WO1994017810A1

WO1994017810A1 - Recombinant cytomegalovirus vaccine

Info

Publication number: WO1994017810A1
Application number: PCT/US1994/002107
Authority: WO
Inventors: Stanley A. Plotkin; Robert P. Ricciardi; Eva Gonczol
Original assignee: The Wistar Institute Of Anatomy And Biology
Priority date: 1993-02-12
Filing date: 1994-02-10
Publication date: 1994-08-18

Abstract

The present invention provides a non-defective adenovirus recombinant expression system for the expression of immediate-early exon 4 proteins, said recombinant HCMV-expressing adenovirus being useful as an immunogenic composition and vaccine.

Description

RECOMBINA T 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.

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 cyto egalic 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, £0.(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 reco binant 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.. (52:129-136 (1988)]; and hepatitis B surface antigen [A. R. Pavis et al, Proc. Natl. 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. _f iB_8_.: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 an immediate-early exon-4 (IE-exon-4) 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 IE-exon 4 subunit 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 using the recombinant adenovirus containing the subunit gene encoding IE-exon-4, in the manufacture of a vaccine composition useful against HCMV infection. The inventors have found that presenting these HCMV subunit proteins expressed by in vivo transcription of the gene to a vaccinate is particularly capable of eliciting a protective immune response. In yet a further aspect the invention provides an adenovirus-produced HCMV IE-exon-4 subunit, which subunit may also form vaccine compositions to protect humans against HCMV. In still a further aspect, the present invention provides a novel murine model useful for demonstrating cytotoxic T lymphocyte (CTL) response to individual HCMV proteins.

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

Petailed Pescription of the Invention The present invention provides novel immunogens and vaccine components for HCMV which comprise an adenovirus expression system capable of expressing a selected HCMV subunit gene in vivo. Alternatively the selected subunit for use in an immunogenic composition, i.e., a composition which elicits a humoral and/or a cell-mediated immune response, may be expressed in, and isolated from, the recombinant adenovirus expression system. Such an immunogenic composition may be used in a vaccine for protecting against human CMV infection. As provided by the present invention, any adenovirus strain capable of replicating in mammalian ceils 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.

The presently preferred subunit protein for use in the present invention is the HCMV IE-exon 4 subunit. The full length IE1 gene was reported by Stenberg et al, J. Virol.. 41:190-199 (1984). An Xbal E fragment containing the exon 4 subunit of the IE (or IE1) gene of the Towne strain of HCMV was reported to GenBank, Los Alamos, New Mexico in September 15, 1989 by Stenberg et al. The nucleic acid sequences of the coding region of the IE-exon-4 are provided in SEQ IP NO:l, in which the native TC nucleotides which precede the lysine codon have been modified to the ATG initiation codon. SEQ IP NO:2 provides amino acid sequences of the IE-exon 4 protein. In the practice of one embodiment of this invention the HCMV IE-exon 4 subunit may be produced in vitro by recombinant techniques in large quantities sufficient for use in a subunit vaccine. Alternatively, more than one HCMV subunit may be employed in a vaccine according to the teachings of the present invention. Alternatively, the recombinant adenovirus containing the subunit may itself be employed as an immunogen or vaccine component, capable of expressing the subunit in vivo . One embodiment of the invention provides a replication competent adenovirus-5 vector carrying the HCMV IE-exon 4 gene.

The desired subunit may be isolated from an available strain of HCMV for insertion into the selected adenovirus. A number of strains of human CMV have been isolated. 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. of The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania, and is described in J. Virol.. 11 (6) : 991 (1973) . This strain is freely available from The Wistar Institute or from the American Type Culture Collection (ATCC) , 12301 Parklawn Prive, Rockville, Maryland, USA, 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 addition to isolating the desired IE-exon 4 subunit from an available strain of HCMV for insertion into the selected adenovirus, the subunit sequence can be chemically synthesized by resort to conventional methods known to one of skill in the art and, e.g., SEQ IP NOS: l and 2. Alternatively, the sequence may be 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 or immediate early antigens and subunits together. The sequences of other HCMV subunits of two HCMV strains have been published [See, e.g., Mach et al, J. Gen. Virol..

62:1461-1467 (1986); Cranage et al, (1986) cited above; and Spaete et al, Virol.. 167:207-225 (1987).

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 IE-exon 4 subunit protein is within the skill of the art. Example 1 below describes in detail the construction of a non-defective adenovirus containing the HCMV IE-exon-4 subunit.

