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Home , B type inclusion, Orthopoxvirus, Pathogenesis, Pathology, Rabbitpox

CHAPTER 3

THE , AND OF AND

Contents Page Introduction 122 The portal of entry of variola 123 The respiratory tract 123 Inoculation smallpox 123 The conjunctiva 123 Congenital 123 The spread of infection through the body 124 Mousepox 124 126 126 Variola virus infection in non-human primates 127 Smallpox in human subjects 127 The dissemination of virus through the body 129 The 129 Toxaemia 130 Pathological and of smallpox 131 General observations 131 The skin 131 Lesions of the mucous membranes of the respiratory and digestive tracts 138 Effects on other organs 139 The of vaccinia and vaccinia1 complications 140 Normal vaccination 140 Postvaccinial 142 Viral persistence and reactivation 143 Persistence of variola virus in human patients 144 Persistence of in 144 Epidemiological significance 146 The immune response in smallpox and after vaccination 146 Protection against reinfection 146 Humoral and cellular responses in 148 Methods for measuring to orthopoxviruses 149 The humoral response in relation to pathogenesis 152

121 122 SMALLPOX AND ITS ERADICATION

Page The immune response in smallpox and after vaccination (cont.) Methods for measuring -mediated immunity 155 Cell-mediated immunity in relation to pathogenesis 156 The immune response in smallpox 157 The immune response after vaccination 158 Cells involved in immunological memory 160 Reducing the risks of vaccination 161 Inactivated virus vaccines 162 Non-specific mechanisms involved in host defence 163 Body temperature 163 164 Age 164 Hormonal effects 164 Interferon 165 Genetic aspects of resistance to smallpox 165 for resistance to smallpox 165 Summary : the pathogenesis of smallpox 166 Viral entry and infection 166 Spread through the body 167 The immune response 167 or recovery 167

INTRODUCTION animals and such experiments could only be undertaken in microbiologically highly The term pathogenesis is used to describe secure laboratories. For want of a better alter- the mechanisms involved in the production native, therefore, most investigations that of , including the spread of virus are relevant to an understanding of the through the body and the physiological pathogenesis of smallpox have been carried responses of the host to the infec- out with other orthopoxviruses, especially tion, of which the most important is the mousepox virus, since so much is known of immune response. The pathogenesis of small- the and immune response in mice. could be studied in three ways: (1) by But mice are not men and mousepox is not using material from human patients, which smallpox. Arguments by analogy must there- had the advantage of direct relevance but the fore be developed cautiously. The results of disadvantage that planned experiments were these diverse investigations will be summar- not possible; (2) by conducting experiments ized in this chapter, and the information with variola virus infections of non-human collated to provide an integrated picture of primates; and (3) by conducting experiments the pathogenesis and immunology of with “model” infections-ectromelia (mouse- smallpox. pox) in mice, vaccinia (rabbitpox strain) in Following the analysis of the pathogenesis rabbits, and monkeypox in monkeys. Each of of smallpox, we proceed to a description of the these approaches had advantages and disad- pathology and histopathology of smallpox vantages. With human subjects, investigation and vaccinia, which have been studied with during life was limited to the of material from on fatal cases and, in a body fluids, skin (rarely), and the few cases, by taking from skin lesions. examination of postmortem material. No Most of this work was carried out early in this investigations were possible during the incu- century; the most recent and most compre- bation period, when most of the critical hensive descriptions of the pathology and events in the spread of the virus through the histopathology of smallpox were published body and the initiation of the immune by Bras (1952a,b). response occurred. Monkeys that developed a Finally, this chapter presents an account of rash after infection with variola virus by the the immune responses in smallpox and vac- respiratory route probably provided the best cinia, which provides the rationale of the use model system, but monkeys are expensive of the tool that made the eradication of 3. PATHOGENESIS. PATHOLOGY AND IMMUNOLOGY 123 smallpox possible-Jennerian vaccination. Virus did not spread beyond the respiratory Immunological intervention designed to pre- tract until the 3rd day after infection, when it vent human began in the 10th was found in free macrophages in the drain- century, with variolation, and entered a new ing lymph nodes. At no stage did a substantial “scientific” phase when vaccination against “primary ” develop in the respiratory smallpox was introduced in 1798 (see Chapter tract, like that found, in both mousepox and 6). Over the past three-quarters of a century smallpox, after infection by cutaneous an enormous literature has accumulated des- inoculation. cribing the mechanisms by which vacci- nation induced immunity to smallpox. Unfor- Inoculation Smallpox tunately, because methods of studying cellular aspects of immunity were developed relative- The clinical picture of inoculation small- ly recently, most of these investigations were pox, which sometimes occurred accidentally concerned only with humoral immunity. In but was usually due to variolation by the spite of its long history, the immunology of cutaneous route, is described in Chapters 1 both smallpox and vaccination against it are and 6 (see Plates 6.1-6.3). A local skin lesion still imperfectly understood. appeared by the 3rd or 4th day. and constitutional symptoms began on the 8th day, and the rash, which was usually much less THE PORTAL OF ENTRY OF severe after variolation than in naturally VARIOLA VIRUS acquired smallpox, appeared on the 10th or 11th day. Thus the incubation period ap- Infection with variola virus occurred via peared to be 2-3 days shorter than in “natur- the respiratory tract, by inoculation through al” smallpox (see Chapter 1, Fig. 1.3). Obser- the skin, or, rarely, via the conjunctiva or the vations on mice infected with mousepox by placenta. scarification (Roberts, 1962b) suggest that the shorter incubation period may have been due The Respiratory Tract to the fact that after dermal infection infected macrophages were transported to the local Epidemiological evidence indicates that lymph nodes and thus to the circulation the usual mode of entry of variola virus was within the first 24 hours, whereas after via the respiratory tract and that respiratory infection viral dissemination by from the and nose, rather than scab macrophages was delayed until the 3rd day. material, were the most important source of Viraemia and rash would therefore occur a infectious virus. In theory, infection by in- few days earlier after dermal infection. haled virus could have occurred via the mucous membranes of the mouth, the nasal The Conjunctiva cavity, or the oro- or nasopharynx; or via the alveoli of the . However, careful study of If infection via the conjunctiva occurred at these sites in fatal cases of smallpox failed to all in smallpox, it was very rare (Rao, 1972). disclose any evidence of a “primary lesion” However, Kempe et al. (1969) noted that there. Nor were patients infectious before the occasionally variolous conjunctivitis, con- enanthem appeared, at the end of the incuba- firmed by viral isolation, occurred at or even tion period. It seems, therefore, that the before the onset of the pre-eruptive fever. In primary infection in the mouth, pharynx or no case was the interval between conjuncti- respiratory tract did not produce a sizeable vitis and rash as long as that found between lesion nor did the lesion of infection ulcerate the appearance of the primary lesion and the and release virions on to the surface. rash in inoculation smallpox, which makes it In the absence of data from human small- pox it is pertinent to look at an model. difficult to decide whether the conjunctiva Observations by Roberts (1962a) on mouse- was actually the portal of entry. pox suggest a possible sequence of events. Fluorescent- staining of sections Congenital Infection taken after exposure of mice to aerosol infec- tion showed that the first cells to be infected Variola major was always severe in preg- were mucosal cells of the upper and lower nant women (see Chapter 1). In Rao’s (1972) respiratory tract and alveolar macrophages. series, abortions or stillbirths occurred in 124 SMALLPOX AND ITS ERADICATION

35% of those in whom pregnancy terminated got smallpox, so that in order to study the and observations were possible. The majority spread of infection through the host it was (55 yo) of 113 babies born in died necessary to use animal models. Four such within 15 days, usudly within 3 days. Con- models have been studied : mousepox in mice, genital smallpox was recognized in only 10 of rabbitpox in rabbits, and monkeypox and these, but some children may have died before smallpox in monkeys and apes. a rash appeared. Nevertheless, at least half of the babies did not acquire infection in utero. Being so much milder than variola major, Mousepox cases of variola minor in pregnant women provided better data on congenital infections. The pioneering work on the pathogenesis There were 150 pregnant women in Mars- of generalized orthopoxvirus infections, den’s (1936) series of 13 686 cases of variola which provided a model that proved useful in minor. Only 6 abortions are known to have understanding the pathogenesis of chicken- occurred, but Marsden thought that smallpox pox (Grose, 1981) as well as that of smallpox, sometimes played a part in the induction of was carried out with mousepox (Fenner, labour during the last 2 months of pregnancy. 1948a,b). Mice that did not die from acute Sixteen women were confined in the smallpox viral hepatitis developed a generalized pustu- hospital and bore 16 live infants (including 1 lar rash (Fenner, 1948~)and the symptoms of pair of twins) and 1 stillborn fetus with a the naturally occurring disease could be papulo-vesicular eruption. Twenty-seven reproduced by the footpad inoculation of women were delivered of live infants just mice with a small dose of virus. before admission to hospital, labour having The course of events after footpad inocu- begun at the time of the pre-eruptive fever. lation was followed by sacrificing mice at Marsden & Greenfield (1934) reported an frequent intervals and titrating the viral analysis of 34 of the cases concerned. In 17 content of certain organs-the inoculated cases the baby escaped in utero infection. In 2 of foot, the regional , the , the these babies, born during the mother’s con- skin, and the blood. The results indicated that valescence, the vaccination did not take; the the sequence of events during the incubation rest were successfully vaccinated, but 4 of period in mousepox followed a consistent them later contracted smallpox. Only 2 of the pattern (Fig. 3.1). If the appearance of the 17 babies who contracted variola minor in primary lesion was taken as the end of the ukro died. The course of the disease was never incubation period, it was evident that this coincident in mother and baby; symptoms in symptom-free period was occupied by a the baby usually occurred 9-11 days after complex series of events in which the virus those in the mother, an interval similar to the passed in a stepwise fashion through the incubation period of inoculation smallpox. body: infection, replication, and liber- The fetus was presumably infected by the ation-usually accompanied by cell necro- growth of virus in the placenta, which would sis-first at the site of inoculation, then in the have been infected during the stage of second- regional lymph node, and then in the deeper ary viraemia (see below). lymph nodes or perhaps directly into the Observation of a laboratory model (preg- bloodstream. It seems from the work of Mims nant mice infected with an attenuated strain (1964), who used fluorescent-antibody stain- of mousepox virus) revealed that the placen- ing to identify virus-infected cells, that in the tas were infected and the virus grew exten- liver and spleen the phagocytic cells were the sively in the fetuses (Mims, 1969). Some live first infected. When infection had breached births occurred, but all such mice had wide- the macrophage barrier the virus replicated spread lesions which proved lethal, although very extensively in the parenchymal cells of the mothers had suffered only a mild disease. the liver and in the spleen, both of which usually showed semiconfluent . A day THE SPREAD OF INFECTION or so after infection of the spleen and liver, THROUGHTHEBODY large amounts of virus were liberated into the bloodstream, and during this secondary virae- The only sources of virus accessible for mia focal infection of the skin, kidneys, lungs, study in human smallpox during the incu- intestines and other organs occurred. There bation period were various of was again an interval during which the virus contacts of cases, some of whom ultimately replicated to reach a high titre before visible 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 125

Days Ectromelia Vaccinia Variola by Variola Days (in mice) inoculation \

d (I- k! I I- I- Regional Regional lymph , Legional lymph -I node node 3. Upper or qslower 2- 4 respiratory tract -2 0 0 3- 3. 3. 3. -3 Replication -;*: Replication liver -:*: Regional lymph node -4 4- in lymphoid organs ilymphoid organs 5- .1 -5 3. Replication Viraemia /iraemia.1 @ 6- focal in lymphoid organs - infec 7 ___ "3. 3. .1 -7 Swelling of foot: ;eneralized rash Seneralized rash @ Viraemia -8 8 - primary lesion (very rare) (common) 9- 0 3. -9 10 - 0 -10 G \#I Early rash papule -0-,,. 3. I1 - @Local secondar Flat and Ordinary- -1 I 0 lesions haemorrhagic type 12 - rash -12 I3 - ulceratioi 0 - I3 1111') 0 14 - - 14 IS - 0 -IS 16 - -16 17 - - I7 18 - -18

- 0 _. 19 I\ - I9 \lo permanent Always ,I '-,' 20 - scar a scar 0 - 20 Variable Always 21 - scarring a scar -21

22 - - 22

23 - Macule - 23 24 - - 24 \'I Papule 25 - ;:: - 25 26 - @ Vesicle -26 27 - - 27 @ Pustule 28 - - 28 29 - Scab - 29 30 - - 30 0 Scar 31 - - 31 32 - 0 - 32

Fig. 3. I. The spread of virus around the body and the and healing of skin lesions in a model system (ectrornelia. after Fenner. I948b), and in vaccinia, inoculation smallpox, and "natural" smallpox in man. For vaccinia and inoculation smallpox, the right-hand column represents lesion at site of inoculation, left-hand column represents generalized skin lesions. (Based on Dixon. 1962.) 126 SMALLPOX AND ITS ERADICATION

changes were produced, so that a period of ensuing infection of the skin and other organs about 2 or 3 days usually elapsed between the and tissues. appearance of the primary lesion and the focal Subsequent research into the pathogenesis lesions of the secondary rash (Fig. 3.1). The of smallpox was carried out with other secondary viraemia was mainly cell-associated laboratory models-rabbitpox and monkey- and only small amounts of virus could be pox in rabbits and monkeys respectively, and recovered from plasma or serum. Mims (1964) non-human primates experimentally infected demonstrated by fluorescent-antibody stain- with variola virus. ing that circulating lymphocytes and mono- cytes contained viral antigen. Rabbitpox Although this work provided a general explanation of what happened during the Studies on the pathogenesis of rabbitpox, a long incubation period of the generalized severe disease of rabbits produced by certain exanthemata (Fenner, 1948b), mousepox is strains of vaccinia virus, did not add much to not a satisfactory model for smallpox. Apart the picture that had emerged from experi- from the difficulty of comparing events in ments with mousepox. After rabbits were mice and men, the usual mode of infection in infected by the respiratory route (Bedson & mousepox is via skin abrasions (it is thus Duckworth, 1963 ; Westwood et al., 1966), analogous to inoculation smallpox), rather virus spread through the body in a stepwise than by the oropharynx or respiratory tract. manner. Viral isolations could always be made More important, mousepox appears to be from the lungs, and very high titres occurred unusual among the generalized orthopox- in the gonads and the adrenal glands. The virus infections, and certainly different from incubation period, from infection until the human smallpox, in that the spleen and liver onset of fever, was 4-6 days. A rash usually are major target organs for viral repli- appeared on about the 6th day, although often cation. In mousepox these organs are clearly rabbits died before the rash became evident the “central foci”, established during the in- (“pockless” rabbitpox ; Christensen et al., cubation period, from which virus is contin- 1967). ually released into the bloodstream, with In spite of the early replication of rabbitpox virus in the , rabbits were not infectious until nasal and conjunctival dis- charges appeared, and environmental con- tamination appeared to be maximal 6-7 days after infection. Examination by fluorescent- antibody staining revealed that after aerosol infection primary lesions developed at two distinct sites in the respiratory tract-the bronchioles and the alveoli. It was also found that as well as being spread to the bloodstream via the lymphatics, rabbitpox virus could spread directly from the bronchiolar or alveo- lar lesions to adjacent blood vessels. Viraemia was mainly leukocyte-associated. Animals that died early, before the rash appeared, had viraemias which increased exponentially to reach very high titres at the time of death ;those that died later had much lower titres (Westwood et al., 1966). This situation invites comparison with the levels of viraemia in haemorrhagic-type and dis- Plate 3.1. Frank Fenner (b. I914). Formerly Pro- crete ordinary-type smallpox respectively (see fessor of in the John Curtin School of below). of the Australian National Univer- sity, Canberra. He is shown here inoculating eggs on the chorioallantoic membrane, a method used for Monkey pox assays of orthopoxviruses and neutralizing antibodies to them, and for the differentiation of variola virus Cynomolgus monkeys are highly suscep- from other poxviruses. tible to monkeypox and usually develop a 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 127 generalized rash (Magnus et al., 1959). virus died, but all developed fever on the 5th Wenner and his colleagues (review: Cho & day and a rash between the 6th and 11th days Wenner, 1973) studied the pathogenesis of (usually on the 7th or 8th day). Assay of monkeypox in cynomolgus monkeys infected various organs revealed high titres in the by intramuscular inoculation. These investi- lungs and skin, with occasional isolations gations confirmed that in orthopoxvirus in- from the blood and spleen. Large amounts of fections of primates, as well as in those of mice virus were recovered from all organs tested in and rabbits, there was a stepwise progression a monkey that died on the 11th day. of infection, a generalized rash developing Rao et al. (1968b) showed that cortisone after viraemia had been established owing to greatly increased the severity of smallpox in the replication of virus in the internal bonnet monkeys (Mama radiata) inoculated organs. Prior to the development of the rash intradermally with variola virus. None of 14 the virus replicated in the spleen, tonsils and controls died, whereas 12 of the 16 animals lymph nodes, without producing extensive or given cortisone and 1 pregnant monkey died severe lesions. Generalized lymphadenopathy after suffering from a much more severe developed in the 1st week after infection and primary lesion and generalized rash than were persisted until the end of the 3rd week. seen in the control animals. Virus could be Enlargement of the cervical and inguinal readily recovered from most of the internal lymph nodes is a feature of monkeypox organs of the cortisone-treated animals at the infections in chimpanzees (McConnell et al., time of their death. This increased severity is 1968) and humans (see Chapter 29). comparable to that seen in pregnant women who contracted variola major (Chapter 1). Orang-utans (Gispen, 1949) and chimpan- zees (Kalter et al., 1979) can be naturally Variola Virus Infection in Non-human infected with variola virus (see Chapter 30), Primates and may suffer a severe disease with an extensive rash (see Plate 30.1). Baboons (papi0 The monkey was the first animal to which gnocephalus) inoculated with variola virus by variola virus was transmitted (Zuelzer, 1874) scarification developed a rather “dry” local and monkeys were extensively used in early lesion but no rash; virus was recovered from studies of cross-immunity between variola the blood, throat swabs and rectal swabs and vaccinia (Brinckerhoff & Tyzzer, 1906 ; between the 4th and the 7th days after Horgan & Haseeb, 1939). Experimental infection (Heberling et al., 1976). studies of the pathogenesis of smallpox in the cynomolgus monkey (Mama irus) were car- ried out by Hahon & Wilson (1960) and Noble & Rich (1969). After an incubation Smallpox in Human Subjects period of about 6 days, inoculated animals usually developed a generalized rash that was The 4th and 5th columns of Fig. 3.1 sparse on the face, hands and feet-sites illustrate diagrammatically, on the basis of where the rash was usually more intense in available data, the sequence of events in human smallpox. After infection by aerosol, human smallpox after intradermal inocu- variola virus replicated in the lung, and lation and after infection via the respiratory secondary sites of replication were estab- tract. Investigations in human subjects rel- lished in the lymph nodes before viraemia evant to the pathogenesis of smallpox have occurred. Although the study was complicat- been limited to virological and serological ed by pre-existing bronchopneumonia, Ha- tests carried out in hospitalized patients or hon & Wilson (1960) failed to demonstrate case contacts. Such studies were limited to the any major focus of local examination of blood, , and throat in the lungs even though they yielded a washings in cases after the onset of fever and substantial amount of virus during the incu- throat washings from case contacts during bation period. Natural transmission to other what was potentially the incubation period. monkeys could occur by the airborne route, These investigations were important because with an incubation period of 8-16 days (see they provided information that permitted Chapter 30, Fig. 30.1). arguments about the pathogenesis based on Only 2 out of 109 rhesus monkeys (Mama model systems to be developed with more mulatta) infected by aerosol with variola major confidence. 128 SMALLPOX AND ITS ERADICATION

