Relationship between Preexisting Anti-Varicella-Zoster Virus (VZV) Antibody and Clinical VZV Reactivation in Hematopoietic Stem Cell Transplantation Recipients

Reactivation of latent varicella-zoster virus (VZV), presentingas localized zoster or as disseminated infection, is a commonand potentially serious complication in hematopoietic stem celltransplantation (HSCT) recipients. We retrospectively studiedanti-VZV immunoglobulin G titers by the immune adherence hemagglutinationmethod after HSCT and also studied VZV DNA by real-time PCRduring clinical VZV reactivation using cryopreserved serum samples.No significant difference was found between anti-VZV titersin 13 patients with VZV infection (localized zoster in 11 patientsand disseminated zoster in 2 patients) and in 13 subjects withoutVZV infection at each time point after HSCT. Preexisting anti-VZVtiters of disseminated zoster cases tended to be lower thanthose of localized zoster cases (P = 0.10). Serum VZV DNA copynumbers at the onset of disseminated zoster cases tended tobe higher than those of localized zoster cases (P = 0.09). Astrong inverse correlation was found between preexisting anti-VZVtiter and serum VZV DNA at onset (r = –0.90, P = 0.006).In HSCT recipients, preexisting antibody does not prevent thedevelopment of VZV reactivation but may contribute to decreasedviral load at onset, resulting in a mild clinical course.

INTRODUCTION

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Introduction

Reactivation of varicella-zoster virus (VZV) is a common eventin patients undergoing hematopoietic stem cell transplantation(HSCT) (1, 4, 6, 9). In post-HSCT recipients, VZV reactivationcan occur frequently as localized zoster and sometimes as disseminatedcutaneous lesions resembling varicella with or without visceralinvolvement, which results in a high mortality rate (1, 4, 6,9). Previous studies revealed that VZV-specific memory T cellsplay a crucial role in suppressing reactivation (1, 8). Althoughall recipients experience a T-cell immunodeficient period afterHSCT, the clinical severity of VZV infection after HSCT variesfrom self-limiting cases to fatal disseminated cases. The riskfactors that determine clinical severity have not been elucidated.Despite current use of varicella-zoster immunoglobulin (VZIG),the role of humoral immunity against VZV infection has beenpoorly evaluated. The aim of this study was to determine therelationship between sustained anti-VZV antibody titers andthe clinical manifestation of the VZV reactivation in HSCT recipients.

MATERIALS AND METHODS

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Materials and Methods

Patients. Patients who had undergone HSCT in Hokkaido University Hospitalduring the period from February 1995 to March 2004 were enrolledas subjects of the present study. Thirteen patients with VZVinfection after HSCT were enrolled. Eleven patients (allogeneic[allo], seven; autologous [auto], four) suffered localized zoster.Two patients (both allo) suffered disseminated zoster with visceralinvolvement (one with acute abdomen and one with interstitialpneumonia). Thirteen patients who had been monitored for atleast 3 years after HSCT without clinical VZV infection werestudied as control subjects. Patient characteristics are presentedin Table 1. The backgrounds of the two groups, including age,male/female ratio, autologous/allogeneic HSCT, total body irradiationregimen, and graft-versus-host disease (GVHD), are almost thesame. In an allo-HSCT setting, acyclovir was administered orallyat a dose of 1,000 mg/day from day –7 to day 35 to preventherpes simplex virus infection. Ten grams of immunoglobulinwas administered intravenously on day 0 and every other weekuntil day 100 for prophylaxis of opportunistic viral infectionin patients who had undergone allo-HSCT. In an auto-HSCT setting,acyclovir was administered orally at a dose of 1,000 mg/dayfrom day –7 to engraftment, and 2.5 g of immunoglobulinwas administered on days –7, 0, 7, and 14. We retrospectivelystudied the transition of anti-VZV-immunoglobulin G titer bythe immune adherence hemagglutination (IAHA) method (beforeand at 3, 6, 12, and 24 months after HSCT) and VZV DNA by real-timePCR during clinical VZV infection (onset and paired serum) usingcryopreserved serum samples.

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TABLE 1. Patient characteristics

Diagnosis of clinical VZV infection. VZV infection was defined by the appearance of typical cutaneousvesicular lesions or the detection of VZV antigen. Localizedzoster was defined as the presence of vesicular lesions in adermatomal distribution. Disseminated zoster was defined asa generalized vesicular eruption that is identical to that ofvaricella. Visceral dissemination was defined as clinical evidenceof internal organ involvement in the absence of other identifiedpathogens that might have accounted for the clinical syndrome.

