Varicella-zoster virus infection and immunization in the healthy and the immunocompromised host

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V A R I C E L L A - Z O S T E R VIRUS I N F E C T I O N AND I M M U N I Z A T I O N IN T H E HEALTHY AND THE IMMUNOCOMPROMISED HOST Authors:

Charles Grose Roger H. Giller Divisions of Pediatric Infectious Diseases and Hematology/Oncology Department of Pediatrics University of Iowa College of Medicine Iowa City, Iowa

Referee:

Anne A. Gershon Division of Pediatric Infectious Diseases Department of Pediatrics Columbia University College of Physicians and Surgeons New York. New York

I. I N T R O D U C T I O N ~Varicella-zoster virus (VZV) is one of the more reclusive human herpesviruses. The other members of this family of DNA viruses include herpes simplex virus (HSV) types 1 and 2, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). The emphasis of this review is on the two VZV infections in the human host: chickenpox and zoster. Because of the nature of this journal, particular attention is focused on diseases caused by VZV in the patient with cancer. The review begins with a description of the virus and its major glycoproteins; then it provides a schema for pathogenesis and a survey of the clinical diseases; finally, it covers newer treatment regimens. This review is very timely because a decade has now passed since the initiation of clinical trials of a live-attentuated varicella vaccine, which has been administered to both healthy individuals as well as children with leukemia. Thus, VZV is the first herpesvirus vaccine to be tested in human populations on a large scale. I II. T H E VIRUS A N D ITS G E N O M E The herpesviruses share a characteristic morphology, although the size of the particle differs among the members of the family. The VZV particle is composed of an icosahedral nucleocapsid which is about 95 nm in diameter; within the capsid is the electron-dense DNA core. The capsid is covered by an innermost layer called the tegument, which is in turn covered by an envelope. The envelope may consist of several lamellae. The diameter of the enveloped particle varies from 150 to 175 nm. An electron micrograph showing numerous unenveloped VZV nucleocapsids within the nucleus of an infected cell is illustrated in Figure 1. The viral genome is housed within the nucleocapsid. The VZV genome is a linear duplex molecule with a molecular mass of about 80 Mdaltons. Since the conversion factor between megadaltons and kilobase pairs is 1:1.5, the number of kilobase pairs is about 120 in the VZV DNA molecule. The DNA molecule is organized in two segments: by convention, the two segments are designated long (L) and short (S). The short segment is arranged in either orientation relative to the long segment. Therefore, the genome is considered to have two isomeric configurations. ~ Recently, the sequence of the entire VZV genome was published by Davison and Scott.-" They found a total of 124,884 bp, among which were 71 open reading frames which may encode viral proteins. Five of the open reading frames probably code for glycoproteins, which have been designated by Roman numerals as gpI through gpV.

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CRC Critical Reviews in Oncology/Hematology

FIGURE I. Electron micrograph. Numerous VZV nucleocapsids are visible within the nucleus of an infected cell. III. T H E G L Y C O P R O T E I N S O F V Z V The glycoproteins are discussed in some detail because they are important structural constituents of the viral particle; in addition, they are important antigens by which the host regulates the immune response after infection. Table I contains a list of published articles about the VZV glycoproteins; these papers are the sources for the following descriptions of the individual viral products. The glycoproteins are located within the envelope of the virion; they are also inserted into the plasma membrane of the infected cell. The predominant viral glycoprotein is gpl, which is a Mr 98,000 product. Of all the viral glycoproteins, this is the major component within the membrane of the infected cell. This glycoprotein is easily identified in cells removed from a chickenpox or zoster vesicle. Therefore, it is an ideal antigen for rapid diagnosis of VZV infection with a monoclonal antibody probe. The biochemical attributes of this glycoprotein have also been studied; they demonstrate that the mature product is composed of N- and O-linked glycans attached to a Mr 70,000 polypeptide backbone. Another unusual characteristic of this glycoprotein is phosphorylation of some of its serine and threonine residues. 28 Glycoprotein gpII is a M r 140,000 product which is the second most prominent constituent of the infected cell membrane. This glycoprotein is assembled on a Mr 100,000 backbone. The glycans include both N- and O-linked oligosaccharides. After the mature glycoprotein is formed, it is cleaved into two lower molecular weight products (M r 68,000 and 66,000) in the presence of a reducing agent such as 2-mercaptoethanol. Antibodies to this glycoprotein

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Table 1

N O M E N C L A T U R E F O R T H E VZV G L Y C O P R O T E I N S 27 Gene

gpl

gpll

gplll

gplV gpV

New nomenclature

Old names of glycoproteins

Ref.

gpl(98),gpl(62) gpl(94), gpl(83), gpl(55), gpl(45) gpl(92), gpl(83), gpl(55), gpl(45) gpi(90), gpl(80), gpI(60) gpl(92), gpl(59), gpI(47) gplI(140),gplI(66) gpll(116), gpll(106), gpll(64) gpll(115), gpll(62), gpll(57) gpll(130), gpll(125), gplI(62) gpll(120), gpll(118), gpll(65) gpll(125), gpll(63) gplll(ll8) gplll(115) gplll(105) gpllI(118) gplV(55),gplV(45) Hypotheticalgene

gp98, gp62 gp2 gC:gp92, gp83, gp55, gp45 90K, 80K, 60K gp92, gp59, gp47 gpl40, gp66 gp3 gB:gp115, gp62, gp57 gp 1, gp3 120K, 118K, 65K gp125, gp63 gpll8 gpl gA:gp 105 118K gp55. gp45

3--8 9--12 13--14 15--16 17 18--20 11, 12 13 21, 22 16 17, 23 3, 4, 5, 7, 24, 25 13 15. 16 26

exhibit the biological activities of complement-independent neutralization and inhibition of cell-to-cell fusion. -'9 Glycoprotein gplII is a M r 1 18,000 product. The backbone is a M r 79,000 protein which contains mainly N-linked oligomoieties. This glycoprotein is barely detectable in the membrane of infected cells. However, of the three major VZV glycoproteins, it appears to possess the most immunogenic neutralization epitope. Monoclonal antibodies to VZV gplII have complement-independent neutralization titers as high as 1:40,000. The least information is available about VZV glycoproteins gplV and gpV. The former is a Mr 55,000 protein species, while the latter is a hypothetical gene product based on a DNA sequence which is compatible with a viral glycoprotein. A recent important finding has emerged from the DNA sequence data collected on several herpesviruses. These results demonstrate that different herpesviruses have similar genomic structure. For example, several glycoprotein genes are conserved in both VZV and HSV. These include VZV gpI, gplI, and gplII, which are homologous to HSV type 1 glycoproteins gE, gB, and gH, respectively. Thus, information gained about the role of a glycoprotein antigen in one herpesvirus system may be relevant to the homologous glycoprotein of a different herpesvirus. IV. P A T H O G E N E S I S O F C H I C K E N P O X The pathogenesis of VZV infection has remained poorly understood because viral replication is usually restricted to the human host and some human cell lines. 3° VZV grows poorly, if at all, in most nonhuman cell substrates with the exception of guinea pig embryonic cells. 3~ The guinea pig also appears to be an animal model for experimental VZV infection. 32 When VZV infects the human, it causes chickenpox as the manifestation of primary infection. When chickenpox abates, the virus enters the dorsal root ganglia where it remains in a latent state, usually for decades. Reactivation of latent virus is associated with a dermatomal exanthem called shingles or zoster. A schema for the pathogenesis of chickenpox has been constructed based on the animal model for exanthematous diseases first proposed by Fenner (as cited in Reference 33). This model includes two viremic stages, with the exanthem appearing after the second viremia

30

CRC Critical Reviews in Oncology/Hematology PATHOGENESIS OF CHICKEN POX

Infection of conjunctivae and/or mucosa of upper respiratory tract

DAY

0

DAY

4-6

DAY

14

Viral replication in regional lymph nodes

/

Primary viremia

-4

Viral replication in liver. spleen and (?) other organs

Secondary v=remia

Infect=on of skin and appearance of ves=cular rash

FIGURE 2. Schema for the pathogenesis of chickenpox. The pathogenesis during the incubationperiod probablyinvolvesa biphasiccoursewith a primary and secondary viremia prior to appearanceof the exanthem. (Figure 2). Chickenpox is acquired from small virus-laden droplets which are carried by air currents from an infected patient to a nonimmune individual. The site of infection is probably the conjunctivae or the nasal passages, whence the virus travels to a local site (possibly the regional lymph nodes) for a primary cycle of replication. After 4 to 6 days, virus is released into the bloodstream during the primary viremia. At this time, some individuals, especially older children, manifest a brief scarlitinaform rash and an occasional vesicle about the head or neck. After the virus is disseminated throughout the body, a second cycle of replication occurs at various sites, such as the epithelium of the respiratory and gastrointestinal tracts: the glandular epithelium of the liver, pancreas, and adrenals; the epithelium of the renal tubules; and the endothelial lining of the blood vessels. About 1 week later, the second (but major) viremia seeds the capillaries and then the epidermis. This viremic phase has been well documented. 34-38 The vesicle formation is due to the ballooning degeneration caused by virus infection within the epidermis. The entire incubation period, from the time of infection to appearance of the vesicular rash, is usually 14 to 15 days. V. C L I N I C A L F E A T U R E S OF C H I C K E N P O X A. The Exanthem In most healthy children, chickenpox is a relatively mild illness. The disease is characterized by a vesicular rash which emerges in successive crops over the first week of illness. Each vesicle appears after a rapid progression from macule to papule and attains a final diameter of about 3 mm. The fluid within the nascent vesicle is initially clear; however, within 3 to 4 days, the vesicle fluid becomes cloudy because of the ingress of phagocytic cells. A crust forms after 4 to 5 days and remains for 1 to 2 weeks• The exanthem usually begins about the head, especially along the hairline (Figure 3). The rash spreads across the trunk and arms (Figure 4); finally it appears over the legs. Typically, lesions of all stages, including macules, papules, vesicles, and crusts, are present over the same region of the skin.

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FIGURE 3. Chickenpoxin an infant. The vesicular lesions initially appear on the face, especially along the hairline. The average course of chickenpox has been delineated in American children. 39 The illness often begins with a mild prodrome which consists of malaise, pharyngitis, and rhinitis. The fever, which peaks by day 4 of rash, is usually below 102°F. The index case within a family develops an average of 207 to 258 vesicular lesions (Table 2). Subsequent cases of chickenpox within the same family unit tend to be more severe, presumably because the amount of inoculum virus during exposure is greater. These secondary cases may have 500 or more vesicles. B. Extracutaneous Manifestations of Chickenpox Chickenpox, on occasion, is also associated with a variety of other extracutaneous viral manifestations. 4° These include pneumonitis, arthritis, hepatitis, orchitis with or without epididymitis, appendicitis and glomerulonephritis, as well as the hematological problems of thrombocytopenia and purpura fulminans. Chickenpox has also been associated with the subsequent development of Reye's syndrome. 4~ Other neurological sequelae include the broadly descriptive meningoencephalitis, encephalomyelitis, and polyneuritis. 42"43The single most striking neurological complication is acute cerebellar ataxia. The ataxia usually appears during the latter half of the first week and second week of clinical chickenpox, but can also precede the appearance of the skin rash. The signs of neurological involvement usually resolve over a period of months with no permanent residue. The extent of central nervous system (CNS) invasion by virus can be monitored by magnetic resonance imaging (MRI). In the MRI scan shown in Figure 5, a lesion is visible in the cerebellar peduncle of the brain in a 5-year-old boy with chickenpox-associated cerebellar ataxia. Within 2 months of the acute CNS disease, the symptoms abated and the lesion on MRI scan disappeared. Thus, MRI is preferred over computed tomography (CT) for assessment of CNS disease caused by VZV and other human herpesviruses. 44 C. Mortality Statistics Chickenpox is generally not considered a serious illness of childhood. 45 However, mortality is very dependent upon the age of the individual with chickenpox (Table 3). In children

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CRC Critical Reviews in Oncology/Hematology

FIGURE 4. Chickenpox in an older child. The pox lesions progress from the face over the trunk and finally to the extremities. The average number of vesicles varies from 200 to 500, depending on the factors described in Table 2.

