Ex Vivo Cytokine responses against Parvovirus B19 Antigens in previously infected pregnant women

Jou nal o Medical Vi ology 70:475–480 (2003) Ex Vi o Cy okine Responses Agains Pa o i us B19 An igens in P e iously In ec ed P egnan Women Amanda Co co an, 1,2 Be na d P. Mahon, 2 Pe e McPa land, 3 Anne Da o en, 4 and Sean Doyle 1 * 1 Bio echnology G oup, Depa men o Biology, Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland 2 Ins i u e o Immunology, Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland 3 Na ional Ma e ni y Hospi al, Holles S ee , Dublin, I eland 4 I ish Blood T ans usion Se ice, S . James’ Hospi al, Dublin, I eland Pa o i us B19 in ec ion is a signi ican cause o e al dea h. The aim o his s udy was o in es iga e he ole o ma e nal immune s a us in modula ing suscep ibili y o e al B19 in ec ion. Pe iphe al blood was ob ained om p egnan women (n ¼199) wi h no clinical e idence o ecen B19 in ec ion. E alua ion o ex i o T cell esponses om 149/199 indi iduals showed signi ican ly highe in e e on-gle els o se o- posi i e indi iduals ollowing VP1 (268 36 e sus 103 19 pg/ml; P¼0.003) and VP2 (242  42 e sus 91 16 pg/ml; P¼0.01) an igen s imu- la ion. Signi ican ly highe le els o in e leukin-2 we e also obse ed in se oposi i e indi iduals ollowing bo h VP1 (P¼0.0003) and VP2 (P¼ 0.0005) s imula ion. The obse ed Th1 cellula esponse is lowe han ha documen ed p e- iously o non-p egnan indi iduals and s ongly sugges s ha diminu ion o he ma e nal an i- i al immune esponse may inc ease suscep - ibili y o e al B19 in ec ion. J. Med. Vi ol. 70:475–480, 2003. ß2003 Wiley-Liss, Inc. KEY WORDS: p egnancy; cellula immuni y; e y h o i us; in e e on-g INTRODUCTION Human pa o i us B19 in ec ion can cause se ious complica ions in he immunocomp omised hos includ- ing p egnan women and indi iduals wi h unde lying blood diso de s such as sickle cell disease [Wool e al., 1989; Se jean e al., 1993; Heegaa d and Ho nsle h, 1995; Ta an ino and Shahidi, 1995]. Exposu e o, and in ec ion wi h, pa o i us B19 du ing p egnancy can lead o e al loss by ei he spon aneous abo ion o non- immune e al hyd ops occu ing in 8–10% o in ec ions [B own, 1989; Jo dan, 1996; Kinney e al., 1988]. B19 is ansmi ed ac oss he placen a du ing ma e nal in ec- ion and subsequen e al in ec ion can lead o se e e anaemia. A e ical ansmission a e o 33–51% has been epo ed [PHLS, 1990; Yaegashi, 2000]. B19 in ec ion du ing p egnancy o en goes unde ec ed as many p egnan women a e asymp oma ic while o he indi iduals expe ience symp oms such as exan hema and a h algia [Komischke e al., 1997]. Fe al loss as a consequence o in au e ine B19 in ec ion is highes in, bu no es ic ed o, he i s 20 weeks o ges a ion [PHLS, 1990]. This suscep ibili y could be a ibu ed, a leas in pa , o he p edilec ion o B19 o apidly di iding e y h oid p ogeni o cells. In he second imes e , he e al ed cell mass inc eases h ee- o ou old and he e ec s o B19 may be u he compounded by he educed li e span o e al ed blood cells. The ela i e imma u i y o he e al immune esponse a his s age may also be a con ibu o y ac o . Cases o la e second and hi d imes e non-hyd opic e al loss, caused by an acu e B19 in ec ion, ha e only been epo ed ecen