756
Immunologic Responses o Vaccinia Vaccines Adminis e ed by Di e en
Pa en e al Rou es
Da id J. McClain, Shannon Ha ison,* Cu is L. Yeage ,*
Di isions o Vi ology and Medicine, US A my Medical Resea ch
Ins i u e o In ec ious Diseases, Fo De ick, F ede ick, Ma yland;
John C uz, F ancis A. Ennis, Paul Gibbs,
B ooke A my Medical Cen e , Fo Sam Hous on, San An onio, Texas;
Michael S. W igh ,* Pe e L. Summe s, James D. A hu ,
Di ision o In ec ious Diseases and Immunology, Depa men o
and Jess A. G aham*
Medicine, Uni e si y o Massachuse s Medical School,
Wo ces e , Massachuse s
To de elop a less eac ogenic bu equally immunogenic accine, his s udy o 91 human olun ee s
compa ed he sa e y and immunogenic po ency o a new, cell cul u e–de i ed accinia i us accine
adminis e ed in ade mally and in amuscula ly wi h he licensed accinia accine adminis e ed by
sca i ica ion. Cu aneous pox lesions de eloped in a highe p opo ion o sca i ica ion accinees.
Sca i ica ion and in ade mal accine ecipien s who de eloped cu aneous pox lesions had mo e
local eac ions bu also achie ed signi ican ly highe cell-media ed and neu alizing an ibody e-
sponses han hose who did no de elop pox lesions. Al hough less eac ogenic, in ade mal o
in amuscula adminis a ion o accinia accine wi hou he concomi an de elopmen o a cu a-
neous pox lesion induced lowe immune esponses.
Vaccinia i us is a membe o he O hopox genus o he P ac ices. Ce ain US mili a y uni s may equi e smallpox ac-
pox i us amily and has li le i ulence o immunocompe en cina ion du ing u u e deploymen s. Apa om mili a y needs,
humans. Apa om i s c i ical ole in he e adica ion o en- he Cen e s o Disease Con ol and P e en ion (CDC) has
demic smallpox, accinia i us has se e al biologic p ope ies ecommended accina ion o pe sons wo king in he labo a-
ha make i an excellen candida e o in oducing o eign o y wi h accinia o ecombinan accinia i uses [7]. The e-
genes ha p omp ed he in es iga ion o ecombinan accinia o e, i is impo an ha mode n p oduc ion echniques be ap-
accines [1–3]. Howe e , cellula immuni y and an ibody e- plied o he manu ac u e o a accinia i us accine o make
sponses o accinia i us adminis e ed by di e en ou es ha e i sa e and e ec i e as a eplacemen o he cu en cal lymph
no been compa ed in humans. These issues bea impo ance smallpox accine. Gi en he a endan isks associa ed wi h he
on how such accines will be used in humans and pe haps adi ional me hod o cu aneous inocula ion o accinia, we
why immune esponses o ecombinan accinia accines ha e sough o e alua e he clinical and immune esponses o al e na-
equi ed mul iple injec ions [4–6]. i e ou es o inocula ion by using a new, cell cul u e–de i ed
The only accinia accine cu en ly licensed in he Uni ed accinia accine.
S a es was p epa ed om cal lymph and is s o ed as a eeze- The accinia accines used in he smallpox e adica ion e o
d ied p oduc . Supplies o his licensed accine canno be e- we e p epa ed on a la ge scale by inocula ing he sha ed abdo-
placed, as he p oduc ion me hod o cal lymph accine is mens o cal es, sheep, o wa e bu alo wi h seed s ocks o
a chaic and he e is no adequa e acili y in which supplies accinia i us, ha es ing he in ec ed exuda i e lymph om
can be egene a ed in compliance wi h Good Manu ac u ing he inocula ion si es, and bo ling he p oduc wi h phenol and
b illian g een as bac e ios a ic agen s [8, 9]. Pa ly because o
he na u e o his p oduc ion, in which bac e ial con amina ion
was expec ed, he accines we e adminis e ed pe cu aneously
Recei ed 5 Augus 1996; e ised 19 No embe 1996.
wi h a bi u ca ed needle, a p ocess ha became known as sca i-
In o med consen was ob ained om human olun ee s in acco dance wi h
human expe imen al guidelines o he US Depa men o Heal h and Human
ica ion because o he pe manen sca ha esul ed. Fo ma ion
Se ices and he O ice o he Su geon Gene al o he US A my ( e : AR 70–
o a cu aneous pox lesion ha healed wi h a sca o med an
25).
The iews, opinions, and/o indings con ained he ein a e hose o he au ho s
impo an me hod o e i ying accina ion s a us du ing he
and should no be cons ued as an o icial Depa men o he A my posi ion,
e a o endemic smallpox. This me hod p o ed e ec i e and
policy, o decision unless so designa ed by o he documen a ion.
success ul when applied by he Wo ld Heal h O ganiza ion o
Rep in s o co espondence: D . Da id J. McClain, Di ision o Vi ology,
USAMRIID, F . De ick, F ede ick, MD 21702-5011.
a campaign o globally e adica e smallpox [10].
