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Subclinical Varicella-Zoster Virus Viremia, Herpes Zoster, and T Lymphocyte Immunity to Varicella-Zoster Viral Antigens after Bone Marrow Transplantation Alexandra Wilson, Margaret Sharp, Celine M. Koropchak, Shirley F. Ting, and Ann M. Arvin

Department of Pediatrics, Division of Infectious Diseases, Stanford University School of Medicine. Stanford. California

Bone marrow transplant (BMT) recipients were evaluated for subclinical varicella-zoster virus (VZV) viremia and symptoms of herpes zoster after transplantation. Viremia was demonstrated by testing peripheral blood mononuclear cells using polymerase chain reaction and was documented in 19% of 37 patients. When reactivation was defined as herpes zoster and/or subclinical VZV viremia, 41% of VZV-seropositive BMT recipients experienced VZV reactivation. None of 12 patients tested before VZV reactivation had T lymphocyte proliferation to VZV antigen (mean stimulation index, 1.0 ± 0.42 [SD] at 100 days [P = .003]). Among patients tested at > 100 days, 5 (63%) of8 with detectable T cell proliferation had subclinical or clinical VZV reactivation compared with none of 6 who lacked VZV T cell responses. Recovery of VZV-specific cytotoxic T lymphocyte function was observed in 50% of BMT patients, but BMT recipients had significantly fewer circulating cytotoxic T lymphocytes that recognized VZV immediate early protein (P = .03) or glycoprotein I (P = .004) than did healthy VZV immune subjects. In vivo reexposure to VZV antigens due to subclinical VZV viremia or symptomatic VZV reactivation may explain the recovery of virus-specific T cell immunity after BMT.

The reactivation of latent varicella-zoster virus (VZV), presenting as localized herpes zoster or as disseminated infection, is a common and potentially serious complication in bone marrow transplant (BMT) recipients. Clinical studies reveal that 23%-40% of patients can be expected to develop VZV infection after transplantation [1-5]. The role of cellmediated immunity in protection against herpesviruses, including VZV, was first suggested by clinical studies showing that herpesvirus reactivation was common in patients receiving cancer therapy or drugs used to prevent transplant rejection. Periods of diminished in vitro T lymphocyte proliferation to VZV antigens have been correlated with an increase in susceptibility to herpes zoster among immunocompromised patient populations, including BMT recipients [6-8]. Clinically, the risk of herpes zoster declines significantly by 1 year after BMT, and most long-term survivors of BMT recover VZV-specific cellular immunity [2, 7]. VZV reactivation, resulting in in vivo reexposure to viral antigens, could playa role in inducing this immune reconstitution. Meyers et al. [7] detected T lymphocyte recognition of VZV antigens by proliferation assay in 16 (89%) of 18 BMT patients who had experienced symptomatic recurrences of VZV, but 15 (52%) of 29 patients who did not have herpes zoster also recovered cellular immunity to VZV. Ljungman et al. [3] described the restoration of cell-mediated immunity to VZV Received I July 1991; revised 16 August 199 l , Grant support: National Institutes of Health (CA-49605; AI-20459). Reprints or correspondence: Dr. Ann M. Arvin. Department of Pediatrics, Stanford University School of Medicine. Stanford. CA 94305.

The Journal of Infectious Diseases 1992;165:119-26 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6501-0016$01.00

