in a way which will make a similar reaction possible. In other words, BCG takes at least 10 days after inoculation to enhance resistance against viral infections, but this state of enhanced resistance is long-lasting, while a mere primary and transient stimulation of the macrophages will only have a short-lived effect on resistance. Repeated administrations of the nonliving immunopotentiating substances may, however, lead to a more prolonged and marked effect. In this connection, one may recall that in the experiments showing a sparing effect of Freund's complete adjuvant on the infection of mice with foot-and-mouth disease virus, this substance, in an oily emulsion, was administered twice, 10 days and 3 days before viral challenge. 3.4. BRUCELLA AND BORDETELLA Microorganisms of the genus Brucella, which cause disease in man, cattle, sheep, swine and other animal species, and Bordetella pertussis, the etiologic agent of whooping cough in man, share with Mycobacterium tuberculosis the capacity of surviving and dividing inside phagocytic cells. Their continued presence within macrophages induces the formation of granuloma and, as a result of interaction between macrophages and sensitized lymphocytes, macrophage 'activation' and delayed type hypersensitivity reactions take place conspicuously. It is not surprising therefore that Brucella and Bordetella organisms, as well as products extracted from them, have been intensively studied as 'immunostimulating' agents. Live Brucella abortus was the first bacterium shown to induce the appearance of circulating interferon in mice and chicks following parenteral administration (Stinebring and Youngner, 1964; Youngner and Stinebring, 1964). In contrast to the short duration (from 6-12 hr to 24 48 hr after injection) of this interferon response, protection of mice infected with B. abortus against viral infections can last several weeks (Billiau et al., 1970). Studies on the mechanism of enhanced resistance of live B. abortus-inoculated mice to i.p. infection with the Mengo strain of encephalomyocarditis virus (Muyembe et al., 1972) have shown that stimulation of the phagocytic and virucidal activity of the peritoneal macrophages and of the blood monocytes was a more valid explanation than serum and tissue interferon induction for this increased resistance. The resistant state lasted for at least 3 weeks; when the bacteria were inoculated by the i.p. route passage of the virus from the peritoneal cavity was inhibited; this was not observed when the bacteria were inoculated intravenously but, in that case, replication of virus in the spleen was decreased. Heat-killed B. abortus was shown to induce in mice, following i.p. inoculation, a 'virus-type' interferon response (with serum peak titers at 6.5 hr) as well as a state of increased resistance against i.p. challenge with the Semliki Forest strain of togavirus (Feingold et al., 1976). Extraction of heat-killed B. abortus with a mixture of chloroform-methanol (CM) yielded an insoluble residue (extracted cells) and a soluble extract: neither of those substances induced interferon production or afforded protection against viral challenge. On the other hand, extraction of live B. abortus with aqueous ether yielded a nonviable insoluble residue with low toxicity, named BRUPEL, which induced interferon and protected mice against viral challenge; extraction of BRU-PEL with CM again destroyed these activities (Youngner et al., 1974). Full activities were restored when the CM extracts were recombined with the extracted cells; furthermore, a CM extract of Escherichia coli was also capable of restoring activity to the extracted B. abortus cells. One may thus conclude that the interferoninducing and antiviral properties of B. abortus reside in a CM-extractable component, which is common to B. abortus and E. coli, and in an unextractable component which is unique to B. abortus. The CM extract from Brucella was shown to contain 92 per

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cent lipids. Since virus challenge, in these experiments, was performed shortly after injection of the preparations, i.e. at the time when interferon was present in the blood, one cannot determine whether these non-viable preparations are as capable as live B. abortus to induce a long-lasting state of antiviral resistance. This was, however, demonstrated to be the case in experiments performed with the BRU-PEL extract mentioned above (Kern et al., 1976). The kinetics of the interferon response to BRU-PEL was similar to that induced by viruses or double-stranded polyribonucleotides and, given shortly before intranasal infection with herpes simplex virus type 2 virus (HSV-2) in 3-week old mice, or i.p. infection with encephalomyocarditis virus (EMC) in adult mice, the substance markedly protected them against mortality. In adult mice infected i.p. with HSV-2, protection by BRU-PEL was significant when it was administered by the same route 1 day before infection, inexistent when treatment was performed 4 days before infection but again significant when injection took place 7, 10 or 14 days before viral challenge. Altogether when BRU-PEL was administered as an interferon inducer (i.e. shortly before infection), its antiviral activity was less marked than that of poly(I).poly(C), but when it was administered several days before infection, its antiviral activity was comparable with that of live Brucella. As in the case of the latter, the conclusion is that this substance (which is insoluble and may therefore persist in macrophages having ingested it) protects against viral infections through a mechanism of stimulation of the phagocytic cells rather than through interferon induction. Intravenous inoculation of mice with Bordetella pertussis vaccine (in which the microorganisms are killed with heat or formaldehyde) causes transient hypertrophy of the spleen and a very striking hyperleucocytosis, in which lymphocytosis predominates (Morse, 1965). The increase in circulating lymph0cytes probably results from release of cells from various lymphoid organs; a lymphocytosis-promoting factor (LPF) has recently been isolated from Bordetella and shown to exert a mitogen-like activity in vitro on murine lymphocytes (Kong and Morse, 1977). Mice inoculated with B. pertussis show enhanced humoral immune responses to heterologous antigens (Floersheim, 1965) while cell-mediated immune responses tend to decrease (Finger et al., 1967); furthermore, an 'endotoxin-like' interferon response is induced (Borecky and Lackovi~, 1967). In spite of the recognition of these various activities, the only investigation we are aware of on the effect of B. pertussis on a murine virus infection was the demonstration (Anderlik et al., 1972) that adult mice infected intracerebrally with 100 LDs0 of lymphocytic choriomeningitis virus (LCM), and treated intravenously on the same day with B. pertussis vaccine, had a definitely longer survival time than control mice similarly inoculated with LCM virus but untreated. Thus, in a particular case where cell-mediated immune (CMI) responses are detrimental to the host by causing encephalitis, an immunopotentiating agent with selective activity (enhancement of humoral immunity, inhibition of CMI) may exert a favorable activity. 3.5. CORYNEBACTERIUMPARVUM The immunostimulating activities of heat--and formaldehyde--killed Corynebacterium paroum or granulosum microorganisms have aroused considerable interest because of their potential application to nonspecific immunotherapy of cancer, which was demonstrated in several experimental and clinical studies. C. parvum is presently used in several oncology centers throughout the world and it is of more than academic interest to know its activity on virus infections. C. parvum injected i.p. (12 or 25 mg/kg) to mice 7, and again 2, days before infection by the same route with encephalomyocarditis virus (EMC) greatly enhanced survival of the animals (Cerutti, 1974). It was shown, in addition, that cell-free peritoneal exudate obtained from C. paroum-treated mice 2-5 days after injection contained a factor capable of inhibiting the replication of EMC and vesicular stomatitis virus in L cell cultures; several characteristics distinguished this factor from interferon. Intraperitoneal injection of C.

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p a r v u m (10 mg/kg) 6-8 days before i.p. inoculation of mice with herpes simplex type 1 virus (HSV-1) protected them from encephalitis and death (Kirchner et al., 1977).

Mice immunosuppressed with cyclophosphamide and showing enhanced susceptibility to HSV-1 infection were also protected by C. p a r v u m treatment; treatments performed shortly before or after virus inoculation were ineffective. Essentially similar results were obtained in another study (Glasgow et al., 1977): mice treated 7-10 days before inoculation of virus were protected against lethal infections with HSV-2, EMC, murine cytomegalovirus and Semliki Forest arbovirus. Moreover, i.p. injection of C. p a r v u m protected mice against i.p. or intranasal infection with EMC virus, showing that the immunostimulant exerts a systemic, rather than local, effect. Enhanced resistance against HSV-2 could be transferred to recipient mice by peritoneal exudate cells from C. acnes-treated animals. The same authors (Morahan et al., 1977) showed that suppression of macrophage function by in vivo treatment with silica increased susceptibility of mice to HSV-2 infection by a 10- to 100-fold factor, but that such a treatment, causing early and sustained viral replication in visceral organs, did not inhibit the antiviral activity of C. acnes. It is interesting to note that a similar observation, suggesting that macrophages may not play a major role in the immunostimulating activity of Corynebacteria, was made with respect to the antitumor activities of C. p a r v u m (Biozzi et al., 1978). Unpublished results from our laboratory are generally in agreement with those of the studies summarized above: we found that adult mice were protected against lethal subcutaneous infection with a mouse-adapted C-type strain of foot-and-mouth disease virus or against lethal intranasal infection with Semliki Forest virus when they received a single i.p. administration from 11 days to 1 day before infection; very small doses of C. p a r v u m (2 mg/kg i.p.) enhanced resistance against murine hepatitis virus, also inoculated i.p., even when treatment preceded infection by only 6hr. With respect to subcutaneous infection of mice with EMC virus, a single intravenous inoculation of C. p a r v u m provided higher enhancement of resistance against lethality when it was performed 6 days, rather than 4 days, before virus inoculation and, in both cases, there was a clear cut dose-effect relationship between the amount of C. p a r v u m injected and the degree of protection. Intranasal administration of minute doses of C. p a r v u m (0.5 or 2mg/kg), 1 day before exposure to an aerosol of A0 influenza virus, significantly enhanced survival of the mice. On the other hand, in the case of i.p. infection with HSV-I, protection of the mice was evident only when treatment with C. p a r v u m was performed at least 5 days before virus inoculation; in vivo transfer of increased resistance could be made with spleen or peritoneal cells from treated mice. Recently, enhanced resistance against infection with Junin virus (an arenavirus which is the etiologic agent of Argentine hemorrhagic fever in humans) was reported in newborn mice treated with C. p a r v u m (Budzko et al., 1978) and it is noteworthy that the kinetics of optimal activity in this system (in which both C. p a r v u m and the virus were injected intraperitoneally) was quite different from that seen in the experiments described above: maximal protection was afforded when C. p a r v u m was administered simultaneously with the virus, a small but significant degree of protection was induced by C. p a r v u m given 3 or 6 days after infection, while treatment 3 days before infection was ineffective. Furthermore, brain Viral titers at various times after infection were comparable in control and C. p a r v u m treated mice. Depression of macrophages by silica injection in vivo was shown to actually enhance resistance to Junin virus infection, suggesting that the protective effect of C. p a r v u m is unlikely to be due, in this system, to its capacity to stimulate macrophages. Neonatal thymectomy had been previously shown to prevent the onset of the disease caused by Junin virus in newborn mice (Weissenbacher et al., 1969) and it is tempting to speculate that, in this system, C. p a r v u m exerts its protective activity by inducing suppression at the level of the thymus. One must also note that, in the case of infection with HSV-2, newborn mice, in contrast to adult mice, were not protected by C. p a r v u m injection. One must therefore conclude that C. p a r v u m may exert its antiviral activities through