*P esen a ilia ions: 465 Pine D ., Jackson Hole, Wyoming (S.H.); MCHJ-
As a consequence o pe cu aneous inocula ion, in ec ious
CI, Madigan A my Medical Cen e , Tacoma, Washing on (C.L.Y.); Abbo
accine i us was p esen in he local lesion a e sca i ica ion.
Diagnos ic Di ision, Abbo Labo a o ies, I ing, Texas (M.S.W.); Depa men
o Radiology, B ooke A my Medical Cen e , Fo Sam Hous on, San An onio,
Subsequen ly, he e we e cases o inad e en au oinocula ion
Texas (J.A.G.).
and inocula ion o suscep ible accinee con ac s. Addi ional
The Jou nal o In ec ious Diseases 1997;175:756–63
complica ions included se e e local sp ead o accine i us in
q1997 by The Uni e si y o Chicago. All igh s ese ed.
0022–1899/97/7504–0003$01.00
pe sons wi h ch onic skin diseases [11]. Consequen ly, i i is
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757JID 1997;175 (Ap il) Pox Lesion Requi ed o Vaccinia Immuni y
sc eened by medical his o y and physical examina ion be o e being
possible o do so wi hou loss o immunogenici y o he ac-
en olled, wi h speci ic a en ion o he p esence o a accinia acci-
cine, i is desi able o adminis e accinia by a ou e ha does
na ion sca o any con aindica ion o accina ion. Labo a o y pa-
no esul in cu aneous lesions con aining ansmissible i us.
ame e s be o e en ollmen included HIV se ologic es , se um
Because o hese sa e y conce ns, human ials wi h ecombi-
chemis ies, a comple e blood cell coun , and a p egnancy es .
nan accinia accines ha e used he injec able ou e.
Subjec s we e accep ed i hey we e in good heal h, had no accinia
We es ed a new in es iga ional accinia accine (BB-IND
sca o his o y o accina ion, and had no signi ican abno mali ies
4984) p oduced in cell cul u e. I was de eloped o pa en e al
ha indica ed an inc eased isk o accinia immuniza ion (i.e.,
injec ion o p eclude he po en ial complica ion o inad e en
ex olia i e skin disease o diso de s o cellula immuni y). The
inocula ion o i us a endan o sca i ica ion. Du ing p eclini-
c i e ia ha excluded a pe son om pa icipa ing in he p o ocol
cal s udies, his cell-cul u ed accinia accine candida e p o ed
we e he same as hose ecommended by he CDC [7]. Volun ee s
who lacked a accina ion sca bu we e subsequen ly p o ed o be
compa able o he Bu eau o Biologics (New Yo k Boa d o
accinia-immune by hei baseline se ologic es (50% plaque-
Heal h) e e ence s ain wi h espec o pock o ma ion on
educ ion neu aliza ion i e [PRNT] o §1:20) we e excluded
cho ioallan oic memb anes o emb yona ed chicken eggs, o -
om s a is ical analyses.
ma ion o lesions a e adul abbi s we e inocula ed in ade -
Vaccina ions and s udy design. Volun ee s we e andomized
mally (id), and in ace eb al and in ape i oneal i ulence in
in o 3 g oups, wi h one- hi d ecei ing licensed accinia accine
adul and suckling ou b ed mice (unpublished da a). A subse-
by sca i ica ion, he cell cul u e–de i ed accinia accine im, o
quen phase I dose-escala ion ial e alua ed his accine in
he cell-cul u ed accinia accine id. In addi ion, one-hal o olun-
humans gi en subcu aneous inocula ions. Howe e , cu aneous
ee s accina ed id we e andomly selec ed o ha e he inocula ion
esicula (pox) lesions de eloped in an inc easing numbe o
si e wiped wi h alcohol immedia ely a e injec ion. This p ocedu e
olun ee s as he subcu aneous dose was inc eased. Neu aliz-
was o de e mine i immune ecogni ion a o ded by p ocessing
ing an ibody and lymphocy e p oli e a ion assays indica ed a
o an igen id could be achie ed wi hou he o ma ion o a cu a-
neous pox lesion. All olun ee s accina ed im unde wen alcohol
highe and ea lie immune esponse in accinees wi h cu a-
wiping a he injec ion si e a e inocula ion o minimize he chance
neous lesions han in accinees who did no de elop pox lesions
o acciden al de mal inocula ion wi h he accine. All inocula ion
(McClain DJ, unpublished obse a ions). Since he majo i y o
si es we e ini ially co e ed wi h a semipe meable d essing un il
pox lesions occu ed in olun ee s ecei ing he highes dose,
any pox lesion had scabbed o un il day 10 a e inocula ion (i
con ounding o pox lesion o ma ion wi h dose could no be
no pox lesion de eloped). Du ing he mon h ollowing accina ion,
excluded in he s a is ical analysis. The e o e, we unde ook a
subjec s we e seen as ou pa ien s wice a week du ing he i s 2
la ge s udy o examine whe he o ma ion o a cu aneous pox
weeks a e accina ion, and hen weekly o he nex 2 weeks.
lesion was c i ical o op imal immune esponses compa ed
These ou pa ien examina ions consis ed o clinical examina ions
wi h injec ion o he cell-cul u ed accinia accine.
and labo a o y es s o assess ad e se eac ions, po en ial complica-
ions, and immunogenici y.