in 22% of BMT recipients who had no preceding clinical signs of herpes zoster. Therefore, ifVZV reactivation is necessary to stimulate the recovery of virus-specific immunity, many patients must experience subclinical episodes of viral replication. Locksley et al. [2] described two BMT patients who had occult, disseminated VZV infection at autopsy and one patient with no cutaneous lesions whose premortem buffy coat cultures were positive for VZV. Nevertheless, technical problems with documenting asymptomatic VZV reactivation have interfered with the investigation ofsubclinical viral replication as a potential mechanism for the restoration of immunity to this herpesvirus. The polymerase chain reaction (PCR) has become an important tool for demonstrating the presence of viral gene sequences that were not detected by conventional methods in clinical specimens. Recently, we found that PCR could be used to demonstrate VZV DNA in peripheral blood mononuclear cells (PBMC) obtained from healthy subjects during the viremic phase of varicella [9]. The first objective of the present study was to determine whether episodes ofsubclinical YZV reactivation could be demonstrated after BMT by testing PBMC from transplant recipients using the VZV peR method. VZV-specific memory T lymphocytes can be demonstrated by assays for cytotoxic function as well as in vitro proliferation, with responses persisting for years after primary infection in healthy individuals [10-13]. The restoration of cytotoxic activity against virus-infected cells after BMT is of particular interest because of the evidence that such responses are critical in protecting these patients from life-threatening cytomegalovirus infections [14, 15]. Therefore, our second major objective was to examine the recovery

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of VZV cytotoxicity in BMT patients by the method that we have used to measure VZV cytotoxic T lymphocyte (CTL) responses in healthy subjects [16]. In our system, autologous lymphoblastoid cells infected with vaccinia recombinants that express the immediate early VZV protein, IE62, or glycoprotein I provide the necessary major histocompatibility complex-matched targets. The need to evaluate the reconstitution of cytotoxicity independently of VZV-specific T cell proliferation is suggested by the lack of correlation between these assays ofT cell immunity in leukemic patients; in this population, VZV cytotoxicity appeared to be more sensitive to immunosuppression [17].

Methods Study populations. The study population consisted of 51 patients who underwent BMT at Stanford University Medical Center between April 1989 and July 1990 and who had serologic evidence of past VZV infection as determined by EIA for IgG antibodies to VZV at the time of transplant. The participants ranged in age from 2 to 54 years (mean, 33). Their underlying diseases were lymphoma (24), leukemia (19), myelodysplastic syndrome (4), aplastic anemia (3), and familial erythrophagocytic lymphohistiocytosis (1). Six of the patients with leukemia had acute nonlymphocytic leukemia (ANLL), 8 had acute lymphocytic leukemia, 3 had chronic myelocytic leukemia, and 2 had acute myelocytic leukemia. All of the patients with lymphoma and 4 of6 patients with ANLL underwent autologous transplantation. The other 23 patients received allogeneic transplants; 20 allogeneic BMT recipients had human leukocyte antigen-matched, related donors, 2 had partially matched, related donors, and 1 had a matched, unrelated donor. The donors were also VZV-seropositive. The conditioning regimen for allograft recipients was total body irradiation (TBI) or busulfan and cyclophosphamide; autologous transplant patients received etopiside, cyclophosphamide, and either TBI or N,N-bis(2chloroethyl)-N-nitrosourea. TBI was given to 11 of 28 autologous and 12 of 23 allogeneic transplant patients. Cyclosporine and prednisone were given to all allograft recipients as prophylaxis against graft-versus-host disease (GVHD) after transplant. In addition, 7 patients received methotrexate and 2 were given antithymocyte globulin. Healthy adults who had serologic evidence of past VZV infection, as determined by EIA for IgG an tibodies to VZV, were used as control subjects for studies of subclinical VZV reactivation and CTL function in BMT recipients. Diagonis of clinical VZV infection. Herpes zoster was diagnosed clinically as a vesicular eruption beginning in a dermatomal distribution. Most cases of VZV infection were confirmed by the Diagnostic Virology Laboratory, Stanford University Medical Center, using an immunofluorescence method to stain vesicular material. Cutaneous dissemination was defined as the appearance of multiple vesicles, distributed randomly outside of the original dermatome. Visceral dissemination was considered probable if the cutaneous lesions were associated with pulmonary infiltrates or abnormal liver function tests that could not be attributed to other clinical causes.

