parvovirus y lupus_relacion

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2010 19: 783LupusM. Pavlovic, A. Kats, M. Cavallo and Y. Shoenfeld

Clinical and Molecular Evidence for Association of SLE with parvovirus B19

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Lupus (2010) 19, 783–792

http://lup.sagepub.com

REVIEW

Clinical and Molecular Evidence for Association

of SLE with parvovirus B19

M Pavlovic1, A Kats2, M Cavallo2 and Y Shoenfeld31Department of Computer and Electrical Engineering and Computer Science, Florida Atlantic University, FL, USA;2Department of Biological Sciences, Florida Atlantic University, FL, USA; and 3Department of Medicine ’B’ and

Center for Autoimmune Diseases, Sheba MedicalCenter (Affiliated to Tel-Aviv University), Tel-Hashomer 52621, Israel

In addition to genetic and environmental factors, viruses have been suspected as causes and/orcontributors to human autoimmune diseases, although direct evidence for the association isgenerally lacking. Parvovirus B19, the cause of Fifth disease in childhood, and possible triggerin the spectrum of autoimmune diseases in adults, has emerged as one of the central viralcandidates within the last few years. In this article we propose a possible model for parvovirusB19 association with systemic lupus erythematosus (SLE). The basis for our model is thesecretion of hydrolyzing anti-ssDNA autoantibodies in 30–70% of cases with SLE, whichin turn can either hydrolyze viral B19 ssDNA in blood and other fluids, or intranuclear,replicated viral ssDNA after re-activation and translocation of the virus into the nucleusof the host permissive cells. Both mechanisms contribute to perpetuation and maintenanceof a ‘vicious cycle’ with concomitant flares in SLE, and involve inevitable TLR9 sensitizationand genetic switch for anti-ssDNA autoantibody production from activated B cells in indivi-duals prone to triggering. This model strongly suggests a major potential impact upon earlyprevention (vaccination) and targeted therapy of this subclass within the SLE spectrum ofdiseases. Incorporated in this new concept is an innovative idea for developing the tool formore precise (individualized) diagnosis, prevention, and therapy. Lupus (2010) 19, 783–792.

Key words: autoimmune diseases; cytokine microarray profiling; hydrolytic anti-DNAautoantibodies; parvovirus B19; systemic lupus erythematosus (SLE)

Introduction

Background

The cause of lupus is unknown but a number ofdisease agents have been implicated.1,2 Epstein–Barr virus, parvovirus B19, bacterial species anddrugs have been connected to the disease.3–8 Thepresence of viruses was sought in a colony ofdogs bred from parents with systemic lupus erythe-matosus (SLE).4 Cell-free filtrates prepared fromthe spleens of these animals were injected into new-born dogs, mice, and rats. The canine recipientsdeveloped antinuclear antibody (ANA) and posi-tive lupus erythematosus (LE) cell tests, proving

that the illness is transmissible4 and, with respectto injection with cell-free filtrate, probably of viralorigin. Lupus patients exhibit an interferon alpha(IFN-a) signature, suggesting an underlying infec-tion of yet unknown origin.9,10 Therefore, the keyquestion is what agent or agents drive the inflam-mation and elicit the inflammatory cascade in SLE?

The parvovirus is at the focus of many derma-tologic and rheumatologic disease-related studiespreviously attributed to unknown factors.11–21

Initially, B19 was identified as the causative agentof erythema infectiosum (erythrovirus), a commonchildhood rash found in outbreaks among school-children during the winter and spring months, latercalled Fifth disease or slapped cheek disease.22–25

Since then, B19 has been shown to be the causativeagent of many diseases, some of them classified asautoimmune in origin, including arthropathy, tran-sient aplastic crisis, persistent anemia, and hydropsfetalis (Table 1). Yet, its best known infectionremains erythema infectiosum or Fifth disease,22

Correspondence to: Dr Mirjana Pavlovic MD, PhD, Research

Professor, Department of Computer and Electrical Engineering and

Computer Science, Florida Atlantic University, 777 Glades Rd,

Boca Raton, FL, 33431, USA

Email: [email protected] or [email protected]

