VECTORS ENCODING HCMV GLYCOPROTEIN AND EXPRESSION PRODUCTS

Abstract: A recombinant expression vector is provided which comprises DNA derived from the genome of human cytomegalovirus (HCMV), such as HXLF 1-5, wherein said DNA encodes a polypeptide which represents at least a portion of the gcII HCMV glycoprotein complex.

Description: VECTORS ENCODING HCMV GLYCOPROTEIN AND EXPRESSION PRODUCTS Field of the Invention This invention relates to a family of anti- genically related glycoprotein complexes contained within the envelope of human cytomegalovirus (HCMV), and in particular to a multi-gene family encoding a principal glycoprotein component of these complexes, gp47-52. This glycoprotein has been shown to elicit humoral and cell-mediated immune responses in humans, and is useful for the diagnosis and treatment of HCMV infection. Background of the Invention Human cytomegalovirus is the most common cause of congenital infection leading to mental retardation, deafness and other neurological handicaps in the United States. Moreover, this virus is frequently associated with life-threatening opportunistic infection in organ and bone marrow transplant recipients, patients with congenital and acquired immunodeficiency diseases, and patients receiving i munosuppressive therapy for cancer, autoimmune diseases and the like. Because HCMV is ubiquitous in nature, measures to limit exposure of individuals at risk of serious infection have met with limited success. Furthermore, antiviral chemotherapy is of little value to infants who have already suffered the devastating consequences of intrauterine HCMV infection, and thus far has shown little promise in ameliorating the progression of systemic HCMV infection and HCMV-associated interstitial pneumonia in the immunocompromised host. Therefore, a safe, effective HCMV vaccine is urgently needed to protect against congenital and acquired HCMV infection. Moreover, improved diagnostic tests can play an important role in detection of sub- clinical HCMV infection, thereby facilitating early therapeutic intervention to decrease the incidence or severity of clinical disease. HCMV is a member of the family herpesviridae. HCMV virions consist of a linear double stranded DNA molecule enclosed within an icosahedral nucleocapsid, surrounded by a tegument and a lipid-containing envelope containing a number of viral glycoproteins and glycoprotein complexes. Three major antigenically distinct sets of HCMV envelope glycoprotein complexes have been described thus far, each made up of two or more glycoproteins associated by disulfide bonds. The first set of complexes, designated gel, contains glyco¬ proteins with molecular weights of 55,000 (gp55], 93,000 (gp93) and 130,000 (gpl30). The gene encoding ■ gp55 has been identified, and exhibits extensive homo- logy with the glycoprotein B gene of herpes simplex virus (HSV) type 1. M. Mach et al., J. Gen. Virol., 67_, 1461 (1986). A second set of antigenically related com- plexes, designated gcll, appears to represent multi- eric forms of one or more gene product(s). The isola¬ tion and characterization of the principal glycoprotein contained within this family of complexes, gp47-52, has been described in detail in U.S. patent application Serial No. 933,789, wherein it is referred to as GLP-B, and in U.S. patent application Serial No. 158,389, filed February 22, 1988, wherein it is referred to as gp52(II). The disclosure of these applications are incorporated by reference herein. Glycoproteins with molecular weights of 86,000 (gp86) and 145,000 (gpl45) have been isolated from a third complex, designated gcIII. The gene coding for gp86 has been identified and exhibits homology with gH of HSV-1. A single monoclonal antibody (mcAb) reactive with the gcll family of complexes, 9E10 (IVI-10118), has been extensively characterized, and is described in detail in U.S. patent application Serial No. 933,789. This monoclonal antibody exhibits cross-reactivity among several strains of HCMV, adenovirus type 2, and herpes simplex virus types 1 and 2. Moreover, this monoclonal neutralizes Towne strain HCMV without com¬ plement, suggesting the possibility that gp47-52 plays an integral role in the biological events required for replication of the virus. Therefore, a need exists to identify and char¬ acterize HCMV glycoprotein complexes and their consti¬ tuent glycoproteins and polypeptide backbones, particu¬ larly those which can elicit protective humoral and cellular immune responses. Summary of the Invention The present invention provides a recombinant expression vector, such as a plasmid, comprising