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Evaluation of anti-parvovirus B19 activity in sera by assay using quantitative polymerase chain reaction
Journal of Virological Methods 107 (2003) 81 /87 www.elsevier.com/locate/jviromet
Evaluation of anti-parvovirus B19 activity in sera by assay using quantitative polymerase chain reaction Takako Saito a, Yasuhiko Munakata b,*, Yi Fu b, Hiroshi Fujii b, Takao Kodera b, Eiji Miyagawa c, Keiko Ishii a, Takeshi Sasaki b b
a Department of Molecular Diagnostics, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan Department of Rheumatology & Hematology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan c New Product Planning and Development Division, Fujirebio Inc., 51 Komiya-cho, Hachioji-shi, Tokyo 192-0031, Japan
Received 19 June 2002; received in revised form 24 August 2002; accepted 27 August 2002
Abstract Human parvovirus B19 (B19) infects cells of erythroid lineage. Production of neutralizing antibodies (Abs) is indispensable for recovery from B19-related disease state. In this study, we used a convenient method to measure neutralizing activities in human sera by using a real-time quantitative PCR based assay. Erythroid cell line KU812Ep6 was incubated with test sera before infection with B19 virus. The copy number of B19-DNA in cultures was decreased in the presence of the sera from patients who recovered from acute B19 infection, whereas no decrease in B19-DNA was in cultures incubated with sera from healthy volunteers who had no B19 infection. The decrease in B19-DNA copy number was calculated and the inhibition percentage was expressed as neutralizing activity to B19. A clinical study showed that the levels of neutralizing ability were high in patients who recovered soon after acute B19 infection, but were low in some patients with a prolonged clinical course for recovery from B19 infection. This method is simple and convenient compared with methods described previously, showing its usefulness to evaluate the neutralizing activity to B19. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Neutralizing antibody; Human parvovirus B19; Quantitative PCR; KU812Ep6
1. Introduction Human parvovirus B19 (B19) infection is most common in children. Acute B19 infection in children causes erythema infectiousum and recovery from the disease is spontaneous with no serious complications (Brown et al., 1994). However, B19 infection in adults often causes serious disease conditions, such as aplastic crisis (Kelleher et al., 1983), hydrops fetalis in pregnant women (Anand et al., 1987), and polyarthritis resembling rheumatoid arthritis (Woolf et al., 1989; Takahashi et al., 1998; Stahl et al., 2000). Persistent infection has also been reported in humans (Kerr et al., 1995). Appearance of the neutralizing IgG antibodies (Abs) specific against B19 is generally considered a leading pathological feature before recovery. In B19 infection,
* Corresponding author. Tel.: /81-22-717-7165; fax: /81-22-7177168 E-mail address: [email protected] (Y. Munakata).
where a cellular immune response is not demonstrable, a humoral response seems to be a crucial factor for recovery from disease (Kurtzman et al., 1989; Sato et al., 1991). Studies on anti-B19 antibodies have indicated the presence of anti-B19 antibodies with a wide neutralizing ability (Bostic et al., 1999; Gigler et al., 1999). This indicates that assessment of B19 neutralizing activity of the serum may be important to monitor the infection condition of patients with B19 infection. So far, neutralizing activity in sera has been measured as its ability to inhibit erythrocyte colony formation by using bone marrow cells in an in vitro assay (Takahashi et al., 1991). The clinical use of this assay, however, has been hampered because obtaining bone marrow cells for each assay is not easy. The test itself also has problems of accuracy and consistency as the bone marrow used often contains precursor cells of different lineages at various stages of differentiation. In other words, the target cell population for B19 infection varies from assay to assay in the colony formation assay.
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To overcome these difficulties, we developed a novel assay to measure neutralizing activity against B19 in sera that is easy to perform, is accurate and is reliable. In this study, an assay is described to measure B19 neutralizing activity in sera by using real-time PCR based on TaqMan chemistry (Gibson et al., 1996; Heid et al., 1996). The assay was done by in vitro culture of the KU812Ep6 erythroid cell line that is highly susceptible to B19 infection (Miyagawa et al., 1999). We evaluated also the disease status and followed the clinical course of the patients with acute B19 infection by using this assay that compares the value of IgG antiB19 antibody measured by enzyme-linked immunosorbent assay (ELISA).
2. Materials and methods 2.1. Serum samples Serum samples were obtained from healthy volunteers (YM, YO, SM, MS, SI, NK, TS) and patients with acute B19 infection (including IM), and six patients (including OA, GF) who showed a prolonged clinical course for recovery from B19 infection. All patients were examined at the Rheumatology and Hematology Clinics of Tohoku University Hospital. The serum samples were kept frozen at /80 8C until use. All samples were obtained following informed consents for performing polymerase chain reaction (PCR) and ELISA of IgM and IgG anti-B19 antibodies. 2.2. Measurement of anti-B19 antibodies The serum samples were tested for IgM and IgG antiB19 antibodies by using a commercial indirect ELISA kit. The sera were tested by using a Parvo IgM-EIA ‘SEIKEN’ kit and a Parvo IgG-EIA ‘SEIKEN’ kit (Matsunaga et al., 1995) (Denka Seiken Inc., Tokyo, Japan) according to the manufacturer’s instructions and by using the microplate reader MPR-A4 (TOSOH Inc., Tokyo, Japan). The titer of anti-B19 antibodies was expressed as an index according to the instructions provided, and an index number over 0.8 was regarded as positive for the presence of anti-B19 Abs. 2.3. Human parvovirus B19 Serum from patient FU with acute B19 infection was used for an in vitro infection assay. The serum contained 2.5 /1014 copies of B19-DNA/ml, and no anti-B19 antibodies was detected by using ELISA. The serum from the healthy individual MO, containing neither B19-DNA nor anti-B19 antibody, was used as a negative control.