NORMAL

COURSE

Table 2 OF CHICKENPOX

Av no. pox per child

Av max temp (°F) Children with chickenpox

Primary cases (322) Secondary cases (209)

I N C H I L D R E N 39

Day 1

Day 2

Day 3

Day 4

Day 6

Children <5 years

Children >5 years

101.2

101.0

100.6

100.2

99.3

207

258

100.7

100.8

100.9

100.5

99.4

310

510

Children with >I000 pox

10

b e t w e e n the ages 1 and 14 years, the death rate is a p p r o x i m a t e l y 1:40,000 cases. In infants, i.e., children throughout the first year o f life, the death rate is three times higher, i.e., 1:13,140. In y o u n g adults o v e r the age of 19 years, c h i c k e n p o x is an e v e n m o r e lifethreatening disease with a death rate o f l: 1460. The morbidity and mortality are particularly high in pregnant w o m e n with c h i c k e n p o x . It is important to r e m e m b e r , h o w e v e r , that the

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FIGURE 5. Magnetic resonance image of the brain. This child developed cerebellar ataxia during the second week of chickenpox. The scan demonstrates a lesion (arrow) in the left cerebellar peduncle. The lesion disappeared on a follow-up magnetic resonance image, which was taken whenthe child's neurologicalexaminationhad returnedto normal. statistics noted here were gathered before the availability of newer antiviral agents which are discussed in the last section of this review. VI. C O N G E N I T A L

VARICELLA INFECTION

Chickenpox in the pregnant woman occasionally causes an intrauterine VZV infection with subsequent embryopathy. This situation was first recognized by Laforet and Lynch, 46 who described a deformed infant born to a mother who contracted chickenpox during the

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CRC Critical Reviews ill Ontology~Hematology

Table3 S T A T I S T I C S ABOUT CASES OF C H I C K E N P O X AND R E L A T E D D E A T H S 4s Age (years)

Estimated no. annual cases

Av no. deaths

Deaths:cases

<1 I--4 5--9 10--14 15--19 >19

98,980 910,686 1.409.889 312.785 55,362 42.353

7 18 38 I0 2 29

1:13,140 1:50.593 1:37,102 1:31.278 1:27,681 1:1,460

eighth week of gestation. At birth the infant was noted to have a shortened atrophic right leg with peglike toes on the right foot. Roentgenographic examination of the right leg revealed underdeveloped long bones and a foot with absent phalanges. Within the first 3 months of life, the infant was found to have diminished vesical and anal spincter function, as well as generalized cortical atrophy and bilateral chorioretinitis. Since the first report, there have been several publications which described the association of congenital defects and maternal chickenpox infection, especially when the mother's illness occurred during the first trimester (reviewed in Reference 47). We have recently reported a severe case of congenital varicella syndrome which followed a second-trimester chickenpox infection in a pregnant woman. 4s The mother of the infant was a 20-year-old primigravida whose pregnancy was complicated by chickenpox at 20weeks gestation. Although the mother had no apparent sequelae of chickenpox, an ultrasound examination of the fetus at 32.5-weeks gestation revealed hydramnios and ventriculomegaly within the fetal brain. A percutaneous cordocentesis was performed and a 5-me sample of fetal blood was obtained. 4~ Chromosome analysis of the cells showed a normal 46XY karyotype. When the infant was delivered by Cesarean section 2 weeks later, his initial physical examination revealed a large head (greater than ninety-fifth percentile) with split sutures, as well as diffuse hypotonia with some spastic movements of the extremities in response to stimulation. Corneal, blink, and Moro reflexes were absent, but grasp and plantar reflexes were present. There was no skin scarring or malformation of the extremities. The infant's subsequent examinations implied severe CNS injury, and his postnatal course became one of increasing inactivity and ventilator dependence. A head computed tomograph delineated extensive hydrocephalus with only a small rim of cortex as well as extensive basal ganglion and brain stem calcifications (Figure 6). A magnetic resonance image of the head revealed massive hydrocephalus with a dependent protein-rich fluid level, suggesting intracranial blood: once again calcifications were observed in the basal ganglion, and little cerebral cortex was seen (Figure 6). An electroencephalogram showed only minimal cerebral activity. An opthalmic examination demonstrated severe bilateral necrotizing chorioretinitis with almost no uninvolved retinal tissue. The infant died on day 24 of life. The initial serological investigations were performed on the in utero cord blood sample obtained 9 days prior to the infant's birth (32.5-weeks gestation). The total IgM value on that sample was 30 mg/d~, and the IgA level was 2 mg/de. By a fluorescent antibody to membrane antigen (FAMA) method, 5° a varicella-specific IgM titer of 1:16 was detected. This value confirmed the clinical diagnosis of congenital VZV infection in the infant since maternal IgM does not cross the placenta. By 2 weeks of age, the infant's total IgM level had fallen to 2 mg/de, and varicella-specific IgM was no longer detectable. The above documentation of the short-lived postnatal VZV-specific IgM response may explain previous

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.8'

¢

FIGURE 6. Postnatal imaging studies of the head. At left, a computed tomograph shows marked hydrocephalus (C) with only a thin mantle of cerebrum (B) persisting around the enlarged ventricles and underlying the skull (A). There are also extensive calcifications in the basal ganglia (D) and brain stem (not shown on this scan). At right, a magnetic resonance image confirmed the presence of hydrocephalus (C) with a fluid-fluid level of different intensity in the dependent occiput (A). Calcifications were again seen in the basal ganglia (D). Also visible is a cranial artifact secondary to a metallic i.v. catheter in a scalp vein (B).

unsuccessful attempts to detect virus-specific IgM in the serum of congenitally affected infants. 47 Moreover, the falling level of IgM in the infant suggests that intrauterine VZV infection may be profoundly immunosuppressive. Table 4 contains a listing of the defects associated with varicella embryopathy. The sequelae of intrauterine VZV infection involve mainly ectodermal derivatives. Upon further consideration, most of the malformations seem to be attributable to destruction of either peripheral nerves or brain tissue, particularly during critical periods of fetal development. These malformations probably are the sequelae of viral destruction within the cervical and lumbosacrai plexi, which innervate the developing limb buds during the first 12 weeks of gestation. Between 12 and 20 weeks of gestation, infections of the optic nerve and retina may impair development of the eye. Likewise, as illustrated by our case report, 48 extensive destruction of brain tissue can occur as late as 20-weeks gestation. VII. C H I C K E N P O X IN T H E I M M U N O C O M P R O M I S E D C H I L D One of the largest surveys of chickenpox in children with cancer was carried out at St, Jude's Children's Hospital in Memphis, Tenn. 5~ The study period began in 1962 and ended in 1973. Altogether 77 cases of chickenpox were documented: 61 in children with acute leukemia or non-Hodgkin's lymphoma, 14 with a solid tumor, and 2 with Hodgkin's disease. The 17 children (11 with acute leukemia and 6 with solid tumors) who were in remission and receiving no further chemotherapy had a typical course of chickenpox with new vesicle formation which lasted an average of 4 days (range, 3 to 7 days). Chickenpox was a more severe disease in the remaining 60 patients still on chemotherapy, e.g., visceral dissemination

36

CRC Critical Reviews in Oncology/Hematology Table 4 N E U R O L O G I C A L S E Q U E L A E OF F E T A L VZV I N F E C T I O N 46-4s Damage to sensory nerves Cutaneous manifestations Zig-zag (cicatricial) skin lesions Hypopigmentation Damage to optic stalk, optic cup, and lens vesicle Microphthalmia Cataracts Chorioretinitis Optic atrophy Damage to cervical and lumbosacral cord Hypoplasia of upper/lower extremities Motor/sensory deficits Absent deep tendon reflexes Anisocoria/Horner's syndrome Anal/vesical sphincter dysfunction Damage to brain/encephalitis Microcephaly Hydrocephaly Calcifications Aplasia of brain

was observed in one third (17/50) of the children with leukemia. One each of the children with solid tumors and Hodgkin's disease also had disseminated disease. The average duration of the rash increased from 4 to 9 days (range, 5 to 14 days) in patients on chemotherapy. This severe disseminated form of VZV infection is often referred to as progressive chickenpox. Of the 19 children with progressive VZV infection, 4 eventually had a fatal outcome. Since all of the deaths were in the leukemia group, the mortality rate of chickenpox in children with leukemia who are receiving chemotherapy is about 8% (4/50). All four deaths occurred in children with radiological signs of varicella pneumonitis (Figure 7). The pneumonitis appeared between 3 and 7 days after the exanthem; all four deaths occurred within 3 days after onset of the lung disease. In two of the four fatal cases, the children also developed varicella encephalitis, which may have contributed to their demise. One half of the cases of progressive chickenpox also had hepatitis, based on elevations in serum transaminase values. Three of the patients were presumed to have pancreatitis because of elevated serum amylase values. Autopsies were performed on two of the four children who died of their VZV infection. When the organs were examined by histopathology for viral-induced inclusions, evidence of infection was seen in the lungs, liver, spleen, gastrointestinal tract, lymph nodes, bone marrow, and brain. The investigators also evaluated the laboratory studies on the patients who died to see if they could establish any risk factors. Neither death nor severity of infection could be correlated with the total white cell counts or absolute neutrophil counts. Absolute lymphopenia (<500 lymphocytes per cubic millimeter) was present in 4/9 children who had progressive chickenpox but in only 5/21 with uncomplicated disease. Although absolute lymphopenia was not related to mortality, these data strongly suggest that immunocompromised patients with lymphocyte counts below 500/mm 3 on the first day of VZV infection are at high risk for dissemination. After the investigators analyzed the chemotherapy and irradiation regimens of the 60 patients, they determined that no individual protocol was associated with more severe chickenpox. There was no difference in mortality or severity of infection as related to the duration of anticancer therapy. However, the risk of dissemination seemed to be diminished by

Volume 8. Issue 1 (1988)

37

Im

\

L FIGURE 7.