ly [Skjo ¨ldeb and-Spa e e al., 2000; Tol ens am e al., 2001]. In he p esen s udy, ollowing an examina ion o incidences o in a-u e ine e al dea h i was ound ha 7.5% (7/93) o hi d i- mes e and 15% (7/47) o la e second and hi d imes e cases con ained B19 DNA in he placen al issue. These cases we e no associa ed wi h hyd ops and no o he explana ions o e al dea h we e e iden . Unusually, none o he p egnan women showed clinical symp oms o B19 in ec ion and many had delayed o absen humo al esponses. Speci ic an i- i al an ibody is conside ed he majo mechanism o immune p o ec ion [Ku zman e al., 1989; Schwa z e al., 1990], howe e , mo e ecen ly he *Co espondence o: D . Sean Doyle, Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland. E-mail: [email p o ec ed] Accep ed 17 Feb ua y 2003 DOI 10.1002/jm .10420 Published online in Wiley In e Science (www.in e science.wiley.com) ß2003 WILEY-LISS, INC. cellula esponse o pa o i us B19 in ec ion has been explo ed. B19 speci ic T-cell p oli e a i e esponses o ecen ly in ec ed and heal hy indi iduals o he capsid an igens [ on Poblo zki e al., 1996; Co co an e al., 2000; F anssila e al., 2001] and o he non-s uc u al p o ein, NS1 [ on Poblo zki e al., 1996; Mi chell e al., 2001] ha e been obse ed. T helpe cells sec e e cy o- kines, which ha e an impo an ole in de e mining he ou come o a p egnancy [Chaoua e al., 1990; Hill, 1991; Tang i and Raghupa hy, 1993]. Th1 and Th2 cells a e he majo subse s o ully di e en ia ed CD4 þ T cells and no mal p egnancy is domina ed by T helpe cells ha sec e e a Th2 ype pa e n o cy okines including in e leukin-4 (IL-4) and in e leukin-5 (IL-5) [Wegmann e al., 1993]. Th1 cy okines, including in e e on-gamma (IFN-g) and in e leukin 2 (IL-2), a e associa ed wi h a poo p egnancy ou come and a e supp essed by placen- al p oduc s such as p oges e one, p os aglandin E2 and cy okines such as IL-4 and IL-10 [Wegmann e al., 1993; Kelly and C i chley, 1997]. Du ing p egnancy, elimina- ion o a i al in ec ion ia a Th1 esponse may esul in endange ing he de eloping concep us. Cy okines ypi- cal o a Th1 esponse, including IFN-g, IL-2, IL-6, and IL-1bha e been epo ed upon s imula ion o T cells o heal hy adul s wi h pa o i us B19 [Wagne e al., 1995; Mo a e al., 1996; Co co an e al., 2000; Jo dan e al., 2001]. Fu he mo e, Co co an e al. [2000] ha e demons a ed ha PBMCs isola ed om no mal heal hy indi iduals (n ¼7, comp ising 3 emales) p oduced IFN-gle els o 610 348 pg/ml and 1765 825 pg/ml when s imula ed wi h VP1 and VP2, espec i ely. In his s udy, we in es iga ed i p egnancy, and he associa ed supp ession o Th1- ype esponses, could modula e ex i o B19-speci ic cy okine p oduc ion om T cells isola ed om p egnan women o elucida e he ole o hos ac o s in de e mining suscep ibili y o B19 in ec ion. MATERIALS AND METHODS S udy Popula ion Hepa inised blood specimens (n ¼199) we e ob ained om p egnan women wi h no e idence o ecen pa - o i us B19 exposu e o symp oms ( imes e 1 (n ¼78), imes e 2 (n ¼80) and imes e 3 (n ¼41)). The age ange o he olun ee s was 15–50 and he mean age was 28 yea s. The ges a ion pe iod a ied om 4 o 42 weeks. No u he clinical da a was a ailable on he olun- ee s. E hical pe mission o his s