2.4. Monoclonal anti-B19 antibody Mouse monoclonal anti-B19 antibody, WP181, was obtained from BALB/C mice immunized with B19 capsid protein. The B19 capsid protein (Matsunaga et al., 1995) was purchased from Denka Seiken Inc. (Tokyo, Japan). 2.5. Construction of a B19-DNA plasmid To calculate the number of VP1/B19-DNA equivalents in the PCR- amplified sample, full length B19 genomic DNA amplified from a patient’s serum by PCR, was inserted into a pGEM-T vector (Promega, Madison, WI). Plasmid DNA was extracted from recombinant bacteria and was purified. The original copy number of the B19-DNA vector was found by comparing the optical density values with the molecular weight of different dilutions of this plasmid. 2.6. Cell culture for the neutralization test The human erythroid cell line KU812Ep6 (Miyagawa et al., 1999) was cultured with RPMI medium containing 10% fetal bovine serum (FBS) and 6 IU/ml of erythropoietin (Kirin Brewery Inc., Tokyo, Japan) at 37 8C in 5% CO2. KU812Ep6 (100,000) cells were preincubated in 500 ml of the medium together with the test serum or WP181 at 37 8C for 30 min, and then 0.50 ml of the serum from FU or MO was added and the cells were further incubated for 2 h. After washing three times with PBS, the cells were cultured for 48 h, and then cells underwent a PCR assay. 2.7. Quantitative PCR assays to detect B19-DNA DNA was prepared from KU812Ep6 cells infected with B19 by using the conventional phenol-chloroform extraction method. A quantitative PCR assay of samples amplified the VP1-region of B19-DNA by using ‘TaqMan PCR reagent kit’ (Roche, Branchburg, NJ). The reaction used a volume of 50 ml consisting of 0.5 mg of sample DNA, 400 mM of dUTP and 200 mM of dATP, dCTP and dGTP, 3.5 mM MgCl2, 200 nM of each primer, 100 nM of TaqMan probe, 0.01 U/ml of AmpErase UNG, 0.025 U/ml of AmpliTaq Gold, and TaqMan buffer A. The sequence of primers, amplifying the nt.2598 /2752 region of VP1 in B19-DNA, used in this study were: forward primer, 5?-CCCTAGAAAACCCATCCTCTGTG-3?; reverse primer, 5?AGGTTCTGCATGACTGCTACTGG-3?. The sequence of the detection probe labeled with fluorescent FAM corresponding to the nt 2692 /2718 region of VP1 of B19-DNA was 5?-TCATGGACAGTTATCTGACCACCCCCA-3?. The thermal cycling conditions were 2 min at 50 8C, followed by 10 min at 95 8C. Thermal
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cycling followed with 40 cycles of 95 8C for 15 s and 60 8C for 1 min. All reactions were done in an ABI/ PRISM 7700 sequence detector system, which contains a GeneAmp PCR system 9600. Each sample was analyzed in duplicate, and the DRn and CT average was calculated from the values from each reaction. Controls were run in duplicates to calibrate a standard calibration curve for each set of analyses. The degree of inhibition of the B19-DNA in the presence of anti-B19 serum was expressed as the inhibition ratio (% inhibition) and was calculated as: 100 /(copy number of B19-DNA without test serum/ copy number of B19-DNA with test serum)/copy number of B19-DNA without test serum. 2.8. Statistical analysis The correlation coefficient was calculated by the program of Cricket graph 1.3.
3. Results 3.1. B19 infection of KU812Ep6 erythroid cells measured by quantitative PCR We reported previously that B19 can be easily propagated in KU812Ep6 cells (Miyagawa et al., 1999). First, it was determined whether an in vitro infection system using KU812Ep6 is suitable for quantitative-PCR. Control plasmids containing full length B19-genomic DNA were diluted in water (105 /10 copies in each sample) and a standard curve was established with CT values plotted against the logarithm of the copy number. The relationship was between the CT and the copy number of control standard B19-DNA was linear (data not shown). The copy number of B19-DNA increased in the KU812Ep6 cells when they were incubated with the serum from FU that contained infective B19 virus, whereas B19-DNA was not detectable in the KU812Ep6 cells when they were incubated with the serum from MO that did not contain B19 virus (Fig. 1A). The increase in B19-DNA in the KU812Ep6 cells was inhibited by the mouse monoclonal IgG antiB19 Ab, WP181, in dose dependently. The antibody concentrations of over 1 mg/ml inhibited effectively the amplification as the increase in B19-DNA was eliminated (Fig. 1B). B19-DNA increased rapidly during 24/ 60 h of incubation. After this period of incubation, the increase in B19-DNA copy number reached a plateau in this culture system. 3.2. Measuring neutralizing activity against B19 in sera We examined the effect of human sera containing anti-B19 IgG Abs on B19-DNA replication in the
Fig. 1. Detection of B19-DNA in the KU812Ep6 cell line. (A) The copy number of B19-DNA was quantitated by amplifying the VP1 region of B19-DNA. Sample DNA extracted from KU812Ep6 cells were incubated with FU serum (m) or MO serum (k) at each time indicated, and underwent quatitative PCR. (B) The inhibitory effect of monoclonal anti-B19 Ab on B19 infection was examined by quantitative PCR at 48 h after infection. Sample DNA was extracted from KU812Ep6 cells and was incubated with FU serum in the presence of anti-B19 Ab, WP181, at the concentration indicated.
