IMMUNOGENIC GLYCOPEPTIDES OF HUMAN CYTOMEGALOVIRUS

Abstract: A monoclonal antibody produced by a process comprising: (a) immunizing a mouse with HCMV virions; (b) fusing spleen cells from said mouse with cells from a myeloma line to produce hybridomas; (c) selecting from said hybridomas a hybridoma which produces a monoclonal antibody (i) which is reactive with HCMV, but does not react with Herpes simplex, adenovirus, Epstein Barr virus or Varicella virus, and wherein said monoclonal antibody is capable of neutralizing HCMV or (ii) which is reactive with HCMV, Herpes simplex and adenovirus and which antibody is capable of neutralizing HCMV in the absence of complement; and (d) clonally expanding said hybridoma.

Description: -I- IMMUNOGENIC GLYCOPEPTIDES OF HUMAN CYTOMEGALOVIRUS Field of the Invention This invention relates to human cytomegalo- virus (HCMV), in particular to glycopeptide components thereof, which are capable of eliciting an immune response. These glycopeptides and antibodies reactive with them are useful in diagnosis and therapy. Background of the Invention Human cytomegalovirus has been associated with a number of clinical syndromes. The infection is usually asymptomatic and self-limiting in the immuno- competent child or adult, but can cause severe congeni¬ tal disease in the fetus or infant and morbidity and mortality in immuno-co promised patients. HCMV is a common cause of mental retardation in children who acquire the infection ir^ utero from mothers carrying an active infection, and is considered the leading oppor¬ tunistic infection causing complications in i muno- suppressed transplant patients. HCMV is also an • important consideration in the treatment of Acquired Immunodeficiency Syndrome (AIDS]. It has recently been suggested that HCMV is the causative agency in Kaposi' s sarcoma, a cancer often seen in AIDS. Much work has been done in recent years on the basic nature of HCMV in order to ameliorate these - problems. HCMV is a virus of the herpes type, composed of a nuclear complex of nucleic acid and proteins, internal capsid proteins with structural or enzymatic functions and an external membrane envelope, containing glycopeptides and glycolipids. The first two groups are sequestered within the virion and may not be available in the intact, infectious virion to react with the immune system or antibodies. Furthermore, their presence in bodily fluids is not diagnostic of active infections as it is typical of herpes-type infections to go into a quiescent phase, where viral products are produced but infectious virions are not released. In the same manner, an initial infection may be abortive, i.e., the host cells begin to produce viral products, but these products are not assembled into virions so that active infection is never established. HCMV can be diagnosed by one or more of the following methods: 1) identification of HCMV in virion bodies or CMV antigens in infected tissues by using indirect immunofluorescent microscopy, 2) detection of the virus in cell monolayers inoculated with infectious tissue or body fluids, 3) identification of HCMV- specific antibody in serum or, 4) hybridization with a complementary-labeled DNA fragment. For a review of current methods of diagnosing HCMV, see Mayo Report (Mayo Clin. Prac, 60, 636, (1985)). The most common method of diagnosing HCMV has been culturing the sa ple In human fibroblasts and • inspection for typical cytopathic effects. This method has the advantage of being useful with a number of different, easily obtainable samples such as urine, throat washings or blood. After two to four weeks of growth, cultures can be Inspected for evidence of HCMV infection, such as inclusion bodies or the typical giant, fused cell. This method has the disadvantage of requiring up to one month before diagnosis is con¬ firmed. Diagnosis utilizing monoclonal antibodies (MoAbs), using ELISA or direct immunofluorescence has been attempted. For example, Schuster et al. (Mayo Clin. Proc, 60, 577-585 (1985)) have produced MoAbs to early and late HCMV nuclear proteins for use in immuno- fluorescence. The MoAb can be labeled with a fluor¬ escent .material and binding to intracellular HCMV in biopsy specimens or cultured cells can be observed. This kind of diagnosis is much more rapid than the culture method. Results may be available in less than one day. Emerging antiviral therapies may become useful in treating HCMV infections. In the case of transplant patients, the im unosuppressant drug regimen can be altered to allow reactivation of latent HCMV. Further¬ more, the symptoms of HCMV may mimic and therefore mask rejection so that corrtrol of transplant rejection requires accurate and rapid diagnosis of possible HCMV- active infections. Antibodies against viruses can be injected directly into the circulation of HCMV patients, where the antibodies will bind to circulating virions and render them incapable of infecting host cells, thereby directly limiting the infection. Additionally, nonexposed adults, especially females of childbearing age, may advantageously be administered a vaccine to confer protection from HCMV infection. U.S. Patent No. 3,959,466 describes such a vaccine, using attenuated HCMV which is passed at least 50 and preferably 125 to 150 times through culture in human lung fibroblasts. Such vaccine is said to induce immunity against HCMV without allowing spread of the virus through the body. However, it is well known in the art that such attenuated viral vaccines, as well as "killed" vaccines, may have a certain low incidents of reversion to a virulent form. This has been well noted in the case of the Salk and Sabin polio vaccines. Recently, subunit vaccines have been success¬ fully constructed. Such subunit vaccine consists of only a portion of the virus and is not capable of causing the production of active virus. Alternatively, genetic material coding for i munogenic gene products can be spliced to a nonvirulent carrier virus, such as Vaccinia. The recombinant virus has the advantage of being reproduced for a time within the body, therefore stimulating immunity to a greater extent than can a killed virus. It is becoming increasingly clear that the research on the immunology of HCMV must look to the membrane envelope components, especially the glycopep¬ tides, as immunogens and markers of active HCMV infec- tion. The starting material for a virus preparation includes, in addition to the desired membrane envelope components, host cell debris, nuclear proteins, capsid proteins and precursor peptides which are not found in the mature, infective virion. Even among the membrane envelope glycopeptides, not all are physically access¬ ible to the immune system because they may reside in a fold or crypt or be sterically hindered from interac¬ tion with host immune responsive cells. Therefore, it is desirable to provide a mem- brane envelope glycopeptide that is physically accessible to interaction with the immune system and with antibodies raised to it, in addition to eliciting antibodies with good in vitro reactivity, e.g., with respect to their binding to the glycopeptide. When