Clinical assessmen s. Volun ee s unde wen semiweekly as-
Me hods
sessmen o local signs o symp oms a ound he inocula ion si e
Vaccines. The licensed accinia (o smallpox) accine (Wy- and o po en ial sys emic symp oms ela ed o accina ion. Any
e h-Aye s Labo a o ies, Philadelphia) is a lyophilized accinia local eac ion a he inocula ion si e was measu ed o de e mine
i us de i ed om he New Yo k Boa d o Heal h s ain [7] and he diame e o any local e y hema and indu a ion and sco ed o
is he only emaining licensed accinia accine in he Uni ed he p esence o absence o wa m h, ende ness, lymphadenopa hy,
S a es. The cell-cul u ed accinia accine was manu ac u ed ac- subcu aneous nodule, o a esicle (pox) lesion. Sys emic eac ions
co ding o Good Manu ac u ing P ac ices a e h ee successi e we e assessed by ques ioning and examining he olun ee s o he
passages in MRC-5 cells. This accine lo was de i ed om he p esence and se e i y o e e , chills, malaise, headache, myalgia,
mas e seed o he smallpox accine p e iously licensed by Con- a h algia, loss o appe i e, nausea, omi ing, dia hea, p u i us,
naugh Labo a o ies (Philadelphia). The Connaugh accine was o ash. These symp oms we e quan i a i ely sco ed based upon
one o se e al licensed accinia p oduc s used in he Uni ed S a es se e i y (0 Åno symp om; 1 Åmild, symp om can be igno ed;
un il he end o smallpox accina ion in he ea ly 1970s. 2Åmode a e, symp om a ec s ac i i y bu is elie ed by analge-
The cell cul u e–de i ed accinia accine was adminis e ed in sics; and 3 Åse e e, symp om canno be elie ed by analgesics).
a dose o 5.1 log
10
p u, ei he as 0.1 mL id o 0.5 mL in amuscu- Da a we e en e ed in o a da abase o subsequen s a is ical analy-
la ly (im). The licensed accinia accine was adminis e ed by sis. The o al local o sys emic symp om sco e o a gi en accinee
sca i ica ion (as di ec ed in he package inse ) by dipping a s e ile was de ined as he sum o all sco es o ei he local o sys emic
bi u ca ed needle in o he accine and hen p icking he skin inocu- symp oms.
la ion si e h ee imes. The licensed accinia accine has a i us Clinical labo a o ies. Blood samples we e ob ained by weekly
i e o Ç10
8
p u/mL, and he adminis a ion o 1 d op ia sca i i- phlebo omy o olun ee s, beginning be o e accina ion un il Ç1
ca ion wi h a bi u ca ed needle is es ima ed o deli e 2.5
m
L. mon h a e accina ion. A comple e blood cell coun wi h a i e-
The e o e, Ç10
5
p u is deli e ed by his me hod. All accina ions pa di e en ial cell coun was done using a cell coun e (CellDyn
we e adminis e ed in o he del oid egion o he a m. 3000; Abbo Labo a o ies, Abbo Pa k, IL). Se um samples we e
Subjec s. The accines we e adminis e ed in an open-label analyzed by an Ek achem 700XR (Eas man Kodak, Roches e ,
NY) o a panel o chemis y measu emen s (sodium, po assium,s udy o heal hy accinia-nai e olun ee s. Volun ee s we e
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758 McClain e al. JID 1997;175 (Ap il)
chlo ide, bica bona e, u ea ni ogen, c ea inine, glucose, calcium, 2% hea -inac i a ed FBS. Cul u es we e incuba ed a 357Cina
humidi ied a mosphe e o 5% CO
2
o 4–6 days. Cul u es we ephospho us, lac a e dehyd ogenase,aspa a e ansaminase, alanine
ansaminase,
g
glu amyl ans e ase, alkaline phospha ase, o al decan ed and ixed wi h 1 mL (200
m
L o he 96-well pla es) o
10% o malin o 15 min a oom empe a u e. Pla es we e incu-bili ubin, and c ea ine phosphokinase). Tes alue means we e
calcula ed o each g oup on a gi en day o measu emen . The ba ed wi h 1 mL (200
m
L o he 96-well pla es) o HBSS con-
aining 1% bo ine se um albumin (BSA) o 30 min. The blocke da a om each hema ologic and se um chemis y es we e ana-
lyzed using S a is ical Analysis Sys em p ocedu e GLM ( e sion bu e was emo ed and 0.5 mL (50
m
L o he 96-well pla es) o
a 1:1000 dilu ion o accinia mouse hype immune asci ic luid6.10; SAS, Ca y, NC) epea ed measu es analysis o a iance
(ANOVA). The o e all di e ences be ween g oups o e he cou se (ATCC, Rock ille, MD) o no mal mouse asci ic luid was added
o he app op ia e wells o 1 h a 357C. A e h ee washes, 0.2o he s udy pe iod we e compa ed by using he uni a ia e es s
o hypo hesis o wi hin-subjec s e ec s. mL (35
m
L o he 96-well pla es) o a 1:2000 dilu ion o pe oxi-
dase-labeled an i–mouse IgG (Ki kegaa d & Pe y) was added oSe ologic assays. Se um specimens om days 0, 10, 14, 20,
and 27 we e ozen o subsequen ELISA and PRNT assay. An all wells o 1 h a 357C. Pla es we e hen washed i e imes and
