Statement Regarding Federally-Funded Research This invention was made with government support under Department of Health and Human Services Grant No. HDMC 5 POl HD19937-03 GT. The government may have certain rights in the invention.

Field of the Invention

The present invention is directed to a 93 kD glycopeptide of the gel complex of human cytomegalovirus (HCMV) having unique B and T epitopes not present in other glycopeptides of gel, to gel glycopeptide fragments which represent the localization and minimization of continuous and discontinuous epitopes present in the major B cell antigenic region of the 55 kD glycopeptide of gcl _f to recombinant expression vectors whose expression products are reactive with gcl- specific monoclonal antibodies and B and T lymphocytes, and to HCMV subunit vaccines and diagnostic methods.

Background of the Invention

Human cytomegalovirus (HCMV) contains several families of antigenically related glycoprotein complexes, each comprised of two or more individual glycoproteins associated by disulfide bonds. These complexes and their constituent glycoproteins are present in the envelopes of HCMV virions, and are also expressed on the surface of HCMV-infected cells during permissive viral infection.

Convalescent sera from human subjects who have recovered from HCMV infection contain antibodies reactive with these envelope glycoproteins. Moreover, peripheral blood mononuclear cells (PBMCs) from seropositive subjects proliferate when stimulated with HCMV glycoproteins. Therefore, it is likely that these glycoproteins play an important role in protective immune responses, including antibody-mediated

neutralization of extracellular virus, B and T cell responses to control or eradicate replicating intracellular virus to prevent cell-to-cell spread, and immune surveillance to prevent reactivation of latent HCMV infection.

Previously described HCMV envelope complexes and their constituent glycoproteins are summarized in Table I, below.

Table I.

Human Cytomeealovirus Envelope Glycoproteins and Glycoprotein Complexes

Complex(es) l_V(daltons) Glycoproteins(s) HW(daltons) Genes(s)

130,000 gpl30 (I) 130,000 gci 180,000 gp 93 (I) 93,000 U _r

( .344- .360)

>450,000 gP 52 (1) 50-52, 000

gCII93 93 , 000 gp 90 (1193) 90,000 gp 52 (1193) 50-52,000

Us gp200 (11200) >200,000 ^• H30.FI-5 gCII200 >200,000 gp 90 (II200) 90,000 (.857-.879) gp 52 (II200) 50-52,000

gpl45 145,000 gCIII 280,000 ϋ, gp 86 86,000 (.450-.470)

Unassociated gp 93* 93,000 with any complex

Egp 48 ² 48,000 U _L -0RF3

(0.054-0.064)

-This glycoprotein is distinct from gp93 of the gel complex. See Kari et al., J. Virol., 60, 345-352, 1986. "Egp48"

² See the simultaneously filed U.S. Patent application S.N._ entitled "An Early Envelope Glycoprotein or Human Cytomegalovirus (HC-V) ; inventor Mark F. Stinski.

The family of antigenically related complexes with molecular weights of 130,000-180,000 and >200,000 daltons have been designated as gel, which contains glycoproteins with molecular weights of 130,000 (gpl30), 93,000 (gp93), and 55,000 (gp55). The glycoproteins were originally identified as gA by L. Pereira et al.. Infection and Immunity. 3j6, 924-932 (1982) (and in U.S. Patent No. 4,689,225 to Pereira, August 25, 1987) and subsequently designated as gB based on their extensive amino acid sequence homology with the gB glycoproteins of Herpes Simplex virus (HSV) and Epstein-Barr virus (EBV) , and gpll of Varicella zoster virus (VZV) .

These gB glycoproteins derive from a single coding region in the unique long (U _L ) region of the HCMV genome between map coordinates 0.344 and 0.380 [or .360 ??] as described by M. Mach et al., J. Gen. Virol., 67, 1461-1467 (1986). The gB gene may represent a primordial gene conserved among herpes viruses involved in biological functions critical to the evolution and survival of these viruses.

The gB gene was originally detected by expression of a cDNA library in λgtll as detected with a monospecific rabbit antiserum reactive with the 55,000 dalton glycoprotein (gp55) of the gel complexes. M. Cranage et al., EMBO J. , 5, 3057-3063 (1986) subsequently expressed the gB gene in vaccinia and determined that gp55 was derived from a precursor with a molecular weight of 145,000. Cranage et al. reported that the gB coding region predicts a polypeptide of 906 amino acids in the AD169 strain of HCMV.

Gretch et al., J. Gen. Virol., 69, 1205 (1988) subsequently reported that the synthesis of the gel family of HCMV glycoproteins involves the glycosylation of a 95,000 dalton polypeptide encoded by the gB open reading frame to a stable simple N-linked glycoprotein intermediate of 138,000 daltons, gpl38. Alternate proteolytic cleavage at several predicted dibasic

protease recognition sites may account for the mature glycoproteins, gp55 and gp93, and possibly for gpl30. Recent reports indicate that gpl30 detected in detergent extracts of extracellular virions is a unique mature glycoprotein that is distinct from the glycosylated precursor, gpl38. Taylor et al. Antiviral Research, 10, 11-26, 1988.

Spaete et al.. Virology, 167, 207 (1988) reported the amino acid sequence of gB from Towne strain HCMV, which predicts a polypeptide of 907 amino acids with 95% homology to the gB gene in AD169. Analysis of the DNA sequence predicted an N-terminal signal sequence and a C-terminal transmembrane region composed of two domains of hydrophobic amino acids. Eucaryotic expression of the gB gene in COS cells (SV40-transformed simian cells) resulted in the synthesis of a gpl30 precursor and a mature gp55 glycoprotein, as determined by reactivity with a neutralizing gel-specific monoclonal antibody. N-terminal sequencing of purified gp55 showed that this glycoprotein is derived by proteolytic cleavage at dibasic amino acids 460-461 and represents the C-terminal region of gpl30. Spaete et al. further localized one neutralizing epitope to a 186 amino acid fragment of gp55 starting at the N-terminal amino acid 461 by expression of a truncated gB gene.

