Methods and compositions for retarding the development of atherosclerotic lesions

A method for preventing or retarding the development atherosclerotic lesions or restenosis involves administering to a subject, preferably a human, an effective amount of an anti-viral composition directed against CMV, and optionally an anti-microbial composition directed against C. pneumoniae. These compositions may be conventional chemical anti-microbial pharmaceutics. Alternatively, the compositions may contain a cytomegalovirus (CMV) protein or fragment thereof (or nucleic acid containing compositions expressing such protein or fragment). Such compositions may contain an immunogenic C. pneumoniae protein or fragment thereof (or nucleic acid containing compositions expressing such protein or fragment). The protein/nucleic acid compositions are administered in an amount capable of inducing cell mediated immunity and/or antibody response in the subject.

FIELD OF THE INVENTION
 The present invention relates generally to pharmaceutical compositions and
 methods of use thereof in treating or preventing atherosclerosis.
 BACKGROUND OF THE INVENTION
 Atherosclerosis (AT) results from an excessive inflammatory and
 fibroproliferative response to vascular insult and has been noted to be a
 principal cause of heart attack and stroke, accounting for up to half of
 all mortalities in the industrialized world including about 13 million
 Americans. Atherogenesis is theorized to follow a response to injury, but
 the agent(s) of injury have yet to be identified fully.
 Viral and/or bacterial infection(s) have been found to be associated in
 some way with the complex process of the development of AT. Particles,
 antigens and DNA sequences of human cytomegalovirus (HCMV), a member of
 the herpesvirus group, have been described in AT plaques of biopsy or
 autopsy material [M. G. Hendrix et al, Am. J. Pathol., 136:23-28 (1990);
 J. L. Melnick et al., BioEssays, 17(10): 899 (October 1995)]. However,
 Melnick, cited above, states that the "observations do not demonstrate a
 viral role in the pathogenesis of atherosclerosis". The possible
 involvement of reactivated HCMV in restenosis of coronary arteries, an
 accelerated form of AT, following angioplastic surgery has been suggested
 [S. E. Epstein et al, Lancet, 348:13-17 (1996); E. Speir et al, Science,
 265:391-394 (1994); Y. F. Zhou et al, N. Engl. J. Med., 335: 624-630
 (1996)]. Seroepidemiologic data show that HCMV infection usually occurs in
 childhood, paralleling the pattern of the appearance of early AT lesions;
 by young adulthood, 50-100% of individuals are HCMV-seropositive. In some
 individuals, the virus is apparently reactivated in artery walls, where it
 may initiate abnormal cell growth that can lead to blocked blood flow and,
 ultimately, heart attack.
 The bacterium Chlamydia pneumoniae is an intracellular bacterium, which has
 been established as an important pathogen in acute and chronic respiratory
 infections [J. T. Grayson, Clin. Infect. Dis., 15:757-763 (1992)] has also
 been associated with AT [J. A. Ramirez, Ann. Intern. Med., 125:979-982
 (1996)]. This bacterium infects about 50% of the population and causes
 flu-like diseases, but also replicates in the arterial wall. C. pneumoniae
 antigens and DNA have been detected in human AT plaques. Population
 antibody prevalence studies have shown that more than 50% of adults
 worldwide have antibody. While antibody is infrequent in children under
 age 5 years, incidence studies have demonstrated antibody conversion of
 6-9% per year in children from the ages 5-14 years. The prevalence of
 antibody continues to increase throughout adulthood, and is highest in the
 elderly.
 Recently, C. pneumoniae, strain TWAR, has been associated with AT based on
 both seroepidemiology and data demonstrating the presence of the organism
 in AT plaques. For example, serologic studies from Finland, the United
 States and other countries have shown that patients with coronary artery
 diseases were significantly more likely to have serologic evidence of past
 infection with TWAR than were controls. Morphologic and microbiological
 evidence of the persistence of TWAR in atheromatous plaques has been
 obtained by electron microscopic studies, immunochemical staining and PCR
 testing of coronary, carotid and aortic atheroma [C.-C. Kuo et al, Clin.
 Microbiol. Rev., 8:451-461 (1995)]. In addition, C. pneumoniae activates
 growth factors involved in inflammatory responses and changes lipoprotein
 metabolism of infected cells. Immune responses to chlamydial infections
 are partly protective but also deleterious, and delayed hypersensitivity
 (DH) is thought to play a pathogenic role in chlamydial disease [R. P.
 Morrison et al, J. Exp Med., 170:1271-1283 (1989)].
 Despite the wealth of reports, no etiological role of HCMV and/or C.
 pneumoniae in the development of AT has been established. Thus, there
 remains a need in the art for reagents and methods useful in ameliorating
 the symptoms and development of atherosclerosis in response to these
 microorganisms.
 SUMMARY OF THE INVENTION
 In one aspect, the invention provides a method for the treatment or
 prophylaxis of atherosclerosis in a mammal, preferably a human, comprising
 administering to a mammal an effective amount of a composition comprising
 a human cytomegalovirus (HCMV) protein or fragment thereof, the amount
 capable of inducing cell mediated immunity and anti-CMV antibody response
 in the mammal. The composition preferably may be administered to infants
 or immunocompromised patients.
 In another aspect the invention provides a method for the treatment or
 prophylaxis of atherosclerosis in a mammal comprising administering to a
 mammal an effective amount of a composition comprising a nucleic acid
 sequence encoding a human cytomegalovirus (HCMV) protein or fragment
 thereof, said composition capable of inducing cell mediated immunity (CMI)
 and inducing an anti-CMV antibody response upon expression of said protein
 in the mammal.
