WO1990000062A1

WO1990000062A1 - Antibody recognition of fragments from cytomegalovirus

Info

Publication number: WO1990000062A1
Application number: PCT/US1989/002613
Authority: WO
Inventors: Bruce E. Kari; Richard C. Gehrz
Original assignee: Childrens Hospital Inc
Current assignee: Childrens Hospital Inc
Priority date: 1988-07-01
Filing date: 1989-06-15
Publication date: 1990-01-11
Anticipated expiration: 1991-01-01
Also published as: IL90833A0; JPH03505457A; EP0429470A1; AU3849989A

Abstract

Disclosed according to the invention are immunogenic proteolytic fragments of a glycoprotein complex (gC-I) of human cytomegalovirus (HCMV). These fragments are useful for production of antibodies to HCMV and for diagnosis of HCMV infections.

Description

ANTIBODY RECOGNITION OF FRAGMENTS FROM

CYTOMEGALOVIRUS

Background of the Invention

Human cytomegalovirus contains several disulfide-linked glycoprotein complexes (Britt, 1984,

Rasmussen et al., 1985b; Farrar and Greenway, 1986;

Kari et al., 1986; Gretch et al., 1988). One of these complexes contains a glycoprotein related to HSV glycoprotein gB (Oranage et al., 1986). This complex has been designated p130/55 or gC-I (Rasmussen et al., 1985b; Lussenhop et al., 1988) and is the most well characterized complex of those found in HCMV. Complex gC-I isolated from whole cells or extracellular virus has been reported to contain as many as three glycoproteins with molecular weights of 160-130,000, 92-93,000 and 50-55,000 (Britt, 1984; Farrer and Greenway, 1986; Kari et al., 1986; Rasmussen, 1988). These glycoproteins are held together by disulfide bonds and contain largely N-linked oligosaccharides (Rasmussen et al., 1988). Complex gC-I can be immunoprecipitated by human serum positive for HCMV and will also stimulate human T cells (Liu et al., 1988).

Several murine monoclonal antibodies which recognize gC-I have been disclosed. Based on a simultaneous two-antibody-binding assay, these antibodies could be grouped into three domains (Lussenhop et al., 1988). Domains I and II are recognized by antibodies which can neutralize Towne strain HCMV in vitro. Antibodies within a single domain were effective at inhibiting each other's binding. Furthermore, as determined by an ELISA assay, antibodies in domain I were found to augment the binding of antibodies in domain II and vice versa. This augmenting activity was also observed in a plaque reduction assay (Lussenhop et al., 1988). Only one antibody (11B4) was placed in a third domain (domain III) based on its unique ability to inhibit the binding of all other antibodies tested.

Antibody 11B4 was also observed to be non-neutralizing and had the ability to inhibit the neutralizing activity of other antibodies in a plaque-reduction assay. These gC-I specific murine monoclonal antibodies were found to recognize several clinical isolates of HCMV as determined by immunofluoresence. This suggested that the epitopes recognized by these antibodies are conserved (Lussenhop et al., 1988).

Since all the murine monoclonal antibodies which we originally characterized showed some type of interaction, i.e., augmentation or inhibition, it seemed possible that the three domains containing the epitopes recognized by these antibodies might be physically close to gC-I. Proteolysis has been one approach used to determine if epitopes are physically close or distant on viral glycoproteins. For example, proteolysis has been used to determine the location of anti- genic determinants on glycoproteins from tick-borne encephalitis virus (Heinz et al., 1983), murine leukemia virus (Pinter et al., 1982) and glycoprotein D from HSV (Eisenberg et al., 1982).

Summary of the Invention

The present invention provides immunogenic proteolytic fragments of gC-I which contained all three immunoreactive domains of gC-I. Using either trypsin or chymotrypsin, a set of fragments could be generated which were recognized by antibodies from all three domains. These fragments were also recognized by several human sera positive for HCMV. Trypsin and chymotrypsin fragments were also examined for T cell reactivity. Lymphocyte proliferative responses were detected with fragments made with either enzyme in the case of individuals who reacted strongly with whole gC-I, but higher responses were consistently obtained with trypsin fragments. Cells obtained from an individual who had low responses to whole gC-I had low responses to the fragments as well. But a T cell clone from this individual was observed to react with trypsin fragments, but not chymotrypsin fragments.