In sum, the gross characteristics of the gC I, II, and III complexes and corresponding glycoproteins have been partially determined. But, the antigenic regions of the complexes and glycoproteins have not as yet been mapped. Such studies would be useful in the development of vaccines and treatment of HCMV infections. Therefore, it is an object of the present invention to characterize biochemically and immunologically the antigenic regions of gp55 and gp93. In particular, another object is the characterization of the epitopes expressed on those proteins.

Summary of the Invention

The present invention is directed to glycopeptides of the HCMV gel complex which have unique B cell epitopes and T cell epitopes, glycopeptide fragments which represent epitopes in the major B cell antigenic region of gp55, recombinant expression vectors, vaccines, and diagnostic methods.

The B cell epitopal glycopeptide according to the invention is substantially pure, is immunogenic, has a molecular weight of about 93,000, and includes one or more unique B cell epitopes which are not present in the gp55 glycopeptide of gel. Those "B cell epitopes" are epitopic sites, regions, antigenic determinants or regions within a polypeptide, glycopeptide or fragment thereof which do not react with or exhibit any substantial response to B lymphocytes, monoclonal antibodies produced by B lymphocytes, antibodies in HCMV seropositive human sera, or the like that are reactive with or respond to gp55. The "gp55" is the 55,000 dalton glycopeptide of the gel complex of human cytomegalovirus.

The T cell epitopal glycopeptide according to the invention is substantially pure, is immunogenic, has a molecular weight of about 93,000, and includes one or more unique T cell epitopes which are not present in the gp55 glycopeptide of the gel complex. These "unique T cell epitopes" are epitopic sites, regions, or antigenic determinants within a polypeptide, glycopeptide or fragment thereof which do not react with or exhibit any substantial response to T lymphocyte cell lines, T helper clones, peripheral blood mononuclear cells (PBMCs), or the like which are reactive with gp55.

The glycopeptide fragments of the antigenic region of gp55 according to the invention are enzymatic digestion products of gel complex, are T cell or B cell immunogenic, have molecular weights of up to about 34 kD. Preferably the fragments include B cell epitopes

composed of the amino acid sequences 560-565, 563-568, and 590-595 as well as amino acid sequence numbered herein as 560-569, these sequences being taken from the entire gel amino acids sequence 1-907. Preferably the enzyme used in the fragment production is chymotrypsin. The present invention is also directed to the provision of recombinant expression vectors. These vectors, which include the gB gene, are capable of expressing polypeptides that can be immunoprecipitated by gel-specific monoclonal antibodies, or that stimulate proliferation of PBMCs or gel-specific T cell lines or T helper clones. Recombinant expression vectors are also provided which are capable of expressing a fusion protein that reacts with gel-specific T cell lines. The present invention also is directed to a recombinant vaccinia virus expression vector capable of expressing a virus which stimulates the proliferation of gel-specific T cell lines or T helper clones. Recombinant vaccinia virus expression vectors are also provided which induce a specific cytotoxic T cell response.

Advantageously, the identification, localization, and minimization of both unique and shared epitopes of the gp93 and gp55 glycopeptides of the gel complex allows for the provision of subunit HCMV vaccines and diagnostic methods which utilize these epitopes.

Brief Description of the Drawings Figure 1 is a depiction of a model of a topographical antigenic map for HCMV glycoproteins in gel complexes. Localization of B cell epitopes on HCMV are also shown in Figure 1.

Figure 2 is a schematic depiction of a molecular analysis of the antigenic domains of the gB (gel) complex of HCMV.

The top panel of Figure 2 depicts the HCMV genome, which includes the unique long (U _L ) coding region, extending from about 0.344 to about 0.360 map units. The second panel of Figure 2 depicts the base pair and amino acid numbering scheme for the gB (gel) complex. The gel glycoprotein designated herein as gpl30, which extends from about amino acid 1 to about amino acid 907, is depicted next. Below gpl30 is depicted the gel glycoprotein complex designated herein as gp93, which has an approximate length of about 630 amino acids. Its N-terminus and the C-terminus positions are not known. The gel glycoprotein designated herein as gp55 is depicted below gp93, and extends from about amino acid 461 to about amino acid 907. A 34,000 dalton chymotrypsin fragment of gel, designated as Chym(R)-gp34 herein, is depicted below gp55, and originates with amino acid 482 at a Tyr-Ala chymotrypsin cleavage site and extends to about amino acid 782.

The last panel of Figure 2 depicts the region of amino acids 461-671, which includes 3 B cell epitopes extending from amino acid 560-569. The three latter epitopes are amino acids 560-565 (18F9); 563-568(34G7) ; and 590-595(41C2) which are located by reactivity with monoclonal antibodies designated herein as 18F9, 34G7, and 41C2 respectively.

Figure 3 is a schematic depiction of a plasmid map for pSP65 (produced by Boehringer Mannheim Biochemicals) , a procaryotic expression vector into which the gB coding region was initially cloned for use in the detailed examples hereinbelow.

Figure 4 is a schematic depiction of a plasmid map for pRIT5 (produced by Pharmacia), a procaryotic expression vector containing a protein A fusion vector having multiple cloning sites, as further described in the detailed examples hereinbelow.

Figure 5 is a schematic depiction of the nucleotide locations of 11 overlapping pRIT5 fusion proteins selectively cloned from the pRIT5 expression vector, designated pRIT5-l through pRIT5-ll, respectively, which were utilized as described in the detailed examples hereinbelow.