Varicella pneumonitis in a young immunocompromised child with chickenpox.

stopping chemotherapy during the incubation period of chickenpox. In a group of 15 patients who had their anticancer medications stopped before onset of chickenpox, 3 disseminated (20%); of 38 patients who continued on their chemotherapy after exposure to chickenpox, 16 had progressive disease (42%) when they themselves contracted the disease. This study was conducted prior to the availability of passive immunization and effective antiviral chemotherapy (described later in this article). Therefore, the above results reflect the outcome of untreated primary VZV infection in childhood cancer patients. VIII. P A T H O G E N E S I S OF Z O S T E R

A. Epidemiological Studies Zoster or shingles is the name of the dermatomal exanthem which represents a reactivation of the VZV strain which originally caused chickenpox in the same patient. The viru~ remains latent, often for decades, in the sensory ganglia alongside the spinal cord. The lc :alization

38

CRC Critical Reviews in Oncology/Hematology Table 5 O C C U R R E N C E OF ZOSTER A C C O R D I N G TO AGE sz

Age (years)

No. cases

Rate per 1000 per annum

0--9 10---19 20--29 30---39 40----49 50---59 60--69 70----79 80~89

6 10 17 18 23 37 38 27 16

O.74 1.38 2.58 2.29 2.92 5.09 6.79 6.42 I0.10

of zoster has been correlated to the areas of the skin which are most heavily involved with pock lesions during chickenpox, the presumption being that the virus has entered a peripheral nerve terminal near a vesicle and traveled via retrograde axonal flow to the dorsal root ganglion. The dermatomal distribution of zoster in 192 cases was carefully charted by HopeSimpson over a 16-year period in Cirencester, U . K ? 3 He observed that most cases occur in sites innervated by the fifth cranial (trigeminal) nerve and the lower thoracic-upper lumbar sensory nerves. For unknown reasons, reactivation usually occurs in only one dermatome and, occasionally, in adjacent dermatomes. The appearance of numerous lesions outside the involved dermatome is very rare in the aged, although many individuals observe a few scattered pox elsewhere on the body. Regions supplied by cervical and lower lumbar-sacral dermatomes are relatively spared. Hope-Simpson also calculated the attack rates of zoster for each decade of life (Table 5). Zoster was unusual in children because many had not yet had chickenpox and therefore could not reactivate VZV. In young adults ages 20 to 49, there were about 2.5 cases per 1000 people. The rate of zoster increased to 5 cases per 1000 by age 50, then to nearly 7 cases per 1000 during the next 2 decades, finally reaching 10 cases per 100 people over the age of 80 years. Hope-Simpson also noted that one episode of zoster did not necessarily confer life-long immunity on the patient. Indeed, 8 of his 192 cases were second episodes, and one was a third occurrence. Four of the nine repeat episodes of shingles were in the same dermatome as the original disease. After reviewing his cumulative data on zoster, Hope-Simpson hypothesized that VZV reactivation is dependent upon a waning of viral immunity over time. He postulated that subclinical reactivation occurs periodically, but the emergent virus is rapidly neutralized by the host's immune system. Eventually, however, the humoral and cellular immune responses are inadequate to restrict VZV replication, and the virus continues to move down the sensory nerve to the skin where it causes the dermatomal exanthem. B. Molecular Studies Molecular techniques have been employed to further define the epidemiology of zoster. For example, VZV isolates collected from the vesicle lesions and the blood from patients with zoster were shown to have identical DNA restriction enzyme cleavage patterns? 3 Likewise, two VZV isolates obtained sequentially from an immunocompromised child who had chickenpox followed by zoster 3 months later were analyzed by restriction enzymes and found to be the same strain. 54 These results support the hypothesis that zoster represents a reactivation of a single VZV strain which caused the original episode of chickenpox. Human trigeminal ganglia have been examined by hybridization techniques to det~:rmine

Volume 8, Issue 1 (1988)

39

FIGURE 8. Zoster in an infant. This infant was exposed to chickenpox in utero because of a maternal infection. When he developed shingles 6 months after birth, the disease was localized in the lower cervical dermatomes which innervate the arm and hand.

whether they contain VZV genomic material, as presumed from the epidemiological studies. In one study, ganglia were removed from five cadavers within 12 hr of death. The investigators employed in situ hybridization to look for VZV RNA in sections of the neurons. 55 The percent of VZV-positive neurons ranged from 0 to 0.3%. Another group looked for VZV DNA in human trigeminal ganglia by blot hybridization techniques. Of ten different trigeminal ganglia, they detected VZV DNA in the total ganglia DNA of three samples. 56 The above two studies strongly suggest that the sensory ganglion is a site of VZV latency. Why VZV was not detected in all patients studied is unclear. It may be related to sampling error or perhaps VZV does not establish latency in all individuals following chickenpox. IX. Z O S T E R IN C H I L D R E N A. Localization of Zoster in Infants When zoster develops in the elderly, the exanthem is usually localized to the lower thoracic and upper lumbar dermatomes, as mentioned previously. In infants and young children, however, zoster frequently occurs over the upper and lower extremities (Figure 8), i.e., in dermatomes supplied by the cervical and lumbosacral plexi. 4° A possible explanation for this divergence in localization between infantile zoster and adult zoster relates to the development of the spinal cord at the time of the initial chickenpox infection. In the infant, the cervical and lumbosacral plexi constitute major portions of the spinal cord because they innervate the developing extremities27 Thus, infants who are infected with VZV either in utero or during the first year or two of life are more likely to establish latency within the cervical and lumbosacral cord. In adulthood, about 70% of the spinal cord is contained within the thoracic and upper lumbar regions. B. Zoster in Children with Cancer Between the years 1975 to 1981, we kept a record of all children with acute lymphocytic leukemia (ALL) who developed zoster28 In our facility, the average yearly census of children with ALL at all stages of treatment was about 100 patients. At the time of diagnosis of

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CRC Critical Reviews in Oncology/Hematology

FIGURE 9. Zoster in a child with leukemia. Shingles appeared over the left buttock in this immunocompromised child.

malignancy, the VZV-immune status of each patient was assessed by the FAMA test to VZV. Additional sera were collected on all patients who developed zoster. During the 7 years of the study, we enrolled a total of 56 patients with ALL into our prospective survey. All of these patients met the criteria for serological evidence of prior chickenpox, i.e., a positive VZV-FAMA assay. A total of 14 children with ALL manifested 17 episodes of zoster during the course of the study. In every instance, the child's FAMA titer post-zoster eventually rose to 1:512 or greater, a value which represented an eightfold or greater rise in VZV-specific antibody. VZV was cultured from the vesicular fluid and/or buffy coat of every child in which isolation was attempted. After the study was completed in 1981, the most recently enrolled patients with ALL were observed for an additional 2 years for the appearance of zoster. Two patients developed multiple episodes of zoster. In both instances, the child was VZVseronegative at the time of diagnosis of malignancy. Each child contracted chickenpox after the diagnosis of ALL. None of the zoster patients with serological evidence of chickenpox prior to malignancy developed a second episode of zoster. These results suggest that the patient with leukemia on chemotherapy is unable to mount as complete an immune response to primary chickenpox infection. The dermatomal distribution of the 17 episodes of zoster in these children with ALL was similar to zoster in the elderly. The majority of cases (nine cases) involved the sensory nerves to the lower thoracic and upper lumbar dermatomes, while four were located on the head and face, and two each were in dermatomes supplied by the cervical and lumbosacral plexi. Figure 9 is an illustration of a child with leukemia who developed zoster over the right buttock, which is innervated from the sacral ganglia. Since the localization was the same as that observed in the elderly, these results suggest that zoster in children with leukemia may simply represent a premature abrogation of viral latency. The episodes of clinical zoster occurred between 4 and 65 months after diagnosis of ALL. Almost 75% of the episodes were diagnosed within 2 years of diagnosis of ALL (Figure 10). There was an even distribution of cases with no obvious clustering over the first 2

Volume 8, Issue 1 (1988) 16

m

j

14 12

8

N

F

/

10

J

i----

r (..)

41

4

I-

2 8.

2

.

. 10. 12

Months

.

14. 16

After

18

. 2 '02' 2 24

Diagnosis

. 26. .

28 30

34

36

of Leukemia

FIGURE 10. Zoster in children with leukemia. The graph shows the cumulative number of cases of zoster which occurred in the first 3 years after diagnosis of leukemia. 14 12 10 O N O Or}

8

6 4

O

2

,?

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i

J

i

r

i

i

.

J

i

10 12 14 16 18 20 22 24

Months After Irradiation Therapy

FIGURE I 1. Zoster following radiotherapy of children with leukemia. The graph shows the cumulative number of cases of zoster which occurred after irradiation in the children with leukemia. years, after which the likelihood of zoster markedly declined. When we analyzed the different therapeutic regimens into which the patients had been randomly assigned after diagnosis of ALL, we did not observe any predilection for a particular orally or intravenously administered chemotherapy protocol. However, we did note an apparent temporal association with irradiation to the head and spine. The relationship of zoster to craniospinal radiotherapy is shown in Figure 11. In the 12 patients who developed zoster after irradiation, 7 episodes occurred within 2 to 6 months after radiotherapy. The association of zoster with the administration of intrathecal medication was also analyzed (Table 6). The patients were assigned to one of four treatment groups: (1) intrathecal methotrexate, (2) intrathecal methotrexate with irradiation, (3) intrathecal methotrexate and hydrocortisone, and (4) intrathecal methotrexate plus hydrocortisone with radiotherapy. The patients at greatest risk of developing zoster were in group 4: those who had received a combination of intrathecal methotrexate and hydrocortisone in addition to craniospinal irradiation. Over one half of the episodes of zoster were in this group. Because of the small number of patients in each of the four groups, however, statistical comparisons were not possible. No A L L patient with zoster died as a sequela of VZV infection; nor did any patient manifest CNS or visceral complaints during acute disease. In only one instance did the

42

CRC Critical Reviews in Ontology~Hematology

Table 6 Z O S T E R IN P A T I E N T S R E C E I V I N G I N T R A T H E C A L (IT) M E D I C A T I O N s8

Group I II II1 IV

Regimen IT methotrexate IT methotrexate irradiation IT methotrexate hydrocortisone IT methotrexate hydrocortisone radiotherapy

No. patients

No. zoster

%

alone with

5 22

0 4

0 18

plus

II

3

27

plus with

18

10

55

vesicular lesions spread beyond the initial or adjacent dermatomes to produce a generalized exanthem over the entire body; this same individual was the only patient who subsequently received i.v. antiviral chemotherapy. Two other patients who were not treated with antiviral medication developed extensive and unsightly keloid scar formation over the involved dermatome. Thus, our study confirms that zoster in ALL is rarely a life-threatening illness; however, the morbidity is appreciable and includes extensive keloid formation. The latter process probably is related to vesicular ulceration at the dermoepidermal junction and may be ameliorated by early antiviral chemotherapy to diminish the extent and duration of cutaneous viral replication. In summary, we have evaluated the incidence of zoster in a population of approximately 100 children under treatment for leukemia. We observed an average of 2 cases per year; this number represents an incidence of 20/1000/year. When Hope-Simpson surveyed the number of cases of zoster in a general population, he found 0.74 cases per 1000 children less than 10 years old. Therefore, VZV-seropositive children under treatment for leukemia are nearly 30 times more likely to develop zoster than healthy children. One remarkable observation of this 7-year appraisal was the finding that multiple episodes of zoster were confined to those individuals who contracted chickenpox while under treatment for their leukemia. This finding strongly suggests that ALL or, even more likely, its chemotherapy regimens interfere with the primary immune response to VZV infection and allow subsequent reactivation. The time interval between varicella and zoster is also much shorter in this subgroup, i.e., < 1 year. The apparent inability of patients with ALL to mount a complete immune response to chickenpox may mimic the previously noted increased likelihood of zoster in infants who have either in utero chickenpox or neonatal chickenpox. X. I M M U N E R E S P O N S E S T O V Z V I N F E C T I O N