Received 27 October 2009; accepted 15 February 2010

! The Author(s), 2010. Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.nav 10.1177/0961203310365715



attacking mostly children of both sexes. The grow-ing evidence of data based on in situ polymerasechain reaction (PCR) demonstrated B19 DNAand tumor necrosis factor alpha (TNF-a) messen-ger ribonucleic acid (mRNA) in endothelia,5 fibro-blasts,5 mast cells,27 lymphocytes,7 synovialtissues,27 and perivascular inflammatory cells in aspectrum of autoimmune diseases, ranging fromSLE and scleroderma, to mixed connective tissuedisease (MCTD).5–8,27

Presently, there is no efficient vaccine for humanparvovirus B19, despite several trials using viralcapsid proteins.23 The reasons for this failure arenot quite clear. However, a new cell line has beendeveloped recently that can produce inactivatedB19 viral capsid protein, specifically VP1, as it isthe primary antigen during native viral infections.28

Based on sequence homology data, a phospholipaseA2 motif has been identified in the VP1 uniqueregion of parvovirus B19, similar toAdeno-Associated Virus-2 (AAV-2).29 It seems tobe a common idea that a successful vaccine basedon an inactivated viral capsid protein would help toprevent outbreaks of Fifth disease in schools, aswell as eliminate any risk associated with infectionof B19 during pregnancy (hydrops fetalis). If par-vovirus B19 is involved in triggering lupus disease,at least in one portion of the lupus spectrum ofdiseases, such a vaccine would be of preventiveimportance for the development of SLE.

Statistics and significance

Infection by parvovirus B19 is most commonbetween 6 and 15 years of age. Some 25–54% ofadults have already had parvovirus B19 infection.30

Estimated incidence of infection shows that 85%people over 70 years are infected, rising to 90%when closer to the end of life.30 Women showhigher prevalence then men. On the other hand, a

small part of the population is naturally immune,since they do not have the receptor for the virus(globoside P), which enables the virus to penetrateinto the cell. This also holds true for p phenotypesof the Amish population.25

Hypothesis

Our hypothesis is that a diverse array of infectiousagents may be involved in eliciting the inflamma-tory symptoms of lupus.31,32 The cause of the dis-ease is specific to an individual and the immuneresponse of the individual. No single agent causesall cases of lupus any more than any single carcin-ogen causes all cases of cancer. Therefore, SLE is aspectrum of diseases (i.e. a syndrome), rather thana single disease. For the reasons explained above,we hypothesize that one of the infectious triggers oflupus could be parvovirus B19, contributing to acertain percentage within the spectrum of SLEsyndrome.31–34

Important viral Features

Parvovirus: details of structure and its use inclinical practice

Human parvovirus B19 can be found in blood, bonemarrow (BM), liver, leukocytes, and synovialfluid.27,35,36 Respiratory droplets and blood pro-ducts are important from an epidemiologicalpoint of view. The virus has an icosahedral shape,forming 18–26 mm diameter particles (50% proteinand 50% DNA, each). Without envelope, B19alludes to the sample from which the parvoviruswas first discovered (5.5 kb ss-DNA). Its DNA haspalindromic sequences with Inverted TerminalRepeats (ITRs) of about 383 nt in length, whichforms hairpins, and therefore the entire structuretends to self-fold (Figure 1). Viral DNA disappearsfrom blood very quickly, but can be found for yearsin BM, synovial tissues and liver, suggesting the per-sistence of the virus in the body and possiblere-activation. Detection of parvovirus B19 DNAin blood is performed by nested PCR with 322 bpnested PCR product. Comparing different autoim-mune diseases (rheumatoid arthritis (RA), Sjogren’ssyndrome (SS), SLE, rheumatoid purpura (RP), pri-mary billiary cirrhosis (PBC), autoimmune inner eardisease (AIE)), only sera of patients with SLE andAIE were found to have B19 DNA.6

The viral capsid is simple and has three coatproteins: VP1 and VP2 (capsid proteins, structural

Table 1 Major diseases caused by parvovirus B19

Disease Actue or Chronic Host

Fifth Disease Acute Normal children

Arthopathy Acute orchronic

Normal adults

Transient aplastic crisis Acute Patients with increasederythropoiesis

Persistent anemia Chronic Immunodeficient andimmunocompromisedpatients

Hydrops fetalis andcongenital anemia

Acute orchronic

Fetus

With courtesy and permission of http://www.aafp.org/afp/20041115/

tips/5.html website.26

Clinical and Molecular Evidence for Association of SLE with parvovirus B19M Pavlovic et al.