such a glycopeptide on the surface of a virion reacts with its corresponding antibody, the virion is rendered incapable of infecting the host cell, that is, it is neutralized. Such a glycopeptide and antibodies raised against it are useful in diagnosing active HCMV infec ¬ tions, treating such infections and, as a component of combined or subunit vaccines, for preventing Infection in susceptible populations. Before such antibody therapy, diagnosis of active HCMV Infection or vaccine can be maαe available, it is necessary to identify and provide those composi¬ tions which stimulate humoral and cellular immunity. The glycopeptides of HCMV and other viruses have proved useful in this respect. For example, detergent extracts of HCMV containing certain membrane envelope glycopeptides can induce humoral and cellular immunity in the guinea pig. MoAbs have been obtained which i munoprecipitate glycopeptides with molecular weights of 50,000-58,000, 93,000 and 130,000 daltons that were associated in disulfide complexes. Some of these anti¬ bodies were observed to have neutralizing activity in vitro but only in the presence of complement. Only one MoAb, which reacted with an 86,000 dalton protein of HCMV was neutralizing in the absence of complement (Ras ussen et al., Proc. Natl. Acad. Sci. USA, 81 , 876-880 (1984)). In 1976, Kim et al. (J. Virol., 20, 604-611 (1976)) were able to find aύ least 33 polypeptides in HCMV, ranging in size from 11,000 to 290,000 daltons. Six of these were shown to be glycopeptides, but the resolution technique, elec'rophαresis in non-reducing detergent gel, aid not resolve these glycopeptides sufficiently to allow characterization of the indivi¬ dual molecules. 0ns viral protein, a glycosylated pro- tein of molecular Weight 66,000 was found to be the most abundant viral polypeotide. Kim later succeeded in partially purifying this glycopeptide and raising MoAbs against it. However, none of his MoAbs was capable of neutralizing HCMV even at very high con- centration. A mixture of nine MoAbs also failed to exhibit virus-neutralizing activity (Kim et al., J_. Clin. Mlcrobiol., 13, 331-343, 1983). Therefore, a need exists for providing those HCMV glycopepricas capable of eliciting a protective humoral response. An additional needs exists for MoAbs directed against such glycopeptides. Brief Description of the Invention The present invention is directed to composi¬ tions of matter which are substantially-pure immuno- genic glycopeptides which are preferably derived from HCMV. These glycopeptides are found on only the mem¬ brane envelope of the intact HCMV virion, where they are associated with other envelope glycopeptides via disulfide linkages. The glycopeptides are physically accessible to the immune system and to antibodies, and are capable of stimulating both humoral and cellular immunity in humans. One embodiment of this invention comprises such a glycopeptide, herein termed GLP-A, which has a molecular weight of about 93,0.00 daltons. GLP-A comprises determinants which react with monoclonal antibodies, which antibodies react with HCMV but do not react with Heroes simplex, adenovirus, Epstein Barr virus or Varicella virus, and which antibodies are capable of neutralizing HCMV, preferably in the pre- sence of complement. A second embodiment of this invention com¬ prises another such glycopeptide, herein term GLP-B, which has a molecular weight of about 50,000 to 52,000 daltons. . GLP-B comprises determinants which react with monoclonal antibodies, which antibodies react with HCMV, Herpes simplex and adenovirus and which anti¬ bodies are capable to neutralizing HCMV in the absence of complement. To obtain the glycopeptides of the present Invention, HCMV from primary clinical isolates or such established strains as Towne or Toledo are first grown in a human cell, most conveniently, primary skin fibroblast cultures. Intact virions shed from the cell accumulate in the supernatant fluid, from which they may be concentrated by any standard means, preferably differential centrifugation followed by sucrose gradient centrifugation. The membrane envelope is stripped from the virion and the disulfide-linked gly¬ copeptide complexes are further purified by high pressure liquid chromatography through an anion exchanger. Immunoprecipitation of the glycopeptide complexes, followed by reduction of the disulfide linkages, alkylation and gel filtration HPLC yielded the substantially-pure glycopeptides of the present invention. Therefore, as used herein, the term "substan¬ tially-pure" is intended to mean that the glycopeptide complexes or the glycopeptides have been extracted from their natural association on the membrane envelope of HCMV with other membrane components and from internal nuclear or capsid comoonents of HCMV. The procedures of the present- invention readily afford a method for producing and purifying polypeptides or polypeptide complexes containing varying degrees of glycosylation, all of which are useful in the production of neutra¬ lizing MoAbs and whicn are also useful for the diagno¬ sis of active HCMV infections. The glycopeptides may be used to immunize mice in the first step of the well-known Kαhler-Milstein method for producing monoclonal antibodies. More con¬ veniently, a composition comprising whole HCMV virions may also be so used. Thus, the present invention also comprises MoAbs which bind to determinants present on GLP-A and GLP-B. Preferred MoAbs of the invention can neutralize HCMV, and can provide the basis for a diagnostic test for intact virions. Therefore, the present invention is directed to a monoclonal antibody produced by a process compris¬ ing: (a) immunizing a mouse with HCMV virions; (b) fusing spleen cells from said mouse with cells from a myeloma line to produce hybridomas; (c) selecting from said hybridomas a hybri¬ doma which produces a monoclonal antibody which is reactive with HCMV, but does not react with Herpes simplex, adenovirus, Epstein 8arr virus or Varicella virus, and wherein said monoclonal antibody is capable of neutralizing HCMV; and (d) clonally expanding said hybridoma. The present invention is also directed to a monoclonal antibody produced by a process comprising: (a) immunizing a- mouse with a HCMV virions; (b) fusing spleen cells from said mouse with cells from a myeloma line to produce hyb s ridqmas; (c) selecting from said hybridomas a hybri¬ doma which produces a monoclonal antibody which is reactive with HCMV, Heroes simplex and adenovirus and which antibody is capable of neutralizing HCMV in the absence of complement; and (d) clonally expanding said hybridoma. Brief Description of the Drawings Figure 1. Unreduced detergent extracts of HCMV Towne strain were fractionated by ion-exchange HPLC. The retained peaks 2-4, which contained glycopeptides and glycopeptide complexes, were examined for their reac¬ tivity to the monoclonal antibodies. Panel (A) Profile of radioactivity obtained by ion-exchange HPLC of unre¬ duced [ 3 HJ glucosamine labeled strain Towne HCMV extract. Panel (B) Glycopeptides from peaks 1-4 were examined by electrophoresis without reduction. Panel (C) Glycopeptides from peaks 2-4 were immunoprecipi¬ tated with monoclonal antibody 'C2 (lanes 2-4, Panel (O) and then ΕlO (lanes '2-'4, Panel (O). The pre¬ cipitates were examined by electrophoresis without reduction. Panel (D) Glycopeptides from peaks 1-4 were examined by electrophoresis after reduction. Panel (E) Glycopeptides from peaks 2-4 were immunoprecipitated with monoclonal antibody 'C2 (lanes 2-4, Panel (E)) and then ' E10 (lanes '2-'4, Panel (E)). The precipitates were examined by electrophoresis after reduction. . Lane numbers in all cases indicate the ion-exchange peak from which the sample was obtained. Numbers to the right of each figure are molecular weights times 10 " . Glycopeptides in all .cases were detected by fluoro- i" t graphy. Figure 2. GLP-A and GLP-B purified by a combination of HPLC and immunoaffinity methods were immunoprecipitated with the monoclonal antibodies to demonstrate retention of their immunαgenicity. Lane 1, glycopeptide GLP-A immunoprecipitated with monoclonal antibody 'C2. Lane 2, GLP-B immunoprecipitated with monoclonal antibody ΕlO. Detailed Description of the Invention EXAMPLE I Growth of Virus and Purification of Glycopeptide A. Materials and Methods 1. Growth, Radioactive Labeling, and Purification of Virus Human primary fibroblast cultures grown in Dulbecco's Modified Eagle's Medium (DMEM) plus 10% fetal bovine serum were infected with Towne strain HCMV at an multiplicity of infec¬ tion (MOD of 1-5. At 1-2 days post infection L 3 Hj glucosamine (5 uCi/ml, 22 Ci/mM, Amersham, Arlington Heights, Illinois) or [ 3s s]methionine (5 uCi/ml, 109 Ci/ mM, DuPont/NEN, Boston, Massachusetts) was added as a marker during subsequent purification. Cells and cellular debris were removed from the medium by low speed centrifugation at 7500 x g for 20 minutes. Virus was collected from the supernatant by centrifugation at 48,200 x g for 1 hour. The viral pellet was resuspendεd in Tris NaCl buffer (50 mM trishy- droxyrnet-hyl^mino methane-HCl, pH 7.4, and 150 mM NaCl) and layered onto 20-60% sucrose gradients made with the same buffer. Velocity sedimentation was done at 131,300 x g for 1 hour. Gradients were collected and moni¬ tored for optical density at 280 nm. A major peak of optical density which banded at 43% sucrose was collected. These fractions were diluted with Tris NaCl buffer and virus collected by centrifugation at 131,300 x g for 2 hours. This purified whole virus prepara- tion was designated "Purified Towne Prep.". 2. Detergent and Chloroform Methanol Extraction Virus detergent extracts were prepared by suspending 10-15 g of purified virus in 3-5 ml of Tris NaCl buffer (50 mM Tris HC1, pH 7.5, 10 mM NaCl and 2 mM PMSF) containing 1.0% Nonidet P-40 (NP-40, polyoxyethylene-jj- t-octylphenol). Detergent-virus suspensions were stirred for 1 hour at room temperature at which time extracted proteins were separated from insoluble proteins by high speed centri¬ fugation for 1 hour. In some cases, purified virus was extracted with chloroform:methanol (2:1, v/v) prior to extraction with detergent to remove lipids. One volume of virus suspen- sion in Tris NaCl buffer was extracted with 20 volumes of chloroform:methanαl by stirring at room temperature for 20 minutes, followed by centrifugation to separate phases and collect delipidated proteins. 3. Isolation of HCMV Disulfide-linked Glycopep¬ tide Complexes bv Ion-Exchange HPLC Disulfide-linked glycopeptide complexes were isolated by s anion-exchange high performance •• liquid chromatography. Separation was achieved using a Varian Model Vista 5500/402 HPLC system consisting of pump, gradient former and UV detector. For anion exchange chromatography, 0.5 ml of unreduced detergent extract was applied directly to an Aquapore AX-300 anion exchange column (Brownlee Labs Inc., Santa Clara, California). The column was washed for 15 minutes with 20 M Tris-HCl buffer, pH. 7.8 containing 2.5 mM NaCl and 0.1% NP-40. Bound glycopeptides were eluted with a 40-minute linear gradient of NaCl from 2.5 mM to 1.0 M in the same buffer as initially used. The column was washed for an additional 15 minutes at 1.0 M NaCl to elute all bound glycopep¬ tides. The flow rate was 1 ml/minute. Frac¬ tions collected were monitored for radio¬ activity. 4. Immunoprecipitation • For Immunoprecipitation, Protein A-Sepharose CL-4B beads (Sigma-Aldrich, St. Louis, Missouri) Were prepared by incubation with goat anti-mouse IgG (H and L) and washed with phosphate-buffered saline (PBS) before use. Whole extract or glycopeptides and glycopep¬ tide complexes obtained by ion-exchange- HPLC were incubated with either monoclonal antibody 1-4S-41C2, 2-29-937 or 2-15-9E10 (preparation, infra) in PBS containing 0.1% NP-40 for 1.5 hours with constant mixing. In some cases, 0.1% sodium dodecylsulfats (SDS) was used in place of NP-40.- The prepared Protein A-Sepharose CL-4B beads were then added to the antigen-antibody solution and allowed to react for an additional 1.5 hours with constant mixing. The beads were washed three times with PBS containing 0.1% NP-40. Following the final wash, proteins associated with the beads were solubilized for SOS-PAGE. 5. Preparation of Monomer Isolated HCMV disulfide-linked glycopeptide complexes were reduced by aαdition of dithio- threitol to a final concentration of 10 M (DTT, United States Biochemicals, Cleveland, Ohio) in the presence of 8M Urea. The reac¬ tion was allowed to proceed at room tem¬ perature for 2-24 hours with constant stirring. Alkylation of the reduced sulfhy- dryl group was done by adding 5-10 mg iodoace- ta ide and allowing the reaction to proceed at room temperature for an additional hour. 6. Gel Filtration HPLC of Individual HCMV Glvcooeptides Samples containing 1% SDS were subjected to gel filtration HPLC using the Varian system described above. Gel filtration was performed - coupling TSK 3000 SW and 4000 S gel filtra¬ tion columns (Toyo Soda, Tokyo, Japan) in series and eluting isocratically with.50 mM soclum phosphate, pH 7.0, containing 0.1% SDS. The flow rate was 0.3 ml/minute. Absorbance was monitored at 275 nm and fractions collected were also monitored for radio¬ activity. 