ANTIBODY RECOGNITION OF FRAGMENTS FROM CYTOMEGALOVIRUS

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: - i - ANTIBODY RECOGNITION OF FRAGMENTS FROM CYTOMEGALOVIRUS Bac αround of the Invention Human cyto egalovirus contains several disul- fide-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 glyco¬ protein gB (Cranage et al., 1986). This complex has been designated pl30/55 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 glycopro- teins 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 glycopro- teins 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 simul¬ taneous 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. Anti¬ bodies within a single domain were effective at inhi¬ biting each other's binding. FurthermQr- , as deter¬ mined by an ELISA assay, antibodies in domain I were -2- 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 acti¬ vity of other antibodies in a plaque-reduction assay. These gC-I specific muriπe 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 con¬ served (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 physi¬ cally close to gC-I. Proteolysis has been one approach used to determine if epitopes are physically close or distant on viral glycoproteins. For example, proteo¬ lysis has been used to determine the location of anti- genic determinants on glycoproteins from tick-borne encephalitis virus (Heinz et al., 1983), murine leuke¬ mia virus (Pinter et al., 1982) and glycoprotein D from HSV (Eisenberg et al., 1982). -3- Summarv 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 indivi¬ dual 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 glycopro¬ tein complex gC-I was digested with chymotrypsin and fragments immunoaffinity purified. Two major glycosy- lated peptides were obtained which had molecular weights (MWs) of 34,000 and 43,000 under πon-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 i munoprecipitated the 34,000 and 43,000 MW frag¬ ments and reacteα with them in Western blot. After -4- 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 condi¬ tions. 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 recogni¬ tion of HCMV, and all of the immunogenic gC-I subunit peptides described hreiπabove 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 ano 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 [ 3 H] arginine, or [ 3 H]G1CN (Amersham). Generation, characterization and purification of mono¬ clonal antibodies. Monoclonal antibodies were gener¬ ated 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 solubi- lizεd with 1.0% NP-40 in 50 M Tris buffer (pH 7.4) containing 150 mM NaCl. Insoluble material was removed by centrifugation at 16,000 x 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. Proteo- lysis was terminated with either enzyme by addition of phenyl ethyl-sulfonyl fluoride (PMSF). Extracts were also digested with pronase (Sigma) using the same con¬ ditions. Pronase digestion was stopped by addition of BSA. -6- Purification of whole gC-I or its proteolytic frag¬ ments. Whole gC-I or its fragments were isolated by a modification of an immunoaffinity method using biotiny- lated monoclonal antibodies and streptavidin agarose (Gretch et al., 1987). Briefly, a biotinylated mono¬ clonal antibody was added to 1.0% NP-40 extracts con¬ taining 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 elutεd from the bound antibody by heating at 100°C 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 immuno- precipitated were separated by SDS-PAGE in 5-15% poly- acrylamide 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 separ¬ ated in straight 10% polyacrylamide gels. Undigested glycoproteins were electroblotted onto nitrocellulose membrane with a .45 micron core size. In order to -7- retain the small glycopεptides 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 con¬ taining 0.05% Tween 20. Phosphate labeled goat anti- mouse 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 visual¬ ization of oaπαs, the reaction was stopped by immersing the paper in water. Deolvcosylation of αlvcooeptides. Chymotrypsin frag¬ ments were digested with t7e enzyme N-Glycanase (Genzyme Corp., Boston, MA) which hydrolyzes asparagine-linked όligosaccharides from glycopεptides to give free oligosaccharide and peptide-contaiπing aspartic acid at the glycosylation site. Fragments obtained with chymotrypsin were eluted from streptava- din agarose affinity columns with 1.0% SDS (w/v). Eluted fragments were reduced with beta-mercaptoethanol and diluted witn phosonate-buffεred saline (pH 8.6) -8- contaiπing 1.0% NP-40 so that the final SDS concentra¬ tion was 0.1%. The protease inhibitor 1,10-phenanthro- line