MINIREVIEW. not (14, 20, 24). In general, there is a grey zone of serological. estimation of antibody titers (Fig. 1) within which it is not

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1 JOURNAL OF VIROLOGY, Apr. 1992, p "Vol. 66, No X/92/ $02.00/0 Copyright X 1992, American Society for Microbiology MINIREVIEW Protective Immune Response against Foot-and-Mouth Disease KENNETH C. McCULLOUGH,l* FRANCO DE SIMONE,2 EMILIANA BROCCHI,2 LORENZO CAPUCCI,2 JOHN R. CROWTHER,3 AND ULRICH KIHM' Institut fur Viruskrankheiten und Immunprophylaxe, Hagenaustrasse 74, CH-4025 Basel, Switzerland'; Istituto Zooprofilattico Sperimentale della Lombardia e dell'emilia, Via Bianchi 7, Brescia, Italy2; Institute for Animal Health, Pirbright, Woking, Surrey GU24 ONF, England3 The causative agents of foot-and-mouth disease (FMD) are small icosahedral viruses of theaphthovirus group within the Picornaviridae family. There is no evidence that these viruses infect cells of the immune system or otherwise interfere detrimentally with their function; additionally, it has not been possible to relate cytotoxicity reactions against virus-infected cells to the efficacy of the immune response against FMD virus infection. In contrast, there is a close association between FMD virus antibody and the protective immune response (10, 14, 15, 20, 24, 25, 29-32). Induction of this antibody is dependent on the structure of the viral antigenic sites (7-9, 11, 18) and on the concomitant presence of Tb-lymphocyte epitopes (4, 5, 7, 8), although a Ti-lymphocyteindependent response has been reported (2). Recent work by Piatti et al. (26) showed that the immune response induced by FMD virus was only Th-lymphocyte dependent when low doses of antigen were used. This latter work was performed in mice, and it is not certain that a similar situation would be found in cattle. As for the major effector immune defense, this relies on the interaction between antibody-virus complexes and the phagocytic cells of the reticuloendothelial system (17, 19). OBSERVATIONS ON THE RELATIONSHIP BETWEEN FMD VIRUS-SPECIFIC ANTIBODY AND PROTECTION The strong association between the capacity of a vaccine to induce FMD virus-specific antibodies and the capacity to protect against challenge has been reported on numerous occasions (10, 14, 15, 20, 24, 25, 29-32). Most laboratories involved in such work have measured this antibody by means of the complement fixation test, serum neutralization test (SNT; also called the virus neutralization test), or the enzyme-linked immunosorbent assay (ELISA). Protection has been measured by using challenge infection experiments, performed normally at 21 days postvaccination, according to the European Pharmacopoeia (12). This challenge is effected by intradermolingual injection of a dose of virus guaranteed to produce the disease in unvaccinated animals. Protection is recorded as the capacity of the vaccination to prevent spread of FMD lesions from the site of inoculation (tongue) to the hooves. The characteristics of the challenge infection and the natural infection (known to involve the respiratory route) would indicate that their main difference lies in the fact that in the natural infection, the virus must overcome the defensive barriers of the mucosal epithelia in the nasopharyngeal region before virus infection can be initiated. It is at the level of the systemic immune response that the two types of infection are related and consequently at this level that the efficacy and potency of vaccination can be determined. A relationship has been demonstrated between a minimum antibody titer detected by the aforementioned assays and protection against FMD virus challenge (10, 14, 15, 20, 24, 25, 29-32). However, this correlation is not precise (Fig. 1). Some animals which possess lower than the minimum desired antibody titer can resist challenge, whereas others do * Corresponding author not (14, 20, 24). In general, there is a grey zone of serological estimation of antibody titers (Fig. 1) within which it is not always certain that the respective animals will be protected against or susceptible to FMD virus challenge (14). The problems surrounding attempts to relate antibody titers as measured by the SNT or ELISA to protection against challenge lie in the different mechanisms involved in vivo and with in vitro assays. The protective immune response against FMD virus must destroy that virus. The SNT and ELISA only measure the capacity of antibody to interact with virus (and in the case of the SNT, to interfere with the infection of cells in