KU812Ep6 cells. When serum from the healty volunteer YM, who had recovered from acute B19 infection, was added to the KU812Ep6 culture system at a dilution of 1:500, the increase in B19-DNA was inhibited until 42 hr of incubation. B19-DNA slightly increased after 42 h of incubation in the presence of serum containing IgG antiB19 Abs (Fig. 2A). The inhibition percentage dramatically increased at 24 h of incubation. The inhibition percentage was optimal at a serum dilution of 1:500 (Fig. 2B). The inhibition percentages at concentrations higher than 1:500 was less than at 1:500. The inhibition percentages at dilutions higher than 1:500 decreased proportionate to the dilution of the test serum as in the case of WP181. The correlation was close between the index of IgG anti-B19 antibodies in the sera from healthy individuals and patients with acute B19 infec-
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Fig. 3. Correlation between anti-B19 IgG Ab and neutralizing ability against B19 in the sera. The titer of anti-B19 IgG Ab measured by ELISA was expressed as an index on the horizontal axis. Then neutralizing ability against B19 in the serum measured by our assay was expressed as the inhibition percentage on the vertical axis. m, samples from healthy individuals and patients from acute B19 infection and k, samples from patients who showed a prolonged clinical course for recovery from acute B19 infection.
normal volunteers, who had no B19 infection, were negative for both IgM and IgG anti-B19 Abs and showed no effect on the copy number of B19-DNA in KU812Ep6 cells (Table 1). However, the neutralizing activity was markedly low in sera from patients who showed a prolonged clinical course for recovery from B19 infection despite the presence of IgG anti-B19 antibodies (Fig. 3). Fig. 2. Effect of serum containing neutralizing anti-B19 Abs on B19 infection of the KU812Ep6 cell line. (A) The copy number of B19DNA and the inhibitory effect of anti-B19 serum (YM) on B19 infection were measured by using the neutralization test. Sample DNA was extracted at each time from KU812Ep6 cells and was incubated with FU serum in the presence (k) or absence (m) of YM serum containing anti-B19 IgG Abs. The inhibition percentage at each time are numbers at the top of the panel. (B) The inhibitory effect of antiB19 serum (YM) on B19 infection was examined at each dilution indicated at 48 h after infection.
tion and the inhibition of B19 replication (Fig. 3). The correlation coefficient was 0.824. Also, sera from Table 1 Neutralizing activity of test sera from normal volunteers who had no B19 infection Donors
YO SM MS SI NK TS
Index of anti-B19 Abs IgM
IgG
0.0 0.0 0.0 0.0 0.0 0.0
0.2 0.4 0.2 0.2 0.0 0.0
Inhibition of B19-DNA replication (%)
0.0 0.0 0.0 0.0 0.0 0.0
3.3. Neutralizing activity against B19 in serum measured during disease recovery In order to evaluate the usefulness of this assay in association with the clinical course of B19-related diseases, we measured the level of anti-B19 antibodies and the neutralizing activities of the sera obtained sequentially from the adult patient with acute B19 infection. In patient IM with erythema infectiousum, the samples from the early phase of infection with severe symptoms of polyarthralgia, skin eruption and cervical lymphadenopathy showed a high titer of IgM anti-B19 antibody. The recovery of symptoms corresponded to the increase in neutralizing activity against B19 in the serum (Fig. 4A). However, patient OA, who was febrile status for more than 3 months, delayed the recovery from acute B19 infection, (Fig. 4B); IgG anti-B19 Ab was detected in this patient during the period of viremia. However, the neutralizing activity in the serum was not detectable in this period, and the patient suffered from fever and skin eruption. After the appearance of neutralizing activity at a detectable level, the symptoms completely disappeared, and the neutralizing activity increased. In patient GF, symptoms relating to B19
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Fig. 4. Anti-B19 Abs in the clinical course of patients with B19 infection. Sample sera were assayed at each time indicated (A) from IM, (B) from OA and (C) from GF. k, titers of anti-B19 IgM Ab measured by ELISA and m, titers of anti-B19 IgG Ab measured by ELISA. The neutralizing activity against B19 in serum was expressed as the inhibition percentage in solid bars and the percentage is shown above the bars.
infection persisted for several months in spite of the presence of IgG anti-B19 Ab (Fig. 4C). Intravenous administration of g-globulin that contained anti-B19 neutralizing activity relieved this patient from B19relating symptoms.