Viraemza 160 7 .. No precise observations have been made on the distribution of variola virions among the various components of the blood in cases of smallpox ; by analogy with other poxvirus infections viraemia would have been expected to be primarily cell-associated. Most assays of viraemia in smallpox were made with serum or lysed whole blood, the material being assayed on the chorioallantoic membrane of chick embryos (Downie et al., 1950, 1953, 024 6 8 10 12 1969b; Mitra et al., 1966). Although viraemia must always have oc- . .:@.em&. ." 0 o curred, virus was only rarely recovered from ++++l I the blood or serum from cases of ordinary- t++ - I ox.. type smallpox. Downie et al. (1950, 1953) .-m and Mitra et al. (1966) recorded one or two E % ++- positive results out of many attempts in such .- > cases and then only in the first few days of the disease. The picture in haemorrhagic-type +- smallpox was quite different. Virus was readi- ly recovered from the blood of all cases and xx &.:m. m Lm 0 x the titres were usually high, as determined by 0 24 6 8 10 12 confluent lesions on the membrane (Downie Day of illness et al., 1953,1969b) or by titration (Mitra et a]., 1966; Sarkar et al., 1969). In these patients Fig. 3.2. Antigenaemia and viraemia in haemor- viraemia usually persisted until the patient rhagic-type smallpox. 0 = early haernorrhagic type: x = late haemorrhagic type; 0, @ = vaccinated; died. Downie et al. (1969b) noted that virae- S = survived (all other cases died). (From Downie mia was consistently much higher in cases of et al., I969b.). early than of late haemorrhagic-type small- pox. They also examined the serum by gel- large amounts of highly infectious virus into precipitation and complement- tests the saliva. The viral titres in throat swabs were for the presence of soluble antigens of variola at their maximum on the 3rd and 4th days of virus ; cases of haemorrhagic-type smallpox the disease (i.e., at the onset of the rash) and usually had an antigenaemia (Fig. 3.2). Com- were highest, and persisted for the longest plement-fixation tests revealed antigenaemia period, in the most severe cases (Sarkar et al., in 31 out of 46 sera tested, the level being, in 1973a ; see Chapter 4). In fatal cases virus was general, proportional to the extent of the usually still present in the throat swabs at the viraemia. The gel-precipitation test was less time of death ; in non-fatal cases it was found sensitive, revealing antigen in only 16 out of for 7-9 days after the onset of fever in discrete 45 sera tested, all from cases of early haemorr- ordinary-type smallpox and sometimes for as hagic-type smallpox with severe viraemia. long as 13 days in non-fatal confluent ordi- Thus haemorrhagic-type smallpox appears nary-type smallpox. to have been associated with overwhelming More important, from the point of view of infection and the continued release of virus pathogenesis, are reports of the recovery of into the bloodstream ; in ordinary-type small- virus in throat washings taken from case pox demonstrable viraemia was usually re- contacts who developed smallpox. Careful stricted to the pre-eruptive and early eruptive laboratory studies by Sarkar and his colleagues stages of the disease. showed that household contacts of cases of smallpox sometimes harboured detectable amounts of variola virus in their throats. Oral and pharyngeal secretions Using a single throat swab, Sarkar et al. Because there is no tough stratum corneum (1973b) isolated variola virus in 34 out of 328 on the oral and pharyngeal mucosae, the subjects who had been in close household lesions of the enanthem ulcerated very soon contact with 52 index cases of smallpox for after their formation (see Plate 3.7), releasing periods of between 4 and 8 days. Only 4 of the 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 129

34 contacts with positive throat swabs subse- bloodstream as cell-associated particles, al- quently developed smallpox, the symptoms though some virus was always found free in appearing 5-6 days after virus was isolated. In plasma. In cell cultures, enveloped forms of a follow-up study (Sarkar et al., 1974), posi- vaccinia virus were more important than tive results were reported in 5 out of 51 close non-enveloped forms in the dissemination of family contacts examined, only 1 of whom virions over a distance (Boulter & Appleyard, subsequently developed smallpox (Table 3.1). 1973), as shown by the “anti-comet’’ test (see Serial testing showed that virus could some- Plate 3.10). Payne (1980) showed that the times be recovered from the throats of vacci- capacity to spread to distant cells in mono- nated contacts, most of whom did not develop layer cultures and to distant organs in the clinical smallpox, for as long as 10 days. mouse was correlated with the production of F. Huq (personal communication, 1982) enveloped virions (Table 3.2). confirmed Sarkar’s findings, but recorded a Unfortunately, no observations were ever much smaller proportion of positive results. made to determine whether enveloped vir- Using a similar laboratory procedure, she ions occurred in infections of monkeys or found 3 positives (all in unvaccinated sub- human beings with variola virus, nor have jects, and all of whom subsequently developed such observations been reported with ectro- smallpox) among 168 contacts who were melia or monkeypox . examined within a week of the development of rash in the index subjects. The finding of virus in throat swabs of THE RASH contacts of acute cases is not altogether surprising ; presumably virus replicating in The primary event for the production of an asymptomatic “primary lesion” in the the focal lesions of the rash in orthopoxvirus fauces or elsewhere in the oropharynx was infections is the localization, in the small released into the oral secretions or could be blood-vessels of the dermis, of virus particles obtained by swabbing the fauces. However, that circulate in the bloodstream, either freely such virus as was present in the oral secretions in the plasma or more commonly with- during the incubation period was of little or in virus-infected leukocytes. Subsequently, no epidemiological importance. The onset of adjacent epidermal cells are infected and the infectivity in the vast majority of cases skin lesion develops as described below. coincided with the development of the rash In the infection of laboratory animals with and was due to the release of virus from the orthopoxviruses, skin areas so treated as to ulcerated surfaces of the lesions of the enan- promote a local inflammatory response are them. associated with an increased density of skin lesions (vaccinia in rabbits : Camus, 1917 ; The Dissemination of Virus through ectromelia in mice : Fenner, 1948a). Numer- the Body ous observations (Ricketts, 1908 ; MacCallum & Moody, 1921) attest to the effect of mild Orthopoxvirions were principally dis- trauma leading to local vasodilatation on the seminated through the body in the lymph and density of skin lesions in different parts of the

Table 3. I. Prolonged presence of variola virus in throat swabs from contacts of cases of smallpox (data expressed as log pock-forming units per swab)

Days after onset of fever in index cased Serial no. of contact Age Vaccination scar 4 6 10 II I2 14 17 19 21 23

55 years + . . 4.0 . . 2.0 Developed smallpox 5 months - 3.0 . . . . 2.0 Developed smallpox 23 years + ...... 1.9 1.9 1.8 1.7 1.5 0 22 years + ...... 3.0 2.9 2.9 1.9 1.6 0 24 years + ...... 3.3 2.9 2.0 2.0 0 45 years + ...... 1.7 . . . . Developed smallpox 28 years + .. 1.8 0 0 ......

a.. = data not recorded. Sarkar et al. ( l973b). CSarkar et al. (1974). dA nurse, revaccinated almost every year, in close contact with a case of haemorrhagic-type smallpox. 130 SMALLPOX AND ITS ERADICATION

The Significance of Enveloped Virions

Viruses belonging to 10 of the 17 viral families that disease in humans are enveloped-i.e., their outer surface is a lipoprotein envelope consisting of cellular and virus-specific polypeptides. Destruction of the envelope, for example by solvents, usually completely destroys infectivity. The orthopoxviruses are exceptional in that although virions released from cells are enveloped (see Chapter 2, Plate 2.9), absence of the envelope is associated with only a slight decrease in the infectivity/particle ratio. Although non-enveloped particles, whose surface consists of the outer membrane (see Chapter 2, Fig. 2.1), have almost the same infectivity as enveloped particles and spread effectively by cell-to-cell contact, they do not spread as well around the body as do enveloped virions (Table 3.2). Because there are two kinds of infectious particle, two kinds of neutralizing antibody can be produced which neutralize enveloped and non-enveloped particles respectively. Both kinds of neutralizing antibody are produced during all orthopoxvirus infections. Antibody that neutralizes only enveloped particles can be produced by immunization with purified viral envelopes. Antibody that neutralizes only non-enveloped particles is produced by inoculation with suspensions of particles obtained by disrupting cells and then inactivated. Neutralizing antibody to non-enveloped virions provides much less effective passive protection against generalized orthopoxvirus infections than does antibody to enveloped virions (see Table 3.5). The two kinds of particle also produce different kinds of cell-mediated immunity, cytotoxic T cells being generated only during infections and not by suspensions of inactivated virions. For these reasons inactivated vaccines provide only partial immunity in experimental animals and have proved useless for vaccination against smallpox. . 1 body in smallpox (the “garter” effect). Vaso- number of skin lesions) might well be dilatation due to sun and wind may have ambulant. played a part in the greater density of lesions No adequate explanation is available to on the face and the extensor surfaces of the elucidate either the toxaemia of variola major hands and arms than elsewhere on the body, or the difference in severity between cases of but this factor does not explain the highly variola major and variola minor with of characteristic “centrifugal” distribution of similar extent. Viral antigen was readily skin lesions in smallpox, for which no satisfac- demonstrable in the plasma of patients with tory physiological explanation has yet been severe smallpox (Downie et al., 1953, 1969b; provided. see Fig. 3.2). The formation of immune complexes between such antigens and IgM antibodies, and the associated activation of TOXAEMIA complement, might have initiated a series of physiological effects that produced the so- All clinical observers have commented on called “toxic” symptoms of variola major. the “toxic” appearance of patients with var- Seeking an explanation of the cause of iola major, especially those suffering from death in smallpox, Boulter et al. (1961a) flat-type or haemorrhagic-type smallpox. examined various physiological parameters in Although very severe cases did occur+x- a model system-rabbitpox. The only consis- tremely rarely-in variola minor, there was tent physiological changes observed in sick usually a great contrast in the general appear- rabbits were extreme hypotension, leading to ance and condition of patients with variola a shock-like syndrome, decreased urinary major and variola minor with approximately output and a rise in blood-urea and plasma- the same numbers of pustules. The patient potassium levels. Death seemed to be due to with acute variola major was usually very sick, lethal concentrations of potassium ion, which whereas the patient with variola minor of occurred possibly as a consequence of the apparently similar severity (in terms of the severe hypotension. No information is avail- 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 131

Table 3.2. Relationship of in vitro virus release to in vitro and in vivo parameters of virus disserninationa

RK 13 cells Mouse vlrulenceb

Vlrus production Vaccinia strain Enveloped Ratlo: Non-enveloped a8comets,,dOf Mortality (Oh) Brain titree virionsc Enveloped

South Africa 5.6 250 0 - Cape Town 5.7 I00 0 - Venezuela 6.0 i 60 0 - Lister 6.3 300 0 - Tashkent 6.6 I60 0 - WR 6.6 300 85 4.7 Lederle 7N 6.7 250 0 - Hall Institute White 6.8 50 0 - Dairen 7.0 16 0 2.7 Lafontaine 7.6 40 0 3.6 IHD-W 8.0 12 25 3.3 Gallardo 8.3 5 25 3.3 IHD-J 8.3 6 70 4.6

a Based on Payne (I980). After incubation with lo6 plaque-forming units intranasally. Titre in log,, plaque-forming unlts per millilitre. cell monolayers with liquid overlay (see Place 3.10). eTitre in log,, plaque-forming unlts per gram of brain. able on blood-pressure or blood-potassium lungs, was complicated by the presence of changes in severe cases of smallpox. secondary infection with streptococci and other (see Councilman et al., 1904). More recent observations have shown that PATHOLOGICAL ANATOMY AND although bacterial infection did sometimes HISTOLOGY OF SMALLPOX complicate smallpox, pustulation was viral and not bacterial in origin, and the high General Observations mortality of variola major was due to viral effects, not secondary bacterial infection. As described in the appropriate chapters of The case-fatality rate was not significantly this book, a wealth of information about the reduced by the use of antibiotics. Bacterial , and control of small- complications were rare in the cases described pox has emerged from the work carried out by Bras (1952a), most of which were treated since the inception of the Intensified Small- with antibiotics. pox Eradication Programme in 1967. How- A striking feature of the pathology of ever, what Bras (1952a) pointed out more smallpox is that although it was a generalized than 30 years ago was still true at the time of disease, often of great severity, specific patho- global eradication : in recent years smallpox logical changes were in large part limited usually occurred, on a large scale, in places to the skin and mucous membranes, except where pathological studies were difficult. for the widespread haemorrhages found in Prior to Bras’s own important work one has to various organs in early haemorrhagic-type go back to Councilman et al. (1904) for a smallpox. comprehensive description of the pathologi- cal anatomy and histology of smallpox. Some years later Lillie (1 930) produced a review of The Skin Lesions published reports on the histopathology of smallpox and vaccinia. A useful description of In order to understand the histopathology the histology of the skin lesions in variola of the skin lesions in smallpox it is necessary major was provided by Michelson & Ikeda to appreciate the relationships between dif- (1927), while MacCallum & Moody (1921) ferent parts of the skin, as illustrated in and Jong (1956) obtained skin biopsies from Fig. 3.3. The epidermis, where the pustule patients suffering from variola minor. develops, contains no blood or lymphatic In earlier studies interpretation of the vessels and its tough outer layer, the stratum pathological changes, particularly in the corneum, is impermeable to viruses. The dead 132 SMALLPOX AND ITS ERADICATION

