EP0708658A1 - Recombinant cytomegalovirus vaccine - Google Patents

Abstract

The present invention provides a non-defective adenovirus recombinant expression system for the expression of an immunogenic fragment of the HCMV gB subunit, said recombinant HCMV-expressing adenovirus being useful as a vaccine.

Description

RECOMBINANT CYTOMEGALOVIRUS VACCINE

This work was performed with government support under National Institutes of Health grants AI-07278 and HD-18957. The U.S. government has certain rights in this invention.

Field of the Invention The present invention refers generally to a recombinant human cytomegalovirus vaccine, and more specifically to a subunit vaccine containing fragments of a HCMV major glycoprotein complex subunit gB gene.

Background of the Invention

Cytomegalovirus (CMV) is one of a group of highly host specific herpes viruses that produce unique large cells bearing intranuclear inclusions. The envelope of the human cytomegalovirus (HCMV) is characterized by a major glycoprotein complex recently termed gB or gCI, which was previously referred to as gA. HCMV causes cytomegalic inclusion disease and has been associated with a syndrome resembling infectious mononucleosis in adults. It also induces complications in immunocompromised individuals.

CMV infection in utero is an important cause of central nervous system damage in newborns. Although the virus is widely distributed in the population, about 40% of women enter pregnancy without antibodies and thus are susceptible to infection. About 1% of these women undergo primary infection in utero . Classical cytomegalic inclusion disease is rare; however, a proportion of the infected infants, including those who were symptom-free, are subsequently found to be mentally retarded. Preliminary estimates based on surveys of approximately 4,000 newborns from several geographical areas indicate that the virus causes significant damage of the central nervous system leading to mental deficiency in at least 10%, and perhaps as high as 25%, of infected infants. Assuming that about 1% of newborn infants per year excrete CMV and that about one fourth of those develop mental deficiency, in the United States this means approximately 10,000 brain-damaged children born per year. This is a formidable number, particularly in view of the ability of these children to survive [J. Infect. Pis.. 123 (5) :555 (1971)].

HCMV in humans has also been observed to cause serious complications and infections in the course of organ transplantations, especially with kidney and liver transplants.

Several HCMV vaccines have been developed or are in the process of development. Vaccines based on live attenuated strains of HCMV have been described. [See, e.g., S. A. Plotkin et al, Lancet, 1:528-30 (1984); S. A. Plotkin et al, J. Infect. Pis.. 134:470-75 (1976); S. A. Plotkin et al, "Prevention of Cytomegalovirus Pisease by Towne Strain Live Attenuated Vaccine", in Birth Pefects, Original Article Series, 20(1) :271-287 (1984); J. P. Glazer et al, Ann. Intern. Med.. 91:676-83 (1979); and U. S. Patent 3,959,466.] A proposed HCMV vaccine using a recombinant vaccinia virus expressing HCMV glycoprotein B has also been described. [See, e.g., Cranage, M. P. et al, EMBO J.. 5:3057-3063 (1986).] However, vaccinia models for vaccine delivery are believed to cause local reactions. Additionally, vaccinia vaccines are considered possible causes of encephalitis. Adenoviruses have been developed previously as efficient heterologous gene expression vectors. For example, an adenovirus vector has been employed to express herpes simplex virus glycoprotein gB [P. C. Johnson et al, Virol.. 164:1-14 (1988)]; human immunodeficiency virus type 1 envelope protein [R. L. Pewar et al, J. Virol.. 63.:129-136 (1988)]; and hepatitis B surface antigen [A. R. Pavis et al, Proc. Nat1. Acad. Sci.. U.S.A.. 82.:7560-7564 (1985); J. E. Morin et al, Proc. Natl. Acad. Sci.. U.S.A.. 84:4626-4630 (1987)]. Adenoviruses have also been found to be non-toxic as vaccine components in humans [See, e.g., E. T. Takajuji et al, J. Infect. Pis.. 140:48-53 (1970); P. B. Collis et al, J. Inf. Pis.. 128:74-750 (1973); and R. B. Couch et al, Am. Rev. Respir. Pis.. J38_:394-403 (1963)].

There remains a need in the art for additional vaccines capable of preventing CMV infection by generating neutralizing antibody and cellular responses to CMV in the human immune system.

Summary of the Invention

In one aspect, the present invention provides a non-defective recombinant adenovirus containing a fragment of a gB subunit of the HCMV free from association with any additional human proteinaceous material. In this recombinant adenovirus, the HCMV subunit is under the control of regulatory sequences capable of expressing the HCMV gB subunit fragment in vitro and in vivo. Another aspect of the present invention is a vaccine composition comprising a non-defective recombinant adenovirus, as described above.

In a further aspect, the invention provides a method of vaccinating a human against HCMV comprising administering to the patient the recombinant adenovirus containing the subunit gene encoding a gB protein fragment in a vaccine composition. The inventors have found that this method of presenting these HCMV gene fragments to a vaccinate is particularly capable of eliciting an immune response.

In still a further aspect the invention provides an adenovirus-produced gB subunit fragment, which fragment may also form vaccine compositions to protect humans against HCMV. Currently, the preferred fragment comprises about amino acids 1 to about 303 of the gB protein SEQ IP NO:2, gB-.._3(a .

Other aspects and advantages of the present invention are described further in the following detailed description of preferred embodiments of the present invention.

Brief Pescription of the Prawings

Fig. IA illustrates diagrammatically the cloning of the gB gene into the early region 3 (E3) transcription unit of Ad5. Represented are the 3.1kb fragment containing the gB gene by the open box; the adenovirus sequences extending from 59.5 to 100 mu (except for the deletion of the 78.5 to 84.7 mu length) by the filled portion of the circle: the large BamHI fragment of the pBR322 by the thin line of the circle.

In the figure, the restriction enzymes are identified as follows: X is Xbal, B is BamHI.

Fig. IB illustrates diagrammatically the construction of the recombinant adenovirus virus Ad5/gB, containing the gB gene of the Towne strain of HCMV described in Example 1. This figure shows the 59.5 mu to 76 mu region where homologous recombination occurs (as indicated by the crossed lines) between wild type Ad5 viral sequence and the adenovirus sequences present on the pAd5 plasmid containing the gB gene. The plaque purified recombinant virus retains the cloning Xbal sites and'the direction of transcription of the gB gene from the E3 promoter is indicated by the bent arrow. Restriction enzymes are as identified above.

Petailed Pescrintion of the Invention

The present invention provides novel immunogenic components for HCMV which comprise an adenovirus expression system capable of expressing a selected HCMV subunit gene fragment in vivo.

Alternatively the selected subunit fragment for use in an immunogenic composition, such as a vaccine, may be expressed in, and isolated from, the recombinant adenovirus expression system. As provided by the present invention, any adenovirus strain capable of replicating in mammalian cells in vitro may be used to construct an expression vector for the selected HCMV subunit. However, a preferred expression system involves a non-defective adenovirus strain, including, but not limited to, adenovirus type 5. Alternatively, other desirable adenovirus strains may be employed which are capable of being orally administered, for use in expressing the CMV subunit in vivo. Such strains useful for in vivo production of the subunit in addition to adenovirus-5 strains include adenovirus type 4, 7, and 21 strains. [See, e.g., Takajuji et al, cited above]. Appropriate strains of adenovirus, including those identified above and those employed in the examples below are publicly available from sources such as the American Type Culture Collection, Rockville, Maryland.

