Comparison of antibody dependent cellular cytotoxicity and complement dependent antibody lysis of herpes simplex virus infected cells as methods of detecting antiviral antibodies in human sera

CopyrightC 1977 AmericanSociety forMicrobiology PrintedinU.S.A.

Comparison

of

Antibody-Dependent Cellular

Cytotoxicity

and

Complement-Dependent Antibody Lysis

of

Herpes

Simplex Virus-Infected Cells

as

Methods of Detecting

Antiviral

Antibodies

in

Human

Sera

THANGAMANI SUBRAMANIAN ANDWILLIAM E. RAWLS* Department of Pathology,McMasterUniversity, Hamilton, Ontario,Canada

Received for publication24January 1977

Anantibody-dependent cellular cytotoxicity (ADCC) assaywasused todetect antibodiestothe herpes simplex virusesinhumansera. The

assay

utilized the

release of 5'Cr fromBHK-21cellsinfected with the viruses, hamsterperitoneal

exudate cells as effector cells, and antiviral antibodies in human sera. The

technique was found to be far more sensitive than complement-dependent antibody lysis of infected cells and virus neutralization. The ADCC assaywas

useful in detecting antibodies in sera that had low titers or no antibodies

detectable by other methods. In a sample of100serafromuniversity

students,

40

were positive by complement-dependent lysis whereas 73 were positive by ADCC. Of400serafromwomenwith cervical cancer, 17didnothave detectable antibodies bymicroneutralization orcomplement-dependent lysis; however,all sera werepositiveby ADCC, suggesting that all of thewomenhad beeninfected inthe past withone orboth types of herpes simplex virus.

The percentage of adults without detectable antibodiestoherpes simplex viruses (HSV) has

been found tovaryin

seroepidemiological

stud-iescarriedout indifferent populations (4, 6,

10-13,15-19, 21, 28, 31). Since minimalfluctuation

in antibody titers has been observed in most individuals followed prospectively (3, 5, 7-9,

25),theabsence of detectable antibodies implies that the individual had not been infectedinthe

past. These seroepidemiological observations suggest population differences in past infec-tions with the viruses; however, it is possible thatantibodies donotpersistat high levels in

all infected individuals (3, 8, 30). Thus, the

differences

in

detectable

antibodies may reflect

notonlydifferencesinpast infection rates, but also differencesin antibody persistence.

Recently, it was demonstrated that cell-me-diated lysis of antibody-coated target cells is a

sensitive method of detecting antibodies to

HSV (20, 24, 27). In the present study, the

antibody-dependent

cellular cytotoxicity (ADCC) techniquewas utilized forquantitation ofHSVantibodiesinhumansera. Asreported by others (24, 27), wefound the ADCC method

tobe much more sensitivethan virus

neutrali-zationorcomplement-dependent antibody lysis of virus-infectedcells. Inaddition, asignificant proportionofserafromadults without

detecta-bleantibody bythelatter methodswere found

to containantibodies detectable by the ADCC

technique.

MATERIALS AND METHODS

Sera. Selectedconvalescent-phase sera from

pa-tients who experienced clinically apparent infec-tions withHSVtype 1(HSV-1)orHSVtype2 (HSV-2)wereusedtoexaminethevariables of the ADCC technique. Serum sampleswerecollected from 100 students attending McMaster University. These samples were collected without regard to specific illness from studentsattending the health clinic and

werefelttoberepresentative of the study group. In addition, selected sera from previously described studieswereutilized(12,22, 29). These included 30

sera(12)and 34 sera(22)found tohavelowtitersor

nodetectableantibodies to HSV-2 by microneutrali-zation, and 60 sera (29) found to have low or no

detectable antibodies by complement-dependent an-tibody lysis of virus-infected cells. The sera were storedat -35°C and heat inactivatedat56°Cfor30 minprior touse in the assay procedures.

Target cells. The KOS strain of HSV-1 and the HV-219 strain of HSV-2 were used (26).Monolayers

of BHK-21 cells in 75-cm2 tissue culture flasks (Corning Glass Works,Corning,N.Y.)wereinfected

atamultiplicity of 3 to 5plaque-forming units per cell, and adsorption of the viruswasfor1h at23°C. Themonolayers were then overlaidwithEagle

min-imal essential mediumsupplemented with5%fetal bovine serum and antibiotics(penicillin, 100U/ml;

streptomycin,100iLg/ml).Tothecultureswasadded 200

IACi

of51Crassodium chromate (NewEngland 551

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Nuclear Corp., Boston, Mass.), and the cultures wereincubatedat37°C. After18hof incubation, the cells were monodispersed by using 0.025% trypsin and0.02% ethylenediaminetetraacetate in buffered saline without calcium and magnesium ions. The cells were then washed five times with tris(hydroxymethyl)aminomethane-buffered saline containing 2% fetal bovine serum. After the final wash, the cellsweresuspended in minimal essential medium with10%fetal bovineserumata concentra-tion of 105 viable cells per ml. BHK-21 cells not infected withviruswerelabeled with 51Crina simi-larfashion and served ascontrols.

