昭和62年8月20日 871

Human Monoclonal Antibody to a 65kDa Structural

Polypeptide of Cytomegalovirus

Nobukazu HOSHI1), Shigeaki TANAKA2), Mikio KIMURA1)

and Yasushi WATANABE2)

Department of Pediatrics1) and Molecular Biology2), Tokai University School of Medicine,Isehara, Kanagawa, Japan

Key words: Cytomegalovirus, Human monoclonal antibody, Virion protein

Abstract

A clonal human cell line that produces an antibody (IgG) against a human cytomegalovirus (HCMV)

virion polypeptide of 65 kilo daltons (kDa) was established by transforming lymphocytes from a bonemarrow transplant recipient with Epstein-Barr virus (EBV). Immunofluorescent staining with thisantibody was positive in cells infected with two laboratory strains and several clinical isolates of HCMV,while it was negative in cells infected with herpes simplex viruses type 1 and 2, varicella-zoster virus, orEBV. Kinetic studies of the formation of the 65kDa polypeptide in infected cells showed its appearance inthe cell nuclei early. In infection (12-24 hr after infection) and its later accumulation in the cytoplasm.These results suggest possible use of this monoclonal antibody for identification of HCMV infection.

Introduction

HCMV, a widely disseminated herpes virus, is a cause of life-threatening infection in immunosu-

pressed hosts1). In our study, more than 80% of bone marrow transplant recipients showed seroconversionto HCMV antigens and increase of and-HCMV antibody-producing cells). There have been a number ofreports suggesting that HCMV-induced immunoglobulins prevent severe interstitial pneumonitis due toHCMV in these recipients3). For this reason, many atempts have been made to produce human monoclonalantibodies to HCMV, but to date these attempts have met with only limited success4). In view of possibleadvantages of human monoclonal antibodies over murine ones5) for future use in immunological therapyand for acquisition of a wider range of antibodies against HCMV, we attempted to produce HCMV-specifichuman monoclonal antibodies. In this study, we have established a stable human cell line that produces an

antibody of IgG class reactive with a HCMV-specific polypeptide of 65kDa, which is the most abundantcomponent of HCMV virions and has been implicated in the induction of neutralizing antibodies6).

Materials and Methods

Cells and virusesTwo laboratory strains of HCMV, Towne and Tanaka7), and three clinical isolates from a congenitally

infected newborn and bone marrow transplant recipients were used. They were propagated in humanembryonic lung (HEL) cells grown in Eagle's minimum essential medium containing 10% fetal calf serum

(FCS)8). Herpes simplex virus type 1 (HSV-1) strain F and type 2 (HSV-2) strain HG 52 obtained from S. Niiat Okayama University and varicella-zoster virus (VZV) strain Oka were propagated by infecting HEL cellsat a multiplicity of infection of 0.5-1.0, An EBV-producing cell line, B95-8, obtained from Y. Ono at Nihon

別刷請求先:(〒259-11)伊 勢原市望星台

東海大学医学部小児科学教室

星 伸和

872 感染症学雑誌 第61巻 第8号

University was clutured in RPMI-1640 with 10% FCS.

Transformation and cloning of lymphocytes

The procedure was described previously9). Briefly, 10 to 20ml of heparinized blood were obtained from

three bone marrow transplant recipients who had elevated antibody titers against HCMV (CF titers

1:32-256)2) and three healthy persons (CF titers 1:8-16). Mononuclear cells obtained by centrifugation of

ficollpaque (Pharmacia, Sweden) were mixed with a Millipore membrane-filtered culture fluid of B95-8 cells

and incubated at 37•Ž for 2hr. Cells were then collected and 5•~104 cells each were plated in wells of a

96-well tissue culture plate. After 4 weeks, culture fluid of individual wells that contained transformed

colonies was assayed for HCMV-specific antibodies by an enzyme-linked immunosorbent assay (ELISA).

Antibody-producing cells were cloned twice by limiting dulution in a conditioned medium composed of the

same volumes of RPMI 1640 with 20% FCS and culture fluid of Daudi cells without feeder cells.

