Characterization of monoclonal antibodies and polyclonal immune sera directed against human cytomegalovirus virion proteins

VIROLOGY

132, 325-338

(1984)

Characterization of Monoclonal Antibodies and Polyclonal Immune Sera Directed against Human Cytomegalovirus Virion Proteins B. NOWAK,* C. SULLIVAN,+ P. SARNOW,? J. C. NICOLAS,$ B. FLECKENSTEIN,*

R. THOMAS,t F. BRICOUT,* AND A. J. LEVINE?

‘Institut fur Kliniache Virokqie, Universitat Erlangen-Nurnber~, 85m Erhngen, Weat Germany; $5%&e University of New York at Stony Brook, School qf Medicine, Llepartment sf Mhobi&gg Stony Brook, New York 1179.& and Snapital Tr ousseau, 24 rue Arwld Netter, 75012 Paris, Fmnce Received July 11, 1989; accepted

October

20, 1989

Four classes of monoclonal antibody-producing cell lines have been obtained that detect human cytomegalovirus virion structural proteins. These antibodies react with (1) a major outer membrane virion glycoprotein(s) gp56-gp136, whose molecular weight varies between strains of cytomegalovirus, (2) a phosphoprotoin, ~~‘71, localized inside the virion membrane, (3) a phosphorylated nucleocapsid protein, ~~155, and (4) a virion-associated phosphoprotein, pp23. Polyclonal immune human sera react with a large number of virion proteins including those detected by these monoclonal antibodies. These monoclonal antibodies were employed in a radioimmune assay to detect low levels (6 X 108 PFWml) of human cytomegalovirus in solution and human urine. These antibodies were also employed in a fluorescent antibody format to identify cytomegalovirus-infected cells obtained from human urine and nasopharyngeal aspirates. These reagents provide useful tools for studying the molecular biology of virus replication, for diagnosing cytomegalovirus infections, and for studying virus latency and activation. INTRODUCTION

CMV is also often associated with acquired immunodeficiency syndrome (AIDS) patients and the virus may be transmitted venerally or by blood products (Mildvan et aL, 1982; Gottlieb and Ackerman, 1982). It is a matter of intensive debate whether or to which extent the virus may possess oncogenic potential in the human organism. The identification of CMV relies mostly upon the isolation of the virus from urine or saliva and the use of partially characterized polyclonal antisera against CMV for confirmation of this agent. CMV, one of the five human herpesviruses, contains a double-stranded DNA genome of approximately 155 X lo6 Da or 285 kb (Fleckenstein et al, 1982). The linear virion DNA molecule is packaged in an icosahedral core of proteins which is surrounded by a membrane containing virionencoded glycoproteins. It has been estimated that some 29-40 proteins and gly-

Human cytomegalovirus (CMV) has been associated with a variety of different clinical syndromes. A common feature in these disorders is a deficiency of the immune system which, under normal conditions, usually keeps this ubiquitous virus under control. A high incidence of CMV infection, most often occurring as CMV interstitial pneumonia, or an infectious mononucleosis-like syndrome, has been reported after bone marrow transplants (Neiman et aL, 19’77; Meyers et al, 1982) as well as in immunosuppressed patients with renal (Glenn, 1981; Schooley et aL, 1983) and cardiac transplants (Rand et al, 1978). Primary CMV infection of pregnant women, diagnosed by the presence of virus in the urine, can lead to congenital central nervous system impairment and multiple other sequelae after prenatal infection in the fetus (Stagno et cd, 1982). CMV can be transmitted to the offspring in utero or when passing through the birth canal.

coproteins

are virion

associated

(Sarov

and

Abady, 1975; Fiala et al, 1976; Kim et uL, 1976; Gupta et al, 1977; Stinski, 1976; Per325

0042-6822&M Copyright All rights

$3.00

0 1984 by Academic Press. Inc. of reproduction in any form reserved.

326

NOWAK

eira et aL, 1982). CMV strain variations are common and most reliably detected by restriction enzyme polymorphisms between strains (Waner and Weller, 19’78; Huang et aL, 1980). Two of the prototype CMV strains, Ad169 and Towne, are compared in this study. Cell lines producing monoclonal antibodies against CMV virion proteins have been prepared using purified virus particles. One set of monoclonal antibodies detects an outer membraneassociated glycoprotein of about 58,000 Da (gp58). The molecular weight of this glycoprotein, as determined by SDS-polyacrylamide gel electrophoresis, differs slightly between the Ad169 and Towne strains of CMV. These antibodies have been employed in both a radioimmunometric double-monoclonal assay for rapid detection of low levels (6 X lo3 PFU/ml) of CMV in human urine or saliva and fluorescent antibody staining of cells from human urine and nasopharyngeal aspirates. This class of monoclonal antibodies also cross reacts with a related 130,000-Da CMV glycoprotein (gp130). Monoclonal antibody-producing cell lines have also been obtained which synthesize antibodies directed against three internal virion-associated phosphoproteins termed ~~‘71, pp29, and ~~155. Immune sera derived from human patients have been employed to determine which CMV proteins these patients have antibodies against. Most sera reacted with a wide variety of CMV proteins, including those detected by the monoclonal antibodies. These results indicate that these CMV monoclonal antibodies should be useful reagents for the rapid diagnosis of CMV infections. In addition, these antibodies should provide useful tools for elucidation of the molecular basis of virus replication, latency, and activation. MATERIALS