More specifically, the HCMV envelope glycoprotein complex gC-I was digested with chymotrypsin and fragments immunoaffinity purified. Two major glycosylated peptides were obtained which had molecular weights (MWs) of 34,000 and 43,000 under non-reαucing conditions. After reduction, one major glycosylated fragment, with a MW of 34,000, was observed in addition to at least two other peptides with MWs of 30,000 and 28,000. Under non-reducing conditions, monoclonal antibodies (MoAbs) which bind to all three domains of gC-I immunoprecipitated the 34,000 and 43,000 MW fragments and reacteα with them in Western blot. After reduction, the MoAb assigned to domain III and one MoAb in domain I were non-reactive while all other MoAbs reacted with the 34,000, 30,000 and 28,000 MW peptides in Western blot.

Five positive human sera were reactive with whole purified gC-I in Western blot under non-reducing conditions. After reduction, nine MoAbs and one serum reacted with 130,000 and 52,000 MW proteins and four sera reacted with these plus a 93,000 MW protein. The five sera were also reactive with the chymotrypsin fragments in Western blot under non-reducing conditions. After reduction, four sera reacted strongly with the 34,000, 30,000 and 28,000 MW oeptides and one reacted weakly. Thus, the gC-I domains recognizeα by murine MoAbs may be important in human immune recognition of HCMV, and all of the immunogenic gC-I subunit peptides described hreinabove are within the scope of the present invention.

Detailed Description of the Invention

The invention will be further described by reference to the following detailed Example.

EXAMPLE I.

A. Materials and Methods

1. Preparation of Towne and AD169 HCMV strains. Both strains of HCMV were grown on human skin fibroblasts, harvested and purified as described (Kari et al., 1986). Virus was labeled by growing with either [³H] arginine, [¹⁴C]GlcN, or [³H]GlcN (Amersham).

Generation, characterization and purification of monoclonal antibodies. Monoclonal antibodies were generated and characterized as previously described

(Lussenhop et al., 1988). Monoclonal antibodies were purified from mouse ascities fluid by high performance liquid chromatography using a hydroxyapatite column (Bio Rad) as previously described (Juarez-Salinas et al., 1984).

Proteolvsis. Either purified Towne strain HCMV or whole cells at 7 to 14 days post infection were solubilized with 1.0% NP-40 in 50 mM Tris buffer (pH 7.4) containing 150 mM NaCl. Insoluble material was removed by centrifugation at 16,000 × g for 30 min. The extract was collected and protein content determined with the BCA protein assay (Pierce). The extract was digested with either TPCK-Trypsin (Worthington) or TLCK-chymotrypsin (Sigma) at an enzyme-to-protein ratio of 1:50 for 24 to 48 hours at room temperature. Proteolysis was terminated with either enzyme by addition of phenylmethyl-sulfonyl fluoride (PMSF). Extracts were also digested with pronase (Sigma) using the same conditions. Pronase digestion was stopped by addition of BSA. Purification of whole gC-I or its proteolytic fragments. Whole gC-I or its fragments were isolated by a modification of an immunoaffinity method using biotinylated monoclonal antibodies and streptavidin agarose (Gretch et al., 1987). Briefly, a biotinylated monoclonal antibody was added to 1.0% NP-40 extracts containing whole gC-I or its fragments. The antibody was incubated with the extracts for 30 min. before adding streptavidin agarose. This mixture was allowed to react for an additional 45 min. with constant mixing. The agarose beads were pelleted by centrifugation and then washed twice with PBS containing 0.1% NP-40 and twice with PBS. Proteins and peptides were eluted from the bound antibody by heating at 100ºC in a Tris buffer (0.2 M Tris, pH 6.8, containing 4% SDS) for 3 min.

SDS-PAGE and Fluorography. Radioactively labeled glycoproteins or glycopeptides which had been immunoprecipitated were separated by SDS-PAGE in 5-15% polyacrylamide gradient gels using the method of Laemmli, 1970. Radioactivity in these gels was detected by fluorography using Enhance (New England Nuclear).