7. SDS-Polyacrylamide Gel Electrophoresis and Fluoroqraphy SDS-PAGE was done with 5-15% polyacrylamide slab gel gradients following the method of Laem li. Samples were solubilized by boiling for three minutes in the presence of 4% SDS. After the samples were cooled to room tem¬ perature, urea and beta-mercaptoεthanol (BME) were added so that samples contained 5% BME and 2M urea. Tritium was detected by fluorography using Enlighting (DuPont/NEN, Boston, Massachusetts). B . Rεsults 1. Isolation of HCMV Glycopeptide Complexes by Ion-Exchange HPLC It has been known that HCMV contains disulfide- linked glycopeptides which react with mono¬ clonal antibodies. It was of interest to determine if ion-exchange HPLC could be used to separate these or other disulfide-linked glycopeptides of HCMV. When unreduced L 3 HJ glucosamine labelled detergent extracts of HCMV were separated by ion-exchange HPLC, one unretained peak and three retained peaks of radioactivity were detected (Fig. 1 A). The unretained peak contained material which could be extracted into chloroforπ methanol (2:1,v/v) and could not be fixed in polyacry- lamide gels using conditions which fix pro¬ teins (lane 1, Fig. 1 B, D). Moreover, this material could not be labellεd with [ 3s S)meth- ionine (data not shown). Based on these results, it was concluded that this peak con- tainεd glycolipid. Thε glycolipids wεrε not examined for reactivity to the monoclonal antibodies. While the unretained peak contained little or no glycopeptide, the three retained peaks did contain disulfide-linked glycopeptides (Fig. I B, D). This was dεtεrmined by εxa- mining material from each of the retained peaks by electrophoresis with and without reduction of disulfide bonds. Examination of peak 2 by elεctrophoresis without reduction sho-wed that it contained one abundant band with an apparent molecular weight of 93,000. However, after reduction of disulfide bonds, at least two glycopeptides were detected with apparent molecular weights of 50-52,000 and 93,000 (lane 2, Fig. 1 B, 0). Thus, peak 2 5 appeared to contain a glycopeptide complex with a molecular weight of 93,000 and a glyco¬ peptide with a similar apparent molecular weight. Peaks 3 and 4 also containεd disulfide-linked glycopeptide complexes. When 10 material from peak 3 was examined by electrophoresis without reduction, two major bands were detected. One band was observed at the top of the resolving gel and the second major band coverεd molecular weights of 15 130-180,000. After reduction of this material, thrsε glycopeotides were detεctεd (lanε 3, Fig. 1 B, D). The most abundant speciεs had an apparent molεcular weight of 93,000, whilε two less-abundant species had molecular 20 weights of 50,000 and 130,000. Analysis of peak 4 by electrophoresis without reduction showed that the most abundant material in this pεak had a molεcular weight greatεr than 200,000 and formed a smear at the top of the 25 resolving gel (lanε 4, Fig. 1 B). Howεver, therε was material in this peak which had a molecular weight similar to that observed for material in pεak 3. This was most likely pre¬ sent becausε of tailing of pεak 3 into peak 4. 30 Finally, when peak 4 was examined by elεctro- phoresis after reduction of disulfide bonds, lower molecular weight glycopeptides were detected (lane 4, Fig. 1 D). Of the most abundant species, one had an apparent molecu- 35 lar weight of 50-52,000, while another speciεs had an apparent molecular weight of ^•90-130,000. However, even after reduction, some material in peak 4 still formed a smear at the top of the resolving gel. 2. Immunoαenicity of the Isolated Glycopeptide Complexes Examined by Reactivity with Monoclonal Antibodies Monoclonal antibodies,were made as described in the second example of this invention. Material from the three retained peaks from ion-exchange was examined for reactivity with MoAbs 2-29-9B7 ('B7), 1-48-41C2 ('C2) and 2-15-9E10 (ΕlO). Equal amounts of radioac- tivity were used from each peak for immuno- prεcipitation. It was determined that -' B7 and * C2 reacted most strongly with the disulfide- linked glycopeptide complexes found in peak 3, precipitating 19 times more radioactivity from this peak than peak 2 and 6 times morε radioactivity whεn .compared to peak 4. These complexes, when reducεd, appεarεd to contain all the glycopeptides detected in peak 3 (Fig. I f Lane 3, C and E). Among these glyco- peptides was the most abundant glycopeptidε, which had a molecular weight of 93,000. Unlike ' B7 or 'C2, ΕlO reactεd most strongly with peak 4, precipitating 5-6 times more radioactivity from this peak than peak 2 and 11-12 times more radioactivity than from peak 3. However, while ' E10 did react most strongly with the glycopeptide complexes found in peak 4, it also reacted with the 93,000 molecular weight glycopeptide complex found in peak 2 which generated glycopeptides with molecular weights of 50-52,000 after reduction of disulfide bonds (Fig. 1, Lanes 2 and 4, C and D). Moreover, the glycopeptide complexes 5 immunoprecipitated from peak 4 also contained glycop.eptides with molecular weights of 50-52,000 as the most abundant species when the complexεs were εxamined aftεr rεduction of disulfidε bonds. However, while the 50-52,000 10 molecular weight glycopeptides were the most abundant species in glycopeptide complexes from peak 4, there were still a number of other glycopeptides detεcted aftεr rεduction of disulfide bonds. In fact, the complexεs immu- 15 noprεcipitatεd by ' E10 appeared to contain most or all of the glycopeptides detectεd in peak 4. Most of the glycopeptides detectεd' in- - - 20 peaks 2-4 werε found in coflipl.s e.s..xea.ct.ivε ' with either 'B7 and 'C2, or ΕlO. However, the 93,000 molecular weight glycopeptide detectεd in pεak 2 did not appεar to react with any of.ithe antibodies εven though there 25 was a significant amount of this glycopeptide presεnt. This suggεstεd that this glycopep¬ tide was differεnt from thε 93,000 molεcular wεight glycopεptide detectεd in peaks 3 and 4. To further examine this possibility, material 30 from peak 2 was immunoprecipitated first with 'C2 and than '37 using approximately a 15-fold excess of antibody. Protein A sepharose beads wεrε addεd several times until no more radio¬ activity could be immunoprecipitated. The supernatant obtained after these precipita¬ tions was immunoprecipitated with ΕlO follow¬ ing the same procedure. After the final immunoprecipitation, the material left In the 5 supernatant was examined by electrophoresis. When this was done, the 93,000 molecular weight glycopeptide was ' the only glycopeptide detectεd. 10 3. Glycopeptides Reactive with the Monoclonal Antibodies When material from any one of the three ion- exchange peaks was immunoprecipitated, under non-reducing conditions, with any one anti- 15 body, sεveral bands were routinely detectεd. After reduction, these various complexεs gave rise to a number of lower molecular weight glycopeptides. Because of this,- it " was deter- ~ mined whether rεduction prior to immunαpreci- 20 pitation would affect the number- or relative amount of the various glycopeptides immunopre¬ cipitated. To examine antibody reactivity to individual glycopeptides, material from each ion-exchange: peak was reduced with DTT in the 25 presεncε of 8 M urεa and 0.1% NP-40. Aftεr rεacting overnight, glycopeptides were alky- lated by addition of iodoacetamide. As a control experiment, whole extract, whictfcon- tainεd all the various disulfide-linked glyco- 30 peptidεs, was reduced under the samε condi¬ tions and examined by electrophoresis without further reduction with BME. When this was done, the majority of the glycopeptidεs werε detected in bands with molecular weights of 35 50-52,000 and 90-92,000. This suggested that thε reduction done with DTT was effectivε. Based on radioactivity, equal amounts of reduced and unreduced material from peaks 2 and 4 were immunoprecipitated with ' E10 while reduced and unreduced material from peak 3 was immunoprecipitated with 'C2. When reduced material was immunoprecipitatεd, thε amount of radioactivity prεcipitatεd was dεcrεasεd 2- to 3-fold whεn compared to the amount precipi- tated from unreduced material. This occurred with both antibodies. However, the number of glycopeptides immunoprecipitated was not affected. Furthermore, densitometric scans dεmonstratεd that thε relative amounts of the glycopeptides immunoprecipitated wεre not affected by reduction prior to immunoprecipi¬ tation. These results suggested that the gly¬ copeptides found in the various disulfide- linked complexes were immunologically related. 