U.S. patent application number 10/223538 was filed with the patent office on 2003-06-26 for human cytomegalovirus dna constructs and uses therefor.
Invention is credited to Berencsi, Klara, Girerd, Yves, Gonczol, Eva, Haensler, Jean, Kari, Csaba.
Application Number | 20030120060 10/223538 |
Document ID | / |
Family ID | 21773167 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030120060 |
Kind Code |
A1 |
Gonczol, Eva ; et
al. |
June 26, 2003 |
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.
Inventors: |
Gonczol, Eva; (Rosemont,
PA) ; Berencsi, Klara; (Rosemont, PA) ; Kari,
Csaba; (Rosemont, PA) ; Haensler, Jean; (Saint
Genis les Ollieres, FR) ; Girerd, Yves;
(Francheville, FR) |
Correspondence
Address: |
Mary E. Bak
Howson and Howson
Spring House Corporate Center, Box 457
Spring House
PA
19477
US
|
Family ID: |
21773167 |
Appl. No.: |
10/223538 |
Filed: |
August 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10223538 |
Aug 19, 2002 |
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09171699 |
Jan 19, 1999 |
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6448389 |
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09171699 |
Jan 19, 1999 |
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PCT/US97/06866 |
Apr 22, 1997 |
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60015717 |
Apr 23, 1996 |
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Current U.S.
Class: |
536/23.72 ;
424/230.1; 435/320.1; 435/69.1 |
Current CPC
Class: |
A61K 2039/51 20130101;
C12N 2710/24143 20130101; A61K 39/00 20130101; C12N 2740/16222
20130101; C07K 14/005 20130101; A61K 2039/53 20130101; C12N
2710/16122 20130101 |
Class at
Publication: |
536/23.72 ;
435/320.1; 424/230.1; 435/69.1 |
International
Class: |
C07H 021/04; C12P
021/06; A61K 039/245; A61K 039/25; C12N 015/00; C12N 015/09; C12N
015/63; C12N 015/70; C12N 015/74 |
Claims
What is claimed is:
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.DELTA.RC/CMV DNA plasmid, said plasmid comprising the human
cytomegalovirus (HCMV) gB gene and capable of expressing gB.
8. A p.DELTA.RC-gB.sub.680 plasmid, said plasmid comprising the
portion of the human cytomegalovirus (HCMV) gene encoding the
N-terminal 680 amino acids of the gB protein (gB.sub.1-680) and
capable of expressing gB.sub.1-680.
9. A p.DELTA.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.DELTA.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.DELTA.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.DELTA.RC-gB; (b) pTet-gB; (c) p.DELTA.RC-pp65; (d)
p.DELTA.RC-gB.sub.680; (e) p.DELTA.RC-pp150; (f) pCBgB; (g)
pCBgB.DELTA.tm; and (h) p.DELTA.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.DELTA.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.DELTA.RC-gB; (b) pTet-gB; (c)
p.DELTA.RC-pp65; (d) p.DELTA.RC-gB.sub.680; (e) p.DELTA.RC-pp150;
(f) pCBgB; (g) pCBgB.DELTA.tm; and (h) p.DELTA.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 .mu.g to about 160
.mu.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.
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 .sctn.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.DELTA.RC-gB plasmid.
[0009] Yet another plasmid of the invention, p.DELTA.RC-gB.sub.680,
contains the portion of the HCMV genome encoding the N-terminal 680
amino acids of the gB protein (gB.sub.1-680).
[0010] The p.DELTA.RC-pp65 plasmid of the invention contains the
portion of the HCMV genome (UL83) encoding the HCMV pp65 tegument
protein. The p.DELTA.RC-pp150 plasmid contains the portion of the
HCMV genome (UL32) encoding the HCMV pp150 tegument protein.
[0011] The p.DELTA.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.DELTA.RC-gB.sub.680, pTet-gB and/or p.DELTA.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.DELTA.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.DELTA.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.DELTA.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 gB.sub.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.
[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., gB.sub.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.
[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.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 gB.sub.680) 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., gB.sub.680 or gB.DELTA.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.DELTA.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.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.
[0038] Another plasmid of the invention, p.DELTA.RC-gB.sub.680
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.sub.1-680. 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.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.
[0040] 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 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.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.
[0042] 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.
[0043] The DNA molecules, and particularly the plasmids described
herein, may be used for expression of the gB, gB.sub.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.
[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 .mu.g and 10 mg, 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.