Western blot analysis. A mini-gel apparatus (Bio Rad) was used for Western blot. Glycoproteins were separated in straight 10% polyacrylamide gels. Undigested glycoproteins were electroblotted onto nitrocellulose membrane with a .45 micron core size. In order to retain the small glycopeptides obtained by proteolysis, it was necessary to use nitrocellulose membrane with a 0.2 micron pore size. After electroblotting, the paper was blocked with 3% gelatin in Tris buffered saline (TBS 20 mM Tris, 500 mM NaCl pH 7.5). MoAbs in asities fluid were diluted 1/500 and human serum was diluted 1/30 in 1% gelatin in TBS. Diluted MoAbs or human serum was allowed to bind to blotted paper overnight at room temperature. The paper was washed with TBS containing 0.05% Tween 20. Phosphate labeled goat antimouse IgG or goat anti-human IgG (Kirkegaard and

Perry), diluted 1/1000 with 1% gelatin in TBS, was added and allowed to react at room temperature for one hour. The paper was washed and the substrate 5-bromo-4-chloro-3-indolyl phosphate/tetrazolium in 0.1 M Tris buffer (Kirkegaard and Perry) was added. After visualization of bands, the reaction was stopped by immersing the paper in water.

Deolvcosylation of glycooeptides. Chymotrypsin fragments were digested with t7e enzyme N-Glycanase

(Genzyme Corp., Boston, MA) which hydrolyzes

asparagine-linked oligosaccharides from glycopeptides to give free oligosaccharide and peptide-containing aspartic acid at the glycosylation site. Fragments obtained with chymotrypsin were eluted from streptavadin agarose affinity columns with 1.0% SDS (w/v).

Eluted fragments were reduced with beta-mercaptoethanol and diluted witn phosonate-buffered saline (pH 8.6) containing 1.0% NP-40 so that the final SDS concentration was 0.1%. The protease inhibitor 1, 10-phenanthroline hydrate was added according to the manufacturer's instructions. The reaction was done at room temperature for 24 hours with constant mixing. At the end of this time, SDS was added to bring the SDS concentration back to 1.0% and the reaction mixture was dialyzed against 0.1% SDS overnight. Samples were concentrated prior to SDS-PAGE.

Lymphocyte proliferation assays. Lymphocyte proliferation assays were performed as previously described (Liu et al., 1988). Briefly, proteolytic fragments used for T cell analysis were extensively dialyzed against PBS to remove toxic substances. Dialysis membrane with a molecular weight cut out of 10-12,000 was used.

Because of this, fragments with molecular weights of less than 12,000 were lost. The T cell clone used in this study was generated and characterized as previously described (Liu et al.).

Results

Trypsin and chymotrypsin digestion of qC-I. These studies were done to determine which enzyme would most effectively degrade gC-I to smaller fragments. To determine the end point of proteolysis, a kinetic study was done. An NP-40 detergent extract was obtained from infected whole ceils wnich were collected from culture media at 7 and 11 days post infection by low speed centrifugation. Proteins were labeled with C³H] arginine and glycoproteins with [¹⁴C]GlcN. A portion of the extract was immunoprecipitated immediately with a gC-I specific monoclonal antibody (41C2, domain I) to establish the nature of the starting material. The remaining extract was divided into several equal aliquotes. These were subjected to proteolysis using either TPCK-trypsin or TLCK-chymotrypsin. Proteolysis was stopped at 0.5, 2, and 24 hours by addition of PMSF and gC-I fragments immunoprecipitated with 41C2. In addition, an aliquot was allowed to remain at room temperature for 24 hours without exposure to proteolysis. Proteins and glycoproteins immunoprecipitated with 41C2 were examined by SDS-PAGE with and without reduction of disulfide bonds.

The gC-I comDlexes immunoprecipitated by 41C2 from the initial extract or from the same extract after 24 hours at room temperature were similar if not identical regardless of the label used. This demonstrated that gC-I in the absence of trypsin or chymotrypsin was stable in the extract for at least 24 hours. In the absence of proteolysis, complexes typical of gC-I were obtained which had molecular weights from 130,000 to greater than 200,000. The most abundant glycoproteins obtaineα from these complexes after reduction had molecular weights of 130,000, 93 , 000 and 50-52,000 regardless of the radioactive label used. In addition, two [³H] arginine-labeled peptides were detected when gC-I was examined without reduction which had molecular weights of 35,000 and 20,000. These peptides were present regardless of proteolysis and were only clearly detected with [³H] arginine, suggesting that they contained little or no carbohydrate.

Between 30 min. and 24 hrs. of proteolysis with both enzymes, complexes with molecular weights of 106,000 to 130,000 were detected with either radioactive label. When these complexes were reduced, glycoproteins with molecular weights ranging from 35,000 to 93,000 were most abundant. It appears that the degradation of complexes in the 106,000 to 130,000 molecular weight range is slow compared to the initial proteolysis of intact gC-I.