culture). Only one antibody specificity (18) has been shown capable of irreversibly destroying virion structure (21), but at high concentrations; other antibody specificities neutralize virus infectivity in vitro or passively protect in vivo without any evidence for conformational alteration in the virus structure (6, 15, 18, 21, 22, 27, 33). In vivo, the experimental work reported in the literature suggests that it is the reticuloendothelial compartment of the immune system which is primarily responsible for the destruction of FMD virus through phagocytosis of the virus-antibody complexes (15, 17, 19). PHAGOCYTOSIS AND VIRUS-ANTIBODY INTERACTIONS During any virus infection, the known characteristics of the immune responses would suggest that free virus could be phagocytosed by the mononuclear phagocytes (the macrophages) and the polymorphonuclear phagocytes (the polymorphonuclear leukocytes or granulocytes). With respect to FMD, the efficiency of this process is probably at the same level as the phagocytosis of any other foreign protein or cell debris, since the phagocytes do not possess specific receptors for FMD virus (19). The phagocytosis of FMD virus is enhanced when the virus is complexed with antibody of the appropriate specificity and affinity (17, 19); under such

2 1836 MINIREVIEW J. VIROL (a) serum neutralisation test (b) liquid-phase sandwich competition ELISA :J.. ' AL~"k Kgo-binding d= FcR treion with antibody-fc: wek; C Q._ C 40 Cu ~ CL C) S ;" ( E; x:x.., is 0@ i. x..},,-,.x.., Yr.).m 000 i.: FIG. 1. Relationship between the resistance of cattle to challenge using FMD virus serotype 01 Lausanne and the FMD virusspecific antibody titer as measured at 21 days postvaccination by SNT (a) or by liquid-phase sandwich competition (blocking) ELISA (b). Symbols: 0, not protected; 0, protected. conditions, phagocytosis is mediated primarily by means of Fc receptors (FcR) for host immunoglobulin species and receptors for the activated third component of complement (C3bR) on the surfaces of the phagocytes. Although these FcR can bind immunoglobulin which is free within the body fluids, this reaction is of low affinity and of short duration for each immunoglobulin receptor-binding event, in contrast to the more efficient binding of antibody-antigen complexes; this subject has been reviewed in general by Leslie (13), the salient points of which may well be applicable to the phagocytosis of FMD virus. The interaction between antibody and virus is governed by the principles of the chemical -kinetics of reactions. These state that at equilibrium, a maximum of 50% of the virus will be complexed with antibody. Although such interactions would certainly interfere with the capacity of the virus to infect susceptible cells, at the level of either adsorption or postpenetration (as shown for FMD virus by Baxt et al [1]), the fact that the reaction of the antibody with the virus is not a static but a dynamic phenomenon could result in the continuous displacement and replacement of the antibody molecules on the virus. The relative stability of the virusantibody complexes would depend on the affinity of each antibody molecule for the virus antigen, the avidity of the combined affinities during that reaction, and the density of antibody molecules on the virus particles. Consequently, while the virus is in a complex with the antibody, it could retain its potential to infect susceptible cells. It has been reported that phagocytes can stabilize antigenantibody complex formation through the binding of the complexes to the FcR (13). By inference to this work, if two FcR were cross-linked by antibody molecules within the same FMD virus-antibody complex, there should be both a ALbody moluon c.18nm long '- ~~THE ANIJlBODY Fo-PORTION IS ALTERED FMQ Vms 25nm diameler AFTER REACTION VWM[ FMNV FcR rmcbon wit _Ieed anffbody-fc: everuibb. etn; MACROPHAGE c. 2000nm diameter FIG. 2. Macrophages can interact with FMD virus, immunoglobulin G, or complexes of FMD virus with antibody, but it is the reaction with the latter that is most efficient because of changes which occur in the antibody molecule. stabilization of the interaction between virus and antibody and a stimulation of phagocytosis (Fig. 2). Such events would result in an internalization of the complexes; once within the cytoplasm of the phagocyte, the virus would become accessible to the degradative lysosomal enzymes (13). PROTECTWVE IMMUNE RESPONSE AGAINST FMD VIRUS Studies with an in vivo model of FMD virus virulence which mimicked events in the calf (28) demonstrated