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The association of anti-parvovirus B19-VP1 unique region antibodies with antiphospholipid antibodies in patients with antiphospholipid syndrome
Clinica Chimica Acta 411 (2010) 1084–1089
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
The association of anti-parvovirus B19-VP1 unique region antibodies with antiphospholipid antibodies in patients with antiphospholipid syndrome Der-Yuan Chen a,b,c, Bor-Show Tzang d,e,1, Yi-Ming Chen a,b, Joung-Liang Lan a,b,c, Chun-Chou Tsai f, Tsai-Ching Hsu f,g,⁎,1 a
Department of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan, ROC Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC School of Medicine, Chung Shan Medical University and Institute of Biomedical Science, National Chung-Hsing University, Taichung, Taiwan, ROC d Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, ROC e Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC f Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan, ROC g Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC b c
a r t i c l e
i n f o
Article history: Received 31 January 2010 Received in revised form 2 April 2010 Accepted 3 April 2010 Available online 10 April 2010 Keywords: Human parvovirus B19 (B19) VP1 unique region protein (VP1u) Antiphospholid antibody (aPL) Antiphospholipid syndrome (APS) Systemic lupus erythematosus (SLE)
a b s t r a c t Background: Human parvovirus B19 (B19) infection has been identified as a trigger of antiphospholipid syndrome (APS). However, the precise role of B19-VP1 unique region (VP1u) in patients with antiphospholipid syndrome remains unclear. Methods: IgM and IgG against B19-VP, and serum levels of antibodies directed against cardiolipin (CL), beta2glycoprotein-I (β2GPI) and phospholipid (PhL) were determined using ELISA in 45 APS patients. Humoral responses of anti-B19-VP1u were assessed by Western blot and B19 DNA was detected by nested PCR. Absorption experiments were performed using B19-VP1u protein to determine the binding specificity of antiphospholipid antibodies (aPL). Results: One and 18 of 45 APS patients had detectable levels of anti-B19-VP IgM and anti-B19-VP IgG, indicating recent and past infection respectively. All serum samples from APS patients with diagnostic pattern DNA−/IgM−/IgG+ had anti-B19-VP1u activity. APS patients with anti-B19-VP1u antibody had a 4fold increased risk for recurrent vascular thrombosis compared with those without anti-B19-VP1u antibody. The binding inhibition of CL, β2GPI, and PhL by absorption with B19-VP1u ranged from 31.4% to 91.1%, 0.8% to 59.8% and 20.2% to 72.1% respectively. Significantly higher inhibition to β2GPI by B19-VP1u absorption was observed in APS patients with anti-B19-VP1u antibody than in those without anti-B19-VP1u antibody. Conclusions: We show a close association of B19 infection with aPL production and suggest B19-VP1u may be of pathogenetic importance in some patients with APS. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Human parvovirus B19 (B19), which was firstly discovered by Cossart et al. in 1975 [1], is known as an important human pathogen that comprises a small non-enveloped particle enclosing a singlestranded linear 5.6-kb DNA genome [2,3]. The icosahedral capsid of B19 consists of two structural proteins, VP1 (83 kDa) and VP2 (58 kDa), which are identical except for the 227 amino acids at amino-terminal end of VP1-protein, the so-called VP1-unique region (VP1u) [4,5]. Additionally, B19-VP1u has been demonstrated to have phospholipase A2 (PLA2) activity, which is essential for its cytotoxi-
⁎ Corresponding author. Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan, 110 Section 1, Jianguo N. Road, Taichung 402, Taiwan, ROC. Tel.: + 886 4 24730022x11702; fax: + 886 4 23248172. E-mail address: [email protected] (T.-C. Hsu). 1 Dr. Bor-Show Tzang and Dr. Tsai-Ching Hsu contributed equally to this work. 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.04.004
city and infectivity [6–10]. Although B19-VP1 represents only 5% of the capsid, VP1u is critical for eliciting an appropriate and predominate immune response in both humans and animals [11,12]. B19 infection has been recognized as a cause or trigger of autoimmune diseases [13–15] and is associated with the production of various autoantibodies, including anti-β2-glycoprotein-I (antiβ2GPI) and anti-cardiolipin antibody (aCL) [16–20]. Many studies have demonstrated that a remarkable similarity exists in the specificity of antiphospholipid antibodies (aPL) between patients with B19 infection and those with systemic lupus erythematosus (SLE) [13–17]. Previous studies have reported that anti-β2GPI and aCL were detected in patients with B19 infection and SLE [13–17,21]. Moreover, aCL from patients with B19 infection increased their binding to antigens in the presence of β2GPI as a cofactor, similar to aCL from SLE patients, but unlike antibodies from patients with other viral infections [17]. These finding highlight the remarkable similarity in the aPL between SLE and B19 infection.
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The classical “Hughes syndrome”–antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies such as lupus anticoagulant (LA), aCL and anti-β2GPI, and is associated with vascular thrombosis, pregnancy-related morbidity or both [22–24]. B19 infection and APS have been reported to show similar symptoms leading to the hypothesis of a common pathogenic background [14–21]. The PLA2-like activity observed in the B19-VP1u may be related to the induction of aPL [19]. Our recent study indicated that autoantibodies against CL, β2GPI, and phospholipid (PhL) in sera from patients with B19 infection, were cross-reactive with B19-VP1u [25]. Moreover, significantly raised immunoglobulins against B19VP1u, CL, β2GPI and PhL were detected in sera of mice immunized with recombinant B19-VP1u protein, as well as striking thrombocytopenia, prolongation of aPTT, and autoantibodies against β2GPI and PhL were detected in mice treated with anti-B19-VP1u IgG [26]. Although these findings suggested a connection between anti-B19VP1u antibodies in manifestations of APS and development of autoimmunity, the relationship between anti-B19-VP1u antibodies and aPL in patients with APS is still obscure. 2. Materials and methods 2.1. Patients and sera Forty-five patients with APS [27] (36 females and 9 males, mean age 37.0 ± 13.3 y) were enrolled from Taichung Veterans General Hospital. The enrolled subjects with APS were classified into 2 groups: primary APS and SLE [28] with secondary APS, according to the clinical course by ACR criteria [27]. Patients with hepatitis B and C infections and other rheumatic diseases were excluded from this study. Patients were considered to be smokers if they ever smoked ≥100 cigarettes, which was determined using the 1985 Health Promotion Disease Prevention supplement of the Health Interview Survey [29]. Peripheral blood samples were collected into glass tubes that contained no additive or anticoagulant. The clotted blood samples were centrifuged at 1250 rpm for 10 min and the sera were collected and stored at −70 °C until examination. The Clinical Research Ethics Committee at Taichung Veterans General Hospital approved the study protocol, and informed consent was obtained from each participant. 2.2. Detection of IgM and IgG against B19-VP in serum from APS patients The IgM and IgG against B19-VP were analyzed by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (IBL-America, Minneapolis, MN). Serum samples were diluted 1:50 and 1:100 for the IBL-America IgM and IgG ELISA kit in the provided sample diluents (IBL-America) respectively. Standard, control, and diluted samples were included in each plate. The absorbance (O.D.) was read at the wavelength of 450/620 nm after adding 100 ml of stop solution. According to the manufacture's instruction and our experimental test, the sensitivity of anti-VP ELISA is 98 to 100%. Negative values range from 0 to 11 U (B19 IgM and B19 IgG). Positive results are N12 U (B19 IgM and B19 IgG). 2.3. Determination of IgG against B19-VP1u using immunoblotting Western blotting was performed as described in our previous report [25]. Protein samples were separated in 12.5% of SDS-PAGE [30] and electrophoretically transferred to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) according to the method of Towbin et al. [31]. After blocking with 5% non-fat dry milk in PBS for 30 min at room temperature, human or rabbit antiserum against B19VP1u was diluted with 5% non-fat dry milk in PBS, reacted with the nitrocellulose strips and then incubated for 1.5 h at room temperature. The strips were washed twice with PBS-Tween for 1 h and secondary antibody consisting of alkaline phosphatase conjugated
