Human cytomegalovirus DNA constructs and uses therefor

Abstract

Novel DNA molecules for in vitro and in vivo expression of HCMV gB, gB transmembrane-deleted derivatives, pp65, pp150, and IE-exon-4 proteins are described. Preferably, the molecules are plasmids. Also described are methods of using these DNA molecules to induce immune responses to HCMV, and the use of a plasmid of the invention to prime immune responses to HCMV vaccines.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 09/171,699, filed Jan. 19, 1999, which is a §371 of International Patent Application No. PCT/US97/06866, filed Apr. 22, 1997, which claims the benefit of the priority of U.S. Provisional Patent Application No. 60,015,717, filed Apr. 23, 1996.

BACKGROUND OF THE INVENTION

[0002] 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 termed gB or gCI, which was previously referred to as gA.

[0003] Infection with HCMV is common and usually asymptomatic. However, the incidence and spectrum of disease in newborns and immunocompromised hosts establishes this virus as an important human pathogen. HCMV has also been suggested to be an important co-factor in the development of atherosclerosis and restenosis after angioplastic surgery.

[0004] 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. Dis., 134:470-75 (1976); S. A. Plotkin et al, “Prevention of Cytomegalovirus Disease by Towne Strain Live Attenuated Vaccine”, in Birth Defects, Original Article Series, 20(1):271-287 (1984); J. P. Glazer et al, Ann. Intern. Med., 91:676-83 (1979); and U.S. Pat. No. 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 vaccines are considered possible causes of encephalitis. Other recombinant HCMV vaccines have been described. See, e.g., G. S. Marshall et al, J. Infect. Dis., 162:1177-1181 (1990); K. Berencsi et al, J. Gen. Virol., 74:2507-2512 (1993), which describe adenovirus-HCMV recombinants.

[0005] There remains a need in the art for additional compositions useful in preventing CMV infection by enhancing immune responses to HCMV vaccines and generating neutralizing antibody and/or cellular responses to CMV in the human immune system.

SUMMARY OF THE INVENTION

[0006] The present invention provides a series of DNA molecules expressing human cytomegalovirus (HCMV) genome fragments, which are particularly useful in inducing HCMV-specific immune responses.

[0007] Thus, in one aspect, the invention provides a DNA molecule which is non-replicating in mammals and which comprises at least one human cytomegalovirus antigen which is operably linked to regulatory sequences which express the antigen in the mammal. Advantageously, the antigen elicits an immune response in said mammal. In one preferred embodiment, the DNA molecule is a plasmid.

[0008] In another aspect, the invention provides a plasmid, pTet-gB, containing the portion of the HCMV genome (UL55) encoding gB. This plasmid further contains a tetracycline regulatable HCMV-immediate early promoter, which is useful in controlling expression of gB. Another plasmid of the invention encoding the full-length gB subunit protein is a p&Dgr;RC-gB plasmid.

[0009] Yet another plasmid of the invention, p&Dgr;RC-gB680, contains the portion of the HCMV genome encoding the N-terminal 680 amino acids of the gB protein (gB1-680).

[0010] The p&Dgr;RC-pp65 plasmid of the invention contains the portion of the HCMV genome (UL83) encoding the HCMV pp65 tegument protein. The p&Dgr;RC-pp150 plasmid contains the portion of the HCMV genome (UL32) encoding the HCMV pp150 tegument protein.

[0011] The p&Dgr;RC-exon-4 contains the portion of the HCMV genome (truncated UL123) encoding HCMV immediate-early (IE) exon-4.

[0012] In yet another aspect, the present invention provides an immunogenic composition of the invention comprising at least one of the DNA molecules of the invention and a carrier.

[0013] In still another aspect, the present invention provides a method of inducing HCMV-specific immune responses in an animal by administering to the animal an effective amount of an immunogenic composition of the invention. Preferably, this composition contains p&Dgr;RC-gB680, pTet-gB and/or p&Dgr;RC-pp65.

[0014] In yet a further aspect, the present invention provides a method of priming immune responses to a selected human cytomegalovirus immunogenic composition by administering an immunogenic composition of the invention prior to administration of the second immunogenic or vaccine composition.

[0015] Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 illustrates the construction of the pTet-gB plasmid.

[0017] FIG. 2 is a graph illustrating the results of pp65-specific CTL responses in BALB/c mice immunized with p&Dgr;RC-pp65. The circle represents VacWR-pp65-infected MC57 (MHC-mismatched) target cells; the diamond represents WT-Vac-infected P-815 cells; and the square represents VacWR-pp65-infected P-815 (MHC-mismatched) target cells.

[0018] FIGS. 3A-3E provides the full-length DNA and amino acid sequences [SEQ ID NO:1 and 2] of a human cytomegalovirus virus gB gene.

[0019] FIG. 4A-B provide the full-length DNA and amino acid sequences [SEQ ID NO:3 and 4] of a human cytomegalovirus immediate-early exon-4.

[0020] FIG. 5 provides the full-length DNA and amino acid sequences of a human cytomegalovirus phosphoprotein (pp) 65 gene Towne strain on the top line [SEQ ID NO: 5 and 6], and, on the bottom line, the sequence of the pp65-AD169 strain where it differs from the Towne strain [SEQ ID NO: 7 and 8].

[0021] FIGS. 6A-6I provide the full-length DNA and amino acid sequences [SEQ ID NO: 9 and 10] of a human cytomegalovirus phosphoprotein (pp) 150 gene, AD169 strain.

[0022] FIG. 7A provides a circular map of the eukaryotic expression vector pCB11.

[0023] FIG. 7B provides a circular map of pCBgB.

[0024] FIG. 7C provides a circular map of pCBgB&Dgr;tm.

[0025] FIG. 8 provides a schematic representation of the gB protein (top line) and of its homolog which is deleted of the transmembrane domain (bottom line).

[0026] FIG. 9 is a graph illustrating the anti-gB titers in sera of BALB/c mice immunized with plasmids pCBgB and pCBgB&Dgr;tm intramuscularly (IM) and intradermally (ID).

DETAILED DESCRIPTION OF THE INVENTION

[0027] This invention relates generally to compositions useful in preventing and treating human cytomegalovirus infection.

[0028] The present invention provides DNA molecules useful for in vitro and in vivo expression of antigenic fragments of the HCMV genome. Particularly desirable antigens include full-length and transmembrane-deleted fragments of gB such as gB1-680, pp65, pp150, and IE-exon-4. Preferably, the DNA molecules of the invention are plasmids. The inventors have found that these DNA molecules induce HCMV-specific immune responses, including ELISA and neutralizing antibodies and cytotoxic T lymphocytes (CTL), and are further useful in priming immune responses to subsequently administered HCMV immunogens and vaccines.

[0029] Thus, in one embodiment, the present invention provides a DNA molecule containing at least one HCMV antigen under the control of regulatory sequences which express the antigen in vivo or in vitro. Desirably, the DNA molecule is incapable of replicating in mammals. In a particularly desirable aspect of this embodiment, the DNA molecule is a plasmid.

[0030] As defined herein, an HCMV antigen includes a portion of the HCMV genome or a protein or peptide encoded thereby which induces an immune response in a mammal. Desirably, the immune response induced is HCMV-specific and protective. However, non-protective immune responses are also useful according to the invention, e.g., for priming immune responses. Currently, preferred HCMV antigens include full-length gB, a fragment or derivative of gB which lacks at least the transmembrane domain, pp65, pp150, and the immediate-early exon-4. Other suitable antigens may be readily selected by one of skill in the art.

[0031] The exemplary DNA molecules of invention, described herein, have been constructed using gene fragments derived from the Towne strain of HCMV. The Towne strain of HCMV, is particularly desirable because it is attenuated and has a broad antigenic spectrum. This strain is described in J. Virol., 11 (6): 991 (1973) and is available from the ATCC under accession number VR-977. The Ad169 strain is also available from the ATCC, under accession number VR-538. 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, or from commercial sources.