4. Further Purification of Individual Immunoqenic HCMV Glycopeptidεs By ion-εxchangε HPLC, we were able to purify immunoge.nic ,.glycopeptidε complεxes, but not individual glycopeptides. Furthermore, it was clear that the monoclonal antibodies would react with sεvεral glycopeptides. Thus, it was not possible to obtain individual glyco¬ peptides by eithεr ion-εxchange HPLC or immunoaffinity methods alone. Therefore, to obtain individual immunogenic HCMV glycopep¬ tides, a purification method was developεd which combinεd ion-exchange HPLC, immuno- affinity purification, and separation by gel filtration HPLC. To obtain the most abundant immunogenic glycopeptides, GLP-A and GLP-B, disulfide-linked glycopeptide complexes were first separated by ion-exchange HPLC. Glyco¬ peptide complexes containing GLP-B found in 5 peaks 2 and 4 were then further purified by immunoaffinity methods using monoclonal antibody ΕlO while those containing GLP-A in peak 3 were further purified using monoclonal antibody 'C2. Glycopeptide complexes purified 10 by these methods were then reduced with DTT and alkylated with lodacetamide to gεnerate individual glycopeptides. To obtain the 93,000 molecular weight glycopeptides from peak 2, which was not reactive with any . 15 monoclonal antibody, the supεrnatant obtained aftεr immunoprecipitation of peak 2 with both *C2 and ΕlO was also reduced and alkylated. The individual glycopeptidε thus- produced were then subjected to gel filtration HPLC. Glyco- 20 peptidεs obtained in -this manner were- -examined by SDS-PAGE and shown to be individual glyco¬ peptides. The glycopeptide recognized by ' E10 was denoted GLP-B; that recognized by 'C2 was denotεd ; GLP-jA. In addition, thε major glyco- 25 peptides recognized by monoclonal antibodies ' E10 and ' C2 and purified by this method could still be immunoprecipitated by thesε antibo- diεs, suggεsting that the glycopeptides main¬ tained their immunogεnicity through the final 30 step of purification (Fig. 2). 35 EXAMPLE II Monoclonal Antibodies to GLP-A and GLP-B Materials and Methods 1. Generation and Purification of Monoclonal Antibodies Monoclonal antibodies were generated by using Purified Towne Prep, as the antigen. Adult BALB/c mice were immunized intraperitonεally with antigen emulsified in complete Freund's adjuvant, followed with three booster immuni¬ zations using saline-suspended immunogen given at threε-wεek intervals. Threε days following thε final boostεf, the mouse was sacrificed and the spleen cells fused with SP2/0-- - Agl4 myeloma cells (American Tissue Culture Collection) at a ratio of 4:1 using polyethy- lεnε glycol.- Twenty-four hours -later, the - fused cells wεre distributed into 96 wells " in Hepes-buf ered Dulbecco'-s modified Eagle'-s medium (DMEM) supple entεd with 10% fεtal bovine serum, 50μg/ml garamycin, 13.6μg/ml hypoxanthine, 0.4μM aminopterin and 7.6μg/ml thymidine. jAftεr two" weeks in this medium the cells wεre transferred to new medium supplemented as above but without aminopterin. Three to four weeks after cell fusion, super- natants from these cultures were assayed for antibodies specific to HCMV NP-40 extract by the ELISA assay. Cloning of cells from posi¬ tive cultures was performed by limiting dilu¬ tion using mouse thymocytes as feeder cells. Individual wells were screenεd microscopically for single coloπiεs and assayed for antibody to HCMV NP-40 extract as above. Antibody- producing clones were expanded for production of ascites fluid in BAL3/c mice. 2. Neutralization Assays Monoclonal antibodies purified by hydroxylapa- tite HPLC were diluted in DMEM supplemεntεd with 2% fetal bovine serum (FBS). Prior to infection, 600 plaque-forming units (pfu) of Towne and 300 pfu of Toledo strain CMV were added to monoclonal antibody in a total volume of 0.4 ml of DMEM. Guinea pig complement (Pel-Freeze Biologicals, Rogers, Arkansas) was added to a final concentration of 2.0% in half the preparations; thε remaining half received an equivalent volume of DMEM. Viral neutrali¬ zation was left to proceed for 60 minutes at 37°C. The HCMV- onoclonal antibody mixturε was adc ' εd to confluεnt layer of skin fibro- blasts in 6-well plates (Costar, Cambridge, Massachussetts) -and incubated for another 6G minutes " for " the " virus to adsorb. Th virus " mixture was aspirated and 5 ml of DMEM supple- mεnted w-I-th -2% FC-S,- -g-aramycin, -penieilli-n G and 0.5% agarosε (Sεa Plaquε, FMC Co., Rockland, Maryland) was addεd to εach wall. On day 3, the onolayers were fixεd with 10% formali 1^. 0% ethanol, stained with methy- lεnε blue and thε plagues counted. Viral neutralization was evaluatεd using the for¬ mula: % Plaquε Reduction = Avα. '■'ax. ofu - test ofu 7. 1QQ Avc. Max. pfu and is reported as the protein concentration required for a 50% reduction in plaque num¬ bers. 3. Purification of Monoclonal Antibodies Monoclonal antioody from ascites was purified by hign-performance hydroxylapatite chromato¬ graphy, according to juarsz-Sallnas (H. Juarez-Salinas et al., Biotεchniquεs, May/Junε 1984), the teaching of which is hereby incor¬ porated by reference. 4. Immunofluorescence Adenovirus, HSV, VZV, and the wild type HCMV were obtained from clinical isolates. For immunofluorεscence, infected and uninfected skin fibroblast culturεs on glass slidεs were fixed in cold acetone:methanol (v/v, 1:1). Fixεd culturεs were preincubatεd with normal porcine serum and washed with phosphate-buf¬ fered saline (PBS). All subsequεnt stεps were done with PBS containing 0.1% NP-40. Cultures ' wεrε incubatεd with HPLC-purifiεd ascitεs monoclonal antibody or with tissuε culturε fluid for 30 minutes and washed with PBS NP-40 buffer. Cultures were then incubated with fluorescein isothiocyanate (FITC) labeled goat antimouse IgG (Cappel, Malvern, Pennsylvania). Following a final wash, slides werε examined with a Zeiss phase fluorescence microscope. 