Comparative complement fixation and serum neutralization antibody titers to herpes simplex virus type 1 and Herpesvirus simiae in Macaca mulatta and humans

Copyright 0 1977 American Society for Microbiology Printed inU.S.A.

Comparative Complement

Fixation and Serum

Neutralization Antibody Titers to Herpes Simplex Virus Type

1

and Herpesvirus simiae in Macaca mulatta and Humans

G. WILLIAM GARY, JR.,* AND ERSKINE L. PALMER

Department ofParasitology and Laboratory Practice, School ofPublic Health, University ofNorthCarolina at Chapel Hill, North Carolina 27514, and Viral Oncology Branch, Virology Division, Bureau ofLaboratories,

Centerfor Disease Control, Atlanta, Georgia 30333*

Received for publication5January 1977

The serological relationship of herpes simplex type 1 virus and Herpesvirus

simiae was studied. Antibody titers to these viruses were determined in 163

Macaca mulatta sera and 67 human sera by serum neutralization (SN) and

complementfixation (CF)tests.Bothgroupsofsera werealso tested by CF with

envelope and capsidantigens of herpes simplextype 1. By SN, themajorityof

the monkeysand allof the humans hadahigher titertoherpes simplextype 1

thantoH. simiae. ByCF, with crude antigens thetiters in the monkey sera

were greater toH. simiaethantoherpes simplextype 1,although fourserahad

equal titers to both antigens; the titers in the human sera were conversely

higher with the herpes simplextype1 antigen, exceptfor fourserawhich had

equal titerstoboth antigens. The capsid CF antigen of herpes simplextype 1

wasreactive with the human serabut virtually nonreactive with the monkey

sera;theenvelope CF antigen ofherpes simplextype 1 wasreactive with both

monkey and humanserabutwassomewhat less reactive than the crude herpes

simplextype 1CF antigen. Inaddition, serumsamplesfromapatientrecently

infected with H. simiae were examined by CF and SN for antibody to both

herpes simplextype 1andH. simiaeviruses.Theserological profileindicateda

positive correlation with the infecting virus. Although the SN titers did not

conclusively reflect an infection withH. simiae, the CF titers werehigher to H. simiae thantoherpes simplexinlaterseraandthusappearedtobe compat-iblewith H. simiae infection.

The close serological relationship of

Herpes-virussimiaeandherpes simplexviruspresents

difficulties in determining specific antibody

levels toH. simiae (6, 12). Serological studies

are hindered bythe prevalence ofantibodyto

herpes simplex inthe general human

popula-tion(3,22). It is apparent thatantibody against

H. simiae detected in human sera

generally

represents a heterotypic response to herpes simplex (3, 29).

Numerous serological surveys ofnonhuman

primatesinwild and captivepopulations,

par-ticularlyMacaca spp., have been conductedto

assayfor theprevalence ofH.simiaeand other

viruses ofpossible significance to man (8, 10, 11, 14, 16, 21, 25, 26, 30; S. S. Kalter, A. R.

Rodriguez,and J. J.Ratner, Bacteriol.Proc.,p.

127, 1964). In addition, Kalter and Heberling

(15)have compiled data from several

laborato-ries thattested nonhuman primate sera from

various sources for antibody to a number of

viruses including H. simiae and herpes

sim-plex. In mostof thestudies, serum

neutraliza-tion(SN) methodswereused; complement

fixa-tion(CF) dataarelimited(9).Inaddition,

stud-iesof nonhumanprimate serafor antibodiesto

bothherpes simplex andH. simiae have been

few(3,28);generally workers lookedonlyforH.

simiaeantibody, with H. simiae antigen (8, 10,

21,30) orherpes simplexantigen (5).However,

inatleastonestudy, inmonkeys fromUganda

(19), a search was made for herpes simplex

antibodywithherpes simplexantigen.