[0032] Thus, the CMV gene fragment encoding the desired protein (e.g., gB, pp65, pp150) or protein fragment (e.g., gB1-680 or IE-exon-4) may be isolated from known HCMV strains. See, e.g., Mach et al, J. Gen. Virol., 67:1461-1467 (1986); Cranage, M. P. et al, EMBO J., 5:3057-3063 (1986); and Spaete et al, Virol., 167:207-225 (1987), which provide isolation techniques. For example, using a known HCMV sequence, the desired HCMV gene or gene fragment [e.g., pp65 (UL83)] is PCR amplified, isolated, and inserted into the plasmid vector or other DNA molecule of the invention using known techniques. Alternatively, the desired CMV sequences can be chemically synthesized by conventional methods known to one of skill in the art, purchased from commercial sources, or derived from CMV strains isolated using known techniques.

[0033] If desired, the DNA molecules of the invention may contain multiple copies of the HCMV gene or gene fragment. Alternatively, the recombinant plasmid may contain more than one HCMV gene/gene fragment, so that the plasmid may express two or more HCMV proteins. For example, as shown herein, the presence of both gB- and pp65-specific ELISA antibodies and pp65-specific CTL in the mice inoculated with pTet-gB and p&Dgr;RC-pp65 in a mixture indicates that gB and pp65 do not mutually block antigen presentation or B and T cell stimulation when expressed in the same cells or in close proximity. Thus, gB (or gB680) and pp65 proteins are particularly well suited for incorporation into a plasmid which expressed both protein (termed herein a chimeric vector). Thus, one particularly desirable embodiment of the present invention provides a DNA molecule containing the gB and the pp65 antigens. In another particularly desirable embodiment, the DNA molecule contains a transmembrane-deleted gB fragment or derivative (e.g., gB680 or gB&Dgr;tm) and the pp65 antigens.

[0034] In the construction of the DNA molecules of the invention, one of skill in the art can readily select appropriate regulatory sequences, enhancers, suitable promoters, secretory signal sequences and the like. In the examples below, the plasmids have been provided with a tetracycline repressor from E. coli. However, if desired, the plasmid or other DNA molecule may be engineered to contain another regulatable promoter, which “turns on” expression upon administration of an appropriate agent (e.g., tetracycline), permitting regulation of in vivo expression of the HCMV gene product. Such agents are well known to those of skill in the art. The techniques employed to insert the HCMV gene into the DNA molecule and make other alterations, e.g., to insert linker sequences and the like, are known to one of skill in the art. See, e.g., Sambrook et al, “Molecular Cloning. A Laboratory Manual” (2d edition), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).

[0035] In one embodiment, the DNA molecules of the invention are plasmids. One exemplary plasmid is pTet-gB. Construction of this plasmid is described in more detail below. Plasmid TetotTA-gB contains the gene from HCMV (the unique long (UL) 55) encoding the full-length gB subunit protein and a tetracycline regulatable HCMV-immediate early promoter which controls expression of gB. For convenience, the sequences of the HCMV gene fragment encoding the full-length gB protein which were used in the examples below are provided in FIGS. 3A-3E [SEQ ID NO: 1 and 2]. As discussed herein, this invention is not limited to this strain of HCMV. pTet-gB has been found to be useful alone, and in conjunction with the other DNA molecules of the invention, and particularly the p&Dgr;RC-pp65 plasmid described below. pTet-gB is also particularly useful for priming immune responses to subsequently administered HCMV immunogenic compositions and vaccines.

[0036] The pTetotTA-gB plasmid has been deposited pursuant to the Budapest Treaty, in the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va., U.S.A. This deposit, designated ATCC 98029, was made on Apr. 23, 1996 and is termed herein, pTet-gB.

[0037] Other plasmids provided herein, p&Dgr;RC-gB and pCBgB, also contain the HCMV gene encoding the gB protein. As demonstrated below, these DNA plasmids have been found to be highly potent immunogens for HCMV. See Examples 8 and 14.

[0038] Another plasmid of the invention, p&Dgr;RC-gB680 contains the portion of the HCMV gene encoding the N-terminal 680 amino acids of the gB protein and is capable of expressing this fragment in vivo or in vitro. This gB fragment is designated herein gB1-680. As illustrated in FIG. 3A-E [SEQ ID NO:2], the full-length gB subunit protein consists of 907 amino acids. This plasmid, which expresses a secreted form of gB, has been found to be a more potent immunogen than the plasmids expressing the full-length gB.

[0039] Also provided herein is plasmid pCDgB&Dgr;tm, which contains a deletion of the gB transmembrane region. This plasmid has been found to induce HCMV-specific neutralizing antibodies (see Example 14) and to be a more potent immunogen than the corresponding DNA plasmid encoding full-length gB.

[0040] Plasmid p&Dgr;RC-exon-4 plasmid contains the portion of the HCMV immediate-early (IE) gene encoding HCMV IE-exon-4 and is capable of expressing the gene product. The HCMV IE-exon-4 gene fragment has been described in international patent application PCT/US94/02107, published Aug. 18, 1994, which is incorporated by reference herein. The IE gene and the intron/exon junctions for Towne strain HCMV are provided in Stenberg et al, J. Virol., 49:190-199 (1984), and are available from GenBank under accession number K01484, M11828-30. The sequences of the IE-exon-4 gene fragment, Towne strain, are provided in FIG. 4A-B [SEQ ID NO: 3 and 4], for convenience. This invention is not limited to the use of the IE-exon-4 sequences from this viral strain.

[0041] Plasmid p&Dgr;RC-pp65 contains the HCMV gene encoding the HCMV phosphoprotein (pp) 65 tegument protein and is capable of expressing pp65 in vivo or in vitro. As described herein, immunization with p&Dgr;RC-pp65 induced a reduction of virus titers in the mouse lung after intranasal challenge with vaccinia recombinants carrying the pp65 gene, suggesting the protective function of cell-mediated immunity in lung after DNA immunization. Further, in contrast to a prior art pp65-containing plasmid construct which induced ELISA antibodies in only about 60% of inoculation mice, nearly 100% of mice inoculated with p&Dgr;RC-pp65 responded with pp65-specific ELISA antibodies. The sequences of the pp65 gene, Towne and AD169 strains, have been described in H. Pande et al, Virol., 181(1):220-228 (1991) and are provided in FIG. 5 [SEQ ID NO: 5-8] for convenience. pp65 sequences may be readily isolated using known techniques from other HCMV strains, or obtained from commercial sources. The strain from which the pp65 sequences are derived is not a limitation on the present invention.

[0042] Plasmid p&Dgr;RC-pp150 contains the portion of the HCMV gene encoding the HCMV pp150 tegument protein and is capable of expressing pp150 in vivo or in vitro. The sequences of the pp150 gene, Ad169 strain, have been described in G. Jahn et al, J. Virol., 61(5):1358-1367 (1987) and are provided in FIGS. 6A-6I for convenience [SEQ ID NO: 9 and 10]. pp150 sequences may be readily isolated using known techniques from another HCMV strain, or obtained from commercial sources. The strain from which the pp150 sequences are derived is not a limitation on the present invention.

[0043] The DNA molecules, and particularly the plasmids described herein, may be used for expression of the gB, gB1-680 fragment, pp65, pp150, or IE-exon-4 in vitro. The molecules are introduced by conventional means into the desired host cell [see, Sambrook et al, cited above]. Suitable host cells include, without limitation, bacterial cells, mammalian cells and cell lines, e.g., A549 (human lung carcinoma) or 293 (transformed human embryonic kidney) cells.

[0044] The host cell, once transfected with the recombinant plasmid (or other DNA molecule) 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 expressed HCMV protein, e.g., gB, 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.

[0045] Alternatively, transfected host cells are themselves used as antigens, e.g., in in vitro immunological assays, such as enzyme-linked immunosorbent assays (ELISA). Such assay techniques are well known to those of skill in the art.