Subcutaneous fat

Fig. 3.3. The structures of importance in the patho- genesis of the skin lesions of smallpox. The epidermis consists of several layers of living cells, the stratum Malpighii. covered by a layer of dead keratinized cells, the stratum corneum (A). The basal layer of m -m the stratum Malpighii consists of dividing cells, the u stratum germinativum (D). above which there is a variable number of layers of cells, called the stratum spinosum (C). and just below the stratum corneum a Plate 3.2. Gerrit Bras (b. 1913). Professor of Pathology at the National University in Utrecht, layer of cells that contain keratohyalin granules, the stratum granulosum (B). The dermis contains blood Netherlands. Bras carried out a classical study of vessels lymphatic vessels and fibroblasts and the pathological changes in variola major during the (V), (L) macrophages The ground substance contains epidemic in Java after the Second World War. (FM). collagenous, reticular, and elastic fibres. The hair follicle (HF) and sebaceous gland (SG) are appendages keratinized cells of the stratum corneum are of the epidermis. continually being rubbed off and replaced from below, cell multiplication occurring in dermis, followed by swelling of the endotheli- the stratum germinativum, which is separated a1 cells in the walls of these vessels, stasis of from the dermis by a thin basement mem- mononuclear cells within the lumen, and brane. Of the three appendages of the skin- subsequently perivascular cuffing with lym- hair follicles, sweat glands and sebaceous phocytes, plasma cells, macrophagesand occa- glands-only the last are destroyed by variola sionally granulocytes (Plate virus, and then, to any extent, only by variola 3.3A). Biopsies from cases of variola minor major virus. (MacCallum & Moody, 1921)showed that the The appearance of the skin lesions, their affected papillae were oedematous, with ex- temporal development and their character- travasation of erythrocytes and leukocytes, at istic distribution are described and illustrated a time when the overlying epidermis was in Chapter 1. The following description of the unchanged or showed at most only an early histology of the skin lesions at various stages vacuolation of the epidermal cells. of the disease draws mainly on postmortem material from variola major described by Epidermal changes Michelson & Ikeda (1927) and Bras (1952a,b) and biopsy material obtained from cases of Following the early changes in the dermal variola minor (MacCallum & Moody, 1921 ; vessels, lesions developed in the adjacent part Jong, 1956). of the epidermis, where all subsequent changes occurred. The cells of the stratum Malpighii became swollen and vacuolated and Ear4 changes in the dermis underwent what was described as "balloon- Bras (1952a) noted that the histological ing" degeneration. This occurred in a sharply picture in the skin lesions in all types of demarcated area and only a small number of variola major, whether haemorrhagic or not, cells separated the centre of the lesions from was essentially the same. The development the surrounding normal skin (Plate 3.3A and and evolution of a skin lesion are shown in B). The degenerated cells were swollen, they Plate 3.3. The earliest change was a dilatation stained faintly with acid dyes, and the charac- of the capillaries in the papillary layer of the teristic B-type (Guarnieri 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 133 bodies: see Chapter 2, Plate 2.7) could be becoming swollen, homogeneous and refrac- found in the cytoplasm. tile, losing their granular character and stain- Early in the disease, in sections stained ing more intensely with acid dyes. Later they with and eosin, the inclusion lysed, so that the base of the vesicle was bodies appeared as round or oval homogen- provided by the subjacent dermis. eous faintly or acidophilic masses (3) Since a portion of the cytoskeleton lying close to the nucleus. One or more were persisted for a long period after the degener- present in a cell and each was usually sur- ation of the cells of the stratum spinosum, the rounded by an unstained halo. In older lesions cavity of the vesicle contained incomplete the inclusions had a granular appearance and septa, creating a multiloculated appearance irregular outline and sometimes occupied a (Plate 3.4A). Such loculation was never large part of the cytoplasm of infected cells. complete. Often there were heavier septa, Intranuclear inclusions have also been de- which were made up of the coils of sweat scribed in cells infected with variola virus glands traversing the cavity. Like the kerato- (Torres, 1936), but not with other orthopox- hyalin cells in the roof of the vesicle, the cells viruses. They were not a conspicuous feature of sweat glands appeared resistant to the of the lesions, but Downie (1965a) confirmed effects of variola virus. that they did sometimes occur. Their signifi- (4) Fluid accumulated inside the vesicle, cance is unknown. with threads of fibrin and a few lymphocytes. The cells continued to increase in size, the (5) The cells immediately around the vesi- cytoplasm became fainter and the nucleus cle showed decreasing degrees of reticulating usually disappeared by lysis. Soon after this degeneration and the basal layers had often the cell membranes ruptured and the vacuoles proliferated, so that the wall around the coalesced to produce the early vesicle by what vesicle was about twice the thickness of the was called “reticulating” degeneration (Plate unaffected epidermis. The rete pegs in the 3.3C). Because this coalescence occurred very area adjacent to the vesicle were relatively quickly a true papule was rarely seen ; almost deep. from the beginning the lesion was already vesicular. The pustule Pustulation occurred by the migration of Vesiculation polymorphonuclear cells from the subpapil- lary vessels into the vesicle, the dermis being The reticulating degeneration which pro- relatively free of such cells (Plate 3.3D and E). duced the vesicle occurred exclusively in the This response was not due to secondary middle and upper layers of the stratum bacterial infection ; numerous investigations spinosum; the basal cells were at first unaf- showed that pustules containing abundant fected and the keratohyalin and horny layers leukocytes were -free. Councilman et showed no changes. Subsequently the cells of al. (1904) commented on the absence of the lower stratum spinosum and the basal polymorphonuclear leukocytes and the abun- layer underwent a different kind of degener- dance of plasma cells in the adventitial ation ; the nuclei and cytoplasm became sheaths of the vessels in the dermis as condensed, the cells became hyalinized and pustulation occurred-+vidence of the rapid- the nuclei fragmented or lysed. Later these ity and extent of the immune response in basal cells disappeared and the cavity of the ordinary-type smallpox. Unfortunately, the vesicle (or pustule) was then immediately literature lacks descriptions of the histopath- adjacent to the dermis. ology of flat-type smallpox. In such cases one The fully developed vesicle (Plate 3.4) might have expected to find a much less resembled a plano-convex lens with the vigorous cellular response at this stage of the following characteristics : disease. (1) The roof, which was very thin over the summit of the vesicle, consisted of com- Urnbilication pressed cells of the stratum spinosum, kerato- hyalin layer and horny layer. Except on the palms and soles, umbilica- (2) The base consisted at first of cells of the tion was a common feature of the skin lesions stratum spinosum and basal layer, which in smallpox (see Chapter 1, Plate 1.10). It was showed a hyaline fibrinoid degeneration, apparent quite early, then partially disap- 134 SMALLPOX AND ITS ERADICATION

Oedema of_ papilla

Separation of epithelial cells

Polymorpho. nuclear cell. infiltrate . 7 Reticulating degeneration

Ballooning - degeneration

New

Densely massed polymorpho- Proliferated nuclear cells epithelium

Plate 3.3. Stages in the development and evolution of the skin lesion. A: The earliest change was oedema of the dermis leading to the separation of epithelial cells of the papillae and lymphocytic infiltration in the dermis, especially around the small vessels. was seen in a few cells in the lower Malpighian layer. B: These changes progressed and the small vessels became dilated and engorged. Inclusion bodies were also visible adjacent to cells showing ballooning degeneration. In early haemorrhagic-type smallpox, illustrated here, there was pronounced haemorrhaging into the dermis. C: As the pathological process progressed, the epithelial cells broke down by reticulating degeneration to produce a multilocular vesicle. D: The vesicle formed by coalescence of the smaller cavities became infiltrated with polymorphonuclear leukocytes to produce a pustule, around which were cells containing inclusion bodies. E: The fully developed pustule became packed with polymorphonuclear leukocytes and the epithelium on either side of the pustule proliferated. F: Eventually the pustule became a crust, beneath which new epithelium grew in to repair the surface. Such lesions, in which the sebaceous glands were not involved, healed without leaving a pockmark. (From Michelson & Ikeda, 1927.) 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 135

Plate 3.4. Fully developed vesicle. A: Loculated cavities with relatively acellular exudate formed as a result of reticulating degeneration of the middle layers of the epidermis. Unaffected keratohyalin and horny layer form the roof of the vesicle; at its base, cells are undergoing hyaline fibrinoid degeneration, which is best seen in B. the higher power view. Haematoxylin and eosin. A x 130; B x260. (From Bras, I952a.) peared and reappeared during the late stages substance for a long period if not artificially of pustulation. Umbilication was mainly due removed. to the swelling of the cells around the vesicle and the proliferation of basal cells surround- ing the lesion, so that the periphery of the Scarring vesicle was raised above the level of its centre, The face bore the heaviest crop of lesions in as well as above the surrounding unaffected most cases of smallpox (for example, “semi- skin. Often the presence of a hair follicle confluent” ordinary-type smallpox was the within a vesicle anchored the centre part, but term applied to cases in which lesions were umbilication was also found in lesions on the confluent on the face but not elsewhere; see lip and glans penis, where no hairs occur. The Chapter l), and lesions usually appeared first partial disappearance of umbilication was due on the face and evolved the most rapidly to the increase in fluid in the vesicle; as the there. However, if the dermis was not in- contents desiccated prior to healing umbilica- volved such lesions should not have produced tion reappeared. scars, yet scarring (pockmarks) was very Scabbing much more common on the face than else- where (Jeiek et al., 1978d). Bras (1952b) With the development of an effective showed that this occurred because sebaceous immune response, healing began. The con- glands are much larger and more numerous in tents of the pustule became desiccated and re- the facial skin than elsewhere on the body. epithelialization occurred between the cavity Although the cells of other skin appendages of the pustule and the underlying dermis. The (hair follicles and sweat glands) were relatively pustular contents became a crust or scab (Plate unaffected by variola virus, cells of the 3.3 F and Plate 3.5), which was subsequently sebaceous glands were highly susceptible shed ;the newly formed epidermis had no rete (Plate 3.6). Degenerative changes began with pegs. In the absence of secondary infection, cytoplasmic hyalinization accompanied by the dermis showed very few changes and in hyperchromatism of the nuclei, most parts of the body the lesions healed and cytolysis. This degeneration occurred without scarring and thus without pock- simultaneously in several parts of the seba- marks. ceous gland, leading to extensive necrosis in In the soles and palms, where the layer of the subepithelial layer of the skin. When horny cells is very thick, the dried exudate healing occurred, the defect in the dermis was remained enclosed within a mass of horny filled with granulation , which subse- 136 SMALLPOX AND ITS ERADICATION

Plate 3.5. Scabbing. A: Low-power magnification; the pustule has become a crust. B and C: High-power magnification. B: A keratohyalin layer has developed under the outer part of the pustule. C: Recovery is at an earlier stage in the centre; later the keratohyalin layer extends beneath the entire crust. Lesions like this left no scar. (From Bras, I952a.) LEI b30TONnWWI (INV A3OIOHLVd 'SISZlN830HLVd 'E 138 SMALLPOX AND ITS ERADICATION quently shrank, leaving localized facial , and the upper part of the trachea and pockmarks. oesophagus (Bras, 1952a). Lesions of the Descriptions of the histopathology of the lower part of the trachea and the bronchi were skin lesions in variola minor do not include much less frequent, and they were rarely material from the face, since biopsies would found in the intestines, except for rare cases of be difficult to obtain from this part of the haemorrhagic-type smallpox in which the body. It is reasonable to suggest that the rarity mucous membrane of the lower rectum was of facial pockmarks after variola minor shed as a large slough (see Chapter 1). The Ueiek & Hardjotanojo, 1980) was due to the mucosal lesions evolved more rapidly than failure of variola minor virus to cause the those in the skin; healing was usually com- necrosis of cells of the sebaceous glands. plete by the end of the 2nd week of illness. In general, the mucosal lesions of haemor- rhagic-type smallpox were similar to those of Spec& gects of earb haemorrhagic-ppe smallpox ordinary-type smallpox in localization and Twenty-three of the 177 cases studied by character, except for the more pronounced Bras (1952a) were of early haemorrhagic-type submucosal haemorrhages. If, as sometimes smallpox. In these cases the major difference happened, such patients died soon after the from the skin lesions just described was the appearance of the skin lesions, the pharyngeal much more extensive hyperaemia of the mucosa did not show any gross lesions but was dermal vessels, which affected the deeper smooth and glistening, with submucosal plexuses as well as the subpapillary network. haemorrhages. There were also haemorrhages between the In patients dying at a later stage there was collagen fibres of the dermis (Plate 3.3B), and often an adherent pseudomembrane on the occasionally into the papillae and even the lateral wall of the pharynx, which when torn epidermis. Usually the patient died before off revealed a haemorrhagic-hyperaemic base. vesiculation or pustulation occurred. Early Similar pseudomembranes were found- reticulating degeneration was usually present more rarely-on the epiglottis and larynx, but in the epidermal cells, which contained large localized lesions sometimes occurred in the vacuoles. Guarnieri bodies were numerous. trachea. Ulcers also formed on the tongue, The skin lesions were usually extremely although they were often difficult to see diffuse, so that often no normal skin could be because of the pleats and folds of that . found over large areas of the body surface. Very rarely, circumscribed lesions were found on both surfaces of the intestines. Spec@ effects of late haemorrhagic-ope smallpox Histopathology of mucosal lesions Although the skin lesions often seemed to be haemorrhagic pustules (see Chapter 1; Although showing a general resemblance Plate 1.23C), histological examination to the lesions found in the skin, the pathologi- showed that bleeding usually occurred in the cal process in mucous membranes was modi- dermis beneath the pustule, although some fied by the of the tissue: epithelial cells vesicles and pustules did contain erythrocytes. in mucous membranes are not so tightly packed together as in the skin, and there is no horny layer which might have contained the Lesions of the Mucous Membranes of the developing vesicle. There was also a more Respiratory and Digestive Tracts pronounced exudation of fluid into the sub- epithelial tissues. In contrast to the skin, in which the The earliest change in the mucous mem- evolution of vesicles and pustules was ob- branes appeared to be exudation into the vious, the lesions of various mucous mem- epithelium, leading to separation of the cells, branes were less easily observed. which underwent hyaline fibrinoid degener- ation. Guarnieri bodies were numerous and numbers of superficial cells were exfoliated Gross findings from an early stage of development of the The mucous membranes on which vario- lesion. Instead of a vesicle, the extensive lous lesions occurred were, in descending necrosis in the epithelial cells, unrestrained by order of frequency, the pharynx and uvula a horny layer, led to early ulceration (Plate (see Chapter 1, Plate 1.3 C), the larynx, the 3.7). The base of the necrotic mass of cells was 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 139

no reason to doubt that the focal mucosal lesions, like those of the skin, were haemato- genous in origin. By analogy with mousepox and rabbitpox, small, non-destructive pri- mary lesions may have occurred in the oral cavity, pharynx or respiratory tract in small- pox, but they would have been impossible to recognize without recourse to a selective staining method such as fluorescent-antibody staining.

Effects on Other Organs

Death was due to viral toxaemia, exacer- bated by clotting defects in haemorrhagic- type smallpox. Antibiotics were usually given Plate 3.7. Ulcer on oral mucous membrane. The to all patients in the Madras Infectious pustule ulcerates because there is no overlying horny layer. (From Michelson & Ikeda, 1927.) Diseases Hospital from the 1950s onwards (A.R. Rao, personal communication, 1981). Such treatment reduced the case-fatality rate sharply outlined against the hyperaemic tun- by 5-10% in vaccinated subjects, compared ica propria. Occasionally the tunica propria with those not given antibiotics, but had no showed patchy necrosis unrelated to the effect on the outcome among unvaccinated superficial defects. Later there was increasing patients. vascularization under the tunica propria and Councilman et al. (1 904), Lillie (1930) and the tissue took on the appearance of granu- Bras (1952a) each described the gross and lation tissue, with numerous polymorpho- microscopic appearances of the various inter- nuclear leukocytes in the demarcation zone nal organs in fatal cases of smallpox. The beneath the necrotic epithelium. This combi- striking feature about all these reports was the nation produced the pseudomembrane ob- absence of specific lesions anywhere except in served macroscopically, which could be easily the skin and mucous membranes. Readers detached. Because of the numerous bacteria should refer to these papers for detailed on the mucous membrane of the pharynx the descriptions ;comment here is focused mainly necrotic lesions were usually found to har- on pathological findings that might have bour masses of bacteria of various kinds, but been of significance in the pathogenesis of this was a secondary phenomenon. smallpox. Probably the most important Since the mucosal lesions, especially those change from this point of view was the of the oropharynx, were the major source of involvement of the reticuloendothelial sys- infectious virus in smallpox, it is important to tem, noted in particular by Bras (1952a). note the time relationships between the development of mucosal and skin lesions. The Reticuloendothelial ytem mucosal lesions appeared during the early papular stage of the rash and had usually The endothelial cells lining the sinusoids of healed, without scarring, by the end of the the liver were often swollen and occasionally pustular stage. proliferating or necrotic. Such changes were Although the oropharynx or the respir- most commonly found in individuals who atory tract is usually regarded as having been died very early in the course of the disease ; the portal of infection in smallpox, no writer Bras suggests that they may have been even has described lesions in the oropharyngeal more prominent during the pre-eruptive mucosa, or anywhere in the respiratory tract, stage. Reticulum cell occurred in which might be regarded as “primary” lesions. the marrow and spleen. In addition, the The diffuse oropharyngeal lesions, like the spleen was usually engorged and contained circumscribed lesions found on the tongue very numerous large lymphoid cells, the and uvula, appeared at the same time as the morphological sign of a developing immune earliest lesions in the skin. There appears to be response. Few specific changes were found in 140 SMALLPOX AND ITS ERADICATION the lymph nodes, except for small necrotic smallpox, there were strikingly few present in foci in those of the pharynx and in the tonsils. the of primary haemorrhagic- type smallpox, a finding which explains the Kidnys profound and bleeding that occurred in these cases (see Chapter 1). Bras (1952a) described spectacular pelvic haemorrhages in cases of early haemorrhagic- type smallpox, a finding which is in accord- ance with the frequent occurrence of haema- THE HISTOPATHOLOGY OF turia in these cases. VACCINIA AND VACCINIAL COMPLICATIONS Testes Clinical aspects of vaccination and revac- Small foci of necrosis occurred in various cination and the complications associated parts of the testis (parenchyma, mediastinum with vaccination are described and illustrated and epididymis) ; these were usually too small in Chapter 7. Numerous studies have been to be recognized macroscopically. carried out on the histology of cutaneous changes in vaccinated calves and rabbits Liver (reviewed by Lillie, 1930) but few studies Most authors noted that the liver was have been made in man. The most important usually considerably heavier than normal, but of vaccination, postvaccinial found it difficult to ascribe a cause for this; it encephalitis, has been studied by many inves- did not appear to be due to engorgement or tigators, but its pathogenesis remains obscure. fatty infiltration. However, the parenchymal cells usually showed intense cloudy swelling. Normal Vaccination

Brain The aim of vaccination was to bring Encephalitis was an occasional complica- vaccinia virus into contact with cells in the tion of smallpox, which occurred much more Malpighian layer of the epidermis. After frequently than did postvaccinial encephalitis primary vaccination a papule developed in 3- after primary vaccination, but it was of minor 5 days, rapidly became a vesicle and later importance compared with the severe toxae- became pustular, reaching its maximum size mia of variola major. Encephalitis occurred after 8-10 days (see Fig. 3.1). A scab was then more often in variola major than in variola formed, which separated at 14-21 days, leav- minor, but because so few ensued in the ing the typical vaccination scar. latter variety of smallpox it was relatively Vaccination produced a generalized infec- more important. Marsden & Hurst (1932) tion in man, with swelling and tenderness of provided an exhaustive review of the litera- the draining lymph nodes and a viraemia ture and gave detailed histories of 11 cases sometimes detectable between the 3rd and the occurring after variola minor, of which 3 10th day after vaccination, most frequently were fatal. The fatal cases had brain lesions on the 6th day (Herzberg-Kremmer & Herz- like those described for postvaccinial en- berg, 1930a,b; Siegert & Schulz, 1953). Gins cephalitis (see below). et al. (1929) recovered vaccinia virus from tonsillar swabs taken 3, 4 and 5 days after vaccination, and Gurvich et al. (1 979) report- Effects spec& to haemorrhagic-type smallpox ed the isolation of vaccinia virus from the As well as the extensive haemorrhages in pharyngeal swabs of 49% of children with the skin found in all cases of haemorrhagic- postvaccinial tonsillitis between the 7th and type smallpox, both early and late, haemor- the 15th day after vaccination, compared with rhages were often found in other sites, such as 776 of children with uncomplicated vaccinia the gastric mucous membrane, the pelvis of and no evidence of pharyngitis. The level and the , the myocardium and endocar- persistence of detectable viraemia were de- dium, and the submucosa of the pharynx and pendent on the strain of virus used; it was larynx. regarded as rare and transient by Blattner et al. Although megakaryocytes were numerous (1964) and Kempe (1 960), who used the mild in the bone marrow of cases of pustular New York City Board of Health strain. More 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 141 prolonged viraemia occurred in children with immunological deficiencies (Keidan et al., 1953), some of whom suffered from progress- ive vaccinia.