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different sorts of mechanisms, depending on the nature of the host-virus immunological and immunopathological relationship in the experimental systems in which this agent is being tested. What the experiments with Junin virus clearly indicate is that C. paroum can exert a protective effect even against an infection in which immunological reactions, rather than viral replication in target organs, play a major role in the pathology. An insight into one of the possible mechanisms of immunopotentiation by C. p a r v u m is provided by a study of its effect on natural killer (NK) cell activity against tumor cells in mice (Ojo et al., 1978): when given intravenously, C. parvum caused a dramatic decrease in NK cell activity, whereas, administered intraperitoneally, it caused a sharp increase in rapidly cytolytic effector cells, which were also shown to be NK cells, among the peritoneal exudate cells. One may recall that, in most studies on the antiviral activity of C. parvum, this agent was indeed administered by the intraperitoneal route and that NK cells are known to be able to lyse virus-infected cells. Two very recent reports have brought important additional information on the possible mechanism of the antiviral activity of C. parvum. On the one hand, it was shown (Hirt et al., 1978) that mouse spleen cells produced considerable amounts of interferon when tested in vitro 5-20 days after in vivo injection of C. p a r v u m ; the highest levels of interferon were obtained when spleen cells from C. paroum treated mice (10 mg/kg i.p.) were again challenged in vitro with C. p a r v u m (20/~g/ml). The interferon induced by C. p a r v u m behaved like 'immunological' (type 2) interferon with respect to its sensitivity to acid pH and lack of neutralization by anti-'viral' (type 1) interferon serum. On the other hand, evidence has been presented that interferon and interferon inducers (Newcastle disease virus, tilorone, statolon) can markedly enhance NK (natural killer) cell activity in mice (Gidlund et al., 1978). The kinetics of induction of NK cell activity after injection of tilorone or statolon was shown to parallel the known kinetics of interferon induction and injection of potent murine interferon preparations also led to a marked increase of NK cell activity in vivo. Furthermore, anti-interferon globulin, when injected into mice, inhibited the capacity of tilorone and Newcastle disease virus to enhance NK cell activity in vivo; it also inhibited, but to a lesser degree, the NK cell activity of peritoneal exudate cells taken from mice injected with C. parvum. It is therefore possible to conclude that, at least in the experimental systems in which this activity requires injection of C. p a r v u m several days before infection, the antiviral activity is due to stimulation of NK cells, probably mediated in part through the production of interferon rather than stimulation of macrophages. It is also noteworthy that, in the case of infection of mice with HSV-2, two treatments with C. parvum, the first 7 days, and the second 1 day, before infection are more effective than a single treatment 7 days before, while a single treatment 1 day before is ineffective (Zerial and Werner, unpublished). On the other hand, local macrophage stimulation might explain the activity of C. p a r v u m on those viral infections in which it has shown some efficacy even when administered once shortly before infection. Several attempts have been made to isolate from C. p a r v u m cell wall material or other constituents and to demonstrate that they could exert the same immunopotentiating activities as the whole organism. With respect to the activities on tumor development, these attempts appear to have been generally unsuccessful, but watersoluble substances extracted from delipidated cells of C. p a r v u m - - i n c l u d i n g a fraction of relatively low molecular weight (6000 daltons) of peptidoglycan nature--were shown to exert some of the activities of the whole cells, in particular enhancement of resistance of mice against EMC virus infection (when administered intravenously 2 hr before s.c. inoculation of the virus) and against the plasma variant of Moloney sarcoma virus--as judged by inhibition of splenomegaly 3 weeks after virus ino c u l a t i o n - w h e n administered i.v. 4 days before infection (Migliore-Samour et al., 1974). It would be interesting to test the activity of such fractions in a system like

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herpes virus infection, which is known to respond to whole C. p a r v u m preparations only when they are administered several days before infection. It may well be that, in the case of an infection like EMC virus, mere transient stimulation of the macrophages is sufficient to afford increased resistance. In spite of the impressive experimental evidence in favor of an antiviral activity of C o r y n e b a c t e r i u m p a r v u m , but possibly because of the known complexity of its activity mechanisms and of its side-effects, it does not appear that clinical studies have been performed to demonstrate a beneficial effect of this agent in viral illnesses of humans. The only study we are aware of consisted in injecting C. p a r v u m intradermally to asymptomatic chronic carriers of hepatitis B surface antigen (HBsAg), to persons with serum antibodies to HBsAg (anti-HBs) and to control individuals without HBsAg or anti-HBs (Papaevangelon et al., 1977): C. p a r v u m caused a significant increase of anti-HBs titers in persons with such preexisting antibodies, but anti-HBs responses were not induced in carriers and HBsAg was not eliminated, its titer remaining practically unchanged. It has also been suggested, but not established, that the benefit derived from C. p a r v u m treatment in some forms of cancer may to some extent be due to the enhanced resistance of these immunologically compromised patients against viral and other infections. 3.6. TRANSFER FACTOR Strictly speaking, the term transfer factor (TF) refers to a dialysable extract of leucocytes, which can transfer specific cellular immunity from a skin test positive donor to a skin test negative recipient. Although first considered, according to this definition, as a specific agent, transferring reactivity only to antigens or haptens to which the leucocytes donor is sensitive, TF has gradually come to be regarded as being able to enhance in a less specific manner induction or expression of cell mediated immunity ('adjuvant' effect). Several attempts have been made to use TF therapeutically in persistent viral infections and various degrees of success have been reported in progressive vaccinia, herpes zoster, 'giant cell' measles pneumonia, subacute sclerosing panencephalitis, chronic active hepatitis and cytomegalovirus retinitis (see review by Mazaheri et al., 1977). In all such studies, evidence for activity of TF was largely anecdotal, since they were not double-blind controlled trials and since most such diseases go through natural cycles of relapse or remission. A small double-blind trial of TF was performed in eight patients with chronic active hepatitis: all four patients who received TF from donors who had recovered from hepatitis B and A showed biochemical improvement (fall in serum transaminase levels) and some histological improvement, but HBsAg remained present in the serum (Schulman et al., 1976). On the other hand, a double-blind study was undertaken to evaluate TF efficacy in patients with severe recurrent herpes simplex type 1 (six patients) or type 2 (twenty-two patients). TF had been obtained from eight donors who showed evidence of humoral and cellular immunity to HSV-1 and HSV-2 antigens but were free from recurrences. Each patient received TF or saline subcutaneously every 2 months for a total of six doses. Results were entirely negative: TF treatment did not decrease the numbers and severity of recurrences when compared with patients receiving the placebo and, furthermore, TF did not change the in vitro response of the recipients' lymphocytes to HSV antigens (Oleske et al., 1978). Interestingly enough, subjective improvement was reported by two-thirds of the patients both in the placebo and the TF group. The use of experimental models could help in clarifying the possible usefulness of TF in the treatment of some viral illnesses; e.g. marmosets have been protected from fatal HSV-1 infection using dialysable leucocyte extract prepared from a human donor with marked cellular immunity to the viru~ (Steele et al., 1976). 3.7. THYMIC HORMONES (OR FACTORS) There is little doubt nowadays that the thymus fulfils at least part of its action