In view of the fragmentary nature of past

serological studies, we attempted to compare

antibody titersin seraofahuman population

and a Macaca mulatta populationby SN and

CF methods with both herpes simplex type 1

andH. simiaecrude antigens.Inaddition, both

groups ofsera were testedby CF with capsid

andenvelope antigens ofherpes simplextype1

(18). Alimited number ofserumsampleswere

available froma recenthuman caseofH.

sim-iae, and these were also tested against both

virusesbySN and CF.

(Apartofthisstudy comprisedadissertation

465

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submitted by G. W. Gary, Jr., in partial

fulfill-mentofthe requirementsfor theDoctor of Pub-lic Health degree at the University of North

Carolina, Chapel Hill).

MATERIALS AND METHODS

Viruses. The VR3strain(cell culture passage 30) ofherpes simplex virus type 1 (24) and the Lilly E2490 strain (cell culture passage 52) ofH. simiae viruswereobtained from stocks storedatthe Virol-ogyDivision,Center for Disease Control(CDC).The latterstrainhadoriginallybeenobtained from Rob-ertHull, EliLilly andCo., Indianapolis,Ind.

Virus propagation. Both viruses were grown in primary rabbitkidney (PRK)monolayercultures in 32-ounce glass prescription bottles. Culture growth medium consisted of Eagleminimumessential

me-diumwith 10%fetal calfserum. Thesamemedium without calf serum was used on the cell cultures whenthe viruses werepropagated. Monolayer cul-tures wereinoculated withvirus at amultiplicityof 5to 10 and wereincubated for about48 h at37°C. Infectious viruses were usedthroughout this study; therefore, because of the biohazardinvolved, all pro-cedureswith H.simiaevirus wereperformed inthe Maximum Security Laboratory, CDC. This is a

speciallaboratory suitewithgas-tight,glove-ported biological safety cabinets similarto thatdescribed by Kuehne (17).

Crude CFantigens. Crude CFantigensof herpes simplex type1andH.simiaeviruseswereprepared from infected PRK cultures. Afterapproximately48

hofincubation, the supernatant medium was de-cantedfrom eachinfected 32-ounce culture, the cell monolayer was washed with 10 ml of 0.85% NaCl bufferedatpH9with0.05 Mglycine-NaOH,andthe cellswerescraped into10 mlofthe same buffer. This suspension of cells was treated in a Dounce tissue

homogenizertodisintegrate the cells and was

cen-trifugedat200 xgtoremovelarge cellular debris. The supernatant fluidcontaining the antigen was dispensed in 1-ml volumes and frozen at -20°C. Control antigens were preparedinthe same manner fromuninfected cell cultures.

Capsid CFantigens. Aviruscapsid antigenwas

preparedfrom herpes simplex type 1-infected PRK cultures essentially bythemethod of Martinetal. (18). The supernatant medium was decanted from each culture and replaced with 5 ml of distilled

water. After30minofincubationatroom tempera-ture, 5 ml of5% Nonidet P-40 (NP-40) was added,

and the cultures were incubated for30 additional min at36°C. Then the cellsuspension was further

disintegratedby 15strokes of aDouncehomogenizer

andwasclearedofcelldebris bycentrifugingat 200 xg.Thesupernatant was layered over 30% sucrose cushionsandcentrifugedinaBeckman SW40 rotor for60min at30,000 rpm.Thesupernatantfrom this step, containing NP-40and sucrose, wasdecanted,

andthe walls ofthe tubes were wipedwith cotton applicatorstoprevent NP-40 fromdraining into the pellet. The pellet, which consisted of capsids, was resuspendedin0.01 Mbarbital buffer, pH 7.4, 1 ml pertube, and stored frozenat -20°C.

Envelope CF antigen. Avirusenvelope antigen

wasalsoprepared fromPRKcultures infectedwith

herpes

was removed from the cultures, and the infected

cells of eachculture bottlewerescrapedinto 2 ml of distilled water. The cells were disintegrated in a

Dounce homogenizer as above, layered over 30%

sucrose cushions, and centrifuged in a Beckman SW40 rotor for 60 min at 30,000 rpm. The pellet,

which contained the virusparticlesintact with their

envelopes,was resuspendedin 1 ml ofdistilled

wa-terperculture and treated withanequalvolume of

diethyl

bubbling nitrogenthroughthe treated suspension.

