Pa o i us B19 B Cell Memo y •JID 2004:189 (15 May) •1873
MAJOR ARTICLE
B Cell Memo y Is Di ec ed owa d Con o ma ional
Epi opes o Pa o i us B19 Capsid P o eins
and he Unique Region o VP1
Amanda Co co an,
1
Be na d P. Mahon,
2
and Sean Doyle
1
1
Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, and
2
Ins i u e o Immunology, Na ional Uni e si y o I eland, Maynoo h,
Maynoo h, Coun y Kilda e, I eland
Backg ound. Loss o an ibody eac i i y agains linea epi opes o pa o i us B19 (B19) capsid p o eins VP1
and VP2 occu s a e in ec ion; howe e , i is unclea whe he B cell memo y is es ablished agains linea epi opes.
Me hods. B cell enzyme-linked immunospo assay was used o e alua e B19-speci ic B cell memo y in olun ee
dono s ( ).np22
Resul s. B cell memo y is main ained agains con o ma ional epi opes o VP2 and is absen agains linea
epi opes o VP2. Indi iduals se onega i e o IgG agains he unique egion o VP1 ha e de ec able B cell memo y,
wi h he po en ial o moun a humo al esponse on eexposu e o B19. Con e sely, in mice immunized wi h VP2,
long-las ing IgG agains linea epi opes o VP2 and a s ong B cell–memo y esponse a e obse ed.
Conclusions. B cell memo y is es ablished and main ained agains con o ma ional epi opes o VP2 and agains
linea epi opes o VP1 bu no agains linea epi opes o VP2. These indings u he ou unde s anding o he
immune esponse o B19 and sugges ha analysis o B19-speci ic B cell memo y me i s conside a ion o u u e
B19- accine s udies.
Pa o i us B19 (B19) is a small, nonen eloped e y h-
o i us ha usually causes a mild, sel -limi ing disease
(called “e y hema in ec iosum”) in child en. Howe e ,
immunocomp omised pa ien s (such as pa ien s wi h
HIV o pa ien s wi h cance who a e unde going che-
mo he apy), p egnan women, and, inc easingly, ans-
plan ecipien s a e a signi ican isk o B19 in ec ion
[1, 2]. Many such indi iduals a e unable o p oduce
neu alizing an ibodies o clea he i us, and his can
lead o a pe sis en in ec ion esul ing in anemia [1].
I is signi ican ha many blood-p oduc companies
ha e begun o implemen sc eening p o ocols o min-
imize he B19 i al load in such blood-de i ed p od-
uc s as packed ed blood cells, albumin, and in a e-
nous immunoglobulin [3].
Recei ed 25 Augus 2003; accep ed 1 No embe 2003; elec onically published
26 Ap il 2004.
Financial suppo : Commission o he Eu opean Communi ies (g an QLK2-CT-
2001-00877); B.P.M. is a Wellcome T us New Blood Fellow.
Rep in s o co espondence: D . Sean Doyle, Na ional Ins i u e o Cellula Bio-
echnology, Dep . o Biology, Na ional Uni e si y o I eland, Maynoo h, Maynoo h,
Coun y Kilda e, I eland ([email protected]).
The Jou nal o In ec ious Diseases 2004;189:1873–80
2004 by he In ec ious Diseases Socie y o Ame ica. All igh s ese ed.
0022-1899/2004/18910-0011$15.00
B19 encodes 3 main p o eins: a nons uc u al p o-
ein, NS1 (77 kDa), and 2 s uc u al p o eins, VP1 (83
kDa) and VP2 (58 kDa) [4]. The la e 2 p o eins, which
compose he i al capsid (5% VP1; 95% VP2), a e
colinea , excep ha VP1 con ains a 227- esidue amino-
e minal ex ension (he ea e , “unique egion”), com-
pa ed wi h VP2 [5]. A ansien , high i emia is ob-
se ed in indi iduals a e in ec ion wi h B19, ollowed
by he de elopmen , wi hin a numbe o days, o B19-
speci ic IgM [6]; wi hin a numbe o weeks, he p es-
ence o B19-speci ic IgG di ec ed agains linea and
con o ma ional epi opes o i al capsid p o eins VP1
and VP2, in addi ion o he p esence o IgG di ec ed
agains NS1, is e iden [3, 7, 8]. B19 in ec ion leads o
an in iguing immunological phenomenon—a speci ic
loss o IgG, ini ially eac i e agains linea epi opes o
VP1 and VP2, occu s a e in ec ion [8, 9]. In ac ,
speci ic IgG agains linea epi opes o VP2 disappea s
wi hin 6–12 mon hs a e in ec ion [8]. Mo e d ama -
ically, he e is a nea - o al diminu ion o NS1-speci ic
IgG in p e iously in ec ed indi iduals [10]. I is, he e-
o e, i mly es ablished ha de ec ion o B19-speci ic
IgG agains con o ma ional epi opes is cu en ly he
mos eliable me hod o he de ec ion o p e ious in-
ec ion wi h, and p obable immuni y o, B19. Howe e ,
1874 •JID 2004:189 (15 May) •Co co an e al.