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goat anti-human or rabbit IgG antibodies was added. The substrate NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3 indolyl phosphate) was used to detect antigen–antibody complexes. 2.4. DNA extraction, PCR amplification, and detection of B19 genomes DNA was extracted using QIA Amp blood kit (QIAagen, Hidden, Germany) as directed by the manufacturer. Nested-PCR for the detection of B19 DNA in serum was performed as described previously [25], B19 positive and negative reference controls [32] were also included in each PCR reaction. The nested PCR was used as it eliminates non-specific background and thus gives a clearer final product. Because of high sensitivity of the nested PCR reaction, stringent precautions were taken and samples were tested at least 3 times to avoid the risk of false-positive results. 2.5. Preparation of recombinant human B19-VP1u protein Construction of B19-VP1u cDNA into pET-32a expression vector (Novagene, Cambridge, MA) was performed and the recombinant B19-VP1u protein was purified as described in our recent publication [25,26]. The purified recombinant B19-VP1u proteins were also analyzed by HPLC and the purity was nearly 98%. 2.6. Determination of levels of aCL, anti-b2GPI and anti-PhL Previous studies have demonstrated that B19-VP2 capsid protein is known to elicit the IgM response, whereas B19-VP1u protein is known to elicit the IgG response [4,33]. We used direct antigen-specific ELISA kits to detect aCL IgG (QUANTA LiteTM ACA IgG III), anti-b2GPI IgG (QUANTA LiteTM b2GPI IgG) (INOVA Diagnostics Inc., San Diego, CA), and anti-PhL IgG (Louisville APL Diagnostics Inc., GA) according to the manufacture's description. Results were expressed as IgG phospholipid (GPL) units or standard IgG units (SGU) using international reference material. For absorption analysis to assess the effect of binding inhibition, the sera were pre-incubated with 2 mmol/l of purified VP1u recombinant proteins for 1 h at 37 °C before ELISA kits (aCL, anti-b2GPI and anti-PhL) were performed. Negative values range from 0–20 GPL (aCL), 0–20 SGU (anti-b2GPI antibodies) or 0– 15 GPL (anti-PhL). Positive results are greater than 20 GPL (aCL), 20 SGU (anti-b2GPI), and 15 GPL (anti-PhL). 2.7. Statistical analyses Data were analyzed using SPSS 10.0 for windows (Chicago, IL). The χ2 test was used to determine significant differences in categorical variables among groups. The paired t test was used to analyze data for statistical significance. A binary logistic regression analysis was used to evaluate the effect of anti-B19-VP1u IgG on the occurrence of recurrent thrombosis. A p b 0.05 was considered to be statistically significant. 3. Results 3.1. Demographic data, clinical characteristics, and the presence of aPL in APS patients Forty-five patients with APS were classified according to the clinical course of their disease as follows: primary APS (n = 10) and SLE with secondary APS (n = 35). As illustrated in Table 1, the most commonly associated thrombosis was cerebral vascular accident in both primary APS patients (50%) and SLE patients with secondary APS (25.7%). No significant differences in age at study entry, duration of disease, proportion of female, number of thrombotic episodes, or thrombosis subtype were observed between primary APS patients and SLE patients with secondary APS. However, significantly higher
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Table 1 Characteristics of 45 patients with antiphospholipid syndrome (APS).a Characteristics
Primary APS (n = 10)
SLE with APS (n = 35)
Age at study entry (y) Duration of disease (y) Females Ever smoker Number of subjects with ≧2 thrombosis Thrombosis by subtype Cerebral vascular accident Livedo reticularis Deep vein thrombosis Peripheral arterial thrombosis Miscarriage late (≧1 in 2nd or 3rd trimester) Myocardial infarction Other thrombosis Used medications Corticosteroids Hydroxychlorquine Other immunosuppressive agents
35.0 ± 9.3 3.2 ± 4.2 8 (80.0%) 1 (10.0%) 4 (40.0%)
37.6 ± 14.3 6.6 ± 5.3 27 (77.1%) 3 (8.6%) 11 (31.4%)
5 (50.0%) 4 (40.0%) 2 (20.0%) 1 (10.0%) 1 (10.0%) 0 (0.0%) 3 (30.0%)
9 (25.7%) 8 (22.9%) 4 (11.4%) 4 (11.4%) 2 (5.7%) 3 (8.6%) 17 (48.6%)
4 (40.0%) 10 (100.0%) 2 (20.0%)
33 (94.2%)⁎ 34 (97.1%) 30 (85.7%)⁎
SLE: systemic lupus erythematosus. a Data were presented as mean ± SD or number (percentage). ⁎ p b 0.05, vs. patients with primary APS, determined by the paired t test.
proportion of usage of immunosuppressive agents including corticosteroids was found in SLE patients with secondary APS than in primary APS patients. 3.2. Results of B19 DNA, anti-B19 IgM and IgG in sera from APS patients Since parvovirus B19 cannot be grown routinely in vitro, B19 infection is generally diagnosed via direct detection of B19 DNA (virological) and by anti-B19 IgM and IgG (serological). Based on the advances in diagnosis of B19 infection [2,31], 1 (2.2%) and 18 (40.0%) of the 45 patients with APS had detectable anti-B19-VP IgM and anti-B19VP IgG, indicating recent infection and past infection respectively. Table 2 shows the diagnostic patterns of B19 infection in APS patients. Regarding the diagnostic patterns of 10 patients with primary APS, 4 (40%) of them had the DNA−/IgM−/IgG+ diagnostic pattern, 1 (10%) of them had the DNA+/IgM+/IgG+ diagnostic pattern, and 5 (50%) of them had the DNA−/IgM−/IgG− diagnostic pattern. In the SLE patients with APS, 14 (40%) of them have the DNA−/IgM−/IgG+ diagnostic pattern and 21 (60%) of them have the DNA−/IgM−/IgG− diagnostic pattern. There was no significant difference in the distribution of diagnostic patterns of B19 infection between primary APS patients and SLE patients with secondary APS. 3.3. Reactivity of IgG directed B19-VP1u in sera from APS patients The B19-VP1u is known to generate prolonged neutralizing immune response and the epitopes localized in VP1u are predomi-
Table 3 Characteristics of 45 patients with APS according to the presence of anti-B19-VPlu antibodies.a Characteristics
Anti-B19-VPlu (−) (n = 25)
Anti-B19-VP1u (+) (n = 20)
Age at study entry (y) Duration of disease (y) Females Number of subjects with ≧2 thrombosis Positivity for aCL Positivity for anti-β2GPI Positivity for aPhL Absorption inhibition degree, % Inhibition to CL/β2GPI/PhLb Inhibition to β2GPIb
37.5 ± 14.4 59.9 ± 59.0 18 (72.0%)
36.5 ± 12.1 83.7 ± 65.8 147 (85.0%)
5 (20.0%) 0 (0.0%) 9 (36.0%) 0 (0.0%)
10 (50.0%)⁎ 5 (25.0%)⁎ 10 (50.0%) 5 (25.0%)⁎
21.3 ± 11.3 21.3 ± 11.3
45.6 ± 22.3⁎⁎ 38.5 ± 17.0⁎
B19-VP1u: B19-VP1 unique region protein; aCL: anti-cardiolipin antibodies; antiβ2GPI: anti-beta2-glycoprotein-I antibodies; aPhL: antiphospholipid antibodies. a Data were presented as mean ± SD or number (percentage). b Among patients with aCL, anti-β2GPI, or anti-PhL antibodies. ⁎ p b 0.05, vs. patients with patients with anti-B19-VPlu (−), determined by the paired t test. ⁎⁎ p b 0.01, vs. patients with patients with anti-B19-VPlu (−), determined by the paired t test.
nantly linear [34,35]. To clarify the antigenicity of VP1u, this study investigated antibodies directed against B19-VP1u protein in serum samples from APS patients with different diagnostic patterns using Western blot analysis. As illustrated in Table 2, all of serum samples from primary APS patients with diagnostic pattern DNA−/IgM−/IgG+ had anti-B19-VP1u activity. The serum sample from 1 patient with primary APS who had diagnostic pattern DNA+/IgM+/IgG+ showed reactivity to anti-B19-VP1u. Only 1 primary APS patient with diagnostic pattern DNA−/IgM−/IgG− had a serum sample that showed reactivity to anti-B19-VP1u. All of serum samples from SLE patients with APS who had diagnostic pattern DNA−/IgM−/IgG+ (n = 14) showed reactivity to anti-B19-VP1u.