AND

METHODS

Virus and cell culture. Human cytomegalovirus seed stocks, strains Towne and Ad169, were obtained from Dr. S. Plotkin (Childrens Hospital, Philadelphia) and Dr. U. Krech (Kantonsspital, St. Gallen, Switzerland), respectively. Strain Towne was plaque purified, and strain Ad169 was pu-

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rified by DNA transfection before use. Human foreskin fibroblasts were prepared from 3- to 7-day-old infants. The cells were cultivated in minimal essential medium (MEM, GIBCO) supplemented with 5% fetal calf serum without antibiotics. After infection with CMV, the cells were maintained in MEM plus 2% fetal calf serum until 80% of the cells showed cytopathic effects. Virus was purified from the cell culture medium by the procedures described by Ebling et al. (1983). L&eling of virus and antibody. Purified virus was iodinated with Bolton-Hunter reagent (New England Nuclear, Boston) as described by the manufacturers. The specific activity of the virion proteins was 5 x lo7 cpm/pg. Concanavalin A (Amersham-Buchler, Braunschweig) was iodinated using methods described by Burridge (1978). 36S-Labeled virus was otained by incubating the cultures with r5S]methionine (Amersham-Buchler, 1200 Ci/mmol, 50 &i/ml) at 4 days postinfection. Antibody was purified by protein A Sepharose column chromatography and the purified monoclonal antibodies were labeled with Bolton-Hunter reagent. ‘%I-Labeled protein A was purchased from New England Nuclear. Production ducing cell

of mxmoclonal antibody-pm lines. Six-to-ten-week-old

BALB/c mice (Jackson Laboratories) were injected intraperitoneally three times at weekly intervals with a mixture of purified Ad169 and Towne CMV (50 gg each injection in incomplete Freunds adjuvant). Two weeks after the last injection the mice were boosted with a tail vein injection of a mixture of the CMV strains. Four days later the mice were sacrificed, the spleens removed, and the spleen cells fused with the nonsecreting myeloma line P3X63Ag8.653 (obtained from the Salk Institute, San Diego) by polyethylene glycol 1000 treatment. The cells were distributed into 96well plates and selected for growth in RPM1 (GIBCO) medium supplemented with 10% NCTC-109 (Microbial. Assoc.), 20% fetal calf serum, 0.1 mM hypoxanthine, 0.4 mM aminopterin, and 0.01 mlM thymidine (HAT medium). Culture fluids were tested for antibodies to CMV proteins by a plate-binding assay. Positive clones were

CYTOMEGALOVIRUS

ANTIBODIES

subcloned several times until stable subclones were obtained. ImmunologhJ procedures. Plate-binding assays (Kohler, 1980), immunoprecipitation protocols (Sarnow et aL, 1982a), and the double radioimmunometric assay (Thomas et a& 1983) were all done as described previously. SDS-polyacrylamide gel electrophoresis was carried out as described by Laemmli (1970). Western blot hybridization (radioimmunoelectrophoresis) was carried out by the procedures described by Burnette (1981). Samples of fresh human urine were obtained from 100 infants. Nasopharyngeal aspirates were obtained from 250 individuals. Only fresh samples (less than 2-4 hr old) transported at 4” were utilized in these tests. Cells were centrifuged out of solution or suspension, transferred to slides or coverslips, fixed, and stained with antibodies as described previously (Reich et c& 1983). The samples were also cultured with primary human fibroblasts to determine if cytomegalovirus could be detected based upon its characteristic cytopathic effect in these cells. This test was read after 21 days in culture. These tests were carried out in the viral diagnostic laboratories at the Hopital Trousseau, Paris, France. Human antisera. CMV antibody-negative serum was from a healthy, seronegative donor, devoid of detectable titers to CMV antigens as tested by immunofluorescence, KBR, and ELISA testing. CMV antibody-positive sera were from (1) a convalescent adult female (AS/PJ, KBR titer of 164, (2) an adult female with a primary CMV infection (AR/IS, KBR titer of 1:128, IgM positive), (3) a 7-day-old female with a CMV infection (AS/NW, KBR titer of 1:128, IgM positive) and, (4) a series of sera from an 8-year-old male obtained at various times after a primary CMV infection (AS/TBa, KBR titer of 164, IgM positive; AS/TBb, KBR titer of 1:128, IgM negative, AS/TBc, KBR titer of 1:256, IgM negative). RESULTS

Identification of Vi&n Proteins and G/ycoproteins of Cytomegahirus As a preliminary step in characterizing the CMV structural proteins detected by