Similarly, a number of strains of isolated human CMV may be employed from which a desired gB subunit is derived. For example, the Towne strain of CMV, a preferred strain for use in preparation of a vaccine of this invention because of its broad antigenic spectrum and its attenuation, was isolated from the urine of a two month old male infant with cytomegalic inclusion disease (symptoms - central nervous system damage and hepatosplenomegaly) . This strain of CMV was isolated by Stanley A. Plotkin, M.P. and is described in J. Virol.. 11 (6) : 991 (1973) . This strain is freely available from The istar Institute or from the ATCC under accession number VR-977. However, other strains of CMV useful in the practice of this invention may be obtained from depositories like the ATCC or from other institutes or universities.

In the practice of one embodiment of this invention the HCMV subunit may be produced in vitro by recombinant techniques in large quantities sufficient for use in an immunogenic composition or subunit vaccine. Alternatively, the recombinant adenovirus containing the subunit may itself be employed as an immunogenic or vaccine component, capable of expressing the subunit in vivo .

The presently preferred subunit proteins for use in the present invention are the HCMV gB subunit fragments. One embodiment of the present invention provides a replication competent (non-defective) adenovirus vector carrying a fragment of the HCMV gB gene which contains a CTL epitope and/or B cell epitope. A preferred gene fragment encodes about amino acid 1 to about amino acid 303 of the gB subunit protein SEQ IP NO:2. Another suitable fragment of gB SEQ IP NO:2 is the fragment spanning about amino acid 1 to about amino acid 700 of SEQ IP NO:2. Still another suitable gB fragment spans about amino acid 1 to about amino acid 465 of SEQ IP NO:2.

More particularly, it is anticipated that smaller fragments containing all or a portion of the gB fragment spanning amino acids about 155 to about 303 will also be desirable for vaccine use. This region is suspected of containing at least a CTL epitope (see Examples 5 and 6 below) . It is anticipated that in the construction of the adenovirus vectors of this invention, any of the subunits of the HCMV envelope protein may be employed. In a manner similar to the use of the gB fragment in this vaccine, other subunits of CMV which may be employed in the production of a vaccine according to the invention may be selected from the gcll, gcIII, or immediate early subunits of the human virus. Alternatively, more than one HCMV subunit may be employed in a vaccine according to the teachings of the present invention. In addition to isolating the desired subunit from an available strain of HCMV for insertion into the selected adenovirus, the sequences of the subunits of two HCMV strains have been published [See, e.g., Mach et al, J. Gen. Virol.. 67_ι1461-1467 (1986); Cranage et al, (1986) cited above; and Spaete et al, Virol.. 167:207-225 (1987) . These subunit sequences can therefore be chemically synthesized by conventional methods known to one of skill in the art, or the sequences purchased from commercial sources. The recombinant adenovirus of the present invention may also contain multiple copies of the HCMV subunit. Alternatively, the recombinant virus may contain more than one HCMV subunit type, so that the virus may express two or more HCMV subunits, subunit fragments, or immediate early antigens and subunits together.

In the construction of the adenovirus vector of the present invention, the CMV subunit sequence is preferably inserted in an adenovirus strain under the control of an expression control sequence in the virus itself. The adenovirus vector of the present invention preferably contains other sequences of interest in addition to the HCMV subunit. Such sequences may include regulatory sequences, enhancers, suitable promoters, secretory signal sequences and the like. The techniques employed to insert the subunit sequence into the adenovirus vector and make other alterations in the viral PNA, e.g., to insert linker sequences and the like, are known to one of skill in the art. See, e.g., T. Maniatis et al, "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982) . Thus, given the disclosures contained herein the construction of suitable adenovirus expression vectors for expression of an HCMV subunit protein is within the skill of the art. Example 3 below provides construction details for the non-defective adenovirus containing these gB fragments.

The recombinant adenovirus itself, constructed as described above, may be used directly as an immunogen or a vaccine component. According to this embodiment of the invention, the recombinant adenovirus, containing the HCMV subunit, e.g., the gB subunit fragment, is introduced directly into the patient by vaccination. The recombinant virus, when introduced into a patient directly, infects the patient's cells and produces the CMV subunit in the patient's cells. The inventors have found that this method of presenting these HCMV genes to a vaccinate is particularly capable of eliciting an immune response. Examples 5 and 6 demonstrate the ability of a recombinant adenovirus containing the gB fragment, amino acid 1-303 of SEQ IP NO:2, to induce a gB-specific, protective CTL response in mice.

The use of these adenovirus recombinants as immunogens capable of inducing a CTL response is surprising in view of the results obtained in the same assays of the examples with other known virus types, which have been used in vaccines previously. According to another embodiment of this invention, once the recombinant viral vector containing the CMV subunit protein, e.g., the gB^rø subunit fragment, is constructed, it may be infected into a suitable host cell for in vitro expression. The infection of the recombinant viral vector is performed in a conventional manner. [See, Maniatis et al, supra-] Suitable host cells include, without limitation, mammalian cells and cell lines, e.g., A549 (human lung carcinoma) or 293 (transformed human embryonic kidney) cells.

The host cell, once infected with the recombinant virus of the present invention, is then cultured in a suitable medium, such as Minimal Essential Medium (MEM) for mammalian cells. The culture conditions are conventional for the host cell and allow the subunit, e.g., gB_W(β subunit fragment, to be produced either intracellularly, or secreted extracellularly into the medium. Conventional protein isolation techniques are employed to isolate the expressed subunit from the selected host cell or medium.

When expressed in vitro and isolated from culture, the subunit, e.g., gB^^, may then be formulated into an appropriate vaccine composition. Such compositions may generally contain one or more of the recombinant CMV subunits.

The preparation of a pharmaceutically acceptable vaccine composition, having appropriate pH, isotonicity, stability and other conventional characteristics is within the skill of the art. Thus such vaccines may optionally contain other components, such as adjuvants and/or carriers, e.g., aqueous suspensions of aluminum and magnesium hydroxides. Thus, the present invention also includes a method of vaccinating humans against human CMV infection with the recombinant adenovirus vaccine composition. This vaccine composition is preferably orally administered, because adenoviruses are known to replicate in cells of the stomach. Previous studies with adenoviruses have shown them to be safe when administered orally [see, e.g., Collis et al, cited above]. However, the present invention is not limited by the route of administration selected for the vaccine.

When the recombinant adenovirus is administered as the vaccine, a dosage of between 10⁵ and 10⁸ plaque forming units may be used. Additional doses of the vaccines of this invention may also be administered where considered desirable by the physician. The dosage regimen involved in the method for vaccination against CMV infection with the recombinant virus of the present invention can be determined considering various clinical and environmental factors known to affect vaccine administration.

Alternatively, the vaccine composition may comprise one or more recombinantly-produced human CMV subunit proteins, preferably a fragment of a gB subunit. The in vitro produced subunit proteins may be introduced into the patient in a vaccine composition as described above, preferably employing the oral, nasal or subcutaneous routes of administration. The presence of the subunit produced either in vivo or as part of an in vitro expressed subunit administered with a carrier, stimulates an immune response in the patient. Such an immune response is capable of providing protection against exposure to the whole human CMV microorganism. The dosage for all routes of administration of the in vitro vaccine containing one or more of the CMV subunit proteins is generally greater than 20 micrograms of protein per kg of patient body weight, and preferably between 40 and 80 micrograms of protein per kilogram.

