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B R I E F R E P O R T

Varicella-Zoster VirusndashSpeci1047297cAntibody Responses in 50ndash59-Year-

Old Recipients of Zoster Vaccine

Myron J Levin1 Kenneth E Schmader2 John W Gnann3

Shelly A McNeil7 Timo Vesikari8 Robert F Betts4 Susan Keay5

Jon E Stek6 Nickoya D Bundick6 Shu-Chih Su6 Yanli Zhao6

Xiaoming Li6 Ivan S F Chan6 Paula W Annunziato6 and Janie Parrino6

1University of Colorado Denver Anschutz Medical Campus Aurora 2Duke University

Geriatric Research Education and Clinical Center Durham Veterans Affairs Medical

Center North Carolina 3Medical University of South Carolina Charleston4University of Rochester New York 5Veterans Affairs Maryland Health Care

System Baltimore Maryland and 6Merck amp Co Inc Whitehouse Station New

Jersey 7Dalhousie University Halifax Nova Scotia Canada and 8University of

Tampere Finland

Prevaccination and 6-week postvaccination samples from the

immunogenicity substudy (n = 2269) of the zoster vaccine

(ZV) ef 1047297cacy trial (N = 22 439) in 50ndash59-year-old subjects

were examined for varicella-zoster virusndashspeci1047297c antibody

responses to vaccination The varicella-zoster virus geomet-

ric mean titer (GMT) and geometric mean fold rise were

higher in ZV recipients than in placebo recipients (GMT

6600 vs 2931 glycoprotein enzyme-linked immunosorbent

assay unitsmL [P lt 001] respectively geometric mean fold

rise 231 vs 100 [P lt 025]) In each group there was a strong

inverse correlation between postvaccination GMT and risk of

subsequent herpes zoster Although these data provide strong evidence that relates ZV-induced antibody and the risk of

herpes zoster a protective threshold was not determined

Clinical Trials Registration NCT00534248

Keywords herpes zoster zoster vaccine immunogenicity

The live attenuated herpes zoster (HZ) vaccine (zoster vaccine

[ZV] Zostavax TM Merck amp Co Inc) was recommended by

the Advisory Committee on Immunization Practices in the

United States in 2008 for immunocompetent individuals aged

ge60 years based on a large randomized placebo-controlled

trial the Shingles Prevention Study (SPS) [1 2] There is strong

clinical evidence that varicella-zoster virus (VZV)ndashspeci1047297c cell-

mediated immunity (VZV CMI) is both necessary and suf 1047297cient

to prevent HZ and that ZV prevents HZ because it stimulates

VZV CMI [3ndash6] It is believed that there is a causal inverse rela-

tionship between the loss of VZV CMI that occurs with aging

and the age-related increase in HZ In contrast VZV-speci1047297c

immunoglobulin G antibody (VZV antibody) does not decline

with age [3 7] Nevertheless the SPS demonstrated that a mea-

sure of VZV antibody in addition to 2 measures of VZV CMI

correlated with protection against HZ although no quantitative

measure of any of these responses reliably predicted the extent

of protection [8]

A subsequent ef 1047297cacy trial of ZV in 22 439 subjects 50ndash59

years old demonstrated an ef 1047297cacy of 698 for preventing HZ[9] Ten percent of subjects were randomly assigned to an im-

munology substudysubcohort that measured VZV antibody

response to ZV The substudy objectives were to determine if

ZV in 50ndash59-year-olds is immunogenic (as evaluated by glyco-

protein enzyme-linked immunosorbent assay [gpELISA]) and

to assess the association between antibody response at week 6

after vaccination and the risk of HZ

METHODS

Study Design

The methods for this event-driven randomized double-blind

placebo-controlled multicenter study (NCT00534248) are pub-

lished elsewhere [10] Subjects were randomized (11 ratio) to

receive either ZV or placebo To evaluate the correlation of

vaccine-induced VZV antibody responses and subsequent pro-

tection against HZ serum samples were collected from study

subjects before and 6 weeks after vaccination with VZV antibody

concentrations measured by gpELISA in (1) the immunology

subcohort population (10 protocol-prespeci1047297ed randomized

subcohort) and (2) the case-cohort population (immunology sub-

cohort plus all subjects in whom suspected HZ developed)