Effector cells. Peritoneal exudate cells from golden Syrian hamsters (Lakeview/Charles River, Newfield, N.J.) were used as effector cells. Adult hamsterswereinjected intraperitoneally with 5 ml ofsterile paraffin oil. Theanimalsweresacrificed 3 days later, and the peritoneal exudate cells were harvested from the peritoneal cavity by lavage with Hanks buffered saline. The cellswerewashed twice with buffered saline and suspendedatthe required concentration in minimal essential medium con-taining 10% fetal bovineserum.

ADCC assay. The test serum was appropriately diluted in minimal essential medium containing 10% fetal bovine serum. To 0.2 ml of the diluted

seruminglass tubes (12 by75mm)wereadded0.4 ml oftarget cells and 0.4 ml of effector cells. The tubes were gently shaken and then incubated at 37°C inahumidatmosphere of95%air and 5%CO2. Atthe end of the incubation period (usually8or20 h), the tubes werethoroughly agitated and centri-fugedat1,500rpmfor 3min(IEC PR-J centrifuge)to sediment unlysed cells and cell debris. A 0.5-ml samplewascarefully removed from thetopof the 1-mlreaction volume and transferredto aclean test tube. The radioactivity of each of the 0.5-ml frac-tionswascountedinaBeckman automaticgamma counter. The percent specific release of 51Cr was calculatedby the formula:

%specificrelease

2 x cpmoftopfraction

-x 100 cpmoftopfraction

+ cpmofbottomfraction where cpm is counts per minute. As controls, all assaysincludedreactionmixtures to which diluent

was added instead of effectors cells or instead of

serum and in which uninfected cells were substi-tuted fortargetcells.Theassayswereperformedin triplicate, and the valuesfrom the three determina-tionswereaveraged. Analysisof variations of

tripli-catevalues indicated thatspecificrelease ofgreater than5%above control valueswasunlikelytooccur by chance (P<0.01)and,therefore,wereconsidered positive for antibody activity.

For quantitation, the data werenormalized and expressedas percentcytotoxicity. Thiswas accom-plished by assigning the value of 0%tothe releasein control mixtures containing known antibody-nega-tive serum and 100% to the release observed in mixturescontainingexcessantibody. The reciprocal

of theserumdilutionthatproduced50%cytotoxicity was taken as the titer ofantibody in the ADCC

assay. The method of expression of results is exem-plified in the data shown in Table 1. The percent specificrelease forcontrol mixtures was about 37, and that for the positive antibody control was 68. The values of 37 and 68% were taken as 0 and100%,

respectively, and the mean percent specific release forthe unknown,48, represents 35% onthisscale.

Complement-dependent lysis of cells by anti-body. Antibodiesto the surface antigen of BHK-21

cells infected withHSV-1 and HSV-2 were also

as-sayedby addingguinea pigcomplement to sensitized

cells. Detailsofthis procedure were previously

de-scribed (14).

Neutralizingantibodies. Thepresence of neutral-izingantibodies was detected by mixing virus and serumandthen assayingforsurviving

plaque-form-ing virus. Details of the plaque reduction test are

described elsewhere(23). RESULTS

Variablesof the ADCCtechnique. Since we wished to use the ADCC test for assaying anti-body activity, several combinations of readily available targetand effector cells were

exam-ined. These included BHK-21 and RK-13 cells infected with HSV-1 or HSV-2 as target cells and peritoneal exudate cells, peripheral blood lymphocytes, and spleens cells from rabbits and hamsters as effector cells. The most satisfactory resultswereobtained withvirus-infected BHK-21cells and hamster peritoneal exudate cells.