IAHA assay. IAHA analysis was performed as described previously (5).

Real-time PCR assay. VZV DNA extraction from serum was performed by using a QIAampDNA blood minikit (QIAGEN GmbH, Hilden, Germany). The primerpair VZV28-F (5'-CAGATTATCCGACATGCAGTCAA-3') and VZV28-R (5'-ACCGGCAAGTCGCCAAT-3')and the probe VZV28-T (5'-CAACGTCGCTTAACG-3') for real-timePCR were designed in the DNA polymerase gene (gene 28) of VZVby using Primer Express software (Perkin-Elmer, Norwalk, CT).The reaction mixtures (50 µl) contained 2x TaqMan universalPCR master mix (Perkin-Elmer), 15 µl of DNA, the primers(each at 900 nM), and the TaqMan MGB probe (200 nM). After 2min at 50°C, the AmpliTaq Gold DNA polymerase was activatedat 95°C for 10 min, followed by 40 cycles at 95°C for15 s and at 60°C for 1 min. All reactions were carried outsix times with a 7500 ABI Prism sequence detector (Applied Biosystems,Foster City, CA). A real-time PCR standard, pCR2.1/VZV28 plasmid,was constructed by cloning a PCR product containing the VZVgene 28 region to the TA cloning vector pCR2.1 (Invitrogen,Carlsbad, CA). The pCR2.1/VZV28 plasmid was diluted to 5 x 10⁷,5 x 10⁶, 5 x 10⁵, 5 x 10⁴, 5 x 10³, 5 x 10², or 5 x 10¹ copiesin 15 µl, and these dilutions were used to generate astandard curve.

Statistical analysis. Comparisons between different groups of patients or clinicaldata were made by using the Fisher exact test for categoricaldata and the t test or Mann-Whitney U test for numerical variables.Correlation between two values was analyzed by Pearson correlationtest. VZV DNA was analyzed after log transformation. The lowerlimit of the assay was used in the case of negative PCR results.

RESULTS

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Results

VZV infection occurred within 1 year after HSCT in 11 of the13 patients. The onset of VZV infection varied day 56 to day595 (median, day 155) after HSCT. All VZV infections occurredafter cessation of prophylaxis with acyclovir. In 6 of the 10allo-HSCT cases, VZV infection occurred during use of an immunosuppressant.All patients were treated with acyclovir. There were no deathsfrom VZV infection. All patients were seropositive for VZV beforeHSCT (Fig. 1A). None of the patients had a known history ofVZV exposure before VZV infection. All VZV infections were consideredto be a reactivation of innate VZV. One patient with VZV reactivation,the most severe case, had become seronegative before the developmentof disseminated zoster and interstitial pneumonia. VZV reactivationin the other 12 cases occurred during the persistent existenceof anti-VZV. There was no statistically significant differencebetween the anti-VZV titers in the 13 patients with VZV reactivationand those in the 13 controls at each time point after HSCT (Fig.1A). The last results before clinical VZV infection (preexistinganti-VZV) varied widely from <2-fold to >256-fold (median,32-fold). Preexisting anti-VZV titers of disseminated zostercases tended to be lower than those of localized zoster cases(P = 0.10). Increase in titer (IAHA, >4-fold) after VZV infectionwas not observed in any of the eight recipients with VZV infectionwithin the first 6 months after HSCT but was observed in threeof the five recipients with VZV infection after 6 months (P= 0.04) (Fig. 1B). Seven available samples that had been obtainedfrom two patients with disseminated zoster and five patientswith localized zoster at onset of VZV infection (stored on day1 of skin eruption before administration of acyclovir) wereexamined by real-time PCR. VZV DNA was detected in all disseminatedzoster cases and in three of the five localized zoster cases,whereas VZV was undetectable in all paired sera (2 to 9 weeksafter onset [data not shown]). VZV DNA copy numbers at onsetof disseminated zoster cases tended to be higher than thoseof localized zoster cases (P = 0.09). The preexisting anti-VZVtiter and the serum VZV DNA level were inversely correlated(r = –0.90, P = 0.006) (Fig. 1C).