 In still another aspect, the invention provides a method for the treatment
 or prophylaxis of atherosclerosis in a mammal, preferably a human,
 comprising administering to a mammal an effective amount of a composition
 comprising a Chlamydia pneumoniae protein or fragment thereof, the amount
 capable of inducing cell mediated immunity and anti-C. pneumoniae antibody
 response in the mammal. The composition preferably may be administered to
 infants or immunocompromised patients.
 In yet another aspect the invention provides a method for the treatment or
 prophylaxis of atherosclerosis in a mammal comprising administering to a
 mammal an effective amount of a composition comprising a nucleic acid
 sequence encoding a C. pneumoniae protein or fragment thereof, said
 composition capable of inducing cell mediated immunity (CMI) and inducing
 an anti-C. pneumoniae antibody response upon expression of said protein in
 the mammal.
 In another aspect, the invention provides a therapeutic or prophylactic
 composition comprising a Chlamydia pneumoniae protein or fragment thereof
 and an HCMV protein or fragment thereof in a pharmaceutically acceptable
 carrier.
 In still another aspect, the invention provides a therapeutic or
 prophylactic composition comprising an anti-microbial agent effective
 against Chlamydia pneumoniae infection and an HCMV protein or fragment
 thereof in a pharmaceutically acceptable carrier.
 In yet another aspect, the invention provides a method for treating
 atherosclerosis or restenosis by administering to a mammal having physical
 evidence of atherosclerosis or restenosis an effective amount of an
 anti-microbial agent directed against Chlamydia pneumoniae infection.
 In a further aspect, the invention provides a method for preventing
 restenosis after coronary atherectomy or balloon angioplasty comprising
 treating a patient prior to, or after said atherectomy or angioplasty with
 an effective amount of said C. pneumoniae/HCMV composition described above
 or with an effective amount of an anti-microbial agent directed against
 Chlamydia pneumoniae infection.
 Other aspects and advantages of the present invention are described further
 in the following detailed description of the preferred embodiments
 thereof.

DETAILED DESCRIPTION OF THE INVENTION
 The present invention provides compositions and methods which are useful
 for both the treatment and prophylaxis of mammalian subjects against the
 onset or development of atherosclerosis or atherosclerotic lesions
 characteristic of restenosis or associated with other arterial injury. The
 invention involves administering anti-microbial compositions directed
 against HCMV and C. pneumoniae to treat and/or prevent the development of
 atherosclerotic lesions and restenosis.
 I. Compositions of the Invention
 A. CMV-containing Compositions
 The compositions useful according to this invention may contain a CMV
 protein or fragment. Thus, the composition can be an attenuated, live CMV,
 preferably HCMV. The composition may also be a heat-inactivated,
 attenuated HCMV. 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, all incorporated by reference herein.]
 Alternatively, the composition may contain only certain HCMV proteins or
 fragments of these proteins which are characterized by the capacity to
 induce cell mediated immunity (CMI) to CMV infection upon administration
 of the protein in combination with an appropriate adjuvant. This
 composition may induce a low to undetectable anti-CMV antibody response in
 the subject. Desired HCMV proteins for this use include, without
 limitation, phosphoproteins 65, 150, 28 and 52, immediate early protein
 (IE), glycoprotein B (gB), and glycoprotein H (gH). Most desirably, the
 early HCMV proteins IE, gB and pp65 are useful in such a composition. One
 example of a useful fragment for this purpose is the immediate-early
 exon-4 (IE-exon-4) subunit of the HCMV. [See, e.g., International Patent
 Application No. WO94/17810, published Aug. 18, 1994, incorporated by
 reference herein].
 Where the HCMV composition contains nucleic acid sequence(s) encoding a
 cytomegalovirus protein or fragment thereof, the nucleic acid sequence may
 be that of the selected protein itself, that is, a `naked` DNA
 composition. So-called `naked DNA` may be used to express the HCMV protein
 or peptide fragment in vivo under the control of suitable promoter
 sequences. [For a discussion of this technology, see, e.g., J. Cohen,
 Science, 259:1691-1692 (Mar. 19, 1993); E. Fynan et al, Proc. Natl. Acad.
 Sci., 90: 11478-11482 (December 1993); J. A. Wolff et al, Biotechniques,
 11:474-485 (1991), all incorporated by reference herein, which describe
 similar uses of `naked DNA`]. For example, HCMV DNA encoding the IE
 protein under control of the HCMV-IE promoter may be used as an HCMV
 composition and administered according to the method of this invention.
 Other suitable homologous or heterologous promoter sequences may readily
 be selected, and the sequence constructed by methods known in the art.
 Alternatively, the nucleic acid sequence composition may contain a vector
 which carries the CMV protein-encoding DNA under the control of regulatory
 sequences which are capable of directing the expression of the product of
 the sequence. Such vectors may be viral in origin. For example, a proposed
 HCMV vaccine using a recombinant vaccinia virus expressing HCMV
 glycoprotein B has been described. [See, e.g., Cranage, M. P. et al, EMBO
 J., 5:3057-3063 (1986).] Additionally, an adenovirus vector carrying an
 HCMV protein sequence or fragment has been described. [See, e.g.,
 International Patent Application No. WO94/17810, published Aug. 18, 1994
 and International Patent Application No. WO94/23744, published Oct. 27,
 1994]. Other viral vectors, such as canarypox virus or a retrovirus may
 also be employed as carriers for the nucleic acid sequences of HCMV
 proteins or fragments.
