Herpes simplex virus 2 (HSV 2) type specific antibody correlates of protection in infants exposed to HSV 2 at birth

(1)

Herpes simplex virus-2 (HSV-2) type-specific

antibody correlates of protection in infants

exposed to HSV-2 at birth.

R L Ashley, … , K Mohan, L Corey

J Clin Invest. 1992;90(2):511-514. https://doi.org/10.1172/JCI115888.

Western blot analysis was used to compare the herpes simplex virus (HSV)-2 antibody profiles of 40 infants less than 2 wk of age who had been exposed to maternal genital HSV-2 at birth. 4 mothers were HSV seronegative at delivery and seroconverted to HSV-HSV-2

("primary infection"), 9 had HSV-1 antibodies and seroconverted to HSV-2 ("nonprimary first episode infection"), and 27 were HSV-2 seropositive ("recurrent infection"). Neonatal

herpes infections developed in 1 of 4 infants of women with primary infection, in 3 of 9 infants of women with nonprimary first episode infection, and in none of the 27 infants of women with recurrent HSV-2. Antibodies to HSV-2 proteins gG-2, VP5, and ICP35 were detected in 83, 89, and 72% of the 36 uninfected infants, respectively. None of the four infected infants had detectable antibodies to gG-2 and only one (25%) had antibodies to VP5 or ICP35. The more limited profiles of the 13 infants born to mothers with first episodes of HSV-2 were then analyzed separately; these profiles were similar among infected and uninfected infants except for gG-2, which elicits antibodies that are type specific for HSV-2. None of the infected infants versus seven of nine (78%) uninfected infants were gG-2

seropositive. These comparisons suggest that maternal type-specific antibodies may play a role in preventing neonatal infection after exposure to HSV-2.

[…]

Research Article

(2)

Herpes Simplex Virus-2 (HSV-2)

Type-specific

Antibody Correlates

of Protection in Infants Exposed

to

HSV-2

at

Birth

Rhoda L. Ashley,*Julie Dalessio, * SandraBurchett,t Zane Brown, Sylvia Berry, Kathey Mohan, * and LawrenceCorey*

Departmentsof *Laboratory Medicine,tPediatrics, ~ObstetricsandGynecology,andI1Medicine,

Universityof Washington, Seattle, Washington 98105

Abstract

Western blot analysis was used to compare the herpes simplex virus (HSV)-2 antibodyprofiles of 40 infants < 2 wk of age who had been exposed to maternal genital HSV-2 at birth. 4 mothers were HSV seronegative at delivery and seroconverted toHSV-2("primary infection"),9 had HSV-1 antibodies and seroconverted to HSV-2 ("nonprimary first episode

infec-tion"), and 27 were HSV-2 seropositive ("recurrent

infec-tion").Neonatal herpesinfectionsdeveloped in 1 of 4 infants of women withprimary infection, in 3 of 9 infants of women with nonprimaryfirstepisodeinfection, and in none of the 27 infants

ofwomenwith recurrentHSV-2.Antibodies to HSV-2 proteins

gG-2, VP5,andICP35weredetected in83,89,and72% of the

36 uninfectedinfants, respectively. None of the four infected infants haddetectable antibodies to gG-2 and only one (25%) had antibodies to VP5 or ICP35. The more limitedprofilesof the 13 infants born to mothers withfirst episodesof HSV-2 were then analyzed separately; these profiles were similar amonginfectedand uninfected infantsexceptforgG-2, which elicits antibodiesthat are typespecificfor HSV-2.Noneof the

infected infants versus sevenofnine (78%)uninfectedinfants weregG-2 seropositive. These comparisons suggest that mater-naltype-specificantibodies mayplay a role inpreventing neona-tal infection afterexposure toHSV-2. (J. Clin.Invest. 1992. 90:511-514.) Keywords:neonatal-herpeshominis- serology-transplacental immunity

Introduction

Previous studies have shown an inverse correlation between

titers ofpassively acquired maternal neutralizing antibody and acquisition of infection in theneonate (1-3). High levels of acquiredantibodyhavebeen associated with milderoutcomes

afterneonatal infection insome cohorts(1,4, 5) butnotall (6). Inneonatalmice thepresenceof antibodiesthatmediate antibody-dependentcellcytotoxicity(ADCC)'and

neutraliza-A portion of the data in thismanuscript waspresentedat theXVI InternationalHerpesvirus Workshop, 7-12 July 1991,Asilomar, CA,

andappeared in abstract form(Abstract 7B-02).