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through the operations of soluble mediators; thymic hormones (or factors) is the term applied to substances which are produced by the thymus gland and which act within the thymus, or elsewhere, to induce T-cell differentiation (see review by Bach, 1978). Thymic factors comprise polypeptides of various degrees of purity and identification and which are called by the teams investigating them: thymosin (A. L. Goldstein), thymopoietin (G. Goldstein), thymic humoral factor, THF (Trainin) and serum thymic factors, FTS (J. F. Bach). Thymic factors could be useful for treating patients with congenital or acquired defective T-cell mediated immunity which would make them especially prone to some virus infections. The only report to date of such an application described THF therapy in four children who were treated with immunosuppressive cytostatic drugs for lymphoproliferative malignant disease and had come down with varicella (Zaizov et al., 1977). It is known that such cases exhibit an increased risk of developing severe forms of generalized varicella (mortality around 7 per cent, vs 0.1--0.4 per cent in the general population). In this study, the patients received daily intramuscular injections of THF for 5-16 days, starting on the first day of the varicella infection. All four children recovered uneventfully from this infection; therapy was shown to increase significantly the number of peripheral blood lymphocytes and of T-rosette forming lymphocytes. 4. INTERFERON AND INTERFERON INDUCERS The topic of interferon and interferon inducers as antiviral agents is comprehensively treated in other sections. It will suffice to recall here that many of the immunopotentiating substances described in this review are capable of inducing the production or release of interferon, and that, on the other hand, interferon as well as classical interferon inducers are known to exert various immunomodulating effects. Double-stranded interferon-inducing polyribonucleotides, like poly(I).poly(C), exert immunostimulating and adjuvant activities that resemble those of endotoxins; a simple synthetic interferon inducer, like tilorone, has been shown to enhance antibody production while depressing cell-mediated immune responses. Interferon itself has been shown, under some experimental conditions, to inhibit antibody production (Gisler et ai., 1974) and to inhibit DNA synthesis induced in lymphocytes by phytohemagglutinin or by allogeneic cells (Lindahl-Magnusson et al., 1972), while also enhancing the specific cytotoxicity of sensitized lymphocytes for allogeneic target tumor cells (Lindahl et al., 1972) and the expression of histocompatibility antigens on lymphoid cells (Lindahl et al., 1976) and on nonlymphoid cells (Vignaux and Gresser, 1978). On the other hand, murine interferon can enhance the phagocytic activity of mononuclear cells from the mouse peritoneal cavity (Huang et al., 1971) and, more recently, evidence was presented that interferon and some interferon inducers can considerably enhance natural killer (NK) cell activity in mice (Gidlund et ai., 1978). Evidence is thus accumulating that the interferon system plays a major role in many immunological events which are associated with viral infections and the possible interactions between antiviral immunomodulating agents and this system certainly deserves to be studied in great detail, although it is likely that, in most cases, the antiviral activity exerted by immunopotentiating substances is not directly and solely mediated through the interferon they induce.

5. SYNTHETIC SUBSTANCES 5.1. COPOLYMER PYRAN, POLYCARBOXYLATES

Pyran is the name given to a copolymer of divinyl ether and maleic anhydride, to which a high density of negative charges gives a polyanionic nature, as is the case also JPT Vol.6. No. 2--C

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with dextran sulfate or with poly(I).(C). It has been shown to induce interferon in mouse and man, to stimulate or to depress various immune responses, to prevent adjuvant-induced arthritis in the rat and .to enhance macrophage functions (particularly cytostatic activity for tumor cells). It is active, by parenteral routes, against many transplanted, viral or carcinogen-induced tumors in the mouse and is presently undergoing clinical trials in cancer patients (Regelson et al., 1978). It appears that toxicity of pyran preparations is, to a large extent, related to their molecular weight, the polymers with the lowest molecular weight (15,000) being the least toxic, while retaining their antitumor activity (Breslow et al., 1973). A pyran preparation (mol. wt not indicated) was shown to protect both normal mice and mice which had been adult thymectomized, lethally irradiated and bone marrow reconstituted (Tx B mice) against mortality following intravenous (i.v.) or intravaginal infection with herpes simplex type 2 virus (HSV-2) (Morahan and McCord, 1975). Tx B mice were found to be about ten times more sensitive to this infection than normal mice. The protective effect required that the compound be administered i.v. or i.p. 24 hr before viral challenge: in pyran-treated mice, virus appeared later and in lower titers in the spinal cord than in the control animals (McCord et al., 1976). While pyran and Corynebacterium p a r v u m were both effective in protecting mice against i.v. infection with HSV-2, only pyran showed activity against intravaginal infection; depression of macrophage function by in vivo administration of silica, although markedly enhancing the sensitivity of the mice to HSV-2 infection by the i.v. route, did not inhibit the activity of pyran or of C. p a r v u m (Morahan et al., 1977). The possible mechanisms of the protective effect of pyran on intravaginal infection of mice with HSV-2 have been analyzed (Breinig et al., 1978): treatment effectively limited viral replication in the vaginal area and survival of mice was correlated with elimination of the early replication of HSV-2 in this area and thus prevention of its spread to the central nervous system. No evidence was found for increased neutralizing antibody production to HSV-2 in pyran-treated mice; on the contrary, antibody production in these mice was delayed and decreased, as was HSV-2 specific delayed hypersensitivity response. These decreased humoral and cell-mediated immune responses in pyran-treated mice were considered to be a reflection of the early decreased growth of the virus. On the other hand, adherent peritoneal cells stimulated by in vivo i.p. treatment with pyran were found to possess antiviral activity in vitro and to transfer resistance to suckling mice in vivo. Since it is likely that their mode of action is similar to that of pyran, one may also mention here the antiviral activity in vivo of polycarboxylates, such as polyacrylic acid, polymethacrylic acid and chlorite-oxidized oxyamylose. The general structure of polyacrylic acids is: [---CH2---CH(COOH)---]~. Polycarboxylates have been shown to exert many effects on host defense mechanisms, such as interferon induction, pyrogenicity, stimulation of macrophage activities, adjuvant activity on humoral and cell-mediated immune reactions, and, consequently, they protect animals against viral, bacterial, fungal and protozoal infections and against grafted tumors and leukemias (see review by De Clercq, 1973). The fact that their antiviral activity is not solely mediated through their interferon-inducing properties was shown by the kinetics of this activity: for instance, carbopol, a polymer of acrylic acid cross-linked with allylsucrose, imparts resistance to mice which receive an i.p. injection of the compound 1-4 days before intravenous challenge with vaccinia virus or intranasal challenge with herpes simplex virus, whereas a modest peak of interferon activity in the serum occurs 20-40 hr after treatment (De Clercq and Luczak, 1976). The ratios between toxic (i.e. causing significant mortality in uninfected mice) and active doses (enhancing resistance to intranasal challenge with herpes virus), by the i.p. route were found to be 5 for carbopol and polyacrylic acid and 25 fol: chlorite-oxidized oxyamylose. It is noteworthy that, like BCG or C. p a r v u m , polyacrylic acids increased the sensitivity of the mice to the lethal effect of endotoxin injection when they were administered 14 to 7 days before the latter (R. Maral, personal communication). Polymethylmethacrylate has also been reported to exert adjuvant activity on im-

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munization of mice and guinea pigs with influenza vaccines consisting of viral subunits (Kreuter et al., 1976). 5.2. LEVAMISOLE

Tetramisole and its levorotatory isomer, levamisole, have been known for more than a decade for their high effectiveness as anthelmintics in man and domestic animals; they are active against most pathogenic nematodes and levamisole is widely used in man, especially for treating ascariasis. It was more recently found (Renoux and Renoux, 1971) that mice immunized with an only moderately active anti-Brucella vaccine were completely protected against challenge with live Brucella bacilli when they had received levamisole or tetramisole together with the vaccine. This observation aroused considerable interest, since it was the first time that a synthetic agent was reported to exert an immunoadjuvant effect and since it was readily possible to undertake clinical trials with a drug which was already in use in humans. To summarize the mass of experimental and clinical data which have been published about the immunomodulating activities of levamisole would be a formidable task and the reader is referred to recently published reviews (Renoux, 1978; Symoens, 1978); we shall borrow from these reviews the following conclusions: "Levamisole stimulates recruitment and functions of macrophage and T cell under a narrow range of doses and time of administration in normal or immunoimpaired man and animal. Strain, sex, age and antigen modulate the activity of levamisole from enhancement to inhibition; stimulation is mediated through a serum factor able also to promote thymocytes in thymusless mice (Renoux). Studies on isolated phagocytes and lymphocytes show that levamisole influences virtually all cell functions involved in cell mediated immune responses. It seems to be the first member of a new series of simple chemical agents that mimic hormonal regulation of the immune system (Symoens)."