 Similarly, such vectors may be non-viral in origin, such as known
 bacterial-based plasmids, e.g., pBR322 or pUC, or mammalian-based plasmids
 and may be employed to deliver the HCMV sequences to the subject. Many
 suitable plasmids are known to those of skill in the art and may be
 designed to contain the selected HCMV protein-encoding sequences.
 As stated above for the protein compositions, the desired HCMV protein
 sequences or DNA sequences encoding them preferably are those HCMV
 proteins which readily induce CMI. Other HCMV proteins which may be
 desirable for use in this invention may induce low or undetectable amounts
 of antibody in the subject. It is presently preferred to use the HCMV
 protein sequences such as pp65, IE, and fragments thereof, such as IE exon
 4.
 The compositions of this invention may also employ more than one HCMV
 protein or fragment, or proteins and fragments from more than one strain
 of HCMV, or the nucleic acid sequences encoding same.
 B. C. pneumoniae-containing Compositions
 The compositions useful according to this invention are anti-microbial
 compositions directed against a C. pneumoniae infection.
 In one embodiment, such compositions may be conventional antibiotics
 effective against many pneumonias, such as tetracycline, erythromycin and
 other conventional pharmaceutical antibiotic agents known to the skilled
 artisan.
 In another embodiment an anti-microbial composition useful in this
 invention may contain an immunogenic C. pneunoniae protein or fragment,
 e.g., the major outer membrane protein. Thus, the composition can be a
 killed C. pneumoniae of any suitable strain. Vaccines based on killed
 strains of C. pneumoniae may be prepared by conventional techniques of
 heat or irradiation.
 Alternatively, the composition may contain only certain C. pneumoniae
 proteins, e.g., the major outer membrane protein, or fragments of these
 proteins which are characterized by the capacity to induce cell mediated
 or humoral immunity to C. pneumoniae infection upon administration of the
 protein in combination with an appropriate adjuvant. This composition may
 induce a low to undetectable anti-C. pneumoniae antibody response in the
 subject. A desired C. pneumoniae protein or peptides for this use is
 desirably a bacterial cell surface protein, which may be randomly
 fragmented. Random fragments of a C. pneumoniae protein may be readily
 tested for immunogenicity by one of skill in the art, using a variety of
 assay formats. Vaccines containing C. pneumoniae cellular antigens or
 fragments may be obtained conventionally, e.g., by cell lysis and standard
 purification or separation techniques.
 Where the C. pneumoniae composition contains nucleic acid sequence(s)
 encoding a surface protein or fragment thereof, the nucleic acid sequence
 may be that of the selected protein itself, that is, a `naked` DNA
 composition. So-called `naked DNA` may be used to express the C.
 pneumoniae protein or peptide fragment in vivo under the control of
 suitable promoter sequences. [See the references cited above discussing
 naked DNA].
 Alternatively, the nucleic acid sequence composition may contain a vector
 which carries the C. pneumoniae protein-encoding DNA under the control of
 regulatory sequences which are capable of directing the expression of the
 product of the sequence. One particularly desirable embodiment is a vector
 encoding the major outer membrane protein. Suitable vectors may be
 selected from among known viral and plasmid vectors as described above for
 the HCMV compositions.
 As stated above for the protein compositions, the desired C. pneumoniae
 protein sequences or DNA sequences encoding them preferably are those C.
 pneumoniae proteins which readily induce CMI. Other C. pneumoniae proteins
 which may be desirable for use in this invention may induce low or
 undetectable amounts of antibody in the subject.
 The compositions of this invention may also employ more than one C.
 pneumoniae protein or fragment, or proteins and fragments from more than
 one strain of C. pneumoniae, or the nucleic acid sequences encoding same.
 C. Combination Compositions
 The present invention also provides for compositions which comprise both an
 anti-microbial agent directed against a C. pneumoniae infection and an
 anti-viral composition directed against HCMV in a suitable pharmaceutical
 carrier.
 For example, a combination composition of the present invention may contain
 an HCMV protein containing composition such as described above and a C.
 pneumoniae protein composition as described above. Alternatively, a naked
 DNA composition may contain the DNA of an HCMV protein or fragment and a
 C. pneumoniae protein or fragment as described above. Similarly a
 composition may contain a mixture of plasmid or viral vectors individually
 bearing DNA sequences which encode an HCMV protein or fragment and a C.
 pneumoniae protein or fragment, as described above.
 Another alternative combination composition may comprise a polycistronic
 vector, which carries the DNA sequence which encodes an HCMV protein or
 fragment and the DNA sequence which encodes a C. pneumoniae protein or
 fragment. These fragments may be under the control of the same regulatory
 sequence which directs expression of both proteins in vivo. Alternatively,
 the polycistronic vector may contain separate regulatory sequences for
 each protein to be expressed.
 Another embodiment of the composition of the present invention comprises
 the use of a conventional pharmaceutical antibiotic active against C.
 pneumoniae combined with an HCMV protein/DNA compositions as described
 above in a suitable pharmaceutical carrier.
 A similar embodiment of the composition of the present invention comprises
 the use of a conventional pharmaceutical antiviral useful against HCMV
 infection combined with a C. pneumoniae protein/DNA composition as
 described above in a suitable pharmaceutical carrier.
 II. Production of the Compositions
 HCMV isolates, C. pneumoniae strains, proteins and fragments of these
 microorganism, and their DNA sequences may be obtained in a variety of
 conventional ways. For example, strains of HCMV useful in the practice of
 this invention may be obtained from depositories, such as the American
 Type Culture Collection, Rockville, Md. (ATCC) or from other institutes or
 universities. Similarly, strains of C. pneumoniae, e.g., the TWAR strain,
 are available from the ATCC under Accession Nos. VR1310, 1355, 1356, 1360
 and 2282, among others. Other strains may also be obtained from institutes
 or universities.