After 24 hours of proteolysis with chymotrypsin, one major [³H] arginine-labeled peptide was

detected under non-reducing conditions, as well as less abundant peptides with molecular weights of between 43,000 and 34,000 and one at 20,000. With [¹⁴C]GlcN labeling, only the peptides between 43,000 and 34,000 could be clearly detected. After reduction of disulfide bonds, these chymotrypsin fragments generated one major 34,000 molecular weight glycopeptide, but a number of less abundant lower molecular weight peptides were also observed.

Similar, but not identical, results were obtained with trypsin. There was a major glycosylated fragment with a molecular weight of 44,000. However, some of the 106,000 molecular weight glycopeptide remained after 24 hrs and there was a lack of glycosylated peptides between molecular weights of 44,000 to 34,000.

After reduction of peptides obtained with trypsin, two major glycosylated peptides with molecular weights of 47,000 and 35,000 were detected. In addition, a smear of glycosylated material was detected above the 47,000 molecular weight glycopeptide. A number of minor lower molecular weight peptides similar to those detected with chymotrypsin were also present. Finally, the banding pattern observed with either enzyme was not changed by extending the reaction time to 48 hrs or by adding additional enzyme.

Immunoprecipitatipn of chymotrypsin. trypsin and pronase fragments with antibodies from domains I. II and III. For these experiments, gC-I was obtained from purified extracellular virus. Immunoprecipitations were done with monoclonal antibodies selected from domains I (39E11), II (34G7), and III (11B4) to determine if the proteolytic fragments contained epitopes from all three domains. Other antibodies identified as representative of domains I and II can also be used (see U.S. Patent application Serial No. 144,760, filed January 19, 1988, the disclosure of which is incorporated by reference herein.)

The chymotrypsin and trypsin glycopeptide fragments obtained from gC-I isolated from extracellular virus were the same as those immunoprecipitated when gC-I was obtained from infected cells. In addition, antibodies from all three domains were capable of immunoprecipitating the same fragments, suggesting that they contained the three domains previously described (Lussenhop et al., 1988). A similar experiment was done with pronase (a non-specific protease). Pronase fragments were similar to those obtained with chymotrypsin. However, with pronase, little of the 43,000 molecular weight peptides remained and most of the gly- copeptides formed a smear with a molecular weight from 30-34,000. This further demonstrates the resistance of this portion of gC-I to proteolysis.

Western blot analysis of whole gC-I and chymotrypsin fraoments of gC-I reacted with monoclonal antibodies and human serum positive for HCMV. Western blot analysis was done for three reasons. First, with immunoprecipitation, several peptides were detected regardless of reduction of disulfide bonds and it was of interest to determine which of these were recognized by the monoclonal antibodies. Secondly, we wished to extenα our study to additional gC-I monoclonal antibodies to see if they would all react with the fragments generated. In this regard, we focused on chymotrypsin fragments since they were less heterogeneous and smaller in molecular weight. Thirdly, it was of interest to determine whether or not human antibooies would recognize the same fragments as murine antibodies. Six human serum were selected for this study. Five were determined to be positive and one negative for HCMV by complement fixation and indirect Immunofluoresence assays (Table 1). Table 1

Sera CMV Titers* HSV Titers**

A,1 <4/<10 (-) <4 (-)

A,2 32/40 (+) <4 (-)

A,3 256/160 (+) 16 (+)

A,4 32/80 (+) ELISA (-)

I,1 128/640 (+) <4 (-)

I.2 32/160 (+) <4 (-)

* CMV titers were determined by a latex aggultination assay (LA) or immunofluoresences (IF). The first number represents the LA titer and the second the IF titer.

** HSV titers were determined by immunofluoresence.

In addition, since gC-I contains glycoproteins which have homology with gB from HSV, these serum were tested for their reactivity with HSV by immunofluoresence. Of those tested, only one (A, 3) was positive for HSV

(Table I). However, the reactivity of this sera was similar to the others. For Western blot analysis, both whole gC-I and chymotrypsin fragments were immunoaffinity purified with a gC-I specific monoclonal antibody. Because of this, a small amount of monoclonal antiboαy sometimes contaminated the preparations and could be detected in Western blot as a small band at the top of the nitrocellulose membrane or as heavy and light chains.

Under non-reoucing conditidns, two monoclonal antioodies (11B4 and 26B1I) failed to react with whole gC-I in Western blot. This occurred even though these antibodies were capable of immunoprecipitating gC-I.