[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.DELTA.Rc-gB.sub.680 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 gB.sub.680 or gB. Yet another desired embodiment involves
combined administration of p.DELTA.RC-gB.sub.680 and
p.DELTA.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 tet.sup.R-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 .mu.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.DELTA.RC-gB
[0064] p.DELTA.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.DELTA.RC/CMV vector. The resulting plasmid is termed
p.DELTA.RC-gB.
[0065] C. Construction of p.DELTA.RC-gB.sub.680
[0066] p.DELTA.RC-gB.sub.680 expresses the N-terminal 680 amino
acids of the gB protein [SEQ ID NO:2]. The plasmid was derived from
p.DELTA.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.DELTA.RC-pp65 AND p.DELTA.RC-pp150
[0067] A. p.DELTA.RC-pp65
[0068] The plasmid p.DELTA.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.DELTA.RC-pp150
[0071] The plasmid, p.DELTA.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.DELTA.RCd [Virogenetics]. The
insert is approximately 3200 bp [SEQ ID NO: 10].
EXAMPLE 4
Construction of p.DELTA.RC-IE-Exon-4
[0073] The plasmid, p.DELTA.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 .mu.g/3.times.10.sup.5 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 .mu.g tetracycline [Tetracycline hydrochloride, Sigma, St. Louis,
Mo.]. Sixty to seventy percent and 40-50% of cells transfected with
p.DELTA.RC-gB and p.DELTA.gB.sub.680 plasmids, respectively,
expressed gB. pp65 protein was expressed in 70-80% of cells
transfected with p.DELTA.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 .mu.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 .mu.g plasmid
DNA/inoculation. Booster inoculations were given i.m. 2.times.,
without pretreatment with Bupivacaine. Mice immunized with
p.DELTA.RC-gB plasmid were boosted 1.times.. 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.DELTA.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 .sup.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 .sup.51Cr-release was
calculated as [(cpm experimental release-cpm spontaneous
release)/(cpm maximal release-cpm spontaneous release).times.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
.mu.g p.DELTA.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.
1TABLE 1 p.DELTA.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.DELTA.RC-gB plasmid seems to be a highly potent immunizing
construct.
2TABLE 2 pTet-gB and p.DELTA.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.DELTA.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.DELTA.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.DELTA.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 .mu.l/mouse, 50 .mu.l/leg), and 2 days later, with
either a mixture of both plasmids (80 .mu.g of each DNA/mouse, 40
.mu.g of each DNA/leg, 160 .mu.g DNA/mouse) or each plasmid
inoculated into two different legs (80 .mu.g DNA of each
plasmid/mouse, a total of 160 .mu.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.
3TABLE 3 pTet-gB and p.DELTA.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.DELTA.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.DELTA.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.DELTA.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.DELTA.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.DELTA.RC-pp65 and
pTet-gB. When the p.DELTA.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 .mu.g gB/mouse in Alum s.c.) or with the Towne strain of HCMV
(20 .mu.g/mouse in Alum s.c.).
4TABLE 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.DELTA.RC-gB or p.DELTA.RC-pp65 (80 .mu.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.times.10.sup.6 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.
5TABLE 5 Virus titers in the lungs of BALB/c mice immunized with
p.DELTA.RC-gB or p.DELTA.RC-pp65 and challenged i.n. with Vac-gB
days Vac-gB titer (log + SD) in lungs* after p.DELTA.RC-gB-
p.DELTA.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]
6TABLE 6 Virus titers in the lungs of BALB/c mice immunized with
p.DELTA.RC-gB or p.DELTA.RC-pp65 and challenged i.n. with Vac-pp65
days Vac-pp 65 titer (log .+-. SD) in lungs* after p.DELTA.RC-gB-
p.DELTA.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.DELTA.RC-gB (expressing
membrane-bound gB) or with p.DELTA.RCgB.sub.680 (expressing the
secreted form of gB) and ELISA and neutralizing antibody responses
were evaluated as follows.
[0099] Plasmids p.DELTA.RC-gB (expressing the whole gB) and
.DELTA.RC-gB.sub.680 (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 .mu.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.
7TABLE 7 HCMV ELISA antibodies induced by plasmids expressing
membrane-bound or secreted form of gB OD of sera of mice immunized
with p.DELTA.RC-gB p.DELTA.RC-gB.sub.680 # 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.DELTA.RC-gB.sub.680 were positive for stronger gB-specific
antibody responses than mice immunized with p.DELTA.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.gtoreq.12 are considered positive.