incuba ed 30 min a 357C wi h 0.5 mL/well (80
m
L/well o heELISA was pe o med as p e iously desc ibed [12] o assay o
an ibodies eac i e wi h cell lysa e an igens om accinia i us– 96-well pla es) o ABTS (2,2’-azino-di[3-e hyl-benz hiazoline sul-
ona e]) subs a e (Ki kegaa d & Pe y). Tes samples and posi i ein ec ed cells, wi h he modi ica ion o human se a as he es
specimen and goa an i–human IgG as he de ec o an ibody and nega i e con ols we e es ed in duplica e. A posi i e con ol
was p epa ed using 10- old dilu ions o accinia i us in nega i e(Ki kegaa d & Pe y, Gai he sbu g, MD). Gi en a lowe limi o
de ec ion o he assay a a 1:100 es se um dilu ion, nega i e i e s an ibody se um. Using hese ‘‘spiked’’ se um samples, 2 wells
we e inocula ed wi h each dilu ion. Two wells we e also inocu-(i.e., op ical densi y compa able o backg ound) we e epo ed as
equal o 1:50. la ed wi h no mal (nonin ec ious) human se um as a nega i e
con ol. Resul s we e ead isually o spec opho ome ically a PRNT, an in i o es o se um’s abili y o neu alize he Wye h
s ain o accinia i us, was de e mined using a modi ica ion o 414 nm. Ti e s we e calcula ed as TCID
50
alues acco ding o
he me hod o Reed and Muench [16] o as he highes specimen he me hod o Ea ley e al. [13]. B ie ly, each coded se um sample
was incuba ed a 567C o 30 min, hen dilu ed 1:10 in Eagle MEM dilu ion wi h an op ical densi y o 0.2 uni s o e ha o he
nega i e con ols.(EMEM) con aining 10% hea -inac i a ed e al bo ine se um
(FBS). A suspension o accinia i us, calcula ed o yield a dose S a is ical me hods. The PRNT and ELISA an ibody esponses
we e analyzed by a iance wi h epea ed measu es ollowed byo Ç40–100 p u/0.1 mL, was p epa ed in Hanks’ balanced sal
solu ion (HBSS) wi h 40 mMHEPES. Two- old dilu ions o se um mul iple compa isons o he s udy g oups using he Tukey-K ame
adjus men o mul iplici y [17]. The da a om each hema ologicsamples we e hen mixed 1:1 wi h 40–100 p u o accinia i us
suspension and incuba ed a 377C o 1 h. A e incuba ion, es and se um chemis y es and lymphocy e ans o ma ion assays
we e analyzed o each s udy g oup by using SAS p ocedu e GLMsamples and con ols we e inocula ed on o monolaye s o Ve o
cells in 12-well cell cul u e pla es. A e adso p ion o 1 h a ANOVA. The o e all di e ences be ween g oups o e he cou se
o he s udy we e compa ed by using he uni a ia e es s o hypo h-377C, each monolaye was o e laid wi h 1 mL o 0.5% aga ose
(FMC Biop oduc s, Rockland, ME) con aining HEPES-bu e ed esis o wi hin-subjec s e ec s. All s a is ical es s we e pe o med
a he
a
Å.05 le el unless o he wise indica ed.Eagle basal medium wi h Ea le’s sal s, and 5% hea -inac i a ed
FBS. A e incuba ion o 2 days a 377C in a humidi ied a mo-
sphe e o 5% ( ol/ ol) ca bon dioxide (CO
2
), each monolaye was
s ained wi h 1 mL o he aga ose o e lay con aining 0.167 mg/
Resul s
mL neu al ed dye. The pla es we e e u ned o he incuba o o
24–36 h and he plaques we e coun ed. The numbe o plaques
Subjec s. Nine y-one olun ee s pa icipa ed in his phase
o each es sample was en e ed in o a compu e p og am o de e -
II s udy. Da a om 8 olun ee s we e excluded om s a is ical
mine he PRNT using p obi analysis. The end-poin i e was
analysis because o p eexis ing accinia immuni y as de e -
he highes se um dilu ion demons a ing ú50% educ ion in he
mined by baseline se ologies. O he emaining 83 subjec s, 11
numbe o plaques o he a e age dose.
we e emale and 72 we e male. Du ing he s udy, 3 olun ee s
Lymphocy e p oli e a ion assays. P oli e a i e esponses o
de eloped medical p oblems ha we e judged as un ela ed o
pe iphe al blood mononuclea cells (PBMC) o li e accinia i us
and hea -inac i a ed an igen we e es ed as p e iously desc ibed
he p o ocol: 1 olun ee de eloped olliculi is 1 week a e
[14, 15]. Resul s we e exp essed as a s imula ion index, de i ed
inocula ion; ano he de eloped lowe ex emi y celluli is 2
by di iding coun s in wells con aining an igen by coun s in wells
weeks a e inocula ion seconda y o an in ec ed leg lace a ion;
wi hou accinia an igen o i us.
and a hi d de eloped nausea, omi ing, and abdominal pain,
In si u ELISA i emia assay. Volun ee s unde wen pe iodic
which led o subsequen lapa oscopic appendec omy on day 8
se um sampling wi hin he i s 2 weeks a e inocula ion, wi h an
a e inocula ion, wi h no mal appendiceal his opa hology and
aliquo ozen a 0707C un il la e assay o accinia i emia.
e en ual ull eco e y.