The pattern obtained with all other monoclonal antibodies was very similar to that obtained with the positive human serum, including reactivity with the 34,000 and 20,000 molecular weight peptides which were also observed by immunoprecipitation of whole gC-I. After reduction of disulfide bonds, monoclonal antibodies 11B4 and 26B11 still failed to react. All other monoclonal antibodies reacted strongly with the 130,000 and 52,000 molecular weight proteins and weakly with proteins slightly below the 130,000 molecular weight protein and a 50,000 molecular weight protein. Again, the human serum positive for HCMV reacted with these proteins. However, while human sera A, 2 gave almost identical results to the monoclonal antibodies, the other positive serum reacted in varying degrees with the 93,000 molecular weight protein. In addition, the human serum reacted weakly with a 26,000 molecular weight protein which was not recognized by the monoclonal antibodies. None of the proteins recognized by the human serum or the monoclonal antibodies were reactive with the negative human sera (A,1) or with the negative murine antibody control (SP2).

Examination of chymotrypsin fragments under non-reducing conditidns showed that all monoclonal antibodies reacted with 43,000 and 34,000 molecular weight peptides. These included 11B4 and 26B11, which failed to react with whole gC-I under non-reducing conditions. The reactivity of 11B4 and 26B11 was less than that of the other monoclonal antibodies. There were weaker reactions with peptides having an apparent mdlecular weight of 63,000. This may represent a small amount of incompletely digested peptide. These peptides appear to represent only a small portion of the protein present since they could not be detected by Comassie blue staining while the 43,000 and 34,000 molecular weight peptides were clearly visible. Again, the negative murine antibody control was not reactive with the peptides recognized by the monoclonal antibodies. Under non-reducing conditions, the pattern obtained with all positive human serum was identical to the monoclonal antibodies and the negative human sera (A,1) was non-reactive.

After reduction of disulfide bonds, monoclonal antibodies 1184 and 26B11 were not reactive with the chymotrypsin fragments of gC-I. All other monoclonal antibodies reacted strongly with the 34,000 and 30,000 molecular weight peptides and, with the exception of 41C2, reacted strongly with a 28,000 molecular weight peptide. Of the human positive serum tested, four (A,2, A,3, I,1 and I,2) reacted strongly with the

34,000, 30,000 and 28,000 molecular weight peptides. The human positive sera A, 4 was only weakly reactive after reduction of disulfide bonds. There were also weaker bands detected at an apparent molecular weight of 50,000 with the monoclonal antibodies, human serum and the negative controls. One of these was probably the heavy chain contributed by the antibody used to initially purify the fragments since this band was detected in the SP2 negative control. Nonetneless, since these bands were detected by the human negative serum, it is likely that reactivity with them in

Western blot was not specific. Finally, the reactivity of the human serum with either whole gC-I glycoproteins or chymotrypsin fragments of gC-I did not apppear to depend on their HCMV titer since a sera with a titer of 32/40 (sera A,2) reacted as well as serum with titers greater than 100 (serum A,3, I,1 and I,2).

Finally, for comparison, gC-I from HCMV strain AD169 was also digested with chymotrypsin, fragments of immunoaffinity purified and examined by Western blot under non-reducing conditions to determine whether or not all three domains would be present. When this was done, all monoclonal antibodies reacted with the fragments in Western blot, demonstrating the presence of these domains in AD169. However, the pattern was slightly different. A weak band was detected at 63, 000 molecular weight, but the lower molecular weight peptides appeared more diffuse than they did with peptide from Towne strain HCMV. A broad band covering molecular weights from 40,000 to 35,000 was detected along with a band at 31,000 and another diffuse band with a molecular weight of 23,000.

Deglvcosylation of chymotrypsin fragments. After reduction of chymotrypsin fragments, the monoclonal antibodies recognized three peptides in Western blot. The heterogenity in molecular weight may have been due to the extent of glycosylation. To examine this poss ibility, glycopeptides obtained with chymotrypsin were reduced and digested with N-glycanase. Glycopeptides labeled with either [³H] arginine or [¹⁴C] GlcN were used. Since N-glycanase action generates peptides free of carbohydrate, all label should have been lost in the L¹⁴C] GlcN labeled peptides, which provided a way to assay the extent of carbohydrate removal. After digestion with N-glycanase, the major 34,000 molecular weight [³H]arginine labeled peptide was lost and peptides with molecular weights of 30,000 and 28,000 were observed. Comparable bands were not detected when

[¹⁴C] GlcN labeled peptides were digested, suggesting that carbohydrate removal was complete.