A. Serological Methods An early method used to measure VZV antibody in human serum was the complement fixation (CF) test. 59 By the CF test, antibody was usually detectable about 1 week after onset of chickenpox, but the titer of CF antibody declined within 3 months of infection. Thus, CF antibody was an excellent indicator of recent illness but not of long-past VZV infection. The first reliable method of testing for serological evidence of immunity to VZV was the FAMA assay? ° The substrate for the FAMA assay is composed of live VZV-infected cells, which contain multiple VZV antigens in their outer membranes. The infected cells are incubated in serial dilutions of the patient's serum, after which fluorescein-conjugated antihuman globulin is added to the mixture. If the patient's serum contains anti-VZV antibodies, these will bind to the surface of the VZV-infected cells; the fluorescein conjugate will bind in turn to the human antibodies. When viewed by fluorescence microscopy, the

Volume 8, Issue 1 (1988)

43

cells have a halo appearance. By this latter serological method, virologists can diagnose both acute VZV infection as well as infection in the distant past. The sensitivity of the FAMA test has also been compared to the complement-enhanced neutralization test. ~ The two assays exhibit a high degree of concordance; however, the antibody titer by the complement-enhanced neutralization test is usually about twofold higher than the FAMA titer. The similarities in titer suggest that the two tests may be measuring an overlapping spectrum of VZV antibodies. VZV antibodies can also be detected with great sensitivity by both enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). 6~.62

B. Radioimmune Precipitation The assays described in the preceding paragraph measure the entire humoral immune response to VZV infection. However, this response actually consists of a spectrum of antibodies to many individual viral proteins. The viral glycoproteins, in particular, seem to elicit the most vigorous antibody responses. For this reason, the technique of radioimmune precipitation has been used to define the antibody responses to individual VZV glycoproteins. In this technique, VZV-infected cultures are incubated in the presence of a radioactive sugar in order to radiolabel the viral glycoproteins? -5 A detergent-solubilized extract is then mixed with patient's serum in order to precipitate viral glycoproteins, which are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The slab gels are suffused with scintillant, then dried and exposed to radiographic film. After I to 3 weeks, the film (fluorogram) is examined for the presence of bands representing the precipitated radiolabeled proteins. The nomenclature for the VZV-specific glycoproteins seen on the fluorograms has been described in detail in an earlier section of this review. Since the earlier nomenclature was based on the estimated molecular weights ( × 10 -3) of the glycoproteins, the new nomenclature incorporates the same estimated molecular weights in parentheses after the Roman numeral designation, e.g., gpI(98). When multiple serum samples are available from a single patient, it is possible to define the sequential appearance and disappearance of VZV glycoprotein-specific antibodies over long periods of time. These studies are described in the following paragraphs. C. Humoral Immunity after Chickenpox When serum samples are collected on a weekly basis after onset of chickenpox, a sequence of appearance of antibodies to the VZV glycoproteins can be defined. 63 As illustrated in Figure 12, antibody to gpII(66) is the first to appear, followed in rapid succession by antibody to gplII(118), gpI(98), and gplV(62). The identity of the last-mentioned glycoprotein, gp62, remains to be confirmed, but recent results from our laboratory suggest that gp62 is actually gpIV and not a cleavage product of gpI(98), as previously surmised. 99 We also followed the persistence of the antibodies and observed that anti-gpIII(118) and anti-gpII(66) were still found 4 years after chickenpox, while levels of antibody to gpI(98) and gplV(62) were often undetectable by radioimmune precipitation techniques within a few years. Serum samples from immunocompromised children with chickenpox were also analyzed. As compared to healthy children previously described, children with cancer appeared to develop less antibody to gpI(98) and gplV(62) after primary VZV infection, but their response to gpIII(118) and gplI(66) was not diminished. In addition to the VZV glycoproteins, the antibody response to the nonglycosylated VZV-specific proteins were also investigated in both healthy and immunocompromised children with chickenpox. Although several VZVspecific polypeptides were precipitated by acute chickenpox serum, there was considerable variability in the profiles. One polypeptide, the major VZV capsid protein Mr 155,000, was usually present in the precipitation profile. The patterns of antibody formation were also analyzed after subclinical infection. In one instance, a mother with a history of childhood chickenpox was exposed to chickenpox in

44

CRC Critical Reviews in Oncology/Hematology

IW

2W

3W

2W

3W

4W

--118 -- 98

~

L--li

J

,~

,,,,~, ....

-66 62

,It,

FIGURE 12. Humoral response to primary VZV infection as seen by reactivity with viral glycoproteins. Sera collected weekly (W) from two children after the onset of varicella were reacted with [3Hlfucose-labeled VZV antigens, and the precipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). As indicated by the intensity of the bands, antibody to gplI(66) is the preponderant antibody to glycosylated antigens in the initial serum sample from each patient.

her own child (Figure 13). The mother's pre- and post-exposure serum anti-VZV neutralizing antibody titer rose from 1:4 to 1:16. In her immunoprecipitation profiles, antibodies to gplI(66) and gplII(118) were clearly detectable in the pre-exposure serum. Within 4 weeks post-exposure, she developed an anamnestic response which included a heightened antibody titer to gpI(98) and gplV(62). The latter two antibodies slowly disappeared over the following 2 years.

D. Humoral Immunity after Zoster The glycoprotein-specific antibody response was defined in healthy and immunocompromised children with zoster. 64 In the few weeks prior to the onset of zoster, very little anti-VZV glycoprotein antibody was present in the serum. Usually only gplII(118) was easily detected in the immunoprecipitation profiles. Within a few weeks after onset of disease,

Volume 8, Issue 1 (1988)

0

45

2W 4W 273W

gp 118 -- ~ : ~ , . ~ : ~ gp 9 8

. . . . . . . . .

.~.

gp 6 6 , . . gp 6 2 " -

: ....

FIGURE 13. Antibody pattern of subclinical VZV reinfection. Serum samples were collected from a mother at the time of VZV reexposure (0) to chickenpox and at the indicated weeks (W) afterward. When the [~H]fucose-labeled glycoproteins precipitated by these sera were separated by SDS-PAGE, it was apparent that levels of antibodies to gpI(98) and gplV(62) exhibited greater rises and declines. Activity against gplI(66) and glll(118) was more persistent.

precipitating activity against all VZV glycoproteins increased dramatically. High levels of antibody to the VZV glycoproteins persisted for almost 2 years, after which time antibodies to at least two of the VZV glycoproteins, gpI(98) and gplV(62), declined noticeably. The zoster serum samples were also mixed with [35S]methionine-labeled VZV antigen preparations in order to detect antibodies against nonglycosylated VZV-specific proteins. More than ten VZV-polypeptides ranging in molecular weight from 32,000 to 174,000 were precipitated by sera collected in the first few months after clinical zoster (Figure 14). One protein which was easily identified corresponded in molecular weight (155,000) to the VZV major capsid protein• A lower-weight nonglycosylated protein (32,000; p32) was of particular interest because it was prominently seen in the post-zoster immunoprecipitation profiles but then disappeared within a few months time. Thus, the presence of anti-p32 antibody was very characteristic of recent zoster (Figure 15). The Mr 32,000 antigen may represent one of the capsid proteins. ~5

46

CRC Critical Reviews in Oncology/Hematology

2W

5W '~

•

9W ~ 30W 31W 34W

141W

rr u n ~

107

- " '9 8 88

~

L -

45

Cl 3d 2W 5W 9W~30W31W34W50W141W --

HMW

~174

..1... ~~"

O~-~

~"

' m m m t ' l l

~ ~

1

Iml¢ H l i P ~

l~""~, ~ l ~

_~

lt"'l

•

"-" ~

1

IIMll

-- 155 ~145 - 118

-62

"'~

- 34 - 32

b

FIGURE 14. Antibodyspecificities during sequential bouts of chickenpox and shingles. Multiple sera were obtained from an 8-year-oldchild with leukemia who first developed chickenpox followed after 30 weeks by zoster. The onset of zoster is indicatedby an arrow. (a) Profiles of the VZV glycoproteins; (b) profiles of [~sS]methionine-labeledpolypeptides. The ten most prominent VZV polypeptides, includingp155, are designated by closed circles in b.

E. S u m m a r y of V Z V Protein-Specific l m m u n o p r e c i p i t a t i o n Profiles Figure 16 summarizes the results of the immunoprecipitation studies on the sera of children with chickenpox and zoster. The results are presented in a diagrammatic format which distinguishes the profile characteristic of each clinical stage of VZV infection. In acute chickenpo× (stage V1), antibodies to two major VZV glycoproteins, gpII(66) and gpIII(l 18),

Volume 8, Issue 1 (1988)

.Iw

3w

47

7w

155 145 118 98 88 62

45 32

I

FIGURE 15. Immunoprecipitationof VZV p32. A patient with a history of chickenpox at 6 years of age and subsequent leukemia developed zoster at 8 years of age. Sara were collected at 1 day, 3 weeks, and 7 weeks post-zoster. The serum samples were incubated with [~sS]methionine-labeledVZV antigen. The immunoprecipitationprofilesdemonstrate the marked increase in antibody activity against the nonglycosylatedVZV polypeptide p32 in the weeks after zoster. as well as to the capsid protein p155 are detected. A wider spectrum of VZV-protein-specific antibodies are present about 4 to 8 weeks after chickenpox (stage V2). During the late convalescent period (stage V3), many of these antibody responses wane, although antigplII(118) and anti-gplI(66) are still detectable. In the acute zoster period (ZI), levels of antibody rise rapidly. A very broad complement of VZV-specific antibodies is detectable during the early zoster convalescent period (Z2). In particular, p32 is a prominent component of the immunoprecipitation profile. Then, in sera drawn long after zoster (stage Z3), titers of antibody to VZV-sgecific proteins again decline. On the basis of these polypeptide-

48

CRC Critical Reviews in Oncology/Hematology

Vl

V2

V3

Zl

MW

Z2

Z3

~HMW

~174 i

155

I

~145 m

m

126

I

118~ ' ~ 1 '

l

m

m

m

I

I

107 98

~88 ' ~

I I

76

66

m

62

i

, ~

45

• m

37

~

52

m m

I FIGURE 16. Diagrammatic summary representations of the immunoprecipitable VZV polypeptides: VI, acute-phase varicella (up to 4 weeks): V2, convalescent-phase varicella, (4 to 8 weeks); V3, post-varicella quiescence: ZI, acute-phase zoster (up to 2 weeks); Z2, convalescent-phase zoster (2 to 8 weeks); and Z3, post-zoster quiescence. The molecular weights (MW) of the individual polypeptides are designated to the fight of column Z2. (HMW, high molecular weight.)

specific differences in the precipitation profiles, it was possible to distinguish a varicella antibody pattern from a zoster antibody pattern. F. I m m u n o g l o b u l i n

M R e s p o n s e to C o n g e n i t a l V Z V I n f e c t i o n

The IgM response to the VZV glycoproteins has been characterized in a case of congenital varicella infection. 48 Most of the studies were performed on a fetal sample collected at 32.5weeks gestation by a percutaneous umbilical cord sampling (cordocentesis). IgM was sep arated from whole serum by sucrose density gradient ultracentrifugation. The purified IgM fraction was mixed with radiolabeled viral antigen preparations, and the immunoprecipitates were analyzed as previously described. One of the precipitation profiles, as presented in Figure 17, clearly shows in lane 2 a fetal IgM response to all three major VZV glycoproteins: gpI(98), gpII(66), and gpIII(ll8). The total quantity of anti-VZV serum IgM declined considerably between 32.5-weeks gestation and 34-weeks gestation, when the child was born. As seen in Figure 18, the IgM fraction from the postnatal serum sample precipitated lesser amounts of the viral glycoproteins (lane 5). The fetal IgM response is also compared with the IgG response of presumed maternal origin found in the cord blood and infant serum. The IgG profiles clearly show that antibodies are being produced by the mother to the major VZV glycoproteins; in addition, an IgG response is detected against a fourth VZV glycoprotein gpIV (lane 6).