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with no enzymatic activity, but antigenic in naturecausing IgM and IgG formation), and NS1, anon-structural protein produced during virus infec-tion.38 Parvovirus IgM and IgG specific antibodiescan be detected from blood samples by using a vari-ety of methods.38–40

The laboratory diagnosis (Table 2) of parvovirusB19 is problematic due to the false positive IgMand presence of detectable B19 DNA in healthypersons. While IgM response persists 10–12 daysafter exposure, IgG response probably persists forlife.41 Transfection seems to be most directed toerythroid progenitor cells. There is a remarkableviral tropism for erythroid early progenitors, thebasis of which is the globoside, blood group antigenP, tetrahexosoceramide (a glycolipid of erythrocyteP antigen). Some studies suggest tissue tropism ofB19 beyond the erythroid progenitor cells, such asleukocytes.7 Indeed, although in illness they arepresent in giant pronormoblasts, they can also befound in fetal liver, normal leukocytes, synovial,myocardial, and endothelial cells (a cause forurgency) and a few leukemic cell lines. Therefore,different receptors for entry into the cells are sug-gested.29 Yet, it is considered that natural resistanceto the virus occurs in people without blood group Pantigen (natural receptor), even though those indi-viduals are very rare.42 A significant characteristicof parvovirus B19 is to replicate selectively in divid-ing cells of BM, gastrointestinal tract (GIT), andembryonic cells. The virus is sensitive to heat, acid,and alkali denaturation. DNAse 1 degrades B19DNA at 60�C within 10 min (pasteurization).43

Viral capsid is also destroyed by pasteurization.

Culture

There is no animal model for B19, and the virus canonly be grown in culture with difficulty. In vitro stu-dies of B19 in explanted human BM cultures haveconfirmed the erythroid specificity of this virus.44

B19 can be cultured in erythroid progenitor cellsfrom a variety of sources, including human BM,44

fetal liver,45 umbilical blood, and peripheral

blood.41,47 In all culture systems, erythropoietin isrequired to maintain viral replication, probably bysupporting the rapid division of erythroid progeni-tors. However, B19 can also be propagated in a fewspecialized cell lines: two megakaryoblastoid celllines,MB-0248 andUT-7/Epo,49 and twohuman ery-throid leukemia cell lines, JK-150 and KU812Ep6.51

These lines have been used to study mechanisms ofreplication and to develop neutralization52 andinfectivity assays.51 However, the yield of virusfrom all of these cultures is poor, and they cannotbe used as a source of antigen for diagnostic tests.

Viral anti-ssDNA and viral cycle (replication)

General featuresThe replication of the parvovirus is closely linked tohost cellular replication. The virus replicates in thenucleus of erythroblasts or other permissive cells,but initiates replication only after the host cell hascompleted the S phase of the cell cycle, and usescellular DNA polymerases. Studies have shownthat either the a or b polymerases are utilized bybidirectional nucleotide incorporation forming anucleotide single-stranded (ss) sequence, whichforms double strands but not of helicodal conforma-tion, as shown in Figures 2 and 3. The existence of ahitherto unknown cell-specific transcription factor

Generic Parvovirus genome

Terminalpalindrome

Promoter

REPP CAP

PolyANon structural(NS) proteins

Structural (virion)proteins (VP)

Figure 1 Schematic presentationof ParvovirusB19 genome. http://www.medynet.com/usuarios/nnuneza/virologia/parvoviruses.htm.Thanks to kindness of the author, Dr Joey Oakley from his unpublished notes.37

Table 2 Laboratory diagnosis for parvovirus infection

IgM IgGPCR forDNA

Unexposed – – –

Acute Infection(3–7days) þþþ – þþþ

Acute Infection(7–14days) þ þþþþ þþ

Previous Infection – þ –1

Immuno compromised patient – – þþ2

1Increased IgM may be detectable for up to 9 months post –infec-

tion;positive PCR results have been observe up to 9 months

post-infection as well.2Recent versus reactived versus previous infection with parvovinus in

an humorally compromised patient will rely greatly on the clinical

history for proper interpretation of nucleic acid testing for parvovinus.

With kind permission of author,Dr Stephen Dewhurst from Rochester

University,at:http:/path.upmc.edu/cases/case522/images/Table-1.gif46


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utilized by parvovirus B19 is suggested.53 Hairpinstructures are critical for virus replication, althoughthe subtle details of replication are still unknown.Two models are proposed for viral replication, andboth of them use palindromic sequences on terminalhairpin structures as a primer for that purpose.