he eason o he pos in ec ion loss o IgG eac i i y agains
linea epi opes o B19 capsid p o eins has no ye been eluci-
da ed, and ew mechanisms ha would explain his loss ha e
been sugges ed [8].
Recen ly, conside able e o has been di ec ed owa d he elu-
cida ion o he na u e o he T cell esponse o B19 and owa d
he de ini ion o T cell epi opes, ye he na u e o B cell memo y
di ec ed agains B19 emains unclea [11, 12]. Memo y B cells
make a signi ican con ibu ion o p o ec i e immuni y and a e
cha ac e ized in e ms o (1) a apid p oli e a i e esponse,
accompanied by cellula di e en ia ion a e an igen eexpo-
su e, o p oduce a ini y-ma u ed, an ibody-sec e ing plasma
cells; (2) a lowe ac i a ion h eshold, compa ed wi h ha o
nai e B cells, in esponse o cy okine and an igen; and (3) an
absence o spon aneous immunoglobulin sec e ion. Recen ly,
he iden i ica ion o an igen-speci ic memo y B cells agains
accine an igens (diph he ia and e anus), whe eby isola ed
pe iphe al-blood mononuclea cells (PBMCs) we e nonspeci -
ically s imula ed by bac e ial an igens and in e leukin-2 (IL-2),
ollowed by he speci ic de ec ion o an igen-speci ic memo y
B cells, has been desc ibed [13]. This app oach may also p o e
o be use ul in he elucida ion o i us-speci ic B cell–media ed
immuni y.
Al hough B19 linea epi ope–speci ic IgG is absen in p e-
iously in ec ed indi iduals, i is cu en ly unclea whe he B
cell–memo y esponse is e e es ablished agains linea o con-
o ma ional epi opes o B19 p o eins in in ec ed indi iduals.
Fu he mo e, in he absence o eliable cell-cul u e echniques
o he de ec ion o B19, no me hod is cu en ly a ailable o
he con i ma ion o ei he pas exposu e o he i us o se ology
esul s o plasma cell–de i ed B19-speci ic IgG. He e we de-
sc ibe he in es iga ion o B cell memo y agains B19, an in-
es iga ion using a B cell enzyme-linked immunospo (ELISpo )
assay o enume a e ci cula ing B cells ha a e speci ic o ei he
con o ma ional o linea ized B19 capsid p o eins. This ech-
nique could also be used o de ine he immune s a us achie able
wi h candida e B19 accines and o aid in he design o im-
muniza ion p o ocols ha adequa ely induce ma u e immune
esponses and so ensu e long-las ing p o ec ion. Da a a e p e-
sen ed ha u he ou knowledge o B cell memo y di ec ed
agains speci ic i al an igens; he use o a B19-speci ic B cell
ELISpo assay o e s he possibili y ha pas exposu e o, and
in ec ion wi h, B19 can be con i med.
SUBJECTS, MATERIALS, AND METHODS
S udy popula ion. Hepa inized enous-blood samples (10 mL;
) we e d awn om a coho o heal hy adul olun ee snp22
(mean age, 26 yea s; age ange, 21–41 yea s) and we e p ocessed
⭐2 h a e collec ion. P io o he collec ion o samples, consen
was ob ained om all olun ee s, and app o al was ob ained
om he e hics commi ee o he Na ional Uni e si y o I eland,
Maynoo h. The human-expe imen a ion guidelines o he au-
ho s’ ins i u ion we e ollowed in he conduc o his esea ch,
as we e he animal-expe imen a ion guidelines o hego e nmen
o I eland’s Depa men o Heal h and Child en.
An igens. B19 ecombinan p o eins VP1, VP2, and NS1
we e p oduced in, and pu i ied om, Spodop e a ugipe da (S 9)
insec cells in ec ed wi h ecombinan baculo i us encoding ei-
he VP1, VP2, o NS1 open- eading ames, as desc ibed else-
whe e [10, 14–16].