Chemical structure of levamisole, tetramisole

Before reviewing clinical data concerning immunoenhancing activities of levamisole in some viral infections, we shall analyze experimental evidence in favor of such an activity. This can be simply done by stating that, to our knowledge, the only positive result to be reported was that levamisole treatment significantly increased survival rates of suckling rats inoculated intraperitoneally, at 10 days of age, with a strain of herpes simplex type 2 virus (HSV-2) (Fischer et al., 1975; Fischer et al., 1976). Survival, 14 days after infection, was 4 per cent in control rats, 30 per cent in animals receiving levamisole subcutaneously (3 mg/kg, 4 hr and 24 hr after infection), 20 per cent in animals treated with adenine arabinoside, and 11 per cent in rats receiving combined treatments with both drugs--suggesting antagonism between their activities. Splenectomy studies indicated that levamisole requires an intact spleen if it is to provide protection against encephalitis caused by HSV-2 and it is possible that the drug enhances splenic entrapment of the virus, thus preventing its dissemination. On the other hand, studies in mice seem to have been uniformly negative, at least with respect to protection by levamisole of newborn or adult mice infected intraperitoneally or intravaginally with HSV-2 (Start et al., 1976; Morahan et al., 1977). In our own laboratory (unpublished results), using several types of virus infections in adult mice, we have failed to observe any significant or consistent activity of levamisole, except for a slight increase in survival time in animals infected with murine hepatitis virus or with influenza A2 virus and receiving levamisole in their drinking water throughout the experiment. Experiments in larger animals have proven more encouraging. Single intramuscular doses of 1-6 mg]kg levamisole were given to 149 cattle and twenty-six goats affected

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with foot-and-mouth disease (type C virus): within 48 hr, there was dramatic improvement of symptomatology in the treated animals as compared with controls (Rojas and Olivari, 1974). Levamisole was also tested for therapeutic activity in Aleutian disease of mink, a persistent infection caused by a parvovirus. The drug was incorporated into the mink ration (1 mg/kg daily for 14 days, followed by 14 days without treatment and again 14 days of treatment): the health status of the treated animals after 3 months was improved, as indicated by an increased body weight, lower incidence of bile duct proliferation and periportal infiltrates and reduced hypergammaglobulinemia (Kenyon, 1978). As regards clinical evidence on the efficacy of levamisole in human viral diseases, several studies have been performed in patients afflicted with recurrent herpes labialis or recurrent herpes progenitalis. Many of these studies were uncontrolled, i.e. they did not include placebo-treated subjects: clinical improvement and decreased frequency of recurrences were reported in several cases of facial, labial, corneal and genital herpes (Kint and Verlinden, 1974; Kint et al., 1974; Lods, 1976). Clinical improvement was associated with enhanced in vitro response of lymphocytes to herpes virus antigen in one study (O'Reilly et al., 1977) and with a decreased response in another study (Spitler et al., 1975). More recently, the results of a double-blind, controlled trial of levamisole in the treatment of recurrent herpes labialis were reported (Russell et al., 1978). It included ninety-nine subjects with recurrent circumoral herpes at least four times a year; forty-eight subjects received the drug (2.5 mg/kg) on two consecutive days each week for 6 months and the fifty-one controls were given a placebo. During the 6 months of the study, there were no significant differences between the two groups in the duration or severity of the lesions or in the subjective assessment of drug efficacy by the patients; as far as the last parameter is concerned, it is noteworthy that 60 per cent of subjects on placebo evaluated this treatment as excellent! No difference was found between the levamisole and control groups in their lymphocyte responses to the virus or to PHA. It must be noted that, before treatment, the frequency of lesions in the levamisole group was higher than in the control group: when this factor was taken into account, there was a significant difference between the two groups, with the group receiving levamisole demonstrating a greater improvement than controls. Clinical studies have been concluded to show a beneficial effect of levamisole treatment in various other viral diseases: an uncontrolled trial showed rapid regression of warts (due to molluscum contagiosum virus) in nine out of ten children (Helin and Bergh, 1974); a double-blind study on seventy children with frequent respiratory diseases during the winter months (actual viral etiology was not assessed by laboratory tests) showed decreased frequency and severity of these infections in the levamisole-treated group (Van Eygen et al., 1976); a placebo-controlled trial was performed in fifty consecutive patients with acute viral hepatitis: disappearance of antibodies against the core antigen was faster in levamisole- than in placebo-treated patients with type B hepatitis and, by the end of the third month, only one of the twenty-three levamisole-treated patients had not recovered from the acute disease (as judged by transaminases, HBsAg presence and histology), vs eight out of twentyseven patients in the control group (Par et al., 1977). In view of the hypothetical viral etiology of Crohn disease, improvement of this condition by levamisole therapy is worth mentioning (Bertrand et al., 1974). In conclusion, it appears difficult, at the present time, to get a clear and definite view of the possible usefulness of levamisole in immunomodulating therapy of viral illnesses. The contrast between the rather negative results of experiments in laboratory animals and the often encouraging data from clinical trials may be due in part to the fact that, in the first case, the drug was given prophylactically, while it was administered in a therapeutic manner to the patients. In the latter case, levamisole may have exerted its beneficial effect by restoring immune responses which had been impaired by the virus infection; better information on this last point might indeed help in selecting the patients who would be more likely to benefit from treatment with levamisole or other immunomodulating drugs.

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5.3. INOSIPLEX Together with levamisole, inosiplex (Isoprinosine ®) differs from the other immunopotentiating substances described in this review by the fact that it has already been extensively used in man, but, unlike levamisole which began its career as an anthelminthic drug, inosiplex was from the start put on the market in some countries as an antiviral agent. Inosiplex is a compound formed from inosine and the para-acetamidobenzoate salt of N,N-dimethylamino-2-propanol in a molar ratio of 1 : 3 0

\I/ H

CH 3

Cl"~

-o-c

H--~--OH HO-- CH 2

CH3

NH I COCH 3

OH

01-1 Structural formulaof inosiplex

Early studies, showing that inosiplex could inhibit cytopathic effect of a rhinovirus or of influenza A2 virus in cell cultures, led some to consider this compound as an antiviral agent in the usual meaning of the term. Inhibition of cytopathic effect was only moderate however, even at high concentrations of inosiplex, and in spite of daily medium change of the cultures to compensate for degradation of the purine portion (see review by Ginsberg and Glasky, 1977). Evidence for an in vivo antiviral activity of inosiplex in laboratory animals was first contradictory, depending on how the animals were treated with the compound. Negative results were reported when this substance was evaluated in a coordinated study at five different laboratories (Glasgow and Galasso, 1972): no antiviral effect could be demonstrated in mice infected with encephalomyocarditis (EMC), type 2 herpes simplex, influenza and rabies viruses, in rabbits infected with vaccinia virus, in cats infected with feline rhinotracheitis and panleukopenia viruses, in ferrets infected with distemper virus and in swine infected with influenza and transmissible gastroenteritis viruses. The only antiviral activity observed was suppression of fibroma virus lesions in rabbits receiving large i.p. doses of inosiplex. In mice, therapy at a dose of 300 mg/kg had been initiated at various times before or after virus inoculation (in most cases early after) and was administered once daily either i.p. or orally. Suspicion that inosiplex did not behave like a classical antiviral agent arose when it was noticed (Muldoon et al., 1972) that treatment initiated 24 hr prior to infection and continued daily thereafter did not increase survival in mice infected intranasally with A2 influenza virus, while treatment started 24hr a~ter infection resulted in significantly increased survival time. Other experiments suggested that optinial activity of inosiplex, administered in a therapeutic manner, i.e. following virus inoculation, required the presence of adequate immune responses in the animal: in mice challenged with a dose of influenza virus causing 60 per cent mortality in untreated controls, inosiplex treatment reduced mortality to 30 per cent while cortisone increased it to 85 per cent, but combined inosiplex and cortisone treatments led to 100 per cent mortality; similar results were obtained when mice were immunosuppressed with antilymphocyte serum (Ginsberg and Glasky, 1977). Direct evidence for an interaction of inosiplex with the immune system was provided by experiments in which mice were immunized with sheep erythrocytes and their spleen cells assayed 4 days thereafter for production of hemolytic plaques in an erythrocyte-agar overlay in

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the presence of various concentrations of the drug: in this in vitro situation, concentrations of 100, 300 and 500 txg/ml of inosiplex significantly increased the number of plaques (Ginsberg and Glasky, 1977). It was also shown (Hadden et al., 1976) that, in in vitro cultures of human peripheral blood lymphocytes, inosiplex, over a concentration range from 0.2 to 250/zg]ml, slightly but significantly augmented proliferation induced by phytohemagglutinin (PHA); in the absence of PHA the drug had no effect on lymphocyte proliferation. Inosiplex has shown some protective effect in experimental virus infections of laboratory animals when care was taken to administer this drug following a therapeutic schedule (i.e. beginning treatment after the animals had been infected) and at appropriate dosage (in most cases, in drinking water, at a 1% concentration). For instance, A/J mice, which are highly susceptible to the lethal effect of an i.p. inoculation of herpes simplex virus type 1, were converted to moderate susceptibility, similar to that of BALB/c mice, when inosiplex was added to their drinking water starting 24 hr after infection (Hadden et al., 1977). In our own studies (Floc'h and Werner, unpublished), in which inosiplex was administered in the same manner, increased survival as a result of treatment was seen in mice inoculated i.p. with murine hepatitis virus or with two strains of herpes simplex type 1 virus but not in mice infected s.c. with EMC virus or through an aerosol of influenza A2 virus. Enhancement of the partial protective effect of a suboptimal dose of tilorone (given 24 hr before infection) was seen in EMC-virus-infected mice receiving inosiplex in their drinking water from the 1st day after infection. There was some difficulty in confirming these various encouraging results in repeat experiments, the actual degree of efficacy of inosiplex being variable from one test to another. An attempt at integrating the antiviral and immunoenhancing effects of inosiplex has been presented recently (Simon et al., 1978). Using a hemadsorption assay, it was shown that at two distinct concentration ranges (from 0.005 to 1.0 t~g/ml and from 10 to 150/xg/ml) inosiplex inhibited infection of HeLa cells with the A0/PR8 strain of influenza virus and also decreased intracellular levels of the virus. This inhibition was moderate however and rarely exceeded 50 per cent (which, with the hemagglutination technique used to measure virus levels, means a twofold dilution difference). At about the same two concentration ranges, inosiplex increased the proliferating effect of concanavalin A and of A2 influenza virus on murine spleen cells cultured in vitro: again, this stimulation was slight, thymidine incorporation being around 1.5 times higher in cultures containing appropriate amounts of the drug than in cultures stimulated by con A or by virus alone. When the effect of inosiplex on influenza virus replication was compared, by linear regression analysis, with its potentiation of virus-induced splenocyte proliferation, the correlation coefficients obtained at the two concentration ranges suggested that both of these effects must have a common biological basis. Furthermore, the authors showed that intraperitoneal inoculation of mice with live influenza A0/PR8 virus caused, 7 days later, a slight decrease below normal levels of in vitro response of their spleen cells to con A; treatment with inosiplex, initiated after inoculation of the virus, normalized this response and, in addition, considerably increased the degree of spleen cell proliferation induced in vitro by the viral antigen. Mice which had received a high dilution of the virus by the i.p. route showed only partial immunity (30 per cent survival) when they were challenged intranasally 20 days later with a lethal dose of virus; their immunity was much stronger (100 per cent survival) when they had been treated with inosiplex from the time of immunization. It has also been shown that inosiplex was able to greatly enhance the antiviral protection afforded by a single dose of murine interferon against lethal infection with EMC virus (Chany and Cerutti, 1977). In these experiments, mice were infected i.p. with 100 LD~0 of virus; the administration of 20,000 units of interferon 1 hr after infection only slightly delayed death; inosiplex alone exerted no effect, but when the animals received 1000 mg/kg i.p. of inosiplex 24 hr before, and interferon 1 hr after, infection, 80 per cent of the animals survived and were resistant to subsequent