hydrate was added according to the manufacturer's instructions. The reaction was done at room tempera¬ ture 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 prolifera¬ tion 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 weignts of less than 12,000 were lost. The T cell clone used in this study was generated and characterized as pre¬ viously 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 enα point of proteolysis, a kinetic study was done. An NP-40 detergent extract was obtained from infected whole ceils wnich were collected from culture - 9- media at 7 and 11 days post infection by low speed cen¬ trifugation. Proteins were labeled with C 3 H] arginine and glycoproteins with [ 1 '*C]G1CN. A portion of the extract was immunoprecipitated immediately with a gC-I specific monoclonal antibody (41C2, domain I) to estab¬ lish the nature of the starting material. The remain¬ ing 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 immunoprεcipitatεd 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 di¬ sulfide bonds. The gC-I comDlεxes immunoprecipitated by 41C2 from the initial extract or from the same extract after 24 hours at room temperature were similar if not iden¬ tical 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 obtainεα from these complexes after reduction had mole¬ cular weights of 130,000, 93 , 000 and 50-52,000 regard¬ less of the radioactive laoel used. In addition, two L 3 Hj arginine-labelεd peptiαεs were detected when gC-I was examined without reduction which had molecular -10- weights of 35,000 and 20,000. These peptides were pre¬ sent regardless of proteolysis and were only clearly detected with [ 3 HJ arginine, suggesting that they con¬ tained 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 radioac¬ tive label. When these complexes were reduced, glyco¬ proteins with molecular weights ranging from 35,000 to 93,000 were most abundant. It appears that the degra¬ dation of complexes in the 106,000 to 130,000 molecular weight range is slow compared to the initial proteoly¬ sis of intact gC-I. After 24 hours of proteolysis with chymotryp¬ sin, one major L 3 HJ 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 [ 1If CJGlcN labeling, only the peptides between 43,000 and 34,000 could be clearly detected. After reduction of disul¬ fide 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 glycosy¬ lated peptides between molecular weignts of 44,000 to -11- 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 addi¬ tion, 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 pro¬ nase 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 deter¬ mine 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 incor¬ porated by reference herein.) The chymotrypsin and trypsin glycopeptide fragments obtained from gC-I isolated from extracellu¬ lar virus were the same as those immuno-p-r-ecipitated when gC-I was obtained from infected cells. In addi¬ tion, antibodies from all three domains were capable of - 12- i munoprecipitating 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 chymo¬ trypsin. However, with pronase, little of the 43,000 molecular weight peptides remained and most of the gly- copepti εs 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 αC-I and chvmotrvosin fraoments of αC-I reacted with monoclonal antibodies and human serum positive for HCMV. Western blot analy¬ sis was done for three reasons. First, with i munoprε- cipitation, 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 stuαy to additional gC-I monoclonal antibodies to see if they would all react with the fragments gener¬ ated. 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 anti¬ bodies. Six human serum were selected for this study. Five were determined to be positive and one negative for HCMV Dy complement fixation ana inairect Immu- nofluoresencε assays (Table 1). -13- Table 1 Sera CMV Titers* HSV Titers** * 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 immunofluoresencε. In addition, since gC-I contains glycoproteins which have homology with gB from HSV, these serum were tested for their reactivity with HSV by immunofluoresencε. Of those tested, only one (A, 3) was positive for HSV (TaDle I). However, the reactivity of this sera was similar to the others. For Western blot analysis, both whole gC-I and chymotrypsin fragments were immunoaffin- ity purified with a gC-I specific monoclonal antibody. Because of this, a small amount of monoclonal aπtiboαy sometimes contaminated the preparations and could be detected in Western blot as a small band at the top of the nitrocellulose me Drane or as heavy and light chains. Under non-reoucir.g conditidns, two monoclonal