Specific steps of replication

1. Parvovirus DNA replication is accounted for bya single-strand displacement model, similar tothat used to describe adenovirus DNA replica-tion.54 There are two stages of replication, bothusing terminal DNA sequences as primers. Thus,RNA or protein primers or primases are notinvolved. This model predicts site-specific cleav-age of intermediates during the replication.

2. The DNA replication of the parvovirus can alsobe thought of as a modified rolling hairpin. Theends of the viral genome are palindromic.In order to initiate DNA synthesis, the 30 endof the viral genome folds back on itself tobecome a primer.55 After some DNA synthesisalong the complementary strand,56–59 the 30 ter-minus of the complementary strand folds into ahairpin structure to serve as a primer for furtherDNA replication.60 Replication continues alongthe complementary strand. As a next step, anobligatory dimer duplex replicative intermediatestructure is formed. Within the palindromeregions lie small, unpaired sequences whichserve as the cleavage sites for the NS1 nuclease.The nuclease subsequently becomes covalentlyattached to the 50 side of the nick.61 DNA repli-cation continues along the viral progenystrand.62 In the final steps,63–65 capsid proteinrecognizes the 30 hairpin structure and packagesof the single strands. About 18–26 DNA nucleo-tides at the 50 end, along with NS1, are foundoutside the capsid. These extra sequences will becleaved off in the extracellular environment orupon entry into the host cell.

Sequence variability

The nucleotide sequence of B19 was originally estab-lished by sequencing a viral isolate designatedpvbaua obtained from the serum of a child withhomozygous sickle cell disease.64 Since then, alarge number of isolates have been sequencedentirely or in part. Sequence differences can be det-ected by restriction enzyme analysis, single-strandedconformational polymorphism analysis of PCRpro-ducts, and sequencing, by multiple alignments. Thereported B19 isolates are all intimately clustered and

show only 6% divergence among themselves. Notunexpectedly, the NS1 gene is well conservedamong most field isolates, consistent with a requiredrole in virus propagation, while the VP1 and VP2regions may occasionally show a greater variabilityof 2–3%.61,62 No correlation between specific diseasesymptoms and B19 sequence has been observed,63

and the conservation of sequence is such thatsequencing is generally unhelpful in investigatingsingle-source outbreaks. Recently, a B19 isolate,tentatively termed V9, was identified in a Frenchchild with transient aplastic anemia, and onsequence analysis this isolate was seen to be mark-edly (>11%) different from other B19 sequences.64

Standard B19 serological tests failed to demonstratean acute B19 infection, and therefore it was sug-gested that the observed aplastic crisis was due toinfection byV9, a putative emerging virus, which didnot show cross-reactivity with B19-specific tests.

Infection: clinical signs and viral re-activation

Parvovirus B19 can be spread by respiratory andblood route. All parvoviruses require receptor-media-ted endocytosis for cell infection. The human parvo-virus B19 virus replicates only in human elytroidprogenitor cells, and cells that are already knownto be permissive due to the existence of receptor-globoside/antigen P.60,66–68 However, a number ofcells which express globoside on their surfaces arenot susceptible to infection, whichmay be due partlyto an intracellular blocking of the transcription ofviral messages in non-erythroid cells.60,68 It was alsolikely that there is another protein-based receptorfor the parvovirus B19 virus, since Weigel-Kelleyet al. have indicated that P antigen is necessary forparvovirus B19 binding, but not sufficient for virusentry into the cells.66 Indeed, very soon the samegroup of authors has identified a5b1 integrin as acellular coreceptor for human parvovirus B19entry into the cell, using as a model human erythro-leukemia cells (K562) which allow parvovirus B19binding, but not entry.69 They reported that upontreatment with phorbol esters, those cells becameadherent and permissive for parvovirus B19 entry,mediated by a5b1 integrins, only in theirhigh-affinity conformation. Thus, in contrast tomature human red blood cells expressing highlevels of P antigen, but not a5b1 integrins, primaryhuman erythroid progenitor cells express high levelsof both P antigen and a5b1 integrins, and thereforeallow for b1 integrin-mediated entry of parvovirusB19.69 The process of cell infection by parvoviruses


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shows many of the features seen for other viruseswhich replicate in the nucleus, but the small andstable parvovirus particles must undergo moresubtle changes during the various steps involved.70

Incubation time is 7–10 days (range, 4–21 days).Transport of the capsid and/or viral DNA into

the nucleus is an important step in infection ofcells with an intact nucleus and occurs principallythrough the nuclear pore complex (NPC). Althoughsmall macromolecules can diffuse freely through thepore, transport of larger molecules is specific, requir-ing ATP and soluble cytosolic factors, and ismediated by nuclear localization sequences(NLSs).70 Parvovirus capsids appear to be able topass through the NPC intact, and over a period of 2or more hours, microinjected capsids enter thenucleus, where they are recognized by antibodiesthat bind only intact capsids.70 Modification ofviral capsids may allow exposure of NLSs neededfor nuclear targeting and transport of viral particles.