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reinfection. In contrast with what was seen in other experimental systems, therapeutic administration of inosiplex (7 hr after the virus, 1 hr after interferon) had no protective effect whatsoever in this system. It appears possible that inosiplex enhances the response of cells to interferon by interacting with their membranes, in a way similar to its enhancing effect on the response of lymphocytes to some mitogens. As inosiplex is a nontoxic substance, there have been many studies on its antiviral activity in man. We shall first review trials performed in volunteers who were artificially infected. Inosiplex was given orally to twenty-two volunteers who, along with twenty-three other volunteers receiving a placebo, were inoculated intranasally with two strains of human rhinovirus (RV-9 and RV-31); treatment had been started 48 hr before infection, daily treatments consisting of 4 times 1.5 g inosiplex daily. Results of this trial were entirely negative: inosiplex treatment exerted no effect on the average clinical score of the colds induced and on laboratory evidence of infection with either or both viruses (reisolation of virus, seroconversion) (Soto et al., 1973). In another study, fifteen volunteers treated with inosiplex (2.5 g twice daily for 10 days, beginning 48 hr before infection) and fifteen placebo-treated volunteers were inoculated intranasally with A/Hong Kong/8/68 (H3N2) influenza virus: treatment with inosiplex did not result in any appreciable modification of the clinical course of the illness and the only demonstrable beneficial effect of the drug was that the total number of virus isolates was slightly but significantly lower in the volunteers receiving it (decrease in virus shedding); acquisition of neutralizing antibodies was similar in both groups (Longley et al., 1973). Prophylactic efficacy of inosiplex was again evaluated in a double-blind study in which volunteers were challenged intranasaUy with rhinovirus type 32 or type 44 (Pachuta et al., 1974); the drug was given orally, at a daily dosage of 6 g, for 2 days prior to intranasal challenge with either virus and for 7 postchallenge days. Results were unimpressive: in both trials (each including nine volunteers on inosiplex and nine others on placebo), the occurrence and severity of the colds were greater in the placebo group but the difference with the drug-treated group was not considered significant; the average numbers of rhinovirus isolations during the postchallenge days were similar in the placebo and in the drug-treated group; postchallenge antibody titers in serum and nasal secretions were not significantly different between the two groups. In view of the experimental evidence that inosiplex behaves as an immunomodulating agent and is more effective when treatment is initiated after than before viral challenge, a double-blind study was performed more recently on the therapeutic efficacy of the drug in rhinovirus infection of volunteers (Waldman and Ganguly, 1977): thirty-nine volunteers were randomly assigned to inosiplex or placebo groups; drug or placebo was started either at the time o f , or 48 hr after, nasal challenge with rhinovirus type 21. Illness was assessed in terms of typical common cold symptoms (sneezing, sore throat, nasal stuffiness, nasal discharge, cough) and infection was determined by virus isolation from nasal washings and serum antibody rises. Evaluation of individual symptoms revealed.a fairly uniform decrease in mean scores for all symptoms in volunteers taking inosiplex as compared with controls (statistically significant for nasal stuffiness and nasal discharge). The illness rate was reduced in the volunteers receiving inosiplex: eleven volunteers among the twenty on placebo wer.e judged to be ill, vs only three out of ten in volunteers receiving inosiplex from the day of challenge and two out of nine in those who started taking the drug 2 days after challenge. The number of volunteers from whom virus was isolated was nearly the same in the placebo and inosiplex groups, but the mean duration of virus isolation was reduced in the treated group and fewer in the latter had serological evidence of infection as determined by antibody rise: these differences were, however, not statistically significant. Interestingly, peripheral blood lymphocytes of volunteers treated with placebo showed 4, 8 and even 21 days after infection a decreased in vitro response to PHA, by comparison with pre-infection values, whereas in individuals receiving inosiplex, PHA response after infection was actually increased.

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As part of a double-blind study of the therapeutic activity of inosiplex on herpes labialis (type 1) and herpes genitalis (type 2), the cell-mediated immune responses of patients were investigated prior to and after initiation of therapy, using as parameters the PHA response of peripheral blood lymphocytes and their ability to produce lymphotoxin following stimulation by PHA (Bradshaw and Sumner, 1977). After a week of treatment, thirteen patients on inosiplex and nine patients on placebo showed an increased response to PHA (and this increase was greater in the drug-treated group), while seven patients on inosiplex and twelve on placebo showed a decreased response to PHA (this decrease being more marked in the placebo group than in the drug group). With respect to changes in lymphotoxin titers, nine patients on inosiplex showed a marked increase vs four placebo-treated individuals, whose increase was small, whereas ten placebo-receiving patients and five inosiplex-receiving patients showed a decrease in such titers. Interpretation of such data is difficult but they suggest that inosiplex acts as an immunomodulating agent in humans and, furthermore, that actual response to this treatment may well vary from one individual to another. Therapeutic and prophylactic-therapeutic efficacy of inosiplex against influenza A (H3N2) challenge infection in human volunteers has been recently assessed (Waldman et al., 1978). This double-blind study was performed on forty-one adult volunteers who were inoculated intranasally with the A/Dunedin/73 strain. Inosiplex, at a daily dose of 4 g (in six administrations of either 1 g or 0.5 g) was started either 2 days prior to challenge or 2 days following it and treatment was continued in both groups for a total of 9 days. When the number of volunteers with ~clinical illness suggestive of influenza was considered in each group, a lower illness rate was seen in each of the two treatment groups compared with placebo; although the infection rate, determined by an increase in serum antibody titer, was not different in the three groups, both frequency and severity of illness were lower in each of the treatment groups compared with placebo. A marked increase in the in vitro response to PHA of peripheral blood lymphocytes was seen 2, 4 and 21 days following virus inoculation in volunteers receiving inosiplex in a therapeutic manner, this increase being more modest in individuals receiving the drug according to a prophylactic-therapeutic schedule and even more discrete (although still significant) in placebo-treated patients. In addition to studies performed in volunteers, clinical trials of inosiplex have been conducted in several areas. According to a recent clinical overview (Glasky et al., 1978), 165 such studies have now been performed, involving a total of 4259 patients with viral illnesses as diverse as influenza, hepatitis A, recurrent herpes simplex, herpes zoster, varicella, mumps and measles. The majority of these trials consisted of open studies, but a number of controlled double-blind studies have been described (hepatitis A, measles, mumps and varicella); the general conclusion to be derived from the accumulated data is that inosiplex, described as both an antiviral and a 'pro-host' drug, with a virtual absence of side effects, exerted a significantly favorable influence on the intensity and duration of symptoms and accelerated recovery. Of particular interest is the beneficial effect observed following inosiplex therapy in open studies performed in forty-five cases of subacute sclerosing panencephalitis (seventeen such patients exhibiting objective improvement) and in cases of dengue hemorrhagic fever. 6. POSSIBLE MECHANISMS OF THE ANTIVIRAL ACTIVITY OF IMMUNOPOTENTIATING SUBSTANCES It is now pertinent to inquire into the mechanisms through which immunopotentiating substances enhance resistance to virus infections; such knowledge may help in defining the potential practical applications of such substances, as well as t h e i r limitations, and also in designing more active and. better tolerated agents. We have seen in the Introduction that the immunological mechanisms which are part of the natural defenses of the host against virus infections are diverse and complex, that they may vary from one virus infection to another, and that they are closely