An ibody assays. Plasma ob ained om blood samples was
used o e alua e B19-speci ic IgG eac i i y o he i al capsid
p o eins. B19-speci ic IgG agains na i e VP2 (VP2-N) was de-
e mined by a comme cial EIA (Bio in In e na ional) and was
quan i ied using he Wo ld Heal h O ganiza ion B19-speci ic
IgG in e na ional-s anda d p epa a ion (code 93/724; Na ional
Ins i u e o Biological S anda ds and Con ol). IgG eac i i y
agains dena u ed VP1 (VP1-D) and dena u ed VP2 (VP2-D)
was de e mined by EIA o ma s de eloped in-house, as de-
sc ibed elsewhe e [16]. In b ie , mic o i e pla es (Nalge Nunc)
we e coa ed o e nigh a 4C wi h ei he VP1-D o VP2-D.
The mic o i e pla es we e hen washed and blocked be o e
being incuba ed wi h plasma samples ha had been dilu ed
1:100 in PBS con aining 0.05% ( / ) Tween-20 (PBST); he
pla es we e hen incuba ed wi h ho se adish pe oxidase (HRP)–
conjuga ed an i–human IgG (Dako) o 30 min. A e a wash
s ep (4 washes wi h PBST), e ame hylbenzidine subs a e was
added o he wells, which we e hen incuba ed o 10 min. The
enzyma ic colo eac ion was e mina ed by adding 1 NH
2
SO
4
.
The abso bance was ead a 450 nm and a 630 nm. Immu-
noassay cu o s we e de ined as he abso bance plus 2 SDs
g ea e han he mean abso bance, as de e mined om a panel
o B19-speci ic IgG–nega i e specimens. Resul s a e exp essed
as index alues (IV), calcula ed as specimen abso bance di ided
by cu o abso bance (IV !1, B19-speci ic IgG nega i e; IV 11.0,
B19-speci ic IgG posi i e).
A idi y assays. The a idi y o B19-speci ic IgG an ibody
agains VP1 and VP2 was de e mined in he same manne as
was used in he an ibody assays desc ibed abo e, bu wi h a
modi ica ion o he wash s ep. Mic o i e pla es we e coa ed as
desc ibed abo e and we e hen incuba ed wi h plasma samples
in duplica e on 2 sepa a e pla es. A e being incuba ed, he
pla es we e washed di e en ly o de e mine a idi y; one pla e
was washed wi h 4 Mo u ea and PBST o 5 min, whe eas
he o he pla e was washed wi h only PBST o 5 min. Bo h
pla es we e hen washed wice wi h only PBST o 5 min. An i-
gen-bound IgG was de ec ed as desc ibed abo e, by use o HRP
conjuga e. The a idi y index (AI) was calcula ed as he a io
o he abso bance ob ained o he pla e washed wi h u ea and
PBST and ha ob ained o he pla e washed wi h only PBST;
Pa o i us B19 B Cell Memo y •JID 2004:189 (15 May) •1875
AI is exp essed as a pe cen age. High and low AIs a e indica i e
o pas and ecen in ec ion, espec i ely.
Isola ion o PBMCs and s imula ion o cells. PBMCs we e
isola ed by densi y-g adien cen i uga ion, as desc ibed else-
whe e [17]. PBMCs ( cells/mL) we e cul u ed o 5 days
6
5⫻10
in comple e RPMI 1640, in he p esence o hea -killed S aph-
ylococcus au eus Cowan s ain I cells (SACs) (Calbiochem) di-
lu ed 1:5000 and o 10 ng o IL-2/mL (Se o ec). SACs and IL-
2 join ly unc ion o induce gene alized an ibody p oduc ion
in es ing memo y B cells [13].
B cell–memo y ELISpo assay. A e cul u e, cells we e
washed and quan i ied by ELISpo assay using a modi ica ion
o a p o ocol desc ibed elsewhe e [13]. Ni ocellulose-lined 96-
well pla es (Millipo e) we e coa ed wi h ei he VP2-N (10 mg/
mL), VP2-D (10 mg/mL), VP1-D (10 mg/mL), o abbi an i–
human IgG (10 mg/mL; Dako) in 50 mmol o sodium ca bona e
bu e /L (pH 9.6) o e nigh a 4C. Rabbi an i–human IgG
acili a ed de ec ion o he o al numbe o IgG-sec e ing B cells
o each dono , o ensu e ha memo y B cell ac i a ion had
occu ed. Pla es we e washed wi h PBS and hen we e blocked
wi h 20% ( / ) e al cal se um in RPMI 1640. A e s imu-
la ion, cells (a concen a ions o , , and 1⫻10
4
65
1⫻10 1 ⫻10
cells/well) we e added o he pla es, incuba ed in comple e
RPMI 1640 o 18 h a 37C, and hen emo ed by washing
wi h PBS. Sec e ed IgG was de ec ed using bio inyla ed abbi
an i–human IgG (1 mg/mL) and subsequen use o s ep a idin-
conjuga ed alkaline phospha ase (Sigma-Ald ich) a a dilu ion
o 1:1000 (1% [w/ ] bo ine se um albumin in PBS). Spo s
we e de eloped by he addi ion o 5-b omo-4-chlo o-3-indolyl
phospha e/p-ni oblue e azolium chlo ide subs a e (Sigma-
Ald ich) o he wells and we e coun ed a e 15 min by ligh
mic oscopy. The numbe s o spo s in iplica e wells we e a -
e aged, and esul an da a we e epo ed as he mean numbe
o spo - o ming cells (SFCs) pe 1 million s a ing cells (SCs).