The suspension wasthen dialyzed at4°C for 24 h

against0.85% NaClbufferedatpH10.5with 0.05 M

glycine-NaOH, layeredontoa30% sucrosecushion, andcentrifugedfor1hat30,000rpm inaBeckman SW40rotortoseparatethe envelopematerialfrom

capsidsorcapsidswithenveloperemnants. The

su-pernatant, which contained the envelope antigen,

was collected and dialyzedagainst0.01M barbital

buffer, pH7.4, foruseinCF tests.

Sera. Human serum samples were obtained in June 1974 from 60 members of theVirologyDivision,

CDC. Seven additional human sera wereobtained frommembers of the TissueCulture and Media

Sec-tion,Scientific ServicesDivision,CDC. These latter sera had been frozen at -20°C since collection in June 1968.Serumsamplesfrom163M.mulattawere

alsotested;theseserawereobtainedatthe time the

monkeys were trapped in Jammu State, India, in

October 1974toassurethat anyherpes simplexvirus

antibody detected in the animals was acquired in

the naturalstateandnotthroughhuman exposure aftertheyweretrapped. Serialserumsamples from

arecenthuman caseof H. simiaeinfection (1)were

obtainedthroughthegenerosityofRichardEmmons andEdwinLennette of theCalifornia State Health

Department.

CF. CF tests were performed by the microtiter LBCF method(4).Allserawereinactivatedat56°C for 30 minbeforetesting.

SN. SNtests were performedbyamicromethod

essentiallyaspreviouslydescribed(20, 27).Sterile,

96-well, plastic microtitrationplatesandthe FS32

strain ofdiploidhumanfibroblasts derivedatCDC fromnewbornforeskinwere used. The culture

me-dium wasEagle minimum essential medium with

10%fetalcalfserumplus1,400mg of sodium bicar-bonate per litertomaintainthepHinanacceptable range under sealed covers. Trypsinized cells in a concentration of114,000/mlweredispensedinto the wells in 0.1-ml amounts; 20 h later, 0.1 ml (1,000

meantissue culture infectiousdoses) ofvirus dilu-tionorvirus-serum mixturewasaddedas appropri-ate.After4daysofincubationat37°C,the

superna-tantwasdrainedoff,andthe cellswerestainedwith

crystal violet-Formalin for 20 min, rinsed, dried,

and read macroscopically as positive or negative

for viral cytopathic effect. Serum-neutralizing end

points were interpolated by the Reed-Muench

method (23).

RESULTS

CF andSN tests onmonkeysera. Fourteen

ofthe 163 M. mulattaserawere so

anticomple-mentary as to preclude their being tested

by

J. CLIN. MICROBIOL.

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CF. The 149 remainingsera weretested by CF

against the crude H. simiae antigen and the crude, capsid, and envelope herpes simplex type 1 antigens. The distribution of CF titers

obtained is presented inTable 1.Only 1of the

149 sera reacted with the capsid antigen.

Al-though the crude and envelope herpessimplex

type 1antigenswere cross-reactive, theywere

lesssensitivethanthe H. simiae crude antigen.

For example,13 (9%)of the149testserareacted

with the crudeH. simiae antigen atatiterof

64, whereas only 3 (2%) reacted with crude

simplex antigenatthis titer. The envelope

an-tigenwasnotreactive aboveserumdilutions of

1:32.

InSNscreentestsof the163M. mulattasera

atadilution of 1:10, 56 (34.4%) reactedtoboth

H.simiaeand herpessimplextype1.Titrations

of these 56 SN-positive sera showed that 48

(85.7%) had a higher SN antibody titer to

herpes simplex type 1, and 8 (14.3%) had a

higher SN titer to H. simiae. Only 4 of 163

(2.5%)hadatiter >10toherpes simplextype1 with a concurrent titer to H. simiae of <10.