Study Population

Healthy subjects aged 50ndash59 years with a history of varicella or

residence in a VZV-endemic area for ge30 years were enrolled

Exclusion criteria have been described elsewhere and included

evidence of immunocompromise [9] The protocol was con-

ducted in accordance with principles of good clinical practice

and approved by the ethical review committee of each partici-

pating site written informed consent was obtained from each

subject before study entry

Received 17 December 2012 accepted 2 May 2013 electronically published 1 August 2013

Presented in part 48th Annual Meeting of the Infectious Diseases Society of America 21ndash

24 October 2010 Vancouver British Columbia Canada Abstract 3363

Correspondence Janie Parrino PhD Merck amp Co Inc PO Box 1000 UG3CD-28 North

Wales PA 19454-1099 (janie_parrinomerckcom)

The Journal of Infectious Diseases 20132081386ndash90

copy The Author 2013 Published by Oxford University Press on behalf of the Infectious Diseases

Society of America All rights reserved For Permissions please e-mail journalspermissions

oupcom

DOI 101093infdisjit342

1386 bull JID 2013208 (1 November) bull BRIEF REPORT



Intervention

Lyophilized ZV and placebo were supplied in 07-mL single-

dose vials and stored at minus15degC or colder Placebo contained the

same stabilizers as ZV but no live virus or virus components

ZV and placebo were reconstituted with sterile water immedi-

ately before administration All subjects received a single 065-

mL subcutaneous injection of either ZV or placebo in the

deltoid area

Follow-up

Subjects were educated regarding the signs and symptoms of

HZ and instructed to call their study site if HZ symptoms oc-

curred Contact by an interactive voice response system was

undertaken monthly until study completion to ensure that sus-

pected HZ was reported Suspected HZ cases were evaluated by

a site investigator Initiation of treatment with antiviral therapy

and pain medication was determined by the treating physician

Assessment of Suspected HZ Cases

All HZ rash characteristics were recorded and lesion swab

samples were submitted for detection of VZV herpes simplex

and human β-globin DNA using a polymerase chain reaction

(PCR) assay (performed at PPD Vaccines and Biologics) [10]

Determination of Con1047297rmed HZ Cases

Suspected HZ cases were de1047297ned as ldquocon1047297rmed HZrdquo if VZV

DNA was present in skin lesion samples If the PCR assay was

positive for β-globin or herpes simplex virus DNA and nega-

tive for VZV DNA then the case was de1047297ned as ldquonot HZrdquo

When there was no specimen or the specimen was inadeq-

uate case determination was decided by a clinical evaluation

committee [9]

VZV-Speci1047297c Antibody Assay

The gpELISA for VZV-speci1047297c immunoglobulin G antibody

(performed at PPD Vaccines and Biologics) detects antibodies

to puri1047297ed VZV glycoproteins from MRC-5 (normal human

lung 1047297broblasts Medical Research Council 5 cell line) cells in-

fected with VZV (KMcC strain) using methods described else-

where [11] First VZV glycoproteins or uninfected MRC-5

lysates were adsorbed to polystyrene microtiter wells Experi-

mental control and standard curve serum samples were incu-

bated in coated tissue culture wells in duplicate at 23degC for40ndash80 minutes until the difference in optical density (OD)

between the VZV glycoproteinndashcontaining (positive) wells and

control (negative) wells was gt0700 as measured in the plate

reader at 405 nm approximately every 5 minutes after an initial

40-minute incubation Color development was stopped with

50 μL of 3N sodium hydroxide Delta OD was calculated for

each serum sample as the difference between the average OD of

the 2 VZV antigen wells and that of the 2 MRC-5 control wells

Quantitation was performed by comparing sample delta OD

with a standard curve with results reported as concentrations

of antibody in gpELISA units per milliliter

Statistics

The immunogenicity objectives were (1) to determine whether

administered ZV is immunogenic and (2) to assess the associa-

tion between antibody response 6 weeks after vaccination and

the risk of HZ To show a signi1047297cantly higher geometric mean

titer (GMT) in VZV antibody titers at 6 weeks after vaccinationin the ZV group compared with the placebo group 2230 sub-