that mice could be protected against FMD virus pathogenicity when the virus was complexed with monoclonal antibodies (MAb), although only certain MAb were effective in this sense (17). The MAb could be related to three distinct antigenic sites containing one or more antigenic determinants (18), work which has been supported by topological investigations from other groups (6, 22, 27, 33). The question arose as to whether this protection was due to the direct effects of the antibody on the virus or to an enhancement of the phagocytosis of virus by cells of the reticuloendothelial system. Using different concentrations of the MAb, it was observed that protection of the animals occurred under conditions wherein the virus was fully infectious for susceptible cell lines in vitro. That is, the antibody could protect in vivo at concentrations which did not neutralize virus infectivity in vitro (in the SNT) (17). This result suggested that the cells of the reticuloendothelial system were indeed playing an important role in the immune defense against FMD virus. Although it was possible that high levels of specific antibody might interfere directly with virus infection of the cells, the lower concentrations which were nonneutralizing (in vitro) but protective were presumably reflecting antibody-dependent enhancement of phagocytosis of the virus. The potential role of phagocytes in the immune defense against FMD virus was analyzed by blocking the phagocytotic capacity of the mice through treatment with silicon

3 VOL. 66, 1992 MINIREVIEW 1837 dioxide or removal of the Fc portion of the antibody before it was complexed with the virus. Both manipulations impaired the protective capacity of the MAb (17). Under such conditions, the mice could be killed by virus-mab complexes wherein the virus infectivity had been neutralized, as determined by its capacity to infect BHK cells in vitro (17). This finding demonstrated that the direct neutralization by antibody of the capacity of FMD virus to infect susceptible cells (the property measured by the SNT) was not of primary importance for the protection of animals against FMD. Certainly, when the antibody concentrations were high, there was an apparent direct neutralization of the capacity of the virus to infect susceptible cells in mice, since this could be observed when using F(ab')2 fragments of the antibodies (17). Nevertheless, between 10 and 500 times more F(ab')2 fragments (depending on the MAb) were required for protection in vivo than for neutralization of virus infectivity in vitro (17). Such in vivo effects may employ one of the mechanisms of virus neutralization alluded to by Baxt et al. (1). Alternatively, the in vivo observations may be reflecting the different capacities for phagocytosis of whole immunoglobulin and F(ab')2 fragments by cells of the reticuloendothelial system. Removal of the Fc portion of the antibody would influence binding of the virus-antibody complexes to the FcR of the phagocytes but not the other phagocytic mechanisms independent of and probably less efficient than that mediated through the FcR. Higher concentrations of F(ab')2 fragments of antibody when complexed with FMD virus may form aggregates which are more readily phagocytosed; indeed, most of the MAb which had been used for the reported studies do aggregate FMD virus at the concentrations found to be protective in vivo when F(ab')2 fragments were used (14a). Evidence in favor of such a proposal of differential phagocytosis of whole immunoglobulin or F(ab')2 fragments complexed with virus can be alluded to by using the work with silicon dioxide-treated mice (17). This methodology directly impairs all types of phagocytic activity, whether mediated through the FcR or by other means. Under these conditions, not even the highest concentrations of antibody could protect all of the mice. At these high concentrations (100- to 1,000-fold greater than that required to protect untreated animals), between 20 and 90% of the animals could resist the virus in the complexes, but this reaction was variable both between experiments and the antibodies used (17). The observation that a few animals could resist the virus when complexed with high levels of specific antibody may be explained by the mechanisms known to be operating in the development of immunocompetent cells. Although silicon dioxide treatment of mice is an efficient antiphagocytic method, impairment of the phagocytotic potential of the animal is not absolute; in addition, new populations of macrophages can be derived from circulating