Evaluation of a killed Rocky Mountain spotted fever vaccine in cynomolgus monkeys

0095-1137/79/11-0719/05$02.00/0

Evaluation

of

a

Killed Rocky

Mountain

Spotted

Fever

Vaccine

in Cynomolgus

Monkeys

JANET C. GONDER,t* RICHARD H. KENYON, ANDCARLE. PEDERSEN, JR.:

U.S.ArmyMedicalResearchInstitute of Infectious Diseases,Fort Detrick,Frederick,Maryland21701 Received for publication27 August1979

Anonhuman primate model of Rocky Mountain spotted fever infectionwas

developed in cynomolgus monkeys (Macaca fascicularis) infectedby the

subcu-taneous routeorbyaerosol.Clinicalresponses,hematologyandserumchemistry

values, and pathological findingswere similartothosefound in humansill with Rocky Mountain spotted fever. The clinical model was then used to test the efficacy of a killed Rocky Mountain spotted fever vaccine grown in chicken

embryo cells. Monkeys were immunized with varying dilutions of the vaccine withatwo-dose schedule andthenchallengedat2months with virulentRickettsia

rickettsiibythe subcutaneousrouteorby aerosol. The undilutedvaccine totally

protectedmonkeys against both challenges, evenatextremelyhigh doses.

RockyMountain spotted fever (RMSF) is an acutefebrileinfectious disease caused by

Rick-ettsia rickettsii that is endemic throughoutthe continental UnitedStates. The infectionis

nat-urally transmitted by the bite of a tick, but

studies in ourlaboratoryindicatedthat aerosol

exposureisapotentialrouteofinfection in

lab-oratoryworkers (8). Atthe presenttime, there

isno commercial RMSF vaccine available.

An inactivated chicken embryo cell-grown RMSF vaccine has beendevelopedin this

labo-ratory (4). A guinea pig model was used for

preliminary studies withthevaccine (5).

Vacci-nated guinea pigs challenged by the subcuta-neous (s.c.) route or byaerosol werefully

pro-tected. RMSFinfection has beenstudied

exten-sively in rhesus monkeys (7, 9, 12, 15), and a

suitable nonhuman

primate

model has been

de-scribed (10). The vaccine has been tested and foundtoprotectrhesusmonkeyschallengeds.c.

(11). A two-dose schedule

provided

the best

protection

(4, 11). However,due to the limited availability of therhesusmonkey,a

cynomolgus

(Macaca

fascicularis)

monkey

model was

de-veloped for further vaccinetesting.

The purpose ofthis study is todescribe the

cynomolgus

monkeymodel of RMSF infection andto testtheefficacyofourvaccineagainsts.c.

andaerosolchallenges.

MATERIALS AND METHODS

Laboratory animals. Forty-three healthy cyn-omolgusmonkeysof bothsexesweighing 3to 4.5kg

tPresent address: 3976 Sunny Vale Dr., DeForest, WI 53532.

tPresentaddress:HQDASGRD-PL(LTCPedersen),Fort

Detrick, Frederick,MD 21701.

wereused.Theywerehoused in individual cages and fedacommercial ration(RalstonPurinaCo.,St.Louis,

Mo.) and water ad libitum. All sampling was done

between 8and9a.m. (before feeding). Monkeys were observed severaltimesdaily for anorexia and depres-sion. Changes in normalaggressive reactionsto han-dling were noted. Base-line clinical and laboratory valueswereestablished duringa2-weekperiod before infection. Meanrectal temperatureswerebasedon 5 sampling days, and base-linehematology and serum chemistry valuesweredetermined from four samples. For3weeks afterchallenge,rectal temperatureswere recordeddaily, and blood sampleswereobtained from thefemoral veinatselectedintervals forrickettsemia, hematology, serumchemistry, andserology.

Rickettsiae and experimental infection. The

Sheila Smith strain of R. rickettsii was propagated

andassayedaspreviouslydescribed(9).Fors.c. infec-tion,yolksac-grown rickettsiaewereinoculated(1ml/

animal)atdosesof101,103,or105plaque-formingunits

(PFU)/ml.Aerosol-infectedmonkeyswereexposedfor 10mintosmall-particleaerosols of103or 105PFU of rickettsiaebyamethodpreviouslydescribed(3). Aer-osol sampleswerecollectedduring eachexposure in anAGI-30glass impingerandtitratedbyplaqueassay (13), and the total inspired dose was calculated for each monkey. Rickettsemias were confirmed by the plaqueassaymethoddescribedbyWikeand

Burgdor-fer (14).