4. Discussion B19 infection is self-limiting in most cases and the recovery from active infection by B19 is generally thought to be dependent on the production of IgG
anti-B19 antibodies (Kurtzman et al., 1989; Erdman et al., 1991; Sato et al., 1991). In virus neutralization, IgG and IgA antibodies appear to have a role in the resistance to natural infection by the nasopharyngeal route (Erdman et al., 1991). In persistent B19 infection, impairment in the production of anti-B19 antibodies with high avidity has been suggested (Kurtzman et al., 1989; Kerr et al., 1995). We reported previously two cases developing rheumatoid arthritis from acute B19 infection (Murai et al., 1999) where we detected B19DNA in their bone marrow even though IgG anti-B19 antibodies were present in the serum. These results
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Fig. 4 (Continued)
suggest that evaluation of the neutralizing activity against B19, which may be contributed by antibodies of all isotypes in the serum, is important to monitor the infection state and the patient care. The assay used to measure the neutralizing activity of the sera is the measurement of the inhibition of erythrocyte colony formation by bone marrow cells (Takahashi et al., 1991). Bostic et al. (1999) reported a sensitive test to measure B19 neutralization of sera by using a RT-PCR based system. In this method, the test serum needs to be diluted to quantify the neutralizing activity. However, this may be inconvenient for practical use, because it requires several assays for one specimen. For the followup of patients with B19 infection, evaluating the disease state by absolute values of the neutralizing activity in the serum is useful. The transcription of RNA occurs without viral replication (Liu et al., 1992; Pallier et al., 1997; Brunstein et al., 2000; Gallinella et al., 2000), which may be responsible for variability of the results. In this study, we developed a novel assay to quanitify the neutralizing activity against B19 in serum based on real-time PCR on B19-DNA by using an in vitro culture system of the KU812Ep6 erythroid cell line. KU812Ep6 is a high B19-infective clone that is cloned by limiting dilution from the B19-susceptible cell line KU812 by using a medium containing erythropoietin (Miyagawa et al., 1999). B19 replicates in this cell line and the infectivity are found by suppression of colony formation by the erythroid colony forming unit (Miyagawa et al., 1999). Using this cell line, we quantitated the neutralization effect of the test serum that inhibited B19DNA replication in the KU812Ep6 cells. In this assay, we incubated KU812Ep6 cells with test serum before being challenged with the virus, because it was intended
to reconstitute the physiological condition in vivo as well. The target cells for B19 infection in vivo may be protected by pre-existing anti-B19 antibodies in serum from invading B19. The dilution of the test sera and incubation time are important to minimize the effect of cytokines and other serum components. Some serum components, such as cytokines, interfere with viral infection (Pasquetto et al., 2002). In the case of influenza A virus, serum amyloid P inhibits its infection (Horvath et al., 2001). The expression of the viral receptor is possibly affected by the serum component (Wang et al., 2002). The serum concentration in the assay should be lower as possible. The dilution of the sera was found to be 1:500, as optimal neutralization was observed at higher than this dilution. We found the incubation time of 48 h, because the maximum inhibitory effect was during 30 /48 h. In sera from healthy volunteers who recovered from acute B19 infection, we detected high titers of IgG anti-B19 antibodies and neutralizing activity against B19 (Fig. 4A). However, in sera from individuals who had no B19 infection, neither IgG antiB19 antibodies nor neutralizing activity was detected against B19. In healthy individuals who recovered from acute B19 infection, neutralization of B19 evaluated by this assay and the level of IgG anti-B19 antibodies measured by ELISA showed a good correlation. Important may be that the neutralizing ability was negative or low at the initial period of B19 infection in some who had a prolonged clinical course (Fig. 4B,C). The patient symptoms disappeared when the levels of neutralizing ability increased in the serum, indicating the importance of monitoring the evaluation of neutralizing activity against B19 infection in a clinical course. Why a low or no neutralizing ability against B19 instead of a high titer
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of IgG anti-B19 antibodies were in patients OA and GF is unknown. However, we suggest that this may be due to impairment of the antigen presentation system in hosts as with impaired vaccination of hepatitis B virus in some cases (Hatae et al., 1992). The antigenic peptide that efficiently induced neutralizing antibodies may hardly be recognized in the host immune system. In summary, a novel assay was developed to measure the B19 virus neutralizing activity of the sera by using quantitative-PCR and an in vitro culture system of the KU812Ep6 erythroid cell line. By using this assay, it may be possible to find the state of B19 infection more precisely, which may help to clarify the clinical state of persistent infection of B19.
Acknowledgements This work was supported by grant-in aid for scientific research (A) from the Ministry of Education, Science, Sports and Culture in Japan, and by grants to T.S. awarded from the Kurozumi Medical Foundation. Informed consent was obtained from all patients in this study.
