Antibody neutralization of picornaviruses: can fever help?

OPINION

Antibody neutralization of picornaviruses: can fever help? Albert BoeyQ, lngrid Delaet and Paul Brioen

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variety of molecular mechanisms underlie the neuof viruses by tralization antibodies. The neutralization of enveloped viruses is often easy to explain, for example, when it depends on interaction of the antibodies with functional envelope glycoproteins, such as the hemagglutinin of the influenza viruses. However, the mechanism of neutralization of naked viruses, such as the picornaviruses, is not so obvious. The picornavirus group includes, among others, poliovirus, rhinovirus (which causes common cold), hepatitis A virus and, in the veterinary field, foot-and-mouth disease virus (FMDV). All picornaviruses have an icosahedral capsid with a diameter of roughly 30 nm, which consists of 60 copies of a structural unit of four proteins (VPl, VP2, VI’3 and VP4), and which encloses a single positive-stranded RNA molecule1~2. Each structural unit has four areas (antigenic sites) to which the great majority of antibodies bind3,4; however, these areas have no known function. The only part of the viral surface that has a known function is a depression into which the virus receptor binds. For rhinovirus and poliovirus, this depression takes the form of a canyon, which is so narrow that antibodies cannot enters-‘, but may bridge itR. Mechanisms of picomavirus neutralization A monoclonal antibody can bind to each of the 60 copies of its own epitope on the surface of the virus. An immunoglobulin G (IgG) molecule may bind simultaneously to two neighboring copies of the epitope (bivalent monogamous binding), if they are suitably spaced. The minimum distance between the two copies is given by the thickness of the Fab regions themselves, that is, 34 nm (Refs 9.10) (Fig. lc). The maxi-

Binding of bivalent antibody can neutralize picornaviruses by a variety of mechanisms. Some monoclonal antibodies can irreversibly neutralize the virus at temperatures that are higher than physiological by disrupting the virion, leading to ejection of the RNA. Fever may enhance this process in vivo, confirming the popular belief in the virtues of fever. A. BoeyB, I. Delaet and P. Brioen are in the Dept of Microbiology and Hygiene, Vrije Universiteit Brussel, Laarbeeklaan 103, B-l 090 Brussels, Belgium.

mum distance is probably considerably less than the 14-15 nm reach of a fully extended IgG molecule9J1. All the epitopes on poliovirus and rhinovirus are ‘conformational’, consisting of discontinuous stretches of amino acids that belong to one or several capsid proteins. A Fab fragment bound to an epitope located on a sphere is expected to stand out radially (Fig. 1). Whether an IgG molecule can grasp two copies of an epitope then depends on the bending of the ‘elbow’ between the variable and constant modules of the Fab (Refs 12,13), which should considerably reduce the maximum distance between the two epitope copies (Fig. lc). However, Fab fragments attached to human rhinovirus were shown to lean towards each other, so as to favor bivalent binding*. Additional flexibility is gained when the epitope is continuous and consists of a single stretch of amino acids that loops far out from the viral surface, as in FMDV (Ref. 4). Antibodies that cannot meet the requirements of monogamous bivalency can still participate in neutralization through bigamous binding, by forming bridges between virions. Multiple bridgeslq

(Fig. la) will cluster the virions into stable aggregates and thus reduce the number of infective units. At least with poliovirus, the chance that a cluster of two or three virions will start an infectious center is no larger than that of a single virion, so that the specific infectivity of the virus is reduced by a factor equal to the number of virions in the cluster14J5. Aggregation has been observed with most monoclonal antibodies directed against poliovirusIs and FMDV (Ref. 16); antibodies that do not cause aggregation appear to be incapable of bigamous binding”. A large excess of antibody may cover the whole virion with monovalently bound molecules (Fig. 1 b), as was first observed with tobacco mosaic virus18, and steric hindrance from this antibody coating may hinder adsorption19,20. However, full covering of the virion is not always required to disable the function of a virion. Unaggregated virions have been described bound to various amounts of a single monoclonal IgG antibody; virions carrying an average of only four molecules of antibody had lost two-thirds of their infectivity2’. The antibody may stabilize the virion (Fig. lc) and inhibit a conformational modification that is essential for penetration or uncoating22,23. Some antibodies may still be able to neutralize the virus after it has been adsorbed onto cells’7,24. Antibody-mediated virion disruption A further possible method of neutralization that we discovered a decade ago involves the antibody physically disrupting the virion (Fig. Id). At that time, poliovirus was thought to be neutralized by a structural modification that lowered isoelectric pH (Refs 19,25). Verifying this theory led to the observation