3.4. The association of B19 diagnostic patterns with aPL in APS patients As illustrated in Table 2, 1 of 4 (25%), 3 of 4 (75%), and 1 of 4 (25%) serum samples showed positivity for aCL, anti-β2GPI, and anti-PhL respectively in 4 patients with primary APS who had diagnostic pattern DNA−/IgM−/IgG+. The serum sample from 1 patient with primary APS who had diagnostic pattern DNA+/IgM+/IgG+ showed positivity for both aCL and anti-β2GPI. Among 14 SLE patients with secondary APS who had diagnostic pattern DNA−/IgM−/IgG+, 2 (14.3%), 6 (43.0%) and 3 (21.4%) serum samples showed positivity for aCL, anti-β2GPI, and anti-PhL respectively. Among 21 SLE patients with secondary APS who had diagnostic pattern DNA−/IgM−/IgG−, 8 (38.0%) serum samples showed positivity of anti-β2GPI, and no any serum sample showed positivity for aCL or anti-PhL.
Table 2 The diagnostic patterns of B19 infection, serology of anti-B19-VP1u, and antiphospholipid antibodies in 45 patients with antiphospholipid syndrome (APS).a Diagnostic patterns of parvovirus B19 infection Primary APS (n = 10) DNA−/IgM−/IgG− (n = 5) DNA−/IgM−/IgG+ (n = 4) DNA+/IgM+/IgG+ (n = 1) SLE with APS (n = 35) DNA−/IgM−/IgG− (n = 21) DNA−/IgM−/IgG+ (n = 14) DNA+/IgM+/IgG+ (n = 0)
Anti-B19-VP1u (n = 20)
Positivity of aCL
Positivity of anti-β2GPI
Positivity of anti-PhL
1 4 1
1 (25%) 1 (25%) 1 (100%)
1 (25%) 3 (75%) 1 (100%)
1 (25%) 1 (25%) 0 (0%)
0 14 0
0 (0%) 2 (14.3%) 0
8 (38%) 6 (43%) 0
0 (0%) 3 (21.4%) 0
SLE: systemic lupus erythematosus; B19-VP1u: B19-VP1 unique region protein; aCL: anti-cardiolipin antibodies; anti-β2GPI: anti-beta2-glycoprotein-I antibodies; anti-PhL: antiphospholipid antibodies. a Data were presented as number (percentage) among the diagnostic patterns of B19 infection.
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3.5. Clinical characteristics of APS patients with anti-B19-VP1u antibodies As shown in Table 3, APS patients with anti-B19-VP1u antibodies were more likely than those without anti-B19-VP1u antibodies to have recurrent thrombosis (50.0% vs. 20.0%, p b 0.05), positivity for aCL (25.0% vs. 0.0%, p b 0.05) and positivity for anti-PhL (25.0% vs. 0.0%, p b 0.05). However, no significant differences in age at study entry, duration of disease, proportion of female, or thrombosis subtype were observed between patients with and without anti-B19-VP1u antibodies. To further verify the clinical and laboratory significance, we used binary logistic regression analysis to investigate the effect of anti-B19-VP1u positivity on the occurrence of recurrent thrombosis and clinical features in APS patients. Compared with APS patients without anti-B19-VP1u antibody, those with anti-B19-VP1u antibody had significantly increased risk of recurrent vascular thrombosis, with an odds ratio of 4.000 (95% CI: 1.074–14.896). In addition, high sensitivity (100%) and negative predictive value (100%) of anti-B19VP1u IgG were observed for predicting the reactivity to CL and PhL in APS patients. However, there was no significant connection between the presence of anti-B19-VP1u IgG and clinical features in APS patients. 3.6. Absorption analysis using purified B19-VP1u for the sera from APS patients To further elucidate the role of B19-VP1u in the reactivity to CL, β2GPI and PhL in APS patients, absorption analysis was performed on the sera from 20 APS patients who had positive IgG aPL using ELISA. As illustrated in Fig. 1, a significant decrease in the levels of aCL, antiβ2GPI, and aPhL antibodies after absorption with B19-VP1u was observed in 20 patients with APS. The inhibition of reactivity to CL, β2GPI and PhL by absorption with purified B19-VP1u was 31.4–91.0%, 0.8–59.8% and 20.2–72.1% respectively. Among APS patients with positivity for aPL, significantly higher degree of inhibition to CL/ β2GPI/PhL by B19-VP1u absorption was observed in sera from those with anti-B19-VP1u antibodies than from those without anti-B19VP1u antibodies (45.6% vs. 21.3%, p b 0.05, Table 3). Moreover, significantly higher degree of inhibition to β2GPI by B19-VP1u absorption was observed in sera from those with anti-B19-VP1u antibodies when compared to those without anti-B19-VP1u antibodies (38.5% vs. 21.3%, p b 0.05, Table 3). No significant difference in the degree of inhibition to CL, β2GPI or PhL was found in sera from
Fig. 1. Changes in the levels of anti-cardiolipin antibodies (aCL), anti-beta2glycoprotein-I (anti-β2GPI), and antiphospholipid antibodies (aPhL) in 20 patients with antiphospholipid syndrome. Data are expressed as median levels (the 25th to the 75th percentile). *p b 0.05, #p b 0.001, vs. before absorption, determined by the Wilcoxon signed rank test.
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patients with primary APS when compared to SLE patients with APS. Additionally, the inhibition of reactivity to CL, β2GPI, and PhL by absorption with 10 μg of B19-NS1 was not found in patients with APS (data not shown). 4. Discussion Consistent with the results of previous studies [14–21], our data show an association of parvovirus B19 infection with the presence of aPL in patients with APS. The presence of anti-B19-VP1u IgG was closely associated with the reactivity to CL and PhL. We also showed that anti-B19-VP IgM may have disappeared but B19 infection can be still be recognized by B19-VP1u IgG immunoblots in APS patients. In addition, our results showed lower positive rates of B19-VP1u IgG in SLE patients with APS than in those with primary APS (40% vs. 60%). The true prevalence of B19 infection in SLE patients may be underestimated as the immunosuppression associated with this disease and the usage of immunosuppressive agents that can inhibit reactivity to B19 infection [36,37]. Many viral infections may be accompanied by the production of aPL [15,16,18,19,38,39]. The widely held belief that the majority of infectious aPLs are usually transient and are not associated with thrombosis [39,40] has been challenged by several reports [17,41–43]. Our previous study showed that the BALB/c mice treated with antiB19-VP1u IgG developed APS-like autoimmunity [26]. In the present study, the presence of anti-B19-VP1u antibodies was closely associated with recurrent vascular thrombosis. Our results and those of previous studies indicate that an increase of aPL may be accompanied by clinical manifestations of APS in B19-infected subjects. However, further studies are required to identify B19 infection responsible for the production of the pathogenic aPL along with APS manifestations. Although APS is known to be an important autoimmune manifestation and has been associated with B19 infection [14–21], the precise role of B19-VP1u in the induction of APS is still obscure. Recent studies indicate that B19-VP1u is linked to PLA2-like activity and is critical for eliciting production of autoantibodies [6–11]. Our results showed that significantly higher seropositivity for aPL was observed in APS patients with anti-B19-VP1u antibodies when compared to those without anti-B19-VP1u antibodies, supporting the recent findings [6–11,17]. To clarify the relationship between B19VP1u and aPL production, we used purified B19-VP1u for absorption analysis of sera from APS patients. We showed an inhibition of binding to CL, β2GPI, and PhL by absorption with B19-VP1u was 31.4 to 91.0%, 0.8 to 59.8% and 20.2 to 72.1% respectively. Our results suggest that similar conformation or epitopes may exist between B19-VP1u and phospholipid antigens. The partially reduced reactivity to CL, β2GPI and PhL after absorption with B19-VP1u suggests that aPL production may be related to the underlying B19 infection in APS patients. Interestingly, significantly higher degree of inhibition was found in sera from patients with antibodies against both B19-VP1u and CL/ β2GPI/PhL when compared to those without B19-VP1u antibodies. Moreover, significantly higher degree of inhibition to β2GPI by B19VP1u absorption was observed in sera from those with anti-B19-VP1u antibodies when compared to those without anti-B19-VP1u antibodies (Table 3). A well-tolerated vaccine consisting of 25% B19-VP1 and 75% B19-VP2 could elicit a strong humoral response [44], and immunoglobulin specific for the B19-VP1u offer life-long protection against re-infection [45]. Our findings in support of a critical role of B19-VP1u in the pathogenesis of APS suggest a novel vaccine for B19 would be intended for patients with autoimmune diseases. Employing the protein database (BL2SEQ), we found that there are some homologous peptides between VP1u (aa 48 to 54-SLDNPLE; aa 117 to 122-KPGQVS) and β2GPI (aa 239 to 245-SLDGPEE; aa 52 to 57KPGYVS) [25]. In favor of the theory of molecular mimicry in the development of APS, affinity-purified anti-VP1 IgG from patients with chronic parvovirus B19 infection has been found to cross-react with