AND

SERA

327

the monoclonal antibodies, total virion-associated proteins and glycoproteins were identified. Purified disrupted virions were iodinated with Bolton-Hunter reagent and analyzed on SDS-polyacrylamide gels. An autoradiogram of such a gel employing the CMV Ad169 and Towne strains is presented in Figs. la and b, respectively. Approximately 25 proteins could be identified to be virion constitutents of both CMV strains, ranging in molecular weight from greater than 200,000 to 14,000 Da. The molecular weights of size standards are indicated on the right side of Figs. la and b. On the left side of the figure the positions of the two glycoproteins gp58 and gp130 and of the ‘71,000-Da phosphoprotein (~~71) are noted; these are the proteins detected by the monoclonal antibodies under study in the first set of experiments. Gp130 is only rarely detected by iodination of virions (compare Figs. la and b with c and d). To identify some of the glycoproteins of CMV, purified Towne CMV virions labeled with [?S]methionine during virus replication (Fig. lc) and unlabeled Towne CMV (Fig. Id) were electrophoresed through SDS-polyacrylamide gels. The virion-associated proteins were reacted with ‘z?-concanavalin A (Con A) which binds to a-methylmannosides and related sugar residues. An autoradiogram of the doublelabeled ([35S]methione and ‘251-Con A) virion-associated proteins and the Con A reactive glycoproteins is presented in Figs. lc and d. Three major virion-associated glycoproteins were detected. These autoradiograms (Fig. 1) also provide evidence for the purity of the CMV virion antigen employed to immunize mice and to screen the resultant monoclonal antibody-producing cell lines. Plate-Binding Assay Screen for Detection of Monoclonal Ant&n&-Producing CeU Lines Mice were immunized with a mixture of purified Ad169 and Towne CMV strain virions. Hybridoma cell lines were produced as detailed under Materials and Methods. After a preliminary screening and subcloning, the culture fluids from nine monoclonal antibody-producing cell lines were tested for activity in a plate-binding assay.

328

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a

gPl=

ET

AL.

b

c

‘130

9P

130 94

PP

68

9P

58

94 PP 71

88

w 58

58

d

43

30

21.5

FIG. 1. Analysis of CMV proteins and glycoproteins from purified virions. Strain Ad169 and Towne CMV were purified. In lanes a (Ad169) and b (Towne) viruses were sonicated, iodinated with Bolton-Hunter reagent, and analyzed on SDS-polyacrylamide gels. An autoradiogram of the labeled proteins from these two strains of CMV is presented. Lane c presents an autoradiogram of Towne [ssS]metbionine-labeled purified CMV proteins after electropboresis on an SDS-polyacrylamide gel. The glycoproteins are labeled in addition by iodinated Con A. Lane d presents an autoradiogram of CMV (Towne) glycoproteins detected by reacting unlabeled CMV proteins with iodinated Con A after gel electrophoresis. The numbers on the right-hand side of each figure represent molecular weights (X10-*) determined by protein standards included in adjacent wells of each gel. The designations on the left of each photograph (gp130, ~~‘71, and gp58) identify the virion proteins against which monoclonal antibodies were prepared in this study.

Individual wells of a microtiter dish were coated with 109 ng of purified virion protein from strain Ad169, Towne, or a mixture of both. After incubation of this antigen with antibody-containing tissue culture fluids, each of the wells was washed extensively. Antigen-antibody complexes were detected by incubation with ‘%I-labeled protein A. The unreacted protein A was removed by washing and an autoradiogram of the mi-

crotiter plate was obtained. Figure 2 presents one such autoradiogram of a screen of nine CMV-specific monoclonal antibodyproducing cell lines. In this experiment the subcloned cell line 42 had stopped producing antibody. All other cell lines made antibodies against both Ad169 or Towne virion proteins and some of these antibodies detected CMV strain differences in a quantitative fashion (clone 331, for example).

CYTOMEGALOVIRUS

-482 -78 -355 42 -243 -85 -331 -255 -215 -2A8

FIG. 2. Autoradiogram of a plate-binding.assay for detection of CMV monoclonal antibodies. Purified unlabeled CMV (166 ng) was coated onto each well of a microtiter plate. Wells received either Ad169 (Ad), Towne (To), or a mixture of both CMV strains (Ad/ To). Culture medium from nine subcloned CMV hybridoma-producing cell lines (492,78,365,42,243,85, 331, 265, 214) and a cell line-producing antibody against adenovirus (2A6) were added to each well. Antigen-antibody complexes were detected by adding lzI-protein A to the thoroughly washed wells. After removing unbound ‘%I-protein A by washing, an autoradiogram was obtained by exposure to X-ray film. A picture of this autoradiogram is presented. Subclone 42 failed to produce detectable antibody in this experiment.