Addresscorrespondenceto RhodaAshley, Ph. D., University of

Washington, Virology Division, c/oChildren'sHospitalandMedical Center,D-536, 4800SandPoint Way NE,Seattle,WA98105.

Receivedfor publication19September1991andinrevisedform10 January1992.

1.Abbreviations used in this paper:ADCC,antibody-dependentcell

cytotoxicity;HSV, herpessimplexvirus.

tion are associated with protection against viral challenge (7,

8).Neutralizing antibody activity and ADCC activityare

pri-marily directed against herpes simplex virus (HSV) glycopro-teins(9). The viralglycoprotein targets ofthese protective anti-body activities are now being elucidated at the epitope level in themousemodel(10).

Ina recentprospective study,weshowed that infants born to motherswith first episodes ofgenital HSV-2had amarkedly higher incidence of infectionthanthose born to mothers with recurrent HSV-2 infections, suggesting that type-specific anti-bodies are particularly important in protecting the neonate after exposure( 1). Women who acquire HSV-2 close to term

andwho areseropositive forHSV- 1 have an equal risk

oftrans-mittingtotheirneonates as do seronegative mothers with pri-maryHSV-2infectionsat term ( 11 ).This findingsuggests that

passively transferred maternal HSV-l antibodies confer little protection againstneonatal HSV-2infection.Sullender andhis

colleagues ( 12) havereported a higher prevalence of antibody to oneHSV-2glycoprotein, gG-2,in healthy infants exposed to

HSV-2atbirth than in infants infectedatbirth.Thepossible protective effect of passively transferred antibodies to other

HSV-2proteins remains unclear. Therefore, weuseda sensi-tiveWesternblot methodtodescribethepassively transferred antibody profiles of 40infantsexposedtoHSV-2 at birth. Nine

major HSV-2 proteintargets wereidentifiedin these profiles and the prevalence ofantibodies to theseproteins was com-pared betweeninfectedanduninfected infantsas well as be-tweeninfants whose mothers had first episode HSV-2

infec-tionsand thosewhosemothershad recurrent HSV-2episodes

atterm.

Methods

Study population.Infants exposed to HSV-2 atdeliverywereidentified

aspart ofaprospective studyof allparturientwomenwithout clinical evidence ofgenital herpeswho entered theUniversityofWashington

Medical Center oraprivatecommunity hospital between 1982 and 1989 ( 1).Cervical and vulvarspecimenswereobtainedfor viral cul-ture. Of 15,923 mothers with viral cultures who delivered live born infants within 48hof culture, 56 (0.35%) had HSV isolated from their

genitaltracts atentry into thedelivery suite. 51of the 56 hadHSV-2 and 5hadHSV-l by monoclonalantibodysubtyping (13).Allinfants

exposedtoHSVwereimmediatelyrecalled for viral cultures andserum collection. Of the 51 infants exposedtoHSV-2,40 hadseraavailable fortesting.Maternalserumwasalso drawnat6wkafter delivery.Sera werecoded and frozen at -20°C.

NeonatalHSVinfectionwasdefinedastheisolation of HSV from theneonateafter24 hof life. All such infantswereimmediatelystarted onsystemicantiviraltherapyaccording totheguidelinesof the Na-tional Institute ofAllergy and Infectious Diseases Neonatal HSVStudy

Group ( 14). Of the 40 infantsonthisstudy,4developedHSV-2 infec-tionsand36remained uninfected.

Westernblotanalysis. HSV-2-infectedcellproteinswere electro-phoreticallyseparated and transferredasdescribedpreviously( 15,16).

J.Clin.Invest.

0021-9738/92/08/0511/04 $2.00

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Briefly, detergentlysate of infected cells was denatured and electro-phoresedfor 1.5 h at 25 mA per gel in a minigel apparatus (Hoefer

ScientificInstruments, SanFrancisco,CA) thentransferredto nitrocel-lulose(Schleicherand Schuell, Keene, NH) in Towbin buffer for 2 h at 200 mAin aTransphor apparatus(Hoefer Scientific Instruments).

Nitrocellulose was cut intostrips("blots") for the solid-phase immuno-assay.

Amodification of the immunostaining protocol was used to in-creasesensitivityand decrease the volume of sera needed for the test.