nonphagocytic monocytes, which may not have been influenced by the silicon dioxide treatment, or from myeloid cell precursors in the bone marrow. It is possible that high concentrations of anti-fmd virus antibody may interfere with the capacity of the virus to infect in vivo such that in certain animals which had received the silicon dioxide treatment, new phagocyte populations would have the time to develop and attack the virus in the complexes. Support for these conclusions was obtained from in vitro observations on the phagocytotic phenomenon (19) which are discussed in the next section. Consequently, the cells of the reticuloendothelial system play a central role in resistance to FMD virus infection. Only with a fully active phagocytic system can an efficient protective immune response against FMD virus be seen (17, 19). This could explain why low levels of specific antibody can be effective in protecting animals against FMD (14, 17, 20, 25, 26, 29, 30, 32). The conclusion from the work on phagocytosis (17, 19) was that the major arm of the protective immune response against FMD virus was effected through opsonization of virus by antibody, resulting in augmented phagocytosis by cells of the reticuloendothelial system. Antibody could be considered as having an additional role at high concentrations, at which the infectious capacity of the virus may be retarded or directly inhibited; however, without phagocytic activity, the efficiency of the immunological protection is considerably reduced, or even annulled. MACROPHAGE ACTIVITY AGAINST FMD VIRUS In vitro experiments demonstrated how macrophages could be responsible for the in vivo observations which had been made on the protective immune response against FMD virus (17, 19). Both the kinetics and degree of phagocytosis were greater with virus-antibody complexes than with free virus (19). This could occur under conditions in which the virus within the complexes was still fully capable of infecting susceptible cell lines. The capacity to phagocytose the complexes was impaired whether macrophage phagocytosis was blocked by silicon dioxide treatment or whether adsorption of the complexes to the FcR of the phagocytes was prevented by the removal of the Fc portion of the antibody before complex formation. Removal of antibody Fc reduced the phagocytic index to that observed with virus alone; silicon dioxide treatment reduced phagocytosis (of complexes or antigen alone) to between 20 and 40% of that observed using normal phagocytes and antigen alone. This finding related directly to the in vivo experiments; removal of antibody Fc would only interfere with FcR-mediated (and perhaps also C3bR-mediated) phagocytosis but not with phagocytic events independent of these receptors; silicon dioxide was effective against a much wider range of phagocytotic events while probably permitting the continued maturation of monocytes into macrophages. Such results confirmed that it was indeed macrophages which were phagocytosing antibody-opsonized virus and that macrophages were probably responsible for both the destruction of the majority of the FMD virus and the ultimate protection of the animals. IMMUNOLOGICAL DESTRUCTION OF FMD VIRUS The efficiency with which macrophages can phagocytose FMD virus is clearly dependent on opsonization (17, 19). When antibody would react with the virus, an alteration in the structure of the Fc portion of the antibody should occur (Fig. 2). Provided the antibody-antigen reaction is of a minimum affinity, the antibody Fc portion will be altered such that the phagocytes will retain the antibody-antigen complexes on their surfaces (by inference to the general review on the subject of phagocytosis by Leslie [13]). In general, once an antibody-antigen complex is bound to an FcR, the antibody reaction with the antigen appears to become stabilized; other antibody molecules or antibodyantigen complexes in the vicinity may also bind in what appears to be an irreversible fashion to the FcR-held complexes (13). When a second antibody molecule within the complex reacts with an adjacent FcR, a cross-linking event occurs. Such a cross-linking of two FcR will induce the trans-membrane signal necessary for initiation of the phago-