The monoclonal antibody 2A6 was employed as a negative control. It was previously prepared against a human adenovirus protein (Sarnow et aL, 1982b). Identification of the Vi&n Proteins Detected by the CMV Mcrnodmal Antibodies After the monoclonal antibody-producing cell lines were subcloned several times and shown to be stable antibody-secreting

ANTIBODIES

AND SERA

329

cell lines, a more detailed analysis of the proteins detected by these antibodies was undertaken. Purified CMV virions were sonicated to partially disrupt intact virions in the preparation. The partially disrupted virions and disrupted virions (soluble antigens and protein aggregates) were then iodinated with Bolton-Hunter reagent. These antigens were incubated with antibodies from different hybridoma clones. Antigen-antibody complexes were immunoadsorbed with protein A on Staph&+ coccus aureus (Cowens strain) bacteria and the complexes collected by centrifugation. After several washings, the immunoprecipitate was analyzed on SDS-polyacrylamide gels. An autoradiogram from such a gel is presented in Fig. 3. Ad169 and Towne strains of CMV were employed as markers (without immunoprecipitation) and were also alternated in the first and second wells, respectively, as antigen sources for each monoclonal antibody tested. Hybridoma cell lines 355 and 331 produced antibodies that detected a single polypeptide of 71,000 Da (~~‘71) in the immunoprecipitation reaction. In contrast to this observation, hybridoma-producing cell lines 492, ‘78, 243, 85, 255, and 214 immunoprecipitated all of the CMV virion proteins, but specifically enriched for the 58,000-Da glycoprotein as compared to the levels of gp58 observed when Ad169 or Towne virions were analyzed without immunoprecipitation (Fig. 3). Gp58 was observed to migrate with a different apparent molecular weight in the Ad169 and Towne strains of CMV. Because the iodinated antigens were a mixture of intact and disrupted virions it may be suggested that gp58 is on the surface of virions, and antibodies to gp58 react with intact CMV, coimmunoprecipitating all associated virion proteins, while enriching for gp58 by also immunoprecipitating the soluble form of this antigen. By the same argument pp71 (71,000 Da) would be located inside the virion and therefore only the soluble pp71 would be detected by immunoprecipitation (Fig. 3). This hypothesis was confirmed by additional experiments. Purified CMV virions were disrupted by NP-40 (0.5%) and Na-deoxycholate (0.5%), and the virion core was separated from the

NOWAK

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AL.

FIG. 3. Immunoprecipitation of CMV virion-associated proteins from strains Ad169 and Towne. Purified Ad169 (Ad) and Towne (To) strains of CMV were sonicated to partially disrupt the virus and solubilize some proteins. This mixture of intact virions and soluble CMV proteins was iodinated and then incubated with monoclonal antibodies from subclones 492, 78,355,243,85,331,255, and 214, as well as 2A6 which produces anti-adenovirus monoclonal antibody (negative control). The lanes marked Ad and To were not incubated with antibody but represent the total viral proteins in the antigen preparation. For each monoclonal antibody the first well contains Ad-CMV and the second well To-CMV as antigen. Antigen-antibody complexes were collected by immunoadsorption to S oureua protein A and centrifugation. The immunoprecipitates were analyzed on SDS-polyacrylamide gels. An autoradiogram of the gel is presented. Molecular weight markers (XIOea) are indicated at the right side of the figure and the migration of gp130, gp58, and pp71 is noted at the left side of the picture. Monoclonal antibodies 355 and 331 detect only pp71 while monoclonal antibodies 492,78,243,35,255, and 214 enrich for gp53, which shows a molecular weight heterogeneity between Ad and To, and coimmunoprecipitate associated virion proteins.

solubilized virion membrane proteins by centrifugation. Gp58 was solubilized and did not sediment with the high-molecularweight virion core. In contrast, part of the pp71 protein was associated with the rapidly sedimenting core particles, while some pp71 remained in the supernatant. Under the same conditions, nucleocapsid constituents are strongly associated with the insoluble pellet, suggesting that pp71 is an internal envelope protein in contact with nucleocapsids. The soluble antigens were then diluted out of the detergent (l/10)

and could be immunoprecipitated with antibodies directed against pp71 or gp58. These [asS]methionine-labeled antigens were analyzed on SDS-polyacrylamide gels (Fig. 4). When the labeled CMV-soluble antigens were incubated with a monoclonal antibody not directed against CMV proteins (Fig. 4d, Pab 421) small levels of the major CMV structural protein (65,000 Da) nonspecifically contaminated the immunoprecipitated S. aureus-antibody complex. The soluble CMV proteins incubated with antibody 355 (Fig. 4~) immunopre-

CYTOMEGALOVIRUS

a

b

c

ANTIBODIES

331

and a diffuse set of proteins in the range of 130,000 Da (designated as gp130). gp130 is poorly iodinated (Fig. 1)

d

Towne strain)