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VZV polymerase chain reaction. PCR was done as described previously, using oligonucleotide sequences complementary to regions of VZV gene 31, which codes for the viral glycoprotein II (gp II) [9]. Two 20-base primer sequences and a third 20-base probe sequence were synthesized (Operon Technologies, Alameda, CA) after excluding homologies with other viral, human, and unannotated DNA sequences currently available in GenBank. Amplification with the upstream and downstream primers yielded a PCR product of249 bp. Synthetic oligonucleotide primers specific for human actin were used as controls for the VZV PCR. PBMC were recovered from whole blood by ficoll-hypaque gradient separation, prepared in aliquots of 2 X 105 and 4 X 105 cells, and frozen at -20°C. After being thawed, PBMC were treated with proteinase K, incubated at 60°C for 1 h, and boiled for 10 min. PCR-sterile conditions were used throughout all steps of sample preparation. The PCR was carried out under standard reaction conditions with denaturation at 95°C for I min, reannealing at 60°C for 30 s, extension at 72°C for 1 min, for 35 cycles, and a final 7-min extension interval. All reactions were carried out in a programmable cyclic reactor (Ericomp, San Diego). PCR controls included human cellular DNA extracted from PBMC of healthy adults, purified DNA from a plasmid with VZV HindlIl fragment D, which includes the gpII open reading frame (ORF), and the reaction mixture alone. The amplified product was detected by dot blot hybridization using the probe oligonucleotide labeled with 32p [9]. T lymphocyte proliferation assay. T lymphocyte proliferation was determined by incubating PBMC with serial dilutions ofwhole VZV antigen made from sonicated, VZV-infected melanoma cells or with an uninfected cell control [18]. The cells were cultured at a concentration of 3 X 105 cells/0.15 mljwell in RPMI with 30% human sera and pulsed with [3H]thymidine after 5 days. The stimulation index (SI) was calculated as the ratio of mean counts per minute in duplicate antigen-stimulated wells to the mean counts in duplicate control wells, with an SI >3.0 considered positive. Each assay included positive control wells stimulated with phytohemagglutinin. T cell cytotoxicity assay. Vaccinia recombinants expressing the VZV IE62 protein and gp I were provided by J. Hay, W. Ruyechan, and P. Kinchington (Uniformed Services University of the Health Sciences, Bethesda, MD) [16]. The IE62 vaccinia recombinant has an insert of nucleotides 105,200-109,133 from VZV ORF62; the gpl recombinant has nucleotides 115,808-117,679 from ORF68. The control vaccinia virus used in the CTL assays was vSC8, which contains p 11, b-galactosidase, p7.5, and vaccinia thymidine kinase sequences but has no foreign viral DNA insert. Autologous Iymphoblastoid cell (LCL) targets for the CTL assay were made from B lymphocytes transformed with Epstein-Barr virus. LCL (2 X 106 cells) were infected with the IE62, gpI, or control recombinants (5 pfu/cell) and incubated for 14 h at 37°C. Before use as targets, LCL were labeled with 300 JLCi of 51Cr. Virus-specific and control LCL targets were added to microtiter plates at 3 X 103 cells/well in 0.1 ml of media. Lymphocytes were recovered from PBMC by ficoll-hypaque centrifugation and separation with Lyrnphokwik-T (One Larnda, Los Angeles). Limiting dilution cultures were prepared with lymphocytes resuspended in RPMI contain-

VZV Reactivation and Immunity after BMT

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A

B

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F

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2 Figure 1. Detection of polymerase chain reaction product after amplification of DNA from peripheral blood mononuclear cells. Samples IA, IC, ID: three bone marrow transplant (BMT) patients who had subclinical cell-associated viremia; samples IB, 2CF: five BMT patients without detectable VZV viremia; samples 1F, 2B: varicella-zoster virus DNA controls; sample 2A: healthy immune donor control.