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human tissue antigens including CL [46]. In addition, naïve BALB/c mice were immunized with microbial pathogens that share structural homology with the TLRVYK hexapeptide, which is specially recognized by the pathogenic H-3 anti-β2-GPI antibodies, and has been proven to induce APS in experimental models [47]. These observations support the hypothesis of molecular mimicry mechanism as a triggering event in the development of aPL. However, further investigation may be needed to confirm this hypothesis. In the present study, we showed reactivity to CL always occurred in APS patients with autoantibodies against β2-GPI, but was not found in those without anti-β2-GPI antibodies. Our results were in accordance with the findings of Loizou et al. who showed that aCL binding in B19-infected patients is enhanced in the presence of extrinsic β2GPI, which bears a remarkable similarity to that found in SLE with APS [17]. A hydrophobic sequence at position 313–316 (Leu– Ala–Phe–Trp) in the fifth domain of β2-GPI has been demonstrated to be essential for the binding of CL [48]. Moreover, an epitope for aCL antibodies has been identified in the COOH-terminal (domain IV) of β2GPI by serial deletion [49]. Structure simulation and prediction of conformational cryptic epitopes of both β2-GPI and B19 VP1u will be crucial for further clarifying the relationships among induction of anti-β2-GPI and aCL antibodies by B19 VP1u. In conclusion, our experimental results indicate that there is a high frequency of recognition of B19-VP1u in APS patients, and B19-VP1u may be a causal link to aPL production. As shown in a study by Rayment et al. [50], incorporation of B19-VP1u IgG immunoblots into serological assays would be essential in APS patients. Although the sample size of this study was too small to obtain a definitive conclusion, our findings may provide a clue for understanding the roles of B19-VP1u in the production of aPL along with APS. Our findings not only increase knowledge about the immune response to B19 virus, but also offer a foundation for the development of novel therapeutic and preventive strategies [47,51]. In addition, awareness of probability of B19-related aPL and APS will allow clinicians to test for parvovirus B19 and avoid the use of aggressive immunosuppressive agents in B19-infected patients with APS. Acknowledgement This study was supported by grants NSC-97-2314-B040-009 and NSC-98-2314-B040-008-MY3 from the National Science Council, Taiwan, Republic of China. References [1] Cossart YE, Field AM, Cant B, Widdows D. Parvovirus-like particles in human sera. Lancet 1975;1:72–3. [2] Corcoran A, Doyle S. Advances in the biology, diagnosis and host-pathogen interactions of parvovirus B19. J Med Microbiol 2004;53:459–75. [3] Young NS, Brown KE. Parvovirus B19. N Engl J Med 2004;350:586–97. [4] Anderson S, Momoeda M, Kawase M, Kajigaya S, Young NS. Peptides derived from the unique region of B19 parvovirus minor capsid protein elicit neutralizing antibodies in rabbits. Virology 1995;206:626–32. [5] Modrow S, Dorsch S. Antibody responses in parvovirus B19 infected patients. Pathol Biol 2002;50:326–31. [6] Zádori Z, Szelei J, Lacoste MC, et al. A viral phospholipase A2 is required for parvovirus infectivity. Dev Cell 2001;1:291–302. [7] Dorsch S, Liebisch G, Kaufmann B, et al. The VP1 unique region of parvovirus B19 and its constituent phospholipase A2-like activity. J Virol 2002;76:2014–8. [8] Canaan S, Zádori Z, Ghomashchi F, et al. Interfacial enzymology of parvovirus phospholipases A2. J Biol Chem 2004;279:14,502–8. [9] Lu J, Zhi N, Wong S, Brown KF. Activation of synoviocytes by the secreted phospholipase A2 motif in the VP1-unique region of parvovirus B19 minor capsid protein. J Infect Dis 2006;193:582–90. [10] Filippone C, Zhi N, Wong S, et al. VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones. Virology 2008;374: 444–52. [11] Musiani M, Manaresi E, Gallinella G, Venturoli S, Zuffi E, Zerbini M. Immunoreactivity against linear epitopes of parvovirus B19 structural proteins. Immunodominance of the amino-terminal half of the unique region of VP1. J Med Virol 2000;60:347–52.
[12] Bönsch C, Kempf C, Ros C. Interaction of parvovirus B19 with human erythrocytes alters virus structure and cell membrane integrity. J Virol 2008;82:11,784–91. [13] Hsu TC, Tsay GJ. Human parvovirus B19 infection in patients with systemic lupus erythematosus. Rheumatology (Oxford) 2001;40:152–7. [14] Lehmann HW, von Landenberg P, Modrow S. Parvovirus B19 infection and autoimmune disease. Autoimmun Rev 2003;2:218–23. [15] Von Landenberg P, Modrow S. Human parvovirus B19 infection and antiphospholipid-syndrome: the two sides of one medal? J Vet Med B Infect Dis Vet Public Health 2005;52:353–5. [16] Von Landenberg P, Lehmann HW, Modrow S. Human parvovirus B19 infection and antiphospholipid antibodies. Autoimmun Rev 2007;6:278–85. [17] Loizou S, Cazabon JK, Walport MJ, Tait D, So AK. Similarities of specificity and cofactor dependence in serum antiphospholipid antibodies from patients with human parvovirus B19 infection and from those with systemic lupus erythematosus. Arthritis Rheum 1997;40:103–8. [18] Chou TN, Hsu TC, Chen RM, Lin LI, Tsay GJ. Parvovirus B19 infection associated with the production of anti-neutrophil cytoplasmic antibody (ANCA) and anticardiolipin antibody (aCL). Lupus 2000;9:551–4. [19] Von Landerberg P, Lehmann HW, Knoll A, Dorsch S, Modrow S. Antiphospholipid antibodies in pediatric and adult patients with rheumatic diseases are associated with parvovirus B19 infection. Arthritis Rheum 2003;48:1939–47. [20] Lehmann HW, Plentz A, Von Landenberg P, Muller-Godeffroy E, Modrow S. Intravenous immunoglobulin treatment of four patients with juvenile polyarticular arthritis associated with persistent parvovirus B19 infection and antiphospholipid antibodies. Arthritis Res Ther 2004;6:R1–6. [21] Kalt M, Gertner E. Antibodies to beta 2-glycoprotein I and cardiolipin with symptoms suggestive of systemic lupus erythematosus in parvovirus B19 infection. J Rheumatol 2001;28:2335–6. [22] Hughes GR, Harris NN, Gharavi AE. The anticardiolipin syndrome. J Rheumatol 1986;13:486–9. [23] Shoenfeld Y, Gharavi A, Koike T. β2GP-I in the antiphospholipid (Hughes') syndrome-from a cofactor to an autoantigen-from induction to prevention of antiphospholipid syndrome. Lupus 1998;7:503–6. [24] Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306. [25] Tzang BS, Tsay GJ, Lee YJ, Li C, Sun YS, Hsu TC. The association of VP1 unique region protein in acute parvovirus B19 infection and antiphospholipid antibody production. Clin Chim Acta 2007;378:59–65. [26] Tzang BS, Lee YJ, Yang TP, et al. Induction of antiphospholipid antibodies and antiphospholipid syndrome-like autoimmunity in naive mice with antibody against human Parvovirus B19 VP1 unique region protein. Clin Chim Acta 2007;382:31–6. [27] Wilson WA, Gharavi AE, Koike T, et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome. Arthritis Rheum 1999;42:309–11. [28] Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40: 1725–9. [29] National Center for Health Statistics. Health promotion and disease prevention, Unites States, 1985. (DHHS pub. No. PHS 88-1591). Washington, DC: US Government Printing Office; 1988. Vital and Health Statistic Series 10: Data from the National Health Survey no. 1630. [30] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [31] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350–4. [32] Gallinella G, Zuffi E, Gentilomi G, et al. Relevance of B19 markers in serum samples for a diagnosis of parvovirus B19-correlated diseases. J Med Virol 2003;71:135–9. [33] Kurtzman GJ, Cohen BJ, Field AM, Oseas R, Blaese RM, Young NS. Immune response to B19 parvovirus and an antibody defect in persistent viral infection. J Clin Invest 1989;84:1114–23. [34] Saikawa T, Anderson S, Momoeda M, Kajigaya S, Young NS. Neutralizing linear epitopes of B19 parvovirus cluster in the VP1 unique and VP1–VP2 junction regions. J Virol 1993;67:3004–9. [35] Musiani M, Manaresi E, Gallinella G, Venturoli S, Zuffi E, Zerbini M. Immunoreactivity against linear epitopes of parvovirus B19 structural proteins. Immunodominance of the amino-terminal half of the unique region of VP1. J Med Virol 2000;60:347–52. [36] Lehmann HW, Plentz A, Von Landenberg P, Küster RM, Modrow S. Different patterns of disease manifestations of parvovirus B19-associated reactive juvenile arthritis and the induction of antiphospholipid antibodies. Clin Rheumatol 2008;27:333–8. [37] Crowson AN, Magro CM, Dawood MR. A causal role for parvovirus B19 infection in adult dermatomyositis and other autoimmune syndromes. J Cutan Pathol 2000;27:505–15. [38] Hansen KE, Amason J, Bridges AJ. Autoantibodies and viral illness. Semin Arthritis Rheum 1998;27:263–71. [39] Dalekos GN, Kistis KG, Boumba DS, et al. Increased incidence of anti-cardiolipin antibodies in patients with hepatitis C is not associated with aetiopathogenic link to antiphospholipid syndrome. Eur J Gastroenterol Hepatol 2000;12:67–74. [40] Uthman IW, Gharavi AE. Viral infections and antiphospholipid antibodies. Semin Arthritis Rheum 2002;31:256–63. [41] Shoenfeld Y, Blank M, Cervera R, Font J, Raschi E, Meroni PL. Infectious origin of the antiphospholipid syndrome. Ann Rheum Dis 2006;65:2–6.