Figure 6 is a schematic depiction of a plasmid map for pSCll (a gift from B. Moss, NIAID), a eucaryotic vaccinia virus vector into which the gB coding region was cloned as described in the detailed examples hereinbelow.

Figure 7 illustrates the amino acid sequence of the gel complex and the codon sequence of the gB gene (Spaete et al., Virology, 167, 207, 1988). Figure 8 is a table indicating antibody reactivity with HCMV-gcI glycoproteins/peptides.

Detailed Description of the Invention

In co-pending U.S. patent application Serial No's. 933,789 and 83,502, filed November 24, 1986 and August 7, 1987, respectively, the disclosures of which are incorporated by reference herein, substantially pure immunogenic 93 kD and 55 kD glycopeptides on the membrane envelope of HCMV and their derivation from gpl30, a precursor glycoprotein, were disclosed. These glycopeptides are associated with other envelope glycopeptides by means of disulfide bonds. The 93 kD glycopeptide immunoreacts in Western Blot with CMV- seropositive human sera but does not react in Western Blot with monoclonal antibodies that immunopreeipitate gel complexes containing gp93. This indicates that certain human antibodies recognize unique B cell epitopes on gp93.

The present invention is based in part upon the discovery that gp93 contains unique amino acid sequences expressing antigenic determinants recognized by B and T lymphocytes which are different from the antigenic

determinants expressed on gp55. The glycopeptides disclosed in the foregoing section constitute the results, in part, of this discovery.

In co-pending U.S. Patent Application Serial No. 07/214,302, filed July 1, 1988, the disclosure of which is incorporated herein by reference, antibody and T cell responses to unreduced and reduced gel glycopeptide fragments obtained by proteolytic digestion of native complexes with chymotrypsin or trypsin were characterized. The results of this study are depicted in Table II, below.

Table II.

HCHV gel (Glyco)peptide Cotnplexeβ and Individual (Glyco)peptideB

Obtained by Proteolytic Digestion of HCHV gel and Detected by HCHV-Specific HcAbs and/or HCHV-(-l-) Human Sera ¹

Unreduced gel Glycopeptide Reduced gel Glycopeptide Complexes: Complexes:

10

Treatment of gel Glycosylated Non-Glycosylated Glycosylated Non-Glycosylated Complexes

Undigested > 200, 000 ² 35,000 130,000

15

130,000 20,000 93,000 12,000 190,000

50,000 - 52,000

20

Chymotrypsin Fragments 43,000

34,000 20,000 34,000 Several

25 43,000

Trypsin Fragments 106,000 34,000 47,000 52,000

30

44,000 20,000 47,000 Several

35,000

'Detection method was immopreclpltation and/or Western Blot

35 ² Molecular weights expressed in daltons

In those applications, it was indicated that the eight epitopes of the major B cell antigenic region of gp55 are contained within these proteolytic fragments. Human convalescent sera from seropositive individuals also react with these fragments.

Using polyclonal and monoclonal gel-specific T cell lines, it has now been found according to the present invention that gel complexes contain T cell epitopes expressed within and outside of the gp55 region. Therefore, the present invention is directed to further localization of T cell epitopes of gel glycoproteins. More specifically, the present invention contemplates glycopeptides with epitopes which are unique to amino acid sequences in the N-terminal region of the gB polypeptide that are contained within gp93 but not within gp55.

In co-pending application U.S. Patent Application Serial No. 83,502, filed August 7, 1987, the disclosure of which is incorporated by reference herein, there is disclosed the generation of several murine B- cell hybridomas producing monoclonal antibodies (McAbs) specifically reactive with gel complexes containing three glycoproteins gpl30, gp93, and gp50-52. The individual gel-specific McAbs were shown to inhibit or augment the simultaneous binding of other of the gcl- specific McAbs in a manner which suggested at least 7 distinct antigenic sites in 3 separate domains of gel. It was concluded that these three domains were the major B cell antigenic region of gp55. The present invention is based in part upon the elucidation of these domains. It has been discovered that these domains contain at least eight epitopal sites which are B cell reactive. In particular, three of these eight epitopes have now been particularly identified and sequenced as the glycopeptide fragments representing localized, minimal antigenic structures of gp55. These three immunoreact with monoclonal

antibodies 18F9, 34G7 and 41C2 as mentioned above. The fragments compose the continuous and discontinuous epitopes in the major B cell antigenic region of gp55. The invention will be further described by reference to the following detailed examples, wherein the methodologies are as described below. The disclosures of the cited references are incorporated by reference herein.

Methods;

Immunoprecipitation. For immunoprecipitation. Protein A-Sepharose C1-4B beads (Sigma-Aldrich, St. Louis, Missouri) were prepared by incubation with goat anti-mouse IgG (H and L) and washed with phosphate- buffered saline (PBS) before use. Whole extract or glycopeptides and glycopeptide complexes obtained by ion-exchange HPLC were incubated with monoclonal antibody (preparation, infra) in PBS containing 0.1% NP- 40 for 1.5 hours with constant mixing. In some cases, 0.1% sodium dodeeylsulfate (SDS) was used in place, of NP-40. The prepared Protein A-Sepharose CL-4B beads were then added to the antigen-antibody solution and allowed to react for an additional 1.5 hours with constant mixing. The beads were washed three times with PBS containing 0.1% NP-40. Following the final wash, proteins associated with the beads were solubilized for SDS-PAGE.