T cell recognition of unreduced trypsin and chymotrypsin fragments. Peripheral blood mononuclear cells (PBMC) were obtained from three seropositive donors. As we previously showed, cells from these individuals were reactive with whole gC-I (Liu et al., 1988).

Cells from individuals which were sercnegative to HCMV did not proliferate when exposed to purified gC-I.

Proliferative responses were obtained with PBMCs obtained from seropositive individuals with either trypsin or chymotrypsin fragments (Table 2). Table 2

A6 (-) A4 (+) A5 (+) A7 (+)

MNC* 2,359 1,303 60 314

MNC+HCMV** 232 42,481 9,020 81,689 MNC Neg Con*** 1,111 7,039 231 1,272 MNC Try Frag.**** 1,099 74,642 586 84,327 MNC Chv. Frag.***** 654 22,224 208 6.260

* MNC Mononuclear cell culture only.

** MNC plus whole virus.

*** MNC plus negative control containing buffer but no virus or fragment.

**** MNC plus trypsin fragments.

***** MNC plus chymotryosin fragments.

Proliferative responses of mononuclear cells (MNC) from HCMV negative (-) and HCMV positive (+) individuals. Cultures containing 10⁶ cells per well were exposeα to antigens for six days prior to being plused with [³H]- thymidine for 24 hrs before harvesting cultures.

Numoers represent average counts per min. from triplicate cultures.

However, with the same amount of protein present, responses were greater with trypsin fragments than with chymotrypsin fragments. This suggested that some T cell epitopes may have been lost by the more complete proteolysis when chymotrypsin was used. In addition, one individual which had a low response to whole gC-I (A5) was observed to have very low or no response to fragments obtained with either enzyme. However, a T cell clone from this individual was observed to have a very strong response to the trypsin fragments, but not to the chymotrypsin fragments. This further demonstrated the loss of T cell epitopes from the chymotrypsin fragments and suggested that the number of T cells recognizing the epitopes on the trypsin fragments was low in the population from which this clone was obtained.

Discussion

The primary purpose of this study was to examine the possibility that a small portion of gC-I would contain the three domains which we detected in our original studies using a simultaneous two-antibody-binding assay. While both trypsin- and cnymotrypsin-generated fragments were recognized by antibodies from all three domains, chymotrypsin was capable of generating the smallest fragments. Because of this, we focused further studies on the chymotrypsin fragments. After proteolysis of [³H] arginine labelled gC-I with chymotrypsin, there appeared to be only one major peptide present under non-reducing conditions which had a molecular weight of 43,000. With Comassie blue staining, the 43,000 molecular weight peptide was still most abundant, but a 34,000 molecular weignt peptide coulc be more clearly detected. Both of these unreduced peptides were glycosylated as determined by incorporation of [¹⁴C] GlcN and were recognized by all monoclonal antibodies and human serum positive for HCMV. There are probably other β cell epitopes in gC-I which are bound by human antibodies, e.g., those which appear to be on the 93,000 molecular weight glycoprotein. The chymotrypsin fragments also appear to contain sites for neutralization since all antibodies in domains I and II neutralized Towne strain HCMV either individually or in combination. However, with all monoclonal antibodies, complement was required for neutralization (Lussenhop et al., 1988). The chymotrypsin fragments also contained T helper cell epitopes; however, more of these appear to remain in the trypsin fragments. That T cell epitopes were deleted by chymotrypsin was clearly demonstrated by the positive reactivity of a T cell clone with the trypsin fragments, but not with chymotrypsin fragments. Furthermore, the individual from which the T cell clone was obtained showed a very low response to either trypsin or chymotrypsin fragments when mononuclear cell cultures were used. Thus, the population of T cells present in the mononuclear cell cultures from this individual recognizing the T cell epitope present in the trypsin fragments must have been low.