jects for the 10 subcohort (1115 randomly selected in each

group) would provide an overall power of approximately 98

at the 025 signi1047297cance level (1 sided noninferiority criterion

lower bound of 2-sided 95 con1047297dence interval for GMT ratio

[ZVplacebo] gt14) This assumed that the true GMT ratio is

17 [12] the standard deviation of the natural-log-transformed

titers is 11 and there would be a 10 nonevaluable rate for im-

munogenicity measurements The immunogenicity summaries

and analyses were based on a per-protocol approach Subjects

and observations with protocol deviations that might invalidate

the evaluation of VZV-speci1047297c gpELISA antibody response

were excluded from the immunogenicity analyses

RESULTS

There were 22 439 subjects randomly assigned to receive ZV or

placebo (Supplementary Figure 1 - CONSORT diagram) Serum

samples were obtained in all subjects before and 6 weeks after vac-

cination VZV antibody was measured in the 10 immunology

subcohort and in patients with suspected HZ The ZV and placebo

recipients in the immunology subcohort were well matched by sex

age race and study completion Most subjects (94) were whiteand 62 were female (Supplementary Table 1 - demographics)

gt94 of subjects completed the study

At baseline (day 1) 5 subjects (3 ZV and 2 placebo recipients)

did not have VZV-speci1047297c antibody measured by gpELISA The

2 treatment arms were well matched before vaccination in the

distribution of high and low antibody titers (P = 84 χ2 test

for homogeneity) (Table 1) Six weeks after vaccination the

GMT was 660 gpELISA unitsmL in ZV recipients versus 293

gpELISA unitsmL in placebo recipients for an estimated GMT

ratio (ZVplacebo) of 23 (95 con1047297dence interval 22ndash24

P lt 001) which met the prespeci1047297

ed statistical criterion Half of the ZV recipients had at least a doubling of VZV antibody

titer The geometric mean fold rise (GMFR) in titer in ZV

recipients was 231 compared with no fold rise in placebo

recipients (P lt 025)

During the study 30 ZV and 99 placebo recipients developed

HZ 6 ZV and 10 placebo recipients developed HZ before col-

lection of their postvaccination blood sample and thus were ex-

cluded from the immunogenicity analyses (Table 2) For the

subjects included in the immunogenicity analyses HZ was

BRIEF REPORT bull JID 2013208 (1 November) bull 1387



identi1047297ed by PCR in 19 of 24 ZV and 78 of 89 placebo recipi-

ents for the rest HZ cases were con1047297rmed by the clinical evalu-

ation committee assessment

In each treatment arm after vaccination the GMT for sub-

jects who did not develop HZ was signi1047297cantly higher than for

subjects who developed HZ although the GMT for the placebo

Table 1 VZV-Speci1047297c gpELISA Titers in ZV and Placebo Recipientsa

Immunogenicity End Point

ZV Recipients (n = 1 136) Placebo Recipients ( n = 1133)

No ()b 95 CI No ()b 95 CI

Titer at day 1 gpELISA unitsmLcd

lt125 3 (03) 01ndash08 2 (02) 00ndash06

ge125 to le100 161 (143) 123ndash165 157 (140) 120ndash161

gt100 to le300 469 (418) 389ndash447 453 (403) 374ndash432

gt300 to le500 174 (155) 134ndash

177 192 (171) 149ndash

194

gt500 316 (281) 255ndash309 320 (285) 258ndash312

GMT gpELISA unitsmL 2836 2657ndash3028 2928 2744ndash3123

Titer at week 6 gpELISA unitsmLef

lt125 0 (00) 00ndash03 2 (02) 00ndash07


gt100ndashle300 201 (185) 162ndash209 443 (408) 378ndash437


gt500 667 (613) 583ndash642 313 (288) 261ndash316


Fold rise from day 1g

ge2 541 (498) 468ndash528 36 (33) 23ndash46

ge3 330 (304) 276ndash

332 4 (04) 01ndash

09ge4 221 (203) 180ndash228 3 (03) 01ndash08

ge5 166 (153) 132ndash175 3 (03) 01ndash08

GMFRg 231 220ndash243 100 098ndash102

Abbreviations GMFR geometric mean fold rise GMT geometric mean titer gpELISA glycoprotein enzyme-linked immunosorbent assay VZV varicella-zoster

virus ZV zoster vaccinea Immunogenicity subcohort population does not include all subjects who developed suspected HZb