Changes in the skin Howard & Perkins (1905) studied the histological appearance of vaccination lesions in biopsies taken from 12 subjects. The earliest changes were cytoplasmic and perinuclear vacuolation in the epithelium, accompanied by cloudy swelling, coagulation necrosis, intercellular oedema and vesicle formation. Within 48 hours, a cup-shaped vesicle tra- versed by an eosinophilic reticular network had appeared, of which the stratum corneum formed the roof, and the floor, centrally, was bare dermis. At the sides were cells in hyaline degeneration, which were intensely eosino- philic, with shrunken pyknotic nuclei. There were marked oedema of the papillae and free erythrocytes, mononuclear and polymor- phonuclear cells, as well as perivascular infil- tration. Subsequently, leukocytes invaded the vesicle, and the necrosis of epithelium and leukocytes produced a crust of dense, homo- geneous, deeply staining reticulum. Guarnieri bodies were present in the epithelial cells. Epithelial outgrowth occurred beneath the crusts. Plate 3.8. Section of a vesicle produced in human In the primary vesicle skin by vaccinia virus inoculated in a child who de- failed to heal and the usual lymphocytic veloped progressive vaccinia. There are no inflamma- infiltration failed to occur because the per- tory cells in the dermis. (From Keidan et al., 1953.) sons involved had defective cell-mediated immune responses. In one such case, Keidan et al. (1953) noted that germinal centres and nodes showed marked lymphocytes were completely absent from the with large germinal centres. There was a axillary lymph node and spleen of the patient, conspicuous proliferation of large pale cells although many plasma cells were present among the lymphocytes of the cortical and (Plate 3.8). paracortical regions-morphological indica- tors of an active immune response-and The draining bmph nodes Guarnieri bodies were seen in one case. A rare sequel of vaccination, persistent Successful primary vaccination produced lymphadenitis, was sometimes confused with moderately enlarged, painful regional lymph if biopsies were performed for the nodes. This reaction was first apparent on diagnosis of painless lymphadenopathy. Hart- about the 5th day as the skin lesion became sock (1968) records that a diagnosis of malig- vesicular, and most pronounced on about the nant lymphoma was mistakenly made in 9 out 10th day. The lymph nodes often remained of 20 such cases; all eventually subsided enlarged and tender for 2-4 weeks after the completely. The lymph nodes in these cases skin lesion had healed. showed diffuse or follicular hyperplasia, an In 2 persons who had been killed in an increased number of reticular lymphoblasts, accident while at the height of a primary which gave a mottled appearance to the vaccinia1 reaction, it was noted (W.E.D. sections, and a mixed cellular response, with Evans, unpublished observation, 1960-cited varying numbers of , plasma cells by Symmers, 1978) that the axillary lymph and mast cells. The morphological changes in 142 SMALLPOX AND ITS ERADICATION the lymph nodes indicate a vigorous immune predominantly encephalitic or myelitic, was response to vaccinial infection. abrupt and took place 10-13 days after vaccination-i.e., at a time when both the Postvaccinial Encephalitis cell-mediated and humoral immune responses were well developed. A striking feature was The incidence and clinical features of this the astonishingly rapid and complete recov- rare but important complication of vaccina- ery of apparently moribund subjects, but the tion are described in Chapter 7. Postvaccinial case-fatality rate was usually between 25 yo encephalitis (or encephalomyelitis) is charac- and 50%. terized by acute perivascular demyelination. Vries (1 960) distinguished two pathologi- Similar lesions sometimes occurred after cal groups, which he characterized as postvac- smallpox (Marsden & Hurst, 1932). Postinfec- cinial encephalopathy in infants, and post- tion encephalitis is a well-recognized compli- vaccinial encephalitis in older persons. In cation of and varicella and a similar infants under 2 years of age the changes seen disease sometimes occurs after vaccination in the brain were essentially vascular :oedema, against . One case has been recorded as a either general or perivascular ; mild lympho- complication of in a human being cytic infiltration of the meninges and some (Verlinde, 1951). The histopathology of post- perivascular spaces ;widespread degenerative infection and postvaccination encephalitis, changes in the ganglion cells ; and sometimes from whatever cause, is identical. perivascular haemorrhages. Older persons ex- Although Kaiser & Zappert (1938) noted hibited the features characteristic of all post- that 35 cases of disease of the central nervous infection encephalitides, as described by system were recorded among 10 090 persons Turnbull & McIntosh (1926) and reviewed by vaccinated in Bohemia in 1801 and 1802, Hurst (1 953). Meningeal was postvaccinial encephalitis was not recognized slight and irregular ; the nerve cells were little as a serious problem until the 1920s. The first affected ; and advanced neuronophagia did clear account of the clinical picture and not occur. The dominant feature was perivas- histological changes was given by Turnbull & cular demyelination of the medullary sheaths, McIntosh (1926). After that time accompanied by destruction of the axis cylin- became more aware of its occurrence and ders (Plate 3.9). The perivascular space con- postvaccinial encephalitis presented a serious tained many lymphocytes and the demyelin- problem, especially in some European coun- ated areas contained lymphocytes and highly tries, in the period between 1930 and 1960 pleomorphic microglia. Conybeare (1964b) (see Chapter 7). and a number of Soviet authors confirmed the Theonset of nervoussymptoms, which were differentiation between encephalopathy in

Plate 3.9. Lesions in the brain in postvaccinial encephalitis. A: Perivascular demyelination. 6: Perivascular cellular infiltration. 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 143

infants and postvaccinial encephalitis in older being a gradation in frequency : variola major, persons, although in older infants the con- variola minor, more virulent strains of vac- ditions merged and early perivascular demye- cinia virus, less virulent strains of vaccinia lination was seen. virus. Although vaccinia virus was occasionally demonstrable in the brain or cerebrospinal VIRAL PERSISTENCE AND fluid (Turnbull & McIntosh, 1926), most REACTIVATION investigators failed to recover virus from the brains of fatal cases. Kurata et al. (1977), for Different viruses of behave in example, were unable to recover vaccinia characteristic ways after the host animal virus from any of 5 reported fatal cases, or recovers from an acute infection, and the from 30 other cases examined during the precise mode of behaviour has important previous 20 years. However, they demon- epidemiological implications. Five situations strated vaccinia1 antigen in the leptomen- can be distinguished : inges and, complexed with IgM, in the areas of perivascular cellular infiltration in 3 out of (1) The acute infection is followed by the 5 cases studied. The consensus is that apart clinical recovery and the virus is completely from postvaccinial encephalopathy in infants, eliminated from the body ;it cannot therefore all the postinfection encephalitides, whether be reactivated by or any occurring as a result of smallpox, vaccination, other means, because it is no longer present in measles or other viral infections, are essen- the host organism. Mumps and poliomyelitis tially an allergic response to some viral or are examples. The smallpox eradication cam- virus-induced antigen(s) Uohnson, 1982). paign was undertaken on the assumption that The most puzzling feature about postvac- smallpox also fell into this category. cinial encephalitis in persons over 2 years of (2) The acute infection is followed by age was the great variability in its frequency clinical recovery but the virus persists in the in different countries and at different times body. It may be reactivated by a variety of (see Chapter 7). There appears to have been stimuli and can then cause recurrent illness, some relationship between the virulence of with the shedding of virus. This is the the virus (variola or vaccinia) and the fre- characteristic mode of behaviour of herpes- quency of postinfection encephalitis. Rao viruses-e.g, in , varicella- (1972) observed frequencies of 1 in 500 zoster and infections. variola major cases and 1 in 100 000 primary (3) The infection is followed by clinical vaccinations in Madras, and Marsden (1936) 1 recovery and in the vast majority of individ- in 2000 cases of variola minor in London. The uals the virus is completely eliminated. Occa- incidence after primary vaccination in differ- sionally, however, it may persist and cause ent countries and at different times varied chronic disease, but it persists in a sequestered from 9 per million in the USA in 1968 to 1219 site and is not shed, so that persistence is of no per million in Austria for the period 1948- epidemiological significance. Measles, with 1953 (see Chapter 7, Table 7.8). In several its rare complication, subacute sclerosing countries in which the incidence was high the panencephalitis, is a good example. rate fell substantially when the strain of virus (4) The infection is usually inapparent, but used for vaccination was changed. virus may persist in sequestered sites for long Postinfection encephalitis in smallpox and periods without ever causing symptoms, un- after vaccination was clearly due to an un- less it is reactivated by procedures such as usual host response, probably to poxvirus renal transplantation. Certain human papova- antigens or antigen-antibody complexes that viruses behave in this way (Gardner, 1977); localized in neural tissue. It is significant they may also be reactivated and excreted in that postvaccinial encephalitis was almost urine during normal pregnancy, with no unknown as a complication of revaccination, harmful effects (Coleman et al., 1980). an observation which led to the promotion (5) After the patient has recovered from the in the 1960s of a variety of schemes of “pre- acute infection the virus persists as a chronic immunization” in countries from which infection which may sometimes be associated smallpox had been eliminated (see Chapter 7). with chronic disease. Such cases may be The likelihood of neural localization of the infectious; hepatitis B is an example. relevant antigen was, in part, determined by From the point of view of the smallpox the virulence of the virus concerned, there eradication programme, the question of viral 144 SMALLPOX AND ITS ERADICATION persistence was of importance from two from infection in various animal species is points of view. First, if reactivation and re- more difficult to evaluate than the persistence of virus could occur in a person who of variola or vaccinia viruses in man. had recovered from smallpox, under any circumstances, such a person would constitute Mousepox a potential source from which smallpox could re-emerge. Secondly, the likelihood of success Several authors have suggested that ectro- in a search for virus in an animal reservoir of melia virus could produce a latent infection in variola or monkeypox viruses is greatly in- mice, but most examples that have been fluenced by whether these viruses persist in quoted could be explained as plausibly by the tissues of infected animals for prolonged persistent infection in the , rather periods. If it occurred, persistent infection than prolonged viral persistence in individual could be of epidemiological importance in mice. However, there are a few examples of human monkeypox (see Chapter 29), for the persistence of for at least persistent virus, even if it were not excreted, several weeks in a sequestered site in healthy could be a source of infection to humans mice and also of persistent infection with handling or eating such animals. shedding. For example, Fenner (1948c) noted the recurrence of foot swelling, and the Persistence of Variola Virus in isolation of virus from the swollen foot, in 2 Human Patients mice that had been infected 2 and 7 months earlier and had not been subsequently exposed The evidence against the persistence of to infection. In other experiments Fenner variola virus in man, after recovery from (1948d) demonstrated the presence of ectro- infection, is strong but circumstantial ; it is melia virus in the lungs and spleen of 2 mice impossible to prove that cases of persistent that had recovered from infection acquired infection never arose. In the whole history of about 45 and 93 days earlier ; 112 other mice smallpox, no case has occurred which could be that were tested more than 5 weeks after recovery gave negative results. There was no unequivocally traced back to infection ac- evidence that such mice shed virus. However, quired from a person who had recovered from because mouse tissues or tumours are passaged the disease months or years earlier, nor has any in other mice, such persistent infections, suspicion of the recurrence of symptoms or of especially if they occurred in animals that had the circulation of variola virus ever arisen in a just recovered from a mild or inapparent person who had recovered from smallpox and infection, could constitute a source for the subsequently undergone immunosuppressive dissemination of mousepox (see Laboratoy treatment. Nor has vaccinia virus, used until animal science, 198 1). recently on a large scale in Europe and North America (where immunosuppression ther- A more significant observation in relation apies are practised more widely than in the to possible persistent infection with ortho- developing countries), ever shown any indi- poxviruses was that recorded by Gledhill (1962a,b), who demonstrated that mice in- cation that it could cause a latent infection in man. Since alert clinicians and virologists fected by the oral route could sustain a chronic infection of the intestinal tract, with have recognized the occurrence of latent infections with a wide variety of viruses, and excretion of virus in the faeces and scabs on the tail near the anus. He was unable to their reactivation after immunosuppressive “activate” acute mousepox in such carriers, treatment, this negative evidence is impor- nor did they cause infection in susceptible tant. It is reasonable to agree with the mice placed in the same cages. conventional belief that variola virus be- longed to the first group of viruses listed-i.e., infection was followed by death or recovery Isolation of other orthopoxviruses from natural4 with complete elimination of the virus; infected normal animals persistent infection did not occur. A number of reports of the recovery of orthopoxviruses from the tissues of appar- Persistence of Orthopoxviruses in Animals ently normal animals have been published (Table 3.3). There are two problems in The evidence that some orthopoxviruses evaluating the significance of these findings, might persist for long periods after recovery in terms of persistent infection. First, it is 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 145

Table 3.3. Recovery of orthopoxviruses from tissues of healthy animals thought to have been naturally infecteda

Example No. Virus Circumstances 10) ow pox ( strain+ From lungs of 5 and kidneys of 14 out of I13 white in an interepidemic period. I (ii) Cowpox (Turkmenia strain)c From kidneys of great gerbil (Rhombomys opimus) and yellow suslik (Citellus fulvus) captured in Turkmenistan, USSR. 2 Monkeypoxd From kidneys of several apparently healthy cynomolgus monkeys that had been exposed to infection in an outbreak in a laboratory colony. 3 Taterapoxe From Iiver/spleen suspension of I of 95 wild Tatera kempi captured in Benin. 4 Ectromeliah From brains of apparently normal mice in a colony enzootically infected with ectromelia virus. s(i) Vacciniah Rabbitpox vlrus recovered from kidney of an apparently healthy Macaca rhesus. S(ii) Vaccinia1 Vaccinia virus recovered from kidney of Cercopichecus ascanius killed in Zaire.

a Excluding “whitepox” viruses (see Chapter 30, Table 30.2). bMarennikova (I979); Shelukhina et al. (1979b).

Table 3.4 Recovery of orthopoxviruses from animals that had been experimentally infected some weeks earlier and showed no clinical signs at time of recovery of virus

Example No. Virus Circumstances

I Ectromeliaa From lungs and spleen of 2 out of I 14 mice tested 5 weeks or more after recovery. 2 Monkeypoxb From kidneys and lungs of hamsters up to 6 weeks after intracardiac injection. 3 Cowpoxb From kidneys and lungs of cotton rats and rats inoculated intranasally up to 6 weeks earlier. 4 ow pox (rat strain)b>C From several organs of white rats and Rattus norvegicus inoculated intranasally 4 weeks earlier. 5 Cowpox (Turkmenia strain)d From kidneys and testes of great gerbils and yellow susllks inoculated 5 weeks earlier. 6 Vaccinlae From spleen and testes of rabbits inoculated intradermally with neurovaccinia virus I I4and I33 days earlier. 7 Vacciniaf From brains of mice pre-treated with cyclophosphamide 60 days after Inoculation with vaccinia virus; by co-cultivation only. 8 Variolag From brains of mlce that had been inoculated intracerebrally as infant mice up to 62 days earlier.

a Fenner ( 19484. bSheiukhina et al. (1979b). Maiboroda ( 1982). dMarennikova et al. (1978b). eOlitsky 8 Long (1929). fGinsberg 8 Johnson (1977). gSarkar et al. (1959). possible that most or all of the positive results poxviruses, there are several reports of the were obtained with animals that were exper- recovery of orthopoxviruses from the tissues iencing an inapparent infection at the time or of laboratory animals that had been infected were convalescing from infection (examples several weeks earlier and were apparently l(i), l(ii), 2, 3 and 4 of Table 3.3). Secondly, normal at the time of isolation of the viruses the virus apparently isolated €rom a normal (Table 3.4). The results with mousepox have animal may have been a laboratory contami- been described. The most systematic studies nant (examples 5(i) and 5(ii) of Table 3.3). were those reported by Marennikova and her colleagues with monkeypox and cowpox viruses (examples 2-5 of Table 3.4). The Isolation orthopoxviruses from ((normal)’animals of animals suffered inapparent infections and after recovery from experimental infection the relevant virus was recovered from some Although most investigators have failed to animals for up to 6 weeks after inoculation, demonstrate persistent infection with ortho- when the experiment was terminated. Great 146 SMALLPOX AND ITS ERADICATION gerbils and yellow susliks suffered severe such an animal during an ecological survey in disease with high mortality after inoculation tropical forest areas, with their abundance and with the Turkmenia strain of cowpox virus, diversity of animals, is quite small, which but virus could be recovered from the kidneys may account for the failure so far to recover and testes of animals that survived for 5 weeks from a wild animal (except (example 5, Table 3.4). in one instance from a sick squirrel), and in Positive results were also reported with part for the rarity of cases of human vaccinia virus in rabbits and mice (examples 6 monkey pox. and 7, Table 3.4) and variola virus in mice (example 8, Table 3.4). THE IMMUNE RESPONSE IN SMALLPOX AND AFTER Epidemiological Significance VACCINATION