Todetermineadose-responsetoR.rickettsii,

mon-keysweredivided into threegroupsofseveneach: low

dose

(101

PFU), medium dose (103 PFU), and

high

dose (105PFU). Three monkeysineach groupwere inoculated s.c., and fourmonkeysineachgroupwere infectedbyaerosol.

Vaccine andimmunization. Thevaccine tested inthisstudywas aninactivated chickenembryo cell-grown product prepared and tested

by

Kenyon and Pedersen (4)containing1.3x

10W

R.rickettsiiperml. The vaccine was administered in two doses (0.5 ml each), 28 daysapart, with undilutedand 1:10, 1:100,

719

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and 1:250 dilutions.Monkeyswerechallenged1month

after the seconds.c.inoculation.

Inthefirstvaccinestudy,10monkeysweredivided

into five groupsof 2 each for vaccination and

subse-quents.c.challenge.Twogroupswereimmunized with

the undiluted vaccine, and one group each was

im-munized with the three dilutions. One group

immu-nized with undilutedvaccinewaschallengedwith 105

PFU of R. rickettsii, and the other was challenged

with103PFU. In thesamestudy, four nonvaccinated

control monkeys were inoculated s.c., two with 103

PFU andtwowith105 PFU.

In the second vaccine study, four monkeys were

vaccinated with undiluted vaccine. Two were then

challenged with 103 PFU and two were challenged

with 10' PFU of R. rickettsii givenin small-particle

aerosol. Four nonvaccinated monkeyswere similarly

infectedtoserveascontrols.

Hematology andserumchemistry.Blood

sam-ples collected in ethylenediaminetetraacetic acidwere

analyzed fortotal (CoulterElectronics,Hialeah, Fla.)

and differential leukocyte counts, hematocrit, total

protein (TS Meter, American Optical, Atlanta, Ga.),

and fibrinogen (1). Blood urea nitrogen, serum

glu-tamic oxalacetic transaminase, and serum alkaline

phosphatase values were determined with

commer-cially available kits (Mallinckrodt, Inc., Hazelwood,

Mo.).

Serology. In thes.c. and aerosol challengestudy,

serumsampleswereobtained beforechallengeand21

days after infection. Inthevaccinestudy,serum

sam-pleswerecollected beforevaccination, 1and 2months

after thefiLrstvaccinedose,and1, 3,and 6 weeksafter

challenge.Themicroagglutinationassayforrickettsial

antibody was performed as describedby Fiset etal.

(2). The rickettsialtestantigen waspreparedas

pre-viously described (11).Allanimals wereseronegative

atthestartof thestudy.

Pathology. Monkeysthatdiedwereexaminedfor

gross and microscopic lesions. Tissues were fixed in

neutral phosphate-buffered Formalin, embedded in

paraffin,sectioned,andstained withhematoxylinand

eosinfor examination.

Statistical analyses.Analysis ofvariance for

re-peated measurementswas usedto detectdifferences

significantly greater than base-line variability. The

lowest level ofsignificancewasset atP<0.05.

RESULTS

Clinicalresponse. Theclinicalresponsesof

21 cynomolgus monkeys infected with R. rick-ettsii are summarized in Table 1. One monkey

did not become ill when challenged with 10' PFU by aerosol. All other monkeys became ill withanorexia, depression, and fever. One

mon-key in each of the two higher-dose groups

de-veloped a rash 10 days after aerosol exposure;

dyspneawasobservedonly occasionally.

In general, the mean incubation time

de-creasedwith anincrease in infecting dose. The

time to onset of fever wasslightly longer after

aerosol exposure than afters.c. challenge with

101 and 103 PFU; at 105 PFU, no difference in

incubation timewasnoted.Therewasno

differ-enceinincubation time between dying and

sur-viving monkeys in each dosegroup.