Briefly,sera were diluted 1:200 inI mlofBlotto(1% nonfat milk in

PBS)and incubatedat roomtemperature overnight with individual

HSV-2 blots. Strips were then washed with PBS containing 0.5% Tween 20, rinsed with PBS, and incubated 1.5 h withhorseradish

per-oxidase-conjugated goatanti-human IgG (diluted 1:10,000 in PBS

containing4% goat serum;BoehringerMannheim Biochemicals,

In-dianapolis, IN). After washingandrinsingasabove,TMB Membrane

Peroxidase Substrate (3', 3, 5', 5-tetramethylbenzidine; Kirkegaard

Perry Laboratories, Gaithersburg, MD)wasmixed accordingtothe

manufacturer'sinstructions and addedtotheblots for 5 min. The reac-tionwasstoppedbyrinsingwith distilledwater.

Allsera weretestedonthesamedayusingthesamelotof HSV-2 Western blots.Additionalblotswerestained usingmousemonoclonal antibodiestogG, VP16,andgD.Reactive bandsweredetected with horseradish peroxidase-conjugated goatanti-mouseIgG ( 1:1,000in PBS;Boehringer Mannheim Biochemicals)toconfirmtheidentityof

immunoreactive bands( 17). Glycoprotein gI was identifiedby the

relative migration characteristics ofaglycosylated protein( 18); the

identityofthisbandis, therefore, consideredpreliminary.

Nucleocap-sidproteinsVP5andICP35wereidentifiedbytheir lack of

glycosyla-tionand by theirmigration characteristics in bis-acrylamide cross-linkedgels ( 19). Serawerecodedsothat bandswerescored without knowledge of either infantoutcome ormaternalserostatus.

Statisticalmethods.Fisher'sexactanalysiswasusedtoanalyze

dif-ferences inprevalenceof antibodies between infants whodeveloped

infectionsandthose whodidnot.Chi square withYates' correctionwas usedtoanalyze antibody prevalencedata between infants of mothers with recurrent HSV-2 infection and those whose mothers had first

epi-sode HSV-2 infectionatdelivery.

Results

Classification ofmaternal HSV-2 infections. All 40 mothers

had HSV-2isolated fromgenitalsecretionssampled in the 48

hours before delivery. Comparison of theHSVserologic

pro-files of the motheratthe time of delivery and postpartum

re-vealed seroconversionto HSV-2in 13 and persistently stable HSV-2 antibodies in 27.4ofthe 13womenwhoseroconverted postpartum had eithernoHSVantibodiesorevidence of early HSV-2 seroconversion ("primary infection"). Nine women

hadafull complement of HSV-I antibodies in theirentry sera

andseroconvertedtoHSV-2-("nonprimaryfirstepisode HSV-2"). One of the four infants born to amother with primary

HSV-2 subsequently developedneonatal herpes asdidthreeof the nine infantsborn tomothers with nonprimary first episode HSV-2 infections.Theremaining27mothershad HSV-2 anti-bodiesonentryintothedeliveryroom,shed HSV-2atdelivery,

and hadunchangedWestern blotprofilesatpostnatal

follow-up indicating they had reactivation oflatent genital HSV-2

infectionsatterm. None ofthe infants ofthese 27 mothers with recurrentHSV-2episodes developed neonatalherpes.

PrevalenceofHSV-2

protein-specific

antibodies in infants.

Toevaluate theassociationbetween HSV-2antibodiesand dis-ease acquisition, we analyzed theWestern blotprofiles from

thefirstavailable infantsera.The time from deliveryto collec-tionofsera was 1-7 d in 28 infants, 8-14 d in 10, and 15din2

infants (median5.5d). Onset of infection occurredat3, 5, and

1 I dofagein threeneonates. Afourthwasculturepositiveone

day after delivery butdidnotdevelop symptomsbeforebeing

lostto follow-uponday4. Amongthe threesymptomatic in-fected infants,sera weretestedat3, 7,and14d oflife,

respec-tively. The fourth,

asymptomatic

infant hadserumcollectedat

day 3 of lifebeforehis releasetocareinanothercity.