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Heid, C.A., Stevens, J., Livak, K.J., Williams, P.M., 1996. Real time quantitative PCR. Genome Res. 6, 986 /994. Horvath, A., Andersen, I., Junker, K., Lyck Fogh-Schultz, B., Holm Nielsen, E., Gizurarson, S., Andersen, O., Karman, J., Rajnavolgyi, E., Erdei, A., Svehag, S.E., 2001. Serum amyloid P component inhibits influenza A virus infections: in vitro and in vivo studies. Antiviral Res. 52, 43 /53. Kelleher, J.F., Luban, N.L., Mortimer, P.P., Kamimura, T., 1983. Human serum ‘parvovirus’: a specific cause of aplastic crisis in children with hereditary spherocytosis. J. Pediatr. 102, 720 /722. Kerr, J.R., Curran, M.D., Moore, J.E., Coyle, P.V., Ferguson, W.P., 1995. Persistent parvovirus B19 infection. Lancet 345, 1118. Kurtzman, G.J., Cohen, B.J., Field, A.M., Oseas, R., Blaese, R.M., Young, N.S., 1989. Immune response to B19 parvovirus and an antibody defect in persistent viral infection. J. Clin. Invest. 84, 1114 /1123. Liu, J.M., Green, S.W., Shimada, T., Young, N.S., 1992. A block in full-length transcript maturation in cells nonpermissive for B19 parvovirus. J. Virol. 66, 4686 /4692. Matsunaga, Y., Takeda, N., Yamazaki, S., Kamata, K., Kurosawa, D., 1995. Seroepidemiology of human parvovirus B19 using recombinant VP1/VP2 particle antigen. Kansenshogaku Zasshi 69, 1371 /1375(in Japanese with English abstract). Miyagawa, E., Yoshida, T., Takahashi, H., Yamaguchi, K., Nagano, T., Kiriyama, Y., Okochi, K., Sato, H., 1999. Infection of the erythroid cell line, KU812Ep6 with human parvovirus B19 and its application to titration of B19 infectivity. J. Virol. Methods 83, 45 / 54. Murai, C., Munakata, Y., Takahashi, Y., Ishii, T., Shibata, S., Muryoi, T., Funato, T., Nakamura, M., Sugamura, K., Sasaki, T., 1999. Rheumatoid arthritis after human parvovirus B19 infection. Ann. Rheum. Dis. 58, 130 /132. Pallier, C., Greco, A., Junter, J.L., Saib, A., Vassias, I., Morinet, F., 1997. The 3’ untranslated region of the B19 parvovirus capsid protein mRNAs inhibits its own mRNA translation in nonpermissive cells. J. Virol. 71, 9482 /9489. Pasquetto, V., Wieland, S.F., Uprichard, S.L., Tripodi, M., Chisari, F.V., 2002. Cytokine-sensitive replication of hepatitis B virus in immortalized mouse hepatocyte cultures. J. Virol. 76, 5646 /5653. Sato, H., Hirata, J., Furukawa, M., Kuroda, N., Shiraki, H., Maeda, Y., Okochi, K., 1991. Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19. J. Virol. 65, 1667 /1672. Stahl, H.D., Pfeiffer, R., von Salis-Soglio, G., Emmrich, F., 2000. Parvovirus B19-associated mono- and oligoarticular arthritis may evolve into a chronic inflammatory arthropathy fulfilling criteria for rheumatoid arthritis or spondylarthropathy. Clin. Rheumatol. 19, 510 /511. Takahashi, M., Koike, T., Moriyama, Y., Shibata, A., 1991. Neutralizing activity of immunoglobulin preparation against erythropoietic suppression of human parvovirus [letter]. Am. J. Hematol. 37, 68. Takahashi, Y., Murai, C., Shibata, S., Munakata, Y., Ishii, T., Ishii, K., Saitoh, T., Sawai, T., Sugamura, K., Sasaki, T., 1998. Human parvovirus B19 as a causative agent for rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 95, 8227 /8232. Wang, J., Crawford, K., Yuan, M., Wang, H., Gorry, P.R., Gabuzda, D., 2002. Regulation of CC chemokine receptor 5 and CD4 expression and human immunodeficiency virus type 1 replication in human macrophages and microglia by T helper type 2 cytokines. J. Infect. Dis. 185, 885 /897. Woolf, A.D., Campion, G.V., Chishick, A., Wise, S., Cohen, B.J., Klouda, P.T., Caul, O., Dieppe, P.A., 1989. Clinical manifestations of human parvovirus B19 in adults. Arch. Intern. Med. 149, 1153 / 1156.
Evaluation of anti-parvovirus B19 activity in sera by assay using quantitative polymerase chain reaction Takako Saito a, Yasuhiko Munakata b,*, Yi Fu b, Hiroshi Fujii b, Takao Kodera b, Eiji Miyagawa c, Keiko Ishii a, Takeshi Sasaki b b
a Department of Molecular Diagnostics, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan Department of Rheumatology & Hematology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan c New Product Planning and Development Division, Fujirebio Inc., 51 Komiya-cho, Hachioji-shi, Tokyo 192-0031, Japan
Received 19 June 2002; received in revised form 24 August 2002; accepted 27 August 2002
Abstract Human parvovirus B19 (B19) infects cells of erythroid lineage. Production of neutralizing antibodies (Abs) is indispensable for recovery from B19-related disease state. In this study, we used a convenient method to measure neutralizing activities in human sera by using a real-time quantitative PCR based assay. Erythroid cell line KU812Ep6 was incubated with test sera before infection with B19 virus. The copy number of B19-DNA in cultures was decreased in the presence of the sera from patients who recovered from acute B19 infection, whereas no decrease in B19-DNA was in cultures incubated with sera from healthy volunteers who had no B19 infection. The decrease in B19-DNA copy number was calculated and the inhibition percentage was expressed as neutralizing activity to B19. A clinical study showed that the levels of neutralizing ability were high in patients who recovered soon after acute B19 infection, but were low in some patients with a prolonged clinical course for recovery from B19 infection. This method is simple and convenient compared with methods described previously, showing its usefulness to evaluate the neutralizing activity to B19. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Neutralizing antibody; Human parvovirus B19; Quantitative PCR; KU812Ep6
1. Introduction Human parvovirus B19 (B19) infection is most common in children. Acute B19 infection in children causes erythema infectiousum and recovery from the disease is spontaneous with no serious complications (Brown et al., 1994). However, B19 infection in adults often causes serious disease conditions, such as aplastic crisis (Kelleher et al., 1983), hydrops fetalis in pregnant women (Anand et al., 1987), and polyarthritis resembling rheumatoid arthritis (Woolf et al., 1989; Takahashi et al., 1998; Stahl et al., 2000). Persistent infection has also been reported in humans (Kerr et al., 1995). Appearance of the neutralizing IgG antibodies (Abs) specific against B19 is generally considered a leading pathological feature before recovery. In B19 infection,
* Corresponding author. Tel.: /81-22-717-7165; fax: /81-22-7177168 E-mail address: [email protected] (Y. Munakata).
where a cellular immune response is not demonstrable, a humoral response seems to be a crucial factor for recovery from disease (Kurtzman et al., 1989; Sato et al., 1991). Studies on anti-B19 antibodies have indicated the presence of anti-B19 antibodies with a wide neutralizing ability (Bostic et al., 1999; Gigler et al., 1999). This indicates that assessment of B19 neutralizing activity of the serum may be important to monitor the infection condition of patients with B19 infection. So far, neutralizing activity in sera has been measured as its ability to inhibit erythrocyte colony formation by using bone marrow cells in an in vitro assay (Takahashi et al., 1991). The clinical use of this assay, however, has been hampered because obtaining bone marrow cells for each assay is not easy. The test itself also has problems of accuracy and consistency as the bone marrow used often contains precursor cells of different lineages at various stages of differentiation. In other words, the target cell population for B19 infection varies from assay to assay in the colony formation assay.