5. Immunoprecipitation For immunoprecipitation protein A sepharosε CL-4B bεads (Sigma-Aldrich, St. Louis, Missouri) were prepared by incubation with goat antimouse IgG (H and L) and washed with phosphate-buffered saline (PBS) beforε usε. Wholε εxtract or glycopεptidεs and glycopεp- tidε complεxεs obtained by ion-exchange HPLC were incubated with either monoclonal antibody 'C2, 'B7, or ΕlO in PBS containing 0.1% NP-40 for 1.5 hours with constant mixing. in some cases, 0.1% SDS was used in place of 0.1% NP-40. The prepared protein A sepharose CL-4B beads were then added to the antigen antibody solution and allowed to react for an additional 1.5 hours with constant mixing. The beads were washed three times with PBS containing 0.1% NP-40. Following the final wash, proteins associated with the beads were solubilized for SDS-PAGE. B. Results 1. Glycopeptidεs Recognized by Groups A and B MoAbs Monoclonal antibodies were obtained which reacted with the glycopeptide bands. Based on reactivity, they could be classified into two groups, A and B. MoAbs 2-29-9B7 ('37) and 1-48-41C2 ( * C2) were " representative o " Group A. MoAb 2-15-9E10 (ΕlO) was rεprεsεn- tativε of Group B. Sincε HCMV containεd disulfide-linked glycopeptide complexes, monoclonal antibodies were used to immunopre- cipitatε both rεducεd and unrεducεd crudε dεtεrgεnt extracts to detεr ine if glycopep- tidεs precipitated because they were recog¬ nized by the antibodies or if any glycopep¬ tides precipitated because they were associ¬ ated by disulfidε bonds to othεr glycopeptides which were recognized by the antibodies. Therεfore, part of an NP-40 extract of Towne strain HCMV was reduced with DTT and all available sulfhydryl bonds alkylated with iodoacetamidε to prεvεnt rε-polymεrization. Extracts rεducεd in this manner were examined by electrophoresis to assure that they had been reduced. All 3 antibodies werε usεd to immunoprecipitate both reduced and unreduced extract as described in materials and methods. MoAbs '37 and 'C2 immunoprecipitated one abun- 5 dant glycopeptide which formed a broad band with an approximate molecular weight of 93,000. In addition, there were 3 lεss- abundant glycopeptides with molεcular wεights of 130,000, 52,000 and 50,000. Thε numbεrs 10 and molecular weights of glycopeptides preci- pitatεd were not affected by reduction prior to immunoprecipitation. Howevεr, whilε thε rεlative amounts of most glycopeptides werε also unaffεctεd, there was some difference in 15 the intensity of the 50,000 to 52,000 molecu¬ lar weight glycopeptides immunoprecipitatεd by 'C2 aftar rεduction. Examination of 'ElO immunαprεcipitatas showed 20 that the pattern of glycopeptides immunopreci¬ pitatεd was vary different when compared to that obtained with ' B7 and 'C2. When 'ElO was used, one abundant glycopeptidεs could be detected with apprpximate mplεcular weight cf 25 50,000 t 52,000. Both these glycopeptides formed broad bands in the gel. Howevεr, thεrε wεre also at least 4 less-abundant glycopep¬ tides with molecular weights of 90-93,00U, 116,000, and 130,000 as well as some material 30 which had a molecular weight greater than 200,000 and smeared at the top of thε gel. However, like '87 and 'C2, the pattern of gly- coproteins and the relativε amounts prεcipi- tated by 9E10 was not affected by reduction 35 prior to precipitation. 2. Neutralization and Immunofluorescent Staining of Infected Cells with Monoclonal Antibodies of Groups A and B Neutralization of HCMV strains Towne and Toledo by ΕlO did not require the addition of complement, whereas ' B7 at similar concentra¬ tions (less than lOug/ml) was dependent upon the addition of 2% guinea pig complement (Table 1). However, at higher concentrations (greater than 30ug/ml) 9B7 could neutralize virus in the absence of complement. In the presencε of 2% guinea pig complement, ' C2 did neutralize Toledo (Table 1), but required much higher antibody concentrations to neutralize this strain in the absence of complemεnt. Furthεr ore, this antibody did not display any neutralizing activity against strain Towne at antibody concentrations less than 200 ug/ml. TABLE I In Vitro Neutralization of HCMV Laboratory Strains Towne and Toledo Microgram per mT for 50X PFU reduction HCMV Strain: TOWNS* TOWNE TOLEDO* TOLEDO Moπocoloπal Antibody +c' -c' +c' -C 9E10 4 8 4 10 4IC2 >200 >200 2 100 987 2 35 2 48 ♦ Average number of PFU in control cultures were 600 for Toledo and 300 for Towne when inoculated with virus and complement, but no antibodies. The MoAbs were also examined for their reac¬ tivity to skin fibroblast cells infectεd with HCMV laboratory and wild type strains as well as Herpes Simplex (HSV), Varicella zoster (VZV), Epstein Barr virus (EBV) and adeno¬ virus. Both MoAbs ' B7 and 'C2 were found to be specific to HCMV infected cells, reacting with both laboratory and wild type strains, but not with HSV, VZV, EBV or adenovirus- infεctεd cells (Table 2). However, MoAbs ΕlO reacted with HCMV-infected cεlls and rεactεd with HSV- and adenovirus-infected cells, but not with VZV-infected cells. All three anti¬ bodies were non-reactive to control skin fibroblast cells. TABLE 2 Reactivity of Monoclonal Antibodies to Infected Cells Immunof1uorescence* Antibody Class Towre AQ169 A# £ HSV Adenovirus VZLI E3V SF Control 9E2Q IgS3 +* + + + N3 N3 + *+Q - fϋ - 41C2 Ig2 +* +_► ++ +* ++ ++ _. - - - - Q β J. IgQl -H- +4- ++ -H- -H. -H ' - - _ - . *Cytoplasmic immunofluorescence of acεtone- methanol fixed, CMV-, HSV-, adenovirus-, and VZU-infected human skin fibroblasts (SF) was performed as described under materials and methods. +A-D are HCMV clinical isolates. §Moπoclonal antibody 9E10 also reacted with adenovirus grown in Hep2 cells. Uninfected Hep2 cells were negative. ND, Not Done. 3. Reactivity of Isolated Glycopeptides and Glycopeptide Complexes with MoAbs 'ElO, *B7 and ' C2 The glycopeptides immunoprecipitatεd by the MoAbs were not affected by reduction prior to immunoprecipitation. Therefore, it was desirable to determine the relatedness of the glycopeptides, both associated in the com¬ plexes by disulfide bonds and those which were 5 not. Equal amounts of radioactivity from peaks 2-4 were collected and serially immuno- prεcipitatεd with 'C2 and *B7, and then ΕlO. When fractions from pεaks 2-4 were immunopre¬ cipitated with 41C2, it was observεd that this 10 antibody rεactεd most strongly with material from peak 3, precipitating 19 times more radioactivity from peak 3 than peak 2 and 6 timεs more radioactivity when compared to peak 4 (See Figure 1). 15 After immunoprecipitation with 'C2 or 'B7, the ion-exchange fractions were immunoprεcipitatεd with monoclonal antibody ΕlO. Immunαprecipl- tation with this monoclonal antibody sho εd 20 that it reacted most strongly with peak 4, precipitating 5-6 times more radioactivity from this peak than peak 2 and 11-12 times more radioactivity than from peak 3. When ' ElO immunogrecipitates from peak 2 werε εxa- 25 inεd by electrophoresis without reduction of disulfide bonds, a 93,000 molecular weight band was detεctεd as wall as two lεss intεnse bands with molecular weights of 140,000 and 180,000. This pattern was very similar to 30 what was observεd whεn material from peak 2 was examined by electrophoresis without reduc ¬ tion. When ' ElO immunoprεcipitates of peak 2 werε examined by electrophoresis after reduc¬ tion, glycoproteins with molecular weights of 35 50-52,000 were detectεd as well as a very small amount of glycopeptide with a molecular weight of 90,000. Howevεr, no 93,000 molεcu¬ lar weight glycopeptide was detected. Immuno- precipitates from peak 3 contained very little radioactivity. Unreduced, this material was in high molεcular weight bands and most of the radioactivity was detected in a glycopeptide with a molecular weight of 50,000 after rεduc¬ tion. However, a very small amount of a 10 93,000 molecular weight glycopeptide