ing I%whole VZV antigen, 15%fetal calf serum (FCS) and 15% interleukin-2 (IL-2), which were added at concentrations of 105 , 5 X 104 , 104,5 X 103 , 103 , orO cells/well to microtiterplates, along with autologous feeder cells (1 X 104 cells/well), in 24 replicate wells/cell concentration. Limiting dilution cultures were incubated for 12 days with addition of media containing whole VZV antigen and IL-2 every 3-4 days. The phenotypes of the effector lymphocytes derived under limiting dilution conditions were assessed by cytofluorometry using CD4, CD8, and CD 16 monoclonal antibodies. Effector cells from the limiting dilution cultures were prepared by adding 0.1 ml offresh RPM I with 50%FCS to each well, agitating to resuspend the cells, and transferring cells from each well in aliquots of 80 ~l to the microtiter plates containing LCL targets. The plates were centrifuged and incubated at 37°C for 4 h; 100 ~l of supernatant was removed from each well and counted in a gamma counter. The replicate wells prepared at each of the effector cell concentrations were scored as positive if the counts per minute were >3.0 SD above the mean for the control wells in the same assay. The data from each assay were evaluated using a computerized analysis of standard limiting dilution plots [19]. Statistical analysis. The software package Epi-info version 5 (Centers for Disease Control, Atlanta) was used to examine clinical correlations with the occurrence of herpes zoster; this program incorporates analysis with Fisher's exact test for population size less than five. 4th Dimension (ACI/ACIUS, Cupertino, CA) and Statview II (Abacus Concepts, Calabasas, CA) programs were used for analysis of immunologic data.

Results Occurrence of symptomatic and subclinical VZV reactivation. Of 51 BMT recipients, 16 (31 %) developed herpes zoster after transplant, during a mean follow-up period of 314 days (range, 54-604). The median interval from transplantation to symptomatic recurrent VZV infection was 142 days (range, 17-395). Ninety-eight percent of the episodes of herpes zoster occurred during the first year after transplantation. No statistically significant associations were found when

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variables including pretransplant diagnosis and disease status, preparative regimen, GVHD prophylactic regimen, or GVHD were examined by univariate analysis for their predictive value in relation to VZV reactivation. Concurrent clinical problems were noted in 7 patients who developed herpes zoster, including GVHD (3), persistent lymphoma ( I), bacteremia (I), esophagitis and duodenitis of unknown etiology ( I ), and drug-induced pneumonitis ( I). All patients with herpes zoster were treated with acyclovir, given intravenously or as oral antiviral therapy. Clinical complications included cutaneous dissemination (I), cutaneous and visceral dissemination (2) and relapsing infection with appearance of new lesions 2 weeks after the initial episode (2). Two patients had severe post-herpetic neuralgia, persisting for 2 to 4 months. Of the 51 BMT recipients who were evaluated for clinical episodes ofVZV reactivation, 37 were also tested for subclinical, cell-associated VZV viremia using the VZV PCR method; 5 of these patients were tested twice after BMT. The patients were tested for cell-associated viremia at time points ranging from 17 to 183 days after transplant (mean interval, 94). PBMC from 7 (19%) of the 37 patients were positive by VZV PCR (figure 1). The overall frequency of positive PBMC samples, including the samples from the 5 patients who were tested twice, was 7 (17%) of 42 (table I). Subclinical VZV reactivation was detected in 3 (27%) of 11 allogeneic BMT recipients who were tested < I 00 days after transplant, at a mean of 78 days, compared with I (II %) of 9 patients tested at a mean of 125 days (table 1). Fewer autologous transplant recipients had viremia at < 100 days (14%), but the difference from allogeneic patients was not statistically significant. Overall, 19% of the allogeneic BMT recipients had VZV detected in PBMC by PCR compared with 14%ofthe autologous BMT recipients (not significant). None of the 7 patients with cell-associated viremia had GVHD at the time of subclinical infection. None of the healthy, VZV-immune adults who were tested ~20 years after primary VZV infection had subclinical VZV viremia. Based on the occurrence of symptomatic herpes zoster, subclinical VZV viremia, or both, a total of 21 (41 %) of 51 VZV-seropositive BMT recipients had VZV reactivation.

Table 1. Relationship between episodes of subclinical varicellazoster viremia, interval after transplantation, and whether the patient received an autologous or allogeneic transplant. Type of transplant Interval after transplant

Autologous

Allogeneic

Total

17-99 days 100-183 days Total

2115 (13) 1/7 (14) 3/22

3111 (27) 1/9 (II) 4/20

5/26 (19) 2/16(12)

NOTE.