HCMV antigens

Virions and capsids of HCMV were purified from culture fluid of infected cells by an established

ultracentrifugation procedure10). For preparation of target antigens for ELISA, HEL cells were infected

with HCMV strain Tanaka, at a multiplicity of infection of 0.5-1.0, and after seven days cells were

collected and sonicated three times for 5 sec each in 0.05M Tris-HC1 buffer, pH 8.6, containing 0.35M NaCl

and 1 mM phenylmethylsulfonylfluoride (Sigma Chemical Co. Ltd., USA). The lysate was kept on ice for 3

min with gentle shaking and then centrifuged at 15,000•~g for 30 min. The protein content was adjusted to

about 1 mg/ml and this supernatant was used as target antigens for ELISA. This antigen preparation will

be referred to as late antigens.

ELISA

HCMV antigens in 0.05M sodium bicarbonate-carbonate buffer, pH 9.6, were allowed to adsorb to the

wells of flat bottomed microtiter plates (Sumitomo Bakelite Co. Ltd., Japan) for overnight at 4s•Ž. Wells

were washed three times with phosphate buffered saline (PBS) containing 0.1% Tween 20 and then treated

with 1% bovine serum albumin (BSA) Fraction V (Sigma Chemical Co. Ltd., USA) in PBS for 1 hr at 37•Ž.

Antibody samples to be assayed were added and the plates were incubated for 1 hr at room temperature and

then washed. Peroxidase-conjugated anti-human IgG (Dakopatts a/s, Denmark) diluted 1:2000 with PBS-

Tween 20 was added and incubated for 1hr at room temperature. The wells were washed six times and the

peroxidase retained was assayed with an ABTS kit (Amersham International Plc. UK) according to the

manufacturer's protocol. Absorbance was measured at 410 nm.

Immunobloting analysis

Proteins separated by electrophoresis on 4-30% gradient polycarylamide gels (Pharmacia, Sweden) in

the presence of 1% sodium dodecyl sulfate (SDS) were transferred to Durapore membranes (Millipore Co.,

USA). The membranes were treated with 5% BSA in PBS for 1hr at 37•Ž, washed twice with PBS, and

then incubated with undiluted culture fluid of clonal cell lines for 2hr at 37•Ž. After washing three times

with PBS-Tween 20, the membranes were incubated with biotinylated anti-human IgG (Vector Lab., USA)

at a dilution of 1:500 for 1 hr at room temperature and then stained with a 1:400 dilution of streptavidin-

biotinylated horseradish peroxidase complex (Amersham International Plc., UK) for 30 min at room

temperature according to the method described").

Immunofluorescent staining

Infected cells were fixed with acetone, treated with antibodies to be tested, and then stained with

fluoresceinisothiocynate-conj ugated goat anti-human IgG (Med. Bio. Lab. Nagoya) at a dilution of 1:20 in

PBS.

Results

Isolation of clones producing anti-HCMV antibodies

昭和62年8月20日 873

Peripheral lymphocytes from three bone marrow transplant recipients and three healthy persons were

infected wiyth EBV, and plated in the wells of culture plates. After three weeks, colonies of transformed

cells were seen in nearly all the wells. Culture fluid of individual wells was assayed for HCMV-specific

antibodies by ELISA with late antigens as target. Antibody (IgG)-positive wells were 21-90% for cell

samples from bone marrow transplant recipients, whereas they were less than 7% for cell samples from

healthy persons (Table 1). Ten wells that showed O.D.>1.5 in ELISA were selected and cells contained

were examined for their antibody-producing ability for several weeks. These were all derived from bone

marrow transplant recipients. Cells obtained from 7 out of 10 wells turned out to be non-producers after 4

weeks. Cells from three wells continuously produced HCMV-specific antibodies for over 4 weeks without

any reduction in titers. These cells were then coned by limiting dilution and three cell lines (WT-3, WT-5,

WT-6) were established. These three cell lines produced 1-2 ,ug/m1 human IgG with K light chains. These

antibodies are called mAb WT-3, WT-5, and WT-6. In this study, mAb WT-5 was characterized further.