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interrelated; furthermore, some immune reactions do contribute to the pathogenesis of some viral infections. The purpose one has in mind when using immunopotentiating substances for combating viral illnesses is to enhance those reactions which play a favorable role in the outcome of the infection without stimulating the detrimental ones. One must admit, however, that for most of the immunopotentiating substances which have shown antiviral activity, the precise immunological mechanisms through which they act are not known. Lipopolysaccharides, for instance, cause a variety of reactions, usually 'toxic' in nature, such as stimulation of lysosomal enzymes in macrophages, production of fever, release of interferon from phagocytic cells, thus temporarily creating a situation which is not favorable to viral multiplication and spread in the treated organism. This state of enhanced resistance to viral infection is rather short-lived and requires administration of the LPS a few hours or, at most, a few days before virus inoculation. Intact microorganisms, whether live like BCG or killed like Corynebacterium parvum, cause the formation of granuloma from which, through interaction with the microorganism of specifically sensitized lymphocytes, substances are released which nonspecifically activate cells of the mononuclear phagocyte system: as a result, these cells, in which virus replication normally takes place, oppose a barrier to virus multiplication and spread. This state of enhanced resistance may be long-lived since the mechanism of macrophage activation is self-perpetuating as long as the injected microorganism, or its products, persist in the tissues. In this situation again, injection of the microorganism and sensitization to it must precede virus inoculation, at least by a few days, for effective protection against infection. Interferon inducers may protect against virus infections through local and systemic stimulation of interferon production, but, like lipopolysaccharides, some of them, such as the double-stranded polyribonucleotides, also stimulate macrophage functions and cause hyperthermia, thus creating a temporarily unfavorable environment for the infecting virus. Tilorone, a simple synthetic interferon inducer, has been shown, in addition to causing greatly enhanced levels of circulating interferon within 24 hr after its administration, to stimulate B lymphocytes and antibody production, and to stimulate macrophages, but it also inhibits production of delayed type hypersensitivity reactions by T lymphocytes: do all these mechanisms come into play in the antiviral activity of tilorone? It is interesting to note that tilorone, as well as pyran copolymer (another interferon inducer and immunopotentiating agent) cause the appearance, as early as 48 hr after i.p. injection in the mouse, of cytoplasmic granules in monocytes and polymorphonuclear leukocytes of the peripheral blood and that these granules persist for long periods, although the antiviral state is not as long-lived as in the case of a persisting microbial infection such as BCG. With microorganisms like BCG, with endotoxins or some interferon inducers, the antiviral state appears to be essentially a nonspecific expression of a more or less prolonged general solicitation of the immune system, an indirect benefit from a situation which may be described as temporarily pathological, an increased ability to cope with viral intruders in a h o s t w h i c h has been put in a state of alarm. It is, of course, more difficult to explain in similar terms the much more discrete antiviral effects of immunomodulating substances such as levamisole or inosiplex, which are also more difficult to demonstrate in laboratory animals. Inosiplex, for instance; is known in most cases to be more active when administered shortly after Viral inoculation than before, in contrast to other immunopotentiating substances which, at least in experimental infections, are completely inactive when administered to an animal already infected. It may be therefore that inosiplex enhances defense mechanisms which have been already triggered by the viral infection itself: it would be fruitful, with this and other immunomodulating drugs showing antiviral activity, to analyze their effects on the responses of T and B lymphocytes t o t h e viral infection, i.e. cytotoxic T cells, NK cells, delayed type hypersensitivity and antibody production. Two aspects of the host's response to many immunostimulating substances are

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relevant to their antiviral activity and deserve special consideration: they are the production of delayed type hypersensitivity (DTH) and the inflammatory activity. The latter has been rather poorly documented with respect to protection against virus infections; it is noteworthy that a bacterial phospholipid extract which enhances resistance of mice against infections with extracellular and intraceUular bacteria, but is devoid of inflammatory activities and does not cause the production of granuloma, does not induce resistance against virus infections or against the take and growth of grafted tumors, in contrast to what is seen with BCG, C. p a r v u m or LPS (see review by Fauve, 1978). This finding led its author to study the effect on bacterial, fungal and parasitic infections of the mouse of inflammatory reactions caused locally by subcutaneous injection of a non-diffusible irritating substance (magnesium silicate embedded in a gel of calcium phosphate): such a manipulation greatly enhanced the host's resistance against virulent bacteria and parasites and against malignant cells. Since the pathogens and tumor cells used in that study could not be in direct contact with the inflammatory focus itself, the conclusion is that one or several of the many soluble 'mediators' of inflammation (bradykinin, histamine, prostaglandins, serotonin . . . . ), or still other substances released from the inflammatory focus, were able to stimulate in a nonspecific way the effector cells in favor of the host's resistance against bacterial infections and tumors. Since it appears likely that all granuloma-inducing agents and substances causing inflammation can in part stimulate immune defenses by this common mechanism, the effect of local inflammation on virus infections would be worth studying. Delayed type hypersensitivity (DTH) reactions with an antigen unrelated to the infecting virus is another mechanism of local or systemic enhancement of resistance and, as already mentioned, it may at least partly explain the effect of microbial agents or products (BCG, C. p a r v u m ) which are able to sensitize and to persist in the sensitized host. For instance, it has been shown that in rabbits immunized with Freund's complete adjuvant, induction of a DTH reaction by tuberculin purified protein derivative (PPD) at the site of dermal vaccinia virus infection accelerated elimination of the virus and led to clinical recovery (Lodmell et al., 1976). Low concentrations of acid-labile 'immunological' interferon were found in the skin of uninfected tuberculin-sensitized animals challenged with PPD; high concentrations of acid-stable ('viral') interferon were found in the skin of tuberculin-sensitized rabbits infected with vaccinia virus and challenged with PPD or saline, but the time of appearance of acid-stable interferon was greatly accelerated in the animals challenged with PPD instead of saline. Increased resistance to the lethal effect of a subcutaneous (s.c.) inoculation of encephalomyocarditis virus (EMC) was observed in mice which had been sensitized to sheep red blood cells (SRBC) by s.c. inoculation and challenged by the same route 6 days later with SRBC one day before virus inoculation (Floc'h and Werner, unpublished). It would be erroneous to believe that only those substances which stimulate, i.e. increase or elicit immune reactions in a broad sense, are capable of enhancing resistance to virus infections. Selective depression of some of these immune reactions can also be beneficial, in keeping with the notion that not all the immune reactions to the infecting virus are favorable to the host's survival. For instance, repeated injections of rabbit anti-mouse thymocyte globulin were found to increase the survival rates of mice infected with low doses of influenza A2 virus (Suzuki et al., 1974); and nude mice, which are deficient in T lymphocytes, die later than normal mice following respiratory infection with A0 influenza virus (Sullivan et al., 1976). Such nude mice show also minimal antibody response to the virus and, interestingly, those which do not die do not eliminate the virus, which persists 2-3 weeks in their lungs and spleen. On the other hand, a drug which inhibits B lymphocytes, cyclophosphamide, was shown to lower the mortality of mice infected with a virulent influenza A0 virus (Singer et al., 1972), while administration of the same drug converted a relatively harmless infection with an avirulent strain into a fatal pneumonic illness (Hurd and Heath, 1975). Thus, depending on the nature of the host-virus relationship (of which

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virulence is one aspect), T and B cell responses may be considered as beneficial or harmful to the final outcome of the infection and stimulation or depression of these responses may have widely different results. A better insight into the mechanisms of the antiviral activities of immunomodulating substances (i.e. stimulating, restoring or depressing, according to the case) will be gained through experiments in which such substances are administered, before or after virus infection, to animals in which defined compartments of the immune system are selectively suppressed, e.g. through treatment with immunosuppressive drugs, as a consequence of a genetic defect, following surgical ablation of thymus or spleen or. killing of macrophages with silica. Very important also will be in vivo-in vitro experiments in which, following in vivo administration of the immunomodulating agent to an animal subsequently or simultaneously infected with a virus, lymphocytes and cells of the monocyte-macrophage system will be studied in vitro with respect to a number of functions known to play a role in natural resistance to, and recovery from, that infection:phagocytic activity, toxicity for virus-infected cells, hypersensitivity to the viral antigen (i.e. in vitro proliferation in the presence of this agent), antibody production. Antiviral activity of normal, stimulated or activated mouse peritoneal cells can, for instance, be measured in vitro, by absorbing them to mouse embryo fibroblasts infected with herpes simplex virus, removing nonadherent cells by washing and then measuring the virus yields in the supernatant fluids (Morahan and Kaplan, 1978); using this technique, it was found that stimulation of the peritoneal cells by i.p. injection of glycogen exerted a weak effect on their antiviral activity, while activation with Corynetracterium parvum or pyran copolymer strongly enhanced it. 7. POSSIBLE T H E R A P E U T I C APPLICATIONS OF I M M U N O P O T E N T I A T I N G DRUGS WITH ANTIVIRAL ACTIVITY Thus far, with relatively few exceptions, experimental studies on the antiviral activity of immunopotentiating substances have mostly consisted in treating animals with these substances and demonstrating that they were more resistant than untreated controls to a subsequent virus infection. If we try to deduce directly from the results of such studies the kinds of application to human or veterinary medicine immunopotentiating substances could have in the field of viral illnesses, we are forced to consider these substances as being nothing more than 'nonspecific vaccines', with the added restriction that specifc viral vaccines generally induce long-lasting immunity, while the enhanced resistance against viral infections afforded by nonspecific immunostimulators is of relatively short duration (except in the case of a living infectious agent like BCG). Practically, it appears difficult to envisage frequent and repeated administrations of such substances to healthy subjects in order to maintain constantly their resistance at a high level, inasmuch as many of the immunopotentiating agents presently available are not devoid of often serious side-effects (see review by Werner et al., 1977). There m a y b e some exceptions to this rule, however, such as the possibility of frequent stimulation, by intranasal administration of nonirritating and nonallergenic drugs, of the local immune defense mechanisms against infection with respiratory viruses. Nontoxic drugs which would exert their immunopotentiating activity following oral administration could also be used to raise the level of rdsistahoe of susceptible subjects, young children for instance, against virus infections; for example, as regards the controversial participation of vitamin C (ascorbic acid) in protection against some virus infections, it is noteworthy that inclusion of this vitamin in the drinking water of mice, while exerting no effect on their humoral antibody response, increases T-lymphocyte response to concanavalin A and enhanced response to interferon induction (Siegel and Morton, 1977). Other possibilities for short-term prophylactic use of immunostimulating substances exist in the veterinary field, as a means of protecting young animals (especially cattle and swine) against virus illnesses of complex and multiple etiology which commonly affect them and cause severe