DNA derived from the HCMV genome. This HCMV DNA can code a polypeptide which reacts with gcll-specific monoclonal antibody 9E10 (IVI-10118), and corresponds to a portion of gp47-52, an im unogenic glycoprotein present in the major HCMV envelope glycoprotein complex gcll. Weston and Barrell, 3. Mol. Biol., 192, 177 (1986), have recently sequenced the short unique (U s ) region of the HCMV genome (AD169 strain), revealing at least 38 open reading frames (ORFs) that potentially code for (glyco)proteins of HCMV. One gene family, designated HXLF (Hind III X left reading frame), con- sists of 5 ORFs that lie in tandem with varying degrees of homology. The products of this gene family were predicted to have potential glycosylation sites for both N-linked and 0-linked oligosaccharidesj signal sequences for membrane insertion; and C-terminal hydro- phobic domains for anchoring in cell membranes. Of particular interest, clusters of serine/threonine were predicted by the sequences of HXLF 1 and 2, which in turn suggested that the gene product might contain a high amount of 0-linked oligosaccharides, which is atypical of most viral glycoproteins. The present invention is based upon our dis¬ covery that the gcll glycoprotein, gp47-52, is encoded by o e or more of the genes encompassed by the HXLF gene family. Therefore, the present invention is also directed to the isolated gene family, e.g., to a sub¬ stantially pure composition of matter consisting essen¬ tially of at least one of the members of the HXLF gene family of HCMV, e.g., HXLF1, HXLF2, HXLF3, HXLF4 and HXLF5. As used within the context of the present invention, the term "DNA derived from the HCMV genome" refers to both DNA which is isolated from HCMV virions, such as Towne strain virions, and to DNA- which is chemically synthesized. The term "a portion of gp47-52" refers to either fully-, partially-, or non-glycosylated polypep¬ tide segments of gp47-52 which comprise epitopes which are reactive with 9E10 or with patient sera comprising antibodies reactive with gp47-52. Non-glycosylated segments of gp47-52 are also referred to as parts of the "polypeptide backbone" of gp47-52. The present recombinant expression vectors can be employed to produce immunogenic polypeptides which formally correspond to a portion of gp47-52. For example, a plas id containing all or a portion of the HXLF gene family can be linearized, mRNA synthesized in vitro, and then translated under conditions whereby the corresponding polypeptides are either glycosylated, or nonglycosylated. The translation products can be either analyzed by SDS-PAGE and extracted therefrom, or solubilized for immunoprecipitation as described in the examples hereinbelow. Therefore, the present invention also provides a number of new gp47-52-related polypeptides (p20, p21-25) and glycoproteins (gp3Q, gp25) which comprise epitopes which react with 9E10, as well as with anti¬ bodies in the HCMV-positive sera of human patients. The role of HCMV gp47-52 in virus infection is unknown, as is the role of its parent glycoprotein com¬ plex, gcll. However, gcll is a major component of the virion envelope and is a target for antibody-mediated neutralization. Taken together, these data strongly support the inclusion of gp47-52 and/or the present "subunit proteins" of gp47-52 into HCMV vaccines, which may also include other classes of HCMV envelope glyco- proteins, such as gel. The gp47-52-related polypeptides and glycopro¬ teins can be readily prepared in quantity by transfor¬ mation or transfection of suitable host cells with the present vectors; or by known chemical methods of poly- peptide synthesis. After isolation and purification of the polypeptide, an immunologically-effective amount thereof is combined with a pharmaceutically-acceptable vehicle, such as a sterile liquid carrier, and paren- terally administered to a mammal, in order to raise the titer of anti-HCMV antibodies in the blood of the recipient. For a description of a putative vaccine employing "glycoprotein A" of HCMV, see L. Pereira (U.S. Patent No. 4,689,225), the disclosure of which is incorporated by reference herein. Apart from their use in vaccines, the present compositions are useful in the production of monoclonal antibodies, which in turn can be used either directly or indirectly to diagnose HCMV, or to treat HCMV infec¬ tions. The present immunogenic compositions can also be used as diagnostic reagents, e.g., to detect anti¬ gen antibodies in physiological fluids, e.g., by RIA or ELISA procedures. The present compositions are also useful to produce clonal populations of antigen- specific