It is difficult to assess the epidemiological Variolation, the ancient practice whereby significance of these observations. Only in smallpox was transmitted to a susceptible Gledhill’s experiments with ectromelia was person by the inoculation of material from there any evidence that virus shedding oc- smallpox scabs or vesicles, was based on curred, but even then the infection of suscep- observations that pockmarked persons never tible contact mice was not observed (Gledhill, suffered from smallpox a second time. It 1962a,b). In all other cases the virus was in a provided the foundation on which the science sequestered site and its presence was revealed of immunology was built (Needham, 1980). only by laboratory manipulations. It is reason- The next major landmark in immunology, as able to suggest that the persistent carriage and Pasteur (1881) recognized when he general- shedding of virus is not a factor of epidemi- ized the use of the term vaccination, was ological significance in orthopoxvirus infec- Jenner’s substitution of an antigenical- tions in the way that is so important in ly related non-virulent agent (cowpox/ and herpesvirus infections. vaccinia virus) for the virus of smallpox. With smallpox, there is a further epidemi- Although immunology arose from obser- ological observation of great significance. vations of protection from infection or re- Over a period of some 50 years, during this infection, the immune response also plays an century, smallpox was progressively elimi- important role in the process of recovery in an nated from every country in the world. In infected person. In the following pages both no instance was a “spontaneous” outbreak aspects are reviewed. identified after smallpox had been eliminated from a region or country. All outbreaks of Protection against Reinfection which the source was found could be traced to the introduction of virus from known After vaccination infected areas by known infected individuals. These results are also relevant to the The responses obtained on revaccination searches for the natural reservoir animal or with vaccinia virus at various intervals after animals of monkeypox in Africa (see Chapter primary vaccination or earlier revaccination 29) and the claims that variola virus has been are described in Chapter 7. Immunity may be isolated from various healthy primates and manifested by a complete absence of reacti- rodents (see Chapter 30). It is clear that vity, by an allergic reaction or by an acceler- orthopoxviruses have only very rarely been ated reaction which may nevertheless pro- recovered from wild animals, even when tests gress to vesiculation and which involves at were carried out in areas and with species in least local replication of the virus used for which there was serological evidence that revaccination. Skin site appears to play a role orthopoxvirus infection was widespread. It is in the severity of the response to revaccina- likely that virus isolation in such circum- tion. In revaccination was often per- stances usually depends on the chance selec- formed on the ventral surface of the forearm tion of an animal suffering or convalescing because positive reactions were more fre- from infection rather than a long-term car- quently obtained there than in revaccination rier. This appears to have happened with over the deltoid muscle. Even more striking rodent strains of cowpox virus (examples l(i) were the finger lesions sustained by workers and 1(ii) of Table 3.3). The chance of catching in vaccine production laboratories, who often 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 147

Cells Involved in the Immune Response

The immune response is a complicated process about which a great deal has been discovered since most of the work on immunological responses in smallpox and vaccinia was carried out. Understanding of cell-mediated immunity, in particular, has burgeoned in recent years. A summary of current views on the cells involved in the immune response as they relate to viral infections is shown in Fig. 3.4. Very briefly, three types of cell are involved-macrophages and two types of lympho- cyte. Certain kinds of macrophage process antigens for presentation to lymphocytes. Lymphocytes belong to two main classes : T cells (meaning -derived cells, of which there are several subclasses), and B cells (coming mainly from the bone marrow in ), which produce antibodies. B cells have antibody-like receptors on their surfaces, one or a few cells having receptors of every imaginable specificity. When an antigen is appropriately presented to such a cell by a macrophage, the B cell differentiates to form an antibody-synthesizing plasma cell and at the same time divides to form a population (clone) of identical cells. Later, some members of this clone are sequestered as long-lived memory B cells, which are of critical importance in mounting a secondary response when reinfection occurs. T cells have different kinds of antibody-like receptors on their surface, and they also react specifically to different antigens, expand clonally, liberate active substances called lymphokines, and sequester a small proportion of each clone as long-lived memory T cells. There are several subclasses of T cells. Two of these modulate the activity of the antibody- producing B cells and of other T cells: Th or helper T cells and Ts or suppressor T cells. Other kinds of T cells are responsible for the two main components of the cell-mediated immune response: Tc or cytotoxic T cells, which actively destroy cells bearing complementary virus-induced antigens on their surface, and Td cells, which differentiate on contact with the specific antigen, release lymphokines and produce delayed hypersensitivity reactions. T cells also produce one class of interferon (gamma-interferon), and there is another poorly understood class of lymphocytes (natural killer (NK) cells), which appear to kill certain host cells non-specifically.

suffered from vaccinia1 whitlows in spite of was rather high. Although collectively they the fact that they were revaccinated annually saw many thousands of cases of variola major, (Horgan & Haseeb, 1944). Such cases often it was very rare indeed to find one in a exhibited enlargement of the epitrochlear and pockmarked person. axillary lymph nodes and sometimes the reinfection progressed like a primary vaccin- Heterologous protection ial reaction, with maximum vesiculation on the 9th or 10th day. Depending on the closeness of the anti- genic relationship and the degree of general- ization of the infection (and thus the intensity After smallpox of the immune response), homologous protec- It is obviously difficult to obtain precise tion would be expected to be greater than figures on the incidence of second attacks of protection induced by a heterologous agent. smallpox. Dixon (1962), quoting from the Experiments in animals reveal that infection observations of Barry (1 889) in Shefield, and with any one orthopoxvirus produces sub- Rao (1972), whose views were based on his stantial protection against disease produced own experience supported by laboratory evi- by any other orthopoxvirus (vaccinia virus dence, suggested that about l in 1000 pock- against ectromelia in mice : Fenner, 1947a ; marked persons suffered a second attack of ectromelia virus against rabbitpox in rabbits : smallpox. Epidemiologists working in the Christensen et al., 1967 ;vaccinia virus against field during the Intensified Smallpox Eradi- monkeypox in monkeys: McConnell et al., cation Programme believed that this figure 1964; vaccinia virus against cowpox in rab- 148 SMALLPOX AND ITS ERADICATION

Cells Antiviral effects Immunoglobulins 6 cell Plasma cell Neutralize viral Kill infected cells Act as opsonins Mediate K cell action

Give enhanced (secondary) response Progeny@ \@Infecting virions viriit Memorya T cell

a Interferon ~ @ Non-specific killing of infected host cells NK cell

Non-specific killing of infected P host cells

placroDhaee Activated macrophage

Fig. 3.4. Cells involved in antiviral immune response . Tc = cytotoxic T cell; Td = delayed hypersensitivity T cell; K cell = killer cell; NK cell = natural killer cell. bits: Downie, 1939b; vaccinia virus against was not widely practised until much later in variola in monkeys : Horgan & Haseeb, 1939 ; Great Britain. Detailed information on the taterapox virus against monkeypox in mon- persistence of immunity to smallpox after keys: Lourie et al., 1975). Likewise, a prior vaccination with vaccinia virus is presented attack of smallpox gave some protection in Chapters 1 and 7. In general, vaccination against vaccination with vaccinia virus, within the previous 5 years protected persons which was much less prolonged after variola exposed to smallpox against clinical disease minor than after variola major (see Chapter 1, and some protection was evident for over 30 Table 1.10). Persons with active variola major, years. In countries in which smallpox was vaccinated in order to prevent smallpox endemic, estimation of the duration of pro- infection in case of a misdiagnosis, never tection was complicated by the fact that exhibited a major reaction (Zikmund et al., subclinical smallpox, recognizable by rises in 1978). the titre of complement-fixing antibodies However, the classic example of cross- (which persist for only a few months after protection, on which the success of the global infection), occurred in a substantial number eradication campaign depended, was that of vaccinated individuals who were in close described by Jenner (1798). Although Jenner contact with cases of smallpox (Heiner et al., realized that cowpox inoculation did not 1971a; see Chapter 1). always result in a take (Jenner, 1799,1804), he maintained that successful vaccination pro- Humoral and Cellular Responses in duced lifelong immunity to smallpox. That Orthopoxvirus Infections view was clearly wrong, and revaccination In orthopoxvirus infections the humoral became an accepted practice early in the 19th response may result in the production of century in continental Europe, although it short-lived IgM or persistent IgG, and it may 3. PATHOGENESIS. PATHOLOGY AND IMMUNOLOGY 149

be elicited by inactive virions or viral antigens nate between different but related antigens. and by both non-enveloped and enveloped Although they can be refined, if used with infectious virions. The cellular response also monoclonal antibodies or with antibodies may be evoked either by inactive antigens produced by immunization with purified (which result in delayed hypersensitivity antigens, the first four tests described below reactions without cytotoxic effects) or by cell ordinarily register reactions between several surface membranes altered by viral infection, or all of the large number of antigens when both cytotoxic responses and delayed produced in orthopoxvirus infections and hypersensitivity may be involved. certain classes of all the antibodies, of varying In the analysis of the scientific literature specificity, that are produced during immuni- which follows, the humoral and cellular zation or infection. Other serological tests are immune responses will be discussed separ- more discriminative, in that they involve only ately ; however, in any animal inoculated or one or a few of the antigens produced during infected with viral antigens or virus prep- viral infection and the corresponding anti- arations, the reactions described, and many bodies, or else the mixed antigen-anti- others, go on simultaneously. In each section body complexes can be separated by diffusion technical methods will be described first and or electrophoresis in gels. then the results of experiments on orthopox- virus infections in laboratory animals and on Complement-fxation (CF) test smallpox and vaccinia in man will be reported. Many poxvirus antigens react in CF tests, including an early antigen located on the surface of vaccinia-infected cells (Ueda et al., Methods for Measuring Antibodies to 1972). However, only antibodies of certain Orthopoxviruses classes and subclasses (in man: IgM and to a At various times almost every method that lesser extent IgG,, IgG2and IgG,) participate has been developed for detecting antibodies in CF reactions; in general, such antibodies has been employed for titrating antibodies to are short-lived so that a positive CF reaction is orthopoxviruses, especially in the case of an index of recent infection (in man, within vaccinia virus, the prototype virus of the 12 months ; Wulff et al., 1969). This property genus. In the following paragraphs the meth- was exploited by Heiner et al. (1971a) in their ods more commonly used are listed, with study of subclinical infections in vacci- comments about particular features of each, nated household contacts of smallpox cases notably its sensitivity, the persistence of the (see Chapter 1, Fig. 1.2). antibodies detected by particular tests, the Because so many antigens are common to potential of the test for distinguishing all orthopoxviruses, the CF reaction is useless between different species of orthopoxvirus, for discriminating between different species and the relation of antibodies detected by of the genus, unless used with an antigen that various tests to protection. is species-specific. With the discovery of monoclonal anti- Immunofuorescence test bodies and the subsequent development of this new tool, serological methods achieved Like the CF test, can a quantum leap in their power to detect and be used to detect many different orthopox- discriminate between antigenic sites (epi- virus antigens. However, in contrast to the topes). In principle, monoclonal antibodies antibodies active in complement fixation, the can be used to titrate either antigens, by any of IgG antibodies involved in immunofluores- the methods outlined below, or antibodies, by cence reactions are long-lived (Gispen et al., using them in blocking tests. At the time this 1974). Combined with serial absorptions of chapter was written no use had been made antisera with suitable suspensions of virus- of monoclonal antibodies in orthopoxvirus infected tissue, immunofluorescence can be research. used to recognize species-specific orthopox- virus antibodies in sera of animals caught in Sensitiuit_y and spec@cit_yof dgerent serological tests the wild (Gispen et al., 1976). However, it Different serological tests differ consider- suffers from the disadvantage of being rela- ably in both sensitivity-i.e., the amount of tively insensitive. Immunofluorescence has antibody required to give a positive result- proved useful in the study of the sequence of and specificity-i.e., their ability to discrimi- intracellular events in orthopoxvirus infec- 150 SMALLPOX AND ITS ERADICATION tions (Ueda et al., 1972) and in studies of the serum mixtures in experimental animals (rab- pathogenesis of poxvirus infections in exper- bit skin : Parker, 1939 ;mouse brain : Bronson imental animals (Mims, 1964, 1966), for it & Parker, 1941), or by looking for plaque or provides a method of demonstrating the focus reduction in cultured cells (McNeill, cellular and intracellular localization of viral 1965; Kitamura & Shinjo, 1972) or pock antigens. reduction on the chorioallantoic membrane (Keogh, 1936; McCarthy et al., 1958a). All Radioimmunoassay species of Orthopoxvirus cross-react in neutral- The sensitivity of several serological meth- ization tests, although titres are highest with ods can be greatly enhanced by tagging the homologous virus (Downie & McCarthy, relevant antigens or antibodies with radio- 1950; McNeill, 1968). In man, neutralizing isotopes. Radioimmunoassay, which is about a antibody persists for many years after recov- thousand times more sensitive than immuno- ery from both smallpox and vaccination with fluorescence, for example, can be used for the vaccinia virus (McCarthy et al., 1958a,b; detection of antigen-antibody reactions in Downie & McCarthy, 1958). tubes or plates, or it can be combined with gel Traditionally, neutralization tests have precipitation and autoradiography to discri- been carried out with suspensions of virions minate between antigens produced in ortho- obtained by disrupting infected cells. Since poxvirus infections (radioimmunoprecipita- such virions lack the envelope antigens found tion, see below). in naturally released virions (see Chapter 2), Hutchinson et al. (1977) developed a these neutralization tests do not measure all radioimmunoassay test for detecting species- the antibodies that are important in protect- specific orthopoxvirus antibodies in absorbed ing against infection or acting against circu- sera, which because of its sensitivity could be lating virions. Antibodies that neutralize the applied to sera obtained during field surveys. infectivity of enveloped virions can be assayed by the “anti-comet” test of Appleyard et al. ELISA method (1971) (Plate 3.10). In essence, this test in- volves the use of a liquid overlay for cell Like the complement-fixation, immuno- monolayers infected with suitable concentra- fluorescence and radioimmunoassay tests, the tions of a strain of an orthopoxvirus, such as ELISA (-linked immunosorbent as- rabbitpox virus, that produces many enve- say) method provides a sensitive way of loped virions. These migrate in the liquid recognizing antigen-antibody reactions. It overlay to produce comet-shaped areas of cell was developed rather too late to have been damage. Antibodies that neutralize the infec- much used in research on smallpox and tivity of envelope antigens, added after viral vaccination, but it constitutes a potentially adsorption, prevent the formation of the useful field test for ecological studies of “comets” but not the development of plaques. monkeypox, particularly if it can be used with Antibodies to non-enveloped virions have no a monkeypox-specific antigen or, in blocking such effect, but prevent plaque production in tests, with suitable monoclonal antibodies. orthodox neutralization tests, in which virus- serum mixtures are added to the monolayer. NeutraliZation test Haemaggltrtination-inhibztzon (HI) test Neutralization of infectivity is the tra- ditional discriminative test in animal viro- All orthopoxviruses, but no other members logy, and is used to distinguish between differ- of the family , produce a haemag- ent species of and Flavivirus, for glutinin which agglutinates cells from select- example, or different strains of Inflttenzavirus. ed chickens. Because of its simplicity, the HI The antigens which evoke neutralizing anti- test has been widely used in serological bodies after infection with orthopoxviruses, surveys in man and in animals (e.g., for however, show a great deal of overlap (i.e., elucidating the of monkeypox virus, several epitopes are shared by all members of see Chapter 29). The haemagglutinin appears the genus), so that, as ordinarily performed, early in the course of infection as one of neutralization tests detect genus-specific several new components of the surface mem- rather than species-specific antibodies. brane of infected cells (Blackman & Bubel, The neutralization of infectivity of ortho- 1972), where it may be recognized by haemad- poxviruses can be carried out by testing virus- sorption tests (Driessen & Greenham, 1959). 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 151

Plate 3.10. The "anti-comet'' test. Strains of virus which produce substantial numbers of extracellular enveloped virions produce "comet tails" from the initial plaques when suitable dilutions are inoculated in mono- layers with a liquid overlay medium (A). Addition to the overlay medium of antibody to viral envelopes or enveloped virions, or of antiserum from an animal that has been infected with vaccinia virus, prevents the production of the "comet tails" but not of the initial plaques (B). Antiserum to surface tubular elements or inactivated virions, although it neutralizes the infectivity of non-enveloped virions, does not inhibit "comet" formation. (From Appleyard et al., I97 I .)