[0046] In yet another embodiment, one or more of the DNA molecules (e.g., plasmids) described herein may be used directly as immunogens in an immunogenic composition or directly for priming the immune response to a subsequently administered immunogenic or vaccine composition. According to this embodiment of the invention, the DNA molecule (e.g., plasmid) containing the HCMV gene or gene fragment is introduced directly (i.e., as “naked DNA”) into the animal by injection. The DNA molecule of the invention, when introduced into an animal, transfects the host's cells and produces the CMV protein in those cells. Methods of administering so-called ‘naked DNA’, are known to those of skill in the art. [See. e.g., J. Cohen, Science, 259:1691-1692 (Mar. 19, 19930; E. Fynan et al, Proc. Natl. Acad. Sci., 90:11478-11482 (December 1993); J. A. Wolff et al, Biotechniques, 11:474-485 (1991); International Patent Application No. PCT WO94/01139, which are incorporated by reference herein for purposes of described various ‘naked DNA’ delivery methods.]

[0047] The preparation of a pharmaceutically acceptable immunogenic composition, having appropriate pH, isotonicity, stability and other conventional characteristics is within the skill of the art. Currently, in a preferred embodiment, one or more of the recombinant plasmids (or other DNA molecules) of the invention is suspended in an acceptable carrier such as isotonic water, phosphate buffered saline, or the like. Optionally, although currently less preferred, such a composition may contain other components, such as adjuvants, e.g., aqueous suspensions magnesium hydroxides.

[0048] An effective amount of an immunogenic composition of the invention preferably contains between 10 &mgr;g and 10 mg, and preferably between about 80 &mgr;g and 150 &mgr;g of DNA of the invention per inoculation. Desirably, for each inoculation, the DNA of the invention is formulated in about 100 &mgr;l of a suitable carrier. In a particularly preferred embodiment, each patient is administered 100 &mgr;g DNA, which is administered three times at about 4 week intervals. Alternatively, the dosage regimen involved in the method for immunizing with the recombinant DNA molecule (e.g., plasmid) of the present invention can be determined considering various clinical and environmental factors known to affect vaccine administration. For example, following a first administration of an immunogenic composition of the invention, boosters may be administered approximately 2- to 15-weeks later. These boosters may involve an administration of the same immunogenic composition as was first administered, or may involve administration of an effective amount of another immunogenic composition of the invention. Additional doses of the vaccines of this invention may also be administered where considered desirable by the physician.

[0049] In another aspect, the present invention provides a method of inducing HCMV-specific immune responses in an animal. The method involves administering to an animal an effective amount of an immunogenic composition containing one or more of the DNA molecules of the invention, as described above. The immunogenic composition is administered by any suitable route, including oral, nasal routes, subcutaneous and intraperitoneal. However, currently preferred are the intramuscular and intradermal routes of administration.

[0050] In a particularly preferred embodiment of this aspect, the method of inducing an HCMV-specific immune response of the invention involves the administration of one or more immunogenic compositions of the invention. These compositions may be formulated so as to contain a single DNA molecule of the invention, or may contain mixtures of the DNA molecules of the invention. In one desirable embodiment, the composition contains p&Dgr;Rc-gB680 or pCBgB&Dgr;tm. In another desirable embodiment, the composition contains a plasmid containing pp65 according to the invention. As illustrated in the examples below, administration of p&Dgr;RC-pp65 has been found to induce a potent HCMV-specific immune response. In another desirable embodiment of the invention, the combined administration of pTet-gB and p&Dgr;RC-pp65 invention (which may be formulated in a single composition, or preferably, administered separately) induces potent HCMV-specific ELISA and neutralizing antibodies to both proteins. In yet another desirable embodiment, the present invention provides a composition containing a chimeric plasmid which expresses pp65 and gB680 or gB. Yet another desired embodiment involves combined administration of p&Dgr;RC-gB680 and p&Dgr;RC-pp65.

[0051] In another aspect of this invention, a method of priming immune responses to a human cytomegalovirus immunogenic or vaccinal composition is provided. This method involves administering an immunogenic composition of the invention prior to administration of a second immunogenic or vaccinal composition. Desirably, an effective amount of an immunogenic composition of the invention, e.g., containing pTet-gB, is administered between about 4 and 15 weeks prior to administration of the immunogenic or vaccinal composition. The second immunogenic or vaccinal composition, for which the immune response is enhanced or primed by the method of the invention, may be an immunogenic composition of the invention or a conventional immunogenic or vaccine composition. For example, such a composition may contain one or more HCMV proteins (e.g., the isolated, purified gB protein described in the examples below), a whole virus (e.g., semipurified Towne strain HCMV virion), or recombinant HCMV viruses. Suitable recombinant viruses are well known to those of skill in the art and include, e.g., the Ad-gB virus [G. Marshall et al, (1990), cited above, and EP 389 286; the Ad-gB-IE-exon-4 virus [WO 94/17810]; the Ad-gB fragment viruses [WO 94/23744]. Other suitable HCMV vaccinal compositions are well known to those of skill in the art.

[0052] These examples illustrate the preferred methods for preparing and using the plasmids of the invention. These examples are illustrative only and do not limit the scope of the invention.

EXAMPLE 1

Construction of pTet-gB Plasmid

[0053] The full-length HCMV-gB gene was obtained from the plasmid pAd-gB [Marshall et al., J. Infect. Dis., 162:1177-1181 (1990)] by XbaI-XbaI-digestion.

[0054] The full length HCMV-gB was inserted into the plasmid pUHD10-3 [Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 12:5547-5551 (1992)]. This plasmid contains:

[0055] (a) a tetracycline regulatable promoter (HCMV minimal promoter, −53 relative to the start site, with heptamerized tet-operon derived from the regulatory region of tetR-gene of transposon −10);

[0056] (b) a multiple cloning site (including an XbaI site); and

[0057] (c) an SV40 polyadenylation signal downstream of the polycloning site.

[0058] After inserting the HCMV-gB (referred to as pTetO-gB), the plasmid was digested with Hind III followed by blunt-ending, then digested with PvuI and the fragment containing the tetracycline regulatable promoter-HCMV-gB-SV40 polyA signal sequences was isolated and inserted into the plasmid pUHD15-1 [Gossen and Bujard, cited above]. This latter plasmid (hereafter referred to as ptTA) contains the HCMV-IE promoter-enhancer which constitutively drives the tTAgene followed by the SV40 polyA signal. The tTA-gene codes for a fusion protein consisting of the tetracycline repressor from E. coli and the carboxy-terminal 130 amino acids of the herpes simplex virus protein 16 gene (HSV VP-16). This fusion protein is a powerful transactivator of the tetracycline regulatable promoter of pTeto (which drives the HCMV-gB gene), because of the specific and high affinity attachment of the tetracycline repressor to the tetracycline operator sequences ensures the activation of transcription from the minimal HCMV promoter by the transactivator domain of HSV VP-16 gene (fused to the tetracycline repressor). The gene activation is specific for the pteto promoter. In the presence of low, non-toxic concentration of tetracycline (1 &mgr;g/ml or less), however, the transactivation is switched off, since tetracycline prevents the attachment of the tetracycline repressor to the teto sequences and no or very low gene expression is allowed (i.e., only the minimal HCMV promoter basal activity which is negligible in almost all cell types investigated so far).

[0059] To obtain the gB-expression plasmid regulatable by tetracycline, ptTA was cut just upstream of the HCMV-IE promoter/enhancer by XhoI, blunt-ended and cut with PvuI. The large fragment containing the HCMV-IE promoter-enhancer-tTA fusion protein gene followed by the SV40 polyA signal and the E. coli sequences of the plasmid (i.e., the replication origin and the beta-lactamase genes) were isolated. This isolated fragment was ligated with the fragment of pUHD10-3 containing the gB gene by the competent blunt-end and PvuI ends, resulting in the plasmid pteto-gB-tTA. The resulting plasmid contains both the transactivator and the HCMV-gB gene. The structure of the plasmid is, in addition to the E.coli-part, tetracycline-regulatable promoter (7 teto+minimal HCMV promoter) followed by the HCMV-gB gene, followed by the SV40 polyA signal, followed by the HCMV-IE promoter-enhancer, followed by the tTA gene and ending with the SV40 polyA signal.