Se um was assayed o i emia by a modi ica ion o an in si u
Clinical assessmen s. ANOVA o accine e ec s a each
ELISA. B ie ly, Ve o cells g own in 24- o 96-well pla es we e
week yielded no signi ican di e ences be ween he s udy
inocula ed wi h 0.1 mL o 50
m
L, espec i ely, o a se um sample.
g oups’ empe a u e p o iles ( empe a u e maximums eco ded
A e adso p ion o 1 h a 357C, he 24-well pla es we e e- ed
wi h 1 mL (100
m
L o he 96-well pla es) o EMEM con aining
each week).
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759JID 1997;175 (Ap il) Pox Lesion Requi ed o Vaccinia Immuni y
The accinees inocula ed id who ecei ed alcohol wiping a eas o con ac de ma i is om d essing adhesi e we e ob-
se ed.a e inocula ion and he accinees inocula ed im did no sig-
ni ican ly di e wi h espec o incidence o pox lesion (25% Clinical labo a o ies. Labo a o y indings we e based on
he analysis o he weekly mean o each subjec o weeks s. 4.5%, PÅ.141). Howe e , ailu e o wipe wi h alcohol
inc eased he incidence in he g oup inocula ed id o 62.5%, 01, 0, 1, 2, 3, and 4. Due o some missing alues when olun-
ee s omi ed scheduled phlebo omies, no epea ed measu eswhich was signi ican ly highe hen ha in he g oup inocula ed
im (Põ.001). This was signi ican ly less han he incidence ANOVA could be done. Hence, an ANOVA was done a each
week. Only spo adic di e ences we e de ec ed o any pos -o pox lesions in he sca i ica ion g oup (96.6%), which was
highe han ha in all he o he g oups (Põ.001). ea men week (no ed in able 1.)
Se ologic and i ologic assays. PRNTs we e signi ican lyThe sca i ica ion g oup had he highes incidence o local
eac ions, he g oup inocula ed im expe ienced he ewes , and g ea e o he sca i ica ion g oup han o he g oups inocula ed
ei he im o id beginning a day 13 a e inocula ion (Põ.001) he subg oups inocula ed id we e in e media e and indis in-
guishable om each o he . Local symp om analysis indica ed using a epea ed measu es ANOVA. Mo eo e , olun ee s
om ei he sca i ica ion o g oups inocula ed id who de elopedhighly signi ican s a is ical di e ences be ween he s udy
g oups, using bo h ANOVA and nonpa ame ic Wilcoxon anal- cu aneous pox lesions had signi ican ly highe neu aliza ion
i e s han hose wi hou pox lesions in compa isons adjus edyses (Põ.001). The e was a highe o al symp om sco e o
he sca i ica ion g oup (mean, 169.2) and a signi ican ly lowe o p e accina ion baseline. The e was no signi ican di e ence
be ween olun ee s inocula ed by sca i ica ion o id who hadsco e o he g oup inocula ed im (mean, 3.0). Mean o al sco es
o accinees inocula ed id wi h and wi hou alcohol wiping de eloped a cu aneous esicle. Howe e , he e was a s a is i-
cally insigni ican end owa d highe i e s in he sca i ica ionwe e 61.7 and 90.4, espec i ely. Addi ional analysis exclusi e
o he g oup inocula ed im e ealed ha he sca i ica ion g oup (Wye h accine) ecipien s. These PRNT esponses a e illus-
a ed in igu e 1.s ill di e ed om he subg oups inocula ed id (Põ.001 by
ANOVA o nonpa ame ic analysis). When he wo subg oups As illus a ed in igu e 2, accinia ELISA an ibody esponses
we e signi ican ly highe o he sca i ica ion g oup a day 27inocula ed id we e compa ed, no di e ences we e ound in
local symp om sco es (PÅ.215 by ANOVA, PÅ.406 by a e accina ion han o ei he o he g oups inocula ed id o
im (Põ.001). In con as o PRNT esponses, signi ican lynonpa ame ic analysis). When local symp om sco es we e
compa ed be ween olun ee s who did and did no de elop highe ELISA i e s we e ound in sca i ica ion accinees han
in hose inocula ed id who o med a cu aneous pox lesion (Pcu aneous pox lesions, o e all and weekly sco e di e ences
we e signi ican (PÅ.0001, nonpa ame ic Wilcoxon es ). õ.001). The e was g ea e s anda d e o in his assay han in
he PRNT assay. No i emia was de ec ed in any olun ee .Sys emic symp om analysis indica ed no signi ican di e -
ences in he s udy g oups wi h espec o o al symp om sco es Lymphocy e p oli e a ion assays. Lymphocy e p oli e a-
ion assays examined s imula ion indices be o e and a e acci-ei he by ANOVA (PÅ.352) o by nonpa ame ic Wilcoxon
es (PÅ.412). Howe e , highe mean sys emic sco es oc- na ion o bo h li e accinia i us and hea -inac i a ed an igen.