Preparation of Monomer. Isolated HCMV disulfide-linked glycopeptide complexes were reduced by addition of dithiothreitol to a final concentration of 10 mM (DTT, United States Biochemicals, Cleveland, Ohio) in the presence of 8M Urea. The reaction was allowed to proceed at room temperature for 2-24 hours with constant stirring. Alkylation of the reduced sulfhydryl group was done by adding 5-10 mg iodoacetamide and allowing the reaction to proceed at room temperature for an additional hour.

Gel Filtration HPLC of Individual HCMV Glycopeptides. Samples containing 1% SDS were subjected to gel filtration HPLC using a Varian system. Gel filtration was performed coupling TSK 3000 SW and 4000 SW gel filtration columns (Toyo Soda, Tokyo, Japan) in series and eluting isocratically with 50 mM sodium phosphate, pH 7.0, containing 0.1% SDS. The flow rate was 0.3 ml/minute. Absorbance was monitored at 275 nm and fractions collected were also monitored for radio- activity.

SDS-Polyacrylamide Gel Electrophoresis and Fluorography. SDS-PAGE was done with 5-15% polyacrylamide slab gel gradients following the method of Laemmli. Samples were solubilized by boiling for three minutes in the presence of 4% SDS. After the samples were cooled to room temperature, urea and beta- mercaptoethanol (BME) were added so that samples contained 5% BME and 2M urea. Tritium was detected by fluorography using Enlighting (DuPont/NEN, Boston, Massachusetts).

Preparation of Viruses. HCMV Towne strain and AD169 strain were grown with or without 3H-glucosamine on human skin fibroblast cultures, harvested and purified on sucrose gradients as described previously (Kari et al., J. Virol., 60, 345-352 (1986)). The purified virus was resuspended in Tris-NaCl buffer (50 mM Tris hydrochloride, 150 mM NaCl pH 7.4), and extracted with 1% Nonidet P-40 (NP-40, Sigma Chem. Co., St. Louis, MO) in Tris-NaCl buffer (50 mM Tris hydrochloride, 10 mM NaCl buffer (50 mM hydrochloride, 10 mM NaCl. , 2 mM phenylmethyl sulfonylfluoride pH 7.5) as described by Kari et al., in J. Virol. , 60, 345-352 (1986). Uninfected skin fibroblasts were extracted in a similar fashion for use as negative controls. Reduction and alkylation of HCMV Towne NP-40 crude extracted material was performed as described by Kari et al. , J. Virol. , 60, 345-352 (1986). All detergent extracted

viral or control fibroblast materials were passed over an Extracti-Gel D column and eluted with water to remove the detergent.

Generation of Murine Monoclonal Antibodies to HCMV. The production of mouse hybridomas secreting mcAbs to HCMV proteins was performed as previously described (Kari et al., J. Virol., 60, 345-352 (1986)). The antibodies produced involved three separate fusion experiments using either AD169 or Towne strain purified HCMV virions. ^' Balb/C mice were immunized for 2, 5 or 10 months. Spleen cells from immunized mice were fused with Sp2-2-Agl4 myeloma cells (American Tissue Culture Collection) using polyethylene glycol as the fusing agent. Resulting hybrid cells were screened for specific antibody production to HCMV using an enzyme- linked immunosorbent assay (ELISA) . Antigens used in the ELISA assay were either purified HCMV Towne Strain or AD169 strain whole virions or HCMV Towne NP-40 extracted material or NP-40 extracted material from uninfected skin fibroblasts. Ascites fluids from expanded clones were purified for IgG using high performance hydroxyapatite chromatography (HPLC) (Juarez-Salinas et al., Biotechnigues, 2_, 164 (1984)). The titer of fractions collected from the column was measured with respect to HCMV-specific activity using the ELISA assay. The protein content of the fractions was determined using the BioRad protein assay (BioRad) and purified mouse IgG as a standard. Only fractions with the highest titers from each run were used for subsequent experiments.

Immunoprecipitation. Monoclonal antibodies were allowed to immunopreeipitate ³ H-glucosamine-labelled HCMV Towne NP-40 extracted proteins which were unreduced or reduced and alkylated prior to immunoprecipitation. Proteins were solubilized in sodium dodecyl sulfate (SDS) and separated by SDS polyacrylamide gel

electrophoresis. The tritium-labelled glycoprotein bands in the gel were identified by fluorography as described (Kari et al., J. Virol., 6_0, 345-352 (1986)). Western Blot. For Western Blot assays, purified HCMV Towne strain whole virus was solubilized with SDS and separated by 5-15% gradient polyacrylamide gel electrophoresis.

The proteins on the gel were subsequently electroblotted onto nitrocellulose paper with a BioRad transblot apparatus. The paper was blocked with 3% gelatin in Tris buffered saline (TBS, 20 mM Tris, 500 mM NaCl pH 7.5). The mcAbs in ascites fluid were diluted 1/500 in 1% gelatin in TBS, and allowed to bind to the paper overnight at room temperature. The paper was washed with PBS-0.05% Tween 20 (polysorbate 20), and alkaline phosphatase-labelled goat anti-mouse IgG (KPL) diluted 1/2000 with 1% gelatin in TBS was added and allowed to incubate for one hour at room temperature. The paper was washed once again and the substrate 5- bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium in 0.1 M Tris buffer solution (KPL) was added. Reaction of the antigen-antibody complex with the substrate resulted in the formation of an insoluble purple product. The reaction was stopped by immersing the paper in water.

ELISA Assay for Detection of Glycoprotein in Tissue Culture Supernatants. Mierotiter wells in 96- well mierotiter plates are pre-coated with HCMV glycoprotein-specific monoclonal antibody I as the capture antibody. Included in each plate are a blank well, a well for an uninfected fibroblast control and a glycoprotein positive control and an glycoprotein positive control well in addition to wells for patient samples. After coating with the capture antibody, the mierotiter wells are washed once with buffer (PBS-0.5% Tween) . Sample solutions are solubilized in PBS-1% Np- 40 in each tissue culture well, and are then dispensed

into individual mierotiter wells. Blank, negative skin fibroblast and glycoprotein positive control samples are included with each clinical sample determination.