None of the 163 sera had a titer >10 to H.

simiae withaconcurrenttiterof <10toherpes

simplextype 1.

Fifty (33.6%) of the 149 sera thatwere not

anticomplementary in the CF test had either

SNorCFtiters; the relationships of thesetiters

inrespect tothe H. simiae and herpessimplex

viruses andantigens arepresented in Table2,

along with the titers for human sera. None of

these 50 serahada CF titergreater to herpes

simplextype1crude antigen thantoH. simiae

crude antigen, although four of the sera had

equal titers to the two antigens. ByCF, none

reacted only to the herpes simplex antigen;

however, nine (18%) reacted only to the H.

simiae antigen. Thirty-four (68%) reacted to

bothviruses, bySN aswell asCF.

The age and sexdistribution of theM.

mu-TABLE 1. Distribution of CF titers in 149 Macaca mulatta sera against indicated antigens

H. simiae Herpessimplextype1 Titer Crude Crude Capsid Enve_lope

<8 lOOa 111 148 129

8 4 11 1 11

16 10 15 7

32 20 9 2

64 13 3

128

256 1

512 1

a Number of sera withindicatedtiteragainst an-tigen.

latta monkeys is presented in Table 3. Monkeys

were considered subadult ifthey were almost

adult size but had not yet sexually matured.

The seropositive monkeys were primarily

adults; although adult monkeys represented

only 49% of the test subjects,they accounted for

92% of the seropositives. Positivity was not

re-lated to sex.

CF and SN tests on human sera. CFtests

were performed on sera of 67 CDC personnel

against the crude H. simiae antigen and the

crude, capsid, and envelope herpes simplex

type 1 antigens as on the M. mulatta sera. The

results are presented in Table 4.Thecapsid and

envelope antigens of herpes simplex virus type

1wereless sensitive than thecrude antigen of

the same virus inreactions with human sera;

nevertheless, they were reactive to a

considera-ble degree. However, the capsid antigen

reac-tivity was much greater against the human

sera than against the monkey sera (Table 1),

where it was virtually nil.

Twenty-seven of the 67 human sera (40.3%) had titers of <10 to both viruses by SN and <8

to both crude antigens by CF, 33 (49.3%) had

TABLE 2. Relationship oftiters inseropositive Macacamulatta and humanseraagainst indicated

antigens

SN CF Sera (%)

H. Herpes H. Herpes

simiae sim- simae slm- M.mulatta Human

plex1 crude crude

a - +b + 2/50(4.0) 0/40 (0.0)

_ + _ _ 1/50(2.0) 0/40 (0.0)

_ + + + 2/50(4.0) 29/40 (72.5)

_ + + _ 1/50(2.0) 0/40 (0.0)

- + _ + 0/50(0.0) 4/40 (10.0)

+ + _ _ 1/50(2.0) 0/40 (0.0)

+ + + _ 9/50(18.0) 0/40 (0.0)

+ + + + 34/50(68.0) 7/40 (17.5)

a -,Titers <10 in SN tests and <8 in CF tests.

a+,

<8

TABLE 3. Age andsexdistribution ofMacaca mulatta monkeys usedinserologicalanalysis

Results ofserologicalanalysisofmonkeys (%)

Category Instudypopulation Inseropositives

Adults 73/149a (49.0) 46/50b (92.0)

Subadults 13/149 (8.7) 2/50 (4.0)

Juveniles 63/149 (42.3) 2/50 (4.0) Male 35/149 (23.5) 11/50(22.0)

Female 114/149(76.5) 39/50 (78.0)

a Denominator is total number of monkeys in studywhosesera were notanticomplementary.

bDenominator isnumberofmonkeys seropositive

byCFand/orSN.

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468

neutralizing titers .10 to herpes simplex type 1

but a titer <10 to H. simiaevirus, and 7 (10.4%)

had neutralizing titers .10 to herpes simplex

type 1 and also had titers of.10 to H. simiae.