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To overcome these difficulties, we developed a novel assay to measure neutralizing activity against B19 in sera that is easy to perform, is accurate and is reliable. In this study, an assay is described to measure B19 neutralizing activity in sera by using real-time PCR based on TaqMan chemistry (Gibson et al., 1996; Heid et al., 1996). The assay was done by in vitro culture of the KU812Ep6 erythroid cell line that is highly susceptible to B19 infection (Miyagawa et al., 1999). We evaluated also the disease status and followed the clinical course of the patients with acute B19 infection by using this assay that compares the value of IgG antiB19 antibody measured by enzyme-linked immunosorbent assay (ELISA).
2. Materials and methods 2.1. Serum samples Serum samples were obtained from healthy volunteers (YM, YO, SM, MS, SI, NK, TS) and patients with acute B19 infection (including IM), and six patients (including OA, GF) who showed a prolonged clinical course for recovery from B19 infection. All patients were examined at the Rheumatology and Hematology Clinics of Tohoku University Hospital. The serum samples were kept frozen at /80 8C until use. All samples were obtained following informed consents for performing polymerase chain reaction (PCR) and ELISA of IgM and IgG anti-B19 antibodies. 2.2. Measurement of anti-B19 antibodies The serum samples were tested for IgM and IgG antiB19 antibodies by using a commercial indirect ELISA kit. The sera were tested by using a Parvo IgM-EIA ‘SEIKEN’ kit and a Parvo IgG-EIA ‘SEIKEN’ kit (Matsunaga et al., 1995) (Denka Seiken Inc., Tokyo, Japan) according to the manufacturer’s instructions and by using the microplate reader MPR-A4 (TOSOH Inc., Tokyo, Japan). The titer of anti-B19 antibodies was expressed as an index according to the instructions provided, and an index number over 0.8 was regarded as positive for the presence of anti-B19 Abs. 2.3. Human parvovirus B19 Serum from patient FU with acute B19 infection was used for an in vitro infection assay. The serum contained 2.5 /1014 copies of B19-DNA/ml, and no anti-B19 antibodies was detected by using ELISA. The serum from the healthy individual MO, containing neither B19-DNA nor anti-B19 antibody, was used as a negative control.
2.4. Monoclonal anti-B19 antibody Mouse monoclonal anti-B19 antibody, WP181, was obtained from BALB/C mice immunized with B19 capsid protein. The B19 capsid protein (Matsunaga et al., 1995) was purchased from Denka Seiken Inc. (Tokyo, Japan). 2.5. Construction of a B19-DNA plasmid To calculate the number of VP1/B19-DNA equivalents in the PCR- amplified sample, full length B19 genomic DNA amplified from a patient’s serum by PCR, was inserted into a pGEM-T vector (Promega, Madison, WI). Plasmid DNA was extracted from recombinant bacteria and was purified. The original copy number of the B19-DNA vector was found by comparing the optical density values with the molecular weight of different dilutions of this plasmid. 2.6. Cell culture for the neutralization test The human erythroid cell line KU812Ep6 (Miyagawa et al., 1999) was cultured with RPMI medium containing 10% fetal bovine serum (FBS) and 6 IU/ml of erythropoietin (Kirin Brewery Inc., Tokyo, Japan) at 37 8C in 5% CO2. KU812Ep6 (100,000) cells were preincubated in 500 ml of the medium together with the test serum or WP181 at 37 8C for 30 min, and then 0.50 ml of the serum from FU or MO was added and the cells were further incubated for 2 h. After washing three times with PBS, the cells were cultured for 48 h, and then cells underwent a PCR assay. 2.7. Quantitative PCR assays to detect B19-DNA DNA was prepared from KU812Ep6 cells infected with B19 by using the conventional phenol-chloroform extraction method. A quantitative PCR assay of samples amplified the VP1-region of B19-DNA by using ‘TaqMan PCR reagent kit’ (Roche, Branchburg, NJ). The reaction used a volume of 50 ml consisting of 0.5 mg of sample DNA, 400 mM of dUTP and 200 mM of dATP, dCTP and dGTP, 3.5 mM MgCl2, 200 nM of each primer, 100 nM of TaqMan probe, 0.01 U/ml of AmpErase UNG, 0.025 U/ml of AmpliTaq Gold, and TaqMan buffer A. The sequence of primers, amplifying the nt.2598 /2752 region of VP1 in B19-DNA, used in this study were: forward primer, 5?-CCCTAGAAAACCCATCCTCTGTG-3?; reverse primer, 5?AGGTTCTGCATGACTGCTACTGG-3?. The sequence of the detection probe labeled with fluorescent FAM corresponding to the nt 2692 /2718 region of VP1 of B19-DNA was 5?-TCATGGACAGTTATCTGACCACCCCCA-3?. The thermal cycling conditions were 2 min at 50 8C, followed by 10 min at 95 8C. Thermal
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cycling followed with 40 cycles of 95 8C for 15 s and 60 8C for 1 min. All reactions were done in an ABI/ PRISM 7700 sequence detector system, which contains a GeneAmp PCR system 9600. Each sample was analyzed in duplicate, and the DRn and CT average was calculated from the values from each reaction. Controls were run in duplicates to calibrate a standard calibration curve for each set of analyses. The degree of inhibition of the B19-DNA in the presence of anti-B19 serum was expressed as the inhibition ratio (% inhibition) and was calculated as: 100 /(copy number of B19-DNA without test serum/ copy number of B19-DNA with test serum)/copy number of B19-DNA without test serum. 2.8. Statistical analysis The correlation coefficient was calculated by the program of Cricket graph 1.3.