Volume 8, Issue 1 (1988)

ANTENATAL

1

2

POSTNATAL

3

t !•

4

5

6

gplll :gp,

• gplll • gpl • gpl

•gpl gpll

°gpll •gplV

•gplV

•gplV

m

IgG Con

IgM

49

g

IgM IgG

IgG

FIGURE 17. Viral glycoproteins precipitated by IgM. The IgM and IgG fractions were isolated from serum samples collected pre- and postnatally after congenital varicella infection. Aliquots of the immunoglobulin fractions were incubated with radiolabeled VZV antigens, and the immunoprecipitates were subjected to SDS-PAGE. Lane 1, [3Hlfucose-labeled VZV antigen control; lane 2, [~4C]glucosaminelabeled VZV glycoproteins precipitated by fetal lgM; lane 3, 13Hlfucose-labeled VZV glycoproteins precipitated by IgG in cordocentesis sample; lane 4, [~H]fucose-labeled glycoproteins precipitated by IgG in infant's postnatal serum sample; lane 5, [~4C]glucosamine-labeled VZV glycoproteins precipitated by IgM in infant's serum sample; lane 6, [~4C]glucosamine-labeled VZV glycoproteins precipitated by lgG in infant's serum sample. The results in lanes 2 and 5 show that the fetal IgM response is directed against the VZV glycoproteins.

G. Cell-Mediated Immunity Generation of virus-specific cell-mediated immunity involves a complex sequence of events. Initiation of the immune response is dependent on adherent cells of the mononuclear phagocyte system which process and reexpress viral antigens in association with major histocompatibility complex (MHC) determinants. 66 Following effective antigen presentation and production of interleukin-I (IL-I) by mononuclear phagocytes, T 4 + helper-inducer cells proliferate and elaborate soluble mediators, such as interleukin-2 (IL-2), which amplify responses by other lymphocytes. In turn, cytotoxic T8 + and T4 + lymphocytes proliferate and acquire the ability to lyse virus-infected targets in an HLA-restricted manner. 67"68Clonal expansion of virus-specific T cells, boosted by repeated antigen exposure, is thought to engender lasting immunity to the virus. T lymphocytes seem to play a pivotal role in recovery from primary infection and in prevention of subsequent reactivation of the herpesviruses. This is most likely related to the varied functional capacities of T cells as helpers for antibody production by B cells, as

50

CRC Critical Reviews in Oncology/Henlatology I0 s --1

I 13.

10 4

0

II

<] ¢)

0..

=3 10 3 C "10

E

L

tI

"1- 1 0 ~ A

10 ~ |

FAMA 6) Age-Related Controls (n:10)

1

FAMA ® Leukemic Children (n:16)

1

FAMA® Leukemic Children (n:4)

FIGURE 18. Cell-mediated immunity to VZV antigen. The lymphocyte proliferative responses to VZV antigen were measured in both VZV-immune (FAMA (~)) healthy and leukemic children, as well as in VZV-susceptible (FAMA O ) children. The data for the seropositive children are presented as the mean --- 2 SD. The figure indicates that mean peak proliferation in seropositive children with leukemia was significantly lower than in seropositive age-related controls (p = 0.014).

promoters of cytotoxic T lymphocyte (CTL) and natural killer (NK) cell development, as effector cells capable of destroying virus-infected targets, and finally as purveyors of immunological memory. Prospective studies 69 have shown that diminished in vitro T lymphocyte proliferation to herpesvirus antigens (VZV, CMV, and HSV) correlates with increased risk of recurrent infection with these viruses in lymphoma patients during treatment. Reactivation occurred despite the maintenance of stable titers of antibody to the respective viruses. Results of other studies (Figure 18) suggested that cell-mediated immunity to VZV frequently diminishes during chemotherapy for acute lymphoblastic leukemia. 7° Children with decreased cell-mediated immunity to the virus were at increased risk of developing zoster; although all patients remained seropositive to VZV, antibody alone was inadequate to prevent viral

Volume 8, Issue 1 (1988)

51

reactivation. Further studies showed decreased numbers of VZV-specific T cells in these patients and provided a possible explanation for the diminished proliferative responses to VZV antigens. Lysis of herpesvirus-infected cells has been shown to occur experimentally via a number of different mechanisms which depend upon recognition of virus-specified surface antigens on infected cells. In the case of VZV, antibody-dependent cellular cytotoxicity (ADCC), NK cells, and CTLs have been shown to kill virus-specific cells in vitro. 71-73 NK cells as well as lymphocytes, monocytes, and neutrophils which mediate ADCC recognize their targets and cause cell lysis without restriction by HLA determinants. In contrast, CTLs operate most effectively when viral antigens are expressed on infected target cells in conjunction with class I or II HLA antigens which are compatible with the effector lymphocytes. NK lysis, a relatively nonspecific mechanism, is augmented by IL-2 production from antigenspecific T cells. There is also growing evidence that lymphocyte responses are directed against proteins encoded by the viral genome. For example, in the case of VZV, immunoaffinity-purified preparations of all three major VZV glycoproteins (gpl, gplI, and gptII) induced T-cell proliferative responses from seropositive individuals in a fashion similar to that which occurred following stimulation with crude VZV antigen. 74.75However, resolution of primary VZV infection was not always accompanied by host cell responses to all three glycoproteins in each individual tested. XI. V A R I C E L L A V A C C I N A T I O N

A. Pathogenesis The schema for pathogenesis of disease caused by live attenuated varicella vaccine virus is based on the pathogenesis model for natural chickenpox (Figure 19). However, the incubation period following varicella vaccine has a wider range than the usual 2-week period for chickenpox. The length of the incubation period appears to be determined, at least in part, by the dose of vaccine virus. The number of infectious viral particles is expressed by the term plaque-forming unit (PFU; Figure 20). For example, in an early trial of an attenuated VZV strain, Hilleman inoculated volunteers with varying doses of virus. TM Vaccinees who received a large inoculum (7000 PFU) developed a mild rash within 7 to 10 days, whereas recipients of either 70 or 7 PFU did not develop an exanthem until 12 to 22 days had passed. The shorter incubation period was also seen in human studies conducted at the turn of the century by physicians who inoculated children with vesicular fluid removed from other children with active chickenpox (reviewed in Reference 35). We now know that vesicular fluid is an excellent source of high-titer virus. One explanation for the inverse relationship between titer of inoculum virus and length of incubation period is suggested in the schema. A high input of inoculum virus increases the likelihood of immediate dissemination via the bloodstream to sites of internal replication, and subsequent major viremia and exanthem within 7 to 10 days. However, a small dose of vaccine virus is more likely to be phagocytosed at the site of entry, and local replication would be required before a primary viremia occurred. Thereafter, further cycles of replication would be necessary before an exanthem appeared. Examples of these delayed varicella vaccine rashes are described in the following sections. B. The Live Attenuated Variceila Vaccine The live attenuated varicella vaccine currently undergoing clinical investigations was developed by Takahashi and colleagues at the Research Insitiute for Microbial Diseases in Osaka, Japan. 77 The vaccine virus was designated Oka strain after the name of the 3-yearold boy with chickenpox from whom it was isolated. The virus has an interesting history of attenuation in cell culture. The virus was first isolated by inoculation of vesicle fluid into human embryonic lung (HEL) cells. After the eleventh passage in HEL cells, the infected

52

CRC Critical Reviews in Ontology~Hematology

FIGURE 19. Immunization with VZV. After inoculation of live virus, the sequence of events appears to be determined by the dose. After a small inoculum, the interval between injection and clinical disease is usually 14 or more days. (A) Virus therefore must replicate locally before a primary viremia occurs. (B) If a large number of infectious particles are injected, the incubation period is as short as 7 to 8 days, a time course most compatible with initial viral multiplication in the internal organs. (C) Since recipients of VZV vaccine were able to transmit the infection, virus also traveled to and replicated in the pharynx.

cells were trypsin-dispersed and inoculated onto guinea pig embryonic (GPE) cells, which were prepared from the skin and muscle tissues of 3- to 4-week-old fetal guinea pigs. After the twelth passage in GPE cells, the virus was further passaged in the human diploid cell line WI-38 another two to six times. Because VZV is maintained in culture by passage of live infected cells, the vaccine strain was cultured in the WI-38 cell line in order to remove any guinea pig determinants acquired during subculture in GPE cells. The Oka strain of varicella vaccine has since been licensed for manufacture in Europe and in the U.S. Although the vaccine is now being distributed in Europe, it has not received final approval by the U.S. Food and Drug Administration as of November 1987.

C. Japanese Clinical Studies The first human studies of the Oka strain of varicella vaccine were described in the original report from Takahashi et al. 77 After the virus had been subcultured 1 1 times in HEL cells

V o l u m e 8, Issue 1 (1988)

cell -i control I0

-2 I0

-4 I0

I0 3

53

I0

FIGURE 20. Plaque assay of infectious virus. Serial tenfold dilutions of VZV are seeded onto monolayers, and the assays are incubated at 32°C. After 5 days, the monolayers are stained and the plaque-forming units are enumerated.

EARLY

CLINICAL

Vaccine HEL HEL HEL HEL HEL HEL HEL

11/GPE 11/GPE 11/GPE 11/GPE 11/GPE 11/GPE I I/GPE

Note:

6 6 6 6 12/WI 2 12/Wl 2 12/WI 2

Table 7 TRIALS OF VARICELLA

V A C C I N E 77

Vaccine dose (PFU)

No. tested

No. positive

Response (%)

1000 500 200 100 2000 1000 200

10 20 12 9 10 I1 9

10 19 11 7 10 11 9

100% 95% 92% 78% 100% 100% 100%

HEL, human embryonic lung cells; GPE, guinea pig embryonic cells; WI, human diploid cells; and PFU, plaque-forming units of virus.

and 6 times in G P E cells, he inoculated 51 healthy children with different numbers o f P F U . O f those w h o r e c e i v e d at least 200 P F U o f v a c c i n e virus, m o r e than 90% r e s p o n d e d with an antibody titer w h e n tested 1 m o n t h after i m m u n i z a t i o n (Table 7). He also passaged the virus six m o r e times in G P E cells and twice in h u m a n W I - 3 8 cells and retested its potency. All 30 healthy children w h o r e c e i v e d doses o f 200 P F U , 1000 P F U , or 2000 P F U d e v e l o p e d a V Z V antibody response.