T-helper lymphocytes, which in turn can be used for HCMV therapy. The present invention also provides RNA probes, a number of which are exemplified hereinbelow, which are complementary to sequences in the HXLF gene family. These probes can be used to detect HCMV in clinical specimens using hybridization methods known in the art. In a preferred embodiment of the present invention, the five HXLF genes were cloned into an SP6 expression vector in both the sense and antisense orientations (Fig. 1C). An abundant 1.62 kb bicistro- nic mRNA, predicted to originate from HXLF1 and HXLF2, was detected, in the cytoplasm of infected human fibro- blast cells by Northern blot analysis. Less abundant RNAs of 1.0 and 0.8 kb, predicted to originate from the HXLF5 and HXLF2 genes, respectively, were also detected. Monocistronic, bicistronic, and polycistronic RNAs synthesized in_ vitro using SP6 polymerase were translated in rabbit reticulocyte lysates with or without canine pancreatic microsomal membranes. The HXLF1 or the HXLFl plus HXLF2 translation products were detected when using the above mRNAs. However, the HXLF3, 4 and 5 gene products were not detected by in vitro translation of the SP6 derived polycistronic mRNA. Nonglycosylated or glycosylated HXLFl and HXLF2 gene products were immunoprecipitated by mono¬ clonal antibody 9E10 which is specific for a virion envelope glycoprotein complex (gc) designated gcll. In addition, the monoclonal antibody 9E10 immunoprecipi¬ tated a diffuse glycoprotein (gp) band designated gp47-52 from HCMV-infected cell lysates. The amino acid composition of gp47-52 purified from virion envel¬ opes has the highest similarity to the predicted amino acid composition of the product of the HXLFl plus HXLF2 open reading frames, but it is more similar to that of HXLF2 than to that of HXLFl. The Northern blot results imply that gp47-52 is synthesized predominantly from the abundant 1.62 kb bicistronic mRNA encoded by the HXLFl and HXLF2 genes. However, the glycoprotein could also be synthesized by the monocistronic 0.8 kb mRNA encoded by the HXLF2 gene as well as the mRNAs pre¬ dicted from the other HXLF genes. Brief Description of the Drawing Figure 1 schematically depicts the location of the HXLF homologous gene family of human cytomegalo¬ virus and its cloning into an SP6 expression vector. Panel (A) depicts the prototype arrangement of the human cytomegalovirus (HCMV) genome showing large uni¬ que (U [ _), short unique (U s ), terminal repetitive (TR) and internal repetitive (IR) regions. Panel (B) depicts the Hindlll-Xbal fragment derived from the U s region and designates the map units on the viral genome. The HXLF family of homologous ORFs are depicted by closed bars labelled HXLFl through HXLF5. Dashed arrows indicate predicted mRNA transcripts with approximate sizes in kb based on the locations of TATA boxes and polyadenylation signals (stars) relative to the ORFs. Beneath each bar representing an ORF is the molecular weight (X10 3 ) of the protein product pre¬ dicted by the respective ORF. Restriction endonuclease sites are indicated by arrows. Panel (C) depicts the HXLF gene family cloned into an SP6 expression vector in both the sense (SP65IT) and antisense (SP64IT) orientations. Abbreviations? Bs (BstE II); HIII (Hindlll); Xb (Xbal); Xh (Xhol); SP (SP6 promoter); Amp (ampicillin resistance gene). Deposit Plasmid pSP65IT has been deposited at In Vitro International, 611(P) Hammond's Ferry Road, Linthicum, MD 21090, under the access code IVI-10168. Detailed Description of the Invention The invention will be further described by reference to the following detailed examples wherein the methodologies were as described hereinbelow. The disclosures of the cited publications are incorporated by reference herein. Virus and cells. The culturing of human foreskin fibroblasts and the propagation of human cytomegalo¬ virus Towne strain have been described previously by M. F. Stinski in J. Virol., 26, 686 (1978). The viral DNA polymerase inhibitor phosphonoacetic acid was used as described by M. W. Wathen et al., 3. Virol., 41, 462 (1982). Expression vectors. The pSP64 and pSP65 cloning vec¬ tors contain the Salmonella typhimurium phage SP6 pro¬ moter upstream of a polylinker sequence containing unique restriction sites for insertion of DNA sequences. The bacteriophage SP6 RNA polymerase ini¬ tiates transcription exclusively at an SP6 promoter, and thus allows for synthesis of pure, full-length single stranded RNA from a DNA template cloned down¬ stream of the SP6 promoter. The polylinker is cloned in opposite directions relative