Establishment of Functional B Cell Memory Against Parvovirus B19 Capsid Proteins May be Associated With Resolution of Persistent Infection

Jou nal o Medical Vi ology 78:125–128 (2006) Es ablishmen o Func ional B Cell Memo y Agains Pa o i us B19 Capsid P o eins May be Associa ed Wi h Resolu ion o Pe sis en In ec ion A. Co co an, 1 B. C owley, 2 C. Dewhu s , 3 B.L. Pize , 3 and S. Doyle 1 * 1 Depa men o Biology and Na ional Ins i u e o Cellula Bio echnology, Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland 2 Depa men o Clinical Mic obiology, S . James Hospi al, Dublin, I eland, and Na ional Vi us Re e ence Labo a o y, Uni e si y College, Dublin, I eland 3 Depa men o Pedia ic Oncology, Royal Li e pool Child en’s Hospi al-Alde Hey, Li e pool, UK Pa o i us B19 (B19) in ec ion can occu du ing acu e lymphoblas ic leukemia and pe sis en i al in ec ion can occu despi e in a enous immunoglobulin adminis a ion. He e, e idence is p esen ed ha esolu ion o pe sis en B19 in ec ion in an acu e lymphoblas ic leukemia pa ien may be associa ed wi h he simul aneous s eng hening o an igen-speci ic B cell memo y agains he B19 capsid p o ein VP2 and diminu- ion in he memo y esponse agains he B19 non-s uc u al p o ein 1 (NS1). De e mina ion o an igen-speci ic B cell memo y s a us may enhance he se ological and molecula analyses o pe sis en B19 in ec ion. J. Med. Vi ol. 78: 125–128, 2006. ß2005 Wiley-Liss, Inc. KEY WORDS: ALL; immunocomp omised; B cell ELISpo ; leukemia; e y h o- i us INTRODUCTION Pa o i us B19 (B19) is he causa i e agen o disease in bo h immunocompe en and immunocomp omised indi iduals [B oliden, 2001; Co co an and Doyle, 2004]. Speci ically wi h espec o he immunocomp omised s a us, he e ha e been a numbe o ecen epo s o B19-associa ed disease in acu e lymphoblas ic leukemia o child en [Heegaa d and Schmiegelow, 2002; Sa asan and Ozdei , 2003; Ke e al., 2003a; Fa e e al., 2004]. Al hough i has been sugges ed ha childhood acu e lymphoblas ic leukemia may be associa ed wi h p io B19 exposu e [Sa asan and Ozdei , 2003], his causal link emains unp o en. In ac , ecen wo k by Isa e al. [2004] has shown ha B19 exposu e, in u e o, is unlikely o be associa ed wi h he subsequen onse o acu e lymphoblas ic leukemia, as judged by he absence o de ec able B19 DNA om Gu h ie ca ds o child en (n ¼54) who subsequen ly wen on o de elop acu e lymphoblas ic leukemia. Adminis a ion o in a enous immunoglobulin is he main ea men o he symp oms associa ed wi h pe - sis en B19 in ec ion, such as pancy openia and ch onic anemia, in immunocomp omised pa ien s [B oliden, 2001] al hough he bene i o in a enous immunoglo- bulin adminis a ion in acu e lymphoblas ic leukemia pa ien s has ecen ly been ques ioned [Fa e e al., 2004]. Concei ably, he cu en lack o s anda diza ion o B19 IgG le els p esen in in a enous immunoglobu- lin may be esponsible o his unce ain y wi h espec o e icacy. To da e, mos s udies on he pa e n o B19 se ology and i aemia du ing acu e lymphoblas ic leukemia ha e ocused solely on he de ec ion o B19 IgG and IgM and i al DNA [Heegaa d and Schmiege- low, 2002; El-Mahallawy e al., 2004; Fa e e al., 2004]. Two ecen epo