The utility of the recombinant adenoviruses of the present invention is demonstrated through the use of a novel mouse experimental model which characterizes cytotoxic T lymphocyte (CTL) responses to individual proteins of strictly human-restricted viruses. For example, the model as used herein is based on the use of two types of recombinant viruses, an adenovirus and a canarypox virus, both expressing a gene of the same HCMV protein. This model is useful in identifying immunodominant HCMV proteins and immunodominant epitopes of individual proteins to incorporate into an appropriate immunizing vector, analysis of proteins of various HCMV strains, immunization protocols and the longevity of cell-mediated immunity to individual proteins or epitopes; and investigation of the optimal vector for effective introduction of a certain antigen or epitope to the host immune system. According to this model, mice are immunized with one recombinant of the invention, and CTL activity is tested in target cells infected with the other recombinant. Specifically, Examples 4-6 below provide a murine model of the cytotoxic T lymphocyte (CTL) response to the amino acid 1-303 fragment of the glycoprotein B (gB) gene [SEQ IP NO:2] of human cytomegalovirus (HCMV) based on the use of gB-expressing adenovirus (Ad-gB) and several poxvirus recombinants. Using this model, it has been demonstrated that the human CMV subunit gB (HCMV-gB) amino acid 1-303 fragment can elicit a major histocompatibility complex (MHC) class I-restricted HCMV-gB-specific CTL response in mice.

The following examples illustrate the construction of a non-defective adenovirus strain capable of expressing the HCMV major envelope glycoprotein gB_j^ fragment and the efficacy of these compositions as an HCMV vaccine. These examples are illustrative only and do not limit the scope of the present invention.

Example 1 - Construction of a Non-defective Adenovirus - αB fAd-αB) Recombinant

The gB gene was cloned from the Towne strain of HCMV [Wistar Institute] as follows. The gB gene was first mapped to the 20.5 kb Hind III P fragment of HCMV using oligonucleotides that corresponded to the 5' and 3' termini of the published AP-169 gB sequence [See, Cranage et al (1986), cited above]. The Hind III fragment was cut with Xbal to generate a 9.8 kb fragment. This fragment was then cut with X alll to generate a 3.1 kb fragment. The 3.1 kb Xmalll fragment which contained the gB gene, had Xbal linkers attached to its 5' and 3' termini.

An adenovirus type 5 plasmid, pAd5 Bam-B, which contains the 59.5 - 100 mu region of the Ad5 adenovirus genome cloned into the BamHI site of pBR322 [See, R. L. Berkner et al, Nucl. Acids Res.. 1^:6003-6020 (1983) and M. E. Morin et al, cited above] was digested with Xbal to remove the 78.5 mu - 84.7 mu sequences of the Ad5 genome. The 78.5 to 84.7 mu deletion removes most of the coding region of the E3 transcription unit of Ad5 but leaves the E3 promoter intact. The Xbal-linked 3.1 kb fragment of CMV containing the gB gene was inserted into this Xbal site of pAd5 Bam-B. Fig. IA provides a diagrammatic illustration of the above description. To generate recombinant virus, the 0-76 mu fragment of wild type Ad5 virus was isolated by digesting the viral PNA with EcoRI [See, U. Petterson et al, J. Mol. Biol. , 72:125-130 (1973)]. This fragment was co- transfected with the 59.5 to 100 mu BamHI fragment of pAd5 Bam-B containing the gB gene as described above into human embryonic kidney 293 cells, available from the American Type Culture Collection. The Ad-gB recombinant was generated by overlap recombination between the viral sequences as illustrated in Fig. IB. The gB recombinant virus was plaque purified on human lung carcinoma A549 cells [ATCC CCL185] using standard procedures. Viruses containing both orientations of the gB gene, as determined by Southern blotting, were isolated. The recombinant containing the gB gene in the same 5• to 3' direction as the adenovirus E3 promoter of the adenovirus type 5 strain is under the transcriptional control of the E3 promoter. The plaque purified recombinant virus retains the cloning Xbal sites. The above-described cloned gB gene is devoid of its natural promoter according to the PNA sequence of gB identified in Spaete et al, (1987) , cited above.

Example 2 - Production of the Full-Length gB Subunit The adenovirus gB plasmid construct and the Ad5 mu 0-76 PNA of Example 1 were cotransfected into 293 cells, human cells transformed by adenovirus 5 early genes [See, Graham et al, J. Gen. Virol. ,36:59-72 (1977); and ATCC CRL1573] employing conventional procedures. This transfection generated a functional recombinant virus by homologous overlap recombination as shown in

Fig. IB.

Southern blot analysis confirmed the presence of an adenovirus, type 5, containing the HCMV gB subunit (referred to as either Ad-5/gB or Ad-gB) recombinant virus which was subsequently purified by plaque purification using standard procedures.

The recombinant virus AP-5/gB, expresses gB subunit protein as determined by conventional assays, i.e., immunofluorescence on fixed cells and by Western blot using monospecific guinea pig antiserum and monoclonal antibodies to gB protein [See, e.g., T. Maniatis et al, cited above]. The Ad-5/gB recombinant, also referred to as Ad-gB, is also described in applicant's publication [Marshall et al., J. Infect. Pis.. 162:1177-1181 (1990)] published after the filing date of the original parent application from which this application claims priority.

Example 3 - Construction of the gB gene fragments

Ad-gBi.₃₀₃ and Ad-gB_M55 recombinant viruses were constructed by overlap recombination as described for Ad- gB in Example 2 above. Briefly, in order to clone the subfragments of the gB gene, five oligonucleotide primers for polymerase chain reactions (PCR) were synthesized.

The primers were designed to anneal with various portions of the gB PNA sequence and promote amplification of the gene. In addition, all of the oligonucleotide primers were engineered to contain an Xba I site so that the PCR product could be digested with this enzyme in order to facilitate cloning into the pAd-5 vector. 5' gB primer : SEQ IP NO:3:

4889: 5'-ACACGCAAGAGA TCTAGA CGCGCCTCAT 3' primer at amino acid 700 of gB protein: SEQ IP N0:4: 5'-TCGTCCAGAC TCTAGA GGTAGGGC

3' primer at aa 465: SEQ IP NO:5:

5'-CGACTCCAT TCTAGA TTAATGAGTTGCATT 3' primer at aa 303: SEQ IP NO:6:

5'-CAAAGTCGGAG TCTAGAG TCTAGTTCGGAAA 3' primer at aa 155: SEQ IP NO:7:

5'-CAGATAAGTGG TCTAGA TCTAAGCGTAGCTACG The above oligonucleotides correspond to the following nucleotide positions of the HCMV gB gene (Towne strain) as reported by Spaete et al, Virology. 167:207-225 (1988). SEQ IP N0:3 corresponds to nucleotide positions 895 to 922 in the sense orientation; SEQ IP NO:4 to nucleotide positions 3090 to 3067 anti-sense; SEQ IP NO:5 to nucleotide positions 2375 to 2350 anti-sense; SEQ IP NO:6 to nucleotide positions 1877 to 1847 anti-sense; and SEQ IP NO:7 to nucleotide positions 1432 to 1400 anti- sense. These immediately preceding nucleotide numbers are not identical to those of SEQ IP NO: 1 because the Spaete et al sequence, to which these numbers correspond, contains additional 5' non-coding sequence while SEQ IP NO: 1 reports only the PNA sequence corresponding to the coding region of the gB protein [SEQ IP NO: 2].

The specific segments or fragments of the gB gene were amplified using the Perkin-Elmer Amplitaq™ kit by mixing 400 ng of the 5' gB primer with each of the 3' primers separately (400 ng of each) and 0.1 μg of purified HCMV genomic PNA or 0.1 μg of previously cloned intact gB gene (see Example 2) . The final reaction mixture was 100 _/xL and the thermocycling conditions were 94°C, 1 minute; 52°C, 1 minute; 72°C, 1 minute, repeated for a total of 35 cycles. Amplified PNA was purified by cutting the proper PNA fragment out of a 1.2% agarose gel, digested with Xbal. repurified by cutting the digested fragments out of a 1.2% agarose gel and then ligated into the Xbal site of the cloning vector pAd-5. Positive recombinants were verified by PNA sequence analysis and sequence analysis confirmed the orientation of the clones.