8TABLE 8 p.DELTA.RC-gB.sub.680 expressing secreted form of gB
induce stronger neutralizing antibody responses than p.DELTA.RC-gB
expressing membrane-bound gB NA titers of sera of mice 1 and 2
month after booster immunized with p.DELTA.RC-gB p.DELTA.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.DELTA.RC-gB.sub.680-immunized mice developed gB-specific
antibodies, but only 3 of 10 responded in the
p.DELTA.RC-gB-immunized group. HCMV-neutralizing antibody titers
were also higher in the p.DELTA.RC-gB.sub.680-immunized mice, 9 of
10 developed significant NA responses versus 3 of 10 in the
p.DELTA.RC-gB-immunized group (Table 8).
[0104] These data show that the p.DELTA.RC-gB.sub.680 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.DELTA.RC-gB plasmid
expressing the full gB.
EXAMPLE 13
P.DELTA.RC-gB.sub.630 Mixed with p.DELTA.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.DELTA.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.DELTA.RC-gB.sub.680
(expressing the secreted form of gB) is suitable for immunization
in a mixture with p.DELTA.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
.mu.g of each in 200 .mu.l: 100 .mu.l (50 .mu.g) into the left leg,
100 .mu.l (50 .mu.g) into the right leg; or the two different
plasmids were inoculated separately: one kind of DNA (100 .mu.l/50
.mu.g) into the left leg, the other kind of plasmid (100 .mu.l/50
.mu.g) into the right leg. A booster immunization was given 1 month
later. The plasmids used in this study were p.DELTA.RC-pp65,
p.DELTA.RC-gB, and p.DELTA.RC-gB.sub.680. 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.DELTA.RC-gB and p.DELTA.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.DELTA.RC-gB.sub.680 either given in mixture with p.DELTA.RC-pp65
or at separate sites were stronger than in mice immunized with the
full-gB-expressing p.DELTA.RC-gB (these results confirm that the
secreted form of gB is a stronger immunogen than the membrane-bound
form).
9TABLE 9 p.DELTA.RC-gB.sub.680 mixed with p.DELTA.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.DELTA.RC-gB and p.DELTA.RC-pp65 mice inoculated
with at p.DELTA.RC-gB.sub.680 and p.DELTA.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.DELTA.RC-gB.sub.680 and p.DELTA.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).
10TABLE 10 p.DELTA.RC-gB.sub.680 mixed with p.DELTA.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.DELTA.RC-gB and p.DELTA.RC-pp65 mice
inoculated with at p.DELTA.RC-gB.sub.680 and p.DELTA.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.DELTA.RC-gB.sub.680 and p.DELTA.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.DELTA.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.DELTA.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.DELTA.tm expressing the full-length gB and its truncated
version, respectively, are shown in FIG. 8. Protein expression from
pCBgB and from pCBgB.DELTA.tm was confirmed by immunofluorescence
and immunoprecipitation after transfection into cultured CHO-K1
cells. The immunoprecipitation experiment indicated that only
pCBgB.DELTA.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.DELTA.tm in 6-8 week old female BALB/c mice. Anesthetized
(xylazine+ketamine) mice (8 per group) received three
administrations of 50 .mu.g pCBgB or pCBgB.DELTA.tm at three week
intervals (days 0, 21 and 42) either intramuscularly (IM) or
intradermally (ID). For IM administration, DNA in 50 .mu.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 .mu.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.
11TABLE 11 INDIVIDUAL ELISA TITERS IN MICE IMMUNIZED WITH HCMV GB
PLASMID VECTORS Intramuscular Intradermal neg. # pCBgB
pCBgB.DELTA.tm pCBgB pCBgB.DELTA.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.DELTA.tm plasmids induced serum IgGs against recombinant gB
protein after IM or ID administration in BALB/c mice
[pCBgB.DELTA.tm/ID.gtoreq.pC-
BgB.DELTA.tm/IM>>pCBgB/ID.gtoreq.pCBgB/IM]. pCBgB and
pCBgB.DELTA.tm plasmids induced detectable neutralizing antibodies
to hCMV (in vitro assay) after IM or ID administration in BALB/c
mice [pCBgB.DELTA.tm>pCBgB].
[0117] pCB-gB and pCB-gB.DELTA.tm have been observed to induce a
strong and long-term antibody response. pCBgB and especially
pCB-gB.DELTA.tm induce neutralizing antibodies.
[0118] The nature of the response (IgG.sub.1/IgG.sub.2a,) differs
between pCB-gB and pCB-gB.DELTA.tm. Particularly, pCB-gB has been
observed to induce an IgG.sub.1 (T.sub.H2) response which is
approximately equivalent to the IgG.sub.2a (T.sub.H1) response
induced. In contrast, pCB-gB.DELTA.tm has been observed to induce
an IgG.sub.1 response that is significantly stronger that the
IgG.sub.2a 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.
Sequence CWU 1
1
* * * * *