udy was ob ained om he Na ional Ma e ni y Hospi al, Dublin, I eland and w i en consen was ob ained om all olun ee s p io o specimen collec ion. An igen Exp ession and Pu i ica ion Pa o i us B19 ecombinan VP1 and VP2 capsids and NS1 we e exp essed in he baculo i us exp ession sys em using Spodop e a ugipe a cells [B own e al., 1990; B own e al., 1991; Ennis e al., 2001]. Capsid and NS1 pu i ica ion ha e been desc ibed p e iously [Ke e al., 1999; Ennis e al., 2001]. Pa o i us B19 IgG Enzyme Immunoassays Plasma specimens we e analysed o B19 IgG speci ic o he capsid an igens VP1 and VP2 in bo h hei na i e (N) and dena u ed (D) con o ma ions as p e iously desc ibed [Co co an e al., 2000]. An enzyme immu- noassay was used o measu e he le el o eac i i y agains B19 NS1. Resul s a e exp essed as index alues (I.V.) ep esen ing specimen abso bance di ided by cu -o abso bance. The cu -o was es ablished as he abso bance þ2 SD g ea e han he mean abso bance ob ained om a panel o B19 nega i e samples. An IV o <1.0 was conside ed se onega i e. B19 IgG eac i i y o con o ma ionally in ac VP1 (VP1-N) was de e mined by a comme cial quali a i e immuno luo escen assay (IFA). The deg ee o luo escence was g aded on a scale o 0-4 acco ding o manu ac u e s’ ins uc ions (Bio in, Dublin, I eland). T cell p oli e a ion assays. Isola ed pe iphe al blood mononuclea cells (PBMCs) we e cul u ed in iplica e wi h pu i ied ecombinan VP1 (10 mg/ml), VP2 (10 mg/ ml) and NS1 (10 mg/ml) o 72 h . Cells cul u ed wi h medium alone o wi h phy ohaemagglu inin (PHA: 2 mg/ ml) se ed as nega i e and posi i e con ols, espec- i ely. The le el o B19-speci ic T cell p oli e a ion was measu ed depending on he amoun o [ 3 H]- hymidine inco po a ion du ing he inal 4 h o cul u e [Co co an e al., 2000]. Cy okine enzyme immunoassays. Supe na an s we e emo ed om PBMCs cul u es, a op imal imepoin s, o de e mine he concen a ion o IL-2 (24 h ) and IFN-g, IL-4 and IL-5 concen a ions (72 h ). Le els o cy okine sec e ed we e de e mined by EIA using comme cially a ailable an ibody pai s (Pha Mingen, San Diego, Cali o nia, USA). Concen a ions we e de e mined by compa ing he abso bance a 405 nm o es samples wi h a s anda d cu e o ecombinan cy okines o known concen a ion. Le els o IL-2 a e exp essed in e ms o index alues (specimen IL-2 (U/ml) di ided by he mean nega i e con ol IL-2 le el (U/ml) plus 2 s anda d de ia ions) o compensa e o di e ences in con ol IL-2 le els be ween expe imen s. S a is ical Me hods Cy okine sec e ion da a om di e en ea men g oups we e compa ed by use o S uden ’s - es . RESULTS Se o eac i i y o S udy Popula ion Pa o i us B19 se op e alence in he adul popula- ion has been epo ed o be 60–70%. In his s udy, whe e B19 se oposi i i y was de ined by he p esence o an ibody agains VP2-N (capsid VP2), all imes e g oups had expec ed le els o eac i i y agains VP2-N (63–71%) (Table I), hus gi ing a ep esen a i e sample popula ion in e ms o pas B19 in ec ion o each o he imes e s. Le els o eac i i y agains VP1 anged om 59–68% (Table I). The low le el o an ibody eac i i y obse ed agains linea epi opes o VP1 (35–52%) and 476 Co co an e al. VP2 (3–15%), in he se oposi i e g oup, is indica i e o pas in ec ion o