4 1838 MINIREVIEW J. VIROL. phagosome containing FMD viruslantibody complexes f ox ases YSproteasee LYSOSOUIE 'I1 esterases phagosome-qysosome fusion FIG. 3. cytotic process; these possibilities are shown in Fig. 3 with respect to how they may be involved in the protective immune response against FMD. The formation of this phagosome around an FMD virusantibody complex and the subsequent fusion with lysosomes to produce the phagolysosome would result in destruction of the virus. The FMD virus within the antibody-antigen complexes would then become exposed to the degradative capacity of the lysosomal enzymes (it has not been possible to demonstrate an inhibitory effect of FMD virus on the formation of the phagolysosome or on the functioning of the lysosomal enzymes, as can be found with certain bacteria and viruses such as mycobacteria and African swine fever virus). This degradation of FMD virus in phagocytes has been monitored kinetically in vitro (19); analysis of the fate of virus after phagocytosis showed a time-dependent destruction of virus infectivity, an event which did not occur in the absence of functional macrophages (19). MONITORING OF THE PROTECTIVE IMMUNE RESPONSE Since the protective immune response against FMD virus requires antibody-dependent opsonization-enhanced phagocytosis of the virus, the detection of specific antibody alone is incomplete as a measure of that protective immune response. Assays such as the complement fixation test (3), SNT (25, 29, 30), and the various forms of the liquid-phase ELISA (10, 14, 16, 31) may measure the opsonization events Protective immune response against FMD virus. Formation of the phagolysosome N-iozyme pemxidase esterases oiddases which are central to the process of efficient immunological protection (17, 19). The problem with these assays is that they measure all opsonization events. Antibody isotype is also of importance. It is known that different immunoglobulin isotypes have different capacities to interact with FcR on phagocytes and also differ in their capacity to fix complement, which can further enhance phagocytosis. The recent observations by Mulcahy et al. (23) demonstrated that the induction of different isotypes in cattle may be crucial to the outcome of a particular vaccination procedure and suggested that vaccine potency may be related to the different effects of the induced isotypes on phagocytosis. Consequently, the measurement of the protective immune response against FMD virus should employ assays which analyze the phagocytosis of opsonized virus. The mouse model of FMD (2, 28, 29) would appear capable of fulfilling this role (17, 19), although an in vitro assay, perhaps using phagocyte cultures (19), would be more desirable. CONCLUDING REMARKS Within recent years a greater understanding of the protective immune response against FMD has been achieved. Induction of the immune response can often be seen as a T-lymphocyte-dependent phenomenon, while the major arm of the effector immune response against FMD virus requires both specific antibody and macrophage activity. From the work reported on MAb-enhanced phagocytosis of FMD

5 VOL. 66, 1992 virus (17, 19), the levels of specific antibody most often observed in immune animals would probably not be capable alone of destroying the virus or removing the threat of disease induction; it would be the macrophages which could achieve these goals, but the efficiency of macrophage activity is dependent on the interaction of antibody with virus. Direct neutralization of FMD virus infectivity appeared to be of importance only when the concentration of antibody was high. This conclusion comes from observations that high concentrations of antibody appeared capable of protecting in vivo when the functioning of phagocyte FcR was impaired (17); however, it is not certain whether this was solely due to the antibody or to FcR-independent phagocytosis. Certainly, when the phagocytic activity of the reticuloendothelial system was inhibited, not even the highest concentrations of antibody employed were able to prevent disease, even when that virus had been neutralized as determined by in vitro assays of its capacity to infect susceptible cells (17). Consequently, the major arm of the protective immune response against FMD virus would appear to require specific antivirus, antibody-dependent, opsonization-enhanced phagocytosis by cells of the reticuloendothelial system. Vaccine efficacy studies and in vitro analyses of the immune defense against FMD should take into consideration the central role played by the reticuloendothelial system in this protective immune response. Only then will it be possible to achieve a clear appreciation of the immune response following challenge infection, natural infection, and vaccination. REFERENCES 1. Baxt, B., D. 0. Morgan, B. H. Robertson, and C. A. 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