OPINION

(d)

Fig. 1. Picornavirus neutralization mechanisms. Poliovirus (diameter of spheres 30 nm) and immunoglobulin G (IgG) antibodies are shown to scale in cartoon form. In (a), aggregation is due to bigamous binding and the virus is neutralized because there are fewer infective units. Infectivity is restored after deaggregation by papain cleavage of the IgG molecules, or by dissociation of immune complexes at pH 2. In (b), at high molecular IgG:virion ratios, aggregation is reduced, in agreement with immune lattice theory. Virions may be covered with antibody, which prevents contact with host cell. Neutralization is reversible at pH 2. In (c), a limited (noncovering) number of IgG molecules bind to single virions (presumably monogamous bivalency) and stabilize the viral capsid (symbolized by the staples) against uncoating. Infectivity is restored by treatment with papain or incubation at pH 2. In (d), the nature of the interaction between virus and IgG is unknown, but requires destabilization of the virion, by incubation either at extremely low ionic strength or at temperatures above 37°C. The neutralization is irreversible as the RNA is released from the viral capsid.

that one particular monoclonal antibody (3.5lf4) destroyed the virus at low ionic strengths, causing the RNA to be expelled from the poliovirions and reducing them to empty capsids that were physically and antigenically identical to those formed when poliovirus is heated to 56°C (Ref. 26). An important observation concerned the reversibility of poliovirus neutralization. At normal ionic strength, neutralization by antibody 35-lf4 was due exclusively to aggregation of otherwise undamaged virions, so that full infectivity was restored when the aggregates were dispersed by papain cleavage of the IgG moleculesi or by dissociation of the immune complexes at pH 2 (Ref. 27) (Fig. la). In contrast, the neutralization at low ionic strength could not be reversed by acid treatmenP (Fig. Id). Since the antibody molecule is released from the empty capsid after disrupting the virionLh, it was

presumed originally that it would be able to interact with a second virion, and so on, so that the process was expected to be catalytic. However, the antibody disruption was found to be stoichiometric: each 35-lf4 antibody molecule modified only two virions. When the antibody reacted with more than two virions, it became inactive, although its activity could be restored by heating to 56°C. The loss of activity is probably due to conformational modification, and the thermal reactivation to restoration of the original conformatiorP. Temperature dependence of virus disruption Study of the temperature requirements of this restoration of activity led to another unexpected observation: control virus that had reacted with antibody 35-If4 at 39°C was antigenically modified even at normal ionic strength. The phenomenon proved to be reproducible:

at 39°C or even at 38°C (but not at 37”(Z), antibody 35-lf4 disrupted the virus at normal ionic strength’“. Two other monoclonal antibodies (out of 39 studied) have been found that neutralize the virus reversibly (that is, by aggregation) at 37°C in normal media, but irreversibly (that is, by disruption) at 39°C or at low ionic strength. In all cases, virus disruption and irreversible neutralization could be prevented by disoxaril, a capsidstabilizing compound, showing that special physical conditions are required to destabilize the virus30. Not all observations fit the destabilization theory equally well. Monoclonal-antibody-mediated disruption of FMDV at 37°C in normal media has been reported31; and we found recently that antibody F7.12 (Ref. 21) disrupts poliovirus equally well at 37°C or 39°C. Virus disruption by this antibody is also temperature dependent, but in the 33-37°C rather than the 37-39°C range (I. Delaet et al., unpublished). Beneficial effects of fever? ‘Weakened by fever, disease germs become easy targets for the blood’s heat-energized antibodies’ (cited in Ref. 32). Fever appears to have beneficial effects in several bacterial and mycotic diseases. In a classic study, rabbits were infected with type III pneumococci by inoculation into the skin. The lesions subsided after the onset of fever, and all animals survived3”, the first example of a pathogen being destroyed in vivo by the raised body temperatures resulting from fever. The negative effect of hypothermia and the positive effect of artificial fever or hyperthermia on the recovery of animals infected with pneumococci, cryptococci, mycobacteria, myxoma, ectromelia, vaccinia or influenza has been clearly demonstrated. In humans, artificial-fever therapy is beneficial in neurosyphilis and gonorrhea32+34. Antipyretics significantly increase the duration of disease due to measles in children, and also increase the frequency of respiratory complications, such as pneumonia, bronchopneumonia, bronchitis and laryngitisi’. However, we found no similar reports for other viral

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diseases and, in the case of poliomyelitis, we know of only one recorded instance of a physician expressing concern about the aggravating effects of hypothermia (Abstr. 5th International Conference on Poliomyelitis, Copenhagen, Denmark, July 26-28 1960, published by Lippincott, 1961). (A sweeping statement about the negative effect of antipyretics on poliomyelitis in children36 could not be traced to any published evidence.) In experimental infections of animals by intracerebral inoculation of picornaviruses, beneficial effects of hyperthermia have been reported in monkeys infected with po1iovirus3’ and in mice infected with poliovirus, coxsackie B and encephalomyocarditis viruses38-42. However, none of these experiments considered the immune status of the animals. The beneficial effects of hyperthermia in experimental poliomyelitis were not only attributed to increased thermodenaturation of the virus, but also to inhibition of viral multiplication at the higher temperature, as assessed in tissue cultures43. Lack of data means that the possible importance of antibodymediated, fever-dependent virus killing in poliomyelitis cannot be assessed. Knowledge of clinically important factors is much more advanced in foot-and-mouth disease. According to a recent review16, resistance to a challenge infection depends exclusively on the ability of phagocytic cells of the animal to destroy the virus, with antibodies being merely accessories to phagocytosis. The hypothesis that anti-

body alone might destroy virus was not even considered, nor was the possibility that fever might enhance this process. We hope that this review will direct future attention to these possibilities. References 1 Rossmann, M.G. et al. (1985) Nature 317,145-153 2 Hogle, J.M., Chow, M. and Filman, D.J. (1985) Science 229,1358-1365 3 Page, G.S. eta/. (1988)l. Virol. 62, 1781-1794 4 Boeye, A. and Rombaut, B. (1992) Prog. Med. Viral. 39, 139-166 5 Rossmann, M.G. and Palmenberg, A.C. (1988) Virology 164,373-382 6 Colonno, R.J. et al. (1988) Proc. Nut/ Acad. Sci. USA 85,5449-5453 7 Pevear, D.C. et al. (1989) J. Viral. 63, 2002-2007 8 Smith, T.J. et al. (1993) J. Viral. 67, 1148-1158 9 Sarma, V.R. et al. (1971) J. Biol. Chem. 246,3753-3759 10 Colman, P.M. et al. (1976) /. Mol. Biol. 100,257-282 11 Huber, R. et al. (1976) Nature 264, 415-420 12 Harris, L.J. et al. (1992) Nature 360, 369-372 13 Lesk, A.M. and Chothia, C. (1988) Nature 335,188-l 90 14 Thomas, A.A.M., Brioen, P. and Boeye, A. (1985) 1. Viral. 54 ,7-13 15 Thomas, A.A.M., Vrijsen, R. and BoeyC,A. (1986) J. Virol. 59,479-485 16 McCullough, K.C. et al. (1992) 1. Viral. 66,1835-1840 17 Vrijsen, R., Mosser, A. and Boeyi, A. (1993) J. Viral. 67,3126-3133 18 Van Regenmortel, M.H.V. and Hardie, G. (1976) Immunochemistry 13, 503-507 19 Emini, E. et al. (1983) J. Viral. 46, 466-474 20 Mandel, B. 11967) Virology 31,238-247