Example 4 - CTL Assays A. Recombinant Viruses Used

The following recombinant viruses were used in the CTL assays of Examples 5-6 below to demonstrate the immunogenicity and vaccine utility of the recombinant adenoviruses of the present invention. Wild-type human adenovirus type 5 (WT-Ad) and the Ad-gB recombinant were propagated in human lung carcinoma A549 cells [ATCC CCL185], as described in Example 1. An E3-deleted adenovirus type 5 mutant lacking the Xbal P fragment of adenovirus PNA (Ad5ΔE3) was constructed by overlap recombination, using plasmid pAd-5 mu 59.5-100, which was deleted in E3 sequences (mu 78.5-84) using the techniques described in Example 1, and pAd-5 mu 0-75.9 [G. S. Marshall et al, J. Infect. Pis.. 162:1177-1181 (1990), hereby incorporated by reference]. A vaccinia virus recombinant containing the gB subunits (VacC-gB) described previously in Gonczol et al, Vaccine. :631-637 (1991) and the parental Copenhagen strain of vaccinia, VC-2 (also known as wild-type vaccinia (WT-Vac) ) were grown in Vero cells [E. Gonczol et al, Vaccine. J3:130-136 (1990); J. Tartaglia et al, Crit. Rev. Immunol.. i :13-30 (1990)].

The vaccinia WR strain [obtained from Pr. Enzo Paoletti, Virogenetics Corp, Troy, NY] was used to develop a recombinant expressing HCMV-gB ( (VacW)-gB) . This recombinant was derived using a strategy similar to that described for the VacC-gB recombinant (Gonczol et al. , cited above) . A canarypox recombinant [ALVAC-CMV (VCP139) which is subsequently referred to as Cp-gB] expressing the HCMV-gB gene was constructed using a strategy similar to that described for a canarypox-rabies recombinant in Taylor et al., Vaccine. 9.:190-193 (1991) [also obtained from Pr. Enzo Paoletti] . Briefly, the gene encoding the HCMV (Towne strain) glycoprotein B was inserted into a canarypox donor plasmid consisting of a polylinker flanked by genomic sequence from which a nonessential gene was specifically deleted (at a unique EcoRI site within a 3.3 kbp PvuII subgenomic fragment of canarypox PNA) . Expression of the gB protein gene was placed under the transcriptional control of an early/late vaccinia virus promoter (H6) previously described [Percus et al., J. Virol.. .62:3829-3835 (1989)]. Cp-gB was derived and plaque-purified by standard methods [Panicali and Paoletti, Proc. Natl. Acad. Sci. USA. 29:4927-4931 (1982)]. The Cp-gB recombinant and parental canarypox virus (WT-Cp) were propagated on primary chick embryo fibroblasts. B. Expression of the gB protein in Cp-gB recombinant virus Chicken embryo fibroblast (CEF) cells [ATCC CRL 1590] infected with either Cp-gB or with the parental wild-type canarypox (WT-Cp) virus preparations were analyzed by Western blot assay using the 4A guinea-pig serum directed against the gB protein. Western blot assays and the 4A guinea-pig serum, used as gB-specific antibody, were described previously in Gonczol et al., J. Virol.. 58:661-664 (1986). Uninfected and HCMV-infected MRC-5 cell [ATCC CCL 171] lysates were included as controls.

A diffuse band at the 140 kPa position and a double band of 55 and 58 kPa were detected in both Cp-gB-infected CEF cells and in HCMV-infected MRC-5 cells. The presence of these gB-specific proteins presumably representing the glycosylated 140 kPa precursor and the differentially glycosylated cleavage products (55 and 58 kPa) indicates that the Cp-gB recombinant expresses the inserted gB gene. The slight difference between the mobility of 55 and 58 kPa cleavage products of control and recombinant gB may reflect different glycosylation patterns.

C. Murine Model and CTL Assay

For immunization of mice, Ad-gB and WT-Ad were purified by CsCl gradient centrifugation. VacC-gB, VacW-gB and WT-Vac were purified by sucrose gradient centrifugation, and Cp-gB and WT-Cp were concentrated on sucrose cushion.

Six- to 8-week-old female BALB/c and CBA mice (from Harlan Sprague-Pawley and Jackson) and 12-week-old male BALB/k mice (from The Wistar Institute Animal Facility) were immunized intraperitoneally (i.p.) with the recombinant viruses described above at 1-5 x IO⁸ pfu unless otherwise stated. One to 12 weeks later, spleens were aseptically removed and cell suspensions were prepared by gently pressing the spleens through a stainless steel mesh. Cells were suspended at 2.5 x 10⁶ viable cells/ml in RPMI 1640 medium containing 5% FBS (Gibco) , 2 x 10"⁵ M 2-mercaptoethanol, 14 mM HEPES buffer, glutamine and 50 μg/ml gentamicin. Spleen cell cultures were restimulated in vitro with Ad-gB (multiplicity of infection (m.o.i.) = 10) or VacC-gB (m.o.i. = 0.5 ) infected autologous spleen cells for 5 days in 24-well plates. Cytolytic activity of nonadherent spleen cells was tested in a chromium release assay which was performed as follows.

1. T-cell subset depletion

For in vitro depletion of CP4 or CP8 cells, 3 x 10⁶ spleen cells were incubated with anti-mouse CP4 monoclonal antibody (MAb) [Pharmingen; Cat.3:01061 P; 20 μg/3xl0⁶ cells] or CP8 MAb [Accurate; Cat.#:CL-8921; diluted 1:4] for 60 minutes at 4°C, and further incubated in the presence of rabbit complement [Accurate; Low-tox M; diluted 1:10] for 30 minutes at 37°C. The cells were washed twice and used as effector cells in a ⁵¹Cr-release test.

2. Chromium release assay

P815 (H-2^d) [ATCC TIB 64], mouse MC57 (H- 2^b) cells [also termed MC-57G, P.P. Aden et al, Im unogenetics, 2:209-221 (1976)] and mouse NCTC clone 929 (H-2^k) cells [ATCC CCL 1] were used as target cells. The HCMV neutralization titer of mouse sera was determined on MRC-5 cells [ATCC CCL 171] by the microneutralization method as described in Gonczol et al., J. Virol. Methods. 14:37-41 (1986).

The target cells were infected with Ad-gB or Ad-5ΔE3 (multiplicity of infection (m.o.i.) = 40-80, 40 hours) or with Vac-gB or WT-Vac (m.o.i. = 5-10, 4 hours). Target cells were washed in the modified RPMI 1640 medium described above and 2 x 10⁶ cells were labeled with 100 μCi of [⁵¹Cr]NaCr04 [Amersham, specific activity 250-500 mCi/mg] for 1 hour. The labeled target cells were washed 3 times in phosphate-buffered saline (PBS) and then mixed with the effector cells at various effector:target ratios in triplicate using 96-well U-bottomed microtiter plates and incubated for 4 hours.

Percentage specific ⁵¹Cr release was calculated as: [ (cpm experimental release - cpm spontaneous release) / (cpm maximal release - cpm spontaneous release)] x 100. Standard deviation of the mean of triplicate cultures was less than 10%, and spontaneous release was always less than 25%.

This CTL assay is a system in which two types of viral expression vectors, poxvirus and adenovirus, carrying the same fragment of the HCMV-gB gene, are alternately used for immunization of animal or for infection of target cells to show that HCMV-gB fragment is an inducer of CTL in mice. Using this model system, the relative immunogenicity of the gB fragment expressed by different recombinant viruses has been evaluated.

Example 5 - CTL Responses to Adenovirus Containing gB Fragments

Ad-gB_j.₃₀₃ and Ad-gB_WJ5 recombinant viruses were constructed as described in Example 3 above. In CTL experiments performed as described in Example 4, CBA mice were immunized i.p. with IO⁸ pfu of the Ad-gB, Two weeks later spleen cells were restimulated in vitro with Ad-gB infected autologous spleen cells and tested for ability to lyse Wt-Ad, Vac-gB or Wt-Vac infected L929 (MHC-class I matched) cells.