Values represent No () of subjects except in rows for GMT and GMFRc

Prevaccination 1123 ZV and 1124 placebo subjects contributed to this analysisdP = 84 (χ2 test for homogeneity in distributions of baseline titers between vaccine and placebo arms)

eWeek 6 1088 ZV and 1087 placebo recipients contributed to this analysis

fP lt 025 (χ2 test for homogeneity in distributions of week 6 titers between vaccine and placebo arms)

gFold rise 1087 ZV and 1086 placebo subjects contributed to this analysis

Table 2 Relationship of HZ to gpELISA Titers 6 Weeks After Vaccination


ZV Recipients (n=1164)a Placebo Recipients (n=1223)a

Nob Observed Response (95 CI) Nob Observed Response (95 CI)

GMT

Developed HZ 24 4541 (3002ndash6870)c 89 1783 (1400ndash2271)c

Did not develop HZ 1086 6593 (6241ndash6966) 1079 2942 (2757ndash3139)

GMFR from d 1

Developed HZ 24 16 (12ndash19) 89 10 (09ndash10)


Abbreviations CI confidence interval GMFR geometric mean fold rise GMT geometric mean titer gpELISA glycoprotein enzyme-linked immunosorbent assay

HZ herpes zoster ZV zoster vaccinea Case-cohort population which includes the 10 immunogenicity subcohort plus all subjects who developed suspected HZb Number of subjects contributing to the immunogenicity analysis subjects who developed HZ before the 6-week date were excluded from this analysisc In both arms GMT differed significantly between subjects whodeveloped HZ andthosewho didnot (ZV group P = 02 placebo groupP lt 01 1-sided2 sample t test)




recipients who did not develop HZ was much lower than the

GMT for the ZV recipients who did develop HZ The GMFR

was also signi1047297cantly lower for the ZV recipients who devel-

oped HZ than for those who did not In the placebo recipients

VZV-antibody did not increase

DISCUSSION

This trial con1047297rms that persons who indicate that they hadprior varicella andor had resided in the United States for ge30

years have serologic evidence of prior varicella infection (only 5

of 2369 individuals lacked antibody at baseline by gpELISA) In

the previous trial of subjects ge60 years old this was true of all

1395 samples tested with gpELISA [8] The 3 subjects in the

current trial who were seronegative at the time of ZV adminis-

tration did not develop any serious adverse events or VZV-like

rashes thereby adding to the safety data available from seroneg-

ative ZV recipients [13]

In subjects 50ndash59 years old ZV was immunogenic as mea-

sured by a signi1047297cant rise in VZV antibody titer The postvacci-nation GMT was 660 gpELISA unitsmL versus 293 gpELISA

unitsmL in the control group and the 6-week GMFR was 23

This response was greater than that observed in the trial of

older subjects [12] in whom the postvaccination GMT and

GMFR were 4784 and 17 respectively These results indicate a

more robust VZV antibody response to ZV in younger vacci-

nees (50ndash59-year-olds) and is consistent with greater ef 1047297cacy

for HZ prevention (698) in 50ndash59-year-olds than in older

subjects (64 and 38 for the 60ndash69- and ge70-year age

groups respectively) in the SPS [1 9]