Haemadsorption tests are useful in confirm- ages were tested for HI and neutralizing ing whether suspicious cytopathic effects in antibodies, more than GOO/, gave positive tissue culture are caused by an orthopoxvirus. results by one or the other test. Most of these The haemagglutinin also occurs as a compo- were positive by both tests but some gave nent of the viral envelope (Payne & Norrby, positive results by HI but not by neutral- 1976), but separately from infectious non- ization, and vice versa. enveloped virions in extracts of infected cells. Non-specific inhibitors of orthopoxvirus Haemagglutinins produced by different haemagglutinin occur in some sera, especially orthopoxviruses cross-react, although titres if the specimens are old, have been improperly are usually higher with the homologous virus. stored or were collected during . They According to Downie and his colleagues can usually be removed from human sera (McCarthy et al., 1958b; Downie & McCar- without loss of specific antibody by treatment thy, 1958), antibodies with HI activity persist with potassium iodate (Espmark & Magnus- for varying periods after recovery from ortho- son, 1964). poxvirus infection, usually for only a few Some strains and mutants of orthopox- months but somewhat longer than CF anti- viruses fail to produce a haemagglutinin or to bodies(seeFig.3.6 and 3.7). Thus theywereuse- promote the production of HI antibodies ful in determining whether recent infection (Cassel, 1957 ; Fenner, 1958). Experiments with smallpox had occurred and were so used with these viruses, and other evidence, show by Heiner et al. (1971a), but have limitations that HI antibodies are unrelated to those when used for epidemiological or ecological involved in neutralization reactions, or to surveys aimed at determining the prevalence protection against infection, although the of orthopoxvirus infections. However, using a presence of HI antibodies is evidence that the somewhat different protocol for the prep- antigens that do evoke the production of aration of vaccinia haemagglutinin, Nakano protective antibodies have been produced. (1985) found that HI antibodies were more persistent than previously thought. In tests on Precipitation testj African subjects who had recovered from monkeypox, he found positive results in some Much of the early work on the soluble individuals more than 4 years after recovery. antigens produced in orthopoxvirus-infected Further, when sera from 600 Africans of all cells, involving both the time course of their 152 SMALLPOX AND ITS ERADICATION production (e.g., Appleyard & Westwood, During infection with a virus as complex as 1964a) and comparisons between strains and an orthopoxvirus, antibodies of many differ- mutants of orthopoxviruses (e.g., Gispen, ent specificities are generated. Most of these 1955; Rondle & Dumbell, 1962), utilized are probably irrelevant, as far as the patho- simple gel-precipitation tests. With the use of genesis of poxvirus infections, recovery and absorbed sera, reactions can be detected which protection are concerned. The relevant anti- differentiate variola, monkeypox and vaccinia bodies belong to three classes: (1) antibodies viruses (Gispen & Brand-Saathof, 1974 ; that neutralize viral infectivity, of which Esposito et al., 1977a). However, the sensi- there are two subclasses, directed respectively tivity of simple gel-precipitation tests is low ; against non-enveloped and enveloped virions they require highly potent sera and are not (review: Boulter & Appleyard, 1973); (2) readily applicable to sera obtained from those that, with complement, lyse virus- animals that have recovered from natural infected cells (review: Sissons & Oldstone, infections. 1980); and (3) antibodies that combine with Gel precipitation used to be recommended circulating antigens to produce immune com- as a method for rapid smallpox diagnosis, plexes, which might have been responsible for using vaccinia-immune sera and vesicle fluid some of the “toxic” symptoms in smallpox. or crust material as a source of antigen (World It has long been believed that specific Health Organization, 1969a; see Chapter 2). It antibodies generated by the humoral immune was widely used for rapid presumptive diag- response played important roles in both nosis in laboratories which lacked facilities protection against orthopoxvirus infections for electron microscopic diagnosis (Rao, and recovery from established infections. The 1972 ; A.W. Downie, personal communica- protective effect of antibodies is most clearly tion, 1981), but was rarely employed in WHO demonstrated by passive immunization ;their collaborating centres after electron micro- putative role in recovery was based mainly on scopy by negative staining had been de- temporal relationships observed during the veloped. Further, although it was almost course of established infections. always positive with fresh material (Noble et a]., 1970), its efficiency was much lower (only about 7004) in material that had been dis- Passive immunity patched from the field to a WHO collabo- Passive immunization, either natural, by rating centre (see Chapter 2, Table 2.10). the transmission of antibodies from mother to The sensitivity and discriminative power progeny, or by the inoculation of antisera, of gel precipitation were greatly enhanced by provides a means of examining the influence two modifications: (1) electrophoresis of the of antibodies on the disease process uncompli- viral antigens or antigen-antibody complexes cated by cell-mediated immunity. The effect in SDS-polyacrylamide gels, and (2) radio- of passive immunization in protecting against isotopic labelling of the antigens. The radio- infection with vaccinia virus was recognized immunoprecipitation test was used by Ikuta as long ago as 1877 (Raynaud, 1877). The et al. (1979) to demonstrate the presence effectiveness and the limitations of passive of serologically related antigens among pox- immunization in generalized poxvirus infec- viruses of the same and different genera. tions are well illustrated in experiments on mousepox (Fig. 3.5). Active immunization The Humoral Response in Relation to with ectromelia virus (not illustrated) usually Pathogenesis inhibited viral replication in the inocu- lated foot and always prevented generaliza- Until comparatively recently, the “immune tion of the disease. Active immunization with response” was equated with the production of vaccinia virus (Fig. 3.5B) had little effect on antibodies. It is now clear that cell-mediated viral replication in the foot but greatly immunity is of even greater importance than diminished generalization, although a tran- antibodies in the pathogenesis of many infec- sient rash sometimes occurred. The antibody tious diseases, and in every infected animal or titre rose and the relative titres against human being with a normal ectromelia and vaccinia haemagglutinins humoral and cellular immunity always oper- were reversed between 7 and 8 days after ate simultaneously. It is convenient, however, infection. Passive immunization was much to consider these two aspects of the immune less effective in modifying the course of the response separately. disease. Vaccinia-immune serum (Fig. 3.5D) 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 153

I A. Normal mice B. Vaccinated mice _____ Virus in foot c - Virus in skin

inoculation

vm -5 C. Ectromelia-immune serum D. Vaccinia-immune serum TuZlOL ,--. - ,\ $91 / \ c I \ C \ 640- 8 ,' \\ €-HI I '/- 320 7 r // .' \

Days after inoculation

Fig. 3.5. The spread of ectromelia virus through the organs of mice, either unprotected (A), or protected by active immunization with vaccinia virus (B). or by passive immunization with ectromelia-immune serum (C) or vaccinia-immune serum (D) obtained from convalescent mice. E-H I and V-H I = haemagglutinin-inhibitinganti- bodies to ectromelia (E) and vaccinia (V) antigens. (From Fenner. I949b.)

had almost no effect and several mice died formed antibodies, even when the antibody with acute hepatitis at the same time as the titres in the passively immunized mice were controls. Ectromelia-immune serum (Fig. higher than those in the actively immunized 3.5C) was more effective but did not influence animals. The reason for the difference is that viral replication in the inoculated foot, and a active immunization with infectious virus low level of replication in the spleen and skin provokes the complete range of cell-mediated was found in most animals. In passively and humoral immune responses; passive im- immunized mice the antibody level rose about munization provides only the antibodies pre- 2 days later than it did in the vaccinated mice. sent in the convalescent animals from which These experiments illustrate two import- the sera were obtained. Secondly, passive ant features of passive immunization that are immunization with the homologous antibody relevant to vaccination and smallpox in man. provided much greater protection than did First, active immunization, whether heter- heterologous antibody, even though the sera ologous (by vaccination) or homologous cross-reacted extensively in neutralization (after recovery from mousepox and, by ana- tests. In relating these experiments to human logy, from smallpox), provided much greater vaccination and infection with variola virus it protection than the administration of pre- is important to realize that vaccinia and 154 SMALLPOX AND ITS ERADICATION variola viruses cross-react to a greater degree circulate in this form during poxvirus infec- than do vaccinia and ectromelia viruses. tions ; they are either cell-associated, in cells Experiments on passive immunization in whose surface membranes may be altered by rabbitpox (Boulter et al., 1961b) confirmed the incorporation of virus-specific antigens, the protective value of potent homologous some of which are similar to those found on antiserum, which protected rabbits from the viral envelope, or they occur outside cells death, even when treatment was delayed until (in plasma or tissue fluids) as enveloped overt disease had developed. Subsequent ex- particles. periments (Boulter et al., 1971 ; see Table 3.5) Antibody which specifically neutralizes showed that antisera produced after infection enveloped virions can be detected by the with live virus had a much greater protective “anti-comet” test (see Plate 3.10). The differ- effect than antiserum produced by immuni- ences between neutralizing antibodies to zation with inactivated virus, even when enveloped and non-enveloped virions are the neutralizing titre, measured by orthodox particularly important in relation to efforts to neutralization tests, was much lower. produce inactivated vaccines for protection Thus antibodies as such do have an effect against smallpox (see below). The relevant on viral replication and spread in general- features of the experiments by Boulter, ized poxvirus infections, although, as de- Appleyard and their colleagues (Appleyard scribed below, cell-mediated immunity is et al., 1971 ; Boulter et al., 1971 ; Turner & probably more important. The influence of Squires, 1971) and later experiments by Payne congenital passive immunization and the (1980) can be summarized as follows : administration of antisera to smallpox cases or (1) Sera of rabbits which had recovered contacts is discussed in Chapter 1 ; the use of from infection with rabbitpox virus con- anti-vaccinia1 gamma-globulin in generalized tained antibodies that neutralized enveloped vaccinia is dealt with in Chapter 7. virions; sera from rabbits inoculated with inactivated virions lacked such antibodies. The antibod_ response during infections (2) Sera from both groups of rabbits neutralized non-enveloped virions. In almost every kind of viral infection that (3) Cross-absorption of the two types of has been studied, there is a relationship sera with concentrated enveloped or non- between the time at which circulating neu- enveloped virions selectively removed neu- tralizing antibodies first become detectable tralizing antibodies of the appropriate and the progressive decrease and eventual specificity. disappearance of virus in the blood and (4) Antibody to isolated envelopes was able parenchymal tissues. However, the simul- to neutralize the infectivity of enveloped taneous activation of cell-mediated immunity virions and to protect mice against the spread and the possible earlier effects of interferon of infection. production (see below) make interpretation (5) Rabbits immunized with inactivated difficult. rabbitpox virus (i.e., with inactivated non- enveloped virions) had high levels of neutral- Neutralizing antibodies and protective immunig izing antibodies to non-enveloped and none to enveloped virions, but showed only partial From the point of view of protection immunity on challenge inoculation, whereas against natural infection, the antibody that rabbits immunized with enveloped virions neutralizes enveloped virions is theoretically were fully protected. more important than the antibody that neu- tralizes non-enveloped virions (review : Boulter & Appleyard, 1973). However, almost Antibod_-mediated complement-dependent bsij all reported studies of the role of neutralizing antibodies in the pathogenesis of poxvirus Oldstone and his collaborators (reviews: infections were carried out before the demon- Oldstone & Lampert, 1979; Sissons & Old- stration of the significance of enveloped stone, 1980) have shown that antibodies can virions, and assays of neutralizing antibodies exert a number of significant effects on virus- have been performed with virus suspensions infected cells. In conjunction with comple- obtained by disrupting infected cells, and thus ment or cytotoxic lymphocytes, IgG antibody have measured antibody to non-enveloped can mediate the destruction of virus-infected virions. It is likely that relatively few virions cells. In experiments with vaccinia in humans, 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 155

Perrin et al. (1977) suggested that lysis of that may require the prior processing of virus-infected cells was more likely to be due antigens in macrophages; (2) a further com- to non-specific “killer” lymphocytes (NK ponent, consisting of antigen recognition and cells) acting in the presence of antibody to the proliferative response of T cells, with viral antigens located in the cell membrane release of lymphokines; and finally (3) an than to specifically sensitized cytotoxic inflammatory response which is amplified by lymphocytes. chemotactic factors. Delayed hypersensitivity can be passively transferred by suspensions of Immune complexes as a cause 4 toxaemia lymphoid cells, but not by antiserum. Two subclasses of T cell may be involved in delayed Soluble orthopoxvirus antigens and im- hypersensitivity reactions (Fig. 3.4) : (1) cyto- mune complexes could be readily demon- toxic T cells (Tc) are evoked by viral infection strated in the plasma of severe cases of and have cytotoxic activity, reacting specifi- smallpox (Downie et al., 1953). It is possible cally with virus-induced antigens on cell that these immune complexes played a part in membranes ; (2) delayed hypersensitivity T the production of the toxaemia that was so cells (Td) are evoked by antigen presented in a characteristic of variola major. non-multiplying form, such as inactivated vaccines, as well as during viral infections; Methods for Measuring Cell-Mediated these T cells are not cytotoxic (Ada et al., Immunity 1981). Delayed hypersensitivity is recognized by Nature 4 the cells involved the accelerated response to inoculation of the antigen(s) into the skin; its development Although observers had long been con- during vaccine inoculation of man was recog- cerned with the frequent lack of correlation nized by Jenner (1798) and has been repeat- between circulating antibodies and recovery edly demonstrated since then (e.g., Pincus & from infections, “cellular immunity”, in- Flick, 1963). It was commonly used as a voked in such situations to explain recovery method of assessing immunity to smallpox, from disease, had no precise meaning until the but its reliability for this purpose depended complex immunospecific responses of T cells on which kind of T cells produced the were recognized during the 1970s, distinct reaction. If they were cytotoxic T cells and independent of the antibody-producing produced by prior infection with active virus, B cells. The T-cell responses, other than those delayed hypersensitivity was a good index of involved in modulation of the humoral re- resistance. If they were the kind of T cell (Td) sponse (see Fig. 3.4), constitute cell-mediated provoked by a non-multiplying antigen, immunity. The mechanisms by which T which lacked cytotoxic capacity, there was lymphocytes exercise antiviral functions are usually no correlation between delayed hyper- complex; they may involve both the direct sensitivity and resistance-i.e, delayed hyper- effects of T cells on cells with virus-modified sensitivity is an indicator of resistance only surface membranes and the effects of their when it is an indicator of the presence of secreted products, which are called lympho- cytotoxic T cells (Ada et al., 1981). kines and include, among others, gamma- interferon. In vitro techniques for anahsing T-cell

The delayed bypersensitivit3, reaction There is no satisfactory in vitro assay of delayed hypersensitivity ; the reaction in- The classical method of measuring cell- volved is a particular kind of inflammatory mediated immune responses is the skin test response which must be tested in intact for delayed hypersensitivity, an expression animals. However, in experimental systems used to contrast the time course and nature of there is a good assay for cytotoxic T cells. A the reaction with that of “immediate” hyper- known number of 5’Cr-labelled, virus-in- sensitivity, which comes on within minutes of fected target cells are cultured together with exposure to the relevant antigen and is varying numbers of lymphocytes obtained mediated by IgE (Gel1 et al., 1975). It is a from the spleen or lymph nodes. The release of complex reaction involving three compon- the radioactive label is an index of cytotoxic ents: (1) an initial one in which antigen- T-cell activity; proof that it is due to T cells is sensitive T cells are sensitized, a procedure provided by the absence of lysis if the 156 SMALLPOX AND ITS ERADICATION lymphocyte preparation is treated with anti- while delayed hypersensitivity is detectable theta serum and complement. by the footpad test 5-6 days after infection. In In vitro experiments with cells infected with contrast, significant neutralizing antibody is ectromelia virus (Ada et al., 1976 ;Jackson et not detectable in the circulation until the 8th al., 1976) and with vaccinia virus (Koszinow- day. ski & Ertl, 1976) showed that some of the Mice pretreated with anti-thymocyte cell-surface changes relevant to T-cell-medi- serum, which acts specifically on T lympho- ated lysis occurred before viral DNA replic- cytes, die from otherwise sublethal doses ation had begun-i.e., they were “early” of virus, because of uncontrolled viral synthetic functions coded for by the input growth in target organs. Such mice have DNA. impaired cell-mediated responses but their The mechanism of cytotoxic T-cell lysis neutralizing antibody levels are normal, inter- was shown by Zinkernagel & Althage (1977) feron levels in the spleen are elevated, and the to be a direct interaction between the appro- innate resistance in target organs is un- priate T cells and cells infected with vaccinia changed. virus. Recognition by T cells depended on the Very large doses of interferon or immune presence on the membranes of infected cells of serum transferred to previously infected re- both virus-specified antigens and the appro- cipients are relatively ineffective against the priate major histocompatibility gene pro- established infection in target organs, al- ducts. The early lytic effect ensures that cells though high levels of interferon and high are lysed before progeny virions are assembled antibody titres can be demonstrated in the and thus accounts for the efficiency of cell- sera of the recipients. On the other hand, mediated immunity in the control of estab- immune spleen cells harvested 6 days after lished infections with orthopoxviruses (see active immunization of the donor transfer below). specific and highly efficient antiviral mech- anisms which rapidly eliminate infection Cell-Mediated Immunity in Relation to from the target organs of the recipients, in Pathogenesis whose serum neither antibody nor interferon is detectable. The active cells in the immune There is even more need to make use of population can be identified as cytotoxic T model systems in laboratory animals to eluci- cells. Mononuclear phagocytes of immune T- date the role of various components of the cell recipients, labelled with tritiated thymi- immune response in smallpox than in studies dine before T-cell transfer, appear in foci of of the spread of infection during the incu- infection in the liver after T-cell transfer, and bation period. Mice are particularly suitable, prior irradiation of immune T-cell recipients since so many genetically defined mouse lines in a regimen designed to reduce blood are available. Mousepox, which proved so monocyte levels significantly reduces the useful for studying other aspects of patho- antiviral efficiency of the transferred cells. genesis, is the system most likely to provide These findings support the idea that blood- clues as to the relative importance of humoral borne cytotoxic T cells with immunologi- and cell-mediated immunity in smallpox, and cal specificity for virus-induced antigenic has been extensively exploited for this pur- changes in infected cell surface membranes pose (Blanden, 1970, 1971a,b; reviews: Blan- enter infectious foci and retard viral spread by den, 1974; Cole & Blanden, 1982). lysing infected cells before the maturation and assembly of progeny virions. This T-cell Experiments with mousepox activity attracts blood monocytes which con- tribute to the elimination of infection by Mechanisms controlling viral growth in phagocytosis and intracellular destruction of the major visceral target organs (liver and virus. Macrophage activation and locally pro- spleen) become operative 4-6 days after duced interferon may increase the efficiency primary infection by the natural route, which of virus control and elimination, but are less in the experimental studies was simulated by important than T cells. subcutaneous inoculation into the footpad. Recognition of the importance of cytotoxic Cell-mediated immune responses occur soon T cells in controlling the replication of after infection : virus-specific cytotoxic T ectromelia virus in foci of infection does not cells are detectable 4 days after infection and mean that humoral antibodies do not also play reach peak levels in the spleen 1-2 days later, a role in pathogenesis. Indeed, the early 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 157 experiments of Fenner (see Fig. 3.5) with izing antibodies were much lower than those convalescent antiserum from ectromelia-im- in non-haemorrhagic smallpox and the reac- mune animals showed that antisera do have an tion was often still negative when the patient effect on the progression of infection, prob- died. Cases of haemorrhagic-type smallpox, ably in controlling the viraemia. but not other types, always had high and sustained viraemia and antigenaemia (see Fig. The Immune Response in Smallpox 3.2). Another important aspect of the humoral Because it is so easy to obtain serum, response was the persistence of antibodies because many serological tests for antibodies after recovery from smallpox, as detected by are simple to perform, and because the different tests. Downie & McCarthy (1958) development of knowledge about cell-medi- provide relevant data on some of the sera from ated immunity is relatively recent, studies of 32 British cases of variola major (of whom 25 the immune response in smallpox and after survived) and 19 cases of variola minor. The vaccination are dominated by reports on the results (Fig. 3.6), expressed on several time humoral component of the immune response frames and on a logarithmic time scale, and very deficient in observations on cell- showed that the levels of neutralizing and HI mediated immunity. antibodies rose on about the 6th day of illness and the level of CF antibodies about 2 days Antibodl, production in cases of smallpox later. Neutralizing antibody titres usually persisted for several years, HI titres usually The most comprehensive studies on the fell to low levels by the 5th year after serological responses to smallpox are those infection, and CF antibody titres rarely per- reported by Downie and his colleagues in sisted for as long as 1 year. 1958 and 1969. Working with variola major patients in Madras, India, Downie et al. (1969a,b) examined the sera of 151 patients Cell-mediated immtmit_y in smallpox with ordinary-type and modified-type small- Understanding of the importance of cell- pox, 37 patients with early haemorrhagic- mediated immunity in the recovery process in type smallpox and 40 patients with late poxvirus infections came too recently for haemorrhagic-type smallpox. appropriate studies to have been conducted in In non-haemorrhagic smallpox haemag- cases of smallpox, although it would have glutinin-inhibiting (HI) and neutralizing been of considerable interest to see whether antibodies showed rising titres from the 6th the T-cell responses were defective in flat- day of illness (i.e., approximately 18 days after type and haemorrhagic-type smallpox. The infection) and most patients developed anti: extent of the rash precluded studies of delayed bodies demonstrable by gel precipitation or hypersensitivity by skin tests. The only report by complement fixation (CF) some 2 days on cellular immunity in smallpox is that of later. Most of the patients studied were adults Jackson et al. (19771, who determined the who had scars attributed to childhood vacci- proportion of T and B cells in the peripheral nation; the antibody response usually oc- blood of 17 smallpox patients in Bangladesh, curred a few days later in unvaccinated than at times varying between 3 and 21 days from in vaccinated patients. the onset of illness. The T-cell counts were Most of the patients with haemorrhagic- consistently lower in smallpox patients than type smallpox were adults with old vacci- in the controls; in 2 out of 4 fatal cases the B- nation scars (Rao, 1972). Their antibody re- cell counts were lower than in any of the sponses were much lower, and occurred later, controls. The 2 patients who had the highest than those of patients suffering from ordi- null cell counts (lymphocytes not identified as nary-type or modified-type smallpox. Only 5 either T or B cells) died, while 5 patients with patients, all with late haemorrhagic-type consistently low null cell counts survived. smallpox, developed antibody that reacted in Since no effort was made to determine the the precipitation test ; 4 of these patients and nature of the T cells studied, this result can 2 others were the only ones with CF antibody. only be regarded as preliminary. If it were Tests for HI antibody, on the other hand, possible to carry them out, studies on were almost always positive and the titres patients with human monkeypox would were comparable to those found in non- provide the only opportunity left to extend haemorrhagic smallpox. The titres of neutral- these investigations. 158 SMALLPOX AND ITS ERADICATION