Fo he hea -inac i a ed an igen, esponses o sca i ica ion we ecu ed 2 weeks a e inocula ion in olun ee s who de eloped
a cu aneous pox lesion (PÅ.038).
All olun ee s ini ially had hei immuniza ion si es co e ed
wi h a apo -pe meable su gical d essing un il pox lesions
Table 1. S a is ically signi ican di e ences in labo a o y alues o
g oups o subjec s inocula ed agains accinia i us in ade mally (id)
scabbed. Howe e , 4 olun ee s wi h p ima y esicles om
o in amuscula ly (im) o by sca i ica ion (sca ).
hei accina ions subsequen ly de eloped seconda y pox le-
sions adjacen o he inocula ion si e, unde nea h he d essing.
id
We no ed ha he d essings we e occlusi e enough o accumu-
la e pe spi a ion unde nea h and a ound he inocula ion si e.
S udy Wi h Wi hou
Tes week wipe wipe im Sca P
Consequen ly, in he majo i y o olun ee s wi h pox lesions,
esicula exuda e accumula ed unde hese d essings despi e
C ea inine (mg%) 1 1.2 1.08 1.12 1.18 .0175
equen d essing changes. This i us-con aining exuda e
Po assium (mEq/L) 3 4.2 4.49 4.25 4.49 .0043
seeped unde he d essing and co e ed a eas much g ea e han
Phospho us (mEq/L) 4 4.05 4.09 4.57 4.45 .0274
ha o he p ima y pox lesion, wi h subsequen seconda y o
% monocy es 2 7.23 8.53 7.6 9.34 .0032
% monocy es 1 6.16 8.1 8.24 7.54 .0392
au oinocula ion. Subsequen ly, a nonocclusi e and smalle
d essing (i.e, d y gauze) was applied o e he inocula ion si es
NOTE. Da a we e analyzed o each s udy g oup by using SAS P ocedu e
o he emaining olun ee s. D essings we e changed i he e
GLM analysis o a iance. O e all di e ences be ween g oups o e s udy
pe iod we e compa ed by using uni a ia e es s o hypo hesis o wi hin-sub-
was a scheduled clinical check, he bandage became we , o
jec s e ec s (
a
Å.05). Monocy es a e exp essed as % o o al whi e blood
he e we e any isible signs o d ied exuda e on he ex e io
cells in comple e blood cell coun . Wi h wipe Åinocula ion id wi h local
su ace o he bandage. A e he ins i u ion o he d y gauze
alcohol wiping a e inocula ion; wi hou wipe Åinocula ion id wi hou alcohol
wiping a e inocula ion.
d essings, no u he inciden o seconda y pox lesions o la ge
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760 McClain e al. JID 1997;175 (Ap il)
accines is no a new idea. Be ween 1930 and 1975, a leas
8 s ains o accinia i us we e de eloped o pa en e al admin-
is a ion in o de o dec ease i ulence [18]. All we e a enua ed
when compa ed wi h s anda d lymph s ains, and some we e
possibly o e a enua ed, p oducing low neu alizing an ibody
le els a e p ima y and boos e inocula ions [19]. Wi h he
e adica ion o smallpox ollowing closely upon ini ial de elop-
men o mos o hese accines, compa able sa e y and e icacy
da a we e no ob ained on a scale compa able o ha o lymph
accines. In addi ion, da a abou cell-media ed esponses we e
no documen ed o hese injec able p oduc s.
In his s udy, accina ion eac ions we e eadily appa en
om clinical obse a ion alone. Ex ensi e labo a o y moni o -
ing o accinees iden i ied no speci ic sa e y conce ns o issues
abou he ou es o accine adminis a ion. The e was no e i-
dence o i emia in any olun ee . Se um chemis y o hema ol-
ogy alue di e ences be ween s udy g oups we e spo adic and
ela i ely small. Gi en he numbe o da a poin s and compa i-
sons in his s udy, hese we e likely o be ype I e o s. As
Figu e 1. Vaccinia plaque- educ ion neu aliza ion i e (PRNT)
hese s a is ical di e ences do no ep esen medically im-
50% esponses as illus a ed by geome ic mean i e s (GMTs).