[0060] The tetracycline-controllable expression system has been found to work correctly in vivo in the mouse as well [J. Dhawan et al, Somatic Cell and Molecular Genetics, 21:233-240 (1995)]. The pTet-gB plasmid is suitable to control naked DNA immunization. It is possible to give tetracycline to mice in their drinking water in concentrations not toxic for the animals but reaching sufficient levels able to regulate expression in muscle tissues [J. Dhawan et al., Somatic Cell and Molecular Genetics, 21: 233-240 (1995)]. By tetracycline treatment of transfected cultures or inoculated mice the time of antigen exposure can be manipulated. The silent presence of the inoculated plasmid can be tested. Without tetracycline treatment, however, this plasmid simply serves as a plasmid DNA immunogen or vaccine.

EXAMPLE 2

Construction of Further Plasmids

[0061] A. Construction of pRC-gB

[0062] pRC/CMV (Invitrogen Corporation) contains the HCMV-IE promoter. The full length gB gene (XbaI-XbaI fragment from pAd5-gB) was obtained using conventional techniques [SEQ ID NO:1] and inserted into pRC/CMV according to manufacturer's directions. The resulting plasmid is termed herein pRC-gB.

[0063] B. Construction of p&Dgr;RC-gB

[0064] p&Dgr;RC/CMV was derived from pRC/CMV plasmid by deleting the PvuII 1290-PvuII 3557 fragment to obtain more unique restriction sites. The full gB [SEQ ID NO:1], derived from the plasmid pAd-gB [Marshall et al., J. Infect. Dis., 162:1177-1181 (1990)], was subcloned using conventional techniques, inserted into pUC-8 (commercially available), then obtained as a HindIII-BamHI fragment and inserted into the HindIII-BamHI digested p&Dgr;RC/CMV vector. The resulting plasmid is termed p&Dgr;RC-gB.

[0065] C. Construction of p&Dgr;RC-gB680

[0066] p&Dgr;RC-gB680 expresses the N-terminal 680 amino acids of the gB protein [SEQ ID NO:2]. The plasmid was derived from p&Dgr;RC-gB, by deleting the C-terminal 227 amino acids of the gB by Xho-digestion, Klenow polymerase filling, removing the C-terminal portion of the gB gene, and religation of the 5400 bp fragment. The insert is approximately 2200 bp.

EXAMPLE 3

Construction of p&Dgr;RC-pp65 AND p&Dgr;RC-pp150

[0067] A. p&Dgr;RC-pp65

[0068] The plasmid p&Dgr;RC-pp65, which expresses the pp65 tegument protein of HCMV, was constructed as follows. H. Pande et al, Virology, 182(1):220-228 (1991), which provides the nucleotide sequences of the pp65 gene, is incorporated by reference herein [SEQ ID NO: 5 and 6].

[0069] The pp65 gene was isolated from the HCMV genome using conventional polymerase chain reaction techniques and inserted into a suitable expression plasmid. In this experiment, the 1696-bp pp65 gene was excised from the pUC-8-pp65 expression plasmid [Virogenetics] by NruI-BamHI digestion. The vector was blunt-ended with Klenow polymerase, digested with BamHI, and the pp65 gene inserted.

[0070] B. p&Dgr;RC-pp150

[0071] The plasmid, p&Dgr;RC-pp150, which expresses the pp150 tegument protein of HCMV, was constructed as follows. The pp150 gene was isolated from the HCMV genome using conventional polymerase chain reaction techniques and inserted into a suitable expression plasmid. One of skill in the art can readily isolate this gene from a desired HCMV strain making use of the published sequences in G. Jahn et al, J. Virol., 61(5):1358-1367 (1987) (which provides the nucleotide sequences of the Ad169 HCMV pp150 gene and is incorporated by reference herein). See, also FIG. 6A-6I herein [SEQ ID NO: 9 and 10].

[0072] In this experiment, the isolated HCMV-pp150 gene was inserted into the XbaI-restricted p&Dgr;RCd [Virogenetics]. The insert is approximately 3200 bp [SEQ ID NO: 10].

EXAMPLE 4

Construction of p&Dgr;RC-IE-Exon-4

[0073] The plasmid, p&Dgr;RC-IE-Exon-4, which expresses the HCMV-IE exon4 product [SEQ ID NO:4], was constructed as follow. The gene was obtained from pAd5-IE-Exon-4 [International Patent Application WO94/17810, published Aug. 18, 1994 and Berencsi et al., Vaccine, 14:369-374 (1996)], by XbaI-digestion [SEQ ID NO:3]. The insert is 1230 bp.

EXAMPLE 5

Production of Plasmid Preparation Stocks

[0074] E. coli DH5alfa competent cells (Gibco BRL, Gaithersburg, Md.) were transformed with the constructed plasmids. Purified plasmid preparations were prepared on Plasmid Giga Kits (Qiagen Inc. Chatsworth, Calif.).

EXAMPLE 6

Expression of HCMV-Proteins After Transient Transfection of 293 Cells with the Purified Plasmid Preparations

[0075] Transient transfections were performed by the purified plasmid preparations, 1.5 &mgr;g/3×105 cells, using lipofectamine (Gaithersburg, Md.). Cells were tested for HCMV-protein expression 2 days after transfection by an immunofluorescence test as described in E. Gonczol et al, Science, 224:159-161 (1984). The antibodies used in this test include the monoclonal pp65-specific Ab [VIROSTAT, Portland, Me., stock #0831], monoclonal gB-specific Ab [Advanced Biotechnologies, Columbia, Md.], and anti-pp150 monoclonal Ab [Virogenetics Corporation]. The IE-Exon-4-specific monoclonal Ab P63-27 was provided by W. Britt, University of Alabama at Birmingham.

[0076] The pTet-gB plasmid expresses the full-length HCMV-gB gene under the control of a tetracycline regulatable HCMV-IE promoter. The other plasmids express the inserted gene in transfected 293 cells under the control of the HCMV-IE promoter. Expression of gB, pp65 and pp150 was found to be strong using all plasmids.

[0077] After transfection with pTet-gB, 10-12% and <1% of cells expressed gB protein in the absence and presence, respectively, of 1 &mgr;g tetracycline [Tetracycline hydrochloride, Sigma, St. Louis, Mo.]. Sixty to seventy percent and 40-50% of cells transfected with p&Dgr;RC-gB and p&Dgr;gB680 plasmids, respectively, expressed gB. pp65 protein was expressed in 70-80% of cells transfected with p&Dgr;RC-pp65.

EXAMPLE 7

Immunization Procedures and Assay Methods

[0078] A. Immunization Procedure

[0079] BALB/c or CBA mice were first pretreated i.m. with 100 &mgr;l of Bupivicaine HCl [0.25% Sensorcaine-MPF (ASTRA Pharmaceutical Products, Inc. Westborough, Mass.)]. In some experiments, identified below, no Bupivicaine pretreatment was used. One day later DNA was inoculated i.m. on the site of Bupivacaine infiltration. The dose for mice was 50-80 &mgr;g plasmid DNA/inoculation. Booster inoculations were given i.m. 2×, without pretreatment with Bupivacaine. Mice immunized with p&Dgr;RC-gB plasmid were boosted 1×. Mice were bled by retroorbital puncture at the indicated times.

[0080] B. ELISA

[0081] Semipurified HCMV virions and purified gB proteins may be prepared by immunoaffinity column chromatography as described in E. Gonczol et al, J. Virol., 58:661-664 (1986). Alternatively, one of skill in the art can readily obtain suitable virions and gB proteins by alternative techniques.