Five μl of biotinylated glycoprotein-specific monoclonal antibody which recognizes a different epitope on gel than that recognized by first monoclonal antibody, are then added to each mierotiter well as a detection antibody. The mierotiter plate is then placed in a plastic bag and incubated at 37°C for 1 hour. Wells are then washed 4 times with wash buffer (PBS-

0.05% Tween), 100 μl of streptavidin-peroxidase is added and the plate is incubated for 30 minutes at 37 °C in a plastic bag. Wells are then washed 4 times with wash buffer and then one time with distilled water. Ortho- phenylenediamine (OPD) substrate (100 μl ) is added and reacted for 15 minutes at room temperature. The reaction is then stopped with 25 μl of 5 N H _j SO^. After 10 minutes, the mierotiter plate is read with a Dynatech plate reader at 490 nm or by visual colorometric reading. This method has been shown to detect glycoprotein at a total protein concentration of 10 nanograms.

Example 1. Initial Characterization of B and T Cell Responses to HPLC-Purified gel and

Proteolytic Cleavage Fragments

A. B Cell Recognition

As described in detail in U.S. Patent

Application Serial No. 83,502, the disclosure of which is incorporated herein by reference, murine gcl-specific monoclonal antibodies recognize multiple epitopes in three domains clustered in a single major antigenic region containing: (a) continuous neutralizing and non- neutralizing epitopes; and (b) conformation-dependent epitope(s) eliciting monoclonal antibodies which block neutralization by other monoclonal antibodies.

Antibody Reactivity with HCMV-gcI Glycoproteins/Peptides ^"

A simultaneous competitive ELISA assay was used to identify eight gcl-specific monoclonal antibodies (mcAbs) reactive with unique continuous or conformation- dependent epitopes in three domains comprising a major B cell antigenic region of gel (Lussenhop et al.. Virology, 160, 308, 1988). Figure 8 summarizes the results. Individual mcAbs in domains I and II either augment or inhibit the binding of other gcl-specific mcAbs, and exhibit similar effects on virus neutralization in a plaque reduction assay. McAbs reactive with conformation-dependent epitope(s) in domain III inhibit binding and neutralizing activity of all other gcl-specific mcAbs.

All of the gcl-specific mcAbs antibodies recognizing continuous epitopes immunopreeipitate gel complexes and react with gpl30, gp55, and proteolytic cleavage fragments obtained by digestion with chymotrypsin in Western blot analysis. However, these same mcAbs are either non-reactive or minimally reactive with gp93. gcl-specific mcAbs recognizing conformational epitopes react with unreduced gel complexes and chymotrypsin fragments, but not with reduced gel glycoproteins or proteolytic fragments.

Human HCMV-positive convalescent sera also recognize B cell epitope(s) on gel complexes and proteolytic fragments. All react with gpl30 and gp55 in Western blot. In addition, some but not all sera react with gp93.

All of the gcl-specific monoclonal antibodies listed in Fig. 8 immunoprecipitated gel complexes and unreduced proteolytic cleavage fragments, and reacted with gpl30 and gp55 in Western Blot analysis. However, these same monoclonal antibodies were either non- reactive or minimally reactive with gp93 in Western Blot, suggesting that gp93 may not share the major antibody recognition site with gp55.

The results summarized in Figure 8 indicate that human HCMV-positive convalescent sera immunoprecipitated gel complexes as well as proteolytic fragments containing the major B cell antigenic domain. All sera reacted with gpl30 and gp55 in Western Blot analysis; however, some but not all sera reacted with gp93. This result suggests that unique determinants on gp93 are involved in the human antibody response which are not recognized by any of the murine gcl-specific monoclonal antibodies described thus far.

B. T Cell Responses to HCMV-qcI Glycoproteins

The experiments summarized at Table III show that peripheral blood mononuclear cells (PBMCs) from all HCMV-seropositive adults proliferate when stimulated with whole HCMV virions, whereas only two-thirds respond to HPLC-purified gel (Liu et al., J. Virol., 62, 1066, 1988). gel-responsive PBMCs also responded to gpl30, gp55, and gp93 obtained by electroelution from polyacrylamide gels. HCMV-specific T helper (T _h ) clones reactive with gel as well as clones reactive with other structural (glyco)proteins are detected in gel responders (data not shown) , suggesting that (glyco) roteins other than gel are important in cell- mediated immunity.

20

Table III. T Cell Responses to HCMV-qcI Glycoproteins

HCMV-Specific T Cell Lines/Clones

KM-3UR WRCT3#3 RC 26

(Polyclonal (Non-gcl 0 gcl-specific (gel-specif :ic specific T cell line) T _h clone) _h clone)

5 Medium control 3,808 142 61

HCMV virions ^a 36,610 12,835 3,785 gcl ^b 20,128 13,997 74 g _P 55 ^c 10,643 6,590 108 gp93 ^c 18,293 11,438 106 0 gpl30 ^c 13,340 11,299 82

BSA 2,337 118 57

Acrylamide 2,382 56 63

25 Virions purified by sucrose gradient ultracentrifugation of extracellular virus from supernatants of HCMV-infected fibroblasts

'unreduced gel complexes separated from detergent extracts

30 of HCMV virions by anion exchange HPLC and immunoprecipitated with gcl-specific mcAb 41C2

"individual gel glycoproteins separated from reduced gel complexes in SDS-PAGE and isolated by electroelution.