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losses; most such illnesses occur, however, in animals which are immunologically immature and it should first be demonstrated that immunopotentiating agents can exert their activities in such animals. On the basis of available experimental data, one can only speculate as to the possible therapeutic uses, stricto sensu, of immunopotentiating drugs endowed with antiviral activities. As stated previously, the great majority of experiments have been performed in systems in which, because of the rapidly lethal character of the infection, administration of the drug after inoculation of the virus would be ineffective. Although the mechanisms of recovery from a virus infection are not essentially different from those underlying resistance to this infection, one cannot be sure that substances increasing nonspecifically the resistance would facilitate recovery if they were administered to the host after it has been infected. Clinically, BCG and levamisole may have shown some activity in cases of recurrent herpes simplex, when administered during the periods free of overt disease and, to our knowledge, inosiplex is the only immunopotentiating drug which has been claimed to show efficacy when administered in a truly therapeutic way, i.e. after infection with the virus and even after appearance of symptoms. Many virus infections cause at least transient disturbances in the immune system of the infected host and this fact must be kept in mind when attempting to visualize the value of treating viral illnesses with immunopotentiating drugs. Immunologic dysfunction induced by virus infection in man and animals include suppression or inhibition of cell-mediated immunity (temporary depression of delayed type hypersensitivity, enhanced allograft survival, inhibition of in vitro reactivities of lymphocytes), abnormalities in antibody response (suppression or enhancement of antibody production, changes in serum immunoglobulin levels, suppression of tolerance induction) and there are many possible ways through which viruses can cause these dysfunctions: alteration of macrophage functions by viruses replicating within them, destruction of B and T lymphocytes, inhibition of T-cell activation (through cell destruction, receptor blockade, or arrest of macromolecular synthesis), alteration of lymphocyte traffic or stimulation of suppressor cells. All these situations are well documented with several examples (see review by Woodruff and Woodruff, 1975). For instance, in man, measles has been known for a long time to cause temporary loss of tuberculin skin reactivity and, following immunization with live measles vaccine, this anergic state may last several weeks; similarly, rubella infection or vaccination with live virus suppresses for several days the in vitro response of peripheral blood lymphocytes to phytohemagglutinin (McMorrow et al., 1974); during acute influenza illness, lymphopenia is observed, which may last up to 4 weeks, and blastogenic responses of lymphocytes to phytohemagglutinin (PHA) and concanavalin A are depressed and remain so for 4 weeks after infection (Dolin et al., 1977). In patients with hepatitis A or B, PHA-responsiveness of peripheral blood lymphocytes was impaired during the first week after the onset of jaundice and there was less marked, but prolonged, impairment for a further period of 6-10 weeks; serum from patients with hepatitis A or B was found to contain an inhibitor of lymphocyte response to PHA (Newble et al., 1975). Among the many investigations performed in animals in order to analyze immunological disturbances caused by virus infection, we shall refer to only two studies, because of the light they throw on possible mechanisms and pathological consequences. In the mouse, infection with murine hepatitis virus (MHV-3) modified the humoral immune response to sheep red blood cells (SRBC): infecting mice before antigen administration caused immunodepression, simultaneous injection of virus and SRBC resulted in immunostimulation; on the other hand, persistent MHV-3 infections (which may be induced in some mouse lines, like C3H or A2G) were associated with a chronic state of immunodepression. Moreover, the presence of circulating interferon (the immunomodulating properties of which are well documented) was well correlated with these modifications: interferon peaking before antigen administration was associated with immunodepression, interferon production after antigen administration was associated with immunostimulation,

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whereas low and permanent levels of circulating interferon were associated with chronic immunodepression (Virelizier et al., 1976). On the other hand, mice lethally infected with street rabies virus failed to develop cytotoxic T lymphocytes specific for rabies virus-infected target cells, while high level cytotoxicity was generated after nonfatal infection with attenuated rabies virus strains; furthermore, concurrent infection with street rabies virus suppressed development of a cell-mediated cytotoxic response specific for influenza virus-infected cells (Wiktor et ai., 1977): it thus appears that street rabies virus (which is not known to replicate in cells of the immune system) induces a general defect in cell-mediated cytotoxic response and development of fatal rabies may reflect the operation of this selective immunosuppressive mechanism. What could be the benefit to be gained from treating virus-infected patients with immunopotentiating drugs, on the basis of the knowledge summarized above about virus-induced disturbances of the immune system? Theoretically, there should be wide possibilities in this area: virus-induced immunodepression is likely to play a role in the severity of the disease itself and it may, in addition, increase the susceptibility of the host to other viral illnesses, to bacterial, fungal or parasitic infections and possibly to malignancies. Compensating through immunomodulating drugs the immunological dysfunctions occurring in many viral infections should therefore have interesting therapeutic applications, but it remains to be proven that this is indeed feasible. There is clearly a need for testing immunopotentiating substances in experimental systems in which such effects would be demonstrable. This brings us naturally to another possible field of application for antiviral immunopotentiating drugs: enhancing resistance to virus infections in immunologically compromised patients or animals. In such situations, these drugs could conceivably be administered in a prophylactic manner. The increased severity of some viral diseases--notably measles, varicella-zoster, cytomegalovirus infection, herpes simp l e x - i n immunologically deficient patients has already been stressed, in the Introduction, as indirect evidence for the importance of an intact immune system in resistance to viral infections. In the realm of congenital immunodeficiencies, such as thymic dysplasia or severe combined immunodeficiency, immunopotentiating drugs may have little to offer, except for those agents, like thymic hormones or transfer factor which, in principle, should substitute for missing immunological functions in such patients. The situation appears quite different, however, with respect to acquired immunodeficiencies, be they due to malignant conditions (leukemia and lymphomas) or to immunosuppressive therapy with cytostatic drugs. For instance, herpes zoster is a frequent complication of lymphoreticular malignancy, and increased susceptibility to clinical infection with varicella-zoster virus (VZV) correlates with deficiencies in in vitro lymphocyte responses to VZV antigen (Arvin et al., 1978). Severe forms of cytomegalovirus infection have been shown to occur in patients receiving immunosuppressive therapy for rheumatological disorders or before kidney or bone marrow transplantation and it appears that most such patients are at risk from endogenous infection (Anonymous, 1977). Deficiencies in cell-mediated immunity to cytomegalovirus, in the presence of normal antibody levels, have been observed in cardiac transplant patients; such deficiencies took up to 3 years after transplant to resolve and were associated with a syndrome of unexplained fever, hepatitis, pneumonitis and leukopenia (Pollard et ai., 1978). Whether enhancement of cytomegalovirus infection was due to immunosuppressive therapy or to graft-vs-host reactions (GVHR) is not established, but it has been shown in mice that GHVR alone can enhance murine cytomegalovirus infection in a chronically infected host (Dowling et al., 1977). With respect to deficiencies in humoral immunity, it is noteworthy that patients with lepromatous leprosy have an impaired immune response that diminishes their efficiency in terminating hepatitis B virus infection with the production of circulating antibodies (Serjeantson and Woodfield, 1978). In all such acquired immunodeficiencies, selective stimulation of the impaired immune response through administration of an appropriate immunopotentiating drug should help in preventing the occurrence, or in decreasing the severity, of virus