The VZV antibody response 6 weeks after vaccination in this

younger group was strongly inversely correlated (P lt 001) with

the likelihood of developing HZ as demonstrated elsewhere in

the ZV trial in older subjects but neither trial established a titer

of VZV antibody that would serve as a surrogate of protection

[8] The lack of a quantitative surrogate of protection is demon-

strated in the current 1047297ndings VZV antibody titers measured

in the placebo recipients who did not develop HZ were lower

than those achieved by ZV recipients who did develop HZ

This con1047297rms that VZV antibody should not be considered

directly responsible for the ef 1047297cacy of ZV against HZ rather

VZV CMI is necessary and suf 1047297cient for preventing HZ This

essential role of VZV CMI has previously been established by (1) substantial clinical observations indicating that HZ occurs

in immunocompromised patients with high levels of VZV anti-

body [4ndash6] and (2) the relationship between the increasing inci-

dence of HZ with increasing age and the decline in VZV CMI

[14] whereas there is no such relationship with VZV antibody

[7] In addition the trial in older subjects did not demonstrate

any correlation between VZV antibody and VZV CMI This

lack of correlation between these 2 classes of immune

responses which has been con1047297rmed [15] may represent the

detection of different VZV epitopes unique to each class of

immune response

The absence of paired VZV CMI and VZV antibody data is a

limitation of our study Another limitation is the lack of data

on chronic pain which may have been related to the magnitude

of the immune response Postherpetic neuralgia greatly affects

quality of life and is the most common complication of HZ but

the role of the immune response to HZ and the subsequentdevelopment of postherpetic neuralgia are poorly understood

In addition the study was performed almost entirely in white

subjects immune response to HZ may differ by racial origin just

as the incidence of HZ is lower in blacks than in whites [16]

The practical implication of the study data is that although

this speci1047297c antibody measure is predictive of a ZV response and

is a suitable immunogenicity marker for comparative studies of

ZV it does not provide a precise threshold for protection Given

that protection from HZ depends on VZV-speci1047297c CMI gpELISA

may be inadequate for assessments among individuals with

altered immune function in whom there may be a lack of cor-

relation between cellular and humoral responses Also impor-

tant when considering comparative immunogenicity studies is

the relationship between gpELISA GMT and GMFR and cli-

nical ef 1047297cacy which may be speci1047297c to ZV a vaccine that

contains the entire Oka strain virus These immunogenicity

measures may not be correlated with the ef 1047297cacy of alternative

HZ vaccines based on different formulations (such as subunit

or recombinant vaccines) that may be developed in the future

SupplementaryData

Supplementary materials are available at The Journal of Infectious Diseasesonline (httpjidoxfordjournalsorg ) Supplementary materials consist of

data provided by the author that are published to bene1047297t the reader The

posted materials are not copyedited The contents of all supplementary data

are the sole responsibility of the authors Questions or messages regarding

errors should be addressed to the author

Notes

Acknowledgments The authors thank all the subjects who participated

in this study The Zostavax Protocol 022 Study Group included the follow-

ing members by country Belgium G Leroux-Roels P Van Damme

Canada R Girard J McElhaney and S McNeil Finland T Haapaniemi

J Immonen K Ivanitskiy T Karppa A Karvonen S Kokko T Korhonen

K Kuismanen P Lagerstrom-Tirri I Seppa and M Virta Germany

B Bergtholdt P Kindermann C Klein A Labitzke R Schaetzl I Schen-

kenberger H Stahl and V von Behren United States M Adams

R Baxter H Bays M Berger B Berwald S Block D Bolshoun B

Bowling D Brandon D Classen L Cohen M Cooperman Cuevas D

DeSantis F Dunlap J Earl W Ellison R Feldman T Fiel C Fisher

N Fraser H Geisberg J Geohas G Gerhard L Gilderman H Gillum

R Haselby J Hoeksrta W Jennings G Juriansz S Keay K Kempf

J Kirstein J Lawless M Levin T Littlejohn F McCarty D McCluskey

J McGettigan R Mills W Miser N Misra A Murray L Murray

M Noss J Pappas C Petit S Powell A Pragalos A Puopolo G Raad

K Reisinger M Reynolds E Riffer G Risi S Rodstein P Rogge Rosen




J Rubino K Schmader D Schumacher B Seidman J Seiler R Severance

S Sharp G Shockey J Stringer C Strout M Throne K Tyring M van

Cleeff and C Woodruff The Data Monitoring Committee included

C Crumpacker S Gravenstein J Neaton H Tilson and J Zaia and the

Clinical Evaluation Committee R Betts J Gnann M Levin V Morrison

K Schmader and D Weber

Author contributions M J L K E S J W G S A M T V R F B

and S K were responsible for subject enrollment data collection and data

interpretation X L Y Z I S F C P W A and J P were responsible for

study conceptdesign and data analysisinterpretation J E S N D B and

S C S were responsible for data analysisinterpretation All authors were

responsible for manuscript preparation

Sponsor rsquo s role This study was funded by Merck amp Co Inc (sponsor)