Another interesting observation was that the chymotrypsin fragments from gC-I also contained the epitope recognized by antibody 11B4 which was placed in domain III. This was established by the ability of 11B4 to immunoprecipitate the chymotrypsin fragments, and its reactivity in Western blot under non-reducing conditions. Antibody 11B4 is a non-neutralizing antibody which blocks binding of neutralizing antibodies (Lussennop et al., 1938). From this perspective, anti bodies directed toward this epitope would have benefit for the virus, but not the host. It would seem that the chymotrypsin fragments could be used as part of a synthetic vaccine, although the epitope recognized by 11B4 should be avoided in any attempt to make a synthetic vaccine. However, 11B4 appears to recognize a conformational epitope since its reactivity with the chymotrypsin fragments was lost after reduction of disulfide bonds. Unlike 11B4, those antibodies which neutralized Towne strain HCMV and the human serum were reactive after reduction of disulfide bonds. Furthermore, after reduction, three peptides were detected by our monoclonal antibodies and human serum. One of the major differences between these three peptides was the extent of glycosylation. The 34,000 molecular weight peptide was most heavily glycosylated, but could be reduced to at least 30,000 molecular weight by action of N-glycanase. The presence of polysaccharides on the 34,000 molecular weight peptide could have some impact on antibody binding as has been demonstrated with other glycoproteins (Alexander and Elder, 1984; Caust et al., 1987). However, our preliminary results (data not shown) show that removal of carbohydrate did not prevent binding of neutralizing monoclonal antibodies and in some cases binding appeared to increase. Moreover, the 28,000 and 30,000 molecular weight peptides were under- or non-glycosylated as compared to the 34,000 molecular weight peptide, but they still bound antibody in Western blot. Thus, a synthetic vaccine might include only the linear amino acid sequences of the peptides in the chymotrypsin fragments. Such subunit vaccines are within the scene of this invention.

Several observations suggest that the fragments of gC-I obtained by digestion with chymotrypsin are important to the function of gC-I. First, the monoclonal antibodies reactive with these fragments also recognize several laboratory and wild type strain of HCMV (Lussenhop et al., 1988). In fact, conformational and non-conformational epitopes were shown to be present in HCMV strains AD169 and Towne. These results suggest that the epitopes are conserved. Secondly, a blocking epitope in domain III was present in these fragments. This epitope may help survival of the virus by preventing other antibodies from binding to important sites for gC-I function. Thirdly, the gC-I chymotrypsin fragments were glycosylated. One important function of glycosylation is to prevent the action of proteases (Iwase, 1988). In our experiments, even pronase (a non-specific protease) was prevented from generating fragments smaller than those obtained with chymotrypsin. These results suggest that this part of gC-I is well protected from proteolysis. Finally, the maintenance of the higher order structure of the chymotrypsin fragments seems to be independent of the rest pf the gC-I molecule and resistant to denaturation with detergents. This Indicates a very stable structure. REFERENCES

The disclosures of the papers cited below are incorporated by reference herein.

1. S. Alexander et al., Science, 226, 1328-1330

(1984).

2. J. B. Britt et al., Virology, 135, 369-378 (1984).

3. J. Caust et al., Arch. Virol., 96 , 123-134 (1987).

4. M. Cranage et al., EMBO J. , 5 , 3057-3063 (1986).

5. R. J. Eisenberg et al., J. Virol., 41, 478-488

(1982).

6. G. H. Farrar et al., J. Gen. Virol., 67, 1469-1473 (1986).

7. D. R. Gretch et al., Anal. Biochem., 163, 270-277 (1987).

8. D. R. Gretch et al., J. Virol., 62, 875-881 (1988).

9. F. X. Heinz et al., Virology, 130, 485-501 (1983).

10. H. Iwase, Int. J. Siochem., 20, 479-491 (1988).

11. H. Juarez-Salinas, Biotechniques, 2 , 164-169

(1984).

12. B. Kari et al., J. Virol., 60, 345-352 (1986).

13. Y.-N. Liu, J. Virol., 62, 1066-1070 (1988).

14. N. 0. Lussenhop et al., Virology, 164, 362-372

(1988).

15. A. Pinter et al., Virology, 116, 499-516 (1982).

16. L. Rasmussen et al., Virology, 163, 308-318 (1988a)

17. L. Rasmussen et al., J. Virol., 55, 274-280

(1985b).

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. An immunogenic peptide having a molecular weight of about 28,000, 30,000 or 34,000 which is prepared by a process comprising:

(a) digesting HCMV envelope glycoprotein complex gC-I with chymotrypsin to yield glycosylated peptide fragments having molecular weights of 34,000 and 43,000; and

(b) reducing the disulfide bonds of the peptide fragments to yield reduced peptides having molecular weights of 28,000, 30,000 and

34,000.