D.-Y. Chen et al. / Clinica Chimica Acta 411 (2010) 1084–1089 [42] Pierangeli SS, Harris EN. Clinical laboratory testing for the antiphospholipid syndrome. Clin Chim Acta 2005;357:17–33. [43] Shoenfeld Y, Blank M, Krause I. The relationship of antiphospholipid antibodies to infections—do they bind to infecting agents or may they even be induced by them? Clin Exp Rheumatol 2000;18:431–2. [44] Ballou WR, Reed JL, Noble W, Young NS, Koenig S. Safety and immunogenicity of a recombinant parvovirus B19 vaccine formulated with MF59C.1. J Infect Dis 2003;187:675–8. [45] Saikawa T, Anderson S, Momoeda M, Kajigaya S, Young NS. Neutralizing linear epitopes of parvovirus B19 cluster in the VP1-unique and VP1–VP2 junction regions. J Virol 1993;67:3004–9. [46] Lunardi C, Tiso M, Borgato L, et al. Chronic parvovirus B19 infection induces the production of anti-virus antibodies with autoantigen binding properties. Eur J Immunol 1998;28:936–48.
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[47] Blank M, Shoenfeld Y, Cabilly S, Heldman Y, Fridkin M, Katchalski-Katzir E. Prevention of antiphospholipid syndrome and endothelial cell activation by synthetic peptides. Proc Natl Acad Sci USA 1999;96:5164–8. [48] Mehdi H, Naqvi A, Kambon MI. A hydrophobic sequence at position 313–316 (Leu–Ala–Phe–Trp) in the fifth domain of apolipoprotein H (β2-GPI) is crucial for cardiolipin binding. Eur J Biochem 2000;267:1770–6. [49] Igarashi M, Matsuura E, Igarashi Y, et al. Human beta2-glycoprotein I as an anticardiolipin cofactor determined using mutants expressed by a baculovirus system. Blood 1996;87:3262–70. [50] Rayment FB, Crosdale E, Morris DJ, Pattison JR, Talbot P, Clare JJ. The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens. J Gen Virol 1990;71:2665–72. [51] Blank M, Shoenfeld Y, et al. Beta-2-glycoprotein-I, infections, antiphospholipid syndrome and therapeutic considerations. Clin Immunol 2004;112:190–9.
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
The association of anti-parvovirus B19-VP1 unique region antibodies with antiphospholipid antibodies in patients with antiphospholipid syndrome Der-Yuan Chen a,b,c, Bor-Show Tzang d,e,1, Yi-Ming Chen a,b, Joung-Liang Lan a,b,c, Chun-Chou Tsai f, Tsai-Ching Hsu f,g,⁎,1 a
Department of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan, ROC Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC School of Medicine, Chung Shan Medical University and Institute of Biomedical Science, National Chung-Hsing University, Taichung, Taiwan, ROC d Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, ROC e Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC f Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan, ROC g Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC b c
a r t i c l e
i n f o
Article history: Received 31 January 2010 Received in revised form 2 April 2010 Accepted 3 April 2010 Available online 10 April 2010 Keywords: Human parvovirus B19 (B19) VP1 unique region protein (VP1u) Antiphospholid antibody (aPL) Antiphospholipid syndrome (APS) Systemic lupus erythematosus (SLE)
a b s t r a c t Background: Human parvovirus B19 (B19) infection has been identified as a trigger of antiphospholipid syndrome (APS). However, the precise role of B19-VP1 unique region (VP1u) in patients with antiphospholipid syndrome remains unclear. Methods: IgM and IgG against B19-VP, and serum levels of antibodies directed against cardiolipin (CL), beta2glycoprotein-I (β2GPI) and phospholipid (PhL) were determined using ELISA in 45 APS patients. Humoral responses of anti-B19-VP1u were assessed by Western blot and B19 DNA was detected by nested PCR. Absorption experiments were performed using B19-VP1u protein to determine the binding specificity of antiphospholipid antibodies (aPL). Results: One and 18 of 45 APS patients had detectable levels of anti-B19-VP IgM and anti-B19-VP IgG, indicating recent and past infection respectively. All serum samples from APS patients with diagnostic pattern DNA−/IgM−/IgG+ had anti-B19-VP1u activity. APS patients with anti-B19-VP1u antibody had a 4fold increased risk for recurrent vascular thrombosis compared with those without anti-B19-VP1u antibody. The binding inhibition of CL, β2GPI, and PhL by absorption with B19-VP1u ranged from 31.4% to 91.1%, 0.8% to 59.8% and 20.2% to 72.1% respectively. Significantly higher inhibition to β2GPI by B19-VP1u absorption was observed in APS patients with anti-B19-VP1u antibody than in those without anti-B19-VP1u antibody. Conclusions: We show a close association of B19 infection with aPL production and suggest B19-VP1u may be of pathogenetic importance in some patients with APS. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Human parvovirus B19 (B19), which was firstly discovered by Cossart et al. in 1975 [1], is known as an important human pathogen that comprises a small non-enveloped particle enclosing a singlestranded linear 5.6-kb DNA genome [2,3]. The icosahedral capsid of B19 consists of two structural proteins, VP1 (83 kDa) and VP2 (58 kDa), which are identical except for the 227 amino acids at amino-terminal end of VP1-protein, the so-called VP1-unique region (VP1u) [4,5]. Additionally, B19-VP1u has been demonstrated to have phospholipase A2 (PLA2) activity, which is essential for its cytotoxi-
⁎ Corresponding author. Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan, 110 Section 1, Jianguo N. Road, Taichung 402, Taiwan, ROC. Tel.: + 886 4 24730022x11702; fax: + 886 4 23248172. E-mail address: [email protected] (T.-C. Hsu). 1 Dr. Bor-Show Tzang and Dr. Tsai-Ching Hsu contributed equally to this work. 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.04.004
city and infectivity [6–10]. Although B19-VP1 represents only 5% of the capsid, VP1u is critical for eliciting an appropriate and predominate immune response in both humans and animals [11,12]. B19 infection has been recognized as a cause or trigger of autoimmune diseases [13–15] and is associated with the production of various autoantibodies, including anti-β2-glycoprotein-I (antiβ2GPI) and anti-cardiolipin antibody (aCL) [16–20]. Many studies have demonstrated that a remarkable similarity exists in the specificity of antiphospholipid antibodies (aPL) between patients with B19 infection and those with systemic lupus erythematosus (SLE) [13–17]. Previous studies have reported that anti-β2GPI and aCL were detected in patients with B19 infection and SLE [13–17,21]. Moreover, aCL from patients with B19 infection increased their binding to antigens in the presence of β2GPI as a cofactor, similar to aCL from SLE patients, but unlike antibodies from patients with other viral infections [17]. These finding highlight the remarkable similarity in the aPL between SLE and B19 infection.