PRNTs we e signi ican ly highe o sca i ica ion inoculees on e-
po an di e ences in a con iguous ime ame, we do no a i-
pea ed measu es analysis o a iance (Põ.001) bu no signi ican ly
bu e hem o causal e en s ela ed o espec i e ea men s.
highe han in ade mal (id) injec ion ecipien s who de eloped a
The s udy indica ed ha , al hough no se e e, local eac ions
cu aneous pox lesion (id w/ pox) a e accina ion (PÅ.09). id
co esponded o o ma ion o a cu aneous pox lesion. As ex-
w/o pox Åin ade mal injec ion ecipien s wi hou pox lesion a e
pec ed, pox lesion incidence was signi ican ly highe in he
accina ion; im Åin amuscula injec ion ecipien s.
sca i ica ion g oup han in he o he g oups, as we e local symp-
oms. In addi ion, highe sys emic sco es occu ed 2 weeks
a e inocula ion in olun ee s who de eloped a cu aneous pox
signi ican ly g ea e han hose o ei he subg oup inocula ed
im o id wi h alcohol wiping a e inocula ion (PÅ.0258) bu
no signi ican ly di e en om hose in he g oup inocula ed
id wi hou alcohol wiping (PÅ0.46). Wi h li e accinia i us
as he an igen, sca i ica ion accinees had signi ican ly highe
s imula ion indices (PÅ.0037) han did ei he g oup inocula ed
im o id. Bo h highe indices and g ea e s anda d de ia ion
we e seen in he assaywi h hea -inac i a ed an igen in compa i-
son o he li e i us assay.
Fo olun ee s who de eloped a pox lesion (whe he id o
by sca i ica ion), he s imula ion indices be o e and a e acci-
na ion we e signi ican ly g ea e han o accinees who did
no de elop a cu aneous pox lesion, whe he assayed using li e
i us o hea -inac i a ed an igen (Põ.001). The e was no
signi ican di e ence in lymphocy e s imula ion esponses be-
ween hose who o med pox lesions om he licensed accinia
accine ia sca i ica ion and hose wi h pox lesions om he
cell-cul u ed accinia accine. These mean lymphocy e e-
sponses o hea -inac i a ed an igen a e illus a ed in igu e 3.
Figu e 2. ELISA esponses o accinia-in ec ed whole cell lysa es
Discussion
as illus a ed by geome ic mean i e s (GMTs). ELISA i e s we e
signi ican ly highe o sca i ica ion inoculees on epea ed measu es
This human s udy compa ed he sa e y and immunogenici y
analysis o a iance (Põ.001), e en in compa ison o in ade mal
o adminis a ion id and im o a cell cul u e–de i ed accinia
(id) injec ion ecipien s who o med cu aneous pox lesions (Põ
accine wi h hose o he licensed accine adminis e ed by he
.001). id w/o pox Åid ecipien s wi hou poxlesions a e accina ion;
im Åin amuscula injec ion ecipien s.
adi ional ou e o sca i ica ion. The use o injec able accinia
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761JID 1997;175 (Ap il) Pox Lesion Requi ed o Vaccinia Immuni y
o a dose o 10
7.8
p u, bu wi h an a endan inc eased isk o
a cu aneous pox lesion. Al hough he c ossing o an addi ional
issue plane wi h injec ion im may ha e lessened his occu -
ence, we we e conce ned ha injec ion im o a simila high
dose o accinia i us migh isk i emia.
The s udy also demons a ed ha inocula ion id will no
eliably p e en he o ma ion o a cu aneous pox lesion. Phase
I da a had aised simila conce ns ega ding he subcu aneous
ou e o inocula ion. In a s udy by Conno e al. [21], ypical
Jenne ian esicles we e seen in 4%–9% o child en accina ed
subcu aneously wi h 10
3
,10
4
,o 10
5
p u. Fu he mo e, i is
appa en ha he p esence o such a lesion, al hough undesi -
able o sa e y easons, is necessa y o he mos obus immune
esponses. By cleansing he inocula ion si e a e inocula ion,
we sough o de e mine i immune ecogni ion a o ded by
in ade mal p ocessing o an igen could be achie ed wi hou
he sa e y isks a endan o a cu aneous pox lesion. This con-
cep s emmed om a p e ious obse a ion ha he cu aneous
esicle was p e en ed a e inocula ion id wi h accinia i he
needle wound was immedia ely cleansed wi h alcohol [22].
Figu e 3. Lymphocy e p oli e a ion ans o ma ion esponses o
These esul s indica e ha eplica ion o he i us in he skin,
hea -inac i a ed accinia an igen. S imula ion indices we e signi i-
can ly highe o sca i ica ion inoculees on analysis o a iance (PÅ
no me ely exposu e o an igen-p esen ing cells (i.e., Lange -
.0258) bu no signi ican ly highe han hose o in ade mal (id)
hans cells) in ha loca ion, is essen ial o gene a e op imal
injec ion ecipien s who de eloped cu aneous pox lesions a e acci-
immune esponses o accinia an igens.
na ion (PÅ.46). id w/o pox Åid injec ion ecipien s wi hou pox
Vaccinees who de eloped cu aneous pox lesions had sig-
lesions a e accina ion; im Åin amuscula injec ion ecipien s.
ni ican ly highe ELISA and neu alizing an ibody esponses.