In conjunction with the external investigators this study was designed exe-

cuted and analyzed by the sponsor Although the sponsor formally re-

viewed a penultimate draft the opinions expressed are those of the authors

and may not necessarily re1047298ect those of the sponsor All coauthors ap-

proved the 1047297nal version of the manuscript

Financial support This work was supported by Merck amp Co Inc

Potential con 1047298 icts of interest Other than employees of Merck amp Co

Inc all authors have been investigators for the sponsor Employees may

hold stock andor stock options in the company M J L is a consultant to

the sponsor and shares intellectual property rights for Zostavax TM All

other authors report no potential con1047298icts

All authors have submitted the ICMJE Form for Disclosure of Potential

Con1047298

icts of Interest Con1047298

icts that the editors consider relevant to thecontent of the manuscript have been disclosed

References

1 Oxman MN Levin MJ Johnson GR et al A vaccine to prevent herpes

zoster and postherpetic neuralgia in older adults N Engl J Med 2005

3522271ndash84

2 Harpaz R Ortega-Sanchez IR Seward JF Advisory committee on im-

munization practices (ACIP) centers for disease control and prevention

(CDC) Prevention of herpes zoster Recommendations of the advisory

committee on immunization practices (ACIP) MMWR Recomm Rep

2008 57(RR-5)1ndash30

3 Oxman MN Zoster vaccine current status and future prospects ClinInfect Dis 2010 51197ndash213

4 Hata A Asanuma H Rinki M et al Use of an inactivated varicella

vaccine in recipients of hematopoietic-cell transplants N Engl J Med

2002 34726ndash34

5 Arvin AM Pollard RB Rasmussen LE Merigan TC Cellular and

humoral immunity in the pathogenesis of recurrent herpes viral infec-

tions with lymphoma J Clin Invest 1980 65869ndash78

6 Onozawa M Hashino S Takahata M et al Relationship between preex-

isting anti-varicella-zoster virus (VZV) antibody and clinical VZV reac-

tivation in hematopoietic stem cell transplantation recipients J Clin

Microbiol 2006 444441ndash3

7 Sadaoka K Okamoto S Gomi Y et al Measurement of varicella-zoster

virus (VZV)-speci1047297c cell-mediated immunity comparison between

VZV skin test and interferon-γ enzyme-linked immunospot assay

J Infect Dis 2008 1981327ndash33

8 Levin MJ Oxman MN Zhang JH et al VZV-speci1047297c immune responses

in elderly recipients of a herpes zoster vaccine J Infect Dis 2008 197

825ndash35

9 Schmader KE Levin MJ Gnann JW et al Ef 1047297cacy safety and tolerabil-

ity of herpes zoster vaccine in persons 50 to 59 years of age Clin Infect

Dis 2012 54522ndash8

10 Harbecke R Oxman MN Arnold BA et al A real-time PCR assay to

identify and discriminate among wild-type and vaccine strains of

varicella-zoster virus and herpes simplex virus in clinical specimens

and comparison with the clinical diagnoses J Med Virol 2009 81

1310ndash22

11 Hammond O Wang Y Green T et al The optimization and validationof the glycoprotein ELISA assay for quantitative varicella-zoster virus

(VZV) antibody detection J Med Virol 2006 781679ndash87

12 Levin MJ Oxman MN Johnson GR Zhang JH Hayward AR Wein-

berg A Immune response to a refrigerator-stable zoster vaccine [letter]