s [Ke e al., 2003a; Fa e e al., 2004] ha e alluded o impo ance he o e all indi idual pa ien esponse, and by implica ion he cellula immune esponse, in success ul e adica ion o B19 in ec ion. E idence is p esen ed in his epo ha he es ablishmen o B cell memo y is coinciden wi h he clea ance o ci cula ing B19 and esolu ion o B19- associa ed symp oms. METHODS A p e iously well 5-yea -old gi l p esen ed wi h py exia, cough, b uising, and a pu pu ic ash. Bone ma ow examina ion con i med he diagnosis o CD10 þ G an sponso : Commission o he Eu opean Communi ies; G an numbe : QLK2-CT-2001-00877. A. Co co an’s p esen add ess is Bio in, 93, The Rise, Moun Me ion, Co. Dublin, I eland. *Co espondence o: D . S. Doyle, Depa men o Biology and Na ional Ins i u e o Cellula Bio echnology, Na ional Uni e - si y o I eland, Maynoo h, Co. Kilda e, I eland. E-mail: [email protected] Accep ed 30 Sep embe 2005 DOI 10.1002/jm .20513 Published online in Wiley In e Science (www.in e science.wiley.com) ß2005 WILEY-LISS, INC. acu e lymphoblas ic leukemia and she commenced on induc ion chemo he apy wi h he MRC ALL 97 ( 99) p o ocol ( egimen B). A e induc ion he apy she s a ed consolida ion chemo he apy. She esponded well, bu in week 17 she was anemic and a bone ma ow aspi a e showed e y h oid hypoplasia. O e he ollow- ing 4 weeks she became ans usion-dependen , equi - ing nine packed ed cell ans usions. Subsequen bone ma ow aspi a es con i med e y h oid hypoplasia wi h no elapse o acu e lymphoblas ic leukemia. Chemo he - apy was commenced on T ¼3 mon hs ( ela i e o T ¼0 o ini ial B19 se ological and nucleic acid es ing) and also adminis e ed a mon hs 3, 16, and 19. Pa en al pe mission was ob ained o specimen collec ion. To al B19-speci ic IgG o capsid VP2 (VP2-N) was de e mined by comme cial EIA (Bio in In e na ional, Dublin, I eland). B19 IgM was assessed acco ding o B own e al. [1989]. B19 IgG eac i i y agains linea - ized VP1 (VP1-D), VP2 (VP2-D), and NS1 was de e - mined using immunoassay o ma s as desc ibed p e iously [Co co an e al., 2000]. B19 DNA le els we e assessed as desc ibed p e iously [Hicks e al., 1995; B aham e al., 2004] and we e quan i ied using a B19 DNA In e na ional S anda d p epa a ion [Saldanha e al., 2002]. In addi ion, cell p oli e a ion s udies and cy okine quan i a ion we e ca ied ou as desc ibed elsewhe e [Co co an e al., 2000]. B cell memo y was analyzed using a B19-speci ic B cell ELISpo assay as desc ibed by Co co an e al. [2004]. B ie ly, ni ocellu- lose-lined pla es (96 well) (Millipo e, Bed o d, MA) we e coa ed wi h ei he capsid VP2 (VP2-N; 10 mg/ml), dena u ed VP2 (VP2-D; 10 mg/ml), dena u ed VP1 (VP1-D; 10 mg/ml), NS1 (10 mg/ml), o abbi an i-human IgG (Dako A/S, Glos up, Denma k) (10 mg/ml) in 50 mM sodium ca bona e bu e pH 9.6, o e nigh a 48C ( abbi an i-human IgG acili a es de ec ion o o al IgG- sec e ing B cells o insu e ha memo y B cell ac i a ion, ia S aphylococcus au eus Cowan s ain 1 cells and IL-2, had occu ed). Pla es we e washed wi h phospha e- bu e ed saline (PBS) and blocked (20%( / ) FCS in RPMI medium). Cells (pos -ac i a ion), a 1 10 7 , 110 6 ,110 5 , and 1 10 4 cells/well we e added