54

CRC Critical Reviews in Oncology/Hematology Table 8

S E R O L O G I C A L RESPONSE T O V A R I C E L L A V A C C I N E s° Vaccine dose (PFU)

No. tested

No. positive

Response (%)

500 1500 5000 15000

27 105 29 20

24 104 29 20

89 99 100 100

The Japanese group next investigated the efficacy of the vaccine within the household. TM For this study, they immunized 23 siblings of index cases with acute chickenpox and 3 children exposed to a case of zoster. The dose of infectious virus in the vaccine preparation was 500 PFU or greater. A total of 5 children were immunized on the same day, 11 on the next day, 3 on the second day, and 4 on the third day after diagnosis in the index child. The three children exposed to zoster were immunized 5 days later. Of the 26 immunized contacts, 18 were found to be susceptible on the basis of a negative VZV serological test; all 18 seroconverted post-immunization. Since none of the latter 18 vaccinees developed chickenpox, immunization of susceptibles with live attenuated VZV vaccine up to 3 days after exposure to chickenpox appeared to completely prevent subsequent chickenpox. The Japanese investigators also tested the effectiveness of vaccination in the hospital setting. TM For these studies, they immunized VZV-susceptible contacts who were exposed to cases of chickenpox or zoster which occurred on the same ward. The children had various underlying diseases, including nephrotic syndrome, nephritis, and hepatitis. Altogether, researchers vaccinated 18 children between the ages of 2 and 12 years. The vaccine preparations contained about 1500 PFU per dose. All 18 vaccinees developed VZV antibody. None of the 18 contracted chickenpox after exposure up to 9 months post-immunization. In 1977, their 2-year cumulative results of varicella vaccination of 181 children were published. 8° The vaccinees included 56 healthy children and 125 with illnesses such as nephrotic syndrome and nephritis (68 cases) and malignancy (6 cases). At doses of vaccine virus of 1500 PFU, 99% of the recipients responded with an antibody titer (Table 8). When blood samples were obtained from 51 children 2 years after vaccination, all but 1 (50/51) had a persistent VZV antibody response when measured by a neutralization test. The Japanese investigators have continued their long-term follow-up evaluations. In 1983, Asano et al. 8~ described the results 5 years after immunization of 26 healthy children. The 26 had originally received either 250 PFU or 1000 PFU of vaccine virus. All vaccinees had seroconverted I month after immunization, and all retained measurable levels of VZV antibody 5 years later. The geometric mean titer of FAMA was 1:30 at 1 month post-immunization and 1:12 after 5 years. In 1985, Asano et al. 82 reported the results of a 7- to 10-year follow-up evaluation of another 106 children who had received varicella vaccine (500 to 5000 PFU). The group included 66 healthy children, while the remainder had chronic illnesses such as nephritis and nephrotic syndrome (but not malignancy). The group of vaccinees had been exposed to other children with chickenpox on 147 occasions: 26 children had family exposures, 66 children had school exposures, and 55 children had neighborhood exposures (Table 9). After these exposures, one vaccinee in the family-exposed group and 4 vaccinees in the schoolexposed group developed chickenpox. As shown in Table 9, the attack rates were 4% and 6% for the two groups of vaccinees, respectively. The vaccinee who developed chickenpox after a family exposure was 1 year old when she was immunized with a dose of 4000 PFU. She contracted chickenpox 3 years post-immunization. The 4 vaccinees who developed

Volume 8. Issue I (1988)

55

Table 9 A T T A C K R A T E OF V A C C I N E E S EXPOSED T O N A T U R A L C H I C K E N P O X 82

Place Family School Neighborhood

No. of children

No. who developed chickenpox

Attack rate (%)

26 66 55

I 4 0

4 6 0

chickenpox after school exposures ranged in age from 2 to 6 years. All received 1500 PFU of vaccine virus. The interval between vaccination and chickenpox varied from 4 to 10 months. All had mild disease with less than 20 pox lesions. The apparent vaccine failure in the 1-year-old suggests that very young children may respond less well to live virus immunization than older children. The reason for the failure of protection in the four other children was less clear, although the investigators suggested the possibility of loss of PFU in some vaccine lots due to incorrect handling. Takahashi recently presented an overview of the vaccination studies in Japan. 83 His review of the vaccination of children with malignancies is important because it includes several clinical trials in Japan. By the end of 1983, 326 children with acute leukemia had been immunized in Japan. In one group, anticancer chemotherapy was suspended for 2 weeks for administration of vaccine. In this group of 251 patients, 46 (18%) had mild to moderate sequelae following immunization, usually including fever and rash. In the group in whom chemotherapy was not suspended, vaccination led to clinical reactions in nearly one half of the patients (37/79 = 47%). Although the sequelae were usually not severe, one child with myelocytic leukemia did develop an extensive exanthem with high fever (40°C) after immunization. An antibody response was detected in 91% of the children with suspended chemotherapy and 96% in the other group. The protective effect of vaccination was also estimated on the immunized children. Over an unspecified time interval, chickenpox occurred in 52 of 300 vaccinees. Thus the attack rate for chickenpox in vaccinated children with childhood leukemia may reach 17%. As stated earlier, the symptoms of chickenpox were generally mild. However, two children did develop severe disease. Takahashi also cites another vaccination study in Japanese children with leukemia. In the latter investigation, 3 of 52 vaccinees contracted natural chickenpox. This attack rate of 6% is the same as that described earlier in healthy immunized children. Takahashi also briefly reviews the experience with immunization of children with lymphoma. Although the dose of the vaccine is not given, he mentions that clinical reactions to immunization occurred in 8 of 20 patients. In four patients, the symptoms were described as " s e v e r e " . A seroconversion was documented in 18 to 20 vaccine recipients. Children with solid tumors also were vaccinated. The malignancies included neuroblastoma, hepatoma, rhabdomyosarcoma, and Wilms' tumor. Mild clinical reactions to immunization were noted in 11% of 54 children, while no severe sequelae were observed. Seroconversion was documented in 91% (49/54) of the children. D. The Collaborative NIH Trial One of the largest evaluations of the live attenuated varicella vaccine was carried out in the U.S. by a NIH-sponsored Collaborative Varicella Vaccine Study Group headed by Gershon and associates. 84 These investigators immunized a total of 307 childhood leukemia patients. The criteria for admission into the vaccine study included leukemia in remission for at least 9 months. The average period of remission was 20 months, with a range of 9

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FIGURE 21. Exanthemin a child immunized with live attenuated VZV vaccine. The rash first appeared over the abdomen 3 weeks after inoculation and did not progress beyond that shown in the photograph. (Courtesy of Dr. Anne A. Gershon.) to 52 months. All children lacked detectable antibody to VZV as measured by the FAMA test. The children received the Oka strain at a dosage which varied from 1000 to 2500 PFU. All children who were receiving chemotherapy for their leukemia had the medications stopped 1 week before administration of the vaccine; chemotherapy was begun again 1 week after vaccination. The side effects of vaccination of these children with leukemia included fever, pain at the injection site, and a rash. After the first immunization, a rash developed in 3/ 54 (6%) of the vaccinees who had already completed their chemotherapy and in 100/241 (42%) of the children who were still receiving chemotherapy and had only a temporary stoppage for 2 weeks. A photograph of a child with an exanthem following varicella vaccine is shown in Figure 21. When the study began in 1979, the children received one dose of vaccine. However, 11% of this group never developed VZV antibody. Of those who did develop antibody by 2 months post-immunization, another 30% lost their VZV antibody titer within 1 year. Because of these poor seroconversion rates, vaccinees since 1981 have been given two doses of the VZV vaccine about 3 months apart. Less than 10% of the vaccinees manifested a rash after their second injection, regardless of whether they were still on chemotherapy. The seroconversion rates in the recipients of two consecutive VZV vaccines have improved. When the first 200 dual vaccinees were evaluated, 189/200 (95%) had detectable

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levels of VZV antibody after the second immunization. When late convalescent sera were obtained from the last group of vaccinees, 166/200 (75%) contained VZV antibody at 1 year post-immunization. After 2 years, 105/147 (71%) still had VZV antibody, while 66% (40/ 61) remained antibody-positive 3 years after immunization. Conversely stated, one third of the seropositive VZV vaccinees became seronegative again 3 years after immunization. The next question is whether the vaccine recipients can contract chickenpox after exposure to wild-type disease. The answer appears to be in the affirmative. Gershon et al. 84 documented 23 cases of clinical chickenpox which occurred in vaccine recipients within 3 years after immunization. Of this group, 11 vaccinees had received 1 dose and another 11 had been given 2 doses, while I case had received 3 vaccinations. The number of vesicles ranged from 1 to 640 with a mean of I 11 pox. Wild-type virus was isolated from five of these cases. These cases of clinical chickenpox demonstrate that live VZV vaccine will not fully protect children with leukemia from contracting chickenpox in the community. However, the disease appears to be much milder in children who have been previously immunized. The disease also would be milder in children with leukemia who had experienced no relapses and had completed their chemotherapeutic regimens. Based on the efficacy data derived from the NIH collaborative trial, the vaccine was estimated to be 80% effective in children with leukemia. 85.86 Gershon et al. 84 have also immunized healthy adults who were VZV-seronegative. The first group of 73 adults had a seroconversion rate of only 58% (57/73) after 1 dose of vaccine, while the rate rose to 92% in 36 individuals (33/36) who received 2 sequential doses of vaccine. One vaccinee developed a mild rash in the immediate post-immunization period. The data for persistence of antibody in adults are as follows: 88% (37/42) for 1 year, 80% (28/35) for 2 years, 88% (15/17) for 3 years, and 88% (7/8) for 4 years. Chickenpox has occurred in six adults who received the vaccine and were known to have seroconverted. At least four of the six adults lost their VZV antibody prior to contracting chickenpox. The onset of the rash ranged from 4 to 72 months after immunization (mean, 20 months). Moreover, the exanthem was mild, with an average of only 20 pock lesions. When two of the virus isolates were analyzed by DNA restriction enzyme cleavage, they were confirmed as wild-type strains. In 1987, members of the NIH Collaborative Group published an extensive analysis of the molecular epidemiology of the live VZV vaccine in children with leukemia and in normal adults. 87 These studies complement an earlier case report of zoster in a child who had received variceila vaccine. 88 By restriction endonuclease digestion of DNA extracted from viral isolates, they confirmed that the exanthem which appeared within the first 6 weeks after vaccination was caused by the vaccine strain. They also showed that one case of zoster which occurred in a vaccine recipient was related to reactivation of vaccine virus. Interestingly, zoster occurred in the area of the original vaccine inoculation. However, virus recovered from another vaccinee with zoster did not resemble the Oka strain and was probably wild-type VZV. Thus, this last case confirms the prior protection data which showed that some vaccinees apparently contract chickenpox when exposed to the disease in the community. E. Other Clinical Trials in the U.S. In addition to the collaborative NIH-sponsored vaccine trials, other clinical evaluations of VZV have also been carried out in the U.S. In one early trial in San Antonio, Tex., 22 children with ALL and I child with non-Hodgkin's lymphoma were given the Oka strain vaccine as prepared by Smith Kline-RIT pharmaceutical company. 89 Group I included 12 children who had completed chemotherapy; group II contained 11 children who were in remission but still receiving maintenance chemotherapy with daily 6-mercaptopurine, weekly methotrexate, and in some cases monthly prednisone and vincristine. In the latter group, chemotherapy was suspended for 1 week before and 1 week after vaccination.