 The preparation of heat-inactivated, or live attenuated viral and bacterial
 preparations are known to the art as provided by the publications
 incorporated above. Alternatively, protein sequences may be isolated by
 conventional techniques from the many available strains. The sequences of
 the subunits of two HCMV strains have been published [See, e.g., Mach et
 al, J. Gen. Virol., 67:1461-1467 (1986); Cranage et al, (1986) cited
 above; and Spaete et al, Virol., 167:207-225 (1987)]. These sequences, and
 other known HCMV sequences, can be chemically synthesized by conventional
 methods known to one of skill in the art, [Sambrook et al, "Molecular
 Cloning", 2nd ed., Cold Spring Harbor, N.Y. (1989)] or the sequences
 purchased from commercial sources.
 Similarly fragments of C. pneumoniae surface proteins may be randomly
 fragmented by conventional means, e.g., restriction enzymes, and sequenced
 and chemically synthesized by employing the same protocols.
 In the practice of another embodiment of this invention the HCMV protein or
 fragment and/or the C. pneumoniae immunogenic protein or fragment may be
 produced in vitro by recombinant techniques in large quantities sufficient
 for use in a composition of this invention. Alternatively, a recombinant
 virus or plasmid vector carrying the HCMV protein or fragment and/or C.
 pneumoniae protein or fragment may be prepared by conventional techniques
 of genetic engineering. See, for example, the techniques described in the
 above-cited international patent applications, incorporated by reference
 herein.
 The preparation of a pharmaceutically acceptable composition containing the
 HCMV and/or C. pneumoniae protein(s) or DNA sequence(s), having
 appropriate pH, isotonicity, stability and other conventional
 characteristics is within the skill of the art. These pharmaceutical
 compositions may optionally contain other components, such as adjuvants,
 e.g., aqueous suspensions of aluminum and magnesium hydroxides, and/or
 pharmaceutically acceptable carriers, such as saline.
 It is anticipated that CMV other than HCMV, and various strains of C.
 pneumoniae may be used to design compositions useful to prevent analogous
 conditions in various animal species, i.e., domestic animals and other
 valuable animals.
 III. Methods of the Invention
 Without wishing to be bound by theory, and as described in more detail
 herein, the inventors theorize, and their preliminary results suggest,
 that AT is a multifactorial process, with CMV and/or C. pneumoniae
 infections initiating development of the disease. Reactivation of these
 pathogens as well as genetic factors related to lipid metabolism
 contribute to the disease. CMV is involved in two of the major mechanisms
 that lead to the development of AT. The first is efficient infection of
 cells in the inner layer of arteries in vivo and induction of and injury
 at the area of infection corresponding to early AT lesions. The second
 mechanism is an increase in serum levels of low-density lipoprotein
 cholesterol, a major lipid contributor to AT plaques. It is anticipated
 that chronic infection with periodic reactivations, typical of human CMV
 infections, leads to full-blown AT. Additionally, seroepidemiologic data
 and direct isolation of Chlamydia pneumoniae from human AT plaques also
 suggest the involvement of this bacterium in the development of AT.
 Coinfection or consecutive infection of the arterial wall with CMV and C.
 pneumoniae likely increase the cellular damage of the intima that leads to
 the development of mature AT plaques.
 Thus, the invention provides preventive and therapeutic compositions and
 methods that reduce the incidence of these closely related infections in
 certain subjects and hence those conditions and diseases characterized by
 injury to arterial vessels. Suitable subjects for treatment with the
 compositions and methods of the present invention include mammals,
 preferably humans, (a) who are anticipating immunosuppression due to organ
 or bone marrow transplant (for administration before, during or after the
 transplant); (b) pre-atherosclerotic subjects seeking prophylaxis of
 atherosclerosis, e.g., human children or infants; (c) subjects with
 existing atherosclerosis, e.g., human adults; (d) subjects who have
 restenosis, an accelerated form of AT, which is the narrowing of coronary
 arteries that occurs in about half of patients after coronary atherectomy
 and balloon angioplasty; and (e) subjects seeking to prevent or ameliorate
 the occurrence of restenosis, i.e., patients anticipating coronary
 atherectomy or balloon angioplasty, or post-surgical patients, among
 others. It should be understood that any injury to an arterial vessel,
 either existing or anticipated, can benefit by the use of the present
 invention.
 According to one embodiment of this invention, a subject is administered an
 effective amount of a composition which contains either (a) a human
 cytomegalovirus (HCMV) protein or fragment thereof, or (b) a nucleic acid
 sequence encoding HCMV protein or fragment thereof. In either case, the
 composition is administered in an amount sufficient to induce CMI and
 humoral immunity to CMV infection and prevent, or retard, the development
 of atherosclerotic lesions.
 According to another embodiment of this invention, a subject is
 administered an effective amount of a composition which contains either
 (a) a C. pneumoniae protein or fragment thereof, or (b) a nucleic acid
 sequence encoding said protein or fragment thereof. In either case, the
 composition is administered in an amount sufficient to induce CMI and
 humoral immunity to C. pneumoniae infection and prevent, or retard, the
 development of atherosclerotic lesions.
 According to yet another embodiment of this invention, a subject is
 administered an effective amount of a composition which contains a
 combination of (a) a C. pneumoniae protein or fragment thereof and a human
 cytomegalovirus (HCMV) protein or fragment thereof, or (b) a nucleic acid
 sequence encoding said C. pneumoniae protein or fragment thereof and a
 nucleic acid sequence encoding HCMV protein or fragment thereof. In either
 case, the composition is administered in an amount sufficient to induce
 CMI and humoral immunity to both C. pneumoniae and HCMV infection and
 prevent, or retard, the development of atherosclerotic lesions due to
 either infective agent.