o he pla es and sec e ed IgG was de ec ed using bio iny- la ed abbi an i-human IgG/s ep a idin-conjuga ed alkaline phospha ase (Sigma-Ald ich, Do se , UK 1 ) and BCIP/NBT p ecipi a ing subs a e (Sigma-Ald ich). Spo s we e a e aged om iplica e wells and esul an da a epo ed as he mean numbe o spo o ming cells (SFC)/million s a ing cells. RESULTS Ini ial se ological es ing (T ¼0), using a B19 VP2 capsid-based immunoassay, o B19 IgG/M p o ed nega i e, howe e B19 DNA posi i i y was con i med by bo h eal- ime PCR and by do blo hyb idiza ion [Hicks e al., 1995; B aham e al., 2004] (Fig. 1). T ea men wi h in a enous immunoglobulin was com- menced, and al hough loga i hmic educ ions in B19 i al load we e obse ed pos - ea men , i al clea ance was no achie ed, as judged by a con inual ele a ion in plasma B19 DNA le els, un il mon h 31 (Fig. 1). Se ological analysis o a specimen ob ained om he indi idual a T ¼1 (Fig. 1) demons a ed he p esence o high le els o in a enous immunoglobulin-de i ed B19 VP2 IgG (index alue 5.76; eac i i y >o ¼1.1), in addi ion o de ec ing he p esence o B19 NS1 IgG (Index alue 3.22). No IgG was de ec ed which was eac i e agains linea epi opes o ei he VP1 o VP2 p o eins (VP1-D and VP2-D; da a no shown). A T ¼2, he B19 VP2 IgG index alue was 2.31, while ha o B19 NS1 had dec eased o 0.77 (se onega i e s a us). F eshly isola ed pe iphe al blood mononuclea cells (PBMCs), om he T1 specimen, we e s imula ed ex i o, wi h bo h B19 VP1 and VP2 p o eins. Al hough phy ohaemagglu inin (PHA) s imula ion esul ed in signi ican s imula ion index (SI (mean s anda d de ia ion): 1062.92 508.81, n ¼3), no de ec able T cell s imula ion was e iden ollowing exposu e o PBMCs o ei he VP1 (SI: 0.3 0.2) o VP2 (SI: 1.7 0.7). Cy okine analysis o cul u e supe na an s likewise indica ed a de ici in he p esence o cy okines associa ed wi h ei he a Th1 (in e e on-g) o Th2 (IL-4 and IL-5) immune esponse ollowing B19 an igen s imula ion (da a no shown). In e e on-gwas de ec able in cul u e supe na an s ollowing PBMC s imula ion wi h PHA (da a no shown). Subsequen analysis o a second specimen (T2) ob ained 5 mon hs a e he ini ial sample, demons a ed he con inued absence o a de ec able cellula immune esponse agains bo h B19 VP1 and VP2 p o eins. In e es ingly, signi ican PBMC p oli e a ion was e iden ollowing s imula ion wi h B19 NS1 p o ein (SI: 8.7 1.7) (Fig. 2A). Again, howe e , he e was no e idence o B19-speci ic cy okine p oduc ion (IL-2, in e e on-g, IL-4, and IL-5) agains B19 VP1, VP2, o NS1 p o eins. J. Med. Vi ol. DOI 10.1002/jm 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 1.00E+10 1.00E+11 1.00E+12 0 5 10 15 20 25 30 35 40 Time (Mon hs) B19 DNA (IU/ml) IVIG IVIG IVIG IVIG IVIG T1 T2 Fig. 1. E ec o mul iple in a enous immunoglobulin adminis a- ion on se um pa o i us B19 le els (B19 DNA (IU/ml)). Ini ial se ological and i ological analyses we e pe o med a T ¼0. Speci- mens o analysis o T cell immuni y and B cell ELISpo we e ob ained a T ¼1 (22 mon hs) and 2 (27 mon hs), espec i ely. Chemo he apy was commenced on T ¼3 mon hs ( ela i e o T ¼0 o ini ial B19 se ological and nucleic acid es ing), chemo he apy was also adminis- e ed a mon hs 3, 16, and 19. IVIG: In a enous immunoglobulin. 