Mu ine se ology and B cell–memo y esponse. BALB/c
mice we e immunized in ape i oneally wi h ei he 50 mgo
VP1-D ( ) o 50 mg o VP2-N ( ) in F eund’s com-
np3np3
ple e adju an (Sigma-Ald ich). The mu ine IgG esponse was
analyzed a weeks 6 and 32 a e immuniza ion, by he an ibody
assays desc ibed abo e o he human an ibody esponse,excep
ha an an i–mouse IgG–HRP conjuga e (Bio-Rad Labo a o-
ies) was used. Mu ine B cell ELISpo assays we e also pe -
o med on isola ed spleen cells aken om indi idual mice,
acco ding o he p o ocol desc ibed abo e, excep ha (1) goa
an i–mouse IgG (Sigma-Ald ich) was used o cap u e o al B
cell–de i ed IgG and (2) bio inyla ed abbi an i-mouse IgG (1
mg/mL) was used o de ec sec e ed IgG.
S a is ical analysis. F equencies o he le el o SFCs ob-
ained om he ELISpo assays we e subjec ed o compa a i e
analysis by S uden ’s es ; compa ison o an ibody da a wi h
ELISpo esul s was assessed by Pea son’s co ela ion; i e s o
mouse an ibody we e compa ed by a epea ed-measu e analysis
o a iance. The s a is ical-so wa e package G aphPad InS a
was used o all analyses.
RESULTS
The p esen s udy was designed o in es iga e whe he B cell
memo y is es ablished agains B19. I can be seen om igu e
1 ha all indi iduals who we e se oposi i e o B19-speci ic
IgG ( ; able 1) exhibi s ong e idence o B cell memo y,np17
whe eas indi iduals who we e se onega i e o B19-speci ic IgG
( ; able 1) do no appea o ha bo any signi ican le elnp5
o B19-speci ic memo y B cells. In ac , he di e ence be ween
he le el o SFCs in se oposi i e indi iduals and ha in se o-
nega i e indi iduals was s a is ically signi ican o bo h VP2-N
and VP1-D ( and , espec i ely); a o al o 15Pp.003 Pp.009
se oposi i e indi iduals exhibi ed B19-speci ic memo y (mean
SD, 87 154 SFCs/1 million SCs; ange, 7.8–606 SFCs/1
million SCs) agains VP2-N, and 12 se oposi i e indi iduals
exhibi ed B19-speci ic memo y agains VP1-D (mean SD,
69110 SFCs/1 million SCs; ange, 4.4–406 SFCs/1 million
SCs) ( igu e 1). The e was no s a is ically signi ican di e ence
be ween he numbe o memo y B cells di ec ed agains VP1-
D and ha di ec ed agains VP2-N ( ); howe e , hePp.2
g ea es numbe o SFCs we e speci ic o VP2-N and we e
e iden in 2 indi iduals (S008, mean SD, SFCs/222 16.4
1 million SCs; S014, mean SD, SFCs/1 million606 69
SCs). B cell memo y could no be de ec ed in he absence o
IL-2 and SAC s imula ion p io o ELISpo analysis (da a no
shown); nei he was he e e idence o memo y di ec ed agains
NS1 in 5 indi iduals (S008, S009, S010, S011, and S012) se-
oposi i e o B19-speci ic VP2 IgG, all o whom exhibi ed B
cell memo y agains ei he VP1-D o VP2-N.
When he IgG a idi y o he 5 specimens (S003, S008, S010,
S013, and S014) posi i e, by EIA, o B19-speci ic IgG agains
VP1-D we e analyzed, a s a is ically signi ican posi i e co e-
la ion ( ) was ound be ween he le el o SFCs and p0.96
a idi y. Such a co ela ion was no obse ed o VP2-N–speci ic
a idi y ( ); nei he was he e a co ela ion be ween he p0.27
le el o SFCs and he endpoin i e o B19-speci ic IgG (da a
no shown).