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The classical “Hughes syndrome”–antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies such as lupus anticoagulant (LA), aCL and anti-β2GPI, and is associated with vascular thrombosis, pregnancy-related morbidity or both [22–24]. B19 infection and APS have been reported to show similar symptoms leading to the hypothesis of a common pathogenic background [14–21]. The PLA2-like activity observed in the B19-VP1u may be related to the induction of aPL [19]. Our recent study indicated that autoantibodies against CL, β2GPI, and phospholipid (PhL) in sera from patients with B19 infection, were cross-reactive with B19-VP1u [25]. Moreover, significantly raised immunoglobulins against B19VP1u, CL, β2GPI and PhL were detected in sera of mice immunized with recombinant B19-VP1u protein, as well as striking thrombocytopenia, prolongation of aPTT, and autoantibodies against β2GPI and PhL were detected in mice treated with anti-B19-VP1u IgG [26]. Although these findings suggested a connection between anti-B19VP1u antibodies in manifestations of APS and development of autoimmunity, the relationship between anti-B19-VP1u antibodies and aPL in patients with APS is still obscure. 2. Materials and methods 2.1. Patients and sera Forty-five patients with APS [27] (36 females and 9 males, mean age 37.0 ± 13.3 y) were enrolled from Taichung Veterans General Hospital. The enrolled subjects with APS were classified into 2 groups: primary APS and SLE [28] with secondary APS, according to the clinical course by ACR criteria [27]. Patients with hepatitis B and C infections and other rheumatic diseases were excluded from this study. Patients were considered to be smokers if they ever smoked ≥100 cigarettes, which was determined using the 1985 Health Promotion Disease Prevention supplement of the Health Interview Survey [29]. Peripheral blood samples were collected into glass tubes that contained no additive or anticoagulant. The clotted blood samples were centrifuged at 1250 rpm for 10 min and the sera were collected and stored at −70 °C until examination. The Clinical Research Ethics Committee at Taichung Veterans General Hospital approved the study protocol, and informed consent was obtained from each participant. 2.2. Detection of IgM and IgG against B19-VP in serum from APS patients The IgM and IgG against B19-VP were analyzed by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (IBL-America, Minneapolis, MN). Serum samples were diluted 1:50 and 1:100 for the IBL-America IgM and IgG ELISA kit in the provided sample diluents (IBL-America) respectively. Standard, control, and diluted samples were included in each plate. The absorbance (O.D.) was read at the wavelength of 450/620 nm after adding 100 ml of stop solution. According to the manufacture's instruction and our experimental test, the sensitivity of anti-VP ELISA is 98 to 100%. Negative values range from 0 to 11 U (B19 IgM and B19 IgG). Positive results are N12 U (B19 IgM and B19 IgG). 2.3. Determination of IgG against B19-VP1u using immunoblotting Western blotting was performed as described in our previous report [25]. Protein samples were separated in 12.5% of SDS-PAGE [30] and electrophoretically transferred to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) according to the method of Towbin et al. [31]. After blocking with 5% non-fat dry milk in PBS for 30 min at room temperature, human or rabbit antiserum against B19VP1u was diluted with 5% non-fat dry milk in PBS, reacted with the nitrocellulose strips and then incubated for 1.5 h at room temperature. The strips were washed twice with PBS-Tween for 1 h and secondary antibody consisting of alkaline phosphatase conjugated
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goat anti-human or rabbit IgG antibodies was added. The substrate NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3 indolyl phosphate) was used to detect antigen–antibody complexes. 2.4. DNA extraction, PCR amplification, and detection of B19 genomes DNA was extracted using QIA Amp blood kit (QIAagen, Hidden, Germany) as directed by the manufacturer. Nested-PCR for the detection of B19 DNA in serum was performed as described previously [25], B19 positive and negative reference controls [32] were also included in each PCR reaction. The nested PCR was used as it eliminates non-specific background and thus gives a clearer final product. Because of high sensitivity of the nested PCR reaction, stringent precautions were taken and samples were tested at least 3 times to avoid the risk of false-positive results. 2.5. Preparation of recombinant human B19-VP1u protein Construction of B19-VP1u cDNA into pET-32a expression vector (Novagene, Cambridge, MA) was performed and the recombinant B19-VP1u protein was purified as described in our recent publication [25,26]. The purified recombinant B19-VP1u proteins were also analyzed by HPLC and the purity was nearly 98%. 2.6. Determination of levels of aCL, anti-b2GPI and anti-PhL Previous studies have demonstrated that B19-VP2 capsid protein is known to elicit the IgM response, whereas B19-VP1u protein is known to elicit the IgG response [4,33]. We used direct antigen-specific ELISA kits to detect aCL IgG (QUANTA LiteTM ACA IgG III), anti-b2GPI IgG (QUANTA LiteTM b2GPI IgG) (INOVA Diagnostics Inc., San Diego, CA), and anti-PhL IgG (Louisville APL Diagnostics Inc., GA) according to the manufacture's description. Results were expressed as IgG phospholipid (GPL) units or standard IgG units (SGU) using international reference material. For absorption analysis to assess the effect of binding inhibition, the sera were pre-incubated with 2 mmol/l of purified VP1u recombinant proteins for 1 h at 37 °C before ELISA kits (aCL, anti-b2GPI and anti-PhL) were performed. Negative values range from 0–20 GPL (aCL), 0–20 SGU (anti-b2GPI antibodies) or 0– 15 GPL (anti-PhL). Positive results are greater than 20 GPL (aCL), 20 SGU (anti-b2GPI), and 15 GPL (anti-PhL). 2.7. Statistical analyses Data were analyzed using SPSS 10.0 for windows (Chicago, IL). The χ2 test was used to determine significant differences in categorical variables among groups. The paired t test was used to analyze data for statistical significance. A binary logistic regression analysis was used to evaluate the effect of anti-B19-VP1u IgG on the occurrence of recurrent thrombosis. A p b 0.05 was considered to be statistically significant. 3. Results 3.1. Demographic data, clinical characteristics, and the presence of aPL in APS patients Forty-five patients with APS were classified according to the clinical course of their disease as follows: primary APS (n = 10) and SLE with secondary APS (n = 35). As illustrated in Table 1, the most commonly associated thrombosis was cerebral vascular accident in both primary APS patients (50%) and SLE patients with secondary APS (25.7%). No significant differences in age at study entry, duration of disease, proportion of female, number of thrombotic episodes, or thrombosis subtype were observed between primary APS patients and SLE patients with secondary APS. However, significantly higher
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Table 1 Characteristics of 45 patients with antiphospholipid syndrome (APS).a Characteristics
Primary APS (n = 10)
SLE with APS (n = 35)
Age at study entry (y) Duration of disease (y) Females Ever smoker Number of subjects with ≧2 thrombosis Thrombosis by subtype Cerebral vascular accident Livedo reticularis Deep vein thrombosis Peripheral arterial thrombosis Miscarriage late (≧1 in 2nd or 3rd trimester) Myocardial infarction Other thrombosis Used medications Corticosteroids Hydroxychlorquine Other immunosuppressive agents
35.0 ± 9.3 3.2 ± 4.2 8 (80.0%) 1 (10.0%) 4 (40.0%)
37.6 ± 14.3 6.6 ± 5.3 27 (77.1%) 3 (8.6%) 11 (31.4%)
5 (50.0%) 4 (40.0%) 2 (20.0%) 1 (10.0%) 1 (10.0%) 0 (0.0%) 3 (30.0%)
9 (25.7%) 8 (22.9%) 4 (11.4%) 4 (11.4%) 2 (5.7%) 3 (8.6%) 17 (48.6%)
4 (40.0%) 10 (100.0%) 2 (20.0%)
33 (94.2%)⁎ 34 (97.1%) 30 (85.7%)⁎
SLE: systemic lupus erythematosus. a Data were presented as mean ± SD or number (percentage). ⁎ p b 0.05, vs. patients with primary APS, determined by the paired t test.