126 Co co an e al. I can be seen om Figu e 2B ha no B cell memo y is e iden agains ei he linea epi opes o B19 VP1 o VP2 p o eins, howe e signi ican B cell memo y is de ec - able agains he B19 NS1 p o ein (123.3 20.2 SFC/10 6 cells; mean SD) in specimen T1. In addi ion, a small hough de ec able le el o B cell memo y was de ec able agains B19 VP2 capsid p o ein (5.3 5.7 SFC/10 6 cells; mean SD). In e es ingly, upon analysis o specimen T2, i was obse ed ha he memo y B cell le el agains he NS1 p o ein had diminished ( en old). Howe e , he le el o de ec able memo y agains capsid VP2, and o a lesse ex en he VP1 unique egion, had inc eased wi h espec o he ini ial analysis, (VP2-N: 13 2, VP1-D: 32, VP2-D: 1.7 0.6, NS1-D: 9 3.6 SFC/10 6 cells). This appa en de elopmen o a unc ional hos esponse agains pa o i us B19 capsid p o ein VP2 was accom- panied by a esolu ion o i al disease symp oms and diminu ion o plasma B19 DNA le els o less han 10 2 IU B19 DNA/ml. DISCUSSION Pe sis en in ec ion wi h pa o i us B19 (B19) is desc ibed in an acu e lymphoblas ic leukemia pa ien despi e he adminis a ion o in a enous immunoglo- bulin he apy. The pe sis en B19 in ec ion was asso- cia ed wi h he de ec ion o NS1 IgG eac i i y, in addi ion o e idence o T cell immuni y and B cell memo y di ec ed agains B19 NS1. Fu he mo e, e idence is p esen ed ha he es ablishmen and enhancemen o B cell memo y, di ec ed agains he capsid p o eins o B19, was associa ed wi h diminu ion o B19 i emia and esolu ion o ela ed disease symp oms in he acu e lymphoblas ic leukemia pa ien . I is e iden ha mul iple adminis a ion o in a e- nous immunoglobulin h oughou he ea men pe iod was insu icien o elimina e B19 i aemia, indeed he i al load emained be ween 10 3 and 10 10 IU B19 DNA/ ml un il he inal in a enous immunoglobulin ea - men a mon h 23. The p ecise o igin (i.e., pa ien - o in a enous immunoglobulin-de i ed) o B19 VP2 IgG de ec able in specimen T1 is unclea , al hough he p esence o B19 NS1 IgG does indica e ha pa ien an ibody p oduc ion capabili y was in ac . None heless, he use o in a enous immunoglobulin he apy o he elimina ion B19 i aemia emains unp o en, indeed a ecen s udy [Fa e e al., 2004], which epo ed a iable success wi h in a enous immunoglobulin ea men in acu e lymphoblas ic leukemia, ecom- mended he ea men only in cases o pe sis en pancy openia o clinical mani es a ions o B19 in ec ion. Howe e , in a enous immunoglobulin he apy has been shown o be success ul o he ea men o B19- associa ed ch onic a igue synd ome [Ke e al., 2003b]. Ul ima ely, he e icacy o in a enous immunoglobulin he apy o he elimina ion o B19 i emia will only become appa en when he le el o B19 IgG p esen in in a enous immunoglobulin p epa a ions is epo ed in e ms o B19 IgG IU/ml as p e ious s udies ha e indica ed a iable B19 IgG le els (64.7 17.5 IU/ml; (n ¼30)) in pooled sol en -de e gen ea ed plasma [Daly e al., 2002]. Al hough ini ially hough o be a ma ke o pe sis en in ec ion only [ on Poblo zki e al., 1995], he de ec ion o NS1 IgG has also been shown o be p esen du ing acu e B19 in ec ion