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 IE-exon-4 subunit, 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 a protective immune response. Examples 2 and 3 below demonstrate the ability of the adenovirus recombinant of this invention, Ad-IE, containing subunit IE-exon-4 to elicit a CTL response from immunized mice.

According to another embodiment of this invention, once the recombinant viral vector containing the CMV subunit protein, e.g., the IE-exon 4 subunit, 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 mammalian cells or 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., IE-exon4, 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., IE-exon4, 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 including the IE-exon-4 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, such as that of the invention, and CTL activity tested in target cells infected with the other recombinant. Specifically, Example 2 below provides a murine model of the cytotoxic T lymphocyte (CTL) response to the glycoprotein B (gB) gene of human cytomegalovirus (HCMV) based on the use of gB-expressing adenovirus (Ad-gB) and several poxvirus recombinants.

The following examples illustrate the construction of a non-defective adenovirus strain capable of expressing the HCMV IE-exon-4, and the efficacy of this composition as an HCMV vaccine. These examples are illustrative only and do not limit the scope of the present invention.

Example 1 - Construction of Ad-IE exon-4 recombinant virus

The protection of humans from CMV infection or virus-induced diseases is based on antibody dependent and/or T-cell dependent immune responses. The following experimental data demonstrates that an adenovirus recombinant containing the major immediate early (IE) gene of HCMV elicits a protective immune response in mice. The nucleic acid sequences of the coding region of the IE-exon-4 are provided in SEQ IP NO:l, in which the native TC nucleotides which precede the lysine codon have been modified to the ATG initiation codon.

To construct the IE-exon-4 adenovirus recombinant, the polymerase chain reaction (PCR) technique was used to amplify the exon 4 portion of the IE gene from purified HCMV genomic PNA (Towne strain) . The PCR primers were synthesized so as to incorporate the proper restriction endonuclease cleavage site, Xbal, (underlined in SEQ IP NOS: 3 and 4 below) for insertion into the Xbal site of the adenovirus vector. In addition, the 5' primer was also modified so that an ATG start translation codon was inserted at the first amino acid position of exon 4. The oligonucleotides used as primers were the following: 5' IE-exon 4: SEQ IP NO:3:

5'-TTATCCTCC TCTAGA ATGAAACAGATTAAG 3¹ IE-exon 4: SEQ IP NO:4:

5'-ATATATATAT TCTAGA GTTTACTGGTCGAC The 5' oligonucleotide corresponds to nucleotide positions 1 to 27 (sense orientation) and the 3' oligonucleotide corresponds to nucleotide positions 1251 to 1222 (anti-sense orientation) of an Xbal E fragment of the HCMV IE1 gene (Towne strain) available from GenBank, Los Alamos, New Mexico (Accession #M11630, Code #8SMIE4) . This fragment was used as an Exon 4 gene template for the PCR reaction. The full length IE1 gene was reported by Stenberg et al, J. Virol.. 4^:190-199 (1984).

In order to clone the IE1 exon 4, the 5' and 3* primers (400 ng each) were mixed with 0.1 μg of purified HCMV genomic PNA and the PNA was amplified using the Perkin-Elmer amplitaq kit. The final reaction volume was 100 μl and the thermocycling conditions were 94°C, l min; 52°C, 1 min; 72°C, 1 min, repeated for a total of 35 cycles. Amplified PNA was purified by cutting the proper size 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. Sequence analysis confirmed the orientation of the clones since the Xbal digested PNA fragments could be inserted into the adenovirus vector in two different orientations.

In this construct the E3 coding region (between map units 78.5 and 84.0) of the adenovirus is replaced by the exon-4 fragment. The correct orientation allows for the proper transcription of the gene fragment (in the sense orientation) from the adenovirus E3 promoter. The exon-4 product of the HCMV-IE gene was shown to be a target for CP8 cytotoxic and CP4 lymphoprόliferative T cell responses in humans. The Ad- IE-exon-4 construct is non-defective in replication (i.e., capable of replicating normally) in tissue culture cells.

This Ad-IE-exon-4 recombinant was used in the in vitro cytotoxic T lymphocyte (CTL) assay and mouse model described below.