to the SP6 promoter in pSP64 and pSP65 to allow cloning of the DNA sequences in sense and anti-sense orientations, respectively. The SP6 expression system used in these studies was adapted from an original cloning vector derived by insertion of the SP6 promoter into pBR322. A particular disadvantage of the original cloning vector was the presence of 250 base pairs upstream of the restriction site used for cloning, resulting in transcription of extraneous sequences between the transcription start site and the restric¬ tion sites of the polylinker. pSP64 and pSP65 have only six bases and nine bases, respectively, between the transcription start site and the restriction enzyme insertion site of the polylinkers. A particular advan¬ tage of these cloning vectors is that the major RNA transcripts are derived from transcription of the DNA template strand to the last and penultimate nucleotide. Nicks in the DNA template do not serve as transcription initiation sites, nor does SP6 RNA polymerase terminate at nicks in the template and thus premature termination resulting in RNA transcripts shorter than full-length, is relatively rare. The construction of the pSP64/65 expression system and its utility for _in vitro synthesis of biologically-active RNA was first described in detail by D. A. Melton et al., "Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter." Nucleic Acids Research, 12, 7035-7056 (1984), the dis¬ closure of which is incorporated by reference herein. The restriction maps and sequences for pSP64 and pSP65 are published in this manuscript (page 7041). In vitro transcription and translation. A 4.5 kb Xbal to Hindlll fragment spanning 0.857 to 0.879 map units of the Towne strain genome was subcloned into SP6 expression vectors SP64 and SP65 (Amersham; Amersham Hts., IL) using the methodology described previously by D. R. Tho sen et al., Gene, 16, 207 (1981). RNA syn¬ thesis from linearized templates was accomplished as described by P. A. Krieg et al., Nucl. Acids Res., 12, 7057 (1984). SP6 polymerase was purchased from Boehringer-Mannheim (Indianapolis, IN). The in_ vitro translation of RNA in rabbit reticulocyte lysates (Promega; Madison, WI) was accom¬ plished as described by M. Stinski et al., 3. Virol., 46, 1 (1983). Translation products were radiolabelled with 35 S-methionine (>1000 Ci/mmol; Amersham). For some experiments, translation was supplemented with canine pancreatic microsomal membranes as recommended by the manufacturer (Amersham). Immunoprecipitation and SDS-PAGE. Infected or mock- infected cells were pulse-labelled at various times after infection with 40 μCi/ml 3 H-glucosamine (20-40 Ci/m ol; Amersham) as described previously by M. F. Stinski et al., J. Virol., 26, 686 (1978). In certain examples, cells were pretreated for 4 hr with 1.5 μM monensin (a gift from C. Grose, U. of Iowa) before pulsing in the presence of monensin. Antigens were solubilized in RIPA buffer (PBS, pH 7.4, containing 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 100 μg/ml PMSF and 0.02% sodium azide) and immunoprecipitated with monoclonal or polyclonal antibodies plus protein A sepharose (Pharmacia; Uppsala, Sweden) as described by D. R. Gretch et al., Anal. Biochem., 163, 270 (1987). The analysis of radiolabelled antigens by SDS-poly- acrylamide gel electrophoresis (SDS-PAGE) and auto- radiography has been described by M. F. Stinski et al., 3. Virol., 1 , 594 (1976). 1,f C-labelled protein mole¬ cular weight standards were purchased from Bethesda Research Laboratories (Gaithersburg, MD). The production and characterization of mono¬ clonal antibodies 41C2 (IVI-10119) and 9E10 has been described by B. Kari et al., 3 . Virol., , 60, 345 (1986) and monoclonal antibody 1G6 was a gift from L. Rasmussen, Stanford University. Human convalescent serum (218) was a gift from L. Frankel, U. of Cincinnati. Northern blot analysis. The purification of cytoplas- mic RNA from HCMV-infected or mock-infected cells and Northern blotting conditions were performed as des¬ cribed previously by M. W. Wathen et al., 3. Virol., 41, 462 (1982). E_. coli riboso al RNA standards were purchased from Promega (Madison, WI). Antisense RNA probes were synthesized from linearized pSP64IT tem- plates as directed by the supplier (Amersham) except that 150 μCi of α 32 P-UTP (800 Ci/mmol; Amersham) was supplemented with cold UTP to a final concentration of 50 μM to ensure full length probe synthesis. Amino acid composition analysis. gp47-52 was purified from virion and dense body envelopes using ion exchange and gel exclusion high performance liquid chromato- graphy (HPLC) as