Title:  Human cytomegalovirus DNA constructs and uses therefor

United States Patent:  6,448,389

Issued:  September 10, 2002

Inventors:  Gonczol; Eva (Rosemont, PA); Berencsi; Klara (Rosemont, PA); Kari; Csaba (Rosemont, PA)

Assignee:  The Wistar Institute of Anatomy and Biology (Philadelphia, PA)

Appl. No.:  171699

Filed:  January 19, 1999

PCT Filed:  April 22, 1997

PCT NO:  PCT/US97/06866

371 Date:  January 19, 1999

102(e) Date:  January 19, 1999

PCT PUB.NO.:   WO97/40165

PCT PUB. Date:  October 30, 1997

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.

DETAILED DESCRIPTION OF THE INVENTION

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 gB_1-680, 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.

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.

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.

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.

Thus, the CMV gene fragment encoding the desired protein (e.g., gB, pp65, pp150) or protein fragment (e.g., gB_1-680 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.

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.DELTA.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.DELTA.tm) and the pp65 antigens.

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).

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.DELTA.RC-pp65 plasmid described below. pTet-gB is also particularly useful for priming immune responses to subsequently administered HCMV immunogenic compositions and vaccines.

The pTetotTA-gB plasmid has been deposited pursuant to the Budapest Treaty, in the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., U.S.A. This deposit, designated ATCC 98029, was made on Apr. 23, 1996 and is termed herein, pTet-gB.

Other plasmids provided herein, p.DELTA.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.

Another plasmid of the invention, p.DELTA.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 gB_1-680. As illustrated in FIGS. 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.

Also provided herein is plasmid pCDgB.DELTA.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.

Plasmid p.DELTA.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 FIGS. 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.

Plasmid p.DELTA.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.DELTA.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.DELTA.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.

Plasmid p.DELTA.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.

The DNA molecules, and particularly the plasmids described herein, may be used for expression of the gB, gB_1-680 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.

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.

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.

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 PCT WO94/01139, which are incorporated by reference herein for purposes of described various `naked DNA` delivery methods.]

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.

An effective amount of an immunogenic composition of the invention preferably contains between 10 .mu.g and 10 .mu.g, and preferably between about 80 .mu.g and 150 .mu.g of DNA of the invention per inoculation. Desirably, for each inoculation, the DNA of the invention is formulated in about 100 .mu.l of a suitable carrier. In a particularly preferred embodiment, each patient is administered 100 .mu.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.

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.

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.DELTA.Rc-gB₆₈₀ or pCBgB.DELTA.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.DELTA.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.DELTA.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.DELTA.RC-gB₆₈₀ and p.DELTA.RC-pp65.

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.

Claim 1 of 3 Claims

What is claimed is:

1. A p.DELTA.RC-pp65 plasmid, said plasmid comprising the human cytomegalovirus (HCMV) gene encoding the HCMV pp65 tegument protein under the control of regulatory sequences which direct expression of the pp65 antigen in mammalian cells.
 

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