Example 2 - CTL Assay-Murine Model

This CTL assay is a system in which two types of viral expression vectors, poxvirus and adenovirus, carrying the same HCMV IE-exon 4 subunit gene, are alternately used for immunization of animal or for infection of target cells to show that HCMV IE-exon 4 subunit is an inducer of CTL in mice. Using this model system, the relative immunogenicities of both a gB antigen expressed by different recombinant viruses and the IE exon 4 subunit antigen has been evaluated. A. Recombinant Viruses Used in CTL Assays The following recombinant viruses were used in the CTL assay of Example 3 below to demonstrate the vaccine utility of the recombinant adenoviruses of the present invention.

Wild-type human adenovirus type 5 (WT-Ad) and the non-defective adenovirus-gB recombinant (Ad-gB) (prepared as described in European Patent Publication No. 389,286 (Sept. 26, 1990) and G. S. Marshall et al, J\_ Infect. Pis.. 162:1177-1181 (1990)) were propagated in human lung carcinoma A549 cells [ATCC CCL185] , using standard procedures.

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 EP No. 389,286 and Marshall et al, cited above, and pAd-5 mu 0-75.9.

A vaccinia virus recombinant containing the gB subunits (VacC-gB) described previously in Gonczol et al. Vaccine. 9_.: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. 8.:130-136 (1990); J. Tartaglia et al, Crit. Rev. Immunol.. 10:13-30 (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. :190-193 (1991) [also obtained from Or. 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. 79:4927-4931 (1982)]. The Cp-gB recombinant and parental canarypox virus (WT-Cp) were propagated on primary chick embryo fibroblasts (CEF) cells [ATCC CRL 1590].

B. CTL Response of Ad-qB Recombinant 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 10⁸ 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 ) cells [also termed MC-57G, P.P. Aden et al, Immunoqenetics. 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%.

C. CTL Response of Ad-IE exon-4 recombinant The CTL-assay was carried out as described above for gB. In this CTL assay mice were immunized with Ad-IE-exon-4 recombinant virus and target cells were infected with Vac(WR strain)-IE recombinant virus or parental vaccinia virus. Briefly, mice were immunized i.p. with Ad-IE-exon-4 at 1-2 x 10⁸ plaque forming units (p.f.u). These spleen cell cultures were restimulated in vitro with Ad-IE-exon-4 or Vaccinia (Copenhagen strain)- IE-exon-4 (Vac-IE-exon-4)-infected autologous spleen cells for 5 days. Cytolytic activity of non-adherent spleen cells was tested in a chromium release assay. The vaccinia recombinants were provided by Or. Paoletti, Virogenetics Corporation, Troy, New York.

For the chromium release assay, MHC class-I matched and mismatched target cells were infected with Ad-IE-exon-4, or parental adenovirus, or with Vac-IE- exon-4 or parental vaccinia virus. Percentage specific ³¹chromium release was calculated as : [ (cpm experimental release-cpm spontaneous release)/(cpm maximal release-cpm spontaneous release)] x 100.

When tested in the CTL assay described above, the CBA mice immunized with the Ad-IE-exon-4 recombinant developed a HCMV-IE-exon-4 specific cytotoxic T cell response.

Example 3 - Protection Study using Ad-IE exon-4 HCMV-protein-specific protection was demonstrated in Ad-HCMV immunized mice from a vaccinia- HCMV recombinant-induced encephalitis/meningitis and death, as follows. The model is described above.

In this experiment, CBA mice were immunized i.p. with 2 x 10⁸ p.f.u. of the Ad-HCMV subunit protein recombinant virus, e.g. Ad-IE-exon 4 of Example 1, and 5- 18 days later were challenged intracerebrally (i.e.) with a lethal dose of a vaccinia(WR strain)-HCMV recombinant virus (e.g. Vac(WR)-gB). Vaccinia(WR strain)-IE or vaccinia(WR strain)-gB recombinant viruses were obtained from Or. Paoletti, Virogenetics Corporation, Troy, NY. The WR-strain of vaccinia is neurovirulent for mice.