[0082] Semipurified HCMV virions (Towne strain) or purified gB protein preparation were used as coating antigen for detection of gB-specific antibodies. OD values higher than mean OD values±2SD of preimmune sera were considered positive, or OD values >0.05, whichever was higher. Lysates of 293 cells transiently transfected with p&Dgr;RC-pp65 were used as coating antigen for detection of pp65-specific antibodies, lysates prepared from untransfected 293 cells served as control antigen. OD values obtained on control antigen-coated wells were subtracted from OD values obtained on pp65 antigen-coated wells and were considered positive if the resulting value was higher than 0.05.

[0083] C. Microneutralization Assay

[0084] This assay was performed as described in E. Gonczol et al., J. Virol. Methods, 14:37-41 (1986). A neutralizing titer higher than 1:8 was considered positive.

[0085] D. Cytotoxic T Lymphocyte Assay

[0086] This assay was performed as described in K. Berencsi et al., J. Gen Virol., 74:2507-2512 (1993). Briefly, spleen cells of immunized mice were restimulated in vitro with VacWR-pp65-infected (m.o.i.=0.2-0.5) autologous spleen cells (effector:stimulator ratio, 2.:1) for 5 days in 24-well plates. Cytolytic activity of nonadherent spleen cells was tested in a 4-h 51Cr-release assay. Target cells (P815 MHC class I-matched, MC57 MHC class I-mismatched) were infected with VacWR-pp65 or VT-Vac WR (m.o.i.=4-8). Percentage of specific 51Cr-release was calculated as [(cpm experimental release−cpm spontaneous release)/(cpm maximal release−cpm spontaneous release)×100]. A pp65-specific cytotoxicity higher than 10% was considered positive.

EXAMPLE 8

Induction of HCMV-Specific Immune Responses by the Plasmid Constructs Expressing the gB Protein

[0087] BALB/c mice were inoculated 2 times at 0 and 5 weeks with 80 &mgr;g p&Dgr;RC-gB preparation. Serum samples at 5, 9 and 19 weeks after the first inoculation were tested for HCMV-specific ELISA antibodies and neutralizing antibodies (NA). The results are provided in Table 1 below, in which the ELISA antigen used was semipurified virions. The OD of responders is provided as the mean±SD at a serum dilution of 1:80. Mean±2SD of the 6 preimmunization sera at a dilution of 1:80 gave an OD value of 0.080. “GM” indicates the geometric mean. 1 TABLE 1 p&Dgr;RC-gB induces HCMV-specific ELISA and neutralizing antibodies (antigen: semipurified virion). weeks after No. of ELISA GM of first responders/ OD of resp. No. of NA NA inoculation total dil 1:80 resp. resp. 0 0/6 0.036 ± 0.022 0/6 NA 5 5/6 0.314 ± 0.188 2/2 19 9 6/6 1.387 ± 0.810 6/6 34 19 ND ND 4/4 22

[0088] These data demonstrate that all mice responded with both ELISA antibody and NA after the booster inoculation. The p&Dgr;RC-gB plasmid seems to be a highly potent immunizing construct. 2 TABLE 2 pTet-gB and p&Dgr;RC-pp65 induces insert-specific ELISA antibodies Weeks after # ELISA Mice Immunized first responders/ OD* With: Inoc. total responders pTet-gB 4  1/10 0.062 8  9/10 0.277 ± 0.257 13  7/7 0.530 ± 0.625 21  6/6 0.503 ± 0.682 31  5/6 0.451 ± 0.505 p&Dgr;RC-pp65 4  5/10 0.168 ± 0.070 8 10/10 0.568 ± 0.387 13  4/4 1.076 ± 0.216 *Mean OD ± SD of serum samples at dilution 1:40.

[0089] HCMV-specific ELISA antibodies were detected in 9 of 10 mice at 8 weeks after the first inoculation with pTet-gB (Table 2). HCMV neutralizing antibodies were detected in 4 of 10 mice, with titers between 1:16 and 1:48 (not shown). All mice immunized with the p&Dgr;RC-pp65 responded with pp65-specific ELISA antibodies. At 13 weeks (pp65- and gB-specific) and up to 31 weeks (gB-specific), OD values remained positive. In a separate experiment pp65-specific ELISA antibodies were also detected during the whole observation period (31 weeks) in 10 of the 10 immunized mice.

EXAMPLE 9

Induction of HCMV-Specific Immune Responses by the Plasmid Constructs Expressing pp65

[0090] To test whether the combination of the pTet-gB and p&Dgr;RC-pp65 results in reduced responses to the individual components, mice were immunized with both plasmids mixed together or inoculated separately. Groups of mice were inoculated with Bupivacaine (100 &mgr;l/mouse, 50 &mgr;l/leg), and 2 days later, with either a mixture of both plasmids (80 &mgr;g of each DNA/mouse, 40 &mgr;g of each DNA/leg, 160 &mgr;g DNA/mouse) or each plasmid inoculated into two different legs (80 &mgr;g DNA of each plasmid/mouse, a total of 160 &mgr;g DNA/mouse inoculated in left and right legs). A similar booster was given 4 weeks later. The time course of both the gB- and pp65-specific ELISA antibody response was very similar in both groups, with nearly all mice developing antibodies by 8 or 13 weeks after the first inoculation (Table 3). In another experiment using the combination of the two plasmids, comparable OD values were observed up to 31 weeks after the first inoculation. 3 TABLE 3 pTet-gB and p&Dgr;RC-pp65 inoculated into the same animal induce gB and pp65-specific antibodies Weeks # gB- # pp65- Antigen, after ELISA ELISA Inocu- 1st resp./ OD* of resp./ OD of lation Inoc. Total responders Total Responders pTet-gB + 34 4/10 0.087 ± 0.024  5/10 0.078 ± 0.033 p&Dgr;RC- pp65, mixed 8 10/10 0.220 ± 0.143 10/10 0.400 ± 0.321 13 10/10 0.392 ± 0.152  9/10 0.303 ± 0.224 pTet-gB + 4  8/10 0.076 ± 0.021  6/10 0.210 ± 0.124 p&Dgr;RC- pp65, separately 8  9/10 0.202 ± 0.268  8/10 0.452 ± 0.333 13 10/10 0.309 ± 0.202  8/10 0.308 ± 0.212 *The mean OD ± SD of serum samples at dilution 1:40.

[0091] Of six mice inoculated with p&Dgr;RC-pp65 alone at a single site, 3 mice responded with pp65-specific lysis of target cells (FIG. 2). In a second similar experiment, 3 of 9 mice immunized with p&Dgr;RC-pp65 alone showed strong pp65-specific CTL responses. pp65-specific CTL were also detected in 4 of 5 tested mice inoculated with the mixture of p&Dgr;RC-pp65 and pTet-gB. When the p&Dgr;RC-pp65 and pTet-gB were inoculated separately into two different legs, 4 of 6 mice tested developed pp65-specific CTL response. These results establish that: 1) pp65-specific CTL responses are induced after DNA immunization; 2) there is no antigenic competition between the gB and pp65 proteins in the induction of antibody and CTL responses; and 3) gB protein expression in the cells at the inoculation site does not interfere with the presentation of pp65-specific T cell epitopes by MHC class I molecules to T cells.

EXAMPLE 10

Priming Effect of pTet-gB

[0092] One inoculation of naked plasmid DNA in mice did not result significant antibody responses in a high percentage of mice. To find out whether the immune system of the nonresponder mice was specifically primed by the DNA inoculation, mice inoculated with pTet-gB were boosted 4 weeks later with either purified gB protein (5 &mgr;g gB/mouse in Alum s.c.) or with the Towne strain of HCMV (20 &mgr;g/mouse in Alum s.c.). 4 TABLE 4 Inoculation of mice with pTet-gB primes the immune system wks after No. of NA GM of NA/ Antigen priming responder/all responder Teto−gB/* 4 0/10  5 Teto−gB 8 4/10 21 Teto−gB/* 4 0/10  4 gB+Alu 8 8/10 77 -/* 4 0/10 NA gB+Alu 8 1/10 16 Teto−gB/** 12 1/5 16 Towne+Alu 14 5/5 97 -/** 12 0/5 NA Towne+Alu 14 3/5 25 *second inoculations were given 4 weeks after the first inoculation **Towne was given 12 weeks after the first inoculation

[0093] This data demonstrates that pTet-gB inoculation primes immune-responses. In other words, the combination of Teto-gB priming and gB+Alu or Towne+Alu booster gave higher number of responder mice and slightly higher NA titers than TetotTA-gB given 2 times.