 Still another embodiment of this invention is a treatment method by which a
 subject as described above is administered an effective amount of an
 anti-microbial composition effective in treating C. pneumoniae infection.
 Such a method may further include contemporaneous treatment (i.e., before,
 during or after administration of the anti-microbial agent) with a
 composition containing either a cytomegalovirus (HCMV) protein or fragment
 thereof, or a nucleic acid sequence encoding said HCMV protein or fragment
 thereof, the HCMV-containing composition administered in an amount
 sufficient to induce CMI and humoral immunity to HCMV infection. Such a
 method enables the prevention or retardation of the development of
 atherosclerotic lesions due to either infective agent.
 Still another novel treatment or prophylactic method of this invention
 involves the use of a composition which contains a combination of an
 immunogenic C. pneumoniae protein or fragment thereof and an antiviral
 composition directed against human cytomegalovirus. Another embodiment of
 this method may also employ a nucleic acid sequence encoding the C.
 pneumoniae protein or fragment thereof. In either case, the C.
 pneumoniae-containing composition is administered in an amount sufficient
 to induce CMI and humoral immunity to C. pneumoniae. This method enables
 the prevention or retardation of the development of atherosclerotic
 lesions due to either infective agent.
 According to the present invention, the selected composition described
 above is administered preferably as a vaccine to infants. Subsequent
 periodic boosters may be administered as for other vaccines.
 Alternatively, the method involves administering the compositions of the
 present invention to patients prior to, or immediately after, undergoing
 organ or bone marrow transplants, blood transfusions, or other
 immunosuppressive treatments. Organ transplant patients can develop
 atherosclerosis very quickly in a transplanted organ, either through the
 organ itself or through blood transfusions. Thus, the method of this
 invention is useful for treating organ transplant, or otherwise,
 immunosuppressed patients, following the transplantation.
 Still another alternative method of the invention involves administering
 one of more of the described compositions to a balloon angioplasty or
 coronary atherectomy patient, either before, during or after the surgical
 procedure to prevent or reduce the development of restenosis.
 It is anticipated that therapeutic treatments of compositions described
 above to adult subjects having some existing degree of atherosclerosis is
 also useful to slow the advance of the condition and/or prevent or reduce
 further injury, i.e., the occurrence of atherosclerotic injury to blood
 vessels.
 Another clinical setting for which the methods of the present invention are
 useful is for a woman planning pregnancy. To avoid passage of CMV to a
 fetus, which, when it occurs, causes congenital malformations and heart
 complications, a woman planning a pregnancy may be treated with a CMV
 composition according to this invention.
 For the compositions containing proteins, the preferred dosage ranges from
 about 10 to about 80 micrograms of protein. In a combination composition,
 the dosages of each protein are at the lower end of that range. Preferred
 dosages, in general, are the lower end of that range. When a recombinant
 virus vector is administered as the pharmaceutical composition, a dosage
 of between 10.sup.5 and 10.sup.7 plaque forming units of each vector may
 be used. Preferred dosages are the lower end of that range. When naked DNA
 is administered as the pharmaceutical composition, a dosage of between
 50-200 micrograms may be used. Preferred dosages are the lower end of that
 range. Dosages of conventional antibiotics are administered by
 conventional routes and dosages ordinarily prescribed for such
 pharmaceutical compositions and these may also be determined by the
 attending physician.
 Additional similar, repeated doses of the protein/nucleic acid
 sequence-containing compositions of this invention may also be
 administered at a desired interval, i.e., at 1-10 year intervals, where
 considered desirable by the physician. The dosage regimen involved in the
 method for treating or preventing atherosclerosis with such compositions
 can be determined considering various clinical and environmental factors
 known to affect such administration.
 The route of administration may be oral, nasal or subcutaneous,
 particularly where the virus vector composition is used. However, another
 route of administration may include intramuscular injection. The route of
 administration depends upon the particular composition selected for such
 use, and may readily be selected by the attending physician.
 The following examples illustrate various aspects of this invention and do
 not limit the invention, the scope of which is embodied in the appended
 claims. BALB/c and C57BL/6 mice were used in these experiments.
 EXAMPLE 1
 Development of Early Atherosclerotic Lesions in Mice Infected with Murine
 CMV
 A. Infection
 Mice were infected with murine CMV (Smith strain; ATCC VR-194) at a dose of
 1.times.10.sup.6 pfu via the intraperitoneal (i.p.) route on day 1.
 Fifty-five days post infection, the mice were sacrificed and the heart was
 obtained and sectioned above the right and left auricles. These tissues
 were subjected to a conventional immunofluorescence assay using anti-MCMV
 polyclonal antibody.
 Endothelial cells (EC) and smooth muscle cells (SMC) of the mouse aortic
 wall express viral antigens after MCMV infection. Accumulation of
 inflammatory and SMCs in the aortic lumen at the site of viral antigen
 expression has been observed and defined as early atherosclerotic lesions.
 B. Additional Experiments
 In the next series of experiments, mice were infected i.p. with the
 parental strain of MCMV at a dose of 1.times.10.sup.4 pfu,
 .gamma.-irradiated at the time of infection (day 0) or non-irradiated, and
 fed an atherogenic or normal diet. Mice were sacrificed on day 36, hearts
 were obtained and sectioned for viral antigen expression and pathological
 changes.