When tested in this mouse model, CBA mice immunized with the Ad-IE-exon-4 recombinant were protected against a lethal dose of vaccinia WR-IE recombinant virus. The protection was HCMV-IE protein specific. Ninety percent of CBA mice, immunized i.p. with Ad-IE-exon-4 recombinant virus were protected against a lethal dose of Vac(WR-strain)-IE recombinant virus, inoculated intracerebrally. Control mice, immunized with Ad-gB recombinant virus or parental adenovirus and challenged later with the Vac(WR)-IE recombinant, died within 7 days after challenge, demonstrating that protection was IE-exon-4 protein specific. 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 IE-exon- 4 gene may be constructed according to the disclosure of the present invention. 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: The Wistar Institute of Anatomy and

Biology

(ii) TITLE OF INVENTION: Recombinant Cytomegalovirus

Vaccine

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONOENCE APPRESS:

(A) APPRESSEE: Howson and Howson

(B) STREET: Spring House Corporate Cntr,

PO Box 457

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

(E) COUNTRY: USA

(F) ZIP: 19477

(V) COMPUTER REAOABLE FORM:

(A) MEOIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-OOS/MS-OOS (0) SOFTWARE: Patentln Release #1.0,

Version #1.25

(vi) CURRENT APPLICATION PATA:

(A) APPLICATION NUMBER:

(B) FILING PATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION PATA:

(A) APPLICATION NUMBER: US 08/017,130

(B) FILING PATE: 12-FEB-1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Bak, Mary E.

(B) REGISTRATION NUMBER: 31,215

(C) REFERENCE/POCKET NUMBER: WST6BPCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-540-9200

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

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1221 base pairs

(B) TYPE: nucleic acid

(C) STRANOEPNESS: double (0) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cONA

(ix) FEATURE:

(A) NAME/KEY: CPS

(B) LOCATION: 1..1218

(xi) SEQUENCE OESCRIPTION: SEQ IP NO:l:

ATG AAA CAG ATT AAG GTT CGA GTG GAC ATG CTG CGG CAT 39 Met Lys Gin lie Lys Val Arg Val Asp Met Leu Arg His 1 5 10

AGA ATC AAG GAG CAC ATG CTG AAA AAA TAT ACC CAG ACG 78 Arg lie Lys Glu His Met Leu Lys Lys Tyr Thr Gin Thr 15 20 25

GAA GAG AAA TTC ACT GGC GCC TTT AAT ATG ATG GGA GGA 117 Glu Glu Lys Phe Thr Gly Ala Phe Asn Met Met Gly Gly 30 35

TGT TTG CAG AAT GCC TTA GAT ATC TTA GAT AAG GTT CAT 156 Cys Leu Gin Asn Ala Leu Asp lie Leu Asp Lys Val His 40 45 50

GAG CCT TTC GAG GAG ATG AAG TGT ATT GGG CTA ACT ATG 195 Glu Pro Phe Glu Glu Met Lys Cys lie Gly Leu Thr Met 55 60 65

CAG AGC ATG TAT GAG AAC TAC ATT GTA CCT GAG GAT AAG 234 Gin Ser Met Tyr Glu Asn Tyr lie Val Pro Glu Asp Lys

70 75

CGG GAG ATG TGG ATG GCT TGT ATT AAG GAG CTG CAT GAT 273 Arg Glu Met Trp Met Ala Cys lie Lys Glu Leu His Asp 80 85 90

GTG AGC AAG GGC GCC GCT AAC AAG TTG GGG GGT GCA CTG 312 Val Ser Lys Gly Ala Ala Asn Lys Leu Gly Gly Ala Leu 95 100

CAG GCT AAG GCC CGT GCT AAA AAG GAT GAA CTT AGG AGA 351 Gin Ala Lys Ala Arg Ala Lys Lys Asp Glu Leu Arg Arg 105 110 115 AAG ATG ATG TAT ATG TGC TAC AGG AAT ATA GAG TTC TTT 390 Lys Met Met Tyr Met Cys Tyr Arg Asn lie Glu Phe Phe 120 125 130

ACC AAG AAC TCA GCC TTC CCT AAG ACC ACC AAT GGC TGC 429 Thr Lys Asn Ser Ala Phe Pro Lys Thr Thr Asn Gly Cys

135 140

AGT CAG GCC ATG GCG GCA TTG CAG AAC TTG CCT CAG TGC 468 Ser Gin Ala Met Ala Ala Leu Gin Asn Leu Pro Gin Cys 145 150 155

TCC CCT GAT GAG ATT ATG GCT TAT GCC CAG AAA ATA TTT 507 Ser Pro Asp Glu lie Met Ala Tyr Ala Gin Lys lie Phe 160 165

AAG ATT TTG GAT GAG GAG AGA GAC AAG GTG CTC ACG CAC 546 Lys lie Leu Asp Glu Glu Arg Asp Lys Val Leu Thr His 170 175 180