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Each child was administered one dose (500 PFU) of varicella vaccine. All 23 children had detectable VZV antibody 1 month post-immunization. The sequelae of immunization in two patients included biphasic rashes. The initial rash occurred about 1 week postimmunization and consisted of maculopapular erythematous lesions which started on the face and neck and spread to the upper trunk. The rash began as discrete lesions, but became confluent within 1 week. After the first exanthem cleared, a second rash appeared about 3 weeks after immunization. The second exanthem consisted of tiny papules or papulovesicles on an erythematous base 5 to 10 mm in diameter. The appearance of the second rash was also accompanied by a fever and lymphadenopathy. The vesicular lesions never became as prominent as those seen with wild-type chickenpox. Nevertheless, this biphasic rash further supports the dual viremic model for the pathogenesis of chickenpox. When both groups I and II were reevaluated at 6 months, one child had lost his antibody. When 16 of the 21 seropositive vaccinees were tested at ! year after immunization, all had detectable VZV antibody. Taken together, these data indicate that at least 4% (1/23) lost their humoral immunity within 6 months. The protective effect of immunization was also evaluated. For 8 vaccinees, there was a total of 13 exposures to chickenpox or zoster. One of the eight contracted chickenpox which was mild (seven pock lesions). The spread of vaccine virus from a recent vaccinee to a susceptible sibling was also evaluated. Of 21 VZVsusceptible siblings, 1 healthy child developed a fourfold rise in serum VZV antibody during the 6-week period after immunization of the family member with leukemia. Although the healthy child manifestated only seroconversion without disease, this case points out the potential for spread of VZV vaccine strain. Other clinical studies of the Oka strain of varicella vaccine have been carried out in Philadelphia. 9° The vaccinees included healthy children between the ages of 18 months and 16 years. In their original clinical trial, the investigators determined that a dose of 500 PFU was necessary to achieve 100% seroconversion within 6 weeks post-immunization. When the vaccinees were retested 1 year and 3 to 4 years later, persistence of VZV antibody was documented in 100% and 94% of the groups, respectively. Protective efficacy was considered high because only 4 of 112 vaccinees contracted mild chickenpox after 16 household, 74 day care, and 60 playmate exposures. Interestingly, the vaccine can also be administered to susceptible children for post-exposure prophylaxis. In 10 children who received vaccine within 3 days of household exposure to chickenpox, only I developed mild disease (20 pock lesions). In a control group, 92% (12 of 13) of susceptible contacts developed typical chickenpox (60 to 600 skin lesions) after similar household exposures. The ability of varicella vaccine to prevent chickenpox, even when administered post-exposure, probably relates to the dual viremic model of pathogenesis. When vaccine virus in very high quantity (7000 PFU) is administered by injection, the incubation period is shortened by 3 to 4 days since the site of primary replication has been bypassed. Therefore, the body can respond to the vaccine virus within the ensuing week and, in the process, abort the second viremic phase of the wild-type varicella virus which is coinfecting the vaccinee. Without a major viremia from the wild-type virus, there is no subsequent clinical disease. XII. T R E A T M E N T M O D A L I T I E S A. Immune Globulin Other than vaccination, there are several modes by which the physician can either prevent chickenpox in the exposed individual or, alternatively, treat the infection in the patient with active disease. Immune globulin has been used in the past to ameliorate the symptoms and signs of chickenpox. 39 Since the amount of VZV antibody in immune globulin is not constant from lot to lot, the efficacy of the gammaglobulin may vary. As an alternative to immune globulin, the physician can prescribe high-titer varicella-zoster immune globulin (VZIG) to

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Table 10 CANDIDATES TO RECEIVE VARICELLAZOSTER IMMUNE GLOBULIN Immunocompromised susceptible children; immunocompromised adolescents (15 years or older) and adults are likely to be immune, but if susceptible they also should receive VZIG Normal susceptible adolescents and adults (serological determination of immune status advised) Newborn infant of a mother who had onset of chickenpox within 5 days before delivery or within 48 hr after delivery Premature infant ( > 28-weeks gestation) whose mother lacks a prior history of chickenpox Premature infant ( < 28-weeks gestation or less than 1000 g) regardless of maternal history From 1986 Report of the Committee on Infectious Diseases, American Academy of Pediatrics.

prevent or attenuate chickenpox in a susceptible individual.91 VZIG must be administered by i.m. injection within 3 to 4 days after exposure, i.e., based on the schema for pathogenesis (Figure 2) the hyperimmune globulin must be circulating before the virus has an opportunity to spread via the primary viremia. The general criteria for selection of VZIG candidates are described in Table 10. The dose of VZIG is one vial (about 1.25 me) for each 20 pounds of body weight; the cost per vial is about $100. In the older and larger individual, VZIG is difficult to use because multiple vials of hyperimmune gammaglobulin must be injected. The newer preparations of gammaglobulin for i.v. administration may be an acceptable substitute for prevention of chickenpox in the VZV-susceptible adult; however, this approach has not been widely tested. B. Vidarabine If the susceptible patient is beyond the fourth day post-exposure, administration of hyperimmune globulin appears to have no beneficial effect. Therefore, the physician must treat chickenpox when it first appears. In the past, adenine arabinoside (ara-A; vidarabine) was the drug of choice for treatment of chickenpox and zoster in immunocompromised patients. 92 Vidarabine was administered intravenously over a 12-hr period at a dosage of 10 mg/kg/ day. Patients treated with vidarabine had more rapid healing, as documented by cessation of new vesicle formation and more rapid time to total pustulation. The only side effects were nausea and vomiting in about 16% of the treated patients. C. Acyclovir Recent clinical trials indicate that a newer antiviral agent called acyclovir (acycloguanosine; Zovirax) may be preferable to vidarabine for i.v. treatment of VZV infections. Acyclovir is an acyclic nucleoside analog of deoxyguanosine which is phosphorylated by the VZVspecific enzyme thymidine kinase. 93The final product, acycloguanosine triphosphate, blocks new viral DNA synthesis, thereby inhibiting further viral replication in the infected host. Although VZV is approximately two- to eightfold less susceptible to acyclovir than HSV type 1, VZV growth is readily inhibited by the serum levels achieved after i.v. administration of acyclovir. Acyclovir has been studied in a randomized double-blind, placebo-controlled study involving 20 immunosuppressed children with chickenpox. 94 All patients were receiving anticancer therapy prior to signs of chickenpox. Eight children placed in the antiviral treatment

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group received acyciovir at a dose of 500 mg/m 2 every 8 hr (total daily dose, 1500 mg/m2); duration of therapy was 7 days. The placebo group of 12 children initially received no antivirai chemotherapy. Within a few days after enrollment into the study, five patients in the placebo group developed varicella pneumonitis, while none of the recipients of acyclovir developed this omnious manifestation of progressive chickenpox (p = 0.054 by the Fisher exact test). Because all five placebo patients who showed signs of pneumonitis were also begun on acyclovir, a meaningful comparison of total healing time could not be made. Nevertheless, the authors concluded that i.v. acyclovir was highly efficacious in the treatment of VZV infection in the child with cancer. In cases of progressive chickenpox which fail to respond to acyclovir, an alternative diagnosis should be considered. For example, we treated an immunocompromised child with cutaneous chickenpox and bilateral pneumonitis with i.v. acyclovir. 95 Although the vesicular lesions abated, the pulmonary disease worsened and the child died. At autopsy, we discovered that CMV was the causative agent of the pneumonitis. Thus, the child presumably had a reactivation of CMV concomitant with acute chickenpox. Since CMV is not sensitive to acyclovir, the pneumonitis did not improve. A randomized comparison trial of acyclovir and vidarabine has also found acyclovir to be better for the treatment of chickenpox and shingles in patients with cancer. 96 In this trial, 11 patients with active VZV infection were entered into each group. Acyclovir was given at a dose of 500 mg/m 2 every 8 hr by a l-hr infusion, and vidarabine was administered at a dose of 10 mg/kg/day as a single 12-hr infusion. The total duration of therapy was either 7 days or at least 2 days beyond the last day of new vesicle formation. Acyclovir was more effective by several criteria: (1) prevention of cutaneous dissemination, (2) duration of positive viral cultures, and (3) time of cutaneous healing. The only serious side effect of acyclovir therapy was renal insufficiency in three patients. Therefore, the recommendation of the investigators was that acyclovir be considered the current treatment of choice for treatment of VZV infection in immunocompromised patients. This choice of acyclovir is further supported by clinical results of another study which evaluated the management of chickenpox in children with cancer. 97 Although one fourth of the chickenpox patients treated with vidarabine still developed pneumonitis, none of the recipients of i.v. acyclovir had VZV dissemination to the lungs (p = 0.03). Because of the possibility of renal toxicity, all patients receiving i.v. acyclovir should have a baseline serum creatinine level drawn prior to onset of therapy and then repeated on a 3- to 4-day schedule. Acyclovir is also available in a capsule formulation (200 mg per capsule). The main indication for this product is the oral treatment of HSV types 1 and 2 disease. VZV infections can also be treated with orally administered acyclovir, but the dosage regimens are not yet established. Because of the relatively poor adsorption of acyclovir and the lessened sensitivity of VZV to acyclovir (as compared with HSV), doses between 2 and 4 g/day have been given to children with leukemia who contract chicken pox or zoster. 9s With oral doses of 250 to 600 mg acyclovir per square meter, mean peak acyclovir levels in serum ranged from 3.5 to 15.1 ix/mole. Based on studies in VZV-infected cell cultures, acyclovir levels of 3.7 Ixmol or higher are considered to be therapeutically effective. 93 Further studies in more patients are required, however, before firm recommendations can be made about this mode of chemotherapy. ACKNOWLEDGMENT Research performed in the authors' laboratories was supported by USPHS grant AI 22795 from the National Institute of Allergy and Infectious Diseases, and grant MV-359 from the American Cancer Society.