When the 13 infants born to mothers with first episode

HSV-2 infectionswereanalyzed,the incidence ofantibodiesto VP5, gB, gC, gE,gI,VP16, gD, and ICP35 didnotdiffer

signifi-cantly betweeninfectedanduninfectedinfants(P20.05)

(Ta-bleI).However, infected infants hada

significantly

lower inci-dence ofantibodiestogG-2 (0of4versus7of9;P<0.05).As

expected, the 27 infants borntomothers withrecurrent infec-tionhadasignificantly higher prevalenceofantibodiestoVP5

(P < 0.0001), gG (P < 0.001), gC (P < 0.0005), gD (P

= _{0.05), and ICP35 (P} < 0.0001) than did the 13 born to

motherswho hadfirst episode infectionatterm.Ofinterest, the incidence ofantibodies toHSV-2 gB, gE, gI, and VP16 were not significantly different between infants born to mothers

withrecurrentorfirst episode HSV-2 infections(P >0.05).

When theprevalence values of the4infected and 36 unin-fected infants from theentirestudypopulationwerecompared,

the mostdramatic differenceswere seenin theprevalenceof

antibodiestogG-2, VP5,andICP35.Noneof theinfected in-fants had detectableantibodiestogG-2while 34 of 36(94%) uninfectedinfants hadgG-2 antibodies. 1 of4(25%)infected infants had antibodiesto VP5 orICP35versus29of 36 (8 1%) uninfected infants.

Antibody profilesin

infected

versusuninfected infants.

Pas-sivelytransferredantibodyprofileswereverylimited in infants bornto mothers withprimaryfirstepisodeHSV-2 infections (Fig. 1 A). Theinfected infant from thisgrouphaddetectable antibodiesagainstgB, gE, and gD(Fig.1A,lane1).Twoother infantsborn tomothers with primary infections hadantibodies

TableI.AntibodyProfilesfrom Infants with MaternalHistory

ofFirstEpisodeorRecurrentHSV-2Genital Infections

Numberof infants with antibody to HSV-2 protein (%)

Maternal first episode Maternal first episode Maternalrecurrentepisode

Infantinfected Infant not infected Infant not infected

HSV-2

protein n=4 n=₉ n=27

VP5 1(25) 3 (33) 27 (100)*

gB 4(100) 6(67) 21 (78)

gG-2 0(0) 7(78) 27(100)*

gC 2(50) 4(44)t 27(100)*

gE 2 (50) 4(44) 22 (81)

gI 1(25) 2(22) 9(33)

VP16 2(50) 7(78) 19 (70)

gD 3(75) 7(78) 27

(100)'

ICP35 1(25) 3 (33) 26(96)*

*_{P <}

0.001

_{by Chi square with Yates' correction comparing antibody}

prevalences of infants born to mothers with recurrent episodes (n

=_{27) and antibody prevalence of infants whose mothers had first}

episodeHSV-2infection (n = _{13). $ P}_<_{0.05 by Fisher's exact test}

comparingantibody prevalence in infected (n=_{4) versus uninfected}

(n=_{9) infants born to women with first episode HSV-2 infections.}

§ P=_{0.05 by Chi square with Yates' correction between infants of}

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A

Infected Uninfected

gq a-

gG-gE

gB -gG --gE

gG

-VP16 ~-VP16--

qDgD----gD

1 2 3 4 5

B

C

Infected Uninfected Un intected Figure1.Antibody profilesfrom infected anduninfected infants.Antibody profiles '.9* from infected infants(lanes1, 7,and8)

VP5-

_-gs

VP5 _werew _compared _{with those of uninfected} 9G--E^gG-- gB_ Ok infants(lanes3, 5,and9-12).Infants from

gE g gC hi mothers withprimaryHSV-2 infections

-'-VP16_I-VP16-

gE

_VP16-

gE16

by gE - (A), nonprimary firstepisodeHSV-2

in-Om

-gD- gD-- gD-- VP16 " fections(B),and recurrent HSV-2

infec-is

l gD-a tions(C),reflectincreasinglevels of

reac-* i S tive antibodies. Multiple gels were required JICP35S ICP'35 for thisstudy;blotsfromeachgelwere

reacted withmonoclonalantibodies to

gG-iii & 2(lanes2 and6)and

VPl6

(lane4)to

localizeproteins onproilesfrom the

re-6 7 8 9 10 11 12 spectivegels.

togG-2, VP16, andgD(Fig. 1 A, lanes3and 5). Thefourth infant had no detectable antibodies against HSV-2 proteins andremaineduninfected.