 FIGS. 1A-1C show sections of a naive mouse, with no reactivity with the
 anti-MCMV antibody in the immunofluorescence (IF) test and with no
 inflammatory reaction in the aortic wall and lumen. FIGS. 1G-1I show large
 areas of virus antigen expression with adherent inflammatory cells on the
 luminal surface of the aorta from a mouse infected, immunosuppressed and
 fed a normal diet. The inflammatory infiltrate consists of many large
 lymphocytes and plasma cells with a few monocytes and peripheral
 mononuclear cells. Similar expression of viral antigens and inflammatory
 reactions was detected in the aortic wall of a mouse infected,
 immunosuppressed, and fed an atherogenic diet. Small areas of the aortic
 wall with expression of MCMV antigens and accumulation of inflammatory
 cells at a site of viral antigen expression in a mouse infected, not
 immunosuppressed, and fed cholesterol or normal diet were also observed.
 Mice not infected but fed a cholesterol diet, or not infected but
 irradiated, showed neither viral antigen expression nor inflammatory
 changes in the aorta.
 These data show that an acute MCMV infection of mice induces a vascular
 wall inflammation that may play a role in the immune injury of the
 vascular structure and thus initiate the development of AT.
 C. Immunosuppression
 Mice were infected with murine CMV (Smith strain) by the same procedure as
 described above. The mice were exposed to gamma-irradiation (500 rad) at
 the time of infection. Thirty-six days post infection, the mice were
 sacrificed and various tissues were examined as described above.
 Immunosuppression by gamma-irradiation at the time of virus infection
 increased the virus titer in salivary glands from 1-5.times.10.sup.3 to
 0.5-2.5.times.10.sup.4 pfu/salivary glands and extended the size of the
 expression of viral antigens in the aortic wall from about 2/10 of aortic
 wall (FIG. 1J) to 5/10 (FIG. 1G) or 7/10 (FIG. 1D). Accumulation of
 inflammatory cells in the lumen and in the periaortic area was seen at the
 region where viral antigen expression was detected in mice not irradiated
 (FIGS. 1J-1L). More irradiated inflammatory cells were seen throughout the
 aortic lumen.
 D. High Cholesterol Diet
 Another group of mice was fed a high cholesterol Western diet designed to
 elevate plasma cholesterol levels for 1 week, then infected with MCMV as
 described in the assays above. Fifty-five days post infection, the mice
 were sacrificed and vascular tissues were examined as described above.
 The high cholesterol diet had no significant synergistic effect on the
 development of early atherosclerotic lesions at the site of viral
 antigens.
 E. Serum Cholesterol--in vivo Assay
 Three groups of mice were treated as follows: One group was fed with a high
 cholesterol diet and infected with MCMV; a second group was fed with a
 normal diet and infected with MCMV, and a third group was not infected and
 used as a control. After fifty-five days post infection (or on the
 analogous day for the controls), serum from each group of mice was
 collected and assayed for total cholesterol and triglycerides using a
 commercially available (Sigma) kit.
 Analysis of the sera of each group demonstrated that the serum level of low
 density cholesterol (LDC), a major lipid component of lipid-leded
 macrophages and other cells in the atherosclerotic plaques, is
 significantly increased in mice infected with MCMV, either fed with
 atherosclerotic diet (as in Part C above) or not, as compared with
 uninfected mice.
 F. Lipid Metabolism--in vitro Assay
 A human arterial smooth muscle cell line (cells No. 101 obtained from Dr.
 Elliot Levine, The Wistar Institute, Philadelphia, Pa.) was infected in
 vitro with human CMV at a multiplicity of infection (MOI) of 1-2. After
 five days, the cells were extracted with chloroform-methanol 2:1 and lipid
 extract analyzed for total and free cholesterol using standard
 methodology.
 In the infected cells, the esterified cholesterol component of the total
 cholesterol was 29.7%, as compared with the undetectable level of
 esterified cholesterol in uninfected cells. These results, and those of
 part D above, indicate that both HCMV and MCMV disturb lipid metabolism in
 infected cells and change it in the direction of the accumulation of lipid
 elements, contributing to the development of atherosclerotic plaques.
 EXAMPLE 2
 Treating Mice with CMV Compositions
 Tissue culture-adapted (attenuated) MCMV (ATCC No. VR-104) was grown in
 tissue culture fibroblasts prepared from BALB/c embryos by conventional
 techniques. Alternatively, the tissue culture-adapted MCMV was inactivated
 by treatment with heat at 56.degree. C. for 30 minutes.
 BALB/c mice (10 in each group) were immunized i.p. with either the live
 attenuated MCMV at a dose of 1.times.10.sup.6 pfu in a volume of 0.1 ml or
 the inactivated virus (0.1 ml) on day 0. Mice received a booster
 inoculation three weeks after the first inoculation. A group of mice was
 not immunized for use as controls.
 Five weeks after the second inoculation, all mice were challenged with
 3.times.10.sup.6 pfu live virulent MCMV, which was grown in vivo in murine
 salivary glands. On day 10 after the challenge, the mice were sacrificed
 and their vascular tissue examined by immunofluorescence (IF) assay using
 an anti-murine cytomegalovirus (MCMV) polyclonal mouse serum.
 The control mice were found to have viral antigen expression in the aortic
 wall and in smooth muscle cells of the heart, and early atherosclerotic
 lesions. However, mice immunized with either live attenuated, or heat
 inactivated, attenuated MCMV were found to have no such lesions. Thus, the
 live or inactivated attenuated MCMV compositions protected the mice from
 the development of viral antigen expression and early atherosclerotic
 lesions following live virulent viral infection, indicating that the
 development of atherosclerosis is preventable.