The Immune Response after Vaccination A I There have been numerous studies of the immune response after vaccination, in both human subjects and laboratory animals, especially rabbits. Most have been concerned only with humoral antibodies and, when neutralizing antibodies were assayed, only with tests involving non-enveloped virions.

Antibod3, production after vaccination The results obtained by Downie and his 0 2 4 8 16 32 2 4 8 16 2% 5 10 20 colleagues in vaccinated and revaccinated human subjects (McCarthy et al., 1958b) . .... B provide comparability with the antibody responses in smallpox just described. Follow- Lx.- . ing primary vaccination, no antibody was Y detected up to the 10th day, after which neutralizing and HI antibodies were present in the majority of individuals and CF antibodies in less than half (Fig. 3.7). Neutralizing antibodies were clearly much the most persist- X. : ent, sometimes being demonstrable for more than 20 years after primary vaccination. HI xx antibody was less persistent, and its persist- z xhax ence varied more markedly from subject to subject. CF antibody was not found more 0 2 4 8 16 32 2 4 8 16 2’4 5 10 20 than 6 months after primary vaccination. In revaccinated individuals (several of whom 12804 CI possessed neutralizing antibodies before re- vaccination), the antibody titres tended to be 640 ..” .. . higher, and when a response occurred, it .. began earlier, often within 7 days. In several x 4 ..% revaccinated individuals CF and HI anti- bodies failed to appear even when there was xx a substantial rise in neutralizing antibody. It was noteworthy that only about half the x* revaccinated subjects who showed an “early” I or “immediate” type of vaccination response xx 1. developed neutralizing antibody. When these results are compared with I those recorded in Fig. 3.6 for non-haemor- rhagic smallpox, it is clear that, calculated

0 2 4 8 16 32 2 4 8 I6 24 5 10 20 from the time of infection rather than the

Days 7 < Months 5 < Years > onset of disease, antibodies appeared more quickly after vaccination than in an attack of Fig. 3.6. Antibody production and persistence in smallpox. The disparity was even greater in cases of non-haemorrhagic smallpox (variola major revaccinated subjects. This result, not unex- and variola minor, not differentiated). as determined pected in view of the much shorter incubation by various tests. A: Neutralization of variola virus pock production on the chorioallantoic membrane; period and more rapidly progressive disease 8: Complement fixation with vaccinia1 antigens; C: process in vaccinia (see Fig. 3.1 and Chapter 1, Inhibition of haemagglutination by vaccinia haemag- Fig. 1.3), explains why primary vaccination, glutinin. Note that the abscissa has several time given early after exposure, often modified and scales, all logarithmic. and that day zero corresponds sometimes aborted an overt attack of small- to the onset of illness-i.e.. about 12 days after pox. The appearance of neutralizing anti- infection. 0 = vaccinated; x = unvaccinated; @, 8% = fatal cases. (From Downie & McCarthy. 1958.) bodies after vaccination with live virus is an 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 159

Primary vaccination -~ Revaccination 100, 1 I A .-6 90. @@ U m ._ 7 80. 'Q .. .. - I ' .' .' Y3

!601?! .. .-. * 1 I <5OJ , , L,..: .. m,, I I,1 I I k-.,.*, ,. , , , , , , , *( I 0 2 4 8 I6 32 2 4 8 16 2Y2 5 I0 20 40 0 2 4 8 I6 32 2 4 8 16 2% 5 I0 20 40

I Days > Months Years ~ Days 2 ~ Monrhs , Years > 160,

0 2 4 8 I6 32 2 4 8 I6 2Y2 5 10 20 40 0 2 4 8 I6 32 2 4 8 I6 2% 5 I0 20 40

< Days > < Monrhs Years ~ Dais . . Monrhr , ' Years ' 640, I El

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0 , , "y- , , , , , ., , -.., .. , 0 2 4 8 I6 32 2 4 8 16 2x 5 I0 20 40 0 2 4 8 I6 32 2 4 8 I6 211 5 10 20 40

Days > < Months , ' Years Days I ' Monrhs c Years '

Fig. 3.7. Antibody production and persistence after primary vaccination (panel on left: A, C. E) and revaccination (panel on right: B, D, F), as determined by various tests. A and B: Neutralization of variola virus pock production on the chorioallantoic membrane; C and D: Complement fixation with vaccinial antigens; E and F: Haemaggluti- nation inhibition of vaccinia haemagglutinin. Note that the abscissa has several time scales, all logarithmic, and that day zero corresponds to the day of infection with vaccinia virus. Circled dots in A represent cases of . (From McCarthy et al., 1958b.) index of an active immune response which Cell-mediated immunit_y after vaccination would include the development of antibodies Pincus 8c Flick (1963) demonstrated that to all viral antigens, as well as increased cell- delayed hypersensitivity, which is one index mediated immunity. The accelerated immune of cell-mediated immunity, developed rapidly response in revaccination (within a week, after vaccination ; they suggested that it compared with over 10 days in primary played an important role in the pathogenesis vaccination) was of considerable importance of vaccinial lesions, both after primary vacci- in protecting persons by vaccination after nation and after revaccination. By vacci- exposure to smallpox. nating children twice, at intervals of 2 days, 160 SMALLPOX AND ITS ERADICATION they showed that the incubation periods for individuals were vaccinated. Cell-mediated the 2nd vaccination, with regard to both immunity was clearly of major importance in papule and vesicle, were greatly shortened. controlling vaccinial infection, since individ- A delayed hypersensitivity response can be uals with defects in cell-mediated immunity elicited in vaccinated subjects by either killed but intact antibody production (Categories 3 or active vaccine (Benenson, 1950), so that an and 5, Fig. 3.8) suffered from progressive “immediate” reaction to revaccination vaccinia, while those with defects in antibody (reaching its maximum within 72 hours) does production but a satisfactory capacity to not necessarily mean that the subject’s mount a cell-mediated immune response immunity has been boosted by reinfection. (Category 4) usually reacted normally to Nevertheless, it indicates that he possesses vaccination. Freed et al. (1972) suggested that some residual . If a potent live vaccine in some circumstances, especially when the is used, with a satisfactory technique, this may immunological defect was acquired (Category be the only reaction seen in highly immune 6), the administration of vaccinia-immune subjects. In those in whom immunity has globulin (VIG) would allow cell-mediated waned somewhat this allergic response is immunity to recover sufficiently to control followed by an enlarging area of erythema, the vaccinial infection. Also, in some cases of often with vesiculation in the centre, which Bruton’s syndrome a partially deficient cell- becomes maximal earlier than in a primary mediated immune mechanism might have vaccination. The absence of such erythema by been “overwhelmed”, but could be restored to the 3rd or 4th day after primary vaccination is effectiveness by the administration of vac- indicative of a deficient cell-mediated im- cinia-immune globulin. mune response and such patients may suffer Delayed hypersensitivity reactions could from progressive vaccinia (see below). never be evoked in patients with progressive vaccinia, nor could the peripheral blood Immunological dt$cienc_y states in man lymphocytes of such patients be stimulated to by exposure to inactivated vaccinia The effects of immunological deficiency virus (Fulginiti et al., 1968). Although neu- states in human subjects who had been tralizing antibody was sometimes present in vaccinated with vaccinia virus provides the the serum, its presence did not prevent the best available information on the relative development of progressive vaccinia if cell- importance of cell-mediated and humoral mediated immunity was defective (Hansson responses in determining recovery in ortho- et a]., 1966). Kempe (1980) records having poxvirus infections in man. Fulginiti et al. seen 15 boys with very low levels of gamma- (1968) described a number of cases of globulin, who had been routinely vaccinated progressive vaccinia (vaccinia necrosum) in in infancy without complications. All had such infants and children, and Kempe (1980) histories of “enormous and hyperactive” de- has summarized his extensive experience with layed hypersensitivity responses to vaccinial these conditions, in relation to vaccination. In antigens. children with immunological defects in cell- mediated immunity, vaccinia virus replicated without restriction, resulting in a continually Cells Involved in Immunological Memory progressive primary lesion (see Chapter 7, Plate 7.8B), persistent viraemia and wide- By far the most important aspect of vacci- spread secondary viral infection of many nation against smallpox was the fact that the organs, including the skin. This response was vaccinated individual was primed, so that he particularly severe in patients with thymic responded more quickly and effectively than alymphoplasia. In patients with thymic an unvaccinated person to infection with the and partially or completely antigenically related variola virus. Orthopox- intact immunoglobulin-synthesizingcapacity virus-specific T-cell and B-cell memory can (Nezelofs syndrome) the progression of the be envisaged as comprising previously in- primary disease was sometimes slower and less duced lymphocytes, of both T and B classes persistent, but a fatal outcome was usual and all subclasses, which persist as non- (Kumar et al., 1977). functioning long-lived cells in lymphoid Fig. 3.8 sets out various kinds of immunolo- tissues and the recirculating pool of lympho- gical defect schematically and suggests the cytes. In the of mice infected with kinds of response that occurred when such ectromelia virus, the appearance of memory T 3. PATHOGENESIS. PATHOLOGY AND IMMUNOLOGY 161

Immunological Immunological Response to vaccination condition status

I. Normal, vaccinated (+)CMI+; (+)Ab+ CMI+; Ab+ : no change

2. Normal, unvaccinated (+)CMI-; (+)Ab- CMI + Ab+ primary vaccination

3. Thymic dysplasia (-)CMI-; (+)Ab- CMI - Ab + : progressive vaccinia \ VIG L CMI - , Ab + progressive vaccinia

4 Bruton's syndrome (+)CMI-.(-)Ab- CMI + , Ab- primary immunization

or

CMI - .Ab-(CMl"overwhe1med"): progressive \ vaccinia VIG

CMI + , Ab + . CMI restored-recovery

5. Swiss syndrome (-)CMI-: (-)Ab- CMI - , Ab- ' progressive vaccinia

VIG L CMI - , Ab + : progressive vaccinia

6 Acquired deficiencies ( - )CMI - ; ( - )Ab - CMI - ; Ab - : progressive vaccinia (e g . lymphoma) \ VIG 4 CMI+ . Ab+ : CMI restored-recovery

Fig. 3.8. The response of normal individuals and individuals with immunological defects to vaccination. Pro- gressive vaccinia is associated with a defective cell-mediated immune response, but under some circumstances vaccinia-immune globulin can be useful (see text). CMI = cell-mediated immunity: Ab = antibody production: VIG = vaccinia-immune globulin: (+) or (-) = potential for CMI or Ab response; + or - = presence or absence of appropriate response. (Based on Freed et al.. 1972.) cells coincides with the disappearance of primary and secondary infections increases, primary cytotoxic T-cell activity, which falls since as long as pre-existing antibody of the off rapidly after viral clearance (Gardner & correct specificity persists this probably Blanden, 1976). After mediating viral clear- aborts infection at the portal of entry. Later, ance, primary cytotoxic T cells apparently the ability to mount a rapid cell-mediated dedifferentiate and lose their lytic capacity, immune response would assist greatly in and on restimulation with antigen, which limiting the growth of virus and extension of occurs on reinfection, the sequence is re- the infectious foci. versed. In addition, in the primed individual there are memory helper T cells whose functional activity can only be expressed in REDUCING THE RISKS OF the presence of antigen, when they elaborate VACCINATION factors that amplify the functions of B cells and of other subclasses of precommitted T Vaccination was introduced and its efficacy cells. established by the most relevant and stringent The role of memory T cells in persisting criterion, its ability to provide protection immunity to orthopoxvirus infections in- against smallpox, almost a century before creases in importance as the interval between immunological techniques were developed. 162 SMALLPOX AND ITS ERADICATION

After smallpox had been eliminated from enveloped and non-enveloped virions and the Europe and North America in the 1950s, the full range of humoral and cellular immune public in these countries did not readily responses. In contrast, inactivated virions tolerate the degree of illness-not to say the failed to provoke a humoral response to the occasional episodes of severe illness or even envelope antigens (Appleyard et al., 1971 ; deaths, which occurred, for example, in ecze- Turner & Squires, 1971 ;Payne, 1980), nor did matous children and those with an immuno- they stimulate the production of cytotoxic T logical deficiency-that were associated with cells (Ada et al., 1981). standard smallpox vaccination. Two ways Experiments with inactivated vaccines were sought to provide immunization against highlighted the lack of correlation between smallpox without the attendant risks of severe levels of neutralizing antibody (measured disease: the use of attenuated strains of against non-enveloped virions) and protec- vaccinia virus and the use of inactivated tion. Thus immunization of rabbits with a vaccines. vaccinia virus “soluble antigen” gave a good The assessment of the efficacy of either of antibody response and protection against these kinds of vaccine presented obvious intradermal challenge with vaccinia virus, problems, since the real criterion for their but resistance to the more virulent rabbitpox value was the ability of the vaccine to protect virus was less than that induced by live against smallpox and the durability of this vaccinia virus, despite the fact that the live protection. On ethical and practical grounds virus induced much less antibody (Appleyard it was not possible to test new vaccines in this & Westwood, 1964b). Rabbits immunized by way, so two other criteria were used in their multiple intradermal injections followed by 6 assessment : the neutralizing antibody re- intravenous injections of heat-inactivated sponse and protection against challenge in- vaccinia virus developed high titres of neu- oculation of standard vaccine, supplemented, tralizing antibody, but even after this inten- in experiments with animals, by protection sive course there was only partial protection against an otherwise lethal challenge with a against challenge infection with rabbitpox suitable orthopoxvirus. virus (a virulent strain of vaccinia virus) By all three tests the standard vacciiie (Madeley, 1968). Similarly, rabbits immu- strains provided excellent protection against nized with large doses of vaccinia virus smallpox. The situation was less clear-cut inactivated by or ultraviolet with attenuated vaccinia virus vaccines, irradiation developed extremely high titres of which are discussed at some length in Chapter neutralizing antibody (tested against non- 11. There appeared to be serious deficiencies enveloped virions), but they remained sus- in protection when inactivated vaccines were ceptible to generalized rabbitpox infection, used, the reasons for which are outlined although protected from death (Boulter et al., below. 1971). The importance of antibody against enveloped virions was most clearly demon- Inactivated Virus Vaccines strated by experiments on the passive transfer of resistance with antiserum (Table 3.5). The production of an inactivated smallpox Antisera against inactivated virus, with very vaccine seemed a feasible procedure, since it high levels of neutralizing antibody to non- was relatively easy to grow and purify large enveloped but none to enveloped virions, amounts of vaccinia virus. Many methods of provided much weaker protection against inactivation were tested (Kaplan, 1969 ; challenge infection than apparently much Turner et al., 1970), including heat, formal- lower titres of antibody induced by infectious dehyde, ultraviolet irradiation, photo- virus, which, however, contained both kinds dynamic inactivation and gamma irradiation. of neutralizing antibody. As described in Chapter 2, there are major More recently, Olsen et al. (1 977) reported antigenic differences between the surface that an inactivated vaccine prepared by antigens of enveloped and non-enveloped mechanically disrupting monkeypox virions virions, both of which are infectious. Small- protected monkeys against disease when they pox vaccine, however it was grown, was were challenged with active monkeypox prepared in such a way that it consisted virus, although it did not prevent infection. predominantly of non-enveloped virions. The There were two significant features in these infection provoked by vaccination with live experiments. First, immunization was carried vaccine led to the development of both out with the homologous virus. The second- 3. PATHOGENESIS, PATHOLOGY AND IMMUNOL,OGY 163