heal hy indi iduals and also empha- sises he impo ance o using capsid-based de ec ion sys ems o accu a e diagnosis o pas exposu e o B19 in p egnan women. I is known ha an ibodies agains linea epi opes on VP2 a e unde ec able 6 mon hs pos - in ec ion [So ¨de lund e al., 1995]. As specimens om his coho exhibi ed he expec ed low le el o eac i i y agains linea VP2 (Table I), i can be concluded ha 92% o se oposi i e p egnan women in his s udy did no ha e an acu e B19 in ec ion. S ong IFN-gResponses Domina e he Cy okine P o ile No all PBMCs isola ed om whole blood specimens aken om p egnan women exhibi ed eac i i y when s imula ed wi h posi i e con ol (PHA) in he T cell p oli e a ion assay. Thus, subsequen da a e alua ion in ol ed he s udy o T cell esponses om he 147/199 indi iduals wi h PHA-induced esponses. Highe le els o he in lamma o y cy okine IFN-gwe e sec e ed by PBMCs ob ained om se oposi i e indi iduals (n ¼99) han in hose ob ained om se onega i e dono s (n ¼48). Mos signi ican we e le els o IFN-g(268  36 pg/ml) when T cells we e s imula ed wi h he immu- nodominan capsid p o ein, VP1 (P¼0.003). Le els o IFN-gwe e also signi ican when cells we e s imula ed wi h he capsid p o ein VP2 (P¼0.01) and o a lesse ex en wi h NS1 (P¼0.09) (Table II). In o de o de e mine i he e was a imes e - dependen pa e n in he ex i o cy okine esponse, specimens we e e-classi ied based upon he s age o ges a ion. IFN-gwas de ec ed in specimens ob ained ac oss all h ee imes e s, howe e no signi ican di e ence was obse ed in he amoun s o IFN-g sec e ed ex i o, in esponse o all B19 an igens es ed, ac oss he imes e s o p egnancy (Fig. 1). B19-Speci ic IL-2 P oduc ion by P egnan Women Al hough signi ican ly high le els o IL-2 we e p o- duced by PBMCs when s imula ed wi h B19 an igens, ini ially he e was no s a is ically signi ican di e ence be ween le els p oduced by se oposi i e (n ¼99) and se onega i e (n ¼48) specimens due o he ac ha h ee se onega i e specimens p oduced high le els o IL-2 upon s imula ion. Exclusion o hese h ee speci- mens (see Discussion) esul s in he obse ed le els o IL-2 sec e ion being signi ican ly highe in se o- posi i e specimens o bo h VP1 (P¼0.0003) and VP2 (P¼0.0005) s imula ion. S ong IL-2 p oduc ion was obse ed ollowing ex i o s imula ion o PBMCs om se oposi i e p egnan women (n ¼99) wi h VP1, VP2 and NS1, esul ing in IL-2 index alues o 1.43 0.9, 1.36 0.7 and 1.27 0.9, espec i ely (Fig. 2). In com- pa ison, IL-2 index alues o 0.85 0.25, 0.89 0.25 and 0.83 0.29 we e e iden ollowing analysis o T cells om se onega i e indi iduals when s imula ed wi h VP1, VP2 and NS1 (Fig. 2). In ac , 44/99 se oposi i e samples p oduced signi ican ly highe le els o IL-2 compa ed o se onega i e samples (P¼0.004 o VP1, P¼0.003 o VP2) despi e he inclusion o he 3 se one- ga i e/high IL-2 specimens in he analysis (see abo e). Mean alues o IL-2 o hese 44 samples we e 0.407 0.049 U/ml o VP1 and 0.306 0.029 U/ml o VP2. No di e ence was obse ed in he ex en o ex i o IL-2 sec e ion om s imula ed PBMCs be ween ime- s e s. Despi e he bias owa ds Th2 cy okine p oduc ion TABLE I. An ibody Response in Plasma Ob ained F om P egnan Women (n ¼199) Agains he B19 Capsid P o eins VP1 and VP2* De