The HSV-2 antibody profiles of infants born to women with nonprimaryfirstepisodes had more bands than were seen with sera from infants born to mothers who had primary

infec-tions(Fig. 1B). Profiles fromtwoof the three infected infants bornto women with nonprimary first episodes of HSV-2 in-cludeantibodiestogB,VP16, (lanes7and8) and, inonecase,

several other viral proteins (lane 8). Neither had detectable antibodytogG-2. Profiles fromtwouninfectedbabiesrevealed

antibodiestogB,gG-2, gE,and gD(lanes9and10). Antibod-iestootherproteins suchasVP5, VP16, and ICP35were pres-entinsome, but notall,sera(Fig. 1 B, lane10, andTableI). Antibody profiles in infantsborn to womenwithrecurrent

genital HSV-2 infections reflectedthepassive transfer of

abun-dantantibodiesto alargenumberof viralproteintargets(Fig.I

C, lanes 11 and12).Nodifferences in number of HSV-2

pro-teintargets wereobserved inantibody profiles of infants with both HSV-1 and HSV-2 passive antibodies and infants with only HSV-2 antibodies (datanotshown).

Discussion

Two aspectsofthis trialareunique:theabilitytoidentifyand

testinfants exposedtoHSV-2atbirthand theabilitytoidentify passive antibodytoninemajorHSV-2immunogensininfants

whodid ordid notdevelop HSV-2 infections afterexposure. WeusedWesternblottechniquestodetermine whetherpassive antibodyprofilesdiffered in the4infantswhodidandthe 36 infants who didnotdevelop herpes infections afterexposureto

HSV-2 atbirth. We found thattheprevalence of detectable antibodytomostHSV-2proteinswassimilar between infected anduninfected babies. However, antibodiestogG-2wereless likely to bedetected in samples from the infected infantsas

compared with uninfected infants. Ourpreviousstudies of an-tibodyresponses in adults have shown thatantibodiestogG-2,

asdetectedbyWesternblot,donotariseasearlyduring

sero-conversionasdoantibodiestogBorgD(20).Sinceallofthe

infected infantswereborn to mothers whowerein the process

ofseroconvertingtoHSV-2,theseinfantswould be lesslikely

to have these "late" antibodies to gG-2. However, when

in-fected (n=₄₎versusuninfected(n=₉₎infantsoffirst_episode,

seroconvertingmotherswerecompared,the markeddifference

inantibodyprevalence togG-2 remained (0%versus78%for infectedversusuninfected,respectively).

Detectable antibody to gG-2 was highly correlated with protection fromdevelopinginfection after HSV-2exposure.Of

13infantswhose mothers wereseroconvertingto HSV-2, seven hadantibodiestogG-2; none becameinfected.Ofthesix in-fants whowereseronegative for gG-2, four (67%)became in-fected whiletwo(33%)remained uninfected. While it is possi-ble that thesixgG-2 seronegative infantsmayhave had anti-body levelstoolowfor detection by our-Westernblot assay, our

data indicateadefinitequantitative differenceintheantibodies passively transferredtoinfectedversusuninfected infants. Be-causeIgG classantibody hasahalf-life of - 21 d, it isunlikely

thattheapparentdifferencesingG-2antibodyprevalencewere dueto decayof maternal antibody. Our findingsextend those

of Sullenderetal.usinganenzymeimmunoassay for gG-2in which 30of34 (88%) infantsexposed to HSV-2 atbirthhad

gG-2antibody and remained well whileoneinfantwho devel-oped neonatalherpeslackedantibodytogG-2 (12).

Protective factorsother thandetectable levelsof passively transferred antibodytogG-2wereclearlyoperative forthe two

infants inourstudy and thefourinfants in Sullender's study

who were gG-2seronegative anduninfected.Human to mouse

adoptivetransfer studies have shown thatprotective immunity

against HSV infection isamultifactorialsysteminvolving

mac-rophages, CD-4 lymphocytes, lymphokines including IFN-gamma andIL-2,andantibody(21). The roleofantibodyin this modelmaybeviaanantibody-dependent cellcytotoxicity

function (8).High titers of maternally derived ADCC antibod-ies have been associated with protectionininfants exposedto HSV at birth and to local versusdisseminated infections in those infantswhodeveloped disease(22).AntibodiestogG-2

have not been shown to have either neutralizing or ADCC

activityin vitro.