Table 1 Appearance of and-HCMV antibody-producing cells

Fig. 1 Immunoblotting analysis of HCMV antigens with mAb WT-5. Proteins in late antigens, viral capsids, andenveloped virions of strain Towne were separated by SDS-PAGE, transferred to Durapore membranes, and immuno-stained with mAb WT-5. (A) Lane 1: late antigens; Lane 2: viral capsids; Lane 3: enveloped virions. Lanes 4 and 5:enveloped virious and marker proteins, respectively, stained with amidoblack. (B) Lanes 1 and 2: enveloped virionsstained with amidoblack and mAb WT-5, respectively. Lane 3: uninfected cell extracts stained with mAb WT-5. LM:the lower matrix protein; and UM: the upper matrix protein.

(A) (B)

874 感染症学雑誌 第61巻 第8号

The viral antigen recognized by monoclonal antibodies

The target antigen of mAb WT-5 was determined by immunoblotting analysis in which three types of

HCMV antigens, i.e. late antigens, virion capsids, and enveloped virions, were used. As seen in Fig. 1A,

mAb WT-5 recognized a 65kDa polypeptide commonly present in these three types of antigen preparation.

mAb WT-3 and WT-6 similary recognized the 65kDa polypeptide (not shown). Better separation of

Fig. 2 Immunofluorescent staining of HCMV-infected HEL cells with mAb WT-5. a: uninfected; b: Towne; c:

Tanaka; and d-f: resh clinical isolates of HCMV. Cells were stained 48 hr after infection.

Fig. 3 Immunoblotting analysis with mAb WT-5of the 65 kDa polypeptide induced by variousstrains of HCMV. Lane 1: uninfected cell ex-tracts; lanes 2 and 3: enveloped viruons of strainsTowne and Tanaka, respectively. Lanes 4 and 5: lateantigens of strains Towne and Tanaka, respec-tively. Lane 6: late antigens induced by a clinicalisolate (Y).

昭和62年8月20日 875

polypeptides consitituting virions was achieved by reducing the amounts of proteins in SDS-PAGE (Fig.1B); the 65kDa polypeptide recognized by mAb WT-5 apparently corresponded to a lower component of

matrix proteins reported by Gibson and Irmiere et al.1213)

Fig. 4 Immunofluorescent staining with mAb WT-5 of HCMV (Tanaka)-infected HEL cells, acetone-fixed at varioustimes of infection. a) uninfected; b)-f) infected, 1, 24, 48, 72 and 96 hr p.i.

Fig. 5. Time course of the 65 KDa polypeptideformation analyzed by immunoblotting usingmAb WT-5. Samples were prepared from HELcells infected with HCMV (Tanaka) at the timesindicated. Lane 1: uninfected; lanes 2-8: 0, 12,24, 36, 48, 72, 96 hr p.i., respectively. Lane 9:marker proteins stained with amidoblack.

Fig. 6 Time course of the 65 kDa polypeptideformation determined by ELISA using mAb WT-5. HEL cells infected with HCMV (Tanaka) wereharvested at the times indicated and cell extractswere asayed for the 65 kDa polypeptide. ELISAtiters (the vertical axis) are expressed by thedilution factors of each sample that showed 0.3O.D. at 410 nm in ELISA.

876 感染症学雑誌 第61巻 第8号

Specific recognition of HCMV by mAb WT-5mAb WT-5 wa able to immunofluorescent stain HEL cells infected with two laboratory strains of

HCMV, Towne and Tanaka, and three clinical isolates of HCMV; no essential differences were observed inthe fluorescent pattern among these strains (Fig. 2). In contrast, no fluorescence was observed in cellsinfected with HSV-1, HSV-2, VZV or EBV. Immunoblotting showed that all the 65kDa polypetides producedby these five strains of HCMV share the epitope that can be recognized by mAb WT-5. The results obtainedwith strains Towne and Tanaka and one clinical isolate (Y) are shown in Fig. 3.Time course of the 65kDa polypetide formation

Immunofluorescent staining of infected cells with mAb WT-5 showed that the 65kDa polypeptidebecame detectable in the nuclei between 12 and 24 hr after infection and then accumulated predominantlyin the cytoplasm between 72 and 96 hr after infection, suggesting its transport from the nuclei to thecytoplasm at late stages of infection (Fig. 4). This is consistent with the previous observation that virions,moninfectious enveloped particles and dense bodies, all of which contain substantial amounts of the 65kDamatrix proteins, are accumlated in the cytoplasm at late stages of HCMV infectionm). Quantitativ analysisby immunoblotting and ELISA showed that the 65kDa polypeptide was first detected at 12 hr afterinfection, and it increased with time and reached over 100 times as much amount at 72 hr as that at 12 hr

(Fig. 5 and 6).