Data are no. of episodes/no. of patients (%).

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Two of the 7 patients who had subclinical VZV viremia developed clinical signs of herpes zoster subsequently, at intervals of 60 days and 130 days later. Three patients who had subclinical VZV viremia at 17, 63, and 85 days after BMT had cleared the cell-associated viremia when they were retested by VZV PCR at intervals of 119,35, and 50 days later. Two patients were not reevaluated by VZV PCR but did not develop herpes zoster. Thus, five of seven patients (71 %)had subclinical viremia without progression to clinical infection. VZV specific T lymphocyte proliferation in relation to the occurrence of symptomatic and subclinical VZV reactivation. Thirty of the 51 VZV -seropositive BMT patients, including 17 allogeneic and 13 autologous transplant recipients, were evaluated for the recovery ofT cell recognition of VZV antigens using the proliferation assay. T lymphocyte proliferation to whole VZV antigen was significantly decreased in patients who were tested during the first 100 days after transplant, at a mean of 66 days (range, 21-92). The mean SI was 1.0 ± 0.42 (SO) in this group compared with a mean of 12.0 ± 6.03 (P = .003) among patients evaluated at > 100 days (mean, 128). T lymphocyte proliferation responses were not significantly different between allogeneic and autologous transplant recipients when data at all time points after BMT were analyzed by t test for two means. None of 12 patients who were evaluated before the occurrence of herpes zoster or subclinical VZV viremia had detectable VZV-specific T cell proliferation. Three patients who were tested within 15 days after the onset of an episode of herpes zoster also had no detectable T cell responses to whole VZV antigen. However, all 5 patients who were tested between 50 days and 4 months after subclinical or clinical VZV reactivation recovered VZV -specific T cell proliferation. Among all patients tested at > 100 days, 5 (63%) of 8 who had recovered T cell proliferation had experienced subclinical VZV viremia or clinical VZV infection, whereas of 6 patients who lacked detectable T cell recognition of VZV antigen had documented reactivation. CTL recognition of VZV /£62 protein and gpI after transplantation. Eighteen of the 51 VZV -seropositive patients were evaluated for CTL recognition of VZV proteins, including 7 tested < 100 days and 11 tested > 100 days (mean, 245) after BMT; 2 patients were tested twice. Overall, 50% of the BMT patients showed recovery of VZV-specific CTL function at a mean of 155 ± 98 days after transplant. There were no significant differences in cytotoxic responses between patients who received TBI and those who did not or between allogeneic and autologous transplant recipients (table 2). Four (57%) of the 7 BMT patients who were tested within 100 days after transplant had CTL that recognized VZV IE62 protein or gpl, while T cell proliferation was detected in only 5 (19%) of26 patients evaluated at < 100 days. The recovery of VZV -specific CTL function was demonstrated as early as 48 days in one patient. Among BMT recipients, the mean precursor frequency of

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Table 2.

Relationship between cytotoxic responses, whether the conditioning regimen included total body irradiation, and whether the patient received an autologous or allogeneic transplant.

Total body irradiation! No total body irradiation Autologous transplant Allogeneic transplant

Cytotoxic T lymphocyte recognition of varicella-zoster viral proteins*

Natural killer cell-like cytotoxicity!

No cytotoxicity

5/13 (38)

5/13 (38)

3/13 (23)

4/5 (80) 5/11 (45) 4/7 (57)

0/5 4/11 (36) 1/7 (14)

1/5 (20) 2/11 (18) 2/7 (29)

NOTE. Data are no. positive/total (%). * Positive response was defined as lysis of target cells expressing the immediate early (IE62) protein and glycoprotein I (gpl) of varicella-zoster virus. t Natural killer cell-like activity was defined as nonspecific lysis of target cells infected with vaccinia control recombinant as well as those expressing IE62 protein or gpl. ! Six of seven allogeneic transplant recipients and 7 of II autologous transplant recipients were given total body irradiation as part of the conditioning regimen for transplantation.