All recombinants showed an Ad virus-specific CTL response, but only Ad-gB (containing the complete gB coding sequence) and Ad-gB^^ exerted gB-specific CTL, indicating the presence of a CTL-epitope on the N- terminal part of the gB protein between amino acid 155 and 303.

Example 6 - Protection Studies with Adenovirus Containing gB Fragments

Using the murine model described in Example 4, CBA mice were immunized with 1 x 10⁸ pfu of Wt-Ad, Ad5Δ3 (an E3 deleted mutant virus, the parental strain of the recombinant viruses) , Ad-gB, Ad-gB^^ or Ad-gBι_₁₅₅. Five to ten days later the immunized mice were challenged i.c. with VacWR-gB (a neurovirulent vaccinia strain expressing the HCMV-gB protein) . Control mice, immunized with the Wt-Ad or Ad5Δ3 virus died within 4-7 days after the challenge.

Ad-gB and Ad-gB^^-immunized mice survived (92% and 95% survival, respectively) , while all of the Ad-gB_j.i_jj-i munized mice died, indicating a protection epitope on the N-terminal part of the gB protein between amino acid 155 and 303.

Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. For example, use of other appropriate non-defective adenovirus strains for construction of analogous expression systems to express the HCMV gB fragment may be constructed according to the disclosure of the present invention.

Additionally, the other subunits of HCMV major glycoprotein complexes, e.g., gcll or gcIII, or immediate-early antigens, may be expressed in a non- defective adenovirus recombinant in the same manner as described above for subunit gB fragment. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Wistar Institute of Anatomy, Biology Government of USA Pept. Health and Human Services

(ii) TITLE OF INVENTION: Recombinant Cytomegalovirus

Vaccine

(iii) NUMBER OF SEQUENCES: 7

(iv) CORRESPONPENCE APPRESS:

(A) APPRESSEE: Howson and Howson

(B) STREET: Spring House Corporate Center, PO Box 457

(C) CITY: Spring House (P) STATE: Pennsylvania

(E) COUNTRY: USA

(F) ZIP: 19477

(V) COMPUTER REAPABLE FORM:

(A) MEPIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-POS/MS-POS

(P) SOFTWARE: Patentln Release #1.0, Version #1.25

(Vi) CURRENT APPLICATION PATA:

(A) APPLICATION NUMBER:

(B) FILING PATE:

(C) CLASSIFICATION:

(Vϋ) PRIOR APPLICATION PATA:

(A) APPLICATION NUMBER: US 08/048,978

(B) FILING PATE: 16-APR-1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Bak, Mary E.

(B) REGISTRATION NUMBER: 31,215

(C) REFERENCE/POCKET NUMBER: WST6CPCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-540-9200

(B) TELEFAX: 215-540-5818 (2) INFORMATION FOR SEQ IP NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2724 base pairs

(B) TYPE: nucleic acid

(C) STRANPEPNESS: double (P) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cPNA

(ix) FEATURE:

(A) NAME/KEY: CPS

(B) LOCATION: 1..2721

(Xi) SEQUENCE PESCRIPTION: SEQ IP NO:l:

ATG GAA TCC AGG ATC TGG TGC CTG GTA GTC TGC GTT AAC TTG 42 Met Glu Ser Arg lie Trp Cys Leu Val Val Cys Val Asn Leu 1 5 10

TGT ATC GTC TGT CTG GGT GCT GCG GTT TCC TCA TCT TCT ACT 84 Cys lie Val Cys Leu Gly Ala Ala Val Ser Ser Ser Ser Thr 15 20 25

CGT GGA ACT TCT GCT ACT CAC AGT CAC CAT TCC TCT CAT ACG 126 Arg Gly Thr Ser Ala Thr His Ser His His Ser Ser His Thr 30 35 40

ACG TCT GCT GCT CAT TCT CGA TCC GGT TCA GTC TCT CAA CGC 168 Thr Ser Ala Ala His Ser Arg Ser Gly Ser Val Ser Gin Arg 45 50 55

GTA ACT TCT TCC CAA ACG GTC AGC CAT GGT GTT AAC GAG ACC 210 Val Thr Ser Ser Gin Thr Val Ser His Gly Val Asn Glu Thr 60 65 70

ATC TAC AAC ACT ACC CTC AAG TAC GGA GAT GTG GTG GGG GTC 252 lie Tyr Asn Thr Thr Leu Lys Tyr Gly Asp Val Val Gly Val

75 80

AAC ACC ACC AAG TAC CCC TAT CGC GTG TGT TCT ATG GCA CAG 294 Asn Thr Thr Lys Tyr Pro Tyr Arg Val Cys Ser Met Ala Gin 85 90 95

GGT ACG GAT CTT ATT CGC TTT GAA CGT AAT ATC GTC TGC ACC 336 Gly Thr Asp Leu lie Arg Phe Glu Arg Asn lie Val Cys Thr 100 105 110

TCG ATG AAG CCC ATC AAT GAA GAC CTG GAC GAG GGC ATC ATG 378 Ser Met Lys Pro lie Asn Glu Asp Leu Asp Glu Gly lie Met 115 120 125 GTG GTC TAC AAA CGC AAC ATC GTC GCG CAC ACC TTT AAG GTA 420 Val Val Tyr Lys Arg Asn lie Val Ala His Thr Phe Lys Val 130 135 140

CGA GTC TAC CAG AAG GTT TTG ACG TTT CGT CGT AGC TAC GCT 462 Arg Val Tyr Gin Lys Val Leu Thr Phe Arg Arg Ser Tyr Ala

145 150

TAC ATC CAC ACC ACT TAT CTG CTG GGC AGC AAC ACG GAA TAC 504 Tyr lie His Thr Thr Tyr Leu Leu Gly Ser Asn Thr Glu Tyr 155 160 165

GTG GCG CCT CCT ATG TGG GAG ATT CAT CAT ATC AAC AGT CAC 546 Val Ala Pro Pro Met Trp Glu lie His His lie Asn Ser His 170 175 180

AGT CAG TGC TAC AGT TCC TAC AGC CGC GTT ATA GCA GGC ACG 588 Ser Gin Cys Tyr Ser Ser Tyr Ser Arg Val lie Ala Gly Thr 185 190 195

GTT TTC GTG GCT TAT CAT AGG GAC AGC TAT GAA AAC AAA ACC 630 Val Phe Val Ala Tyr His Arg Asp Ser Tyr Glu Asn Lys Thr 200 205 210

ATG CAA TTA ATG CCC GAC GAT TAT TCC AAC ACC CAC AGT ACC 672 Met Gin Leu Met Pro Asp Asp Tyr Ser Asn Thr His Ser Thr

215 220

CGT TAC GTG ACG GTC AAG GAT CAA TGG CAC AGC CGC GGC AGC 714 Arg Tyr Val Thr Val Lys Asp Gin Trp His Ser Arg Gly Ser 225 230 235

ACC TGG CTC TAT CGT GAG ACC TGT AAT CTG AAT TGT ATG GTG 756 Thr Trp Leu Tyr Arg Glu Thr Cys Asn Leu Asn Cys Met Val 240 245 250

ACC ATC ACT ACT GCG CGC TCC AAG TAT CCC TAT CAT TTT TTC 798 Thr lie Thr Thr Ala Arg Ser Lys Tyr Pro Tyr His Phe Phe 255 260 265

GCA ACT TCC ACG GGT GAT GTG GTT GAC ATT TCT CCT TTC TAC 840 Ala Thr Ser Thr Gly Asp Val Val Asp lie Ser Pro Phe Tyr 270 275 280