Al hough 3 indi iduals (S003, S008, and S012) had ci cu-
la ing VP2-D IgG (de ec able by EIA in all 3 [ able 1] and by
Wes e n blo in S003 only), he e was no e idence o any sig-
ni ican B cell memo y agains linea epi opes o VP2 ( ig-
u e 1). Fu he mo e, he le el o memo y B cells obse ed in
his coho was no signi ican ly di e en om ha obse ed
in se onega i e indi iduals ( ). Induc ion o memo yP1.999
agains epi opes o he B19 capsid p o eins is, he e o e, con-
ined o 2 egions—linea epi opes in he unique egion o VP1
and con o ma ional epi opes p esen on VP2-N. VP1 canno
1876 •JID 2004:189 (15 May) •Co co an e al.
Figu e 1. Human pa o i us B19 (B19)–speci ic B cell memo y in blood samples ob ained om se oposi i e (SP) and se onega i e (SN) olun ee
dono s. Resul s o B cell memo y a e exp essed as B19-speci ic spo - o ming cells (SFCs)/1 million s a ing cells (SCs) and a e he mean SD o
iplica e wells. ELISpo mic opla es we e coa ed wi h ei he na i e VP2 (black ba s), dena u ed VP1 (diagonally shaded ba s), o dena u ed VP2
(ho izon ally shaded ba s).
be pu i ied in a con o ma ionally in ac o m, and so no in-
o ma ion wi h espec o he con o ma ional B cell epi opes
o his p o ein is a ailable a p esen .
To es he hypo hesis ha he e is a species-speci ic di e -
ence be ween he an igenici y o linea and con o ma ional epi-
opes o he B19 capsid p o eins and o explo e he app op ia e-
ness o animal-model sys ems in B19 immunogenici y s udies,
a mu ine model was employed. The mu ine B19-speci ic IgG
esponse agains ei he VP1 ( ) o VP2 ( ) was an-np3np3
alyzed a weeks 6 and 32 a e immuniza ion. Equally s ong
esponses we e obse ed agains bo h linea (endpoin i e ,
log
10
4.0) and con o ma ional (endpoin i e , log
10
4.6) epi opes
o VP2 a week 6 ( igu e 2A); he an ibody esponse agains
linea epi opes showed no e idence o waning, because analysis
o VP2-speci ic IgG eac i i y a week 32 e ealed no s a is ically
signi ican di e ence ( ) be ween con o ma ional epi-Pp.1
opes and linea epi opes (VP2-N endpoin i e , log
10
4.9; VP2-
D endpoin i e , log
10
4.2). In addi ion, he e was equally s ong
immuno eac i i y agains VP1-D a week 6 (endpoin i e , log
10
3.9) and a week 32 (endpoin i e , log
10
4.1) ( igu e 2A).
Simila esul s we e ob ained in mu ine B cell ELISpo analysis.
Howe e , unlike he human esponse o B19 in ec ion, whe e
he e was no e idence o memo y agains linea epi opes o VP2,
in he mu ine esponse he e was s ong B cell memo y agains
bo h he con o ma ional and linea epi opes o VP2 (mean
SD, and SFCs/1 million SCs, espec i ely) a 103 55 129 11
week 32 a e immuniza ion ( igu e 2B). B cell memo y agains
VP1-D was also s ongly induced (mean SD, SFCs/104 6
1 million SCs) and was no signi ican ly di e en ( ) omPp.8
he esponse induced agains VP2-N. These da a indica ea s ong,
long-las ing, in insic an igenici y o he B19 capsid p o eins ha
is di ec ed agains bo h he con o ma ional and linea epi opes.
DISCUSSION
The p esen s udy is he i s o demons a e B cell mem-
o y agains B19 VP2 and agains he unique egion o VP1.
We ha e shown ha B cell memo y is main ained agains he
unique egion o VP1, e en in he absence o any de ec able
an ibody agains he unique egion. In addi ion, we ha e shown
a s a is ically signi ican posi i e co ela ion ( ) be ween p0.96
he le el o an ibody di ec ed agains he unique egion o VP1
and he le el o SFCs. Finally, whe eas B cell memo y is no
main ained agains linea epi opes o VP2, we ha e shown ha ,
in immunized mice, B cell memo y does pe sis agains linea
epi opes o VP2. This esul sugges s ha in e ac ion be ween
VP2 i sel and he human immune sys em (o , concei ably, be-
ween o he B19 p o eins [e.g., NS1] and he human immune
sys em) du ing in ec ion may be esponsible o he absence o
B cell memo y agains linea epi opes o VP2.