The influence of different concentrations of effectorcells was examinedby sensitizing 51Cr-labeled BHK-21 cells that had or had not been infected with HSV-1 or HSV-2; sensitization

was carriedout by incubating the target cells

TABLE 1. ResultsofatypicalADCC assayusing 51Cr-labeled BHK-21 cellsinfectedwith HSV-1 Effec- cpmof cpmof Spe- yo

:f ev Antibody bottom top cific toxic cells toHSV-1 frac- frac- release ityo

cells

~~~tion

tion M t %

1:50a None 3,511 792 37

3,482 788 37 0

3,540 796 37

None Positive 3,122 676 36

control 2,936 686 38 0

3,029 681 37 1:50 Negative 3,324 726 36

control 3,566 804 37 0

3,445 765 36

1:50 Positive 2,940 1,520 68

control 2,842 1,484 69 100

2,891 1,502 68 1:50 Unknown 3,158 1,010 48

serum 3,096 1,026 50 35

3,127 1,018 49

aFiftyperitoneal exudatecellsto onetarget cell.

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with 1:50 dilutions of sera for 1 h. Different numbers of hamster peritoneal exudate cells

were added, and 20 h later the lysis oftarget

cellswasassessed. Theoptimum target:effector cellsratio was foundto be1:50 forBHK-21cells infectedwith HSV-1 (Fig. 1)and1:200for

BHK-21 cellsinfected with HSV-2 (Fig. 2).

The rate ofdestruction of sensitized target cellswas examinedbysensitizingthecells with

serum,adding peritonealexudate cells at a ra-tio of 1:50 for HSV-1 and 1:200 forHSV-2, and

then terminating the reaction after various

pe-riods of incubation. Specific destruction was

evident after 4 h ofincubation and increased

rapidly thereafter (Fig. 3). Maximum specific lysis was attained by 8 h ofincubation, after

which increased lysis in the presence of

anti-body wasassociatedwith anincreased nonspe-cific lysis. Somewhat similar results were

ob-tained with cells infected by HSV-2; specific

lysis was initially detectedat 4 hand increased rapidly until8 h. Nonspecificlysisat 20hafter infectionwasless forHSV-2-infected cells than forHSV-1-infected cells (datanotshown). The

differences between lysisinthe presence of

an-tibodies and inthe absence of antibodies were

100

30

20n

K

/,

=

,

j

U a W 75 1W 12i 1W 175 200

TARGET:EFFECTORRATIO

FIG. 1. Effect of varying numbers of effector cells

on the lysis ofBHK-21 cells infected with HSV-1. Symbols: 0, BHK-21 cells infected with HSV-1 and sensitized with serum containingneutralizing

anti-bodies to HSV-1; A, BHK-21 cells infected with HSV-1 and sensitized with serum not containing detectable neutralizing antibodies to HSV-1; 0,

BHK-21cellsinfectedwithHSV-1 and not sensitized; A, uninfected BHK-21 cells sensitized with serum containing neutralizing antibodies to HSV-1.

TARGE: EFFECTOR RATIO

FIG. 2. Effect of varyingnumbers of effector cells on the lysis of BHK-21 cells infected with HSV-2. Symbols: 0, BHK-21 cellsinfected with HSV-2 ancs sensitized with serumcontainingneutralizing anti-bodies to HSV-2; A, BHK-21 cells infected with HSV-2 and sensitized with serum not containing neutralizingantibodies; 0, uninfected BHK-21 cells sensitized withserumcontaining HSV-2 antibodies.

NCAoNTIMEeI)

FIG. 3. Kinetics oflysis of BHK-21 cells infected

with HSV-1 by hamster peritoneal exudate cells.

Symbols: 0,BHK-21cellsinfectedwithHSV-1 and sensitized with serumcontaining neutralizing anti-bodies to HSV-1; A, BHK-21 cells infected with HSV-1 and sensitized with serum lacking neutraliz-ingantibodies toHSV-1; 0, uninfected BHK-21 cells sensitized with serumcontaining neutralizing

anti-bodies toHSV-1. VOL. 5, 1977

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maximal at 8 h forHSV-l-infected cells and at 100 20 hforHSV-2-infected cells.

The influence of decreasingconcentrations of

9o

-antibody isdepictedinFig. 4. Withincreasing serum dilution, there was a decrease in the

percent cytotoxicity. The reciprocal of the se- so / rum dilution that produced a 50% cytotoxicity

wastaken as the antibody titer. In the ADCC 70

assay, the slopes of the dose-response curves

wererelatively flat, andthe titersof sera upon g / repeat assays were more variable than the

titersobtained

by

complement-dependent

lysis.

g

/0

Antibody

titersfor thetype 1 serumusedinthe 0 50

experiment

shown in

Fig.

4 were 1:20

by