Itispossible that gG-2antibodieshaveserved inour West-ernblot system as markersofmoresubtletype-specific antibod-ies with neutralizing, ADCC, or otherbiologic functions

di-rectly involvedin preventing infection. BothgB andgD, for example, haveneutralizingand ADCC functions. Antibodies elicited byHSV- 1infectionsbutdirectedagainsttype-common

epitopesmakeitdifficulttodetecttype-specificseroconversion

toHSV-2gBandgDbyWesternblot(23). Inthisstudy, for example, of nine infantsbornto HSV-1

seropositive

mothers who wereseroconvertingto HSV-2, six (67%)haddetectable

(5)

antibody reactive with HSV-2 gD. These antibodies could be to either type-common epitopes or to type-specific epitopes; West-ern blot cannot discWest-ern antibodies to type-specific epitopes, nor canit detect antibodies to conformational epitopes that may havebiologic significance. Clearly, the relative roles ofantibod-ies totype-specificortype-common epitopes of gG-2, gB, gD, orother HSV-2proteinswill bedetermined only with assays basedontype-specificepitopes from individualproteins.

Whileevidencefrom animal modelsconfirmsthe protec-tiveeffectof passively transferred antibodies to individual pro-teins ( 10), theprotective effectdepends upon the dose of chal-lenge virus and the relative competence of other armsofthe immunesystem. The absenceofmaternallyderived antibody

togG-2 may defineagroup ofinfantsathigherrisk ofinfection after HSV-2 exposure. Yet there is no evidence thatantibodies

togG-2 alone preventinfection.Maternalvirustiter, lengthof time ofvirus exposure, and the presence of genital mucosal

antibodies toHSV (24), are other factors which may affect

transmission.The roleofserumantibodyinpreventing

infec-tionin the human neonate remainsanimportantquestion be-cause antiviral therapy has not been completely effective in

preventing morbidityandmortalityofsymptomaticherpes

in-fections in the neonate.Immunoprophylaxis regimensarean

appealing potential option for exposed neonates. Our work suggests thatantibodiestotype-specific epitopes (which

pre-dominateongG-2)may beofparticularvalue in immunopro-phylaxis. Identification of otherprotein- andepitope-specific

antibodies that blocktransmissionis necessary tocombine op-timalantibodycomponents forprotective passive immuniza-tion.

Acknowledgments

Thisworkwassupportedby Herpes ProgramProjectgrant AI 30731to L.Corey andbyaMarchofDimes BirthDefects Foundationgrantto Z. Brown.

References

1.Yeager,A.S.,A. M.Arvin,L. J.Urbani,and J.A.Kemp.1980. Relation-ship of antibodytooutcome in neonatalherpes simplexvirusinfections.Infect. Immun.29:532-538.

2.Yeager,A.S.,and A. M. Arvin. 1984. Reasonsforabsenceofahistory of recurrentgenital infections inmothersofneonatesinfectedwith herpessimplex virus.Pediatrics. 73:188-193.

3.Prober, C.G.,W.M.Sullender,L.L. Yasukawa, D.S.Au, A. S. Yeager, and A. M. Arvin.1987.Lowriskofherpessimplex virus infectionin neonates exposedtothevirusatthetime ofvaginal deliverytomotherswithrecurrent genital herpes simplex virus infection.N.Engl.J.Med. 316:240-244.

4.Sullender,W.M.,J. L.Miller,L. L.Yasukawa,J.S.Bradley,S. B. Black,

A.S. Yeager, and A. M. Arvin. 1987.Humoralandcell-mediated immunity in neonates withherpessimplex virusinfection.J.Infect.Dis. 155:28-37.

5.Kohl, S., M. S. West, C. G. Prober, L. S. Loo, W. Sullender, and A. Arvin. 1989. Neonatal antibody-dependent cellular cytotoxicity antibody levels are

asso-ciatedwiththeclinicalpresentation ofneonatalherpes simplexvirusinfection.J. Infect.Dis.160:770-776.

6.Whitley,R.J.,A.J.Nahmias,A. M.Visintine,C. L.Fleming,and C.A.

Alford.1980. The naturalhistory ofherpessimplexvirusinfection ofmother and newborn.Pediatrics.66:489-494.

7. Baron, S., M.G. Worthington,J.Williams,and J. W.Gaines.1976. Post-ex-posure serumprophylaxis ofneonatal HerpesSimplex infection ofmice. Nature (Lond.).261:505-506.

8.Kohl, S.,and L. S. Loo. 1982.Protection ofneonatalmiceagainst herpes simplexvirusinfection: probablein vivoantibody-dependent cellular-cytotoxic-ity.J.Immunol.129:370-376.