IgG eac i i y agains con o ma ional epi opes o VP2 is now
accep ed as he mos eliable indica o o pas in ec ion wi h
B19 [9, 18, 19]. In he p esen s udy, 88% (15/17) o indi iduals
demons a ing IgG eac i i y agains VP2-N exhibi ed B cell
memo y agains VP2-N, whe eas only 71% (12/17) showed
e idence o B cell memo y agains he unique egion o VP1.
The absence o de ec able B cell memo y agains linea epi opes
o VP2 was qui e ma ked, especially since 18% (3/17) o se-
Pa o i us B19 B Cell Memo y •JID 2004:189 (15 May) •1877
Table 1. Summa y o dono se ology: IgG esponse, in plasma
ob ained om adul olun ee s, agains he pa o i us B19 (B19)
capsid p o eins VP1 and VP2, in na i e and dena u ed o ms.
Dono ( )np22
Ti e o
B19-speci ic
IgG agains
VP2-N, IU/mL
Index alue
a
VP2-D VP1-D
B19-speci ic IgG se oposi i e
S001 90 0.5 0.5
S002 49 0.5 0.6
S003 122 2.3 1.2
S004 120 0.5 0.6
S005 118 0.5 0.3
S006 21 0.6 0.5
S007 18 0.4 0.4
S008 147 1.8 2.0
S009 27 0.4 0.4
S010 105 1.0 1.2
S011 43 0.6 0.6
S012 53 1.2 0.5
S013 68 0.6 0.8
S014 140 0.6 1.2
S015 160 0.2 0.8
S016 152 0.5 0.7
S017 38 0.2 0.3
B19-speci ic IgG se onega i e
S018 1 0.4 0.4
S019 1 0.5 0.7
S020 3 0.4 0.3
S021 2 0.4 0.3
S022 1 0.1 0.4
NOTE. VP1-D, dena u ed VP1; VP2-D, dena u ed VP-2; VP2-N, na i e
VP-2.
a
Index alues a e calcula ed as specimen abso bance di ided by cu o
abso bance.
Figu e 2. A, Mu ine IgG esponse o pa o i us B19 a e a single
in ape i oneal immuniza ion wi h B19 VP1 and VP2 an igens. A weeks
6 and 32 a e immuniza ion, ho acic-blood samples we e aken om
mice and le els o IgG speci ic o na i e VP2 (VP2-N), dena u ed VP2
(VP2-D), and dena u ed (VP1-D) in se um we e de e mined by ELISA.
Resul s a e exp essed as he mean log
10
endpoin i e s o an ibody,
calcula ed by eg ession o he s aigh pa o he cu e o OD
450/630 nm
e sus se um dilu ion, o a cu o o 2 SDs abo e backg ound con ol
alues ob ained om se um in nai e mice. B, B19–speci ic B cell memo y
in immunized mice a week 32 a e immuniza ion. Resul s o B cell
memo y a e exp essed as B19-speci ic spo - o ming cells (SFCs)/1 million
s a ing cells (SCs) and a e he mean SD o iplica e wells.
oposi i e indi iduals exhibi ed IgG eac i i y agains linea epi-
opes o VP2. To ou knowledge, he p esen s udy p o ides
he i s con i ma ion ha B cell memo y is ne e es ablished
agains linea epi opes o VP2 and adds u he c edence o he
concep ha long-li ed plasma cells and memo y B cells a e
2 sepa a e o ms o independen ly con olled immunological
memo y [20, 21]. A ecen epo o he enume a ion, in he
adenoids o child en, o IgG memo y B cells agains 4 pneu-
mococcal an igens has also shown a b oad an igen-speci ic e-
sponse, which sugges s ha ce ain an igens a e mo e likely
han o he s o elici he es ablishmen o B cell memo y [22].
I is also likely ha he B19-speci ic B cell ELISpo assay could
ind applica ion in he con i ma ion o pas in ec ion wi h B19.