The poo esponses o inocula ion id (in hose wi hou pox
lesions) and im con i m and expand on ea lie obse a ionslesion. Ei he accine dissimila i ies o he ou e o accina ion
could accoun o he symp oma ic di e ences be ween sca i- conce ning injec able accina ions in child en [19]. The in es i-
ga o s o ha s udy concluded ha ‘‘... un il mo e is known ica ion accinees and accinees inocula ed id who de eloped
a pox lesion: Impu i ies and animal p o eins peculia o cal abou he impo ance o neu alizing an ibody in he immuni y
o smallpox, i would seem unwise o adminis e accine bylymph exuda e could explain in pa he eac i i y o he li-
censed accinia accine, o sca i ica ion could pe mi g ea e he sub-cu aneous ou e...’’ [21]. This conclusion was based
on he ela i ely poo a es o se ocon e sion (67%), as mea- i al eplica ion han would in ade mal inocula ion. Gi en
compa able esul s in p eclinical es s and he ac ha bo h su ed by accinia-neu alizing an ibodies, in child en acci-
na ed subcu aneously compa ed wi h hei pe cu aneously ac- accines s udied de i ed om New Yo k Boa d o Heal h
s ains, i is unlikely ha he cell cul u e–de i ed accinia i us cina ed coun e pa s (87%), and he unimp essi e neu alizing
an ibody esponses upon success ul pe cu aneous e accina ionhas enough sequence a ia ion o in insic biologic dispa i ies
om he cal lymph–de i ed Wye h accine o accoun o o child en whose p ima y accina ion was subcu aneous.
In addi ion o an ibody esponses, cell-media ed esponsessuch di e ences. The accinees inocula ed id who ecei ed
alcohol wiping a e inocula ion and hose inocula ed im did we e also supe io in olun ee s who de eloped pox lesions
(whe he inocula ed id o by sca i ica ion). The e was no sig-no signi ican ly di e wi h espec o incidence o pox lesions,
bu he la e g oup had signi ican ly ewe local symp oms ni ican di e ence in lymphocy e s imula ion esponses be-
ween hose who o med pox lesions om he licensed accinia han did hose inocula ed id, who in u n had ewe han he
sca i ica ion accinees. accine by sca i ica ion and hose wi h pox lesions om he
cell cul u e–de i ed accinia accine. In e io cell-media edThis s udy s ongly indica ed ha , al hough less eac ogenic,
accinia accine adminis e ed im a a dose o 10
5
p u ails o esponses o accina ion wi hou a pox lesion, as obse ed in
his s udy, may explain why Conno e al. [21] epo ed ha induce an immune esponse compa able o ha elici ed by
s anda d sca i ica ion. Al hough a highe dose o accinia i us child en who had ini ially ecei edan injec able (subcu aneous)
accine exhibi ed ú30% incidence o ‘‘p ima y- ype’’ e-migh ha e been a emp ed im, he WHO Smallpox E adica ion
Campaign [10] and o he clinical ials [18–21] had suppo ed sponses o pe cu aneous e accina ion. This s udy also con i ms
he obse a ion by Che y e al. [20] o a co ela ion be ween he sa e y o a pa en e al dose o 10
5
p u, as used in his
ial. In a phase I dose-escala ion human ial, he cell cul u e– neu alizing an ibody and lymphocy e s imula ion espon-
si eness o accinia a e accina ion.de i ed accinia accine was used sa ely subcu aneously up
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762 McClain e al. JID 1997;175 (Ap il)
An unexpec ed e en in his phase II s udy was he high us ec o s ha a e incapable o p oduc i e eplica ion in hu-
man-de i ed cell lines [25, 26].incidence o seconda y pox lesions adjacen o he inocula ion
si e in he i s se o olun ee s (4/31). The use o semipe me- When adminis a ion id o he cell-cul u ed accinia esul ed
in a cu aneous pox lesion, immune esponses we e no signi i-able d essings appea ed o ha e caused his complica ion, as
no u he ins ances occu ed a e ins i u ing he use o d y can ly di e en om hose seen wi h sca i ica ion wi h he
licensed accinia accine. Al hough he e is a s a is ically insig-gauze d essings. Al hough apo -pe meable, he semipe me-
able d essings caused accumula ion o pe spi a ion a ound he ni ican end owa ds highe neu aliza ion i e s wi h he la e ,
u u e s udies will examine he cell-cul u ed accine’s immuno-inocula ion si e despi e equen d essing changes, pe mi ing
i us-con aining exuda e o seconda ily inocula e a eas p oxi- genici y by his same sca i ica ion ou e.
ma e o he p ima y pox lesion. The la ge adhesi e a ea o his
d essing caused a ious deg ees o con ac de ma i is in se e al
Acknowledgmen
olun ee s, bu his did no igge au oinocula ion, since he
seconda y lesions we e unde he d essing a ea and no a he
We g a e ully acknowledge he skill ul assis ance o Tama a
poin o adhesi e con ac . I onically, he use o hese semipe -
Lewis in he comple ion o ELISAs.
meable d essings was o iginally manda ed as a p o ec i e mea-
su e agains con ac and en i onmen al sp ead o he accine
Re e ences
i us. Apa om he ou ins ances o au oinocula ion p ecipi-
a ed by semipe meable d essings, he e we e no se ious o
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