These detectable titers to H. simiae occurred

onlyinserawhere the titers to herpes simplex

type 1 were .240 (mean = 314), 7.4- to 25-fold

higher than the H. simiae titers. Of the 40

human sera found positive by one method or

the other, 36 (90%) had a CF titer greater to the

crude herpes simplex type1antigenthan to the

crude H. simiae antigen, and4 had the same

titer toboth. Thus, none had a CF titer greater

toacrude H. simiae than to crude herpes

sim-plex type 1.

The SN and CF titers obtained for the 40

seropositive human sera with the H. simiae

and simplexvirusesand antigensareshownin

Table 2, along with comparable data for the

seropositive monkey sera. Most of the human sera, 33 outof40 (82.5%), reactedwith herpes

simplex by SN and CF; only 7 (17.5%)

cross-reacted with H. simiae by both the SN and CF tests.In contrast, most of the seropositive mon-key sera, 34 out of 50 (68%), cross-reacted with herpes simplex in both tests, and none reacted

only to H. simiae inboth tests.

Eight serial serum samples from a human

TABLE 4. Distribution of CFtiters in 67human seraagainstindicatedantigens

No. of sera with indicated titer against antigen Titer H. simiae Herpes simplex type 1

Crude Crude Capsid lonpe

<8 31 27 44 37

8 11 2 9 13

16 20 14 13 14

32 5 14 1 3

64 10

128

SN

1280- 320- 160- 80-

40-110

caseof H. simiaevirus infection (1)were

stud-ied by SN and CF with both H. simiae and

herpes simplex type 1 viruses and crude

anti-gensasreagents. The results obtainedare

pre-sentedin Fig. 1. By SN, antibody titerstoH.

simiae andherpes simplex type 1 were low to

both viruses early in the disease and rose to

high levels during the course ofthedisease;the

SN titerstoherpes simplex type1were

consist-ently higher than those toH. simiae. By CF,

however, titersweresometimeshigheragainst

H. simiae than againstherpes simplextype 1,

dependingonthelength of time since the

infec-tionoccurred. In the early stagesofthe

infec-tion, CF titers to herpes simplex type 1 were

higher, but in the later stages the H. simiae

titers werehigher.

DISCUSSION

The SN data on humanserapresentedhere

concurgenerallywith the data of others (2, 3,

12, 29) which show that the presence of SN

antibodytoH. simiaeisdependentonand

sec-ondary to the presence of antibody to herpes

simplex. Our findings also indicate that the

level of SN antibody to H. simiae is directly

relatedtothe heightof the SNantibodytiterto

herpes simplex, which further bears out the

findingsof previousauthors. Cabassoetal. (3)

reported one exception to this relationship,

namely,anindividualwith antibodytoH.

sim-iae intheabsenceofantibodytoherpes simplex.

Infection of humans with H. simiae virus

moreoften thannotresultsindeath.Therefore,

comparative studies of H. simiae and herpes

simplex antibodies in humans who have had

overtH. simiae infection are rarely possible. AlthoughSN titersforthesingle humancaseof

H. simiae infection presented in this study

wereconsistently higher to herpes simplex type

1 thantoH. simiae, the CF results showed a

reversing relationship in the convalescent

CF 512 -256 -128

-64

-32

-16 -8

6/23/69 T * f f f

PRE ILLNESS 4/12J73 421V735/2173 5/1S/73 5/24/73 ONSET

OF ILLNESS

FIG. 1. Serological profile ofhuman case of H. simiae.Symbols:herpes simplex type 1, CF(I ), SN (-); H. simiae,CF(0), SN (E).

6/11'73

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phase and thus provide supporting evidence of aninfection with H. simiae. Although we did

notinvestigate dual infections ofH. simiae and

herpessimplex in monkeys, the results of our

CF tests with monkey sera relate well to those with the human case in that the CF titers were

highest with crudeantigen of the suspect

field-infecting virus, H. simiae. To our knowledge this serological study reports, for the first time, profiles of antibody to both viruses by both CF and SN which can serve as a basis for study and evaluation of future cases.