9.Norrild,B.,S. Shore,and A.Nahmias. 1979. Herpes simplexvirus glyco-proteins: participation of individualherpessimplex virustype 1glycoprotein antigensinimmunocytolysisandtheir correlationwithpreviously identified gly-coprotein.J. Virol.32:741-748.

10. Kohl, S., N. Strynadka, R. Hodges, and L. Pereira. 1990.Analysis ofthe roleofantibody-dependentcellularcytotoxic antibodyactivityin murine neona-talherpes simplexvirusinfection withantibodiestosynthetic peptidesof glyco-proteinD andmonoclonal antibodies toglycoproteinB.J.Clin.Invest. 86:273-278.

11.Brown, Z. A., J.Benedetti,R. L.Ashley, S.Burchett,S.Selke,S. Berry, L. A.Vontver, and L.Corey. 1991. Neonatalherpes simplexvirusinfectionin relationtoasymptomaticmaternalinfectionatthetime oflabor.N.Engl.J.Med. 324:1247-1252.

12.Sullender, W. M., L. L. Yasukawa, M. Schwartz, L.Pereira,P.A. Hens-leigh,C. G. Prober, and A. M. Arvin. 1988.Type-specificantibodiestoherpes simplex virustype 2(HSV-2)glycoproteinGin pregnant women,infants

ex-posedtomaternal HSV-2infectionatdelivery,andinfants withneonatalherpes. J.Infect.Dis. 157:164-171.

13. Petersen, E. P.,0.W.Schmidt,L.C.Goldstein,R. C.Nowinski,andL. Corey.1983.Typing of clinicalHSVisolatesusingmousemonoclonal antibodies toHSV-I andHSV-2:comparison with type-specific rabbit antiseraand restric-tion endonucleaseanalysisofviral DNA. J.Clin.Microbiol. 17:92-96.

14.Whitley,R.,A.Arvin,C.Prober, S.Burchett,L.Corey,D.Powell,S. Plotkin,S.Starr, C.Alford,J.Connor, et al. 1991.Acontrolledtrialcomparing vidarabinewithacyclovirin neonatal herpes simplex virusinfection.N.Engl.J. Med.324:44- 449.

15.Ashley, R. L., J.Militoni,F.Lee, A.Nahmias,andL.Corey.1988. Com-parison ofWesternblot(immunoblot)andglycoproteinG-specificimmunodot enzyme assayfordetecting antibodiestoherpes simplexvirus types 1 & 2 in human sera. J.Clin. Microbiol. 26:662-667.

16.Ashley,R.L.1989.GenitalHerpesInfections.InClinicsin Laboratory Medicine: Sexually Transmitted Diseases.Vol.9. F. N.Judson, editor.W. B. SaundersCo., Philadelphia,PA.405-420.

17.Ashley, R., K. Mack,M.Shurtleff,C.Critchlow,and L.Corey. 1988. Differential effect ofsystemic acyclovirtreatmentofgenitalHSV-2infectionson antibodyresponsestoindividualHSV-2proteins.J.Med.Virol. 4908:703-708. 18.Longnecker,R., S.Chatterjee, R. J.Whitley,and B.Roizman. 1987. Identificationofaherpes simplex virus1glycoproteingenewithinagene cluster dispensable for growthincell culture. Proc.Natl.Acad. Sci.USA. 84:4303-4307. 19. Braun, D. K., B. Roizman,and L.Pereira. 1984.Characterization of post-translationalproducts ofherpessimplexvirus gene35proteins bindingto thesurfacesoffull capsidsbutnotemptycapsids.J. Virol.49:142-153.

20. Lopez,C.,A.Arvin,and R. L.Ashley. 1992. Immunity toherpesvirus infectionsinman.InThe Human Herpesviruses: Biology, Pathogenesis,and Treatment. B.Roizmanand C. Lopez,editors.RavenPress, New York.

21.Kohl,S.,L.S. Loo, and S. B. Greenberg. 1982.Protection ofnewborn micefromalethalherpes simplexvirusinfectionby humaninterferon, antibody, andleukocytes.J.Immunol. 128:1107-1111.

22.Kohl, S. 1989. The neonatal human's immune response to herpes simplex virus infection:acriticalreview. Pediatr. Infect.Dis. J. 8:67-74.

23.Ashley, R.,and J.Militoni. 1988.Useofdensitometric analysisfor inter-preting HSV serologiesbasedonWestern blot.J.Infect.Dis.157:319-326.