-ac;

T Cell Responses to Proteolytic Fragments of HCMV- el

The experiments summarized at Table IV show that gcl-specific polyclonal T cell lines proliferate when stimulated with trypsin or chymotrypsin gel fragments as well as whole gel complexes. However, certain gcl-specific T _h clones react with gel complexes and trypsin fragments but not with chymotrypsin fragments. These data suggest that T cell epitope(s) are expressed on gp55 in the region spanning amino acids 482-782 defined by chym(R)-gp34. In addition, T cell epitope(s) are expressed in the C-terminal region of the gB polypeptide sequence contained in gp93 but not gp55. These data suggest that there is a major B cell antigenic region expressed on gpl30 and gp55 recognized by the gcl-specific mcAbs, and in addition, unique determinants on gp93 involved in the human antibody response.

.Table IV.

T Cell Responses to Proteolytic Fraσments of HCMV-qcI

HCMV-Specific T Cell Lines/Clones

SJK WRCT3 3

(Polyclonal

ECMV-specific (gcl-specific

T cell line T _h clone)

Medium control 81 256

HCMV virions ^a 46,055 20,497 gcl 5,954 11,509 trypsin fragments' ¹ 22,961 34,420 chymotrypsin fragments' ¹ 11,119 377

Virions purified by sucrose gradient ultracentrifugation of extracellular virus from supernatants of HCMV-infected fibroblasts

'unreduced gel complexes separated from detergent extracts of HCMV virions by anion exchange HPLC and immunoprecipitated with gcl-specific mcAb 41C2

^" -"unreduced trypsin and chymotrypsin fragments obtained by proteolytic digestion of unreduced gel cσπrolexes purified as above (*)

Example 2. Molecular Analysis of B and T Cell Epitopes on gel A^ Location of Glycopeptide Fragments on the gB Sequence by N-terminal Sequencing

An abundant reduced chymotrypsin gel glycopeptide fragment of molecular weight 34,000 was purified by gel filtration. N-terminal sequencing on a Porton gas phased sequenator located this fragment to the N-terminal half of gp55, starting with amino acid 482 at a Tyr-Ala chymotrypsin cleavage site. The chym(R)-gp34 fragment is depicted in Figure 2. Deglycosylation of chym(R)-gp34 with N-glyconase resulted in a nonglycosylated polypeptide having a molecular weight of about 30,000 daltons. Assuming the average molecular weight of an amino acid is 100, chym(R)-gp34 extends to about amino acid 782 in the predicted transmembrane region of gp55, and includes substantially all of the extracellular domain of gp55.

All monoclonal antibodies as well as seropositive convalescent sera reacted with the 34 kD reduced chymotrypsin fragment as shown in Table IV.

B. Definitive Epitope Analysis to Characterize the Major B Cell Antigenic Domain(s) of gp55

Hexapeptides overlapping by a single amino acid for the region of gel spanning amino acids 461-671 were generated using the pin-synthesis method originally described by Geysen et al., (Proc. Natl. Sci., 81, 3998 (1984)) (see Figure 3). The multi-pin-synthesis technology, manufactured by Cambridge Research Biochemicals under license from Commonwealth Serum Laboratories, was designed to synthesize nanomole amounts of overlapping peptides from known sequences using f-moc chemistry. Reactivity of the gcl-specific monoclonal antibodies with peptides irreversibly bound to the pins was then determined by ELISA according to a procedure provided by the manufacturer.

A B cell epitope was located by reactivity of gcl-specific (Domain II) monoclonal antibody 34G7 to amino acids 563-569, shown in the last panel of Figures 1 and 2. Amino acid 565 within this region is a methionine, which upon cleavage with cyanogen bromide (CNBr) eliminated binding of 34G7 to this epitope.

A second B cell epitope, which overlaps the 34G7 epitope, was located by reactivity of gcl-specific (domain II) mcAb 18F9 to amino acids 560-565 as shown in Figures 1 and 2.

A third B cell epitope has been located by reactivity of gcl-specific (domain I) mcAb 41C2 to amino acids 590-595 as shown in Figures 1 and 2.

The proximity of these epitopes to amino acids associated with loop structures (Proline) and intra- chain disulfide bonds (Cysteine) are consistent with the proposed topographical model depicted in Figure 1. The results of the hexapeptide synthesis confirmed that antibody responses to gp55 involve primarily a series of continuous and spacially related epitopes comprising a single major antigenic domain. Thus, a polypeptide derived by proteolytic cleavage of biochemically purified gel, the molecular expression product of the portion of the HCMV genome coding for this antigenic region, or relevant synthesized peptides in this region can be useful in vaccines to induce neutralizing antibody responses in humans against HCMV, and can also be useful as antigen to detect the presence of antibodies reactive with gp55/gcl in human or animal sera.

Example 3. Expression of gB in Procaryotic and Eucaryotic Expression Vectors for T Cell Epitope Analysis A_ _; _ Procaryotic Expression in pSP65

The gB coding region derived from Towne strain

HCMV, located at about 0.344 to about 0.360 map units in the HCMV genome, was initially cloned into the

procaryotic expression vector pSP65 under the control of an inducible promoter. This promoter facilitates the high level production of mRNA transcripts from the foreign gene of interest in bacteria. Figure 3 depicts the plasmid map for pSP65.

The resulting gB-mRNA was then translated in vitro in rabbit reticulocyte lysates. The p95 polypeptide product of gB was identified by ³⁵ S-met/SDS- PAGE. The gB polypeptide was immunoprecipitated by gcl- specific monoclonal antibodies and stimulated proliferation of PBMCs and gcl-specific T _h clones in proliferation assays. The preparation of these gcl- specific Th clones is described in detail in co-pending U.S. Patent Application Serial No. 07/024,571, filed March 11, 1987, the disclosure of which is incorporated by reference herein. Table V, below, depicts the level of ³ H-thymidine incorporation by the T _h clones with whole HCMV virions, HPLC-purified gel, and various translation lysates of the mRNA of gel.