Discussion

Using the EBV-transformation technique for the production of human monoclonal antibodies9), wehave established human cell lines producing the antibodies against the 65kDa virion protein of HCMV.Three cell lines thus obtained have been cultured for more than 12 months with constant production of theantibody. Of these, the chracteristics of mAb WT-5 was determined here. The use of peripherallymphocytes from bone marrow transplant recipients appeared to be suitable for these purposes.Presumably this is due to the fact that HCMV is reactivated frequently in these immunosuppressed

patients and immune responses to HCMV are potentially stimulated in them1)2).More than 50 species of proteins specified by the HCMV genome are synthesized in infected cells and

about 30 of them are associated with virions12). Historically, one particular species of virion protein was

predominantly detected by immunoprecipitation in which virion components were incubated with humansera of either acute or convalescent infection with HCMV15). This virion component with an apparentmolecular weight 65-69k was found to make up about 70% of the virion and named matrix protein byGibson and Irmiere et al.12)13) This protein was later found to be composed of two species of polypeptideswhich were separable by PAGE, and they were accordingly named the lower and upper matrix proteins13).The 65kDa polypeptide recognized by mAb WT-5 in this study apparently corresponds to the lower matrix

protein (Fig. 1A and 1B).mAb WT-5 appeared to be able to distinguish HCMV from other herpes viruses. So far the most

sensitive method for laboratory diagnosis of active HCMV infection is the virus isolation in tissuecultures16), and at least 7 days are required for initial observation of the HCMV-induced cytopathic effectafter inoculation of clinical specimens onto susceptible cells17). The present study shows that theimmunofluorescent staining with mAb WT-5 can identify HCMV in cell cultures within 48 hr afterinoculation (Fig. 2), and both ELISA and immunoblotting analysis can detect the HCMV-specific antigenbetween 12 and 24 hr after infection (Fig. 5 and 6), suggesting potential usefulness of mAb WT-5 in clinicalapplications.

Our preliminary tests for neutralization of HCMV infectivity with mAb WT-5 has been negative

(unpublished observation). However, there is evidence that the lower matrix protein elicits both humoral18)

昭和62年8月20日 877

and cell-mediated immune response in man19). In view of this, possible use of mAb WT-5 for prophylaxis

and treatment of severe HCMV infection does deserve consideration.

References

1) Meyer, J.D. & Atkinson, K.: Infection in bone marrow transplantation. Clinical Haematol., 12: 791, 1983.2) Kato, S., Hoshi, N., Nagasaka, M., Muranaka, S., Yabe, H., Kimura, M. & Tokai University Bone Marrow

Transplantation Team.: Allogeneic Bone Marrow Transplantation in Children: Tokai Experinece 1982 to 1984:Tokai J. Clin. Med., 10: 147-158, 1985.

3) Condie, R.M. & O'Reilly, R.J.: Prevention of cytomegalovirus infection by prophylaxis with an intravenous,hyperimmune, native unmodified cytomegalovirus globulin. Randomized trial in bone marrow transplantrecipients. Am. J. Med., 76: 134-141, 1984.

4) Emanuel, D., Gold, J., Colancino, J. & Lopez, C.: A human monoclonal antibody to cytomegalovirus. J. Immunol.,133: 2202-2205, 1984.

5) Pereira, L., Hoffman, M., Tatsuno, M. & Dondero, D.: Polymorphism of human cytomegalovirus glycoproteinscharacterized by monoclonal antibodies. Virology, 139: 73-86, 1984.

6) Pereira, L., Hoffman, M., Gallo, D. & Cremer, N.: Monoclonal antibodies to human cytomegalovirus: Three surfacemembrane proteins with unique immunological and electrophoretic properties specify cross-reactive determinants.Infec. Immunol., 36: 924-932, 1982.

7) Takekoshi, M., Ihara, S., Maeda, F., Tanaka, S. & Watanabe, Y.: A new human cytomegalovirus isolate has aninvertible subsegment within its L component producing eight genome isomers. J. Gen. Virol., (in press)

8) Tanaka, S., Ihara, S. & Watanabe, Y.: Human cytomegalovirus induces DNA-dependent RNA polymerases inhumna diploid cells. Virology, 15: 297-304, 1987.