was also detected in immunoprecipitates from peak 3. It was interεsting to notε that most of thε glycopeptides detected in peaks 2-4 reacted 15 with either 'B7 and 'C2, or ΕlO. However, the 93,000 molecular weight glycopeptide detected in peak 2 did not appear to react with any of the antibodiεs εvεn though thεre was a significant amount of this glycopeptide 20 present. This suggested that this glycopep¬ tide was different from the 93,000 molecular weight glycopeptides detected in peak 3. To further examinε this possibility, matεrial from pεak 2 jwas immunoprεcipitated first with 25 'C2 and then ' B7 using approximately a 15-fold excess of antibody. Protein A sepharose beads were added sεvεral timεs until no morε radio¬ activity could bε precipitated. The super¬ natant obtained after these precipitations was 30 immunoprecipitatεd with ' ElO following thε samε procedure. After the final immunopreci¬ pitation, the material left in thε supεrnatant was examined by electrophoresis. When this was done, the 93,000 molecular weight glyco¬ 35 peptide was the only glycopeptide detected. The results of these assays are summarized on Table 3, below. TA3LE 3 Glycopeptide Complexes and Glycooεptides Immunoprecipitated by MoAbs Ion-Exchange HPLC Glycopeptidε Glycopeptides Peak* (MoAb Reac- Complex Immuπo- Immunoprecipitated tivity) precipitated After Reduction ( ol wt. , d) (mol wt. , d) 2 (9E10) 93 50-52 4 (9E10) 450 50-52, 90, 116, 130, >200 2 (41C2, 937) " 130, and >130 50-52, 93 " , 130 2 (none)- 93 Xd Giycopεp- 93 Kd Glycopep¬ tide not Associ- tide not Immuno- atεd in Disulfide- precipitated by linked Complex anv MoAb *Fiαurε 1 EXAMPLE III Stimulation of Human Cellular Immunity A. Materials and Methods 1. Isolation of HCMV-Soecific T Cell Blasts HCMV-specific T cell blasts were prepared in 25 cm 2 upright tissue culture flasks by stimu¬ lating 10 x 10 6 fresh peripheral blood mono- cytεs (MNC) at a concentration of 1 x 10 s MNC/ml with 10.0 μg of heat-inactivated HCMV antigen (Purifiεα Towne Prep., 56°C/1 hr) suspended in RPMI 1640 medium in 10% PHS. After six days at 37°C in 5% C02 atmosphere, the HCMV-specific blasts were clonεd by limit¬ ing dilution in 96 well U-bottomed tissue culture plates at 0.3 cell/well in the pre¬ sence of x-irradiated autologous MNC as feeder cells, heat-inactivated HCMV antigen (lμg/ml), and 10-20% TCGF (Biotest, Frankfurt, Germany). Thereafter, cells were refed every 3-4 days with fresh TCGF-containing media. Weekly, HCMV antigen and autologous x-irradiatεd feeder cells were added to the media. After ' day 21, growing cells were fεd and transferrεd to 24 well plates for further expansion and then resεεdεd into 25 cm 2 tissue culture flasks for large scale production. Some εxpandεd clonεs wεre subcloned by a second limiting dilution to ensure clonality. Lines were developed from 4 different HCMV seroposi- tive donors yiεldiπg more than 100 individual T-hεlpεr (Tj-,) cell clones. 2. Characterization of the HCMV-Th Clones All Tf, clones ware characterized as to pheno- type, proliferative responses, IL-2 produc¬ tion, and cytotoxic activity as follows: A. Phenotvoe analysis Th clones were analyzed for expression of OKT phenotypic determinants using the monoclonal antibodies 0KT3 (total T cells), 0KT4 (helper/inducer T cells), and 0KT8 (cytotoxic/suppressor T cells) (Ortho Pharmaceuticals Inc., Raritan, New Jersey) using an indirect immunofluore- scence assay. Fluorescence was detected either by fluαrεscεncε microscopy using a Zeiss fluorεscent microscope or flow cytometry using the EPICS541 (Coulter Corp., Hialeah, Florida). All Tj-, c ι 0 nes 5 expressed the 0KT3+ 0KT4+ 0KT8~phεπotypε. B. Lymphocyte proliferation T cell clones were rεsted in tissue culture media in the absence of TCGF 10 overnight, and then restimulated for 72 hours with either HCMV antigen or a related herpεs viral antigεn, HSV, to determine the specificity of prolifera¬ tion responsεs to HCMV. Rεsponses to HSV 15 were similar to tissue culture media background control. Thus, all T n clones were HCMV-spεcific. " C. Interleukin-2 (IL-2) orpductipn 20 Tf-, clcnes wεrε stimulatεd with HCMV anti¬ gεn, and thε supεrnatants harvested at 24 hpurs and assayed for IL-2 activity using the murine CTLL-20 (IL-2 dependent) murμne cell linε. All T^ clones werε ' 25 shown to produce IL-2, as seen by the survival and growth of CTLL-20. D. Cytotoxic activity All Tf, clones were tested for cytotoxic 30 activity against the NK targεt cell line, K562, autologous uninfectεd and HCMV- and HSV-infεctεd human skin fibroblasts as target cells. No NK or virus-specific cytotoxic activity was observed with any 35 of the Tf, clonεs. B. Evaluation of T h Cell Recognition of HCMV Immunoqens The early and key step in cellular immunity is recognition of the foreign antigen by the subpopula- tion of whitε blood cells (WBC) called T-helper (T^) cells. The first immunogεnic rεactions of an antigen are dεpεndεnt on its recognition by T^ cells. Aftεr recognition, the T^ cells are stimulated to proliferatε -and to producε lymphokines such as IL-2 which cause proliferation of several types of WBC. The rεlativε proliferation of one of the HCMV-specific T^ cell clones (WRC*3) by purified, unreduced glycopeptide complexεs derived from HPLC peaks 2 ("2UR"), 3("3UR") and 4( " '4UR") is shown in Table 4. TABLE 4 Proliferation of T h Cell Clone WRC#3 3 H-thvmidine incorporated Stimulant (com) WRC*3 (Control) 95 t 36 WRC*3 + WRCMNCl R ?;* (Control) 143 ± 10- WRC*3 + WRCMNClRR + HSV (lμg/well) 336 ± 92 WRC*3 + WRCMNC IRR + Towne CMV 55,080 ± 1,210 (0.5μg/well) WRC*3 + WRCMNC IRR + Triton X - 9,311 ± 2,866 100 εxtract of Towne CMV WRC*3 + WRCMNCl R + 2UR (0. lμg/well) 1,194 t 792 WRC*3 + WRCMNCI RR + 3UR (0. lμg/well) 19,706 + 5,252 WRC»3 + WRCMNCIRR + 4UR [O.luq/well) 25,387 ± 389 ♦Irradiated MNC from donor WRC. Importantly, antigen presεnted on the surface of adherent cells induces the clonal expansion of mono- nuclear lymphocytes that are directed specifically against the triggering antigen. After stimulation sub- sides, survivors of the expandεd clone remain as memory cells in the body, ready to expand rapidly again when the same antigen is presented. The primary memory cells are T^ cells. GLP-A and GLP-B glycopeptides stimulate the memory WBC from serooositive but not seronεgativε donors as shown in Table 5. These results showεd clearly that these glycαoeptidε complexεs are an impor¬ tant immunologic coπoonent of HCMV. TABLE 5 Proliferation of MNC from Seroneqative and Seropositive Donors TL O WC KM (Off-) (OW-) (QW+) (C_W+) Stimulant Me≤iuK 645 _ 145 231 .