Table V.

Comparison of Reactivity of T Helper Cell Clones to HPLC-purlfled gel and Protein Translated from gel mRNA

Antigent ³ H-thymidine incorporation in counts per minute with T _h Clonesi

10 SP-CN/T3-3 SP-CN/T5-10 SP-CN/T5-43 SP-CN/T3-4

Control 311 275 210 122

15

Vhole HCMV virions 33,003 27,079 77 ,933 26,498

HPLC purified gel 19,670 2,822 273 169

20 Translation lysate with no RNA 110 ND 107 ND

N_>

Translation lysate with gel mRNA 4,394 1,784 427 131

Translation lysate with nucleocapsid

25 of black beetle mosaic virus mRNA 234 118 143 ND

Translation lysate with mRNA transcribed from a region upstream of gel 181 347 190 242

30

As indicated by the data of Table V, the HCMV- T _h clones SP-CN/T3-3 and SP-CN/T5-10 which responded to HPLC-purified gel also responded to a polypeptide translated from gel mRNA in a rabbit reticulocyte lysate system. THe HCMV-T _k clones SP-CN/T5-43 and SP-CN/T3-4 which did not respond to HPLC-purified gel did not respond to polypeptide translated from gel mRNA, either. Furthermore, SP-CN/T3-3 and SP-CN/T5-10 did not respond to lysate control nor translated products of other mRNA. Therefore, it was concluded that continuous T cell epitope(s) are expressed on the gB polypeptide backbone. B. Procaryotic Expression in pRIT5

The gB coding region (0.344-0.360 m.u.) was then cloned into the procaryotic expression vector, pRIT5 (Pharmacia, Piscataway, NJ) . Figure 4 depicts the construction of pRIT5. The pRIT5 cloning vector is comprised of a promoter, the open reading frame for staphylococcal protein A, and a multiple cloning site downstream of the staphylococcal protein A ORF which can be used to introduce foreign genes in frame with protein A. The high level expression of hybrid protein and convenient single-step purification using an IgC Sepharose 6FF affinity gel column (Pharmacia) allowed for the isolation of significant quantities of fusion proteins for T cell epitope mapping.

Unique restriction sites were identified in the gB coding region to allow for the selective cloning of 11 overlapping gB fragments representing the entire open reading frame of the gB (gel) gene. Figure 5 depicts the nucleotide locations of these fragments, and the unique restriction enzymes designated as pRIT5-l through pRIT5-ll, respectively. Three fusion proteins derived by expression of the pRIT5-2, pRIT5-4, and pRIT5-9 recombinants were used to locate the immunodominant T cell epitopes of gB. The pRIT5-2 fusion protein designated herein as ProteinA-gcI-2 (base pairs 938- 2136) comprised the N-terminal end of gB outside of

gp55, while the pRIT5-4 fusion protein designated herein as ProteinA-gcI-4 (b.p. 1983-2730) comprised a region including both unique gp93 sequences as well as the N- terminal region of gp55 including much of the major B cell antigenic region. The pRIT5-9 fusion protein designated herein as ProteinA-gcI-9 (b.p. 2786-3791) comprised the C-terminal region of gp55. Based on the reactivity of a gcl-specific polyclonal T cell line, designated herein as KM, with ProteinA-gcI-2 and ProteinA-gcI-4, but not with ProteinA-gcI-9, the T cell epitopes were located in the N-terminal two-thirds of the gB polypeptide.

KM T cell line is a polyclonal T cell line which was produced by repeated stimulation of peripheral blood mononuclear cells obtained from an HCMV- seropositive donor (KM) with HPLC-purified gel complexes. This polyclonal T cell line is comprised of HCMV-specific T cells reactive with all T cell epitopes expressed on gel complexes and its constituent glycoproteins. It is, therefore, useful for screening of peptide fragments of larger gel glycoprotein structures for epitope analysis.

Table VI, below, depicts the proliferation of the KM gcl-specific T cell line in the presence of the various fusion proteins. The level of ³ H-thymidine incorporation was assayed in samples of the various antigen, including the fusion proteins expressed by the pRIT5-2, pRIT5-4, and pRIT5-5 recombinants.

Table VI.

Lymphocyte Proliferation Assay with Protein A Fusion Proteins (KH gel-Specific T Cell Line)

10 Antigen ³ II-Thymidine Incorporation ³ H-Thymidine Incorporation counts per minute

Positive Negative

15

Control 304 + 156

CMV 11,731 + 5,673

20

Vaccinia-gel 1,244 + 420

25 Protein A 329 + 95

ProteinA-gcI-2 2,132 ± 527

30

ProteinA-gcI-4 1,990 + 435

ProteinA-gcI-9 639 + 322

35

The data of Table VI suggest that gel contains T cell epitope(s) in the N-terminal region, perhaps overlapping with the gel major B cell antigenic domain. More specifically, the data suggest that the gcl-specific T cell response includes T _h reactive with epitopes shared among gpl30, gp55, and gp93, presumably in the same region as that containing the major B cell antigenic region of gp55, as well as T _h reactive with unique epitopes expressed only on gp93. In addition, the peptide sequence in pRIT5-2 outside of gp55 (i.e., on gp93) also contains T cell epitope(s), suggesting that this region of the polypeptide may be important in cell-mediated immunity. This can be seen from a comparison of Figs. 2 and 5. Recombinant pRIT5-2 ending at bp2136 does not contain the sequence within gp55 which begins about bp2300. Therefore, the T cell response to pRIT5-2 locates one or more T cell epitopes to a region of the gB sequence outside of gp55, but which is included in gp93.