Clinical studies data indicating the reasons forassociation with SLE

State of the art of SLE and parvovirus B19 infection:pros and consMicroarray studies have been conducted on lupuspatients.71–73 However, these investigations were

limited to expression of human genes. A key molec-ular signature has been identified through theseefforts, the up-regulation of IFN-a.9,10 The likelycandidate source for this molecule is the plasmocy-toid dendritic cell (pDC). However, the agent oragents expected to induce the IFN-a within apatient have remained elusive. No studies havebeen conducted in an effort to develop a genechip or proteonomic display that contains anarray of possible agents, as well as the molecularsignatures of cytokines and antibodies. A morecomplex approach is evidently necessary, to explainthe causative factors of lupus disease.

Our hypothesis is that a diverse array of infec-tious agents may be involved in eliciting the inflam-matory symptoms of lupus and that one of thesescenarios involves parvovirus B19. There are someclinical data which are in agreement with parvo-virus involvement, although the exact mechanismsare not elucidated. For example, the episodes offever, anemia, and arthralgia are found in patientsdiagnosed with SLE, red cell aplasia, and giant ery-throblasts, with positive IgM antibodies in serum.74

In parvovirus infection, on the other hand, symp-toms and signs are similar, as presented in Figure 2.Reactivation of the disease by virus74 was found in42 old women. A 46-year-old woman had B19

B19 Viremia and antibody response

Clinicalfeatures

Fever, chills, Rash &arthralgia

Hematologicalchanges

B19 DNA

B19 Viremia andantibody responses

Reticulocytes

Dot blot

IgMIgG

Days

0 7 14 21 28 2 4 6

Months

IgG

IgM

PCR

Viremia

Hemoglobin

Normal values

headache,myalgia

Figure 2 B19 viremia and antibodies response within the time-course of the disease. Erik D. Heegaard and Kevin E. Brown.‘‘Human Parvovirus B19.’’ Clin Microbiol Rev. 2002 July, 15(3): 485–505. doi: 10.1128/CMR.15.3.485-505.2002. With author’sand Publishers kind permission.24


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infection followed by serious flare of SLE.75 Thisalso raises the question: is there a parvoviral enhan-cer of SLE? In another study, a 68-year-old femalewith SLE and pure red cell aplasia, antiphospholi-pid syndrome, IgG HPV, was B19 viral DNA pos-itive at the time of admission.21 Was it acoincidence or causality? A 26-year-old woman,three years after being diagnosed with SLE, pres-ented with fever, arthralgia, nausea, vomiting, andwas IgG positive to viral DNA.76 The clinicalexamples of the association of SLE and other auto-immune diseases are described by manyauthors.74–77 There are also quite contrasting datastating that there is no correlation between parvo-virus B19 and SLE, as described by Bengtssonet al.’s study in 2000, of a group of 99 SLEpatients.78 The reason for such results (no IgGprevalence in comparison to control) could be inthe difference in sensitivity used in the two assaysapplied,78 immunosupression due to drug (steroid)treatment, or immunosupression due to disease.However, for similar reasons these data are equallyas convincing as those described by Hsu and Tsauin 2001, who have found parvoviral B19 DNA onlyin patients with SLE and AIE between the spec-trum of autoimmune diseases.6 Some of the authorsstate that parvovirus B19 causes symptoms thatonly mimic lupus disease, such as vasculitis,arthralgia, and fever episodes.79,80 Taken together,the clinical data suggest the critical role of uniform,longitudinal studies, with a strong emphasis on auniquely designed approach and evaluation of lab-oratory and clinical signs in order to confirm asso-ciation between parvovirus B19 infection and SLE,beyond reasonable doubt.