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infections. It seems possible that part of the benefit derived from nonspecific immunotherapy by cancer patients is due to an enhancement of their resistance to viral infections, to which treatment with cytostatic drugs makes them prone. In other situations associated with immunodeficiency, usefulness of immunopotentiating drugs for decreasing susceptibility to virus infections can also be suggested, with the proviso that these drugs should not antagonize the beneficial effects expected from immunosuppressive treatment, e.g. stimulate graft rejection. It is probable also that, besides obvious and severe cases of acquired immunodeficiencies, there occur in the life of any individual short or prolonged periods of discrete immunodeficiency, due to many possible reasons, which predispose him to clinically overt virus infections. This may be the case, for instance, in those young children who exhibit excessively frequent infections of the respiratory tract and finally improve as they grow older. Interesting applications of immunopotentiating drugs are provided by such situations, but it will first be necessary to find reliable and sensitive laboratory techniques to detect these discrete and transient states of immunodeficiency. Veterinary studies could be useful to test the possibility of combating through immunopotentiation the susceptibility to virus infections of immunologically deficient animals: immunodeficiency disorders in young horses have been shown to correlate with a high prevalence of adenovirus infections (McGuire et al., 1977)and the outcome of canine distemper virus inoculation in gnotobiotic dogs was shown to be correlated with their cell-mediated immunity level, assessed by skin tests with phytohemagglutinin (Krakowka et al., 1977). The possibility of using immunopotentiating substances for the management of persistent (also called chronic) viral infections is a highly speculative topic, since relatively little is known presently about the various mechanisms of such persistence. These mechanisms may include unique properties of the virus (such as the nonimmunogenicity of viroids implicated in the subacute spongiform encephalopathies), as well as inadequate host defenses. Agents causing latent infections, such as herpes simplex and varicella-zoster viruses, escape immune elimination by remaining in nerve cells during the intervals between disease episodes and, on the other hand, in several chronic infections, the viruses seem to grow mainly in lymphoid tissue, particularly in macrophages (this was shown to be the case with LCM and lactic dehydrogenase viruses in the mouse, aleutian disease virus in minks, and equine infectious anemia virus in horses). Under such circumstances, it is not clear how immunopotentiation could be beneficial, but there are reasons for believing that persistent viral infections may, in some cases, be associated with hyporesponsiveness of humoral (hepatitis B) or cellular immune mechanisms. Burkitt lymphoma, associated with Epstein-Barr virus (EBV) infection, has been attributed to a state of immunodepression perhaps caused by endemic malaria. It remains for the future to explore the clinical value of immunomodulating treatments in persistent viral infections of humans, such as hepatitis B, the rubella syndrome, cytomegalovirus and EBV infections, subacute sclerosing panencephalitis (SSPE) and progressive multifocal leukoencephalopathy (PML). Immunological disturbances are evident in some of these conditions; enhanced susceptibility of immunosuppressed patients to hepatitis B and cytomegalovirus has already been mentioned; antibody levels to measles virus are abnormally high in SSPE, while PML occurs only in subjects whose immunological responsiveness has been severely lowered by malignant disease and/or immunosuppressive drugs. There are also other poorly understood chronic diseases which may be due to persistent virus infections, such as multiple sclerosis and amyotrophic lateral sclerosis. Several animal models of persistent viral infections are available and it should be feasible, albeit not without technical difficulties, to study the effects of immunopotentiating drugs on such diseases. For instance, 'mouse hepatitis virus (MHV-3) exerts different effects on different strains of mice: strain A mice are completely resistant, most strains die of acute hepatitis and, thirdly, in certain strains (such as C3H and A2G) the virus produces a state of persistent infection which leads

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to neuropathological manifestations (Virelizier et al., 1975). It was later shown (Virelizier and Allison, 1976) that MHV-3 replicates freely, with giant cell formation, in macrophages from susceptible mice, that it does not replicate in macrophages from resistant A mice and, finally, that macrophage cultures from C3H or A2G mice showed intermediate susceptibility. It would be highly interesting to see whether treatment of the latter strains of mice with immunopotentiating substances, after they have been infected with the virus, would help them to get rid of the infection. Many, if not most, persistent viral infections, in a wide variety of hosts, are associated with the production of virus-induced immune complexes which are present in the circulation or may deposit in the tissues, thereby causing disease. Glomeruli in the kidney, blood vessels and choroid plexus are the main sites where circulating immune complexes deposit. Immune complexes less often deposit in the joints, heart, lungs and liver. Circulating immune complexes likely represent a quite common phenomenon that occurs whenever the host mounts an antibody response against the infecting virus. In acute viral infections, symptoms like myalgia or joint pain may result from immune complex deposits which locally activate various possible mediators of inflammation; in chronic infections, in which ongoing viral replication coexists with a continuous host immune response, immune complexes commonly cause nephritis and arteritis (see review by Oldstone and Dixon, 1975). As in the case of persistent viral infections, the immunopathology of which is largely dependent on deposition of immune complexes in the tissues, it would be premature to speculate about the possible uses of immunopotentiation for the therapy of immune complex diseases: conceivably further stimulation of antibody production might help to dissolve the circulating complexes before they deposit in critical tissues, or stimulated macrophages might have an increased capacity to clear such complexes. There are several models of immune complex diseases in the mouse (infections with lactic dehydrogenase virus, with lymphocytic choriomeningitis virus, with various oncornaviruses) and the possible impact of immunopotentiating substances on such diseases is amenable to experimental analysis. The provisional conclusion one may reach concerning the possible uses in human and veterinary medicine of immunopotentiating substances endowed with antiviral activity is that the situation may be, for exactly opposite reasons, just as difficult and complicated as in the case of antiviral chemotherapy. In the latter field, nontoxic agents which can effectively inhibit viral replication are usually very narrowly specific, which severely limits their usefulness; on the other hand, while there are many different mechanisms through which viruses are known to replicate, the mechanisms through which the infected host's immune responses cope with virus invasion are equally diverse and they do not always interact harmoniously in order to preserve the host's integrity. Therefore, just as it is difficult to visualize a single broad-spectrum 'antiviral' drug, it appears illusory to envisage an immunopotentiating substance which could be used to stimulate resistance to, and hasten recovery from, all possible virus infections. The contradiction between antiviral chemotherapy and antiviral immunotherapy is only apparent and both approaches could conceivably be used concurrently. It is known, for instance, that effective antiviral substances like amantadine or methisazone, when applied in vivo, do not block totally virus replication in target tissues (such as lungs and brain): they only reduce it to a level which, owing to the intervention of the host's immune response, is compatible with his survival. Combined treatments of virus infections with a selective antiviral drug (when precise diagnosis can be done on time) and with an appropriate immunopotentiating substance could be of definite therapeutic advantage. A great deal of experimental work, based on models of virus infections which would be more relevant than many of those presently in use, is still necessary and we should not forget 'how long and hard the work must be before the really important applications become applicable' (Lewis Thomas, The Lives of a Cell, 1974).

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ADDENDUM Some contributions which are relevant to this review's topics have appeared after we sent in the manuscript and before correcting the proofs. Not unlike other 'defense" mechanisms, interferon has been shown to exert detrimental, rather than beneficial, effects in a particular virus infection; this was well documented in the case of the infection of newborn mice with lymphochoriomeningitis (LCM) virus: administration of anti-murine interferon globulin prevented the appearance of hepatic ahd renal lesions and protected the animals from death. High titers of interferon are induced in the mouse by LCM infection: anti-interferon globulin increases in vivo production of LCM virus but suppresses the lesions (I. Gresser, Symposium on Antiviral Defenses, Pasteur Institute, Paris, October 20-21, 1978). Attempts have been made to influence experimental virus infections (encephalomyocarditis, murine hepatitis, herpes simplex type 1) in the mouse through the production of a strong local inflammatory reaction, such as the sterile subcutaneous granuloma which follows injection of magnesium silicate. This was shown by Fauve (loc. cit.) to markedly enhance resistance of mice against bacterial, fungal or protozoal infections and tumor cell invasion, but this procedure did not alter the susceptibility of mice to the virus infections quoted above (Zerial, Floc'h and Werner, submitted for publication). Mechanisms of nonspecific resistance to virus infections must be basically different from those involved in other situations, which are readily influenced by inflammation. While several soluble mediators of inflammation are known to be released from such an inflammatory focus, interferon is notably absent in this case. Two controlled double-blind clinical studies have been performed on the activity of levamisole in herpes simplex. Mehr and Albans (Lancet ||, 773-774, 1977) saw no significant beneficial effect in twenty-eight patients with recurrent herpes labialis or genitalis. On the other hand, Krueger, Spruance and Overall (18th Interscience Conf. on Antimicrob Ag and Chemother., Atlanta, Ga. October 1-4, 1978) administered levamisole or a placebo to forty-two patients with a high frequency of recurrent herpes labialis and concluded that the levamisole treated patients has less severe (in terms of duration and extent of lesions) but more frequent episodes of disease than those receiving the placebo. In both studies, patients were instructed to initiate a 3-day treatment regimen at the first sign of each herpes episode. Inosiplex was studied for its effect on herpes zoster in a double-blind study involving thirteen children with various forms of cancer (Feldman, Hayes, Chaudhary and Ossi, Antimicrob.. Agents and Chemother. 14, 495-497, 1978). According to multiple criteria (photographic evaluation of the progression of dermatome lesions, in vitro lymphocyte stimulation in response to PHA and VZV antigen, antibodies to VZV) the drug had no demonstrable therapeutic effects on the zoster illness. Since the basis for the antiviral activity of inosiplex appears to be the stimulation of lymphocytes previously sensitized to the viral protein, the authors conclude that, in their study, drug failure might have been related to deficient lymphocytes in patients who had previously received anticancer chemotherapy or irradiation. On the other hand, Huge, Rancurei, Picard and Dechy (Nouv. Presse m~d. 7, 3767, 1978) reported favorable results with inosiplex in three adolescent cases of subacute encephalitis. Signs of clinical improveme]at appeared several months after initiation of treatment with inosiplex (which was administered at a daily dose of 50-100 mg/kg for 5 days followed by 8 days without treatment). A novel synthetic immunomodulating compound has recently been described: it is 1,2 - 0 - isopropylidene - 3 0 - 3' - (N', N' - dimethylamino - n - propyi) - D - glucofuranose, or: SM-1213. This compound enhances the resistance of mice to bacterial and Candida albicans infections and it induces increased acid pbosphatase and peroxidase activities in macrophages as well as increased iysozyme activity in mouse serum and spleen 48 hr after oral treatment with as little as 1 # g per mouse (Gordon, Hashimoto and Majde, 18th Interscience Conf. on Antimicrobial Agents and Chemother., Atlanta, Ga. October 1-4, 1978). SM-1213 was also reported to protect normal and cyclophosphamide-immunosuppressed mice from influenza A and vaccinia virus infections and to protect hamsters from lethal herpes virus encephalitis, possibly through an activating effect on neutrophils (Gordon, personal communication).