T lymphocytes that recognized IE62 protein was 1:233,000 ± 162,000, with a range of 1:7000 to >1:400,000 (figure 2). Ten (66%) of 15 BMT recipients had detectable CTL response to the IE62 protein. The mean frequency of CTL precursors specific for gpl was 1:277,000 ± 142,000 (range, 1:32,000 to > 1:400,000). Limiting dilution cultures from 9 (60%) of 15 BMT patients showed lysis of gpl targets. The mean precursor frequency of CTL specific for each of these two VZV proteins was equivalent (P = .44, t test). In contrast to findings in the BMT population, the mean precursor frequency of CTL specific for IE62 protein in a population of 16 healthy VZV-immune donors was 1:102,000 ± 85,000 (range, 1:13,000-1 :231 ,000) (figure 2). In 11 healthy subjects, the mean frequency ofgpl-specific CTL was 1:121,000 ± 86,000 (range, 1:15,000-1 :228,000). Statistical analysis demonstrated that BMT recipients had significantly fewer circulating CTL that recognized either the IE62 protein (P = .03, t test) or gpl (P = .004, t test) than did healthy VZV-immune subjects. Cultures of PBMC from five BMT patients, obtained a mean of 108 ± 52 days after transplantation, exhibited natural killer (NK) cell-like cytotoxicity, as evidenced by the capacity of effector cells to lyse the vaccinia control target cells as well as those expressing IE62 or gpl (table 2). In contrast, none of the cultures derived by stimulating lymphocytes from healthy immune subjects with whole VZV antigen contained effector cells that lysed the vaccinia control targets. In addition to being used to assess cytotoxic function, the limiting dilution cultures from BMT and healthy immune subjects were analyzed for differences in the phenotypes of lymphocyte subpopulations that predominated after in vitro

liD 1992;165 (January)

VZV Reactivation and Immunity after BMT

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stimulation with whole VZV antigen for 12 days (figure 3). The mean ratio ofCD4+ to CD8+ cells was 0.24 ± 0.24 in cultures from BMT recipients compared with 0.72 ± 0.58 in cultures from healthy immune subjects (P = .002, t test). This difference was due to the significant reduction in the percentage of CD4+ cells in cultures from BMT patients, which had a mean of 12% CD4+ cells, in comparison to healthy subjects, with a mean of 33% CD4+ cells (P < .00 I, t test). The mean number of cells that expressed the CDI6

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surface marker for NK cells was equivalent in cultures from BMT and healthy subjects, with means of I I% and 10%, respectively, when the five BMT patients whose cultures exhibited NK-like lytic activity were excluded. Cultures from this subgroup of BMT recipients contained a mean of 51% CDI6+ lymphocytes. None of the patients who were tested for VZV CTL function were evaluated for subclinical VZV viremia. However, 2 of the 16 BMT patients who had herpes zoster were evaluated for cytotoxic function before the episode of VZV reactivation. One patient had a detectable CTL response when tested 19 days before the onset of herpes zoster, with CTL frequencies of I :272,000 to IE62 and I :332,000 to gpl, but he had developed clinical complications, including chronic hepatitis, in the interim. The second patient had CTL frequencies of 1: 13,000 to IE62 and I :32,000 to gpl 175 days before VZV reactivation; however, he had drug-induced pneumonitis requiring treatment with high-dose prednisone before the onset of herpes zoster. Two BMT patients were evaluated after the occurrence of herpes zoster. One patient had VZV-specific CTL responses of I :55,000 to IE62 and 1:273,000 to gpl 175 days later. The culture from the second patient, who was tested 165 days after the onset of herpes zoster, exhibited NK-like cytotoxicity, with marked lysis of target cells infected with the vaccinia control as well as those expressing VZV IE62 or gpl.