The duration of fever after aerosol exposure

wasshorterat thetwo lower dosages than was

observedat the samedosages given s.c. At the

high dose, the duration of feverwaslonger after

aerosol than after s.c. challenge. Duration of

fever inthe monkeys that diedwasshorter than

inthose thatsurvived.Temperaturesdecreased rapidly 1 to3days before death.

Therewasno difference inthe magnitude of

fever betweenroutesofinfectionordose. Rectal temperatures increased more than 2°C above

base-line values of 38.0to 38.5°C in all febrile monkeys (P<0.001).

Fifty percentofthemonkeys in each aerosol

group died. All of the monkeys inoculated s.c.

with101 PFUdied,asdidtwoofthree in each of the other two dose groups. The mean time to

death in the low- andmedium-dose groups was

approximately 11 days, compared with 8 to 10

days inthehigh-dose group.Therewas a tend-encyfor themonkeysinall dosegroupsexposed

by aerosoltolivelongerthanthoseinfecteds.c.

Pathology.Splenomegalywasnotedongross

pathological examination. The most common

lesions microscopically were vasculitis and

thrombosisof thecapillaries, arterioles, and

ven-ules of the facial skin, ears, and nares. The

lesionswerecharacterizedprimarily by perivas-cular infiltration of lymphocytes and plasma cells. Vasculitis was alsoseen in the palpebral

TABLE 1. Clinical responsesofcynomolgus monkeys to R. rickettsii infection

Challenge No. with Fever(days)

No. No. ill Rick- No. Mean days to

Dose Route etts- Rash Time to onset Duration dead death (range)

emia (range) (range)

10' s.c. 3 3 3 0 6 (5-7) 5.7(4-7) 3 11 (10-11)

Aerosol 4 3 2 0 7.3(7-8) 2.7(2-4) 2 11.5 (11-12)

103 s.c. 3 3 3 0 3 (2-4) 7.3(5-9) 2 10

Aerosol 4 4 4 1 5.7(5-6) 4.0 (3-5) 2 12 (11-13)

105 s.c. 3 3 3 0 4 5 2 8

Aerosol 4 4 3 1 3 6.7 (5-9) 2 10

CLIN. MICROBIOL.

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conjunctiva,testes, uterus,thymus, heart, skel-etal muscle, and abdominalmesentery.Varying degrees of interstitialpneumonia (no difference

was seen in monkeys infected by either route)

and minimal adrenitiswerenoted. Two monkeys

infected s.c. had fibrin thrombi in glomerular

tufts, suggesting disseminated intravascular

co-agulation.

Hematology and serum chemistry. The

total leukocyte counts increased 2,000to 4,000

cellsper/mm3 above base-line values inall but

the afebrile monkey. These increasesweredue

toanabsoluteneutrophilia and occurred during

the febrileperiod. Increaseswerenotsignificant

atP< 0.05. The hematocrit decreased slightly in all groups due to repeated bleeding. There was nosignificant change in total protein values,

although plasma fibrinogen increased signifi-cantly (P <0.01) during the febrile period (as highas700mg/100 ml).

Blood urea nitrogen and serumglutamic

ox-alacetic transaminase valueswereelevated (>2

standard deviations) on days 4 and 6 of the febrile period in the medium- and high-dose

groups.Onlya1-day elevationwasnoted in the

low-dose groups (on day 6). There were no

changesin serum alkaline phosphatase values.

Rickettsemia and serology. All monkeys infected by thes.c.routedeveloped rickettsemia. Themeantimetoonsetinthehigh-dose group

was4days and, in the othertwogroups,6days. The duration of rickettsemiainthetwo surviv-ing monkeyswas4days.Inthose that died after s.c.challenge,rickettsemiapersisteduntil death.

After aerosolexposure,onlytwooffour

mon-keys in the low-dose group developed

ricketts-emia. These two monkeys died. Rickettsemia

wasfirstdetectedonday7.Deaths occurredon

days11and 12 with rickettsemia persisting until death (mean duration=5.5days).

All fourmonkeys in the medium-dose group

demonstrated rickettsemia. Mean timetoonset

was6.2days (range=5to7days). Rickettsemia

persisted until death in the two monkeys that died (duration = 7days). Duration of

ricketts-emia in bothsurviving monkeyswas3days.