ec ion an igen T imes e 1 Posi i e 2 B19 IgG s a us (%) posi i e 3 Posi i e VP1-N 59 65 68 VP2-N 63 65 71 VP1-D 52 51 35 VP2-D 15 6 3 NS1-D 2 4 0 *In con o ma ionally in ac (N) and dena u ed (D) o ms, and o he B19 nons uc u al p o ein, NS1. B19 se oposi i i y was de ined by he p esence o an ibody agains VP2-N (capsid VP2). TABLE II. Ex Vi o IFN-gSec e ion by PBMCs Isola ed F om Se oposi i e and Se onega i e P egnan Women A e S imula ion Wi h Pu i ied VP1, VP2, o NS1 (10 mg/ml) Specimen se o eac i i y B19-speci ic IFN-gp oduc ion (pg/ml þSE) VP1 VP2 NS1 Posi i e (n ¼99) 268 36 242 42 185 34 Nega i e (n ¼48) 103 19 91 16 100 17 IFN, in e e on; PBMCs, pe iphe al blood mononuclea cells. Fig. 1. T imes e dependence o ex i o in e e on-gp oduc ion ollowing PBMCs s imula ion wi h B19 an igens. Isola ed PBMCs we e indi idually s imula ed wi h pu i ied B19 an igens, VP1, VP2 and NS1 o ei he medium alone o PHA as a nega i e o posi i e con ol, espec i ely. Se onega i e (SN) and se oposi i e (SP) specimens we e ca ego ised acco ding o imes e 1 (SP (n ¼39); SN (n ¼20)), 2 (SP (n ¼41); SN (n ¼17)) and 3 (SP (n ¼19); SN (n ¼11)). Resul s ep esen he mean concen a ion o in e e on-gp oduced om iplica e wells (SE). Ex Vi o B19-Speci ic Cy okine Responses 477 du ing p egnancy, PBMC s imula ion wi h B19 an i- gens did no p oduce any signi ican le els o in e - leukin-4 and -5 (da a no shown). DISCUSSION This s udy ep esen s he i s simul aneous e alua- ion o bo h humo al and cellula immuni y agains pa o i us B19 in p egnan women. The popula ion coho exhibi ed he expec ed high le el o se oposi i i y (63–71%) agains B19 and a signi ican ex i o Th-1 esponse agains B19 an igens, hough educed com- pa ed o non-p egnan indi iduals. Fu he mo e, e i- dence is p esen ed o he p esence o a cellula esponse o pas B19 in ec ion in he absence o a de ec able humo al esponse, which has implica ions o cu en B19 diagnos ic p ac ices. O e all he le el o pa o i us B19 se op e alence amongs p egnan women in I eland was 66% (132/199). This e alua ion compa es a ou ably wi h ha epo ed p e iously in bo h Ge man and US (73.2%) popula ions using iden ical es me hodology [Sea le e al., 1997; US Food and D ug Adminis a ion, 1999]. Signi ican ly, a lowe le el o IgG an ibody posi i i y is obse ed agains bo h linea ised B19 VP1 and VP2, con i ming he neces- si y o B19 IgG de ec ion sys ems o u ilise na i e B19 an igens o acili a e op imal es sensi i i y [Ke e al., 1999]. So ¨de lund e al. [1995] ha e shown p e iously ha an ibodies agains linea epi opes a e los wi hin 6 mon hs ollowing B19 in ec ion, hus he low le el o an ibody posi i i y agains hese epi opes, obse ed in he p esen s udy, con i ms u he ha he majo i y o se oposi i e indi iduals we e in ec ed a leas 6 mon hs p io o specimen dona ion. Cases o ma e nal in ec ion and subsequen e al dea h due o B19 in ec ion ha e been epo ed in all h ee imes e s [W igh e al., 1996; Yaegashi e al., 1998; Skjo ¨ldeb and-Spa e e al., 2000]. Al hough he di ec pa hogenic e ec s o B19 on e al de elopmen a e a majo cause o mo ali y, he ole o he ma e nal immune s a us in ei he p o ec ing agains o media ing pa o i us B19–induced e al loss is unknown. I is gene ally accep ed