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Discussion The detection of cell-associated VZV viremia in BMT recipients, using the PCR method, provides the first virologic evidence that these severely immunocompromised patients can experience VZV reactivation in the absence of clinically apparent cutaneous infection and without developing signs ofvisceral dissemination. This cell-associated viremia is most likely to represent the reactivation of endogenous virus rather than reinfection with VZV, because all of the episodes

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were documented within a short time after transplantation while the patients were still hospitalized on the compromised host unit or while they were following precautions to minimize exposure to infectious diseases at home. Of interest, VZV viremia could be detected during the first 3 months after transplantation, whereas the usual interval from transplant to the occurrence of herpes zoster is 4-5 months. The majority of patients with subclinical viremia did not develop clinical signs of VZV reactivation subsequently. However, 31% of the patients in our study had herpes zoster, a rate comparable with other reports about the incidence of recurrent VZV after BMT [1-5]. Although some clinical studies have found a higher risk of VZV infection among allogeneic transplant patients, we found no significant differences in the incidence of either subclinical or clinical VZV reactivation between autologous and allogeneic BMT recipients [2, 20]. Clinical experience concerning the relationship between GVHD and risk of recurrent VZV has also been variable; no significant correlations were noted between GVHD and cellassociated viremia or clinical VZV reactivation in our BMT population [2, 3, 21]. The documentation of cell-associated viremia, in the absence of localized herpes zoster, provides a new perspective about the pathogenesis of recurrent VZV infections that is consistent with the clinical observation that some transplant recipients with serologic evidence of past VZV infection develop a generalized varicella-like disease after BMT. VZV disease was classified clinically as "varicella" in 16% of the BMT patients described by Locksley et al. [2], most ofwhom had serologic evidence of VZV infection before transplant. Herpes zoster, like recurrent herpes simplex virus infection, has been attributed to the spread of the reactivated virus along neural pathways from the site oflatency in dorsal root ganglia, while the viremia that occurs in some immunodeficient patients is considered to result from a failure to restrict local cutaneous replication of the virus [22, 23]. However, the occurrence ofsubclinical, cell-associated viremia in BMT patients suggests that the virus also may be taken up directly by mononuclear cells, presumably at the neuronal site of viral reactivation, without requiring a phase of cutaneous replication. In vitro, the activation of T lymphocytes by stimulation with phytohemagglutinin makes this otherwise resistant cell population permissive for VZV infection at low frequencies [24]. It is possible that cell-associated VZV viremia in BMT patients is potentiated by the characteristic persistence of activated lymphocytes in circulation for a prolonged period after transplant [25-27]. In the present study, the documentation of cell-associated VZV viremia during the time preceding the recovery of lymphoproliferative responses to VZV antigen suggests that subclinical viral reactivation could stimulate the induction or clonal reexpansion of T cells that recognize VZV antigens. Among BMT recipients, the restoration of T lymphocyte proliferative responses to herpesvirus antigens has been

lID 1992; 165 (January)