EXAMPLE 11

DNA Immunization Decreases Replication of the Corresponding Vaccinia Recombinant in Mice

[0094] Vaccinia virus recombinants expressing either HCMV-gB or pp65 were prepared using the methods described in WO 94/17810, published Aug. 18, 1994. Briefly, the VacWR-gB and VacWR-pp65 recombinants were constructed as described [Gonczol et al, Vaccine, 9:631-637 (1991)], using the L variant of the neurovirulent WR strain of vaccinia virus as vector [Panicali et al, J. Virol., 37(3):1000-1010 (1981)] and the gB or pp65 genes (HCMV Towne strain) as inserts cloned into the nonessential BamHI site in the HindIII F region [Panicali and Paoletti, Proc. Natl. Acad. Sci., 79:4927-4931 (1982)] under the control of the vaccinia H6 early/late promoter. Vaccinia recombinant viruses and the parental wild-type WR strain were grown on Vero cells and purified as described [Gonczol et al, cited above].

[0095] After plasmid immunization, vaccinia virus recombinants expressing either HCMV-gB or pp65 were used for challenge in the model described in WO 94/23744, published Oct. 27, 1994. Vaccinia virus WR strain replicates in mouse lung after intranasal inoculation and immune protection can be evaluated by virus titrations of the lung. Eight-week old female CBA and BALB/c mice were first pretreated with Bupivacaine, then 1 day later immunized either with p&Dgr;RC-gB or p&Dgr;RC-pp65 (80 &mgr;g/mouse). Mice were boosted 8 days later with DNA. Eight days after the second DNA dose mice were i.n. challenged either with 5×106 pfu of Vaccinia WR-gB or Vaccinia WR-pp65. Lungs were taken at the time of virus challenge (day 0) and at days 1, 3, 4, 5, and 7 after challenge for virus titration. Lungs were homogenized, freeze-thaw 3 times and virus titer determined on Vero cells by plaque titration. 5 TABLE 5 Virus titers in the lungs of BALB/c mice immunized with p&Dgr;RC-gB or p&Dgr;RC-pp65 and challenged i.n. with Vac-gB days Vac-gB titer (log + SD) in lungs* after p&Dgr;RC-gB- p&Dgr;RC-pp65- Diff. in titer (log) challenge immunized immunized 0 3.29 ± 2.83 3.29 ± 2.83 0 1 2.24 ± 2.9 2.76 ± 2.51 −0.25 3 4.86 ± 4.61 5.60 ± 5.45 0.53 4 4.54 ± 4.47 5.24 ± 4.9 1.13 5 4.33 ± 3.82 5.03 ± 4.9 1.43 7 2.85 ± 2.84 4.17 ± 4.27 1.04 *Mean of titer (log) ± SD of 3 or 4 mice

[0096] 6 TABLE 6 Virus titers in the lungs of BALB/c mice immunized with p&Dgr;RC-gB or p&Dgr;RC-pp65 and challenged i.n. with Vac-pp65 days Vac-pp 65 titer (log ± SD) in lungs* after p&Dgr;RC-gB- p&Dgr;RC-pp65- challenge immunized immunized 0 5.52 ± 4.83 5.52 ± 4.83 1 4.31 ± 4.3 4.56 ± 3.5 3 7.68 ± 6.75 7.15 ± 7.11 4  7.7 ± 7.66 6.57 ± 6.56 5 7.45 ± 6.79 6.02 ± 6.14 7 7.17 ± 6.17 6.23 ± 6.08 *Mean of titer (log) ± SD of 3 or 4 mice

[0097] This data demonstrate that immunization with either plasmid reduced the titer of the corresponding challenge virus by 0.5-1.4 log on days 3, 4, 5 and 7 after the challenge.

EXAMPLE 12

Secreted Form of gB is More Potent Immunogen than Membrane-Bound gB

[0098] To test whether gB bound to the membranes of gB-expressing cells or truncated form of gB lacking the transmembrane region of the molecule (it is secreted from the cell) induce stronger immune responses, mice were immunized with p&Dgr;RC-gB (expressing membrane-bound gB) or with p&Dgr;RCgB680 (expressing the secreted form of gB) and ELISA and neutralizing antibody responses were evaluated as follows.

[0099] Plasmids p&Dgr;RC-gB (expressing the whole gB) and &Dgr;RC-gB680 (expressing N-terminal 680 amino acids of the gB molecule and lacking the transmembrane region) were used in the following immunization protocol. Groups of 10 mice (BALB/c, female, 8 weeks old, purchased from HSD), were inoculated i.m. in the left leg with 50 &mgr;g plasmid DNA/mouse/inoculation. Mice were not inoculated with bupivacaine prior to DNA inoculation. Two months later a booster immunization was given (same dose, route).

[0100] Sera were tested in the gB-specific ELISA assay described above before the booster inoculation and 1 month after booster. The results are shown in Table 7, which shows the OD values of serum dilutions of 1:40 of individual mice. Preimmune serum samples of 40 mice were included. Cut off value: OD=0.15. 7 TABLE 7 HCMV ELISA antibodies induced by plasmids expressing membrane-bound or secreted form of gB OD of sera of mice immunized with p&Dgr;RC-gB p&Dgr;RC-gB680 # of before after # of before after mouse booster booster mouse booster booster 1 0.31 0.55 1 0.83 >3.00 2 0.09 0.10 2 0.52 >3.00 3 0.09 0.13 3 1.65 >3.00 4 0.06 0.08 4 0.06 0.09 5 0.07 0.07 5 1.29 >3.00 6 0.04 0.04 6 1.92 >3.00 7 0.08 0.17 7 2.31 >3.00 8 0.51 1.88 8 1.22 >3.00 9 0.07 0.07 9 0.62 >3.00 10 0.06 0.06 10 1.50 >3.00

[0101] The results in Table 7 show that ten mice immunized with the p&Dgr;RC-gB680 were positive for stronger gB-specific antibody responses than mice immunized with p&Dgr;RC-gB.

[0102] Table 8 provides the results following the immunization protocol above, where the mice had been boosted after 2 months using the same protocol as described for the first immunization. Sera obtained 1 and 2 month after the booster were tested in a HCMV-microneutralization assay. Preimmune sera were included as negative controls, NA titers≧12 are considered positive. 8 TABLE 8 p&Dgr;RC-gB680 expressing secreted form of gB induce stronger neutralizing antibody responses than p&Dgr;RC-gB expressing membrane-bound gB NA titers of sera of mice 1 and 2 month after booster immunized with p&Dgr;RC-gB p&Dgr;RC-gB680 1 M 2 M 1 M 2 M 16 24 128 64  8 <8 64 32  4 <4 256 192  4 8 <4 12  8 4 128 96  4 4 64 64  8 24 64 32 48 48 48 ND  6 4 96 96 <6 4 16 24

[0103] As shown in Table 8, nine of the p&Dgr;RC-gB680-immunized mice developed gB-specific antibodies, but only 3 of 10 responded in the p&Dgr;RC-gB-immunized group. HCMV-neutralizing antibody titers were also higher in the p&Dgr;RC-gB680-immunized mice, 9 of 10 developed significant NA responses versus 3 of 10 in the p&Dgr;RC-gB-immunized group (Table 8).

[0104] These data show that the p&Dgr;RC-gB680 plasmid expressing the N-terminal 680 amino acids of gB (lacking the transmembrane region of the protein) given intramuscularly induces more potent antibody responses to gB than the p&Dgr;RC-gB plasmid expressing the full gB.