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REFERENCES 1. Ecker, J. R. and Hyman, R. W., Varicella-zoster virus DNA exists as two isomers, Proc. Natl. Acad. Sci. U.S.A., 79, 156, 1982. 2. Davison, A. J. and Scott, J. E., The complete DNA sequence of varicella-zoster virus, J. Gen. Virol.. 67, 1759, 1986. 3. Grose, C., The synthesis of glycoproteins in human melanoma cells infected with varicella-zoster virus, Virology. 101, 1, 1980. 4. Grose, C., Edmond, B. J., and Friedrichs, W. E., Immunogenic glycoproteins of laboratory and vaccine strains of varicella-zoster virus, Infect. lmmun., 31. 1044, 1981. 5. Grose, C. and Friedrichs, W. E., Immunoprecipitable polypeptides specified by varicella-zoster virus, Virology, 118, 86, 1982. 6. Weigel, K. A. and Grose, C., Common expression of varicella-zoster virus glycoproteins antigens in vitro and in chickenpox and zoster vesicles, J. Infect. Dis.. 148, 630, 1983. 7. Grose, C., Edwards, D. P., Friedrichs, W. E., Weigel, K. A., and McGuire, W. L., Monoclonal antibodies against three major glycoproteins of varicella-zoster virus, Infect. lmmun., 40, 381, 1983. 8. Montalvo, E. A., Parmley, R. T., and Grose, C., Structural analysis of the varicella-zoster virus gp98gp62 complex: posttranslational addition of N-linked and O-linked oligosaccharide moieties. J. Virol.. 53, 761. 1985. 9. Asano, Y. and Takahashi, M., Studies on the polypeptides of varicella-zoster (V-Z) virus. I. Detection of varicella-zoster virus polypeptides in infected cells, Biken J.. 22, 81, 1979. 10. Asano, Y. and Takahashi, M., Studies on the polypeptides of varicella-zoster (V-Z) virus. II. Synthesis of viral polypeptides in infected cells, Biken J., 23.95, 1980. 1 I. Okuno, T., Yaminishi, K., Shiraki, K., and Takahashi, M., Synthesis and processing of glycoproteins of varicella-zoster virus as studied with monoclonal antibodies of VZV antigens. Virology. 129, 357, 1983. 12. Namazue, J., Campo-Vera, H., Kitamura, K., Okuno, T., and Yamanishi, N., Processing of virusspecific glycoproteins of varicella-zoster virus, Virology. 143,252, 1985. 13. Keller, P. M., Neff, B. J., and Ellis, R. W., Three major glycoprotein genes of varicella-zoster virus whose products have neutralization epitopes, J. Virol., 52, 293, 1984. 14. Ellis, R. W., Keller, P. M., Lowe, R. S., and Zivin, R. A., Use of a bacterial expression vector to map the varicella-zoster virus major glycoprotein gene. gC, J. Virol., 53, 81, 1985. 15. Forghani, B., Schmidt, N. J., Myoraku, C. K., and Gallo, D., Serological reactivity of some monoclonal antibodies to varicella-zoster virus. Arch. ViroL. 73, 311, 1982. 16. Forghani, B., Dupuis, V. W., and Schmidt, N. J., Varicella-zoster glycoproteins analysed with monoclonal antibodies, J. Virol., 52, 55, 1984. 17. Edson, C. M., Hosler, B. A., Poodry, C. A., Schooley, R. T., Waters, D. J., and Thorley-Lawson, D. A., Varicella-zoster virus envelope glycoproteins: biochemical characterization and identification in clinical material, Virology, 145, 62, 1985. 18. Weigle, K. A. and Grose, C., Molecular dissection of the humoral response to individual varicella-zoster viral proteins during chickenpox, quiescence, reinfection and reactivation, J. Infect. Dis.. 149,741, 1984. 19. Grose, C., Edwards, D. P., Friedriehs, W. E., Weigle, K. A., and McGuire, W. L., Varicella-zoster virus specific gpl40: a highly immunogenic and disulfide-linked structural glycoprotein, Virology, 132. 138, 1984. 20. Ito, M., lhara, T., Grose, C., and Start, S., Human leukocytes kill varicella-zoster virus infection fibroblasts in the presence of murine monoclonal antibodies to virus-specific glycoproteins, J. Virol., 54, 98, 1985. 21. Vafai, A., Wrohlewska, Z., WeUish, M., Green, M., and GUden, D., Analysis of three late varicellazoster virus proteins, a 125,000 molecular-weight protein and gpl and gp3, J. Virol., 52, 953, 1984. 22. Wrohlewska, Z., Gilden, D., Green, M., Devlin, M., and Vafai, A., Affinity-purified varicella-zoster virus glycoprotein gpl/gp3 stimulated the production of neutralizing antibody, J. Gen. Virol., 66, 1795, 1985. 23. Edson, C. M., Hosler, B. A., Repress, R. A., Waters, D. J., and Thorley-Lawson, D. A., Crossreactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein, J. Virol., 56, 333, 1985. 24. Friedrichs, W. E. and Grose, C., Glycoprotein gp118 of varicella-zoster virus: purification by serial affinity chromatography, J. Virol.. 49, 992, 1984. 25. Montalvo, E. A. and Grose, C., Neutralization epitope of varicella zoster virus on native viral glycoprotein gp118 (VZV glycoprotein gplll), Virology, 149, 900, 1986. 26. Davison, A. J., Waters, D. J., and Edson, C. M., Identification of the products of a varicella-zoster virus glycoprotein gene, J. Gen. Virol.. 66, 2237, 1986.

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27. Davison, A. J., Edson, C. M., Ellis, R. W., Forghani, G., Gilden, D., Grose, C., Keller, P. M., Vafai, A., Wroblewska, Z., and Yamanishi, K., A new common nomenclature for the glycoprotein genes of varicella-zoster virus and their glycosyiated products, J. Virol.. 57, 1195, 1986. 28. Mootalvo, E. A. and Grose, C., Varicella-zoster virus glycoprotein gpl is selectively phosphorylated by a virus-induced protein kinase, Proc. Natl. Acad. Sci. U.S.A., 83, 8967, 1986. 29. Mootalvo, E. A. and Grose, C., Assembly and processing of the disulfide-linked varicella-zoster virus glycoprotein gp11(140), submiued. 30. Weller, T. H., Serial propagation in vitro of agents producing inclusion bodies derived from varicella and herpes zoster, Proc. Soc. Exp. Biol. Med.. 83, 340, 1953. 31. Edmond, B. J., Grose, C., and Brooell, P. A., Varicella-zoster virus infections of diploid and chemically transformed guinea pig embryo cells: factors influencing virus replications, J. Gen. Virol.. 54, 4031, 1981. 32. Myers, M. G., Stanberry, L. R., and Edmond, B. J., Varicella-zoster virus infection of strain 2 guinea pigs. J. Infect. Dis.. 151, 106, 1985. 33. Grose, C., Variation on a theme by Fenner: the pathogenesis of chickenpox, Pediatrics, 68, 735, 1981. 34. Feldman, S. and Epp, E., Isolation of varicella-zoster virus from blood, J. Pediatr., 88,265, 1976. 35. Meyers, M. G., Viremia caused by varicella-zoster virus: association with malignant progressive varicella, J. Infect. Dis.. 140, 229, 1979.

36. Ozaki, T., l ~ a ,

T.~ Matsui, Y., Nagai, T., Asaoo, Y., Yaminishi, K., and Takahashi, M.,

Viremic phase in nonimmtmocompromised children with vaficella. J. Pediatr.. 104, 85, 1984. 37. Grose, C., Isolation of varicella-zoster virus from blood, J. Pediatr., 105,504, 1984. 38. Asano, Y., ltakura, N., Hiroishi, Y., Hirose, S., Ozaki, T., Kuoo, K., Nagai, T., Yazaki, T., Yamaoishi, K., and Takahashi, M., Viral replication and immunologic responses in children naturally infected with varicella-zoster virus and in varicella vaccine recipients, J. Infect. Dis.. 152, 863, 1985. 39. Ross, A. H., Modification of chickenpox in family contacts by administration of gamma globulin, N. Engl. J. Med.. 267,369, 1962. 40. Grose, C., Varicu~a-zoster virt~s infections: chickenpox (varicella) and shingles (zoster), in Human Herpesvirus Infections. Glaser. R. and Gotlieb-Stematsky, T., Eds., Marcel Dekker, New York, 1982. 41. Linoemann, C. C., Shea, L., Partio, J. C., Shubert, W. K., and Schiff, G. M., Reye's syndrome: epidemiological and viral studies, 1963--1974, Am. J. Epidemiol.. 101, 517, 1975. 42. Underwood, E. A., T~e neurological complications of varicella: a clinical and epidemiological study, Br. J. Child. Dis., 32, 83, 177,241, 1935. 43. Johnson, R. and Milbourn, P. E., Central nervous system manifestations of chickenpox, Can. Med. Assoc. J., 102, 831, 1970. 44. Bale, J. F., Andersm, R. Do, and Grose, C., Magnetic resonance imaging of the brain in childhood herpesvirus infections, Pediatr. Infect. Dis. J.. 6, 644, 1987. 45. Preblud, S. R., Age-specific risk of varicella complications, Pediatrics, 68, 14, 1981. 46. Laforet, E. G. and Lynch, C. L., Multiple congenital defects following maternal varicella, N. Engl. J. Med.. 236, 534, 1947. 47. Hanshaw, J. B., Dudgeon, J. A., and Marshall, W. C., Varicella-zoster infections, in Viral Diseases of the Fetus and Newborn. W. B. Saunders, Philadelphia. 1985, 161. 48. Cuthbertson, G., Weiner., C. P., Gfller, R. H., and Grose, C., Prenatal diagnosis of second trimester congenital varicella syndrome by virus-specific lgM, J. Pediatr.. in press. 49. Weiner, C. P., Cordocentesis for diagnostic indications: two years' experience, Obstet. Gynecol., in press. 50. Williams, V., Gershon, A. A., and Brunell, P. A., Serologic response to varicella-zoster membrane antigens measured by indirect immunofluorescence, J. Infect. Dis., 130, 669, 1974. 51. Feldman, S., Hughes, W. T., and Daniel, C. B., Varicella in children with cancer: seventy-seven cases, Pediatrics, 56, 388, 1975. 52. Hope-Simpson, R. E., The nature of herpes-zoster: a long-term study and a new hypothesis, Proc. R. Soc. Med., 58, 9, 1965. 53. Pichini, B., Ecker, J. R., Grose, C., and Hyman, R. W., DNA mapping of paired varicella-zoster virus isolates from patients with shingles, Lancet, 2, 1223, 1983. 54. Straus, S., Reinhold, W., Smith, H., Ruyechan, W., Henderson, D., Blaese, R., and Hay, J., Endonuclease analysis of viral DNA from varicella and subsequent zoster infections in the same patient, N. Engl. J. Med., 311, 1362, 1984. 55. Hyman, R. W., Ecker, J. R., and Tenser, R. B., Varicella-zoster virus RNA in human trigeminal ganglia, Lancet, 2, 814, 1983. 56. Gildeo, D., Vafai, A., Shtram, Y., Becket, Y., Devlio, M., and Wellish, M., Varicella-zoster virus DNA in human sensory ganglia, Nature (London). 306,478, 1983. 57. Barson, A. J., Differentiation, growth and disorders of development of the vertebrospinal axis, in Scientific Foundations of Paediatrics. Davis, J. A. and Dobbing, J., Eds., W. B. Saunders, Philadelphia, 1974, 577. 58. Grose, C., A prospective study of zoster in children with leukemia, Report to the Thrasher Research Fund, Salt Lake City, Utah, 1982.

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59. Gold, W. and Godek, G., Complement-fixation studies with a varicella-zoster antigen, J. Immunol., 95. 692, 1965. 60. Grose, C., Edmond, B. J., and Brunell, P. A., Complement-enhanced neutralizing antibody response to varicella-zoster virus, J. Infect. Dis.. 139,432, 1979. 61. Forghani, B., Schmidt, N. J., and Dennis, J., Antibody assays for varicella-zoster virus: comparison of enzyme immunoassay with neutralization, immune adherence hemagglutination, and complement fixation, J. Clin. Microbiol., 8, 545, 1978. 62. Friedman, M. G., Leventon-Kriss, S., and Sarov, I., Sensitive solid-phase radioimmunoassay for detection of human immunoglobulin G antibodies to varicella-zoster virus. J. Clin. Microbiol., 9, I, 1979. 63. Weigle, K. A. and Grose, C., Molecular dissection of the humoral immune response to individual varicellazoster virus proteins during chickenpox, quiescence, re-exposure and reactivation, J. Infect. Dis., 149, 741, 1984. 64. 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