Example 4. Eucaryotic Expression of gB in Vaccinia

The gB (gel) coding region was cloned into the eucaryotic vaccinia virus vector pSCll (provided by B. Moss, National Institute of Allergy and Infectious Diseases, Bethesda, MD) at the Smal site. Figure 6, panel A, depicts the construction of the pSCII vector. Panel B of Figure 6 depicts a modification of pSCll, designated pSCllt, in which there has been inserted a universal translation terminator to facilitate transcription and translation of full-length mRNA from foreign genes cloned in frame into this vector. The gene was introduced into vaccinia virus by homologous recombination between the TK gene of the wild strain vaccinia virus and the TK flanking sequences on either side of gB which had previously been inserted into the pSCII plasmid. The resulting recombinant vaccinia virus was identified by expression of the β galactosidase gene

using Xgal from Bethesda Research Laboratories Life Technologies, Inc. (Gaitersburg, MD) , and was purified by three cycles of plaque purification.

The vaccinia-gB recombinant, designated as VAC- gB herein, allows for eucaryotic expression in mammalian cells to allow for post-translational processing such as glycosylation which may be critical for conformation- dependent immune recognition. Expression of gB in fibroblasts infected with the VAC-gB recombinant was confirmed by indirect immunofluorescence using a gcl- εpecific monoclonal antibody. Furthermore, the gcl- specific mcAb 41C2 immunoprecipitated all three gel glycoproteins, gpl30, gp93 and gp55, in lysates of vaccinia-gB infected fibroblasts. A polyclonal gcl- specific T cell line (KM) and several gcl-specific T _h clones (KM-A, KM-B, KM-C, KM-G, KM-H, KM-J, KM-3, AND WRCT3#3) were then stimulated with whole HCMV virions and lysates of fibroblasts infected either with wild strain vaccinia or with VAC-gB. Table VII, below, depicts the results of a lymphocyte proliferation assay for the T cell line and T _h clones, which were labelled with ³ H-thymidine, and added to whole HCMV virions and to lysates of wild strain vaccinia-infected or VAC-gB- infected fibroblasts.

Table VII.

Lymphocyte Proliferation Assay with ³ H-Thymidine-Labelled T Cella in Whole Virions and Wild Strain Vaccinia verstis Vacclnia-gcI Recombinant

T Cells Medium Control HCMV Vaccinia ² Vaccinia-gel ³

10 Rcl-Speclfjc T Cell Line :

KM 304 + 156 11,731 + 5,673 ND 1,244 + 420 counts per minute

HCMV-Specific T _h Clones:

15

KM-A 60 + 7 15,778 ± 1,360 74 + 12 647 + 83

KM-B 59 + 7 1,894 ± 587 57 + 6 465 + 37

20 KM-C 74 + 14 17,071 ± 1,638 89 + 11 4,565 + 834 ϊ

KM-G 195 + 63 3,161 + 519 117 + 26 714 + 204

KM-H 72 + 23 2,007 + 245 77 + 7 67 + 23

25

KM-J 90 + 15 6,252 + 636 55 + 5 2,619 + 246

KM-3 95 + 18 2,530 + 6B8 55 + 2 1,058 + 300

30 VRCT3/3 46 + 10 8,839 + 1,351 ND 5,337 + 970

'Whole HCMV virions

² Lysate of fibroblasts Infected with wild strain vaccinia

³ Lysate of fibroblasts infected with vaccinia-gB recombinant

As shown in Table VII, all the T cell line/clones responded to whole HCMV, and all but one T _h clone (KM-H) also reacted specifically to gel expressed in vaccinia (VAC-gb-infected fibroblasts). HCMV-specific polyclonal T cell lines stimulated by cell-associated HCMV were then tested for cytotoxic activity against autologous fibroblasts infected with HCMV, vaccinia pSCll which did not contain a foreign gene, or vaccinia-HCMV gB recombinants as shown in Table VIII below.

Table VIII.

Cytotoxic Activity of Polyclonal T Cell Structures Stimulated by Cell-Associated HCHV

Z Specific Lysis Measured by ⁵¹ Cr Release ¹ Effector Cells E:T ² HCMV- pSCll Vaccinia- infected Vaccinia gB gene Fibroblasts Recombinant

10

WRC anti-HCMV 50:1 0.4 + 1.5 40.0 + 6.7 19.4 + 13.8 36 . 7 + 5 .1 T Cell Line

25:1 -1.7 + 0.5 33.7 + 2.7 25.3 + 3.0 25 .5 + 1.5 υ_

15 12:1 -1.7 + 0.8 24.0 + 3.1 23.3 + 2.0 23 . 1 + 4 . 0 4

SKJ anti-HCMV 16:1 4.0 + 0.8 ND 3.8 + 4.0 36.4 + 51.0 T Cell Line

8:1 -4.0 + 0.9 ND 6.4 + 1.4 -1.4 + 2.0

20

^• Autologous uninfected or HCMV/vaccinia-infected fibroblasts used as target cells ² Ratio of effector cells to target cells (v/v) in sample.

Products of vaccinia-HCMVgB were shown to induce specific cytotoxic activity, suggesting that gel proteins are targets for the cytotoxic T cell response.

In summary, linear and/or conformational epitopes expressed on the polypeptide encoded by gB, as well as unique and/or shared epitopes expressed on gp55 and gp93, are involved in recognition by antibodies and T _h and T _c cells. Thus, it is likely that gp93, as well as gp55, is an appropriate candidate for a subunit HCMV vaccine.

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

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