antioodies (11B4 and 26B1I) failed to rεact with whole αC-I in estern blot. This occurred even thougn t ese - 14- antibodiεs were capable of immunoprεcipitating gC-I. The pattern obtained with all other monoclonal anti¬ bodies was very similar to that obtained with the posi¬ tive 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 mono¬ clonal antibodies reacted strongly with the 130,000 and 52,000 molecular weight proteins and weakly with pro¬ teins slightly below the 130,000 molecular weight pro¬ tein and a 50,000 molecular weight protein. Again, the human serum positive for HCMV reacted with these pro¬ teins. However, while human sera A,2 gave almost iden¬ tical 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 mono¬ clonal antibodies. None of the proteins recognized by the human serum or the monoclonal antibodies were reac¬ tive with the negative human sera (A,l) 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 incluα ' εσ 11B4 and 26B11, which failed to react with whole gC-I under non-reducing con¬ ditions. The reactivity of 11B4 and 26B11 was less than that of the other monoclonal antibodies. There - 15- were weaker reactions with peptides having an apparent mdlecular weight of 63,000. This may represent a small amount of incompletely digested peptide. These pep¬ tides 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 antibo¬ dies. Under non-reducing conditions, the pattern obtained with all positive human serum was iαentical to the monoclonal antibodies and the negative human sera (A,l) 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 ana 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, 1,1 and 1,2) reacted strongly with the 34,000, 30,000 and 28,000 molecular weignt 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 proDably the neavy chain contriouteα by the antibody used to initially purify the fragments since this Dand was detected in the SP2 negative control. Nonetneless , -16- 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, 1,1 and 1,2). 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 frag¬ ments in Western blot, demonstrating the presence of these domains in AD169. However, the oattern was slightly different. A weak band was detected at 63,C( molecular weight, but the lower molecular eignt oεo- tidεs appeared more diffuse than they did with peotioe from Towne strain HCMV. A broad band covering molecu¬ lar weights from 40,000 to 35,000 was detected along with a band at 31,000 and another diffuse banc with a molecular weight of 23,000. Deαlvcosylation of chvmotrvpsin fragments. After reduction of chymotrypsin fragments, the monoclonal antibodies recognized three peptides in Western olot. The heterogenity in molecular weight may have been cue 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 [ 3 H] arginine or [^c GlcN were used. Since N-glycanase action generates peptides free of carbohydrate, all label should have been lost in the L^CjGlcN labeled peptides, which provided a way to assay the extent of carbohydrate removal. After digestion with N-glycanase, the major 34,000 molecular weight [ 3 Hj arginine labeled peptide was lost and pep¬ tides with molecular weights of 30,000 and 28,000 were observed. Comparable bands were not detected when labeled peptides were digested, suggesting that carbohydrate removal was complete. T cell recognition of unreduced trypsin and chymotryp¬ sin fragments. Peripheral blood mononuclear cells (PBMC) were oDtained 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 sercnεgative 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). -18- MNC* MNC+HCMV** MNC Neg Con*** MNC Try Frag.**** MNC Chv. Fraα.***** * 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 chvmotrvosin fragments. Proliferative responses of mononuclear cells (MNC) f rαm HCMV negative (-) and HCMV positive (+) individuals. Cultures containing 10 6 cells per well were exposeα tc antigens for six days prior to being plused with L 3 HJ - thymidine for 24 hrs before harvesting cultures. Numoers represent average counts per min. from tripli¬ cate cultures. However, with the same amount of protein pre¬ sent, rεsppnses 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 wnen chymotrypsin was used. In addition, one individual which had a low response to whole gC-I (A5) was oDsεrved to have ver_y low or no response to fragments ootaineα with either enzyme. - 19- However, a T cell clonε 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 tryp¬ sin 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-antiboαy- binding assay. While both trypsin- and cnymotrypsin- generated fragments were recognized by antibodies from all three