Detection of the parvoviral infection is also indi-cated by typical hallmarks:

. recent infection (IgM antibody assay the thirdday after symptoms);

. presence of viral DNA (nucleic acid hybridiza-tion, the most sensitive test);

. B19 in leukocytes.81

Indirect experimental proof of possible molecu-

lar association of parvovirus B19 and SLE

Anti-DNA hydrolyzing antibodies and SLE(Abzymes)

The hallmark of SLE is the production of an arrayof IgG and IgM autoantibodies directed against

one or more nuclear components, the most fre-quent of which are double-stranded (ds)DNA and/or ss DNA. Both anti-ssDNAand anti-dsDNA are involved in disease develop-ment and have been eluted from the kidney biopsiesof experimental murine models and SLEpatients.82,83 Rodkey et al.84 showed that mouseanti-ssDNA monoclonal antibody BV04-01 hasbinding specificities for a hairpin-like structure,similar to that which ssDNA human parvovirusB19 DNA has. Furthermore, its DNA sequencehas a number of 5, 6, and 7 thymidine motifs.Knowing that at least 5 T motifs85 of viral DNAare necessary to be recognized by the arginine ofthe anti-ssDNA antibody in order to bind to threeof them, one can visualize the conditions foranti-ssDNA hydrolytic antibody action inthis system.

Real-time fluorescent detection of hydrolysis ofthe synthetic parvovirus ssDNA sequence bypurified anti-ssDNA antibodies

Quite recently, Cavallo et al.86 have designedthe new fluorescence-based real-time method formonitoring anti-DNA antibody hydrolytic activity.By using an oligo-probe mimicking the part of par-vovirus B19 sequence with thymidine pentamerfor recognition (a prerequisite for antibody bindingto the substrate),85 and isolated and purifiedlupus anti-ssDNA antibody using a novel two-stepmagnetic bead method30,87 in appropriate propor-tion and mixture, it was found that hydrolysisof synthetic sequence occurs in different kineticscompared with commercial DNase1. This stronglysuggests that the DNase activity of the antibodyis its intrinsic property and that due to that prop-erty, the antibody can cleave viral DNA. This isalso, to the best of the authors’ knowledge, thefirst indication of a possible associationbetween parvovirus B19 and SLE at the molecularlevel. The hydrolysis of synthetic parvovirusB19 ssDNA sequence by lupus anti-ssDNAantibody could be the molecular manifestationof an acquired antimicrobial attack of the hostsystem, part of the anti-DNA antibody clear-ance strategy from the body, or the direct antinuc-lear DNA cleavage during ssDNA replicationwithin permissive host cells with consecutivecell death and exposure to the immune systemof the new array of antibodies, thereby perpetuat-ing and maintaining the ‘vicious cycle’ in lupusdisease.


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Proposed model of possible parvoviral B19 and

anti-DNA antibody involvement in SLE and

other autoimmune diseases

This work is intended to summarize clinical andexperimental data which would contribute to themodel of possible parvovirus involvement in SLE.Our model is given in Figures 3 and 4, and showstwo possible mechanisms of action of lupusanti-ssDNA hydrolyzing antibodies in serum andsynovial liquid, and at nuclear (cellular) level. Inthe first case anti-ssDNA binding antibody,secreted from activated B cells, attacks free micro-bial (parvoviral) DNA and hydrolyzes it, contribut-ing to the clearance of DNA from plasma throughglomeruli, where they can underline basal mem-brane, alone, or in complex with small pieces ofviral DNA. In the second case, anti-ssDNA hydro-lyzing antibodies, after crossing the cell membranevia cell structural proteins88 and their translocationinto the nucleus, directly bind to, and hydrolyze,replicating viral ssDNA in dividing cells. Thisnewly formed viral DNA, built up by utilizing cell

host enzymes, is created due to bidirectional ds rep-lication, but is not helicoidal in structure. When itbecomes hydrolyzed by antibodies, it causes celldeath, which in turn opens the cell, exposing tothe immune system a new set of fresh antigensand, therefore, perpetuating and maintaining the‘vicious cycle’ in SLE. Figure 4 shows the possibledetails of this interaction, causing flares in SLE inresponse to viral infection or re-activation, withcorresponding clinical signs. The immune com-plexes, or unmethylated CpG motifs of hydrolyzedDNA, sensitize transmembrane Toll-Like Receptor9 (TLR9)90 on the surface of antigen presentingcells (APCs), in this case B cells. This, in turn,causes signaling to nucleus activating nuclear tran-scription factor kappa B (NF-kB)91 or MyD88signaling with subsequent increase in anti-ssDNAantibody synthesis through a genetic switch, thesteps of which are not yet clearly understood.92