9) Kozbor, D. & Roder, J.C.: The production of monoclonal antibodies from human lymphocytes. Immunology Today,4: 72-79, 1983.

10) Huang, E.S., Chen, S.T. & Pagono, J.D.: Human cytomegalovirus 1. Purification and characterization of viral DNA.J. Virol., 12: 473-481, 1983.

11) Adams, J.C: Heavy metal intensification of DAB based HRP reaction product. J. Histochem. Cytochem., 29: 775,1981.

12) Gibson, W.: Structural and nonstructural proteins of strain Colbarn cytomegalovirus. Virology, 111: 516-537,1981.

13) Irimere, A. & Gibson, W.: Isolation and characterization of a virion-like particle from cells infected with humanstrains of cytomegalovirus. Virology, 118: 118-133, 1983.

14) Sarov, I. & Abady, I.: The morphogenesis of human cytomegalovirus. Isolation and polypeptide characterization ofcytomegalovirus and dense body. Virology, 66: 463-473, 1975.

15) Pereira, L., Hoffman, M. & Cremer, N.: Electrophoretic analysis of polypeptides immune precipitated fromcytomegalovirus-infected cell extracts by human sera. Infec. Immunol., 36: 933-942, 1982.

16) Drew, W.L.: Controversies in viral diagnosis. Rec. Infec. Dis., 8: 814-824, 1986.17) Cleaves, C.A., Smith, T.F., Shuster, E.A., Pearson, G.R.: Rapid detection of cytomegalovirus in MRC-5 cells

inoculated with urine speciemens by using low-speed centrifugation and monoclonal antibody to an early antigen. J.Clin. Microbiol., 19: 917-919, 1984.

18) Nowak, B., Sullivan, C., Sarnow, P., Thomas, R., Bricout, F., Nicolas, J.C., Fleckenstein, B. & Levine, A.J.:Characterization on monoclonal antibodies and polyclonal immune sra directed against human cytomegalo virion

proteins. Virology, 132: 325-338, 1984.19) Borysiewicz, L., Morris, S., Page, J. & Sissons, J.G.P.: Analysis of the human cytotoxic and helper T cell response to

cytomegalovirus in vitro. Birth Defects: Orig. Art. Series, Vol. 20: 369-374, 1984.

878 感染症学雑誌 第61巻 第8号

ヒ トサ イ トメガ ロ ウイル スの ウイル ス粒 子 蛋 白(65k)に 対 す る

ヒ ト型 モ ノク ローナル 抗体 の性 状

1)東 海大学医学部小児科学教室

2)同 分子生物学教室

星 伸和1)田 中 重明2)木 村三生夫1)渡 辺 秦2)

(昭和61年12月1日 受付)

(昭和62年2月18日 受理)

骨 髄 移 植 患 者 か ら採 取 した リン パ 球 を用 いEB

ウイ ル スで トラ ン ス フ ォ ー メ ー シ ョンを 行 な い,

ヒ トサ イ トメ ガ ロ ウ イ ル ス特 異 的IgG産 生 ヒ ト

型 モ ノ ク ロー ナ ル 抗 体 産 生 細 胞 が 得 られ た.こ の

モ ノ ク ロー ナ ル 抗 体 は,分 子 量65kの ウイ ル ス粒

子 ポ リペ プ チ ドと反 応 した.こ の抗 体 を 用 い,ヒ

トサ イ トメ ガ ロ ウ イ ル ス に 対 す る特 異 性 を検 討 し

た 所,2つ の 研 究 室 継 代 株 と3つ の 新 鮮 分 離 株 が,

螢 光 抗 体 法 に よ っ て染 色 され,他 の ヘ ル ペ ス ウ イ

ルスでは,螢 光を認めなかった.

この抗体を用いて,分 子量65kウ イルス粒子ポ

リペプチ ド合成動態を検討 した結果,比 較的早期

(感染後12～24時 間)に は,核 内で検出され,後 期

になると細胞質内に漸次蓄積 されていくように思

われた.こ の ことから,こ のモノクローナル抗体

は,ヒ トサイ トメガロウイルス感染の解析に有用

であると思われた.