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FIG. 1. (A) Transition of anti-VZV titer after HSCT (control, ${blacktriangleup}$ ; localized zoster, •; disseminated zoster, ${circ}$ ). In cases of VZV infection, only the results before VZV onset were plotted. (B) Transition of anti-VZV before, at onset of and after VZV infection in 13 cases of VZV reactivation (localized zoster, •; disseminated zoster, ${circ}$ ). VZV infection within the first 6 months after HSCT (continuous line) and VZV infection at more than 6 months after HSCT (dashed line) are also indicated. (C) Correlation of preexisting anti-VZV titer and serum VZV DNA (localized zoster, •; disseminated zoster, ${circ}$ ). Preexisting anti-VZV titer and serum VZV DNA were inversely correlated (determined by Pearson correlation test; r = –0.90, P = 0.006).

DISCUSSION

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Discussion

Reactivation of latent VZV, presenting as localized zoster oras disseminated infection, is a common and potentially seriouscomplication in HSCT recipients. Previous studies revealed that23 to 60% of patients can be expected to develop VZV infectionafter HSCT (1, 4, 6, 9). Analyses of risk factors such as allogeneicversus autologous transplant, GVHD, underlying disease, andpre-bone marrow transplant irradiation have not revealed definitiveassociations (1, 4). Although the importance of cell-mediatedimmunity in VZV infection has been recognized (1, 8), therehas been little investigation of the role of humoral immunity.

Passive immunization in the form of VZIG has been clinicallyused mainly for prophylaxis after exposure and sometimes fortreatment of VZV infection. The Center for Disease Control guidelinerecommends immediate VZIG administration after VZV exposurefor HSCT recipients (3). Anti-VZV can interfere with the initialphases of VZV replication and reduce attack rate (10). Evenif infection occurs, the severity of the cutaneous disease andthe risk for dissemination are modified by VZIG prophylaxis,indicating that specific antibodies present at the beginningof the incubation period can restrict the pathogenic potentialof the virus (2). In contrast, passive antibodies administeredafter the onset of illness do not alter the severity of varicella(2).

In the case of primary VZV infection, no one has preexistinganti-VZV, and the disease is always disseminated. In this situation,early detection of high anti-VZV titers does not predict milderinfection (2). In contrast, in the case of VZV reactivation,loss of specific cell-mediated immunity is observed in all cases,but titers of preexisting anti-VZV vary. Mazur et al. reportedthat the absence or low level of circulating innate anti-VZVwas a significant risk factor for the dissemination of virusin herpes zoster (7). Based on these findings, we speculateda protective role of preexisting anti-VZV titer against VZVreactivation after HSCT.

Although preexisting anti-VZV could not prevent developmentof VZV reactivation in our series, a strong inverse correlationwas found between preexisting anti-VZV and serum VZV DNA atthe onset of VZV infection. Our data suggest that a low levelof preexisting anti-VZV results in a severe and disseminatedclinical course in HSCT recipients. We speculated that circulatinganti-VZV cannot prevent the cell-to-cell spread of VZV, whichleads to development of VZV infection, but that it has a neutralizingeffect against viremia, which determines the severity of VZVdisease.

Our results suggest that monitoring the anti-VZV titer has littlediagnostic value, especially within 6 months after HSCT, becausean increase in anti-VZV titer (IAHA, >4-fold) in paired seraafter VZV infection was not observed in this period. In twoof the three cases with active humoral immune response, anti-VZVwas already increased at clinical onset. Anti-VZV titer at clinicalonset was not correlated with clinical severity, and this maybe the reason why previous studies failed to show a role ofhumoral immunity by analyzing anti-VZV at clinical onset. Activehumoral immune response was dependent not on severity of clinicalpresentation (localized or disseminated disease) but on thetiming of the onset after HSCT. This observation is consistentwith a previous finding that reconstitution of cytotoxic T lymphocyteswas also dependent on the time after HSCT (1).

In conclusion, preexisting antibody does not prevent the developmentof VZV reactivation but may contribute to decreased viral loadat onset, resulting in a mild clinical course in HSCT recipients.These findings should be confirmed in a larger series, alongwith an evaluation of cellular immunity.

ACKNOWLEDGMENTS

We thank Michiaki Takahashi and the staff of The Research Foundationfor Microbial Diseases of Osaka University (Osaka, Japan) fortheir technical support and advice in the analysis of the serumsamples.

FOOTNOTES

* Corresponding author. Mailing address: Department of Gastroenterology and Hematology, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan. Phone: 81-11-716-1161, ext. 5920. Fax: 81-11-706-7867. E-mail: masahiro.onozawa{at}nifty.ne.jp.

Published ahead of print on 11 October 2006.

REFERENCES

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