I is well es ablished ha he humo al immune esponse o
B19 e ol es g adually, om a popula ion o an ibodies wi h
eac i i y agains bo h linea and con o ma ional epi opes o
one wi h p edominan ly con o ma ional-epi ope speci ici y
[8, 9, 23, 24]. In he p esen s udy, al hough only 5 indi iduals
exhibi ed, by ei he mic opla e immunoassay o Wes e n blo ,
IgG eac i i y agains VP1-D, 12 indi iduals displayed a posi i e
B cell–memo y esponse agains he unique egion o VP1. I
has been well documen ed ha he unique egion o VP1 ex-
hibi s cha ac e is ics associa ed wi h immunodominance, in-
cluding he s imula ion o ex i o T cell p oli e a ion and o
cy okine sec e ion [16]. In addi ion, his egion appea s o elici
neu alizing-an ibody esponses mo e e ec i ely han do o he
linea epi opes o B19 [3]—speci ically, mo e e ec i ely han
he VP1 egion co esponding o aa 60–100 [25]. Ou analysis
u he e eals a s a is ically signi ican co ela ion be ween an-
ibody a idi y agains he unique egion o VP1 and he le el o
SFCs speci ic o VP1-D, indica ing ha he s eng h o he an-
ibody esponse agains he unique egion o VP1 may be ela ed
o he induc ion o B cell memo y. Concei ably, he le el o
eexposu e o B19—o , pe haps, B19 pe sis ence—could accoun
o he di e en B cell esponses o B19 in ec ion in p e iously
in ec ed indi iduals.
E en hough 3 o he indi iduals es ed (S003, S008, and
S012) displayed IgG eac i i y agains linea epi opes o VP2
and had high a idi y (AIs, 80.7, 93.2, and 40.2, espec i ely),
1878 •JID 2004:189 (15 May) •Co co an e al.
he e was no e idence, by ELISpo assay, o memo y agains
hese VP2-speci ic epi opes. This phenomenon sugges s ha
he obse ed IgG eac i i y is a p oduc o long-li ed plasma
cells ha a e es ablished ea ly in he esponse and is no a
p oduc o memo y B cells, which a e mos ly es ablished a e
unde going selec ion in he ge minal cen e [26]. The induc ion
o memo y a e in ec ion may a o con o ma ional epi opes
o e linea epi opes because o simple s oichiome y [27]. Lin-
ea epi opes a e less likely o be mul i alen , he eby educing
B cell ecep o c oss-linking; his educ ion, in u n, would lead
o he imposi ion o a selec i e disad an age in he compe i i e
en i onmen o he ge minal cen e [28]. This phenomenon is
exempli ied in equine in ec ious anemia i us in ec ion—al-
hough he ini ial an ibody esponse is p edominan ly di ec ed
agains linea de e minan s o he en elope p o ein, wi hin 1–
2 mon hs a e in ec ion he IgG eac i i y g adually p og esses
o a mo e con o ma ionally dependen an ibody popula ion
[29]. In addi ion, con o ma ional epi opes a e likely o exis
on in ac capsids, which, when in e nalized, a e p ocessed by
speci ic B cells, esul ing in he p esen a ion o a b oade ange
o pep ides o speci ic T cells [16]. The e o e, e en hough he
T cells ecognize only linea pep ides, assis ance would be p e -
e en ially di ec ed owa d B cells ha a e speci ic o con o -
ma ional o discon inuous epi opes. This si ua ion is hough
o occu in immuni y agains polio i uses, whe e B cell memo y
agains con o ma ional epi opes is cen al o p o ec ion bu
equi es suppo by T cells ha ecognize pep ides om ei he
in e nal o ex e nal capsid p o eins [30, 31]. On he o he hand,
he e may be o he explana ions o his phenomenon; o ex-
ample, i is known ha s ong immune-ac i a ion signals can
induce lymphocy e apop osis [32]. Linea epi opes o VP2 can
elici a s ong ini ial IgG esponse, and i is concei able ha such
s imula ion could induce B cell apop osis; howe e , his scena io
seems unlikely, gi en ha no loss o memo y is obse ed in
immunized mice. Al e na i ely, he lack o es ablishmen o B
cell memo y agains linea epi opes o VP2 could be due o he
absence o a “main enance” signal, essen ial o he main enance
o ma u e B cells, om a unc ional B cell ecep o [33], al hough
i is di icul o cons uc a scena io, beyond hose desc ibed
abo e, in which his would occu selec i ely.