+ 57 ~ " 312 + 105 1,129 + 635 ("-" control) Staph (Λage lysa e 123,073 + 25,855 fO* 257,167 + 22,356 182,130 + 22,747 ("+" control for all donors) Twne Ag 442 + 94 109,252 + 7,154 5*,C58 +.10,157 31,224 + 6,381 ** 983 + 69 23,922 + 1,714 3,592 + 42 11,852 + 2,423 3LR 902 + 45 9,277 + 2,331 2,975 + 1,792 39,0Ba + 427 4UR 1,144 + 456 19,781 + 2,839 1,855 + 230 13,(27 + 1,258 * - not determined. + - Mononuclear cells (MNC) isolated from heparinizεd blend of donors on Ficoll-Hypaque density gradients werε assayed for lymphocyte proliferation by the method of R. C. Gehrz et al., J. Infect. Pis., 143, 391 (1981). EXAMPLE IV Diagnostic Assay for Human Cytomegalovirus Using Monoclonal Antibodies from Group A A. Materials and Methods Two monoclonal antibodies directεd against different epitopεs of the group A glycopeptides are used in this enzyme-linked immunoadsorbent assay. The primary or capture monoclonal antibody I is adsorbed onto a solid phase. The secondary monoclonal antibody II is conjugated to any indicator system which will give a read out. In the presence of HCMV, a positive read out will be obtained. Capture antibody I, e.g. *C2, was adsorbed onto Immulon I polystyrene microwells of the Re ovastrip system from Dynatech (Alexandria, Virginia). A carbonate-bicarbonate buffer pH 9.5 was used and the wells werε washεd with phosphate-buffered saline pH 7.4 containing 0.05%-tween 20 (PBS-tween). Purified CMV viruses and virus-infected or uninfected skin fibroblasts call lysatε obtainεd with PBS containing 1% NP-40 wεre added into the microwells and incubated for one hour at 37°C. The wells werε then washed with PBS-tweεn. Biotinylatεd monoclonal antibody II, ε.g., ' B7 was addεd and thε samplεs wεrε iπcubatεd for one hour at 37°C. The wells wεrε then washed with PSB-tween. Streptavidin-conjugated to pεroxidase (Kirkegaard & Perry Laboratories, Inc.) was added- and the samples were further incubated for one hour at 37°C. The wells were then washed with PBS-tweεn and distilled water. Thε substrate ortho-phanylenediaminε in phosphatε-citratε buffεr, pH 5.0, was used with hydrc- gεn perpxide added. Aftεr 15 inutεs pf incubaticn at rpom tεmpεraturε, the reaction was stoppεd with 5N sulphuric acid. A Dynatεch microwεll platε reader was used to read the absorbencε of the product at 490 n.m. As shown in Table 5, the reaction with Towne strain of HCMV was linear above 7.8 nanograms total protein, while there was no mεasurable reaction with thε skin fibroblast control. Patient samples were assayed directly and it was not possible to detεrmine total virus protein. Table 6 shows a typical patient assay. An HCMV-positive sample, verified by cell culture diagnosis, was shown to react strongly over background value. B. Results TABLE 6 Samplε Total Protεin Absorbence HCMV Antigen in (nanoqram) at 490 n. . Towne Strain 0 0.002 0.5 0.051 1 0.060 2 0.088 3.9 0.085 7.8 0.110 15.6 0.146 31.3 0.211 62.5 0.288 125 0.430 250 0.732 500 1.379 Patient Sample Absorbeπcε cell lysate ' . • ■ at 490 n.m. Skin Fibroblasts 1.967 infectεd with HCMV (diluted 1/16) Uninfected Skin 0.117 Fibroblasts HSV-1 Infected 0.101 Skin Fibroblasts HSV-2 Infected 0.1.29 Skin Fibroblasts Patient samples wεrε idεntified by classic cytopathic effect pattern rεcogπition by trainεd clinical virology laboratory personnel. DISCUSSION We have discovered that HCMV contains at least two membrane envεlope glycoprotein complexes which con¬ tain glycopeptides GLP-A, and GLP-B, which are arranged on the surface of the virus particlε in such a way that they are neither encasεd in a fold, buried in a crypt nor sterically hindered from reacting with macrocom- ponεnts of the bodily environment, e.g., antibody and Th cell antigen recognition sites. Thεsε glycopeptides strongly elicit humoral and cellular immune responses, indicating a significant role in recovery from HCMV infection. Futhermore, MoAbs to these glycopeptidεs nεutralize live viruses. This invention provides a process for iso- lating icrogram quantities of substantially purε gly¬ copeptides. It is expected that GLP-A and GLP-B and the MoAbs derived therεfrom will provε usεful for thε diagnosis of HCMV, therapy for infectεd patiεnts and prophylaxis for uninfεctεd pεrsons. MoAbs 1-48-41C2 and 2-29-9B7 arε providεd as ' εxamplεs of antibodies that can be raised against GLP-A. Thεsε antibodiεs are particularly useful for the diagnosis of active HCMV viruses. Those skilled in the art, following the teachings of this invention, ■ may readily producε GLP-A to usε in thε idεntification of such monoclonal antibodies. MoAb 2-15-9E10 is also provided which reacts with GLP-8 and is capable αf neutralizing HCMV w tnout the addition of complεment. Those skilled in the art, following the teachings of this invention, may readily produce GLP-B to use in the identification of other monoclonal antibodies which share this characteristic. Such antibodies are useful in HCMV therapy and the degree of cross reactivity with other viruses of the herpes class confers an additional advantage to this use. The hybridomas producing the MoAbs provided as examples have been deposited with jn Vitro Interna¬ tional, Linthicum, Maryland, and have been assigned the following access codes: Hb 2-29-9B7 = IVI-10117; Hb 2-15-9E10 = IVI 10118; Hb 1-48-41C2 = IVI-10119. Cultures of these deposited hybridomas will be made available to the public upon the grant of a patent based upon the presεnt application. It is to bε under¬ stood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by the United Statεs Government. While certain representations embodied are described herein for the purposes of illustration, it will be apparεnt to those skilled in thε art that mod¬ ifications thεrεin may be made without departing from the spirit and scope-of thε invεntion. Wε wish to thank Childrεn's Hospital, St. Paul, Minnεsota for gεnεrous support and εncouragement in this work.

Claims: WHAT IS CLAIMED ISt 1. A monoclonal antibody produced by a process comprising? (a) immunizing a mouse with HCMV virions; (b) fusing spleεn cells from said mouse with cells from a myeloma line to produce hybridomas; (c) selecting from said hybridomas a hybridoma which produces a monoclonal antibody which is reactive with HCMV, but does not react with Herpεs simplex, adenovirus, Epstein Barr virus or Varicella virus, and wherein said mono¬ clonal antibody is capable of neutralizing HCMV; and (d) clonally expanding said hybridoma. 2. The monoclonal antibody of claim 1 which immunopre- cipitates a 93 kD HCMV envelope-associatεd glyco¬ peptide. 3. The monoclonal antibody of claim 1 which is pro¬ duced by hybridoma IVI-10117. 4. The monoclonal antibody of claim 1 which is pro¬ duced by hybridoma IVI-10119. 5. The monoclonal antibody produced by a process comprising: (a) immunizing a mouse with HCMV virions; (b) fusing spleen cells from said mouse with cells from a myeloma line to produce hybridomas j (c) selecting from said hybridomas a hybridoma which produces a monoclonal antibody which is reactive with HCMV, Herpes simplex and adeno¬ virus and which antibody is capable of neu¬ tralizing HCMV in the absence of complement; and (d) clonally expanding said hybridoma. 6. The monoclonal antibody of claim 1 which immunopre- cipitates a 50-52 kD HCMV envelope-associated gly- coprotein. 7. The monoclonal antibody of claim 5 which is pro¬ duced by hybridoma IVI-10118.

Inventors: GEHRZ RICHARD C, KARI BRUCE E, LUSSENHOP NANCY O, GEHRZ, RICHARD, C., KARI, BRUCE, E., LUSSENHOP, NANCY, O.
Applicants: CHILDRENS HOSPITAL INC
Application year: 1987
Main classification: C12P21
IPC codes: A61K39, C07K1, C07K14, C07K16, C12N5, C12N15, C12P21, C12R1, G01N33