in 61%–69% o indi iduals [Hemaue e al., 2000; Ennis e al., 2001]. Mo eo e , signi ican T cell esponses o NS1 ha e also been epo ed in ecen ly in ec ed indi iduals and pa ien s who de eloped ch onic a h opa hy ollowing B19 in ec ion [Mi chell e al., 2001]. I is clea om he p esen s udy, howe e , ha immuni y o NS1 does no appea o be consis en wi h clea ance o B19 in ec ion, a leas in his indi idual su e ing om acu e J. Med. Vi ol. DOI 10.1002/jm A 0 2 4 6 8 10 VP1 VP2 NS1 PHA S.I. T2 B T1 T2 SFC/ 10^6 cells 0 10 20 30 40 VP2-N VP2-D VP1-D NS1 0 10 20 30 40 VP2-N VP2-D VP1-D NS1 Fig. 2. E alua ion o PBMC p oli e a i e esponse and an igen- speci ic B cell memo y in acu e lymphoblas ic leukemia. A: Speci ic p oli e a i e esponse o human pa o i us B19 p o eins VP1 (VP1-D), VP2 (VP2-N), and NS1 in addi ion o he posi i e con ol mi ogen, phy ohaemaggul inin (PHA), a T ¼2. Resul s a e exp essed as s imula o y indices (SI) and a e he mean SD o iplica e wells. B: Pa o i us B19-an igen-speci ic B cell memo y a T ¼1 and 2. Resul s o B cell memo y a e exp essed as B19-speci ic spo - o ming cells (SFCs)/numbe o s a ing cells and a e he mean SD o iplica e wells. Indi idual ELISpo mic opla es we e coa ed wi h he ollowing B19 p o eins: na i e (capsid) VP2 (VP2-N), dena u ed VP2 (VP2-D), dena u ed VP1 (VP1-D), and NS1. Human IgG de ec ion using abbi an i-human IgG was used o con i m ha o al B cell ac i a ion had occu ed (da a no shown). No e: B19 NS1 p o ein esponse ¼123.3 20.2 SFC/10 6 cells in specimen T1. B19 Memo y and Pe sis en In ec ion 127 lymphoblas ic leukemia. In addi ion, he p esence o high i e B19 VP2 IgG, in he absence o de ec able cellula immuni y, also appea s o be insu icien o comple ely amelio a e B19 i aemia. Howe e , he es ablishmen o B cell memo y agains capsid VP2, and o a lesse ex en VP1, may play a ole in he clea ance o B19 i emia. We ha e p e iously epo ed e idence o B cell memo y agains B19 capsid p o eins in immunocompe en indi iduals [Co co an e al., 2004], and he da a p esen ed abo e ep esen he i s de e mina ion o a simila pa e n o B cell memo y in acu e lymphoblas ic leukemia, coinciden wi h he esolu ion o B19 i aemia and symp oms. Assessmen o an igen-speci ic B cell memo y also acili a es dis- c imina ion be ween in a enous immunoglobulin- and indi idual-de i ed B19 IgG, espec i ely, which is signi ican in de e mining he se ocon e sion s a us o he indi idual. In e es ingly, no e idence o B cell memo y was e iden agains B19 NS1 in i e immuno- compe en indi iduals [Co co an e al., 2004] he eby sugges ing ha he p esence o B cell memo y agains his p o ein may be indica i e o memo y dys unc ion in acu e lymphoblas ic leukemia. Resolu ion o B19 i aemia o less han 10 2 IU/ml B19 DNA in he pa ien was coinciden wi h a h ee old inc ease in de ec able B cell memo y agains capsid VP2 (and a en old educ ion wi h espec o NS1). In conclusion, we sugges ha de e mina ion o an igen-speci ic B cell memo y should accompany he se ological and molecula in es iga ion o pe sis en B19 i aemia, and associa ed disease mani es a ions, in immunocomp omised indi iduals. 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