described previously by B. Kari et al., cited above. After strong acid hydrolysis, amino acid compositions were determined by reverse phase HPLC on a C-18 column (E. M. Science, Elmsford, NY) as des¬ cribed by H. Scholze, 3. Chromat., 350, 453 (1985). Example I. Northern blot analysis of the HXLF steady state mRNA in infected cells. The HXLF gene family, which consists of five homologous ORFs in tandem, was cloned into the SP6 expression vectors in sense and antisense orienta¬ tions as outlined in Figure 1. Four different mRNA transcripts were predicted from the HXLF gene family based on the locations of TATA boxes and polyadenyla- tion signals relative to the ORFs (see Figure IB). A 1.6 kb bicistronic RNA was predicted from the HXLFl and HXLF2 genes while a 2.7 kb tricistronic RNA was pre- dieted from the HXLF3 through HXLF5 genes. In addi¬ tion, monocistronic transcripts of 0.8 and 1.0 kb genes were predicted from HXLF2 and HXLF5 genes, respec¬ tively. Northern blot analysis was performed on steady state cytoplasmic RNA harvested from mock- or HCMV- infected cells at 24 and 72 hr p.i. In addition, RNA was harvested from infected cells treated for 24 hr with phosphonoacetic acid (PAA) which inhibits the replication of HCMV DNA and, consequently, blocks the early to late transition in the viral replicative cycle. In addition, PAA treatment increases the steady state level of viral RNA. A 4.5 kb antisense RNA probe which covered all five HXLF genes was synthesized from pSP64IT by linear- izing the antisense template with restriction endonu- clease Xbal (see Figure 1C). A shorter 3.1 kb probe, which covered HXLF3, 4 and 5 genes only, was synthe¬ sized from a BstE II-linearized template. Both the full length probe and the truncated probe detected a predominant 1.62 kb transcript at 24 hr p.i. and at 24 hr p.i. when PAA was present (data not shown). Reduced levels of the 1.62 kb RNA were detected by both probes at 72 hr p.i. in the absence of PAA. The 1.62 kb transcript is presumably the bicistronic mRNA predicted from the HXLFl and HXLF2 genes. Both probes would be expected to hybridize to the 1.62 kb mRNA given the relative ho ology between the various HXLF genes. The full length probe detected minor mRNAs of 0.8 and 1.0 kb which correspond to the monocistronic RNAs predicted from the HXLF2 and HXLF5 genes, respectively. Neither probe detected the 2.7 kb polycistronic transcript pre¬ dicted from the HXLF3, HXLF4 and HXLF5 genes. Thus, the predominant mRNA observed was a 1.62 kb mRNA predicted to be encoded by the HXLFl and HXLF2 genes which accumulated in the cytoplasm of virus- infected cells at early times after infection. The level of this transcript relative to total cellular and viral RNA is decreased at late times after infection. Example II. In vitro synthesis of the HXLF gene products. To allow for the ij vitro synthesis of mRNA from either the first ORF (i.e., HXLFl), the first two ORFs together (HXLFl plus HXLF2), or all five ORFs (HXLFl through HXLF5), the sense plasmid pSP65IT was completely linearized with either restriction endonuclease Xhol, BstEII or Hindlll, respectively (see Figure 1C). The approximate sizes of the in. vitro synthesized mRNAs were determined by agarose-formaldehyde gel electro- phoresis (data not shown) and the RNAs were translated in rabbit reticulocyte lysates in the presence or absence of dog pancreas membranes (DPM). DPM allow for core glycosylation with N-acetylglucosamine and mannose sugars during glycoprotein translation _in_ vitro. [ . E. Rothman et al., Nature, 269, 775 (1977)] The translation products were either analyzed by SDS-PAGE or solubilized for immunoprecipitation as described hereinbelow. After translation of mRNA from the HXLFl gene in the absence of DPM, a diffuse band ranging in mole¬ cular weight from 21 to 25 kDa (p21-25) was detected. After translation of mRNA from the HXLFl plus HXLF2 genes in the absence of DPM, the diffuse p21-25 plus an additional non-diffuse 20 kDa polypeptide (p20) were detected. The apparent molecular weights of these in vitro translation products are similar to the predicted protein molecular weights from the HXLFl and HXLF2 ORFs (see Fig. IB). The diffuse nature of p21-25 is characteristic of the HXLFl gene product only and may be due to inter¬ nal initiation or premature termination during transla¬ tion of the HXLFl mRNA. Alternatively, the diffuse nature of p21-25 may be due to the higher proline con¬ tent predicted to be present in HXLFl (see Table I). Proline rich regions can disrupt part of the protein's alpha helical structure leading to anomolous mobilities in