Clin Vaccine Immunol 2009 161381 author reply 1381ndash2

13 Diaz C Dentico P Gonzalez R et al Safety tolerability and immunoge-

nicity of a two-dose regimen of high-titer varicella vaccine in subjects gt

or =13 years of age Vaccine 2006 246875ndash85

14 Weinberg A Lazar AA Zerbe G et al In1047298uence of age and nature of

primary infection on varicella-zoster virus-speci1047297c cell-mediated immune

responses J Infect Dis 2010 2011024ndash30

15 Tang H Moriishi E Okamoto S et al A community-based survey of

varicella-zoster virus-speci1047297c immune responses in the elderly J Clin

Micro 2012 5546ndash50

16 Schmader KE George LK Hamilton JD Racial differences in theoccurrence of herpes zoster J Infect Dis 1995 171701ndash5




Intervention

Lyophilized ZV and placebo were supplied in 07-mL single-

dose vials and stored at minus15degC or colder Placebo contained the

same stabilizers as ZV but no live virus or virus components

ZV and placebo were reconstituted with sterile water immedi-

ately before administration All subjects received a single 065-

mL subcutaneous injection of either ZV or placebo in the

deltoid area

Follow-up

Subjects were educated regarding the signs and symptoms of

HZ and instructed to call their study site if HZ symptoms oc-

curred Contact by an interactive voice response system was

undertaken monthly until study completion to ensure that sus-

pected HZ was reported Suspected HZ cases were evaluated by

a site investigator Initiation of treatment with antiviral therapy

and pain medication was determined by the treating physician

Assessment of Suspected HZ Cases

All HZ rash characteristics were recorded and lesion swab

samples were submitted for detection of VZV herpes simplex

and human β-globin DNA using a polymerase chain reaction

(PCR) assay (performed at PPD Vaccines and Biologics) [10]

Determination of Con1047297rmed HZ Cases

Suspected HZ cases were de1047297ned as ldquocon1047297rmed HZrdquo if VZV

DNA was present in skin lesion samples If the PCR assay was

positive for β-globin or herpes simplex virus DNA and nega-

tive for VZV DNA then the case was de1047297ned as ldquonot HZrdquo

When there was no specimen or the specimen was inadeq-

uate case determination was decided by a clinical evaluation

committee [9]

VZV-Speci1047297c Antibody Assay

The gpELISA for VZV-speci1047297c immunoglobulin G antibody

(performed at PPD Vaccines and Biologics) detects antibodies

to puri1047297ed VZV glycoproteins from MRC-5 (normal human

lung 1047297broblasts Medical Research Council 5 cell line) cells in-

fected with VZV (KMcC strain) using methods described else-

where [11] First VZV glycoproteins or uninfected MRC-5

lysates were adsorbed to polystyrene microtiter wells Experi-

mental control and standard curve serum samples were incu-

bated in coated tissue culture wells in duplicate at 23degC for40ndash80 minutes until the difference in optical density (OD)

between the VZV glycoproteinndashcontaining (positive) wells and

control (negative) wells was gt0700 as measured in the plate

reader at 405 nm approximately every 5 minutes after an initial

40-minute incubation Color development was stopped with

50 μL of 3N sodium hydroxide Delta OD was calculated for

each serum sample as the difference between the average OD of

the 2 VZV antigen wells and that of the 2 MRC-5 control wells

Quantitation was performed by comparing sample delta OD

with a standard curve with results reported as concentrations

of antibody in gpELISA units per milliliter

Statistics

The immunogenicity objectives were (1) to determine whether

administered ZV is immunogenic and (2) to assess the associa-

tion between antibody response 6 weeks after vaccination and

the risk of HZ To show a signi1047297cantly higher geometric mean

titer (GMT) in VZV antibody titers at 6 weeks after vaccinationin the ZV group compared with the placebo group 2230 sub-