ATT GAT CAC ATA TTT ATG GAT ATC CTC ACT ACA TGT GTG 585 lie Asp His lie Phe Met Asp lie Leu Thr Thr Cys Val 185 190 195

GAA ACA ATG TGT AAT GAG TAC AAG GTC ACT AGT GAC GCT 624 Glu Thr Met Cys Asn Glu Tyr Lys Val Thr Ser Asp Ala

200 205

TGT ATG ATG ACC ATG TAC GGG GGC ATC TCT CTC TTA AGT 663 Cys Met Met Thr Met Tyr Gly Gly lie Ser Leu Leu Ser 210 215 220

GAG TTC TGT CGG GTG CTG TCC TGC TAT GTC TTA GAG GAG 702 Glu Phe Cys Arg Val Leu Ser Cys Tyr Val Leu Glu Glu 225 230

ACT AGT GTG ATG CTG GCC AAG CGG CCT CTG ATA ACC AAG 741 Thr Ser Val Met Leu Ala Lys Arg Pro Leu lie Thr Lys 235 240 245

CCT GAG GTT ATC AGT GTA ATG AAG CGC CGC ATT GAG GAG 780 Pro Glu Val lie Ser Val Met Lys Arg Arg lie Glu Glu 250 255 260

ATC TGC ATG AAG GTC TTT GCC CAG TAC ATT CTG GGG GCC 819 lie Cys Met Lys Val Phe Ala Gin Tyr lie Leu Gly Ala

265 270

GAT CCT CTG AGA GTC TGC TCT CCT AGT GTG GAT GAC CTA 858 Asp Pro Leu Arg Val Cys Ser Pro Ser Val Asp Asp Leu 275 280 285 CGG GCC ATC GCC GAG GAG TCA GAT GAG GAA GAG GCT ATT 897 Arg Ala lie Ala Glu Glu Ser Asp Glu Glu Glu Ala lie 290 295

GTA GCC TAC ACT TTG GCC ACC CGT GGT GCC AGC TCC TCT 936 Val Ala Tyr Thr Leu Ala Thr Arg Gly Ala Ser Ser Ser 300 305 310

GAT TCT CTG GTG TCA CCC CCA GAG TCC CCT GTA CCC GCG 975 Asp Ser Leu Val Ser Pro Pro Glu Ser Pro Val Pro Ala 315 320 325

ACT ATC CCT CTG TCC TCA GTA ATT GTG GCT GAG AAC AGT 1014 Thr lie Pro Leu Ser Ser Val lie Val Ala Glu Asn Ser

330 335

GAT CAG GAA GAA AGT GAG CAG AGT GAT GAG GAA GAG GAG 1053 Asp Gin Glu Glu Ser Glu Gin Ser Asp Glu Glu Glu Glu 340 345 350

GAG GGT GCT CAG GAG GAG CGG GAG GAC ACT GTG TCT GTC 1092 Glu Gly Ala Gin Glu Glu Arg Glu Asp Thr Val Ser Val 355 360

AAG TCT GAG CCA GTG TCT GAG ATA GAG GAA GTT GCC CCA 1131 Lys Ser Glu Pro Val Ser Glu lie Glu Glu Val Ala Pro 365 370 375

GAG GAA GAG GAG GAT GGT GCT GAG GAA CCC ACC GCC TCT 1170 Glu Glu Glu Glu Asp Gly Ala Glu Glu Pro Thr Ala Ser 380 385 390

GGA GGC AAG AGC ACC CAC CCT ATG GTG ACT AGA AGC AAG 1209 Gly Gly Lys Ser Thr His Pro Met Val Thr Arg Ser Lys

395 400

GCT GAC CAG TAA 1221

Ala Asp Gin 405

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 406 amino acids

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

(ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Lys Gin lie Lys Val Arg Val Asp Met Leu Arg His Arg 1 5 10 lie Lys Glu His Met Leu Lys Lys Tyr Thr Gin Thr Glu Glu 15 20 25