There are few reports of SN tests for both

herpes simplex and H. simiae antibodies

con-ductedconcurrently on the same monkey sera.

In a study of eight monkeys with naturally

acquired SN antibody, Burnet et al. (2)found

thelevels to beeitherthe same to herpes

sim-plexandH. simiae orhighertoherpes simplex,

with one exception where the titer was higher

to H. simiae.Ueda et al. (28),inastudy with 19

monkeys, found that all the monkeys with anti-body to both viruses had higher titers to herpes simplex. Cabasso et al. (3) tested five lots of

simiangamma globulin and found the titers to

be higher to herpes simplex virus infour lots andhigherto H. simiae in onelot. Our results

aresimilar; in 149 M. mulatta, 48 of which had

SN antibody to one or both viruses, wefound

that40had ahigherlevelof antibodytoherpes

simplexand8hadahigher leveltoH. simiae.

Apparently, SN antibodies found in Macaca

spp. will generally neutralize herpes simplex

virus aswell or better than H. simiaevirus,but

exceptions dooccur.Ourdataarealsoin

agree-ment with those of Shah and Southwick (26)

thatantibody incidenceishigherinadult

mon-keys;theydetectedSNantibodiesto H. simiae

in 7of8adultmonkeysbutinnoneof39

juve-niles.

Ueda et al. (28) could not be sure that the

monkeystheystudied had not beenexposedto

herpes simplex virus during theirperiod of

con-tactwith humansafter capture; such exposure

could account for the antibody that was

de-tected. Weavoidedthisproblem byhaving the

seraused in our studycollected at the time the

monkeys werecaptured. We cannot, however,

preclude the possibility that the monkeys had been infected in the wild with an unknown herpes-like virus which cross-reacts with both

herpes simplex andH. simiae.

Some workershave stated that theCF test is

not capable of detecting antigenic differences

betweenH.simiaeandherpessimplex (6, 7, 13)

viruses. However, to our knowledge there is

only onereportof CFtestsperformedwithH.

simiae antigen (9). Falke (9) found

cross-reac-tivitywith CF antigens of three herpes simplex

strainsand one H. simiae strain, but,intesting

the sera of 42 rhesus and cynomolgusmonkeys,

he found thesera to be more reactive with the

H. simiae antigen. Thehighest titers were

ob-tainedwith this antigen. He alsoreportedthat

in one human serum tested, equivalent titers

wereobtained to two herpes simplex antigens

and one H. simiae antigen. Our study with a

larger number of monkeyssupportsthe data of

Falke. We found antibody levels by CF to be

higher against H. simiae antigen in 44 of 48

monkeys. In the other4 monkeys, titers were

equalwith H. simiae andherpes simplextype1

crudeantigens.

In our study of 67 human sera, 40 of which

had CFantibody to one or both crude antigens,

36had titers higher to herpes simplex and4had

equaltiterswith both antigens. It is apparent

that, although cross-reactivity does occur

be-tween the two viruses, the CF antigens react

best with homotypic antibody, that is, with

antibody to the virus which infectedthe

mon-key or human individual. This was also shown

tobethe case with thesinglehumanH. simiae

infection which was studied.

Although the cross-reactivity of theenvelope

antigen of herpes simplex type 1 was

essen-tially the same as with the crude antigen of

herpes simplex type 1, the capsid antigen was

of strikingly low reactivity with the monkey

sera.Theimplications are thatacapsidantigen

ismore specific than either a crude or an

en-velope antigen and that a capsid antigen

pre-pared from H. simiae-infected cells might be

similarly specificfor H.simiaeantibody. Ifthis

isactually so, definition ofH. simiae infection

would be greatly simplified. Obviously, the

next step is to prepare an H. simiae capsid

antigen. Its preparation was notpossible

dur-ingthe course ofthis study becauseof the

bio-hazards involved; however, suitable

contain-mentfor such work withH.simiae is nowbeing

developed at CDC.

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