jects for the 10 subcohort (1115 randomly selected in each

group) would provide an overall power of approximately 98

at the 025 signi1047297cance level (1 sided noninferiority criterion

lower bound of 2-sided 95 con1047297dence interval for GMT ratio

[ZVplacebo] gt14) This assumed that the true GMT ratio is

17 [12] the standard deviation of the natural-log-transformed

titers is 11 and there would be a 10 nonevaluable rate for im-

munogenicity measurements The immunogenicity summaries

and analyses were based on a per-protocol approach Subjects

and observations with protocol deviations that might invalidate

the evaluation of VZV-speci1047297c gpELISA antibody response

were excluded from the immunogenicity analyses

RESULTS

There were 22 439 subjects randomly assigned to receive ZV or

placebo (Supplementary Figure 1 - CONSORT diagram) Serum

samples were obtained in all subjects before and 6 weeks after vac-

cination VZV antibody was measured in the 10 immunology

subcohort and in patients with suspected HZ The ZV and placebo

recipients in the immunology subcohort were well matched by sex

age race and study completion Most subjects (94) were whiteand 62 were female (Supplementary Table 1 - demographics)

gt94 of subjects completed the study

At baseline (day 1) 5 subjects (3 ZV and 2 placebo recipients)

did not have VZV-speci1047297c antibody measured by gpELISA The

2 treatment arms were well matched before vaccination in the

distribution of high and low antibody titers (P = 84 χ2 test

for homogeneity) (Table 1) Six weeks after vaccination the

GMT was 660 gpELISA unitsmL in ZV recipients versus 293

gpELISA unitsmL in placebo recipients for an estimated GMT

ratio (ZVplacebo) of 23 (95 con1047297dence interval 22ndash24

P lt 001) which met the prespeci1047297

ed statistical criterion Half of the ZV recipients had at least a doubling of VZV antibody

titer The geometric mean fold rise (GMFR) in titer in ZV

recipients was 231 compared with no fold rise in placebo

recipients (P lt 025)

During the study 30 ZV and 99 placebo recipients developed

HZ 6 ZV and 10 placebo recipients developed HZ before col-

lection of their postvaccination blood sample and thus were ex-

cluded from the immunogenicity analyses (Table 2) For the

subjects included in the immunogenicity analyses HZ was















lt125 3 (03) 01ndash08 2 (02) 00ndash06



gt300 to le500 174 (155) 134ndash

177 192 (171) 149ndash

194

gt500 316 (281) 255ndash309 320 (285) 258ndash312



lt125 0 (00) 00ndash03 2 (02) 00ndash07




gt500 667 (613) 583ndash642 313 (288) 261ndash316



ge2 541 (498) 468ndash528 36 (33) 23ndash46

ge3 330 (304) 276ndash

332 4 (04) 01ndash

09ge4 221 (203) 180ndash228 3 (03) 01ndash08

ge5 166 (153) 132ndash175 3 (03) 01ndash08













GMT



GMFR from d 1













DISCUSSION











































immune response

























SupplementaryData






Notes














































Con1047298



References



3522271ndash84





2008 57(RR-5)1ndash30




2002 34726ndash34














825ndash35








1310ndash22






























lt125 3 (03) 01ndash08 2 (02) 00ndash06



gt300 to le500 174 (155) 134ndash

177 192 (171) 149ndash

194

gt500 316 (281) 255ndash309 320 (285) 258ndash312



lt125 0 (00) 00ndash03 2 (02) 00ndash07




gt500 667 (613) 583ndash642 313 (288) 261ndash316



ge2 541 (498) 468ndash528 36 (33) 23ndash46

ge3 330 (304) 276ndash

332 4 (04) 01ndash

09ge4 221 (203) 180ndash228 3 (03) 01ndash08

ge5 166 (153) 132ndash175 3 (03) 01ndash08













GMT



GMFR from d 1













DISCUSSION











































immune response

























SupplementaryData






Notes














































Con1047298



References



3522271ndash84





2008 57(RR-5)1ndash30




2002 34726ndash34














825ndash35








1310ndash22
























DISCUSSION











































immune response

























SupplementaryData






Notes














































Con1047298



References



3522271ndash84





2008 57(RR-5)1ndash30




2002 34726ndash34














825ndash35








1310ndash22












































Con1047298



References



3522271ndash84





2008 57(RR-5)1ndash30




2002 34726ndash34














825ndash35








1310ndash22

















j infect dis.-2013-levin-1386-90

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