proportion of usage of immunosuppressive agents including corticosteroids was found in SLE patients with secondary APS than in primary APS patients. 3.2. Results of B19 DNA, anti-B19 IgM and IgG in sera from APS patients Since parvovirus B19 cannot be grown routinely in vitro, B19 infection is generally diagnosed via direct detection of B19 DNA (virological) and by anti-B19 IgM and IgG (serological). Based on the advances in diagnosis of B19 infection [2,31], 1 (2.2%) and 18 (40.0%) of the 45 patients with APS had detectable anti-B19-VP IgM and anti-B19VP IgG, indicating recent infection and past infection respectively. Table 2 shows the diagnostic patterns of B19 infection in APS patients. Regarding the diagnostic patterns of 10 patients with primary APS, 4 (40%) of them had the DNA−/IgM−/IgG+ diagnostic pattern, 1 (10%) of them had the DNA+/IgM+/IgG+ diagnostic pattern, and 5 (50%) of them had the DNA−/IgM−/IgG− diagnostic pattern. In the SLE patients with APS, 14 (40%) of them have the DNA−/IgM−/IgG+ diagnostic pattern and 21 (60%) of them have the DNA−/IgM−/IgG− diagnostic pattern. There was no significant difference in the distribution of diagnostic patterns of B19 infection between primary APS patients and SLE patients with secondary APS. 3.3. Reactivity of IgG directed B19-VP1u in sera from APS patients The B19-VP1u is known to generate prolonged neutralizing immune response and the epitopes localized in VP1u are predomi-
Table 3 Characteristics of 45 patients with APS according to the presence of anti-B19-VPlu antibodies.a Characteristics
Anti-B19-VPlu (−) (n = 25)
Anti-B19-VP1u (+) (n = 20)
Age at study entry (y) Duration of disease (y) Females Number of subjects with ≧2 thrombosis Positivity for aCL Positivity for anti-β2GPI Positivity for aPhL Absorption inhibition degree, % Inhibition to CL/β2GPI/PhLb Inhibition to β2GPIb
37.5 ± 14.4 59.9 ± 59.0 18 (72.0%)
36.5 ± 12.1 83.7 ± 65.8 147 (85.0%)
5 (20.0%) 0 (0.0%) 9 (36.0%) 0 (0.0%)
10 (50.0%)⁎ 5 (25.0%)⁎ 10 (50.0%) 5 (25.0%)⁎
21.3 ± 11.3 21.3 ± 11.3
45.6 ± 22.3⁎⁎ 38.5 ± 17.0⁎
B19-VP1u: B19-VP1 unique region protein; aCL: anti-cardiolipin antibodies; antiβ2GPI: anti-beta2-glycoprotein-I antibodies; aPhL: antiphospholipid antibodies. a Data were presented as mean ± SD or number (percentage). b Among patients with aCL, anti-β2GPI, or anti-PhL antibodies. ⁎ p b 0.05, vs. patients with patients with anti-B19-VPlu (−), determined by the paired t test. ⁎⁎ p b 0.01, vs. patients with patients with anti-B19-VPlu (−), determined by the paired t test.
nantly linear [34,35]. To clarify the antigenicity of VP1u, this study investigated antibodies directed against B19-VP1u protein in serum samples from APS patients with different diagnostic patterns using Western blot analysis. As illustrated in Table 2, all of serum samples from primary APS patients with diagnostic pattern DNA−/IgM−/IgG+ had anti-B19-VP1u activity. The serum sample from 1 patient with primary APS who had diagnostic pattern DNA+/IgM+/IgG+ showed reactivity to anti-B19-VP1u. Only 1 primary APS patient with diagnostic pattern DNA−/IgM−/IgG− had a serum sample that showed reactivity to anti-B19-VP1u. All of serum samples from SLE patients with APS who had diagnostic pattern DNA−/IgM−/IgG+ (n = 14) showed reactivity to anti-B19-VP1u.
3.4. The association of B19 diagnostic patterns with aPL in APS patients As illustrated in Table 2, 1 of 4 (25%), 3 of 4 (75%), and 1 of 4 (25%) serum samples showed positivity for aCL, anti-β2GPI, and anti-PhL respectively in 4 patients with primary APS who had diagnostic pattern DNA−/IgM−/IgG+. The serum sample from 1 patient with primary APS who had diagnostic pattern DNA+/IgM+/IgG+ showed positivity for both aCL and anti-β2GPI. Among 14 SLE patients with secondary APS who had diagnostic pattern DNA−/IgM−/IgG+, 2 (14.3%), 6 (43.0%) and 3 (21.4%) serum samples showed positivity for aCL, anti-β2GPI, and anti-PhL respectively. Among 21 SLE patients with secondary APS who had diagnostic pattern DNA−/IgM−/IgG−, 8 (38.0%) serum samples showed positivity of anti-β2GPI, and no any serum sample showed positivity for aCL or anti-PhL.
Table 2 The diagnostic patterns of B19 infection, serology of anti-B19-VP1u, and antiphospholipid antibodies in 45 patients with antiphospholipid syndrome (APS).a Diagnostic patterns of parvovirus B19 infection Primary APS (n = 10) DNA−/IgM−/IgG− (n = 5) DNA−/IgM−/IgG+ (n = 4) DNA+/IgM+/IgG+ (n = 1) SLE with APS (n = 35) DNA−/IgM−/IgG− (n = 21) DNA−/IgM−/IgG+ (n = 14) DNA+/IgM+/IgG+ (n = 0)
Anti-B19-VP1u (n = 20)
Positivity of aCL
Positivity of anti-β2GPI
Positivity of anti-PhL
1 4 1
1 (25%) 1 (25%) 1 (100%)
1 (25%) 3 (75%) 1 (100%)
1 (25%) 1 (25%) 0 (0%)
0 14 0
0 (0%) 2 (14.3%) 0
8 (38%) 6 (43%) 0
0 (0%) 3 (21.4%) 0
SLE: systemic lupus erythematosus; B19-VP1u: B19-VP1 unique region protein; aCL: anti-cardiolipin antibodies; anti-β2GPI: anti-beta2-glycoprotein-I antibodies; anti-PhL: antiphospholipid antibodies. a Data were presented as number (percentage) among the diagnostic patterns of B19 infection.
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3.5. Clinical characteristics of APS patients with anti-B19-VP1u antibodies As shown in Table 3, APS patients with anti-B19-VP1u antibodies were more likely than those without anti-B19-VP1u antibodies to have recurrent thrombosis (50.0% vs. 20.0%, p b 0.05), positivity for aCL (25.0% vs. 0.0%, p b 0.05) and positivity for anti-PhL (25.0% vs. 0.0%, p b 0.05). However, no significant differences in age at study entry, duration of disease, proportion of female, or thrombosis subtype were observed between patients with and without anti-B19-VP1u antibodies. To further verify the clinical and laboratory significance, we used binary logistic regression analysis to investigate the effect of anti-B19-VP1u positivity on the occurrence of recurrent thrombosis and clinical features in APS patients. Compared with APS patients without anti-B19-VP1u antibody, those with anti-B19-VP1u antibody had significantly increased risk of recurrent vascular thrombosis, with an odds ratio of 4.000 (95% CI: 1.074–14.896). In addition, high sensitivity (100%) and negative predictive value (100%) of anti-B19VP1u IgG were observed for predicting the reactivity to CL and PhL in APS patients. However, there was no significant connection between the presence of anti-B19-VP1u IgG and clinical features in APS patients. 3.6. Absorption analysis using purified B19-VP1u for the sera from APS patients To further elucidate the role of B19-VP1u in the reactivity to CL, β2GPI and PhL in APS patients, absorption analysis was performed on the sera from 20 APS patients who had positive IgG aPL using ELISA. As illustrated in Fig. 1, a significant decrease in the levels of aCL, antiβ2GPI, and aPhL antibodies after absorption with B19-VP1u was observed in 20 patients with APS. The inhibition of reactivity to CL, β2GPI and PhL by absorption with purified B19-VP1u was 31.4–91.0%, 0.8–59.8% and 20.2–72.1% respectively. Among APS patients with positivity for aPL, significantly higher degree of inhibition to CL/ β2GPI/PhL by B19-VP1u absorption was observed in sera from those with anti-B19-VP1u antibodies than from those without anti-B19VP1u antibodies (45.6% vs. 21.3%, p b 0.05, Table 3). Moreover, significantly higher degree of inhibition to β2GPI by B19-VP1u absorption was observed in sera from those with anti-B19-VP1u antibodies when compared to those without anti-B19-VP1u antibodies (38.5% vs. 21.3%, p b 0.05, Table 3). No significant difference in the degree of inhibition to CL, β2GPI or PhL was found in sera from
Fig. 1. Changes in the levels of anti-cardiolipin antibodies (aCL), anti-beta2glycoprotein-I (anti-β2GPI), and antiphospholipid antibodies (aPhL) in 20 patients with antiphospholipid syndrome. Data are expressed as median levels (the 25th to the 75th percentile). *p b 0.05, #p b 0.001, vs. before absorption, determined by the Wilcoxon signed rank test.