denaturing polyacrylamide gels. After translation of mRNA from all five ORFs, a protein profile identical to translation of mRNA from HXLFl and HXLF2 was detected. The HXLF3, HXLF4 and HXLF5 ORFs were predicted to make proteins of 28, 27.5 and 26.2 kDa, respectively (see Figure IB). Thus, the products of the downstream HXLF ORFs were apparently not expressed in_ vitro even though full-length RNA was detected by agarose-for aldehyde gel electrophoresis. It is unlikely that the protein products migrated in gels the same as the HXLFl and HXLF2 products. The HXLF ORFs were predicted by Weston et al., cited above, to have sites for N- as well as 0- glycosylation. Therefore, the _in_ vitro synthesized RNAs were also translated in the presence of dog pancrease membranes (DPM) to allow for core glycosyla- tion of the nascent polypeptides [j. E. Rothman et al., Nature, 269, 775 (1977)]. After translation of mRNA from the HXLFl gene in the presence of DPM, a 30 kDa glycoprotein (gp30) was detected. When mRNA from the HXLFl plus HXLF2 genes was translated in the presence of DPM, a major 30 kDa glycoprotein band was again detected. It is possible that the HXLFl and HXLF2 pro- teins were glycosylated to approximately the same apparent molecular weight, the DPM suppressed transla¬ tion of the HXLF2 mRNA, or the HXLF2 glycosylated pro¬ tein is present in low amounts. A similar result was obtained when mRNA from all five genes (HXLFl through HXLF5) was translated in the presence of DPM. Example III. Immunoprecipitation of the HXLF gene products with a HCMV-specific monoclonal antibody or with human conva- lescent serum. Native polypeptide translation To determine whether or not the HXLF genes specified bonafide HCMV envelope glycoproteins, monoclonal antibodies that react with envelope glycoprotein complexes (gc)I (gp55, gp93-130), II (gp47-52) and III (gp86, gpl45) were used for immu¬ noprecipitation. Monoclonal antibody 9E10, which recognizes a major glycoprotein component of the gcll family, gp47-52, reacted strongly with the diffuse P21-25 translated from the HXLFl mRNA. In addition, monoclonal antibody 9E10 immuno¬ precipitated both the diffuse p21-25 and non-diffuse p20 specified by mRNA from HXLFl and HXLF2. When mRNA from HXLFl through HXLF5 was translated, monoclonal antibody 9E10 again immunoprecipitated p21-25 and p20. However, neither monoclonal 41C2, which reacts with the gcl family, nor monoclonal antibody 1G6, which reacts with the gcIII family, immunoprecipitated the HXLF mRNA translation products. Thus, p21-25 from the HXLFl ORF is gcll speci- fie, since immunoprecipitation with 9E10 detects the entire diffuse band p21-25. The p20 product of the HXLF2 ORF is apparently also recognized by monoclonal antibody 9E10 even though there is only a low degree of homology between these two ORFs. The possibility that p20 was immunoprecipitated via disulfide-linkage with p21-25 is unlikely because immunoprecipitated p20 was also detected by non-reducing SDS-PAGE. Example IV. DPM-mediated translation. To further analyze the HXLF gene products, mRNA from all five ORFs was translated in the presence of DPM and the products were immunopre¬ cipitated with either monoclonal antibody 9E10 or human convalescent serum 218. Both reacted with the 30 kDa product of the HXLFl ORF. In addition, both immunopre¬ cipitated a second 25 kDa specie ' s which may represent either unglycosylated HXLFl ORF product or glycosylated product from the HXLF2 ORF. Monoclonal antibodies to gel or gcIII, as well as HCMV negative human serum reacted weakly or failed to react with these transla¬ tion products. The weak reactivity with monoclonal antibody 41C2 could be prevented by adding 1 mg/ml of ovalbumin (data not shown). These results demonstrate that monoclonal antibody 9E10, which is specific for the envelope gly¬ coprotein gp47-52, immunoprecipitates gene products from the HXLFl and HXLF2 genes. In addition, human convalescent serum contains antibodies which react with the HXLF gene products. Exa ple V. Synthesis of gp47-52 in infected cells. The immunopre¬ cipitation studies presented above suggested that gp47-52 is a product of at least the HXLFl and HXLF2 genes. Gp47-52 resides in virion envelopes as a family of disulfide-linked glycoprotein complexes which are collectively designated gcll. Gp47-52 contains high levels of 0-linked sugars, lower levels of N-linked sugars and multiple polypeptides. The synthesis of gp47-52 was investigated using infected human fibro- blast cells in the presence or absence of the ionophore monensin, which inhibits golgi transport and allows the addition of high mannose N-linked but not 0-linked oli- gosaccharides [D. C. Johnson et al., 3. Virol., 43, 1102 (1982) and Cell, 32, 987 (1983.)]