AAC GGA ACT AAT CGC AAT GCC AGC TAT TTT GGA GAA AAC GCC 882 Asn Gly Thr Asn Arg Asn Ala Ser Tyr Phe Gly Glu Asn Ala

285 290

GAC AAG TTT TTC ATT TTT CCG AAC TAC ACT ATC GTC TCC GAC 924 Asp Lys Phe Phe lie Phe Pro Asn Tyr Thr lie Val Ser Asp 295 300 305 TTT GGA AGA CCG AAT TCT GCG TTA GAG ACC CAC AGG TTG GTG 966 Phe Gly Arg Pro Asn Ser Ala Leu Glu Thr His Arg Leu Val 310 315 320

GCT TTT CTT GAA CGT GCG GAC TCA GTG ATC TCC TGG GAT ATA 1008 Ala Phe Leu Glu Arg Ala Asp Ser Val lie Ser Trp Asp lie 325 330 335

CAG GAC GAG AAG AAT GTT ACT TGT CAA CTC ACT TTC TGG GAA 1050 Gin Asp Glu Lys Asn Val Thr Cys Gin Leu Thr Phe Trp Glu 340 345 350

GCC TCG GAA CGC ACC ATT CGT TCC GAA GCC GAG GAC TCG TAT 1092 Ala Ser Glu Arg Thr lie Arg Ser Glu Ala Glu Asp Ser Tyr

355 360

CAC TTT TCT TCT GCC AAA ATG ACC GCC ACT TTC TTA TCT AAG 1134 His Phe Ser Ser Ala Lys Met Thr Ala Thr Phe Leu Ser Lys 365 370 375

AAG CAA GAG GTG AAC ATG TCC GAC TCT GCG CTG GAC TGT GTA 1176 Lys Gin Glu Val Asn Met Ser Asp Ser Ala Leu Asp Cys Val 380 385 390

CGT GAT GAG GCC ATA AAT AAG TTA CAG CAG ATT TTC AAT ACT 1218 Arg Asp Glu Ala lie Asn Lys Leu Gin Gin lie Phe Asn Thr 395 400 405

TCA TAC AAT CAA ACA TAT GAA AAA TAT GGA AAC GTG TCC GTC 1260 Ser Tyr Asn Gin Thr Tyr Glu Lys Tyr Gly Asn Val Ser Val 410 415 420

TTT GAA ACC ACT GGT GGT TTG GTG GTG TTC TGG CAA GGT ATC 1302 Phe Glu Thr Thr Gly Gly Leu Val Val Phe Trp Gin Gly lie

425 430

AAG CAA AAA TCT CTG GTG GAA CTC GAA CGT TTG GCC AAC CGC 1344 Lys Gin Lys Ser Leu Val Glu Leu Glu Arg Leu Ala Asn Arg 435 440 445

TCC AGT CTG AAT CTT ACT CAT AAT AGA ACC AAA AGA AGT ACA 1386 Ser Ser Leu Asn Leu Thr His Asn Arg Thr Lys Arg Ser Thr 450 455 460

GAT GGC AAC AAT GCA ACT CAT TTA TCC AAC ATG GAG TCG GTG 1428 Asp Gly Asn Asn Ala Thr His Leu Ser Asn Met Glu Ser Val 465 470 475

CAC AAT CTG GTC TAC GCC CAG CTG CAG TTC ACC TAT GAC ACG 1470 His Asn Leu Val Tyr Ala Gin Leu Gin Phe Thr Tyr Asp Thr 480 485 490 TTG CGC GGT TAC ATC AAC CGG GCG CTG GCG CAA ATC GCA GAA 1512 Leu Arg Gly Tyr lie Asn Arg Ala Leu Ala Gin lie Ala Glu

495 500

GCC TGG TGT GTG GAT CAA CGG CGC ACC CTA GAG GTC TTC AAG 1554 Ala Trp Cys Val Asp Gin Arg Arg Thr Leu Glu Val Phe Lys 505 510 515

GAA CTT AGC AAG ATC AAC CCG TCA GCT ATT CTC TCG GCC ATC 1596 Glu Leu Ser Lys lie Asn Pro Ser Ala lie Leu Ser Ala lie 520 525 530

TAC AAC AAA CCG ATT GCC GCG CGT TTC ATG GGT GAT GTC CTG 1638 Tyr Asn Lys Pro lie Ala Ala Arg Phe Met Gly Asp Val Leu 535 540 545

GGT CTG GCC AGC TGC GTG ACC ATT AAC CAA ACC AGC GTC AAG 1680 Gly Leu Ala Ser Cys Val Thr lie Asn Gin Thr Ser Val Lys 550 555 560

GTG CTG CGT GAT ATG AAT GTG AAG GAA TCG CCA GGA CGC TGC 1722 Val Leu Arg Asp Met Asn Val Lys Glu Ser Pro Gly Arg Cys

565 570

TAC TCA CGA CCA GTG GTC ATC TTT AAT TTC GCC AAC AGC TCG 1764 Tyr Ser Arg Pro Val Val lie Phe Asn Phe Ala Asn Ser Ser 575 580 585

TAC GTG CAG TAC GGT CAA CTG GGC GAG GAT AAC GAA ATC CTG 1806 Tyr Val Gin Tyr Gly Gin Leu Gly Glu Asp Asn Glu lie Leu 590 595 600

TTG GGC AAC CAC CGC ACT GAG GAA TGT CAG CTT CCC AGC CTC 1848 Leu Gly Asn His Arg Thr Glu Glu Cys Gin Leu Pro Ser Leu 605 610 615

AAG ATC TTC ATC GCC GGC AAC TCG GCC TAC GAG TAC GTG GAC 1890 Lys lie Phe lie Ala Gly Asn Ser Ala Tyr Glu Tyr Val Asp 620 625 630

TAC CTC TTC AAA CGC ATG ATT GAC CTC AGC AGC ATC TCC ACC 1932 Tyr Leu Phe Lys Arg Met lie Asp Leu Ser Ser lie Ser Thr

635 640

GTC GAC AGC ATG ATC GCC CTA GAC ATC GAC CCG CTG GAA AAC 1974 Val Asp Ser Met lie Ala Leu Asp lie Asp Pro Leu Glu Asn 645 650 655

ACC GAC TTC AGG GTA CTG GAA CTT TAC TCG CAG AAA GAA TTG 2016 Thr Asp Phe Arg Val Leu Glu Leu Tyr Ser Gin Lys Glu Leu 660 665 670 CGT TCC AGC AAC GTT TTT GAT CTC GAG GAG ATC ATG CGC GAG 2058 Arg Ser Ser Asn Val Phe Asp Leu Glu Glu lie Met Arg Glu 675 680 685

TTC AAT TCG TAT AAG CAG CGG GTA AAG TAC GTG GAG GAC AAG 2100 Phe Asn Ser Tyr Lys Gin Arg Val Lys Tyr Val Glu Asp Lys 690 695 700

GTA GTC GAC CCG CTG CCG CCC TAC CTC AAG GGT CTG GAC GAC 2142 Val Val Asp Pro Leu Pro Pro Tyr Leu Lys Gly Leu Asp Asp

705 710

CTC ATG AGC GGC CTG GGC GCC GCG GGA AAG GCC GTT GGC GTA 2184 Leu Met Ser Gly Leu Gly Ala Ala Gly Lys Ala Val Gly Val 715 720 725

GCC ATT GGG GCC GTG GGT GGC GCG GTG GCC TCC GTG GTC GAA 2226 Ala lie Gly Ala Val Gly Gly Ala Val Ala Ser Val Val Glu 730 735 740

GGC GTT GCC ACC TTC CTC AAA AAC CCC TTC GGA GCC TTC ACC 2268 Gly Val Ala Thr Phe Leu Lys Asn Pro Phe Gly Ala Phe Thr 745 750 755