Three of the four monkeys in the high-dose

group developed rickettsemia after aerosol

ex-posure. The mean time to onset was 5.3 days

(range=5 to6days). The rickettsemia persisted

until death in the two monkeys that died (du-ration = 5 and 6 days). The duration of

rick-ettsemia in thesurviving monkeywas5days.

Allmonkeys in thes.c.and aerosolstudies had

microagglutination titers ofatleast 1:64onday

21.Theaerosol-challenged monkey that didnot

becomeill hadatiter of 1:64. Titers correlated

withincreasing challenge doses. The geometric

meantitersof thelow-, medium-, and high-dose groups were 1:64, 1:107,and 1:341,respectively.

Vaccine studies. Resultsofthe vaccine stud-iesaresummarized in Table 2. Alleight

nonvac-cinated, control monkeys became ill andseven

died. All controlshad demonstrableleukocytosis duetoabsoluteneutrophilia. Decreases in hem-atocritoccurred duetorepeated bleeding. Slight increaseswerenoted in bloodureanitrogen and serum glutamic oxalacetic transaminase values,

with no significant changes in serum alkaline

phosphatase.

Onemonkey vaccinated and challenged with

anaerosoldose of105PFUwasmildly febrileon

days2and3(1.0°C above base line). One

mon-key in eachof twogroupsreceivingdiluted

vac-TABLE 2. Clinicalresponsesofvaccinated and unvaccinatedcynomolgus monkeys (n=

2/group)

after challenge with R. rickettsii

No. with Fever (days)

Vaccine androute

Chasekenge

No. dead

dose(PFU) Ricketts- Fev Timeto onset

Duration

emia (range)

s.c.

None 105 2 2 3 oa 2

103 2 2 5.5 9 1

(5-6)

1:250 103 0 1 6 8 0

1:100 0 1 6 6 0

1:10 0 0 0

Undiluted 103 0 0 0

105 0 0 0

Aerosol

None 105 2 2 4.5 oa 2

(4-5)

103 2 2 6 Oa 2

Undiluted 105 0 1 2 2 0

103 0 0 0

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722 GONDER, KENYON, AND PEDERSEN

cine (1:100 and 1:250) became febrile after s.c.

challenge. The magnitude andduration of fever

werecomparabletothoseseeninthe

nonvaccin-ated controls. The three vaccinated monkeys that became febrile showednosigns of anorexia ordepression. None of the vaccinated monkeys

showedanyalterations from base-line values in

hematology or serum chamistry values, except

that the hematocrit decreased due torepeated bleeding.

Before challenge themicroagglutination titers

were relatively high (>1:32) in the monkeys

given undiluted and 1:10 dilutionofthe vaccine. After challenge these monkeys showed a slow

rise to peak titers (>1:100) at 21 days. Those receiving 1:100 and 1:250 dilutionshadnegligible titers (1:4) just beforechallenge, but exhibiteda

pronounced antibody response (>1:32) 7 days

afters.c.challenge with 103 PFU of R. rickettsii.

The rise in titer from1 to6weeks tendedtobe slower in the groupreceivingthe 1:250dilution

of vaccine (Fig. 1).

There were nodifferencesin the

microagglu-tination titers afters.c. or aerosol challenge of

monkeys given the undiluted vaccine. Antibody

response after challenge was morepronounced

in those monkeys challenged with the higher dose

(105),

butby 6 weeks the titers in thetwo

dosagegroupswereessentiallythesame.

No rickettsiae were isolated from peripheral

blood ofvaccinated monkeysatany time after

challenge.

DISCUSSION

RMSF incynomolgusmonkeys closely

resem-bles the disease in rhesusmonkeys (9) and

hu-mans (16). Skin rashes were less frequent in

cynomolgus than in rhesus monkeys andmaybe

a:

E- VACCINATE VACCINATE CHALLENGE

.0

o -8 -4 2 4 6

WEEKS BEFORE CHALLENGE WEEKS AFTER CHALLENGE

FIG. 1. Microagglutination antibodyresponsesof

cynomolgus monkeys vaccinated with undiluted(0),

1:10(-),1:100(A), and 1:250 (V) dilutions of RMSF

vaccine andchallengeds.c.with 103 PFU of R.

rick-ettsii.

due to the former's dark

complexion

and the

resulting difficulty in recognition ofarash.