3. Results 3.1. B19 infection of KU812Ep6 erythroid cells measured by quantitative PCR We reported previously that B19 can be easily propagated in KU812Ep6 cells (Miyagawa et al., 1999). First, it was determined whether an in vitro infection system using KU812Ep6 is suitable for quantitative-PCR. Control plasmids containing full length B19-genomic DNA were diluted in water (105 /10 copies in each sample) and a standard curve was established with CT values plotted against the logarithm of the copy number. The relationship was between the CT and the copy number of control standard B19-DNA was linear (data not shown). The copy number of B19-DNA increased in the KU812Ep6 cells when they were incubated with the serum from FU that contained infective B19 virus, whereas B19-DNA was not detectable in the KU812Ep6 cells when they were incubated with the serum from MO that did not contain B19 virus (Fig. 1A). The increase in B19-DNA in the KU812Ep6 cells was inhibited by the mouse monoclonal IgG antiB19 Ab, WP181, in dose dependently. The antibody concentrations of over 1 mg/ml inhibited effectively the amplification as the increase in B19-DNA was eliminated (Fig. 1B). B19-DNA increased rapidly during 24/ 60 h of incubation. After this period of incubation, the increase in B19-DNA copy number reached a plateau in this culture system. 3.2. Measuring neutralizing activity against B19 in sera We examined the effect of human sera containing anti-B19 IgG Abs on B19-DNA replication in the
Fig. 1. Detection of B19-DNA in the KU812Ep6 cell line. (A) The copy number of B19-DNA was quantitated by amplifying the VP1 region of B19-DNA. Sample DNA extracted from KU812Ep6 cells were incubated with FU serum (m) or MO serum (k) at each time indicated, and underwent quatitative PCR. (B) The inhibitory effect of monoclonal anti-B19 Ab on B19 infection was examined by quantitative PCR at 48 h after infection. Sample DNA was extracted from KU812Ep6 cells and was incubated with FU serum in the presence of anti-B19 Ab, WP181, at the concentration indicated.
KU812Ep6 cells. When serum from the healty volunteer YM, who had recovered from acute B19 infection, was added to the KU812Ep6 culture system at a dilution of 1:500, the increase in B19-DNA was inhibited until 42 hr of incubation. B19-DNA slightly increased after 42 h of incubation in the presence of serum containing IgG antiB19 Abs (Fig. 2A). The inhibition percentage dramatically increased at 24 h of incubation. The inhibition percentage was optimal at a serum dilution of 1:500 (Fig. 2B). The inhibition percentages at concentrations higher than 1:500 was less than at 1:500. The inhibition percentages at dilutions higher than 1:500 decreased proportionate to the dilution of the test serum as in the case of WP181. The correlation was close between the index of IgG anti-B19 antibodies in the sera from healthy individuals and patients with acute B19 infec-
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Fig. 3. Correlation between anti-B19 IgG Ab and neutralizing ability against B19 in the sera. The titer of anti-B19 IgG Ab measured by ELISA was expressed as an index on the horizontal axis. Then neutralizing ability against B19 in the serum measured by our assay was expressed as the inhibition percentage on the vertical axis. m, samples from healthy individuals and patients from acute B19 infection and k, samples from patients who showed a prolonged clinical course for recovery from acute B19 infection.
normal volunteers, who had no B19 infection, were negative for both IgM and IgG anti-B19 Abs and showed no effect on the copy number of B19-DNA in KU812Ep6 cells (Table 1). However, the neutralizing activity was markedly low in sera from patients who showed a prolonged clinical course for recovery from B19 infection despite the presence of IgG anti-B19 antibodies (Fig. 3). Fig. 2. Effect of serum containing neutralizing anti-B19 Abs on B19 infection of the KU812Ep6 cell line. (A) The copy number of B19DNA and the inhibitory effect of anti-B19 serum (YM) on B19 infection were measured by using the neutralization test. Sample DNA was extracted at each time from KU812Ep6 cells and was incubated with FU serum in the presence (k) or absence (m) of YM serum containing anti-B19 IgG Abs. The inhibition percentage at each time are numbers at the top of the panel. (B) The inhibitory effect of antiB19 serum (YM) on B19 infection was examined at each dilution indicated at 48 h after infection.
tion and the inhibition of B19 replication (Fig. 3). The correlation coefficient was 0.824. Also, sera from Table 1 Neutralizing activity of test sera from normal volunteers who had no B19 infection Donors
YO SM MS SI NK TS
Index of anti-B19 Abs IgM
IgG
0.0 0.0 0.0 0.0 0.0 0.0
0.2 0.4 0.2 0.2 0.0 0.0
Inhibition of B19-DNA replication (%)
0.0 0.0 0.0 0.0 0.0 0.0
3.3. Neutralizing activity against B19 in serum measured during disease recovery In order to evaluate the usefulness of this assay in association with the clinical course of B19-related diseases, we measured the level of anti-B19 antibodies and the neutralizing activities of the sera obtained sequentially from the adult patient with acute B19 infection. In patient IM with erythema infectiousum, the samples from the early phase of infection with severe symptoms of polyarthralgia, skin eruption and cervical lymphadenopathy showed a high titer of IgM anti-B19 antibody. The recovery of symptoms corresponded to the increase in neutralizing activity against B19 in the serum (Fig. 4A). However, patient OA, who was febrile status for more than 3 months, delayed the recovery from acute B19 infection, (Fig. 4B); IgG anti-B19 Ab was detected in this patient during the period of viremia. However, the neutralizing activity in the serum was not detectable in this period, and the patient suffered from fever and skin eruption. After the appearance of neutralizing activity at a detectable level, the symptoms completely disappeared, and the neutralizing activity increased. In patient GF, symptoms relating to B19
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Fig. 4. Anti-B19 Abs in the clinical course of patients with B19 infection. Sample sera were assayed at each time indicated (A) from IM, (B) from OA and (C) from GF. k, titers of anti-B19 IgM Ab measured by ELISA and m, titers of anti-B19 IgG Ab measured by ELISA. The neutralizing activity against B19 in serum was expressed as the inhibition percentage in solid bars and the percentage is shown above the bars.