ATC ATC CTC GTG GCC ATA GCC GTC GTC ATT ATC ATT TAT TTG 2310 lie lie Leu Val Ala lie Ala Val Val lie lie lie Tyr Leu 760 765 770

ATC TAT ACT CGA CAG CGG CGT CTC TGC ATG CAG CCG CTG CAG 2352 lie Tyr Thr Arg Gin Arg Arg Leu Cys Met Gin Pro Leu Gin

775 780

AAC CTC TTT CCC TAT CTG GTG TCC GCC GAC GGG ACC ACC GTG 2394 Asn Leu Phe Pro Tyr Leu Val Ser Ala Asp Gly Thr Thr Val 785 790 795

ACG TCG GGC AAC ACC AAA GAC ACG TCG TTA CAG GCT CCG CCT 2436 Thr Ser Gly Asn Thr Lys Asp Thr Ser Leu Gin Ala Pro Pro 800 805 810

TCC TAC GAG GAA AGT GTT TAT AAT TCT GGT CGC AAA GGA CCG 2478 Ser Tyr Glu Glu Ser Val Tyr Asn Ser Gly Arg Lys Gly Pro 815 820 825

GGA CCA CCG TCG TCT GAT GCA TCC ACG GCG GCT CCG CCT TAC 2520 Gly Pro Pro Ser Ser Asp Ala Ser Thr Ala Ala Pro Pro Tyr 830 835 840

ACC AAC GAG CAG GCT TAC CAG ATG CTT CTG GCC CTG GTC CGT 2562 Thr Asn Glu Gin Ala Tyr Gin Met Leu Leu Ala Leu Val Arg

845 850 CTG GAC GCA GAG CAG CGA GCG CAG CAG AAC GGT ACA GAT TCT 2604 Leu Asp Ala Glu Gin Arg Ala Gin Gin Asn Gly Thr Asp Ser 855 860 865

TTG GAC GGA CAG ACT GGC ACG CAG GAC AAG GGA CAG AAG CCC 2646 Leu Asp Gly Gin Thr Gly Thr Gin Asp Lys Gly Gin Lys Pro 870 875 880

AAC CTG CTA GAC CGA CTG CGA CAC CGC AAA AAC GGC TAC CGA 2688 Asn Leu Leu Asp Arg Leu Arg His Arg Lys Asn Gly Tyr Arg 885 890 895

CAC TTG AAA GAC TCC GAC GAA GAA GAG AAC GTC TGA 2724

His Leu Lys Asp Ser Asp Glu Glu Glu Asn Val 900 905

(2) INFORMATION FOR SEQ IP NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 907 amino acids

(B) TYPE: amino acid (0) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( i) SEQUENCE OESCRIPTION: SEQ IP NO:2:

Met Glu Ser Arg lie Trp Cys Leu Val Val Cys Val Asn Leu Cys lie 1 5 10 15

Val Cys Leu Gly Ala Ala Val Ser Ser Ser Ser Thr Arg Gly Thr Ser 20 25 30

Ala Thr His Ser His His Ser Ser His Thr Thr Ser Ala Ala His Ser 35 40 45

Arg Ser Gly Ser Val Ser Gin Arg Val Thr Ser Ser Gin Thr Val Ser 50 55 60

His Gly Val Asn Glu Thr lie Tyr Asn Thr Thr Leu Lys Tyr Gly Asp 65 70 75 80

Val Val Gly Val Asn Thr Thr Lys Tyr Pro Tyr Arg Val Cys Ser Met

85 90 95

Ala Gin Gly Thr Asp Leu lie Arg Phe Glu Arg Asn lie Val Cys Thr 100 105 110

Ser Met Lys Pro lie Asn Glu Asp Leu Asp Glu Gly lie Met Val Val 115 120 125 Tyr Lys Arg Asn lie Val Ala His Thr Phe Lys Val Arg Val Tyr Gin 130 135 140

Lys Val Leu Thr Phe Arg Arg Ser Tyr Ala Tyr lie His Thr Thr Tyr 145 150 155 160

Leu Leu Gly Ser Asn Thr Glu Tyr Val Ala Pro Pro Met Trp Glu lie

165 170 175

His His lie Asn Ser His Ser Gin Cys Tyr Ser Ser Tyr Ser Arg Val 180 185 190 lie Ala Gly Thr Val Phe Val Ala Tyr His Arg Asp Ser Tyr Glu Asn 195 200 205

Lys Thr Met Gin Leu Met Pro Asp Asp Tyr Ser Asn Thr His Ser Thr 210 215 220

Arg Tyr Val Thr Val Lys Asp Gin Trp His Ser Arg Gly Ser Thr Trp 225 230 235 240

Leu Tyr Arg Glu Thr Cys Asn Leu Asn Cys Met Val Thr lie Thr Thr

245 250 255

Ala Arg Ser Lys Tyr Pro Tyr His Phe Phe Ala Thr Ser Thr Gly Asp 260 265 270

Val Val Asp lie Ser Pro Phe Tyr Asn Gly Thr Asn Arg Asn Ala Ser 275 280 285

Tyr Phe Gly Glu Asn Ala Asp Lys Phe Phe lie Phe Pro Asn Tyr Thr 290 295 300 lie Val Ser Asp Phe Gly Arg Pro Asn Ser Ala Leu Glu Thr His Arg 305 310 315 320

Leu Val Ala Phe Leu Glu Arg Ala Asp Ser Val lie Ser Trp Asp lie

325 330 335

Gin Asp Glu Lys Asn Val Thr Cys Gin Leu Thr Phe Trp Glu Ala Ser 340 345 350

Glu Arg Thr lie Arg Ser Glu Ala Glu Asp Ser Tyr His Phe Ser Ser 355 360 365

Ala Lys Met Thr Ala Thr Phe Leu Ser Lys Lys Gin Glu Val Asn Met 370 375 380

Ser Asp Ser Ala Leu Asp Cys Val Arg Asp Glu Ala lie Asn Lys Leu 385 390 395 400 Gin Gin lie Phe Asn Thr Ser Tyr Asn Gin Thr Tyr Glu Lys Tyr Gly

405 410 415

Asn Val Ser Val Phe Glu Thr Thr Gly Gly Leu Val Val Phe Trp Gin 420 425 430

Gly lie Lys Gin Lys Ser Leu Val Glu Leu Glu Arg Leu Ala Asn Arg 435 440 445

Ser Ser Leu Asn Leu Thr His Asn Arg Thr Lys Arg Ser Thr Asp Gly 450 455 460

Asn Asn Ala Thr His Leu Ser Asn Met Glu Ser Val His Asn Leu Val 465 470 475 480

Tyr Ala Gin Leu Gin Phe Thr Tyr Asp Thr Leu Arg Gly Tyr lie Asn

485 490 495

Arg Ala Leu Ala Gin lie Ala Glu Ala Trp Cys Val Asp Gin Arg Arg 500 505 510

Thr Leu Glu Val Phe Lys Glu Leu Ser Lys lie Asn Pro Ser Ala lie 515 520 525

Leu Ser Ala lie Tyr Asn Lys Pro lie Ala Ala Arg Phe Met Gly Asp 530 535 540

Val Leu Gly Leu Ala Ser Cys Val Thr lie Asn Gin Thr Ser Val Lys 545 550 555 560

Val Leu Arg Asp Met Asn Val Lys Glu Ser Pro Gly Arg Cys Tyr Ser

565 570 575

Arg Pro Val Val lie Phe Asn Phe Ala Asn Ser Ser Tyr Val Gin Tyr 580 585 590

Gly Gin Leu Gly Glu Asp Asn Glu lie Leu Leu Gly Asn His Arg Thr 595 600 605

Glu Glu Cys Gin Leu Pro Ser Leu Lys lie Phe lie Ala Gly Asn Ser 610 615 620

Ala Tyr Glu Tyr Val Asp Tyr Leu Phe Lys Arg Met lie Asp Leu Ser 625 630 635 640

Ser lie Ser Thr Val Asp Ser Met lie Ala Leu Asp lie Asp Pro Leu

645 650 655

Glu Asn Thr Asp Phe Arg Val Leu Glu Leu Tyr Ser Gin Lys Glu Leu 660 665 670 Arg Ser Ser Asn Val Phe Asp Leu Glu Glu lie Met Arg Glu Phe Asn 675 680 685