Table 3.5. Comparison of the protective activity of three types of antiserum administered passively to rabbits before challenge infection with rabbitpox virusa

Response to Antiserum challenge

Neutralization Against Source Fever Death titre

Inactivated vaccinia virus Horse 800 OOOb 12/12 5/12 500 OOOb 515 415 Live vaccinia virus Sheep I50 OOOc 415 015 Rabbit I9 oooc 8/10 0110 Live rabbitpox virus Rabblt 32 OOOc 015 015 None - - 18/18 I15/18

a From Boulter et al. ( I97 I ). Measured against non-enveloped virions; no neutralizing antibodies to enveloped virions. Measured against non-enveloped virions; neutrallzlng antibodies to enveloped virions also present. and more important-feature was that, al- Germany during the late 1960s (see Chapter though the inactivated preparation had no 11) for a “priming” vaccination, followed by haemagglutinating capacity, it elicited the vaccination with standard vaccine. Subse- production of haemagglutinin-inhibiting as quently, Marennikova & Macevie (1975) well as neutralizing antibodies, demonstrat- showed that pre-immunization of rabbits ing that the method of inactivation left at with vaccine inactivated by 6oCo gamma- least some of the envelope antigens intact. irradiation greatly enhanced their response to It also appears that inactivated virus pro- vaccination with active vaccine given 7-60 vokes a rather different kind of cell-mediated days later, both in the titre of antibody immune response from that found after produced and in its rate of production. Pre- infection, eliciting delayed hypersensitivity T immunization reduced the incidence of virae- cells but not cytotoxic T cells (Ada et al., mia in the rabbits 4-5 days after vaccination 1981), perhaps because the surface antigens with live virus. Preliminary human trials on involved in infected cells are not produced, or the use of this preparation as a priming because the mode of inoculation results in too antigen were carried out in eastern Europe in localized and immobile an antigenic mass. 1977 (see Chapter 11). Indeed, the delayed hypersensitivity reaction itself was deficient in rabbits immunized with NON-SPECIFIC MECHANISMS inactivated vaccine. For example, Turner et al. INVOLVED IN HOST DEFENCE (1970) and Turner & Squires (1971) found that inactivated vaccines did not produce an The efficacy of vaccination and the obvious delayed hypersensitivity response, increased susceptibility of individuals with although the animals responded more rapidly certain immunological defects illustrate than did the controls to challenge inoculation clearly the great importance of the immune with live virus. response in orthopoxvirus infections. There Thus, inactivated vaccines suffered from are nevertheless a number of defence mechan- two defects: they failed to elicit antibodies isms against viral infections whose activity is that neutralized enveloped virions and they not specific in an immunological sense. Most failed to provoke the production of cytotoxic of these are ill-understood and it is difficult to T cells. In addition, the adverse effects of evaluate their importance in orthopoxvirus inactivated measles virus vaccines, which infections. became evident in the mid-1 960s (Fulginiti et al., 196?), made authorities reluctant to consider another inactivated Body Temperature virus vaccine when there was already a successful live virus vaccine available. Never- As outlined in Chapter 2, determination of theless, in an effort to reduce the incidence the ceiling temperature of viral replication is and severity of postvaccinial encephalitis, a useful laboratory method of distinguishing formalin-inactivated vaccine (“vaccinia-anti- between certain orthopoxviruses and between gen”) was used in the German Democratic variola major and certain strains of variola Republic and the Federal Republic of minor virus. In animal models, body tempera- 164 SMALLPOX AND ITS ERADICATION ture has a dramatic effect on the severity of the leporipoxvirus disease, (Marshall, 1959), and mice housed at 2 "C are about 100 times more susceptible to mouse- pox than those maintained at 20 "C (Roberts, 1964). There is no evidence that raised body temperature affected the progress of variola major ; severe cases (flat-type and haemor- rhagic-type smallpox) were often associated with higher temperatures than those found in ordinary-type smallpox. However, Dumbell & Wells (1 982), comparing variola major and (variola minor) viruses, found that many fewer virions of alastrim (variola mi- nor) virus (which had the lower ceiling temperature) than of variola major virus were released from infected cells when the tem- perature was raised. The decreased dissemina- tion of virus could act in concert with Plate 3. I I. Rijk Gispen (b. I9 10). Formerly Director of the National Institute of Public Health at developing immunity to reduce the severity Bilthoven. Netherlands. Gispen was an important of variola minor. contributor to the immunology of orthopoxvirus infections from the 1950s to the 1970s. He was the first to develop methods of differentiating between Nutrition antibodies due to infection with monkeypox. variola Almost any severe nutritional deficiency and vaccinia viruses. will interfere with the activity of phagocytes, and the integrity of the skin and mucous membranes is impaired in many types of variola minor was much more severe in nutritional deficiency (review : Scrimshaw et malnourished than in well-nourished infants. al., 1968). Immunoglobulin levels, antibody The occurrence of blindness after smallpox responses and the numbers of circulating B is said usually to have been associated with cells are generally normal in cases of moderate secondary bacterial infection or nutritional to severe malnutrition, but cell-mediated deficiencies. immunity is consistently impaired, whether measured by cutaneous delayed hypersensiti- Age vity tests or by the numbers of circulating T cells (Chandra, 1979). The number of null Among unvaccinated persons, smallpox cells-i.e., cells without the surface character- produced its highest mortality in the very istics of T or B cells, which suppress the young and the aged, and its lowest in the age activity of other lymphocytes-was relatively group 5-20 years, but there is no obvious increased in cases of malnutrition. It will be explanation for these age-related effects ex- recalled that the proportion of such cells was cept in so far as the mechanisms of specific substantially increased in patients with small- and non-specific resistance function less ef- pox and that there seemed to be a correlation fectively at the extremes of life. between the height of the null cell count and the Uackson et al., 1977). Little information is available about the Hormonal Effects effect of malnutrition on smallpox, although it seems clear that its effects were not as Pregnancy had a very pronounced effect on dramatic as those seen in measles in young the severity of smallpox. Especially in variola children in many African countries. The major, pregnant women were much more mortality of variola major in unvaccinated likely than any other category to suffer from infants was so high that it was difficult to haemorrhagic-type smallpox (see Chapter 1). determine whether nutritional deficiency was Pregnant women have elevated levels of 17- important. However WHO epidemiologists dihydroxycorticosteroids, which have an working in Ethiopia and Somalia noticed that anti-inflammatory effect, depress the immune 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 165

response and inhibit interferon production. consequent disease was milder than smallpox Studies in rabbits infected with vaccinia virus acquired by the inhalation of virus contained showed that cortisone diminished the local in oropharyngeal secretions. This is unlikely inflammatory reaction and increased viral to be the complete explanation ; intradermal titres in the blood and internal organs (Bug- inoculation was associated with a shorter bee et al., 1960). Vaccination could produce incubation period and the immune response severe effects in humans receiving corticos- would have been differently stimulated, but teroid . interferon in the inoculum and possibly the Rao et al. (1968b) found that cortisone local production of interferon induced by converted experimental smallpox in monkeys inactivated virus in the inoculum may have from a non-lethal into a lethal disease. Virae- played a role. mia was greatly enhanced in intensity and persisted for a longer time, the internal organs contained much more virus than in control GENETIC ASPECTS OF RESISTANCE animals, and there were numerous haemor- TO SMALLPOX rhages in the lungs and in the mucous membrane of the . Because of the availability of lines of mice of known genotypes and of congenic recom- binant strains (Klein, 1975), these animals are Interferon uniquely suitable for the analysis of genetic resistance to viral infection. Studies with Interferons are a family of of low mousepox(Briody et al., 1956 ;Schell, 1960a,b ; molecular weight produced by a wide variety Wallace et al., 1985) demonstrated that gene- of cells. Lymphocytes produce a different tic factors played a major role in determining kind of interferon (gamma-interferon) from their response to mousepox. fibroblastic cells, but all kinds of interferon It is clearly not possible to analyse the may render cells that take them up more genetic component of the resistance of resistant to viral infection. Vaccinia virus was humans to smallpox in this way. Nevertheless, the first virus shown to be sensitive to it is worth examining whether any human interferon in an intact animal. Isaacs & population groups showed unusual resistance Westwood (1959) showed that interferon or susceptibility, independent of the effects of prepared in rabbit cells protected rabbits vaccination. completely against intradermal infection with a large dose of vaccinia virus, when given a day before the virus was administered, Natural Selection for Resistance to and against a smaller dose when both were Smallpox administered intradermally on the same day. However, Blanden (I 970,1971a) showed that Once again, an animal model may provide a passively administered interferon had no useful lead in understanding what may have effect on recovery from mousepox. Interferon happened in smallpox. Myxomatosis, a severe seems unlikely to have played a role in generalized poxvirus disease in European determining differential host responses in rabbits (Oryctolagus cuniculus), provides the best smallpox, but it may have been important in example of rapid enhancement of the level of determining the differences in severity of genetic resistance in a population as a result of inoculation smallpox and the “natural” exposure to the disease (Fenner & Ratcliffe, disease. 1965; Fenner, 1983). Although it was no- where near as lethal a disease as myxomatosis, Interferon and inoculation smallpox smallpox was severe enough to have had a selective effect for resistance among humans Inoculation smallpox (variolation) is much exposed to infection over many centuries, as milder than “natural” smallpox (see Chapters in India and and to a lesser extent in 1 and 6). Following the demonstration Europe. that vaccinia scabs contained interferon, Reports of smallpox in the 16th and 17th Wheelock (1964) suggested that the presence centuries among the Indian tribes of North of interferon in scab material that was used for America (Stearn & Stearn, 1945) and in Brazil variolation might have so interfered with the (Hemming, 1978) describe the extreme sever- replication of the inoculated virus that the ity of smallpox in these hitherto unexposed 166 SMALLPOX AND ITS ERADICATION . Hemming notes that in Brazil correlation between ABO blood groups and the Portuguese colonists observed that Negro the occurrence of neurological or dermal slaves, when they got smallpox, suffered less complications of vaccination (Gurvich et al., severely than the Amerindian slaves ; most of 1980). Most human geneticists now doubt the European invaders themselves were im- whether smallpox was an important selective mune because of infections sustained in their agent in blood group ABO polymorphism childhood. Although several other factors (Mourant et al., 1978). besides lack of genetic resistance could have been involved in exacerbating the effects of the disease among the Amerindians-notably SUMMARY: THE PATHOGENESIS OF the severe social disruption accompanying SMALLPOX the first outbreaks-it is tempting to consider that their extreme susceptibility was in part Although a good deal of speculation and related to the absence of previous selection for extrapolation from various model systems resistance to smallpox. will inevitably be involved, it is worth From what we now know about the attempting to summarize the results of the relation between the major histocompati- work presented in this chapter in the form of a bility genes and resistance to,viral infections comprehensive picture of the pathogenesis of (review: Zinkernagel, 1979), it would have smallpox in man. been interesting to investigate the relation between HLA groups and susceptibility to smallpox ; experiments with mousepox Viral Entry and Infection showed that at least part of the genetic resistance to this disease was associated with Infection usually occurred by the implan- the H-2 complex in the mouse (R.V. Blanden, tation on the oropharyngeal or respiratory personal communication, 1981). The oppor- mucosa of virus released from lesions in the tunity to do this never arose, but Vries et al. mouth, nose and pharynx into the nasal and (1 977) showed that after vaccination with oropharyngeal secretions of the source case vaccinia virus, the lymphocytes of Dutch during the first week of rash. Such material soldiers belonging to a particular HLA group probably consisted of well-dispersed virions (Cw3) responded significantly less effectively and would have been relatively free of inter- than others in an in vztro lymphocyte transfor- feron. In contrast, scab material usually mation test using vaccinia virus as antigen (a consisted of large fragments of inspissated measure of cell-mediated immunity). This material with infectious virions bound within HLA group made up 30% of the general a dense, hard fibrin mesh, which contained a population in the Netherlands, compared substantial amount of interferon, and from with 83% of the low-responder group. which it was difficult to release virus except During the 1950s and 1960s, a great deal of by mechanical grinding. These features prob- information was accumulated on the distri- ably accounted for the much lower transmissi- bution of ABO blood groups in different bility associated with scabs, compared with populations. Pettenkofer et al. (1962) claimed oropharyngeal secretions. that there was a correlation between past The initial site of lodgement was usually histories of smallpox epidemics and the distri- somewhere in the oropharynx, nasopharynx bution of ABO blood group frequencies, and or the lower respiratory tract. Whatever cells that in India there were different degrees of were initially infected, macrophages would scarring in persons of blood groups B and 0 soon have become infected and by about the compared with those of blood groups A and 3rd day would have entered the lymphatics AB. However, investigations by several dif- and thus reached the regional lymph nodes. ferent teams of workers in India (Bhatta- They might also have entered the blood- charyya et al., 1965; Downie et al., 1965b; stream at this stage, or in any case by about the Helmbold et al., quoted by Vogel & Chakra- 4th day, after a brief delay and replication in vartti, 1966 ;Sukumaran et al., 1966), in Brazil the draining lymph nodes. (Krieger & Vicente, 1969), and in Zaire The initial infection in the oropharynx or (Lambotte & Israel, 1967) found no evidence respiratory tract was silent, producing neither of a correlation between the incidence and symptoms nor a local lesion that could be severity of smallpox and the ABO blood recognized clinically, or by autopsy in cases group of the subjects. Nor was there any that died early. 3. PATHOGENESIS, PATHOLOGY AND IMMUNOLOGY 167

Spread through the Body cells in the skin, produced interferon. In cases in which the early cellular immune response The inevitable lack of careful postmortem was vigorous, replication of the virus was examinations of cases of smallpox dying from inhibited and the skin lesions were restricted, other causes during the incubation period of so that a discrete rash developed. the disease, combined with the paucity of If the cellular immune response was grossly careful postmortem or virological exami- deficient, the case may have presented as flat- nations of acutely fatal cases, makes it difficult type smallpox. Haemorrhagic-type smallpox to assess precisely where the virus replicated was associated with unrestricted repli- before the secondary viraemia occurred, at cation of the virus especially in the bone the time of the onset of symptoms. The most marrow, so that a much higher viraemia likely places were the lymphoid organs developed than in most non-haemorrhagic (spleen, bone marrow and lymph nodes), but cases and megakaryocyte destruction in the extensive necrosis did not occur there. Virae- bone marrow led to defects in the blood mia was largely cell-associated and most coagulation mechanism. Further, the immune virions that existed free in the plasma were response, both humoral and cellular, was probably in the enveloped form. defective in haemorrhagic-type smallpox, The reasons for the localization of virus in which was particularly frequent in pregnant the skin and the characteristic “centrifugal” women, probably because of their increased distribution of the rash are unknown. Prob- corticosteroid secretions. ably, infected macrophages migrated from small vessels in the dermis into the epidermis, Death or Recovery where they proceeded to cause infection of the cells of the Malpighian layer. Oedema and The outcome of the infection was either ballooning degeneration followed, with re- death or recovery, with or without sequelae. ticulating degeneration and splitting of the Except in haemorrhagic-type smallpox, the epidermis that produced a multiloculated cause of death was obscure, since none of the vesicle. Later there was a migration of poly- “vital organs” (brain, lungs, heart, kidneys, morphonuclear cells into the lumen of the liver) seemed to have been severely damaged developing vesicle, so that its contents be- in fatal cases. It was ascribed to severe came pustular. toxaemia, perhaps due in part to the effect of circulating immune complexes. Death in very severe cases (confluent ordinary, flat and The Immune Response haemorrhagic types) was associated with a high and prolonged viraemia and a poor The early lodgement of infected macro- humoral antibody response, and probably a phages in the lymph nodes, bone marrow and defective cellular immune response as well. spleen would have stimulated an immediate The commonest sequelae were pockmarks, immune response to the wide variety of viral which could occur all over the body but were antigens produced by infected cells. The first usually most profuse on the face because of component of the immune system to become the large number of sebaceous glands there manifest was the production of cytotoxic T and the deeper pitting associated with their cells, which, because of their affinity to the involvement. Encephalitis, with a patho- early viral antigens found in the cell mem- genesis as obscure as that of postvaccinial branes, promptly destroyed many infected encephalitis, occurred in about 0.2q4 of cases cells before they produced virions. Later, of variola major. It was somewhat rarer in neutralizing antibodies appeared, some of variola minor, but a relatively more import- which were directed against the viral enve- ant cause of death in that disease.. Arthri- lope (which contained several of the antigens tis and osteomyelitis sometimes occurred, also found in the surface membrane of infect- though often they were recognized only after ed cells); others were able to neutralize the recovery. Blindness was an important but rare infectivity of intracellular non-enveloped complication, usually occurring in cases in virions, which were released when necrotic which there was malnutrition and/or second- cells were disrupted. The activity of cytotoxic ary bacterial infection. T cells and the titre of antibodies increased as Recovery was accompanied by long-lasting time progressed. In addition, infected macro- immunity to reinfection with variola virus. phages and lymphocytes, as well as infected Heterologous immunity-e.g., to vacci- 168 SMALLPOX AND ITS ERADICATION nation-was much less prolonged, especially thus, previously infected persons, even when in cases of variola minor. Variola virus did immunosuppressed, have shown no recur- not persist in the body after recovery; rence of infectivity.