infection persisted for several months in spite of the presence of IgG anti-B19 Ab (Fig. 4C). Intravenous administration of g-globulin that contained anti-B19 neutralizing activity relieved this patient from B19relating symptoms.
4. Discussion B19 infection is self-limiting in most cases and the recovery from active infection by B19 is generally thought to be dependent on the production of IgG
anti-B19 antibodies (Kurtzman et al., 1989; Erdman et al., 1991; Sato et al., 1991). In virus neutralization, IgG and IgA antibodies appear to have a role in the resistance to natural infection by the nasopharyngeal route (Erdman et al., 1991). In persistent B19 infection, impairment in the production of anti-B19 antibodies with high avidity has been suggested (Kurtzman et al., 1989; Kerr et al., 1995). We reported previously two cases developing rheumatoid arthritis from acute B19 infection (Murai et al., 1999) where we detected B19DNA in their bone marrow even though IgG anti-B19 antibodies were present in the serum. These results
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Fig. 4 (Continued)
suggest that evaluation of the neutralizing activity against B19, which may be contributed by antibodies of all isotypes in the serum, is important to monitor the infection state and the patient care. The assay used to measure the neutralizing activity of the sera is the measurement of the inhibition of erythrocyte colony formation by bone marrow cells (Takahashi et al., 1991). Bostic et al. (1999) reported a sensitive test to measure B19 neutralization of sera by using a RT-PCR based system. In this method, the test serum needs to be diluted to quantify the neutralizing activity. However, this may be inconvenient for practical use, because it requires several assays for one specimen. For the followup of patients with B19 infection, evaluating the disease state by absolute values of the neutralizing activity in the serum is useful. The transcription of RNA occurs without viral replication (Liu et al., 1992; Pallier et al., 1997; Brunstein et al., 2000; Gallinella et al., 2000), which may be responsible for variability of the results. In this study, we developed a novel assay to quanitify the neutralizing activity against B19 in serum based on real-time PCR on B19-DNA by using an in vitro culture system of the KU812Ep6 erythroid cell line. KU812Ep6 is a high B19-infective clone that is cloned by limiting dilution from the B19-susceptible cell line KU812 by using a medium containing erythropoietin (Miyagawa et al., 1999). B19 replicates in this cell line and the infectivity are found by suppression of colony formation by the erythroid colony forming unit (Miyagawa et al., 1999). Using this cell line, we quantitated the neutralization effect of the test serum that inhibited B19DNA replication in the KU812Ep6 cells. In this assay, we incubated KU812Ep6 cells with test serum before being challenged with the virus, because it was intended
to reconstitute the physiological condition in vivo as well. The target cells for B19 infection in vivo may be protected by pre-existing anti-B19 antibodies in serum from invading B19. The dilution of the test sera and incubation time are important to minimize the effect of cytokines and other serum components. Some serum components, such as cytokines, interfere with viral infection (Pasquetto et al., 2002). In the case of influenza A virus, serum amyloid P inhibits its infection (Horvath et al., 2001). The expression of the viral receptor is possibly affected by the serum component (Wang et al., 2002). The serum concentration in the assay should be lower as possible. The dilution of the sera was found to be 1:500, as optimal neutralization was observed at higher than this dilution. We found the incubation time of 48 h, because the maximum inhibitory effect was during 30 /48 h. In sera from healthy volunteers who recovered from acute B19 infection, we detected high titers of IgG anti-B19 antibodies and neutralizing activity against B19 (Fig. 4A). However, in sera from individuals who had no B19 infection, neither IgG antiB19 antibodies nor neutralizing activity was detected against B19. In healthy individuals who recovered from acute B19 infection, neutralization of B19 evaluated by this assay and the level of IgG anti-B19 antibodies measured by ELISA showed a good correlation. Important may be that the neutralizing ability was negative or low at the initial period of B19 infection in some who had a prolonged clinical course (Fig. 4B,C). The patient symptoms disappeared when the levels of neutralizing ability increased in the serum, indicating the importance of monitoring the evaluation of neutralizing activity against B19 infection in a clinical course. Why a low or no neutralizing ability against B19 instead of a high titer
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of IgG anti-B19 antibodies were in patients OA and GF is unknown. However, we suggest that this may be due to impairment of the antigen presentation system in hosts as with impaired vaccination of hepatitis B virus in some cases (Hatae et al., 1992). The antigenic peptide that efficiently induced neutralizing antibodies may hardly be recognized in the host immune system. In summary, a novel assay was developed to measure the B19 virus neutralizing activity of the sera by using quantitative-PCR and an in vitro culture system of the KU812Ep6 erythroid cell line. By using this assay, it may be possible to find the state of B19 infection more precisely, which may help to clarify the clinical state of persistent infection of B19.
Acknowledgements This work was supported by grant-in aid for scientific research (A) from the Ministry of Education, Science, Sports and Culture in Japan, and by grants to T.S. awarded from the Kurozumi Medical Foundation. Informed consent was obtained from all patients in this study.
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