EXAMPLE 13

P&Dgr;RC-gB630 Mixed with p&Dgr;RC-pp65 and Given at One Site of Inoculated Separately Induce both gB- and pp65-Specific Antibodies

[0105] As shown above, pTet-gB and p&Dgr;RC-pp65 plasmids mixed and inoculated at one site induced immune responses to both gB and pp65 indicating that there is no antigenic competition between gB and pp65. In this experiment whether the p&Dgr;RC-gB680 (expressing the secreted form of gB) is suitable for immunization in a mixture with p&Dgr;RC-pp65 was tested.

[0106] Groups of 10 BALB/c mice (female, HSD, 9-10 weeks old) were inoculated either with a mixture of two plasmids containing 50 &mgr;g of each in 200 &mgr;l: 100 &mgr;l (50 &mgr;g) into the left leg, 100 &mgr;l (50 &mgr;g) into the right leg; or the two different plasmids were inoculated separately: one kind of DNA (100 &mgr;l/50 &mgr;g) into the left leg, the other kind of plasmid (100 &mgr;l/50 &mgr;g) into the right leg. A booster immunization was given 1 month later. The plasmids used in this study were p&Dgr;RC-pp65, p&Dgr;RC-gB, and p&Dgr;RC-gB680. Table 9 shows results obtained with sera taken 8 days after booster. The ELISA antigen was purified gB. Cut off value: 0.081.

[0107] The results show that mice immunized with mixtures of p&Dgr;RC-gB and p&Dgr;RC-pp65 developed both gB and pp65 ELISA antibodies. Similar responses were observed in mice immunized with the two plasmids given at separate sites (Table 10 below). HCMV-gB-specific antibody responses in mice immunized with p&Dgr;RC-gB680 either given in mixture with p&Dgr;RC-pp65 or at separate sites were stronger than in mice immunized with the full-gB-expressing p&Dgr;RC-gB (these results confirm that the secreted form of gB is a stronger immunogen than the membrane-bound form). 9 TABLE 9 p&Dgr;RC-gB680 mixed with p&Dgr;RC-pp65 and given at one site or inoculated separately induce gB-specific antibodies gB-specific antibody (OD at serum dilutions of 1:40) mice inoculated with p&Dgr;RC-gB and p&Dgr;RC-pp65 mice inoculated with at p&Dgr;RC-gB680 and p&Dgr;RC-pp65 one at two at one at two mouse site mouse sites mouse site mouse sites #326 0.085 #356 0.115 #341 1.280 #336 1.058 #327 0.193 #357 0.082 #342 1.070 #337 0.550 #328 0.121 #358 0.099 #343 1.385 #338 0.193 #329 0.060 #359 0.107 #344 1.190 #339 1.039 #330 0.115 #360 0.107 #345 2.588 #340 0.207 #331 0.093 #361 NT #351 1.037 #346 0.288 #332 0.061 #362 0.092 #352 0.771 #347 0.220 #333 0.089 #363 0.065 #353 0.493 #348 0.513 #334 0.078 #364 0.152 #354 0.560 #349 0.223 #335 0.088 #365 0.082 #355 0.933 #350 0.719 Mean 0.098 0.100 1.130 0.521 OD:

[0108] Mice immunized as above with the mixture of p&Dgr;RC-gB680 and p&Dgr;RC-pp65 showed gB-specific antibody responses similar to those observed in mice immunized with the two kinds of plasmids given at separate sites. Results of pp65-specific antibody responses showed that mice responded to the pp65 antigen regardless of immunization with a mixture or with plasmids given at separate sites (Table 10). Table 10 shows results obtained with sera taken 8 days after booster (cut off value: 0.050). 10 TABLE 10 p&Dgr;RC-gB680 mixed with p&Dgr;RC-pp65 and given at one site or inoculated separately induce pp65-specific antibodies pp65-specific antibody (OD at serum dilutions of 1:40) mice inoculated with p&Dgr;RC-gB and p&Dgr;RC-pp65 mice inoculated with at p&Dgr;RC-gB680 and p&Dgr;RC-pp65 one at two at one at two mouse site mouse sites mouse site mouse sites #326 0.037 #356 0.000 #341 0.389 #336 0.276 #326 0.037 #356 0.000 #341 0.389 #336 0.276 #327 0.149 #357 0.000 #342 0.238 #337 0.295 #328 0.002 #358 0.508 #343 0.440 #338 0.000 #329 0.000 #359 0.008 #344 0.077 #339 0.009 #330 0.009 #360 0.176 #345 0.008 #340 0.030 #331 0.007 #361 dead #351 0.081 #346 0.051 #332 0.014 #362 0.009 #352 0.077 #347 0.124 #333 0.000 #363 0.028 #353 0.049 #348 0.281 #334 0.000 #364 0.097 #354 0.016 #349 0.118 #335 0.008 #365 0.201 #355 0.178 #350 0.014 Mean 0.014 0.109 0.154 0.111 OD:

[0109] The data show that mice develop significant immune responses both to gB and pp65 after immunization with a mixture of p&Dgr;RC-gB680 and p&Dgr;RC-pp65, indicating that these two HCMV antigens are able to induce parallel immune responses when introduced by expression plasmids to the immune system.

EXAMPLE 14

Immunization Studies in Mice Immunized with HCMV Plasmid Vectors Expressing Full-Length and Transmembrane-Deleted gB

[0110] As shown in the studies described above, full-length gB and transmembrane-deleted gB have been found to induce a strong and long-term antibody response when delivered by plasmid DNA. The following experiments provide further evidence of this effect.

[0111] A. pCBgB and pCB-gB&Dgr;tm

[0112] The gB open reading frame (ORF, nucleotides 1-2724) was obtained from the CMV Towne strain [SEQ ID NO: 1] using conventional techniques. The gB&Dgr;tm (transmembrane-deleted gB) was obtained from the wild type gene by deleting in frame the sequences coding for the hydrophobic transmembrane domain of the protein [nucleotides 2143-2316 were deleted from the gB ORF, SEQ ID NO:1]. These two coding sequences were introduced into the polylinker of the eukaryotic expression vector pCB11 corresponding to a commercially available pUC backbone with the HCMV IE1 promoter/enhancer sequences and the terminator sequences from the bovine growth hormone gene (FIG. 7A). The resulting plasmids, pCBgB and pCBgB&Dgr;tm expressing the full-length gB and its truncated version, respectively, are shown in FIG. 8. Protein expression from pCBgB and from pCBgB&Dgr;tm was confirmed by immunofluorescence and immunoprecipitation after transfection into cultured CHO-K1 cells. The immunoprecipitation experiment indicated that only pCBgB&Dgr;tm gave rise to a secreted form of gB which could be recovered from the cell culture medium.

[0113] B. Immunization

[0114] The study described below was performed with pCBgB and pCBgB&Dgr;tm in 6-8 week old female BALB/c mice. Anesthetized (xylazine+ketamine) mice (8 per group) received three administrations of 50 &mgr;g pCBgB or pCBgB&Dgr;tm at three week intervals (days 0, 21 and 42) either intramuscularly (IM) or intradermally (ID). For IM administration, DNA in 50 &mgr;l of saline was injected into the quadriceps with a Hamilton syringe equipped with a 20 gauge needle. For ID administration, DNA in a total volume of 100 &mgr;l of saline was injected into 5 sites of shaved dorsal skin with a pneumatic jet injector.