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patients with primary APS when compared to SLE patients with APS. Additionally, the inhibition of reactivity to CL, β2GPI, and PhL by absorption with 10 μg of B19-NS1 was not found in patients with APS (data not shown). 4. Discussion Consistent with the results of previous studies [14–21], our data show an association of parvovirus B19 infection with the presence of aPL in patients with APS. The presence of anti-B19-VP1u IgG was closely associated with the reactivity to CL and PhL. We also showed that anti-B19-VP IgM may have disappeared but B19 infection can be still be recognized by B19-VP1u IgG immunoblots in APS patients. In addition, our results showed lower positive rates of B19-VP1u IgG in SLE patients with APS than in those with primary APS (40% vs. 60%). The true prevalence of B19 infection in SLE patients may be underestimated as the immunosuppression associated with this disease and the usage of immunosuppressive agents that can inhibit reactivity to B19 infection [36,37]. Many viral infections may be accompanied by the production of aPL [15,16,18,19,38,39]. The widely held belief that the majority of infectious aPLs are usually transient and are not associated with thrombosis [39,40] has been challenged by several reports [17,41–43]. Our previous study showed that the BALB/c mice treated with antiB19-VP1u IgG developed APS-like autoimmunity [26]. In the present study, the presence of anti-B19-VP1u antibodies was closely associated with recurrent vascular thrombosis. Our results and those of previous studies indicate that an increase of aPL may be accompanied by clinical manifestations of APS in B19-infected subjects. However, further studies are required to identify B19 infection responsible for the production of the pathogenic aPL along with APS manifestations. Although APS is known to be an important autoimmune manifestation and has been associated with B19 infection [14–21], the precise role of B19-VP1u in the induction of APS is still obscure. Recent studies indicate that B19-VP1u is linked to PLA2-like activity and is critical for eliciting production of autoantibodies [6–11]. Our results showed that significantly higher seropositivity for aPL was observed in APS patients with anti-B19-VP1u antibodies when compared to those without anti-B19-VP1u antibodies, supporting the recent findings [6–11,17]. To clarify the relationship between B19VP1u and aPL production, we used purified B19-VP1u for absorption analysis of sera from APS patients. We showed an inhibition of binding to CL, β2GPI, and PhL by absorption with B19-VP1u was 31.4 to 91.0%, 0.8 to 59.8% and 20.2 to 72.1% respectively. Our results suggest that similar conformation or epitopes may exist between B19-VP1u and phospholipid antigens. The partially reduced reactivity to CL, β2GPI and PhL after absorption with B19-VP1u suggests that aPL production may be related to the underlying B19 infection in APS patients. Interestingly, significantly higher degree of inhibition was found in sera from patients with antibodies against both B19-VP1u and CL/ β2GPI/PhL when compared to those without B19-VP1u antibodies. Moreover, significantly higher degree of inhibition to β2GPI by B19VP1u absorption was observed in sera from those with anti-B19-VP1u antibodies when compared to those without anti-B19-VP1u antibodies (Table 3). A well-tolerated vaccine consisting of 25% B19-VP1 and 75% B19-VP2 could elicit a strong humoral response [44], and immunoglobulin specific for the B19-VP1u offer life-long protection against re-infection [45]. Our findings in support of a critical role of B19-VP1u in the pathogenesis of APS suggest a novel vaccine for B19 would be intended for patients with autoimmune diseases. Employing the protein database (BL2SEQ), we found that there are some homologous peptides between VP1u (aa 48 to 54-SLDNPLE; aa 117 to 122-KPGQVS) and β2GPI (aa 239 to 245-SLDGPEE; aa 52 to 57KPGYVS) [25]. In favor of the theory of molecular mimicry in the development of APS, affinity-purified anti-VP1 IgG from patients with chronic parvovirus B19 infection has been found to cross-react with
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human tissue antigens including CL [46]. In addition, naïve BALB/c mice were immunized with microbial pathogens that share structural homology with the TLRVYK hexapeptide, which is specially recognized by the pathogenic H-3 anti-β2-GPI antibodies, and has been proven to induce APS in experimental models [47]. These observations support the hypothesis of molecular mimicry mechanism as a triggering event in the development of aPL. However, further investigation may be needed to confirm this hypothesis. In the present study, we showed reactivity to CL always occurred in APS patients with autoantibodies against β2-GPI, but was not found in those without anti-β2-GPI antibodies. Our results were in accordance with the findings of Loizou et al. who showed that aCL binding in B19-infected patients is enhanced in the presence of extrinsic β2GPI, which bears a remarkable similarity to that found in SLE with APS [17]. A hydrophobic sequence at position 313–316 (Leu– Ala–Phe–Trp) in the fifth domain of β2-GPI has been demonstrated to be essential for the binding of CL [48]. Moreover, an epitope for aCL antibodies has been identified in the COOH-terminal (domain IV) of β2GPI by serial deletion [49]. Structure simulation and prediction of conformational cryptic epitopes of both β2-GPI and B19 VP1u will be crucial for further clarifying the relationships among induction of anti-β2-GPI and aCL antibodies by B19 VP1u. In conclusion, our experimental results indicate that there is a high frequency of recognition of B19-VP1u in APS patients, and B19-VP1u may be a causal link to aPL production. As shown in a study by Rayment et al. [50], incorporation of B19-VP1u IgG immunoblots into serological assays would be essential in APS patients. Although the sample size of this study was too small to obtain a definitive conclusion, our findings may provide a clue for understanding the roles of B19-VP1u in the production of aPL along with APS. Our findings not only increase knowledge about the immune response to B19 virus, but also offer a foundation for the development of novel therapeutic and preventive strategies [47,51]. In addition, awareness of probability of B19-related aPL and APS will allow clinicians to test for parvovirus B19 and avoid the use of aggressive immunosuppressive agents in B19-infected patients with APS. Acknowledgement This study was supported by grants NSC-97-2314-B040-009 and NSC-98-2314-B040-008-MY3 from the National Science Council, Taiwan, Republic of China. References [1] Cossart YE, Field AM, Cant B, Widdows D. Parvovirus-like particles in human sera. Lancet 1975;1:72–3. [2] Corcoran A, Doyle S. Advances in the biology, diagnosis and host-pathogen interactions of parvovirus B19. J Med Microbiol 2004;53:459–75. [3] Young NS, Brown KE. Parvovirus B19. N Engl J Med 2004;350:586–97. [4] Anderson S, Momoeda M, Kawase M, Kajigaya S, Young NS. Peptides derived from the unique region of B19 parvovirus minor capsid protein elicit neutralizing antibodies in rabbits. Virology 1995;206:626–32. [5] Modrow S, Dorsch S. Antibody responses in parvovirus B19 infected patients. Pathol Biol 2002;50:326–31. [6] Zádori Z, Szelei J, Lacoste MC, et al. A viral phospholipase A2 is required for parvovirus infectivity. Dev Cell 2001;1:291–302. [7] Dorsch S, Liebisch G, Kaufmann B, et al. The VP1 unique region of parvovirus B19 and its constituent phospholipase A2-like activity. J Virol 2002;76:2014–8. [8] Canaan S, Zádori Z, Ghomashchi F, et al. Interfacial enzymology of parvovirus phospholipases A2. J Biol Chem 2004;279:14,502–8. [9] Lu J, Zhi N, Wong S, Brown KF. Activation of synoviocytes by the secreted phospholipase A2 motif in the VP1-unique region of parvovirus B19 minor capsid protein. J Infect Dis 2006;193:582–90. [10] Filippone C, Zhi N, Wong S, et al. VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones. Virology 2008;374: 444–52. [11] Musiani M, Manaresi E, Gallinella G, Venturoli S, Zuffi E, Zerbini M. Immunoreactivity against linear epitopes of parvovirus B19 structural proteins. Immunodominance of the amino-terminal half of the unique region of VP1. J Med Virol 2000;60:347–52.
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