 EXAMPLE 3
 Mice Infected with HCMV
 This example demonstrates the effect on the development of early
 atherosclerotic lesions in the mouse aortic intima of infection of mice
 with HCMV.
 HCMV does not replicate in mouse cells, but expresses immediate early (IE)
 antigens. The expression of CMV IE antigens may be sufficient to cause
 such lesions. This theory is tested by examining attachment of
 inflammatory cells and SMCs to the intima, expression of IE antigens and
 cellular adhesion molecules in the ECs and SMCs.
 BALB/c mice are injected i.v. with live purified HCMV at a dose of 10.sup.4
 -10.sup.6 pfu. Two to twenty days after infection, mice are sacrificed,
 heart sections obtained as described above, and expression of IE antigens
 is tested by IF assay using a monoclonal antibody specific to IE antigens
 (e.g., E13, Chemicon). The presence of early atherosclerotic lesions is
 analyzed on consecutive sections stained with hematoxilin-eosin. The
 presence of significant lesions indicates that IE expression alone can
 cause early atherosclerotic damage.
 EXAMPLE 4
 Latency Murine CMV Model of HCMV Infection
 This experiment describes a latency CMV murine model which mimics the
 course of HCMV infection in humans:
 Mice are inoculated with MCMV, thereby establishing an acute infection, and
 tissue is tested for atherosclerotic lesions and expression of CMV viral
 antigens as described above. After the virus becomes latent, i.e., 3-4
 weeks, the same tests are performed and viral antigens are not expressed.
 Mice are then immunosuppressed, preferably by gamma irradiation to
 reactivate the CMV infection. The same tests as described above are
 performed and viral antigens are again expressed. After the virus again
 becomes latent, the tissues are examined and viral antigens are no longer
 expressed.
 A periodic increase in the severity of atherosclerotic lesions accompanies
 the periodicity of virus reactivation.
 A second set of mice are similarly treated as above, but immunized with the
 selected CMV composition either before acute infection or in the latency
 stage. Comparison of atherosclerotic lesions in control mice vs. immunized
 mice at the various stages of the model demonstrates the efficacy of
 immunizing the mice with a CMV composition of this invention to prevent
 the development or further progression of such lesions.
 EXAMPLE 5
 Rabbit Model of HCMV
 The rabbit is a well-established model for experimental atherosclerosis.
 Rabbits are infected with HCMV or MCMV and the aortic tissue tested as
 above described for expression of IE antigens, as well as for the
 development of fatty streaks, i.e., the early lesions of atherosclerosis
 (AT). Since the rabbit aorta is considerably larger than the mouse aorta,
 the fatty streaks are visible macroscopically.
 Once AT lesions develop in the rabbit aorta after CMV infection, the CMV
 compositions described herein are tested for the ability to protect the
 animal against further progression of AT lesions.
 EXAMPLE 6
 Development of Early Atherosclerotic Plaques and Antigens
 A. BALB/c and C57BL/6 mice were infected i.p. with either 1.times.10.sup.4
 pfu a recombinant MCMV expressing the bacterial .beta.-galactosidase
 (lacZ) gene (MCMV-lacZ) [C. A. Stoddart et al, J. Virol., 68:6243-6253
 (1994)(gift of Dr. E. Mocarski, Stanford University, Stanford, Calif.), or
 with the same dosage of the parental MCMV (Smith strain, ATCC VR-194).
 BALB/c mice were infected i.p. with 1.times.10.sup.4 pfu of MCMV-lacZ
 virus, and immunosuppressed by .gamma.-irradiation (450 rad) at the time
 of infection to slow down virus clearance, and fed either an atherogenic
 diet (8% coconut oil, 2% soybean oil, 0.5% cholesterol) or a normal mouse
 diet. Uninfected mice were treated similarly. Mice were sacrificed 25 days
 after virus infection, and hearts were obtained and sectioned as described
 in B. Paigen et al, Atheroscl., 68:1614-1620 (1987). Sections were treated
 with X-gal to detect lacZ-expressing cells indicating virus replication,
 or stained with H&E for pathological changes or with oil red 0 for lipid
 accumulation in the tissues.
 An inflammatory response developed in the aortic wall of the mice, similar
 to early AT lesions in humans, and lipid metabolism shifted to high levels
 of serum LDL-C, a major contributor to AT plaques. MCMV-lacZ replicated
 and induced inflammatory reactions at the site of replication in the
 aortic wall (FIG. 2A).
 In mice infected with MCMV-lacZ, irradiated and fed an atherogenic diet,
 lacZ was expressed in the endothelial and smooth muscle cells of the aorta
 (FIGS. 2A-2C). An early AT plaque at the site of virus replication with
 inflammatory cells in the lesion and below it through the thickness of the
 aorta was also seen (FIG. 2A-2C). Oil red O staining revealed lipid in
 this same area (not shown). Mice infected with the virus, irradiated and
 fed a normal mouse diet also showed lacZ-expression in the salivary gland
 and arterial adventitia associated with adventitial vacuoles, in a few
 muscle cells in the heart (not shown). Mice infected with the virus,
 irradiated and fed a normal mouse diet also showed lacZ-expression in the
 salivary gland and arterial adventitia associated with adventitial
 vacuoles, in a few muscle cells in the heart (not shown), as well as in
 the subendothelial space of a pulmonary vein (not shown). Uninfected and
 irradiated mice fed either an atherogenic or a normal diet showed no
 pathological changes.
 These results provide the first evidence that acute MCMV infection induces
 an inflammatory response in the aortic wall of mice, resembling early AT
 lesions found in humans.