PP gP

AND SERA

- 58 - 43

- 30

FIG. 4. Immunoprecipitation of detergent-treated CMV virion proteins. Purified Towne CMV-labeled in tivo with [96S]methionine was disrupted by treatment with 0.5% NP40 and 0.5% sodium deoxycholate for 1 hr at 0’. The soluble antigens were then diluted 11 10 into 50 mM Tris-HCI, pH 8.0, 5 mM EDTA, 150 m&f NaCl, and 0.5% NP40 and incubated with either monoclonal antibody 492 (lane a), 85 (lane b), 355 (lane c), or Pab 421 (lane d) which does not detect CMV structural proteins (negative control). The antigen-antibody complexes were adsorbed to S. aureus protein A and the washed immunoprecipitates were analyzed on SDS-polyacrylamide gels. An autoradiogram of a slab gel is presented. Monoclonals 492 (lane a) and 85 (lane b) immunoprecipitated gp130 and gp58. Monoclonal 355 detected the pp?l protein. Pab 421 (lane d) does not detect, any CMV proteins (negative control) but contains a very low level of the major CMV structural protein of 65,000 Da which contaminates all samples of this experiment. Molecular weights (X10-‘) are given, and the position of gp58, gp130, and pp71 are indicated.

cipitated only the pp71 protein while soluble proteins incubated with antibody 492 (Fig. 4a) or 85 (Fig. 4b) immunoprecipitated a major protein with apparent size between 55K and 60K (designated as gp58

and was detected by a reaction with iodinated Con A (Fig. Id). The simultaneous precipitation of both glycoproteins, gp130 and gp58, could be due to a cross-reaction of a common sugar moiety or gp58 could be derived from gp130 via proteolytic processing. Peptide maps of gp130 and gp58 show that these two glycoproteins contain common tryptic peptides (Nowak and Anderson, unpublished results). The specificity of two additional classes of monoclonal antibodies, obtained in an independent B-cell fusion experiment, were tested by employing radioimmunoelectrophoresis or Western blot analysis. Virion proteins from the CMV Towne strain were separated by SDS-polyacrylamide gel (15%) electrophoresis and the proteins were transferred to nitrocellulose paper. The paper was cut into strips and stained with amido black (Fig. 5, lanes a-d). Three of the strips were then incubated with monoclonal antibodies from clone 127 (Fig. 5, lanes b and e), clone 157 (Fig. 5, lanes c and f), and as a negative control Pab 421, a monoclonal antibody known not to react with CMV proteins (lanes d and g). The antigen-antibody complexes were detected A which by incubation with ‘?-protein binds to these antibodies. Figure 5 presents a photograph of the protein-stained filters (lanes a-d) and an autoradiogram of the ‘251-protein A-detected antigens. The monoclonal antibody clone 127 reacted with a 155,000-MW virion antigen termed pp155 (see Fig. 1 for the 145-155K-Da doublet) while monoclonal antibody clone 157 reacted with a 29,000-MW antigen, termed pp29 (Fig. 5, lanes c, f). Table 1 reviews the properties of the four proteins detected by different classes of these monoclonal antibodies and lists the B-cell line clone numbers from independent isolates. CMV Protein Detected by Immune Human Polyclonal Sera

To determine which CMV structural proteins elicit an immune response in humans after an active CMV infection, sera from convalescent patients were employed

332

NOWAK

a

bed

ef9

-155

- 29

FIG. 5. Electrophoretic transfer of proteins from purified virions and subsequent immune reactions with monoclonal antibodies. Virion proteins of strain Towne were separated on a 15% SDS-PAG and transferred to NC paper. The paper was cut in strips. One strip was directly stained with amido black (lane a), the other strips were incubated with monoclonal antibodies. Immune reactions were detected by ‘%I-labeled protein A and subsequent autoradiography (lanes e-g). Then these filter strips were also stained with amido black (lanes b-d). The filter shown in lane b was incubated with the monoclonal antibody 127 and marked at the top with radioactive ink. The autoradiogram is shown in lane e. The 127 antibody was found to react with the upper band of the doublet of 145,000-155,000 Da. The filters c and d were incubated with the monoclonal antibodies 157 and Pab421 (negative control, a monoclonal antibody to the cellular p53 protein), respectively. The result is shown in lanes f and g on the autoradiogram. Molecular weights (X10e3) of viral proteins are indicated.

to either immunoprecipitate or in a Western blot format rified CMV or infected cell source of antigen. Purified ([35S]methionine labeled)