com- u

plement-dependent lysis and 1:290 by ADCC, ; 40 whereas the titers for the type 2 serum were W 1:125 by complement-dependent lysis and c 30

1:5,500byADCC.Thus,the ADCC method was l

found to be more sensitive than the

comple-ment-dependent lysis ofinfected cells by anti- 20

body.

Therate ofbinding ofantibody to the target 10 I

cells was found to be very rapid (Fig. 5). The

maximum rate ofbindingwasobservedinthe

first few minutes of the reaction, andbinding 0 10 20 30 40 50 00

REACTIONTIME(nu_tes)

_100 100 FIG. 5. Kinetics of binding ofantibodyto target

cells. The kinetics of antibodybinding to target cells

90.-90 was assayed by mixing 0.5 mlofa 1:5 dilution of

serumwith107targetcellsin0.5mlandincubating

so. so at4C. Atintervals,10p4ofthe reaction mixturewas

removed and diluted in10 mlof cold diluent. The 70 \ \ 'Q.\ 70 Q cells were washed three times and then mixed with

I:\l

\ > ¢

effector

cells. Thepercentcytotoxicityin these

experi-ments is

expressed

in relation tothe

cytotoxicity

ob-tained whenexcessantibodywas notremoved, which so

50 ~ t \ \ \ - " 2 was taken as 100%. Symbols: 0, BHK-21 cells in-u fected withHSV-2 and reacted with HSV-2 antise-40

o \ \ ' 40 i rum; 0, BHK-21 cells infected with HSV-1 and

re-U acted withHSV-1 antiserum.

~30 30

20 \ \< 20 was

essentially complete by

30 min. For the

!2~ \ \sera tested, 15 to 25% additional cytotoxicity

lo

10 S \D 10 wasobserved ifthe excessantibodies werenot

removed fromthe reaction mixture. The results

10 20 40 00

11,00

°0

I of these experiments also demonstrate that

SERUMDLUnON induction of cell cytotoxicity is brought about

FIG. 4. Effects of serum dilution oncytotoxicity of by antibodybound to thetargetcells and not by

BHK-21 cells infected with HSV-1 or HSV-2. Sym- a direct attachment of free

antibody

to effector bols: 0, BHK-21 cells infected with HSV-1, sensi- cells.

tized with varying dilutions of HSV-1 antiserum, Analysis of sera from college students.

and reactedwith effector cellsat a ratio of 1:50; 0, From the above analysis, a standardized test of BHK-21 cells infected with HSV-1, sensitized with ADCC was adopted in which HSV-1 target cells varying dilutions of HSV-1 antiserum, and lysed were reacted with effector cells at a ratio of 1:50 with complement; A, BHK-21 cells infected with

HSV-2, sensitized with varying dilutions of HSV-2 and

incubated

for 8 h, and HSV-2 target cells antiserum, andreactedwitheffector cells at a ratioof were

acd

withb

effetor

cells

atqaertio

of

1:200; A, BHK-21 cells infected with HSV-2, sensi- 1:200 and incubated for 20 h. Sequential serum tizedwithantiserum toHSV-2, and lysed with com-

samples

collected fromtwo

patients

with acute

plement. ulcerativepharyngitis were

analyzed

for

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ANTIBODIES TO HSV DETECTED BY ADCC ASSAY 555

bodies by the ADCC method and by comple-ment-dependent lysis. HSV-1wasisolated from

throat swabsofboth ofthese patients. The ki-neticsoftheappearanceofantibodieswere

sim-ilar in bothpatients by both techniques;

how-ever, thetiters obtained bythe ADCC method were muchhigher than those obtained by

com-plement-dependent lysis (Fig. 6).

In preliminary analysis ofsera from college

students, wefound a largepercentage

serone-gative by complement-dependent lysis (data

notshown). Therefore,sera werecollected from

100students and tested by

complement-depend-ent lysis and by ADCC at a dilution of 1:10

(Table2). All serapositive by

complement-de-pendentlysiswerealso positivebyADCC;

how-ever, 33 sera positive by the ADCC method

titered lessthan 1:10bycomplement-dependent lysis. These data suggest that a substantial

proportion ofthe student population had been infected with HSV-1 in the past but did not possess antibodies of sufficient titer to be de-tectedbycomplement-dependent lysis.

Analysisofserafrompatients with cervical

neoplasia. Antibodies to HSV-2 havebeen

as-sociated with squamous cell carcinoma ofthe

1/100

DAYSAFt IULNESS

FIG. 6. AntibodytitersdeterminedbyADCCand by complement-dependent lysis in sera ofpatients with acute herpetic pharyngitis. Symbols: 0, sera