. Infected cells were radiolabelled with 3 H- glucosamine at various times after infection and in the presence of monensin, and the resultant antigenic pro¬ ducts were solubilized and immunoprecipitated with 9E10 as described hereinabove. A characteristically diffuse glycoprotein band (gp47-52) was detected at 48 and 72 hr p.i. but not at 24 hr p.i. or in uninfected cells. In the presence of monensin, glycoproteins designated gp32 and gp25 were immunoprecipitated by 9E10. These glycoproteins are probably simple N-linked precursors of gp47-52 since monensin blocks the pro¬ cessing of simple N-linked sugars as well as the addi¬ tion of 0-linked oligosaccharide chains. It is unlikely that gp32 represents golgi-mediated processing of gp25 or vice versa since the concentration of monen¬ sin used completely blocked processing of the HCMV gB homolog precursor glycoprotein (data not shown). These results are consistent with the immunoprecipitation of 30 and 25 kDa HXLF gene products from i vitro transla¬ tions supplemented with DPM since DPM allows simple N- glycosylation i vitro. In addition, the identifica¬ tion of multiple precursors in the presence of monensin implies that gp47-52 is encoded by multiple HXLF genes. Example VI. Comparison of the amino acid composition of gp47-52 with the predicted amino acid compositions of the HXLF gene products. To further demonstrate that gp47-52 is a product of the HXLF genes, this glycoprotein was purified from virion envelopes by a combination of ion exchange and gel exclusion high performance liquid chromatography (HPLC) followed by immunoprecipitation as described by B. Kari et al., J. Virology, 60, 345 (1986), the disclosure of which is incorporated by reference herein. The reduced and alkylated gp47-52 was subjected to strong acid hydrolysis and its amino acid composition was determined by reverse phase HPLC as described by Scholze, in 3. Chromatography, 350, 453 (1985). The observed amino acid composition of gp47-52 was then compared to the predicted amino acid composi¬ tions of the products coded by the HXLF genes as shown in Table I, below. Table I Comparison of the observed amino acid composition of gp47-52 with the predicted amino acid composition of the HXLFl and HXLF2 ORFs a Amino Acid Compo!sitions b/ Pretdieted from ORF Mean of Mean of Mean of Amino HXLFl HXLFl, 2, Observed Acid HXLFl HXLF2 and 2 3, 4 and 5 qp47-52 c / Lys 0.2 0.3 0.2 0.4 0, ,2 His 0.2 0.4 0.3 0.5 0. .2 Asp 1.0 0.3 0.5 0.6 0. .4 Met 0.2 0.1 0.1 0.2 0. .3 He 0.2 0.3 0.2 0.4 0. .4 Gly 0.7 0.7 0.7 0.9 0. .5 Ala 1.0 1.0 1.0 1.0 1. ,0 Pro 1.8 0.8 1.2 1.2 1. ,3 Glu 2.0 0.8 1.2 1.0 1. ,3 Thr 1.2 1.2 1.2 1.0 1. ,0 Val 2.3 0.8 1.4 1.7 1. , 2 Leu 2.8 1.4 1.9 1.9 2. ,1 Tyr 1.8 0.6 1.1 1.0 0. . 7 Arg 2.3 0.7 1.3 1.2 0. 6 Phe 0.5 0.3 0.4 0.5 0. 9 Ser 1.7 0.6 1.0 1.4 0. 5 a/ Amino acid compositions given relative to Ala = 1.0 in all cases. —' Based on DNA sequence by Weston and Barrell, 3. Mol. Biol., 192, 177 (1986). c/ Based on the average of two separate analyses of purified gp47-52. The observed mean amino acid composition of gp47-52 is more similar to the predicted composition of the product coded by HXLFl plus HXLF2 than to the com¬ positions coded by either gene. However, the observed mean composition had more similarities to the predicted composition of HXLF2 than HXLFl, 3, 4 or 5. Although some of the variance may reflect differences between the Towne strain used in this study and the sequenced AD169 strain, the presence of the HXLF3, HXLF4 and HXLF5 gene products, or experimental error, it is likely that gp47-52 is composed of multiple HXLF gene products. The heterogeneous nature of gp47-52 and the different mRNA size classes detected by Northern blot analysis support this possibility. Therefore, gp47-52, which is a major component of the virion complex gcll, is specified by the HXLF gene family between map units of 0.857 and 0.879 in the short unique component of the viral genome. Discussion The virion envelope glycoprotein gp47-52 is a product of the HXLF gene family of human cytomegalo¬ virus. The evidence for this is as follows: (i) mono¬ clonal antibody 9E10, which is specific for gp47-52, immunoprecipitated the ijτ_ vitro synthesized HXLFl and HXLF2 gene products; (ii) the mean amino acid composi¬ tion of gp47-52 is most similar to the predicted compo¬ sition of the HXLFl plus HXLF2 genes; (iii) carbohy¬ drate analysis of gp47-52 has revealed a high level of 0-linked sugars and low amounts of N-linked sugars. This is consistent with the hydroxyamino acid content and number of N-glycosylation sites predicted for the HXLF gene products; and (iv) monoclonal antibody 9E10 immunoprecipitated two precursor glycoproteins of 25 and 32 kDa from monensin-treated infected cells, as well as two HXLF glycoprotein gene products of 25 and 30 kDa from ^n_ vitro translations supplemented with DPM. Taken together, these data indicate that gp47-52 is a product of the HXLF genes. 