CMV proteins employing puextracts as a Ad169 virions and CMV-in-

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fected or mock-infected human fetal fibroblasts labeled with [35S]methionine were resuspended in 0.5% NP40 and sonicated twice at 4” to disrupt virions and aggregates. After centrifugation the supernatant fraction was incubated with immune or nonimmune human sera and the antibody-antigen complexes were collected by adsorption to X aureus protein A and centrifugation. The washed immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis. An autoradiogram of such a gel is presented in Fig. 6. Lanes a and b contain an aliquot of the purified CMV preparation and the [“S]methionine-labeled crude-infected cell extract (the antigens employed) without exposure to antisera. Purified virus incubated with normal human serum (CMV antibody negative) (lane c) or two different CMV antibody-positive sera, AS/TBc in lane d and AS/IS (lane k), show that the human antibodies react with a large number of CMV structural proteins. Proteins from CMV-infected cell extracts (lanes e-g) or from mock-infected cell extracts (lanes hj) were incubated with CMV antibody-negative control sera (lanes e, h) or CMV antibody-positive sera AS/TBc (lanes g, j) and AS/NW (lanes f, i). It is clear from these results that a number of CMV-specific proteins found in virions or infected cells are detected by immune sera but not normal or nonimmune sera. These results are in good agreement with a similar set of experiments carried out by Pereira et al. (1982). One of the difficulties with the interpretation of these results is that CMV virion proteins are known to aggregate, forming oligomeric protein complexes in solution (see Fig. 3). Antibody directed against one protein in the aggregate could coimmunoprecipitate other CMV proteins in the oligomeric protein complex. To avoid this potential problem Western blot radioimmunoelectrophoresis was carried out employing either CMV-infected cell extracts or mock-infected cell extracts as a source of antigen. The total infected or mock-infected cell protein was fractionated by SDS-polyacrylamide gel electrophoresis and the proteins were transferred to nitrocellulose strips which were then incu-

CYTOMEGALOVIRUS

ANTIBODIES TABLE

PROPERTIES OF CMV

PROTEINS

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SERA

333

1

DETECTED

BY MONOCLONAL

ANTIEWJDIES

Immunoreactivity Apparent molecular weight (X10-*) 155 (PP155) 71 (PPW 130-53

(gp58)

29 (PP2%

Modiications proteins’ PO. PO4 Glycoprotein PO4

of

Location in viriona

Polyclonal immune eera

Internal Internal Envelope

+ + +

Internal

+

bated with immune or nonimmune human sera. After exposure to the antibody, the washed strips of nitrocellulose paper were incubated with an anti-human antibody ELISA assay resulting in a brown color staining of the antigenic bands. Mock-infected cell extracts incubated with immune serum AS/TBc (Fig. 7, lane a) or a serum devoid of CMV antibodies (Fig. ‘7, lane 5) failed to detect any antigens in this assay. CMV-infected cell extracts employed as antigen and serum derived from an g-yearold male and obtained at different times after CMV infection (Fig. ‘7, lane b, AS/ TBc; lane c, AS/TBb; lane d, AS/TBa) detected a large number of CMV-specific antigens while CMV antibody-negative sera and the same antigen extract failed to detect any antigen (Fig. ‘7, lane e). Similarly when purified CMV virions (Towne) were employed as an antigen, three different antibody-positive sera, from different patients, AS/TBa (lane g), AWPJ (lane h), and AS/IS (lane i), detected a number of CMV structural proteins while CMV antibody-negative sera (lane j) failed to react with these virions proteins. It is clear from these data that humans respond to CMV infections by producing antibody directed against a wide variety of CMV structural proteins including those proteins detected by the monoclonal antibodies described in the previous section (Figs. 3-5, Table 1). A Lloubk Monoclrmal Raditnhmurumcetric Assay for CMV Virions and Antigens

Monoclonal antibodies to major surface virion glycoproteins (gp130 and gp58)

Monoclonal clone numbers 127 355,331

85,492, 243, 78,155, 214, 150, 3, 31 108, 142, 157

should be useful in clinical diagnosis. To demonstrate this a radioimmunometric assay was developed and tested with CMV virions. Monoclonal antibody 492 (Table 1) directed against gp130 and gp58 was covalently fixed to protein A on S. aureua by glutaraldehyde crosslinking. The S. aureua-protein A/492-antibody reagent was incubated with known concentrations of purified CMV. The antigen-antibody complexes were collected by centrifugation and washed. Purified ‘?-labeled monoclonal antibody 85 was then added. This provided a complex composed of S. aureus-protein A, Ab-492-CMV antigen, and Ab-85 labeled with ‘%I. This complex was pelleted, washed, and counted. Figure 8 presents the results of antibody 85 bound (cpm) as a function of the concentration of antigen. Because purified CMV virions were employed as antigen, the concentration of antigen can be translated into virion particles per milliliter and plaque-forming units per milliliter of CMV detected in this assay. Employing this pair of monoclonal antibodies (492 and 85) it was possible to detect 0.1 bg of protein/ml of virus preparation which is approximately 6 X 10’ particles and (in this preparation) 6 X lo3 PFU/ml. This test may be useful for diagnostic purposes. It has been successful using buffered human urine and saliva, two of the body fluids most commonly tested for the presence of CMV. Second, the sensitivity of the test can be increased by incubating the S. aureus-protein AiAb-492 reagent with 10 ml of urine and CMV. By pelleting the antigen-antibody S. aureua complex and resuspending in a smaller