frompatient1testedbyADCC;0,serafrompatient 1 testedbycomplement-dependent lysis; A,serafrom patient 2 tested byADCC; A, sera frompatient2

testedbycomplement-dependent lysis.

cervix,

yet

asmall percentage of

patients

with

cancer do not have detectable antibodies to

HSV-1orHSV-2. Toevaluate whether the

sero-negativity of thesewomenrepresented the

ab-sence of past infection or levels of

antibody

below detection

by

the usual assay

methods,

sera found

negative

by microneutralization

or

by

complement-dependent

lysis

were

analyzed

by

ADCC

(Table 3).

While4.3%ofwomenwith invasive cancerlacked antibodiestoHSV-2

by

the conventional

methods,

all had antibodies detectable

by

the ADCC method. There was

alsoasubstantial decrease in the percentage of

seronegative

women with carcinoma in situ and of control women when their sera were

analyzed

by

ADCC.

To confirm these

findings,

50 sera from

womenwithcervical

neoplasia

and 50 serafrom control women were each divided into three

samples

and

assayed

for antibodies to HSV-1 andHSV-2

by

three methods. Theseserawere

selected on the basis of low or absent titers of

antibody

toHSV-2asdetermined

by

microneu-tralization or

by

complement-dependent

lysis.

TABLE 2. Seronegativity of college studentsas determinedbyADCC andcomplement-dependent

lysis

Antibodiesto

No. HSV-la

Method teste

Posi- Nega-tive tive

Complement-dependent 100 40 60

lysis

ADCC 100 73 27

aSeratested atadilutionof1:10.

TABLE 3. Seronegativity ofcasesof cervical neoplasia and control women asdeterminedby

ADCC

Negativeforantibodiesto HSV-2b Disease statea

Ni

stud- Conventional ADCC

id methodc DC

No. % No. %

Invasive carci- 400 17 4.3 0 0 noma

Carcinoma in 115 15 13 4 3.5

situ

Controls 523 92 17.6 23 4.4

aStudy subjectswerederived from three studies

and are not comparable with respect to ages and

socioeconomic status.

bNegativeat aserumdilutionof1:10.

cAntibodies analyzed bycomplement-dependent lysisorbymicroneutralization.

VOL. 5, 1977

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556 SUBRAMANIAN AND RAWLS

Theresults of theseassays(Table4) supportthe conceptthatsomeindividuals without

detecta-ble antibody by the neutralization test or by

complement-dependent lysis hadantibodies de-tectable by ADCC. All of the sera that were

negative by complement-dependent lysis but positive by ADCC were assayed by the ADCC

technique, using uninfected BHK-21 cells as

targetcells; nonecontained antibodiesto

unin-fectedcells, suggesting that the lysis of infected BHK-21 cells in the ADCC represented

virus-specific antibody. Furthermore, 21 of the sera

were titrated for antibody by

complement-de-pendent lysis and ADCC. A correlation was

observed between titers obtained by the two methods (Fig. 7). Of the 10 serawith titers of

1:500 or less by ADCC, 7 had no detectable

antibody by complement-dependent lysis. All

serawith ADCCtiters ofgreaterthan 1:500 had antibodies detectable by

complement-depend-entlysis.

DISCUSSION

Lysisof cells byantibodytosurfaceantigens

and effector cells from nonimmune animals is thought to represent theterminal sequence of events initiated by attachment of effector cells through Fcreceptorstotheantibody molecules (20, 27). Pertinenttothisstudyistheexquisite sensitivity of this reaction for detectionof anti-body. Lysis of cells infected with HSV by anti-viral antibodies and mouse peritoneal exudate

cells (20) and humanperipheral blood cells (24, 27) waspreviously described. We found thata

continuous line of baby hamster kidney cells (BHK-21) infected with either HSV-1orHSV-2

canbe readilylysed with human antibody and

hamster peritoneal exudate cells. As reported by others (24, 27), we found the ADCC assay

capable of detectingantibodiestothese herpes-viruses at serum dilutions several hundred

times greaterthanby the standard neutraliza-tiontestorbycomplement-dependentantibody

lysis of virus-infected cells. Shore et al. (27)

were unable to differentiate past infections

with HSV-2 by the ADCC assay, and we also wereunabletoclearly make thisdistinction by

theassay wedeveloped(unpublished data).

Al-though it providesnoadvantageover