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. Intβrnationa! Application No: PCT/ MICROORGANISMS Optional Sheet In connection with the microorganism referred to on page- _, llne- . of the description l A. IDENTIFICATION OF DEPOSIT* Further deposits are Identified on an additional sheat Q» pSF65IT (Plasmid) Name of depositary Institution « IN VITRO INTERNATIONAL, INC. Address of depesitsry Institution (Inciudlng postal coda and country) * 611(F) Hammonds Ferry Road Linthicum, Maryland 21090 Oats of deposit • Accession Number * April 26 , 1988 IVI-10168 ADDITIONAL INDICATIONS ' (leave blsπk If not applicable). This informstion is conUnusd on s sspsrate snached sheet Q C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE > (if the indications sre not for sll dssignstsd SUtes) D. SEPARATE FURNISHING OF INDICATIONS • (Isave blent if not applicable) The indications listsd bslo will be submitted to the Intemstional Bureau later ■ (Specify the general nature of the Indications e.g., ' • Accession Number of Osposit ") E. j This sheet was received with the intsrnβtioπal apoiicstion when filed (to be checked by the receiving Office) (Authorized O fficer) I I The dste of receipt (from the supiicant) by the Internationsl Bureau "> :___• (Authorized Officer) Form PCT.RO 134 (January tββt)

Claims: WHAT IS CLAIMED IS: 1. A recombinant expression vector comprising DNA derived from the HCMV genome, wherein said DNA encodes a polypeptide which reacts with monoclonal antibody IVI-10118, said polypeptide corresponding to a portion of gp47-52. 2. The expression vector of claim 1 which is a plasmid. 3. The expression vector of claim 1 which comprises the five homologous HCMV HXLF genes. 4. The expression vector of claim 1 which is essen¬ tially free of other DNA derived from the HCMV genome. 5. The expression vector of claim 1 which comprises the gene HXLFl. 6. The expression vector of claim 1 which comprises the genes HXLFl and HXLF2. 7. A substantially pure polypeptide which is coded for by the expression vector of claims 5 and 6. 8. A substantially pure, nonglycosylated polypeptide having a molecular weight of about 21-25 kD which is coded by the expression vector of claim 5. 9. A substantially pure, nonglycosylated polypeptide having a molecular weight of about 20 kD which is coded by the expression vector of claim 6. 10. A substantially pure, immunogenic glycoprotein having a molecular weight of about 30 kD, which is coded for by the expression vector of claim 5. 11. A substantially pure, immunogenic glycoprotein having a molecular weight of about 25 kD which is coded for by the expression vector of claim 5. 12. A vaccine against HCMV, comprising an immunologi- cally effective amount of the polypeptide of claim 7 in combination with a pharmaceutically effective vehicle. 13. A method for raisng the titer of an antibody against HCMV in the blood of a mammal, comprising administering to said mammal an immunαgenically effective amount of the polypeptide of claim 7. 14. A method of detecting HCMV-specific antibody in a clinical sample, which comprises contacting the sample with the polypeptide of claim 7, and detecting the resultant polypeptide-antibody com¬ plex. 15. The method of claim 14 wherein the complex is detected by reacting it with a second antibody which incorporates a detectable label or a binding site for a detectable label. 16. The method of claim 14 wherein the polypeptide further incorporates a detectable label. 17. A substantially pure composition of matter consist¬ ing essentially of at least one member of the HXLF gene family of human cytomegalovirus (HCMV). 18. The composition of matter of claim 17 wherein the member comprises the gene HXLFl or HXLF2. 19. The composition of matter of claim 17 wherein the members comprise the genes HXLFl and HXLF2.

Inventors: GEHRZ RICHARD C, STINSKI MARK F, GEHRZ, RICHARD, C., STINSKI, MARK, F.
Applicants: CHILDRENS HOSPITAL INC, UNIV IOWA RES FOUND, THE CHILDREN'S HOSPITAL, INCORPORATED, UNIVERSITY OF IOWA RESEARCH FOUNDATION
Application year: 1989
Main classification: C12N15
IPC codes: A61K39, C07K14, C12N15, C12P21, C12R1, G01N33