ha a success ul p egnancy equi es a Th2 esponse and ha s ong Th1 esponses a e associa ed wi h p egnancy ejec ion [Raghupa hy, 1997; Raghupa hy, 2001]. In e es ingly, ecen wo k by Luppi e al. [2002] p esen s e idence o an inc ease in CD8 þ lymphocy e equency in pe iphe al blood specimens aken du ing he hi d imes e o p egnancy compa ed o a con ol g oup. In his p esen s udy we show ha ex i o s imula ion wi h B19 an igens esul s in signi ican IL-2 and IFN-gp oduc ion om PBMC despi e he p oposed Th2 bias o p egnancy. Compa a i e in o ma- ion on ex i o cy okine sec e ion in esponse o B19 an igen s imula ion is limi ed. Howe e , in he p esen s udy, he le el o ex i o IFN-gsec e ion was educed subs an ially om ha o no mal heal hy in- di iduals s imula ed wi h he same an igens [Co co an e al., 2000] whe e B19 se oposi i e adul s p oduced IFN-gle els o 610 348 pg/ml and 1765 825 pg/ml when s imula ed wi h VP1 and VP2, espec i ely. In e e on-gp oduc ion by PBMCs om p egnan women (p esen s udy) was 205 27 pg/ml wi h VP1 and 176 26 pg/ml wi h VP2 s imula ion. This is sug- ges i e o a p egnancy-associa ed diminu ion o Th1 media ed immuni y. In ac , a ansien down- egula- ion in he Th1 esponse is seen du ing p egnancy in heuma oid a h i is pa ien s and his educ ion is associa ed wi h a s a e o emission as p o-in lamma o y cy okines cause he pa hological damage o join s e iden du ing heuma oid a h i is [Russell e al., 1997]. The dec ease in he cellula immune esponse o B19 obse ed may be ele an o he ad e se ou comes associa ed wi h B19 in ec ion du ing p egnancy. We p opose ha his diminished Th1 esponse may dec ease he a e o B19 clea ance, hus gi ing he i us he oppo uni y o exhibi g ea e in ec i i y. A simila phenomemon has been epo ed wi h Leishmania majo pa asi ic in ec ions o gene ically esis an p egnan mice whe eby cellula esponses agains L. majo we e weakened due o educed IFN-gp oduc ion du ing p egnancy and was associa ed wi h diminished clea - ance o he pa asi e [K ishnan e al., 1996]. In addi ion, L. majo in ec ion in hese mice caused an inc ease in he equency o e al eso p ions. Al hough a sys emic Th1 esponse was bene icial in igh ing in ec ion, i was also de imen al o ges a ion, e en a a dec eased le el. IL-2 is no p esen no mally in subs an ial le els du ing p egnancy a ound he ophoblas [King e al., 1995], howe e , in he p esen s udy, ex i o s imula ion wi h B19 an igens elici ed p oduc ion o his p o- in lamma o y cy okine. IL-2 p oduc ion has been ob- se ed p e iously in women who we e in ec ed wi h B19 du ing p egnancy [Jo dan e al., 2001]. Using immunohis ochemical echniques on placen al issue sec ions (n ¼25), IL-2 was de ec ed a he ma e nal- e al in e ace o all women in he s udy who se ocon e ed du ing p egnancy despi e he ou come o he p egnancy. Howe e , i was p oposed ha in p egnancies wi h a poo ou come IL-2 is de ec able on he e al side o he in e ace, whe eas hose wi h a posi i e ou come end o Fig. 2. Ex i o IL-2 p oduc ion ollowing s imula ion o isola ed PBMC, om se oposi i e (black ba s) and se onega i e (whi e ba s) indi iduals, wi h pa o i us B19 VP1, VP2 and NS1 an igens, espec i ely. IL-2 esul s a e exp essed as index alues (I.V. S.E.M.) ep esen ing he amoun o IL-2 p oduced abo e he backg ound le el o each indi idual (I.V. ¼mean IL-2 concen a ion (U/ml)/mean IL-2 concen a ion o he nega i e con ol þ2 s anda d de ia ions). 