shown to correlate with the occurrence and resolution of recurrent herpesvirus infections. For example, episodes of herpes simplex virus and cytomegalovirus reactivation have been associated with the earlier reconstitution of T lymphocyte proliferation to specific viral antigens [28, 29]. Episodes of subclinical VZV reactivation during the early posttransplant period may also enhance the reconstitution of VZV-specific cytotoxic T cell activity. Our observations indicate that VZV CTL function can be detected during the first 100 days after transplant more often than T cell proliferation to VZV antigen. Even though both assays require the in vitro response of virus-specific T cells to VZV antigen, some discrepancies in the detection of cytotoxicity and proliferation have been noted previously [16, 18]. These differences may be explained by a threshold effect related to low numbers of circulating virus-specific T cells and by diminished in vitro lymphokine production, which could be compensated for by the longer incubation period and the addition of exogenous IL-2 to the limiting dilution cultures. On the basis of the analysis of immunocompromised patients with leukemia by Giller et al. [30], the defect is not likely to result from a failure of VZV antigen presentation. The lymphocyte subpopulations that were detected after stimulating limiting dilution cultures from BMT recipients with whole VZV antigen showed a significant reduction in the outgrowth of CD4+ cells compared with the pattern of cell phenotypes in cultures from healthy subjects. This predominance of CD8+ T cells in antigen-stimulated cultures resembles the typical distribution of peripheral blood lymphocyte phenotypes observed after BMT, which is characterized by an inversion of the CD4+:CD8+ T cell ratio that can last for several years [25-27]. Although CD8+ T lymphocytes have been defined as the "classic" cytotoxic effector cell in animal models of antiviral immunity, evidence is accumulating to indicate that human CD4+ cells effectively mediate the lysis of target cells infected with various human viral pathogens, including the herpesviruses [31, 32]. The failure of the CD4+ T cell population to respond to secondary in vitro reexposure to VZV antigen may reflect a significant deficiency in the capacity of the BMT recipient to control VZV replication by CTL mechanisms, since the precursor frequencies of cytotoxic T lymphocytes that recognized VZV proteins were equivalent within CD4+ and CD8+ lymphocyte subpopulations from healthy immune subjects [15]. The diminished CD4+ T cell response to VZV antigen may explain why the overall frequencies of CTL precursors specific for the IE62 protein or gpl remained significantly lower after BMT than in healthy, VZV immune individuals. In contrast to virus-specific T cell immunity, NK cell numbers increase immediately after BMT, and NK function equivalent to the responses ofhealthy subjects can be demonstrated during the first few months after BMT [33-37]. In our experiments, limiting dilution cultures from five BMT recipients (27%) contained lymphocytes that lysed control

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VZV Reactivation and Immunity after BMT

targets as well as those that expressed VZV IE62 protein or gpI, indicating NK-mediated cytotoxicity. Analysis of the effector cell populations in cultures from these patients showed that a mean of 51 % of cells expressed the CD 16 surface antigen. In contrast, we never observed such a predominance of NK cells when lymphocytes from healthy immune subjects were stimulated with VZV antigen under limiting dilution conditions [16]. A significant association between NK activity and resistance to lethal infection has been shown in murine models of herpesvirus disease [38, 39]. Diminished nonspecific cytotoxicity has also been identified in some patients with severe herpesvirus infections, including BMT recipients with cytomegalovirus disease [17, 40]. This capacity of lymphocytes from some BMT patients to lyse targets expressing VZV proteins by a mechanism that is not antigen-specific may help to limit VZV replication before the recovery of virus-specific T lymphocyte responses. In general, this study as well as earlier analyses of VZVspecific cell-mediated immunity have demonstrated a gradual recovery of T cell proliferation to VZV antigens, with a larger percentage of BMT patients having detectable responses as the interval after transplant increases. Our experiments showed that BMT patients also recover cytotoxic T cells that can recognize and lyse autologous target cells that express VZV proteins. However, the recovery of virus-spec ific cellular immunity is often delayed and may not occur until after the patient has experienced an in vivo reexposure to VZV antigens. In addition, even though episodes ofVZV reactivation can induce virus-specific cell-mediated immunity, its persistence during subsequent immunosuppressive therapy is unpredictable [7, 30, 41 ]. Since inactivated or subunit herpesvirus vaccines could provide an effective substitute for "natural" resensitization by viral reactivation and a means to boost the host response periodically after BMT, the potential for immunoenhancement using such vaccines should be investigated in BMT populations. Acknowledgments

We thank J. Hay, W. Ruyechan, and P. Kinchington (Uniformed Services University of the Health Sciences, Bethesda, MD), who provided the VZV-vaccinia recombinants used for the cytotoxicity assays, the patients who participated, and Karl Blume, Luis Hernandez, and Barbara Stallbaum (Bone Marrow Transplant Program, Stanford University Medical Center) for their generous help with this study. References I. Watson JG. Problems of infection after bone marrow transplantation. J Clin Pathol 1983;36:683-92. 2. Locksley RM, Flournoy N, Sullivan KM, Meyers JD. Infection with varicella zoster virus after bone marrow transplantation. J Infect Dis 1985; 152: 1172-80. 3. Ljungman P, Lonnqvist B. Gahrton G, Ringden 0, Sundqvist V. Wahren B. Clinical and subclinical reactivation of varicella-zoster

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