[0115] In each group, mice were labeled and bled on days 14 (following 1 injection), 35 (following 2 injections), 56 (following 3 injections), 116 and 202. The anti-urease IgG antibody response was followed by ELISA against recombinant gB produced in MRC5 cells infected with ALVAC-gB. The sera collected on days 116 and 202 were analyzed for hCMV neutralization in complement dependent microneutralization assay [Gonczol et al, cited above (1986)]. The data is provided in Table 11 and summarized in FIG. 9. 11 TABLE 11 INDIVIDUAL ELISA TITERS IN MICE IMMUNIZED WITH HCMV GB PLASMID VECTORS Intramuscular Intradermal neg. # pCBgB pCBgB&Dgr;tm pCBgB pCBgB&Dgr;tm serum Day Mouse ELISA ELISA ELISA ELISA ELISA 14 1 50 50 <50 <50 <50 2 <50 200 <50 <50 <50 3 100 9600 100 <50 4 <50 300 <50 <50 5 100 100 <50 <50 6 <50 75 <50 50 7 100 75 <50 <50 8 50 <50 <50 <50 35 1 100 100 75 50 <50 2 150 900 150 600 <50 3 200 12800 6400 2400 4 150 3200 1600 200 5 400 1200 100 1600 6 100 1200 1200 6400 7 150 300 75 100 8 150 100 200 150 56 1 150 1600 200 1200 <50 2 200 2400 200 38400 <50 3 200 38400 6400 12800 4 75 61200 6400 12800 5 400 2400 1200 4800 6 100 38400 3200 9600 7 200 19200 600 1600 8 600 4800 1200 4800 116 1 <50 1200 75 600 <50 2 1600 800 37.5 12800 <50 3 400 9600 1200 640 4 <50 25600 2400 4800 5 25 1600 150 800 6 <50 25600 1600 4800 7 <50 6400 300 800 8 200 1200 200 800 202 1 <50 1000 50 250 <50 2 400 1000 25 8000 <50 3 1600 8000 800 3000 4 <50 64000 1600 1500 5 25 1500 50 500 6 <50 24000 1200 3000 7 <50 4000 200 375 8 1000 150 375

[0116] As illustrated in Table 11 above and in FIG. 9, pCBgB and pCBgB&Dgr;tm plasmids induced serum IgGs against recombinant gB protein after IM or ID administration in BALB/c mice [pCBgB&Dgr;tm/ID≧pCBgB&Dgr;tm/IM>>pCBgB/ID≧pCBgB/IM]. pCBgB and pCBgB&Dgr;tm plasmids induced detectable neutralizing antibodies to hCMV (in vitro assay) after IM or ID administration in BALB/c mice [pCBgB&Dgr;tm>pCBgB].

[0117] pCB-gB and pCB-gB&Dgr;tm have been observed to induce a strong and long-term antibody response. pCBgB and especially pCB-gB&Dgr;tm induce neutralizing antibodies.

[0118] The nature of the response (IgG1/IgG2a,) differs between pCB-gB and pCB-gB&Dgr;tm. Particularly, pCB-gB has been observed to induce an IgG1 (TH2) response which is approximately equivalent to the IgG2a (TH1) response induced. In contrast, pCB-gB&Dgr;tm has been observed to induce an IgG1 response that is significantly stronger that the IgG2a response induced.

[0119] 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. 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.

Claims

1. A DNA molecule which is non-replicating in mammals and comprises a sequence encoding a human cytomegalovirus antigen,

wherein the sequence is operably linked to regulatory sequences for expressing the antigen in mammals and wherein the antigen elicits an immune response in the mammal.

2. The DNA molecule according to claim 1, which is a plasmid.

3. The DNA molecule according to claim 1, wherein said antigen is selected from the group consisting of:

(a) gB;

(b) a gB derivative lacking at least the transmembrane domain;

(c) pp65;

(d) pp150;

(e) immediate-early exon-4; and

(f) combinations of (a)-(e).

4. The DNA molecule according to claim 3, which comprises a sequence encoding the gB and the pp65 antigens.

5. The DNA molecule according to claim 3, which comprises a sequence encoding the gB derivative and a sequence encoding the pp65 antigen.

6. A pTet-gB DNA plasmid, said plasmid comprising the human cytomegalovirus (HCMV) gB gene and a tetracycline regulatable HCMV-immediate early promoter, said promoter controlling the expression of gB.

7. A p&Dgr;RC/CMV DNA plasmid, said plasmid comprising the human cytomegalovirus (HCMV) gB gene and capable of expressing gB.

8. A p&Dgr;RC-gB680 plasmid, said plasmid comprising the portion of the human cytomegalovirus (HCMV) gene encoding the N-terminal 680 amino acids of the gB protein (gB1-680) and capable of expressing gB1-680.

9. A p&Dgr;RC-pp150 plasmid, said plasmid comprising the human cytomegalovirus (HCMV) gene encoding the HCMV pp150 tegument protein and capable of expressing pp150.

10. A p&Dgr;RC-exon-4 plasmid, said plasmid comprising the portion of the human cytomegalovirus (HCMV) gene encoding HCMV immediate-early (IE)-exon-4 and capable of expressing IE-exon-4.

11. An immunogenic composition comprising a carrier and a DNA molecule according to claim 1.

12. A pCBgB&Dgr;tm plasmid, said plasmid comprising the human cytomegalovirus (HCMV) gB gene containing a deletion in the transmembrane domain.

13. An immunogenic composition comprising a carrier and a DNA molecule according to claim 1.

14. The immunogenic composition according to claim 13, wherein the DNA molecule is selected from the group consisting of:

(a) p&Dgr;RC-gB;

(b) pTet-gB;

(c) p&Dgr;RC-pp65;

(d) p&Dgr;RC-gB680;

(e) p&Dgr;RC-pp150;

(f) pCBgB;

(g) pCBgB&Dgr;tm; and

(h) p&Dgr;RC-exon-4.

15. The immunogenic composition according to claim 13, comprising two or more DNA molecules.

16. The immunogenic composition according to claim 14, comprising a first DNA molecule which comprises a sequence encoding the gB antigen or a gB derivative, and a second DNA molecule which comprises a sequence encoding the pp65 antigen.

17. The immunogenic composition according to claim 13, wherein the carrier is selected from the group consisting of saline and isotonic water.

18. A method of inducing human cytomegalovirus-specific (HCMV) immune responses in an animal, comprising the step of administering to said animal an effective amount of a first immunogenic composition according to claim 13.

19. The method according to claim 18, wherein the composition comprises pTet-gB and p&Dgr;RC-pp65.

20. The method according to claim 18, further comprising the step of administering a second immunogenic composition to said animal, said second immunogenic composition comprising a plasmid selected from the group consisting of:

(a) p&Dgr;RC-gB;

(b) pTet-gB;

(c) p&Dgr;RC-pp65;

(d) p&Dgr;RC-gB680;

(e) p&Dgr;RC-pp150;

(f) pCBgB;

(g) pCBgB&Dgr;tm; and

(h) p&Dgr;RC-IE-Exon-4.

21. The method according to claim 18, wherein said second immunogenic composition is administered between about 2 to about 15 weeks following administration of said first immunogenic composition.

22. A method of priming immune responses to a selected human cytomegalovirus immunogenic composition, comprising the steps of:

administering a first immunogenic composition according to claim 13 and administering the selected human cytomegalovirus immunogenic composition.

23. The method according to claim 22, wherein the first immunogenic composition is administered between about 4 and 15 weeks prior to administration of the selected immunogenic composition.

24. The method according to claim 22, wherein the first immunogenic composition comprises pTet-gB.

25. The method according to claim 24, wherein pTet-gB is administered in an amount between about 50 &mgr;g to about 160 &mgr;g.

26. The method according to claim 22, wherein the selected immunogenic composition comprises an immunogen selected from the group consisting of a recombinant virus comprising an HCMV immunogen, an HCMV protein, and HCMV virions.

27. The method according to claim 26, wherein the HCMV protein is gB.

28. The method according to claim 26, wherein the recombinant virus is selected from the group consisting of Ad5.gb and Ad5-IE-exon-4.

29. A DNA molecule which is non-replicating in mammals and comprises a sequence encoding a human cytomegalovirus antigen selected from the group consisting of:

(a) pp65;

(b) pp150;

(c) immediate-early exon-4;

(d) gB and an antigen of (a) to (c);

(e) a gB derivative lacking at least the transmembrane domain and an antigen of (a) to (c); and

(f) a combination of antigens (a) to (c).

30. An immunogenic composition comprising a carrier and at least two DNA molecules, wherein said DNA molecules are selected from the group consisting of:

(a) gB;

(b) a gB derivative lacking at least the transmembrane domain;

(c) pp65;

(d) pp150; and

(e) immediate-early exon-4.