334

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a

b

cdefghij

ET AL. k

71 65 56

40 37

33

-

26

21

FIG. 6. Immunoprecipitation of [?Sknethionine-labeled CMV (AD169) proteins from purified virion preparations and from cell extracts. AD169 virions and CMV-infected and mock-infected HFF cells were labeled with [%]methionine and prepared for immunoprecipitation was described under Materials and Methods. An aliquot of the virus preparation (lane a) and of the infected cell extract (lane b) was electrophoresed directly. Equal amounts of antigen from purified virions were treated either with a CMV-negative human serum (lane c) or with CMV-positive antisera AS/TBc (lane d) and AS/IS (lane k). Proteins from CMV-infected cell extracts (lanes e-g) and from mock-infected cell extracts (lanes h-j) were immunoprecipitated either with a negative control serum (lanes e, h) or with CMV-positive sera AS/NW (lanes f, i) and AS/TBc (lanes g, j). The immune complexes were collected by addition of S uureus. Antigens were eluted in sample buffer (Laemmli, 1970) and separated on a 12.5% (lanes a-j) or 15% (lane k) SDS-polyacrylamide slab gel. Labeled proteins were detected by autoradiography. Molecular weights (X10’) of viral proteins are indicated.

volume for the second antibody (85) reaction, the sensitivity is increased about tenfold (6 X IO6 particles of CMV and 6 X 10’ PFU/ml). Lastly, the use of two different monoclonal antibodies, directed against two different antigenic determinants on the same protein, ensures the specificity of the diagnostic test. This property eliminates a major problem in using monoclonal antibodies for diagnostic purposes. Many monoclonal antibodies

cross react with contaminant antigens. The possibility of two different monoclonal antibodies directed against different antigenie determinants (even on the same protein) both cross-reacting with a contaminant antigen is very low. Fluorescent Antibody Detection of CMV-lnfected Cells from Patients The availability of well-characterized monoclonal antibodies directed against the

CYTOMEGALOVIRUS

ab

c

d

ANTIBODIES

ef

AND SERA Q

h

335 i

66 58

215

FIG. 7. Electrophoretic transfer of proteins from cell extracts and purified virions and subsequent immune reactions with human sera. Proteins were separated on a 15% SDS-PAG and transferred to NC paper. The paper was cut in strips which were incubated with different human sera. Transferred proteins from mock-infected cell extracts were incubated with human CMV-positive serum AS/ TBc (lane a) or a CMV-negative serum (lane f). Transferred proteins from Towne-infected HFF cell extracts were treated with a series of CMV-positive sera, taken at different times from an 8year-old boy, AS/TBc (lane b), AWTBb (lane c), AS/TBa (lane d), or with a CMV-negative serum (lane e). Transferred proteins from purified virions (strain Towne) were incubated with three CMVpositive sera from different patients, AS/TBa (lane g), AWPJ (lane h), AS/IS (lane i), or with a CMV-negative serum (lane j). The antigens were detected by a subsequent ELISA assay, resulting in a brown color staining of the antigenic protein bands. Molecular weights (X10-‘) of marker proteins and of viral protein are indicated.

structural proteins of CMV provides an opportunity to utilize these antibodies in a rapid direct diagnosis of virus infection. To accomplish this an attempt was made to recover cells either from fresh urine samples obtained from infants (Stagno et d, 1982) or from nasopharyngeal aspirates (Neiman et d, 1977; Meyers et al, 1982). Cells were centrifuged out of suspension from 100 fresh urine samples and 250 nasopharyngeal aspirates. These cells were fixed onto slides and stained with the CMV monoclonal antibody 65 preparations which detect the gp58 virion antigen. Fluorosceinlabeled anti-mouse antibody was employed

as a second antibody to detect the binding of the CMV mouse monoclonal antibody. At the same time the urine and nasopharyngeal samples were cultured for infectious CMV to compare results with the fluorescent antibody testing. Out of 100 urine samples tested in this way 16 were positive for infectious CMV and 10 of these were also positive for the presence of gp58 by fluorescent antibody testing. Clone 85 antibodies detected gp58 in the cytoplasm of infected cells (Fig. 9A) from urine samples. The other 64 samples were negative for infectious CMV and antibody-tested infected cells. From the 250 samples of na-