‘Persistent’ forms and persistence of Chlamydia Beatty, Byrne and Morrison’s recent article in TIM’, on chronic inflammation associated with chlamydial infection, reviews the evidence from their group about antigens produced by morphologically atypical chlamydiae that have recently been shown to be induced in tissue culture by nutrient deprivation. Their observations create a powerful case for there being a role for such chlamydial forms as

a source of Hsp60, a protein that has already been established by Morrison and colleagues to be responsible for immunopathological damage in chlamydial ocular infection. There is one point that needs clarification, however, and others that arise in relation to chlamydia-associated reactive arthritis, where pathology is also mediated by the immune response. The authors’ definition of atypical chlamydiae

21 [cenogle, J. et al. (1983) Virology 127, 412-425 22 Wetz, K. (1993) Virology 192,465-472 23 Mosser, A.G., Leippe, D.M. and Rueckert, R.R. (1989) in Molecular Aspects of Picornuvirus infection and Detection (Semler, B.L. and Ehrenfeld, E., eds), pp. 155-167, American Society for Microbiology 24 Mandel, B. (1967) Virology 31,248-259 25 Mandel, B. (1976) Virology 69, 500-510 26 Brioen, P., Rombaut, B. and Boeyk, A. (1985) 1. Gen. Viral. 66,2495-2499 27 Mandel, B. (1961) Virology 14,316-328 28 Delaet, I., Vrijsen, R. and Boeye, A. (1992) Virology 188, 93-101 29 Delaet, I. and Boeyi, A. (1993) J. Viral. 67,5299-5302 30 Delaet, I. and BoeyC,A. (1994) J. Gen. Virol. 75, 581-587 31 McCullough, K.C. et al. (1987) immunology 60,75-82 32 Bennett, I.L. and Nicastry, A. (1960) Bacterial. Rev. 24, 16-34 33 Rich, A.R. and McKee, C.M. (1936) Bull. Johns Hopkins Hosp. 59, 171-207 34 Mims, C.A. and White, D.O. (1984) in Viral Puthogenesis and Immunology, pp. 184-185, Blackwell 35 Ahmady, A.S. and Samadi, A.R (1981) Indian Pediutr. 18,49-52 36 Dianzani, F. and Baron, S. (1991) in Medical Microbiology (3rd edn) (Baron, S., ed.), p. 666, Churchill Livingstone 37 Wolf, H.F. (1934) Proc. SOL.Exp. Biol. Med. 32,1083-1087 38 Walker, D.L. and Boring, W.D. (1958) J. lmmunol. 80,39-44 39 Baron, S. and Buckler, C.E. (1964) ]. lmmunol. 93,45-50 40 Cathala, F. (1967) Rev. Fr. Etud. Clin. Biol. XII, 330-342 41 Lwoff, A. et al. (1960) C.R. Acad. Sci. 250,2644-2645 42 Lwoff, A., Tournier, P. and Carteaud, J-P. (1959) C.R. Acud. Sci. 248,1876-l 878 43 Lwoff, A. (1969) Bucteriol. Rev. 33, 390-403

as ‘persistent’ rather than ‘noncultivable’ is possibly misleading. We know of no direct evidence to suggest that noncultivable chlamydiae are any more able to persist within tissues than the well-defined infectious and reproductive forms. We agree that it is an attractive speculation, particularly given the resistance of starvation-induced bacteria - often themselves noncultivable - to environmental damage2. Chlamydial infection may persist in viva, but noncultivable forms