Com-paredtotherhesusmonkey, themean incuba-tion time tendedtobe shorter and the duration of fever tendedtobe longer afters.c.

challenge

with 103 PFU of R. rickettsii. All

monkeys

in-fecteds.c.in thisstudy became

ill,

whereas2of the12rhesusmonkeysintheprevious

study

did

not. This may reflect the smaller number of animals used here. The mortality rate in

cyn-omolgus

monkeys

infected with 103 PFU was

similartothatof rhesus monkeys. Rectal

tem-peratures decreased rapidly before death, as

seen previously in rhesus monkeys (10), al-though mean time to death was somewhat longerin

cynomolgus monkeys.

The microscopic lesions of vasculitis and

thrombosis ofsmall vesselsinnumeroustissues

seenin thecynomolgus

monkeys

weresimilarto

thosereportedin rhesus

monkeys

(7, 10, 15) and

humans (6). The lack of consistent or severe

respiratoryinvolvementinthe aerosol-infected monkeyshasbeenreported (12, 15).

Thechangesinhematology andserum chem-istry values were similar to those reported by Sammonsetal.

(10),

although

decreases in total protein and serum alkaline phosphatase were notseenin thepresentstudy.

Although three of the vaccinated monkeys becamefebrile, they either had been givenalow antigen concentration (1:100or 1:250

dilutions)

oran

extremely high challenge

dose (105PFU).

It is

important

to note that these monkeys showednootherclinical

signs

of

illness,

nordid

they develop rickettsemia.Thehematologyand serumchemistry values ofall vaccinated

mon-keys remained normal throughout the study. Prechallenge microagglutination titers (after

twodosesofvaccine)werecomparabletotiters observedwiththesameschedule in rhesus

mon-keys (11).

In conclusion, the cynomolgus monkey ap-pearstobea suitable substitute for the rhesus monkey for the study of RMSF in nonhuman

primates. The clinical and physiological re-sponses andpathological changesare similar in the twospecies.RMSFresultingfrom s.c. infec-tion is indistinguishable from the disease

ac-quired byaerosol exposure. The chicken embryo

cell vaccine protects cynomolgus monkeys

againstboth s.c. andaerosol

challenges.

LITERATURE CITED

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andS. H.Spielman.1969. Amicroagglutination tech-J. CLIN. MICROBIOL.

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niquefor detection and measurement of rickettsial an-tibodies. Acta Virol.13:60-66.

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Pedersen, Jr., and E. W. Larson.1979.Cynomolgus

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4. Kenyon, R.H., and C. E.Pedersen,Jr.1975. Prepara-tion ofRocky Mountainspotted fever vaccine suitable forhumanimmunization. J.Clin. Microbiol.1:500-503. 5. Kenyon,R.H., R. A.Kishimoto,and W. C. Hall.1979. Exposure ofguinea pigstoRickettsiarickettsiiby aer-osol, nasal, conjunctival, gastric, and subcutaneous routesandprotectionaffordedbyanexperimental vac-cine.Infect.Lmmun.25:580-582.

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D.C.

7. Moe,J.B., G.L.Ruch,R. H.Kenyon,J. D. Burek

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10. Sammons, L. S., R. H. Kenyon, G. T. Burger, C. E. Pedersen, Jr., and R.0. Spertzel. 1976. Changes in blood serum constituents and hematologic values in Macaca mulattawithRockyMountain spotted fever. Am. J. Vet.Res. 37:725-730.

11. Sammons, L. S., R. H. Kenyon, and C. E. Pedersen, Jr. 1976. Effect of vaccination schedule on immune response of Macaca mulatta to cell culture-grown Rocky Mountain spotted fever vaccine. J. Clin. Micro-biol. 4:253-257.

12.Saslaw, S., H. N. Carlisle, G. L. Wolf, and C. R. Cole. 1966. RockyMountain spotted fever: clinical and labo-ratoryobservations of monkeys after respiratory expo-sure.J. Infect. Dis. 116:243-255.

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14.Wike, D. A., and W. Burgdorfer. 1972. Plaque forma-tion in tissue culture by Rickettsia rickettsii isolated directly from whole blood and tick hemolymph. Infect. Immun. 6:736-738.

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VOL. 10,1979

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