domains, chymotrypsin was capable of gener¬ ating the smallest fragments. Because of this, we focused further studies on the chymotrypsin fragments. After proteolysis of L 3 Hj arginine labelled gC-I with chymotrypsin, there appeared to be only one major peo- tide present under non-reducing conditions which had a molecular weight of 43.00C. With Co assiε blue stain¬ ing, the 43,000 molecular ./eight peptide was still most abundant, but a 34,000 molecular weignt peptide coulc be more clearly detected. Both of these unreduced pec*- tides were gi ccsylatec as Determined by incorporation of ana were recognized by all monoclonal antiDodies anc nu an serum positive for HCMV. There -20- arε 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 con¬ tained 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 dem¬ onstrated 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 mono¬ nuclear 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 ooservation was that the chymotrypsin fragments from gC-I also contained the epitope recognized " by antibody 1134 which was placed in domain III. This was established by the ability of 11B4 to immunoprecipitatε the chymotrypsin fragments, and its reactivity in Western blot under non-reducing conditions. Antibody 11B4 is a non-neutralizing anti¬ body which blocks biπαing of neutralizing antibodies (Lussennop et al., 1938). From this perspective, anti- -21 - bodiεs 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 synthe¬ tic vaccine. However, 11B4 appears to recognize a con- formational epitopε 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. Further¬ more, 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 caroohydratε did not pre- vεnt binding of neutralizing monoclonal antibodies ano in sd e cases binding appeared to increase. Moreover, the 28,000 and 30,000 molecular weight peptides were undεr- or non-glycosylatεd 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 a ino acid sequences of the -22- peptides in the chymotrypsin fragments. Such subunit vaccines are within the scene of this invention. 5everal observations suggest that the frag- ments 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, conforma- tional 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 impor¬ tant sites for gC-I function. Thirdly, the gC-I cny- otrypsin fragments were glycosylated. One important function of glycosylation is to prevent the action of proteases (Iwase, 1988). In our experiments, even pro¬ nase (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 chy¬ motrypsin 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. - 23- REFERENCES The disclosures of the papers cited below are incorpor¬ ated 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. , 3 - , 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. Sioche . , 20, 479-491 (1988). 11. H. Juarεz-Salinas, Biotechniαues , 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 refer¬ ence to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remain¬ ing within the spirit and scope of the invention. Int β rnational Application No: PCT/ MICROORGANISMS Optional Sheet in eonnaetlon with tha mieroorganlem referred to on paoa- . Ilne- ■ ot tha daaertptlon * A. IDENTIFICATION OF DEPOSIT > Further βaeealts ara IderrtlΛeβ on an additional eneet | i > Nama ot αaooaitary Inatituoon * IN VITRO INTERNATIONAL , INC . Addraaa ot βeeoeitary matttutlon (including poatai eooa ana country) * 611 (P) Hammonds Ferry Road Linthicum, Maryland 21090 Data of αaooait * t Accaaaion Numoar . 7-31-88; 8-7-87 ; 9-10-86 JB16-11B4: 2-29-34G7 : 1-48-41C2 •. ADDITIONAL INDICATIONS ' <lea»e blank il not aαpucabla). Thia intαrmattαn la contlnuaβ on a aaoarate attacntα imii C. OESIONATED STATES FOR WHICH INDICATIONS ARE MADE > (II tha indieationa ara not lor all daatonatad Stataa) O. SEPARATE FURNISHING OF INDICATIONS • (leave olank il not appiicaola) Tha indieationa liataβ Balow will oa auBmittad tβ tna International Buraau later • (Specify the general nalu'a at the Indieationa e.g.. " Acceaawn Mumoer αl Oeooait ") t. •7~~- Thia aneet waa received witn the internaiiαnai aαoneation wn.n fil.β (to oa chacneβ by the receiving Offlee) (A ϋut_hoftted Gfflcar) t.JM . Tha date ot receiet (from tne aooiieant) by tne International Bureau 11 ____> (Aumoriied Officer) I Form PCT RO 134 lueπuarv 1M1)

Claims: -27- WHAT IS CLAIMED ISi 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.

Inventors: GEHRZ RICHARD C, KARI BRUCE E, GEHRZ, RICHARD, C., KARI, BRUCE, E.
Applicants: CHILDRENS HOSPITAL INC, THE CHILDREN'S HOSPITAL, INCORPORATED
Application year: 1989
Main classification: A61K39
IPC codes: A61K39, C07K1, C07K14, C07K16, C12P21, G01N33