Despi e he absence o memo y agains linea epi opes o
VP2 in any o he human specimens es ed, in he mu ine model
he e was a s ong B cell–memo y esponse induced agains
VP2-D and VP1-D. The main enance o high i e s o an ibody
a week 32 a e immuniza ion (VP2-D endpoin i e , log
10
4.2;
VP1-D endpoin i e , log
10
4.1) ( igu e 2A), in pa allel wi h a
high le el o SFCs a week 32 o VP2-D (mean SD, 129
11 SFCs/1 million SCs) ( igu e 2B), demons a es ha bo h
VP1 and VP2 possess linea epi opes ha a e in insically im-
munogenic and ha acili a e he es ablishmen o B cell mem-
o y. I is in e es ing o no e ha he le el o SFCs obse ed in
he p esen s udy a e mice we e immunized wi h B19 an igens
is compa able o ha obse ed (50–100 SFCs/1 million SCs)
when mice we e na u ally in ec ed wi h lymphocy echo iomen-
ingi is i us [34]. Al hough his analogous B cell–memo y e-
sponse in in ec ion and in immuniza ion is signi ican , i does
no help explain why speci ic epi ope eac i i y is los a e
na u al in ec ion in humans. Fu he mo e, hese esul s illus-
a e he di e ence be ween he immuni y gene a ed by in ec-
ion in he na u al hos , and he di icul y o using a mu ine
model o assess immunogenici y. The di e en esponses ob-
se ed in he human and mu ine models may be pa ly ex-
plained by he ac ha F eund’s adju an is powe ul and,
he e o e, may ha e induced s onge immuno eac i i y o bo h
linea and con o ma ional epi opes han would be seen in na -
u al in ec ion. In addi ion, he ou e o deli e y was di e en
in each model: in he mu ine model, deli e y was in ape i-
oneal (and, he e o e, sys emic), whe eas he deli e y was mos
likely mucosal in na u al in ec ion. Replica ing i us may be
esponsible o inducing in humans hos -speci ic cellula ac o s
ha cause speci ic B cell dele ion and, he e o e, an ibody loss
agains linea egions o VP2. Because NS1 is a cy o oxic p o ein
[35] ha is capable o inducing apop o ic e ec s in he hos
[36], he possibili y ha NS1 migh play a ole in he loss o
epi ope speci ici y canno be uled ou .
Ini ial B19- accine s udies ha ha e been pe o med in mice,
guinea pigs, and abbi s [37] ha e ocused on he le el o ci -
cula ing B19 an ibody and i s associa ed abili y o neu alize
he i us. Al hough a co ela ion has been obse ed be ween
he de elopmen o an ibody agains he unique egion o VP1
and neu aliza ion o he i us [38], he di e ing na u es o
he human and mu ine immune esponses o B19 an igens
would sugges ha cau ion be aken in he ex apola ion and
applica ion o he esul s o nonp ima e accine s udies o hu-
mans. Mo e ecen ly, he in i o e icacy o a candida e accine
o B19 (MEDI-491; Medimmune) has been demons a ed; in
a double-blind, phase 1 ial, 24 olun ee s all p oduced neu-
alizing an ibody a e 3 immuniza ions [39]. Despi e he de-
elopmen o a candida e B19 accine [37, 39], he e has been
no analysis o he po en ial ole ha memo y B cells may play
in ei he he e icacy o B19-subuni accines agains in ec ion
o in he pe sis ence o immunological p o ec ion. The B19-
speci ic B cell ELISpo assay p esen ed he e would be an ideal
ex i o diagnos ic ool o he e alua ion o whe he candida e
B19-subuni accines ha e he capaci y o es ablish B cell mem-
o y. Such an app oach would be pa icula ly impo an o
unde s anding he in e play be ween indi idual i e s o B19-
speci ic IgG and B19-speci ic memo y B cells, bo h o es ablish
he ela i e impo ance o each pa ame e in comba ing B19
in ec ion in immunized indi iduals and o de ine co ela es o
p o ec ion. I would also ha e impo an implica ions in he
design o boos e s a egies, as has been he case in accine
Pa o i us B19 B Cell Memo y •JID 2004:189 (15 May) •1879
e alua ion o e anus oxoid and diph he ia, whe e i has been
concluded ha he le el o an igen-speci ic SFCs is an indi ec
measu e o immunological memo y [13].
To summa ize, analysis o he induc ion o B cell memo y
a e B19 in ec ion shows ha , al hough B cell memo y is
s ongly main ained agains con o ma ional epi opes o he ma-
jo capsid p o ein VP2, i is no main ained agains i s linea
epi opes. Fu he mo e, al hough p e iously in ec ed indi iduals
may no longe ha e ci cula ing IgG agains he unique egion
o VP1, memo y B cells pe sis and ha e he po en ial o moun
a humo al esponse on eexposu e o B19. Addi ional s udies
o selec ed pa ien coho s (e.g., pe sis en ly in ec ed indi iduals
o indi iduals wi h a B19 in ec ion o de ined du a ion) should
u he elucida e he mechanism and ex en o he absence o
B cell memo y agains linea epi opes o B19 capsid p o eins.
The indings o he p esen s udy ha e implica ions o bo h
he con i ma ion o B19 in ec ion and he de elopmen o a
accine o p o ec agains B19 in ec ion.
Acknowledgmen
We hank Susanne Mod ow (Uni e si y o Regensbu g, Ge -
many) o help ul commen s made du ing he p epa a ion o
he manusc ip .
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