This global concept is in agreement with the newinnate immunity scenario of autoimmune diseases,in which the DNA-sensing cytosolic cellular pro-teins propagate the information on a viral DNAsequence through a signal to TLR9, thereby avoid-ing the antigen presentation loop to T cells throughAPC (B-lymphocyte or dendritic cells). This model,at the same time, states that DNA cannot be pres-ented to the immune system in the classical way,since it is too large for the MHCI and/or II classof molecules in both types of APCs. Rather, itsimmunogenic role is to be sliced into smaller CpGmotifs (by DNases from the blood) which, asunmethylated particles, sensitize transmembrane,surface TLR9 to transduce the signal and informa-tion on the DNA sequence to the correspondingtranscription factors,91 and employ immunoglobu-lin gene machinery for anti-DNA autoantibody

Figure 4 Model of Parvovirus B19 Association with SLE II. Pavlovic, M., Kats, A., Shoenfeld, Y., 2008.32

A. In sera: Antimicrobialstrategy and clearance of

B19DNA

Anti-DNAantibody

B. In cell nucleus: Nuclear DNA hydrolysis byantiss-DNA nuclear autoantibodies with

perpetuation and maintenance of “Vicious cycle”

Figure 3 Model of Parvovirus B19 Association withAutoimmunity. Pavlovic, M., Kats, A., Shoenfeld, Y., 2008.89


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synthesis in the cells of B-series. These are not nowparvovirus B19 specific antibodies designedtowards viral capsids (IgG and IgM isotypes)secreted as a result of primary infection (sincethey have disappeared from plasma), but quitenew, hydrolytic, anti-ssDNA autoantibodies tar-geted against viral and probably even hostssDNA (in replicating cells with inserted viralDNA). So, the cells are going either to apoptosisor necrosis, exposing to the activated immunesystem a new array of autoantigens (especiallynuclear). Meanwhile, serum clearing mechanisms,employed by regular DNases and newly synthesizedanti-ssDNA antibodies, are insufficient to cleanapoptotic waste. This results in precipitation ofantibodies, and their complexes with DNA,beneath basal membranes of small blood vessels,including glomeruli. Increasing evidence of datasuggest that impairment in the uptake of apoptoticcell debris is linked to autoimmunity.93 Thus, theseantibodies, the synthesis of which is triggered byparvoviral infection and re-activation, trigger theflare, and can be both an early predictor and adiagnostic sign of the flare.94 In this way, they per-petuate and maintain the ‘vicious cycle’ in SLE.

Parvovirus B19 and SLE association: possibleimpact and benefits

The primary impact of the above approach is thatphysicians would be able to tailor specific therapiesfor a disease that can have a multitude of symptomsand causes. The patient will benefit because theirdisease and their immune response to the underly-ing disease will be understood in much greaterdepth. Currently, nearly all patients are routinelyplaced on global immunosuppressive regimens,such as corticosteroids and non-specific che-motherapies. With computer-analyzed data sets ofgenes or proteins from possible infectious agents(especially parvovirus B19 as a center of interest)and sophisticated analysis of the immune profileconcordant with each patient readily available tothe physician (microarrays at different levels),appropriate therapeutic strategies can be employed.

Summary

Should an agent, such as parvovirus, be shown tobe involved in eliciting the cyclical infection in asignificant number of patients with lupus, wouldit not be advisable to seek a vaccine to preventand perhaps someday treat the infection? Forexample, human monoclonal antibodies against

parvoviruses, strategically prepared for therapeuticpurposes could be employed in treatment. Webelieve that development of the vaccine based oninactivated capsid proteins (against viral proteins)would be of the primary interest since they are themost probably presented to the immune system inthe classical way, rendering T memory cellsinformed and educated about their features criticalfor T-lymphocyte’s activation. However, since noneof the vaccines based upon that concept did notsucceed so far,23,95 in accordance with innateimmunity scenario and B-cell memory for viralDNA mentioned above,90–94 this loop of viralDNA recognition and memory storage within Bcells should not be ignored, and therefore consid-ered on a larger scale of vaccine spectrum, includ-ing even attenuated virus.

Acknowledgment

The authors want to thank to Mr Alex Kotlarchykfor his careful and helpful proof reading of thearticle.

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