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envelope glycoprotein, gp58, and a second glycoprotein, gp130, which may be a precursor to gp58 or antigenically related to it. The apparent molecular weights of gp58 in SDS-polyacrylamide gels differ when the Towne and Ad169 strains of CMV were compared. This was the major difference in strain variations detected in this study. It may therefore be possible to examine CMV strain variations as a function of dify.-.-. , I ferent CMV-associated diseases or syn-5 -6 -9 dromes using such monoclonal antibodies. LOG [&TEIi~ It is now clear monoclonal antibodies can FIG. 8. Double-monoclonal radioimmunometric as- be employed to determine strain heterosay for CMV. 5! aureas protein A-antibody 492 com- geneity of the CMV structural proteins. plexes were incubated with varying concentrations The three other classes of monoclonal anof purified unlabeled CMV. The S aureus protein Atibodies obtained in this study detected Ab492 antigen complex was then incubated with ‘%I- virion-associated phosphoproteins, pp155 labeled antibody 85 and the antibody-antigen-anti(clone 127), pp71 (clone 355), and pp29 body complex was collected by centrifugation and (clone 108). Two of these antigens could be washed. The pellets were counted and the results plotted as [1261jAb 65 bound (cpm) as a function of detected in their denatured form by Westprocedures. Monoclonal anthe CMV protein concentration. The assay detects 196 ern blotting tibodies directed against an immediate ng protein which is approximately 6 X 10’ virion particles/ml or in this preparation, about 6 X 108 PFU/ early antigen, an early protein, and strucml. The assay can be performed with up to 10 ml of tural polypeptides have been described rehuman urine or saliva without losing this sensitivity. cently (Goldstein et ab, 1982; Pereira et al, The log of the protein concentration is given in grams 1982). Polyclonal immune human sera from of protein. patients recovering from CMV infections reacted with a large number of CMV strucsopharyngeal washings ‘7 were positive for tural proteins including those four antiinfectious CMV and 5 of these also detected gens detected by the monoclonal antibodgp58 by fluorescent antibody testing (Fig. ies. Both immunoprecipitation and West9B). The other 243 samples were negative ern blotting procedures detected CMV for infectious virus and infected cell anvirion proteins from either purified virus tigens. CMV-infected primary human fibroblasts (Fig. 9C), like the cells from the preparations or infected cell extracts when urine or bronchiole washings, showed a human sera were tested. In a series of sera taken over the time course of infection the bright cytoplasmic fluorescent antibody pp155 and pp29 antibodies were present at staining when clone 85 monoclonal antithe earliest time with antibodies directed bodies were used. In order to obtain intact against the major structural protein (~65) cells in the urine and nasopharyngeal developing later in time. washings, only fresh samples (less than Two different assays were developed that several hours after collection) kept at 4’ could be utilized for rapid diagnosis of CMV were employed. These results indicate the infections. The double-monoclonal rafeasibility of using fresh cells from urine assay has been used for and nasopharyngeal washings for the di- dioimmunometric rapid detection of CMV antigens in human agnosis of CMV infections. urine and saliva which are rich sources of the virus in external body fluids. This test DISCUSSION employs two different monoclonal antibodies directed against gp58. In order to Four sets of monoclonal antibodies have obtain a positive result, two different anbeen obtained that recognize four distinct tigenic determinants on the same protein structural proteins of human CMV (Table must be recognized. This requirement re1). Clone 85 antibodies reacted with a virion duces the possibility of a false positive due 20-

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FIG. 9. Fluorescent antibody staining of cells derived from urine or bronchiole washings. Cells from fresh urine or bronchiole washings were centrifuged out of suspension and fixed on slides. Clone 85 monoclonal antibodies were used to detect CMV gp58 antigen in infected cells using an indirect immunotluorescence assay. (A) Cells derived from the urine of a patient that also had infectious CMV in the urine. (B) Cells derived from bronchiole washings of a patient with infectious CMV in the sample. (C) Primary human fibroblasts infected with CMV. Photograph is taken at 20X magnification.

to a cross-reacting contaminant in the sample. The sensitivity of this test, determined in reconstruction experiments, is about 0.1 pg of antigen/ml which is about 6 X 10’ particles/ml or 6 X lo3 PFU/ml. Increasing the volume of the sample tested increased the sensitivity about tenfold. This is the range of virus concentrations detected in urine or saliva of CMV-infected individuals. The second test employed to rapidly diagnose CMV infections also used clone 85 antibodies in a fluorescent antibody mode to detect CMV-infected cells in nasopharyngeal washings or urine. Out of 250 samples from the nasopharyngeal washings, ‘7 contained infectious CMV and 5 of these samples contained cells that gave cytoplasmic fluorescence with clone 85 antibody. The remaining 243 samples were negative for infectious virus and fluorescent cells. From 100 urine samples, infectious CMV was isolated from 16 samples and 10 of those 16 samples contained cells that clone 85 antibodies detected gp58 in the cytoplasm. The remaining 84 samples were negative for both virus and antigen.

In this study it was important that urine samples be kept cold (4”) and not be older than 2-4 hr after collection. When these conditions were not fulfilled, intact cells could not be detected in the samples. These experiments demonstrate that a rapid diagnosis of CMV infections is feasible and fairly reliable. This is the first indication that cells obtained from body fluids will be useful in this process. ACKNOWLEDGMENTS This work was supported by the New York State Science and Technology Foundation, Academic Research Associates, and the Sklarow Foundation, as well as the Bundesministerium fur Forschung und Technologie, Projekttrager Biotechnologie. The excellent technical assistance of Agnes Gmeiner and Gail Urban is greatly appreciated. REFERENCES BURNETTE, W. N. (1981). Western blotting electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112. 195-203.

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