Ser Tyr Lys Gin Arg Val Lys Tyr Val Glu Asp Lys Val Val Asp Pro 690 695 700

Leu Pro Pro Tyr Leu Lys Gly Leu Asp Asp Leu Met Ser Gly Leu Gly 705 710 715 720

Ala Ala Gly Lys Ala Val Gly Val Ala lie Gly Ala Val Gly Gly Ala

725 730 735

Val Ala Ser Val Val Glu Gly Val Ala Thr Phe Leu Lys Asn Pro Phe 740 745 750

Gly Ala Phe Thr lie lie Leu Val Ala lie Ala Val Val lie lie lie 755 760 765

Tyr Leu lie Tyr Thr Arg Gin Arg Arg Leu Cys Met Gin Pro Leu Gin 770 775 780

Asn Leu Phe Pro Tyr Leu Val Ser Ala Asp Gly Thr Thr Val Thr Ser 785 790 795 800

Gly Asn Thr Lys Asp Thr Ser Leu Gin Ala Pro Pro Ser Tyr Glu Glu

805 810 815

Ser Val Tyr Asn Ser Gly Arg Lys Gly Pro Gly Pro Pro Ser Ser Asp 820 825 830

Ala Ser Thr Ala Ala Pro Pro Tyr Thr Asn Glu Gin Ala Tyr Gin Met 835 840 845

Leu Leu Ala Leu Val Arg Leu Asp Ala Glu Gin Arg Ala Gin Gin Asn 850 855 860

Gly Thr Asp Ser Leu Asp Gly Gin Thr Gly Thr Gin Asp Lys Gly Gin 865 870 875 880

Lys Pro Asn Leu Leu Asp Arg Leu Arg His Arg Lys Asn Gly Tyr Arg

885 890 895

His Leu Lys Asp Ser Asp Glu Glu Glu Asn Val 900 905 (2) INFORMATION FOR SEQ IP NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANOEPNESS: single (P) TOPOLOGY: unknown

(ii) MOLECULE TYPE: PNA (genomic)

(xi) SEQUENCE PESCRIPTION: SEQ IO NO:3:

ACACGCAAGA GATCTAGACG CGCCTCAT 28

(2) INFORMATION FOR SEQ IO NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANOEPNESS: single (P) TOPOLOGY: unknown

(ii) MOLECULE TYPE: PNA (genomic)

(xi) SEQUENCE PESCRIPTION: SEQ IO NO:4:

TCGTCCAGAC TCTAGAGGTA GGGC 24

(2) INFORMATION FOR SEQ IO NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANPEPNESS: single (P) TOPOLOGY: unknown

(ii) MOLECULE TYPE: PNA (genomic)

(xi) SEQUENCE PESCRIPTION: SEQ IO NO:5:

CGACTCCATT CTAGATTAAT GAGTTGCATT 30 (2) INFORMATION FOR SEQ IO NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANOEPNESS: single (P) TOPOLOGY: unknown

(ii) MOLECULE TYPE: PNA (genomic)

(xi) SEQUENCE PESCRIPTION: SEQ IP NO:6:

CAAAGTCGGA GTCTAGAGTC TAGTTCGGAA A 31

(2) INFORMATION FOR SEQ IO NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANOEPNESS: single (P) TOPOLOGY: unknown

(ii) MOLECULE TYPE: PNA (genomic)

(xi) SEQUENCE PESCRIPTION: SEQ IO NO:7:

CAGATAAGTG GTCTAGATCT AAGCGTAGCT ACG 33

Claims

WHAT IS CLAIMEP IS:

1. A non-defective recombinant adenovirus containing a human cytomegalovirus protein gene encoding a cytomegalovirus protein gB subunit fragment containing at least one CTL epitope, said gene being under the control of an expression control sequence, said virus capable of expressing said subunit protein.

2. The adenovirus according to claim 1 wherein said fragment is selected from the group consisting of:

(a) the fragment spanning about amino acid 1 to about amino acid 303,

(b) the fragment spanning about amino acid 1 to about amino acid 700,

(c) the fragment spanning about amino acid 1 to about amino acid 465,

(d) fragments spanning about amino acid 155 to about amino acid 303, and

(e) smaller fragments of (a) through (d) of SEQ IP NO:2.

3. An immunogenic composition comprising a a non-defective recombinant adenovirus and a suitable pharmaceutical carrier, wherein said recombinant adenovirus comprises a human cytomegalovirus protein gene encoding a cytomegalovirus protein gB subunit fragment containing at least one CTL epitope, said gene being under the control of an expression control sequence and said virus is capable of expressing said subunit protein in vivo in an animal.

4. The composition according to claim 3 wherein said fragment is selected from the group consisting of:

(a) the fragment spanning about amino acid 1 to about amino acid 303,

(b) the fragment spanning about amino acid 1 to about amino acid 700,

(c) the fragment spanning about amino acid 1 to about amino acid 465,

(d) fragments spanning about amino acid 155 to about amino acid 303, and

(e) smaller fragments of (a) through (d) of SEQ 10 NO:2.

5. The composition according to claim 3 wherein said protein gene encodes an additional cytomegalovirus subunit protein fragment or a selected cytomegalovirus subunit protein.

6. The composition according to claim 3 wherein said gB subunit fragment is about amino acid 1 to about amino acid 303 of SEQ 10 NO:2.

7. The composition according to claim 3 wherein said adenovirus is selected from the group consisting of an adenovirus type 5, adenovirus type 4 and adenovirus type 7 strain.

8. The composition according to claim 7 wherein said gB subunit fragment is obtained from the Towne strain cytomegalovirus, and the adenovirus is type 5.

9. The use of a non-defective recombinant adenovirus comprising a human cytomegalovirus protein gene encoding a cytomegalovirus protein gB subunit fragment containing at least one CTL epitope, said gene being under the control of an expression control sequence and said virus being capable of expressing said subunit protein in vivo in an animal, in the preparation of a CMV vaccine.

10. The use according to claim 9 wherein said fragment is selected from the group consisting of:

(a) the fragment spanning about amino acid 1 to about amino acid 303,

(b) the fragment spanning about amino acid 1 to about amino acid 700,

(c) the fragment spanning about amino acid 1 to about amino acid 465,

(d) fragments spanning about amino acid 155 to about amino acid 303, and

(e) smaller fragments of (a) through (d) of SEQ 10 NO:2.

11. The use according to claim 9 wherein said adenovirus is present in an effective amount of between 10^s to 10⁸ plaque forming units.

12. An immunogenic composition comprising a gB subunit protein fragment containing at least one CTL epitope expressed in a recombinant adenovirus vector.

13. The composition according to claim 12 wherein said fragment is selected from the group consisting of:

(a) the fragment spanning about amino acid 1 to about amino acid 303,

(b) the fragment spanning about amino acid 1 to about amino acid 700,

(c) the fragment spanning about amino acid 1 to about amino acid 465,

(d) fragments spanning about amino acid 155 to about amino acid 303, and

(e) smaller fragments of (a) through (d) of SEQ 10 NO:2.