Lys Phe Thr Gly Ala Phe Asn Met Met Gly Gly Cys Leu Gin 30 35 40

Asn Ala Leu Asp lie Leu Asp Lys Val His Glu Pro Phe Glu 45 50 55

Glu Met Lys Cys lie Gly Leu Thr Met Gin Ser Met Tyr Glu 60 65 70

Asn Tyr lie Val Pro Glu Asp Lys Arg Glu Met Trp Met Ala

75 80

Cys lie Lys Glu Leu His Asp Val Ser Lys Gly Ala Ala Asn 85 90 95

Lys Leu Gly Gly Ala Leu Gin Ala Lys Ala Arg Ala Lys Lys 100 105 110

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

Asn Gly Cys Ser Gin Ala Met Ala Ala Leu Gin Asn Leu Pro

145 150

Gin Cys Ser Pro Asp Glu lie Met Ala Tyr Ala Gin Lys lie 155 160 165

Phe Lys lie Leu Asp Glu Glu Arg Asp Lys Val Leu Thr His 170 175 180 lie Asp His lie Phe Met Asp lie Leu Thr Thr Cys Val Glu 185 190 195

Thr Met Cys Asn Glu Tyr Lys Val Thr Ser Asp Ala Cys Met 200 205 210

Met Thr Met Tyr Gly Gly lie Ser Leu Leu Ser Glu Phe Cys

215 220 Arg Val Leu Ser Cys Tyr Val Leu Glu Glu Thr Ser Val Met 225 230 235

Leu Ala Lys Arg Pro Leu He Thr Lys Pro Glu Val He Ser 240 245 250

Val Met Lys Arg Arg He Glu Glu He Cys Met Lys Val Phe 255 260 265

Ala Gin Tyr He Leu Gly Ala Asp Pro Leu Arg Val Cys Ser 270 275 280

Pro Ser Val Asp Asp Leu Arg Ala He Ala Glu Glu Ser Asp

285 290

Glu Glu Glu Ala He Val Ala Tyr Thr Leu Ala Thr Arg Gly 295 300 305

Ala Ser Ser Ser Asp Ser Leu Val Ser Pro Pro Glu Ser Pro 310 315 320

Val Pro Ala Thr He Pro Leu Ser Ser Val He Val Ala Glu 325 330 335

Asn Ser Asp Gin Glu Glu Ser Glu Gin Ser Asp Glu Glu Glu 340 345 350

Glu Glu Gly Ala Gin Glu Glu Arg Glu Asp Thr Val Ser Val

355 360

Lys Ser Glu Pro Val Ser Glu He Glu Glu Val Ala Pro Glu 365 370 375

Glu Glu Glu Asp Gly Ala Glu Glu Pro Thr Ala Ser Gly Gly 380 385 390

Lys Ser Thr His Pro Met Val Thr Arg Ser Lys Ala Asp Gin 395 400 405

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (O) TOPOLOGY: unknown

(ii) MOLECULE TYPE: CPNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TTATCCTCCT CTAGAATGAA ACAGATTAAG 30

(2) INFORMATION FOR SEQ IP NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANOEPNESS: single (0) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cONA

(xi) SEQUENCE DESCRIPTION: SEQ IP NO:4:

ATATATATAT TCTAGAGTTT ACTGGTCGAC 30

Claims

WHAT IS CLAIMEO IS:

1. A non-defective recombinant adenovirus comprising a cytomegalovirus gene encoding an immediate early antigen exon-4 subunit protein from a human strain of cytomegalovirus, said gene being under the control of an expression control sequence, said virus being a type 5 strain adenovirus capable of expressing said subunit protein in vivo in an animal.

2. The non-defective recombinant adenovirus according to claim 1, wherein the human cytomegalovirus strain is Towne.

3. An immunogenic composition comprising a non-defective recombinant adenovirus comprising a cytomegalovirus gene encoding an immediate early antigen exon-4 subunit protein from a human strain of cytomegalovirus, said gene being under the control of an expression control sequence, said virus being a type 5 strain adenovirus capable of expressing said subunit protein in vivo in an animal, in a suitable pharmaceutical carrier.

4. The composition according to claim 3, wherein the human cytomegalovirus is Towne strain.

5. A human cytomegalovirus immediate early exon-4 subunit protein produced by an adenovirus expression vector.

6. The use of a non-defective recombinant adenovirus comprising a cytomegalovirus gene encoding an immediate early antigen exon-4 subunit protein from a human strain of cytomegalovirus, said gene being under the control of an expression control sequence, said virus being a type 5 strain adenovirus capable of expressing said subunit protein in vivo in an animal, in admixture with a suitable pharmaceutical carrier in the manufacture of a vaccine composition against cytomegalovirus infection.

7. The use according to claim 6 wherein said vaccine is manufactured for oral administration.