478 Co co an e al. ha e he IL-2 on he ma e nal side. I is well es ablished ha some cy okines ha e ad e se e ec s on he ou come o p egnancy, pa icula ly IFN-g, IL-2 and TNF-a. These cy okines ac i a e cy o oxic cells such as na u al kille (NK) and lymphokine ac i a ed kille (LAK) cells which can kill ophoblas cells [D ake and Head, 1989] and when hese cy okines a e adminis e ed o mice hey cause abo ions [Kinsky e al., 1990]. In p egnan women wi h a his o y o ecu en spon aneous abo - ions signi ican ly highe le els o NK cells [Kwak e al., 1995], IFN-gand IL-2 exp ession [Tang i and Raghu- pa hy, 1993] ha e been ound when compa ed o no mal p egnan women. IFN-gp oduc ion by B19-speci ic T cells may also be de imen al o he concep us by inhibi ing g anulocy e-mac ophage colony-s imula ing ac o (GM-CSF), which is in ol ed in ophoblas g ow h and di e en ia ion [Robe son e al., 1994]. Al hough Th1 cy okines a e impo an du ing implan- a ion and pa u i ion o p egnan women [Haimo ici and Ande son, 1993; Aboagye-Ma hiesen e al., 1996], he e may be imes du ing p egnancy when he e us is mo e sensi i e o he le el o p o-in lamma o y cy o- kines. Ou s udy shows ha he le els o Th1 cy okines did no signi ican ly di e o e he h ee imes e s bu he le el o IFN-gp oduced was highes in imes e 1. P e ious s udies ha e shown ha p o-in lamma o y cy okines such as IFN-gcan ha e dele e ious e ec s on he e us du ing p egnancy [Raghupa hy, 1997]. This obse a ion, combined wi h he ela i e imma u i y o he e us du ing imes e 1, sugges s ha an inc eased Th1 esponse o B19 may be de imen al o he e us. O he ac o s o be conside ed when de e mining he e ec o cy okines caused by B19 in ec ion du ing p eg- nancy a e heges a ional pe iod o he in ec ion, he s age o di e en ia ion o he concep us, he le el o soluble cy okine ecep o s and he a io o Th2 cy okines. In e es ingly, h ee o he se onega i e dono s p o- duced high le els o IL-2 upon ex i o B19 an igen s imula ion. The eason o his is unclea bu ecen e idence sugges s ha ce ain indi iduals wi h pas B19 in ec ion may possess an an igen-speci ic cell media ed immune esponse in he absence o a humo al esponse o he i us. Tol ens am e al. [2001] obse ed ou B19 an ibody nega i e cases ha had speci ic T cell memo y p o en by ei he ELISpo assay o e ame binding. These obse a ions ha e signi ican consequences o he design o an e icacious accine o pa o i us B19 in ha u u e subuni accines should con ain bo h B19 T cell and B cell epi opes. P e ious s udies on B19 in ec ion du ing p egnancy ha e ocussed on bo h he di ec e ec s o B19 on he e us and he ma e nal humo al esponse as he majo mechanism o p o ec ion agains B19-induced e al dea h. Ou esul s, which show a signi ican diminu- ion in ex i o in e e on-gsec e ion in esponse o B19 an igen s imula ion, demons a e ha cellula immu- ni y agains B19 is a enua ed du ing p egnancy which may ha e an ad e se e ec on an i- i al Th1- ype esponses he eby inc easing e al suscep ibili y o in ec ion. 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