neutrali-zationassays or assaysinwhichcomplementis

used with respect to ease ofperformance and

cost,the ADCCtest isclearly useful in

detect-ingHSVantibodies atlow concentrations. Antibody titers to the HSV tend to remain relativelyconstant throughout adult life (5, 8,

9, 25, 31). Yet, when comparing populations living in different socioeconomic settings,

dif-ferences areobserved bothinthepercentageof

the populations with detectable antibodies to

the viruses and inthe meanantibody titers of

those who are seropositive (1, 4, 6, 10-13, 15,

17-19, 21, 28, 31). This suggests that environ-mental factors influence the antibody levelsin

humans. A decreaseofantibodiesto

undetecta-blelevels has been observed in a few children

afterprimaryherpetic gingivostomatis (8, 30). Antibody titers appear to rise rapidly among

these children, with the reappearance of virus

intheoral secretions. These observations

sug-gestthat, after aprimary infection, some

indi-viduals do not have a persistence ofantiviral

antibody detectable by standard methods. Itis

possible that establishment oflatent infection

may be required for the maintenance of

anti-bodies at a given level. Individuals not estab-lishingalatent infectionduringaninitial expo-sure to the virus could become susceptible to

subsequentreinfectionsduringwhichtheymay

become latently infected. Reinfections and/or

recurrence maythusresultinthedevelopment ofincreased levels ofantibodies in certain

pa-tients (2, 3, 8, 25, 30). Ifthis istrue, the

envi-ronmentfactorsfostering increasedexposureto theviruses wouldnotonlyinfluencethe proba-bility of being infected initially, but also

in-creasetheprobability ofbeingreinfected. This

couldaccountfortheobserveddifferences in the

percentagewith antibodies and the differences in mean antibody titers among the seroposi-tives indifferent social settings.

TABLE 4. Seronegativityofselectedsera from women withcervical neoplasia and controlwomenas determined bythreetechniques

Negativefor HSVantibodiesa

Disease state No.studied Neutralization Complement-dependent ADCC

lysis

HSV-1 HSV-2 HSV-1 HSV-2 HSV-1 HSV-2

Cervical neo- 50 22 22 21 25 4 4

plasiab

Controls 50 30 30 30 40 9 9

a Allassays wereperformedat aserum dilution of 1:10.

bCases ofinvasivecarcinomaandcarcinoma in situincluded.

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ANTIBODIES TO HSV DETECTED BY ADCC ASSAY

1o'or

I 1moeo

Ic

<10 10 20 40

TITER BYCOMPLEMENT-DEPENDENT LYSIS FIG. 7. Comparison oftitersofantibodiesto HSV-2 obtained by complement-dependent lysis and by

ADCC.

Bycomplement-dependentantibody lysis,we

foundantibodiestothe HSVin 40% ofthe col-lege students examined. This finding is in

agreement with those of Glezen et al. (7) and

Smith et al. (28), who found between 30 and 48% of college studentswithneutralizing anti-bodies totype 1 virus. However, by using the

more sensitive ADCC assay, we were able to

detectantibodies in 73%ofourstudent

popula-tion. Approximately 33% of the students

ap-pearedtohave been infected inthepastbut did

not maintain antibody titers sufficient to be detectedby theless sensitive method.

Seroepidemiological studies have shown an

association betweenantibodiesactivityto

HSV-2andcarcinomaofthe cervix(1,4, 6, 10-12,

16-18, 21). Accordingtothe standard antibody as-say techniques, not all women with cervical cancer show evidence of past infection with

HSV-2 (21). Thisimplies that infection by the virusmaynotbea necessaryfactorinthe

gene-sis of the cancer. However, we were unable to

findasingleserumwithoutdetectable

antibod-iesto HSV-2 among400 cancer cases. This

ob-servation suggests that essentially all women

with cervical cancer have been infected with

eitherHSV-1orHSV-2.If, aspostulatedabove,

establishment oflatency is requiredto main-tain antibody titer levels detectable by

stan-dard assays, thenour datasuggest that some womenmaydevelop cervical neoplasia without

becominglatentlyinfected.

ACKNOWLEDGMENTS

Weareveryappreciative of theassistanceof Toni Fran-cisco,CarolLavery,and ElaineJaggard.

Thestudywasmadepossible thoughagrantfromthe National Cancer InstituteofCanada.

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