 EXAMPLE 7
 Influence of MCMV on Lipid Metabolism
 To test how MCMV infection influences the lipid metabolism of mice fed an
 atherogenic or normal diet, sera were obtained from mice, and total
 cholesterol (TC) and high density lipoprotein cholesterol (HDL-C)
 determined. Results showed that TC levels in mice infected but fed normal
 diet were very similar to those in the naive mice, while TC levels were
 higher in mice fed the cholesterol diet, either infected or uninfected.
 The results also showed that MCMV-infected normocholesterolemic mice had a
 significantly higher percentage of LDL-C in the serum than naive mice.
 Similarly, significantly higher LDL-C levels were found in mice fed an
 atherogenic diet and infected than in mice fed the same diet but
 uninfected. The results indicate that MCMV infection in mice disturbs
 lipid metabolism and shifts it to higher levels of LDL-C.
 EXAMPLE 8
 Effects of C. pneumoniae Infection
 BALB/c and C57BL/6 mice were infected intranasally with C. pneumoniae
 (10.sup.7 TCID.sub.50) and sacrificed on days 3, 7, 11, and 27. Hearts
 were obtained and sectioned for C. pneumoniae antigen expression and
 pathological changes. FIG. 3A shows expression of C. pneumoniae antigens
 in the aortic wall and FIG. 3B shows an inflammatory reaction at the site
 of C. pneumoniae replication.
 Human arterial smooth muscle cells (HASMC, obtained from Dr. E. Levine, The
 Wistar Institute) infected with human CMV (HCMV) or C. pneumoniae
 expressed HCMV antigens (FIG. 4A) or C. pneumoniae antigens (FIG. 4B),
 indicating the replication of each pathogen in these cells.
 EXAMPLE 9
 The Causative Role of Acute and Reactivated CMV Infection in the Initiation
 and Development of Early and Mature AT Plaques
 The following experiment is conducted to establish to what extent latent
 and reactivated MCMV infection, alone or in combination with an
 atherosclerotic diet, increases the number and severity of AT plaques in
 mice, and to analyze the role of CMV and contribution of
 hypercholesterolemia to the development of AT initiated by CMV.
 A. Sixteen groups (see Table 1) of C57BL/6 normal female mice (even-number
 and odd-number groups of 30 and 40 mice, respectively, 8-10 weeks of age,
 Charles Rivers Laboratory) with no genetic disorders are infected i.p.
 with MCMV grown previously in salivary glands, at a dose 1.times.10.sup.4
 pfu/mouse. A group of these mice are immunosuppressed by
 .gamma.-irradiation (450 rads) at the time of infection and a group of
 these mice are not irradiated. Each group is then fed an atherogenic or a
 normal diet throughout the experiment.
 Mice in even-number groups are further irradiated (450 rads) 2 times during
 the observation period of 300 days to reactivate latent CMV infection. The
 in vivo mouse model for the acute (infectious virus detectable), latent
 infectious virus no longer detectable), and reactivated (infectious virus
 detectable again) phases of MCMV infection in the salivary glands, lungs,
 spleens, and kidneys has already been developed.
 Ten mice of odd-number groups (acute infection) are sacrificed on days 25,
 50, 225, and 300, while 10 mice of even-number groups (latent or
 reactivated infection) are sacrificed on days 125, 225, and 300. Hearts
 are then processed: (i) for the qualitative and quantitative evaluation of
 AT plaques and for the determination of lipid accumulation by staining
 sections of aorta with H&E and oil red O; (ii) for the presence of MCMV
 antigens in sections by immunofluorescence assay using hyperimmune serum
 from MCMV-infected mice; (iii) for the presence of viral mRNA and DNA
 during acute, latent, and reactivated stages of infection using RNA and
 DNA extracted from the upper part of the hearts (not sectioned) for RT-PCR
 and PCR analysis with appropriate primers for the immediate early or late
 genes; (iv) for the expression of mRNA and/or proteins of the Hsp family,
 and growth factors, cytokines, adhesion molecules, e.g., vascular
 endothelial growth factor, Le.sup.x, ICAM-1, VCAM-1, NO, IL-12, IL-8,
 IL-10, and IFN-.gamma..
 Mouse sera are tested for the percentage of LDL-C by determining TC and
 HDL-C, as well as for serum ICAM-1 and serum VCAM-1 by ELISA.
 Analysis of the aorta obtained from genetically normal mice reveals the
 extent to which acute MCMV infection initiates the development of early AT
 plaques; whether a latent MCMV infection with periodic reactivations, a
 common characteristic of HCMV infections in humans, furthers early plaques
 into mature, complicated plaques. The influence of a cholesterol diet
 applied throughout the experiment is evaluated, and immunosuppression
 applied at the time of infection to prolong initial virus replication, on
 the number and severity of AT plaques in the aorta.
 Finally, the extent to which MCMV immediate early and late gene DNA and
 mRNA are present in the acute, latent, and reactivated stages of infection
 in the aortic wall of the mice is observed and the cellular mechanisms
 involved in the development of AT.
 Analysis of mouse sera provides further information about the
 pathomechanisms of this complex disease, and is predicted to reveal that
 acute and chronic MCMV infection shifts lipid metabolism to a high
 percentage of LDL-C in genetically normal mice, as suggested by the
 preliminary experiments.
 B. ApoE-deficient and/or LDL receptor-deficient mice (Jackson Laboratories)
 are infected or uninfected, and treated as described for normal mice in
 odd-number groups in Table 1. Each group consists of 10 mice, of which 5
 are sacrificed on day 25 and 5 are sacrificed on day 50 after initial
 infection and treatment. Hearts and sera are obtained and examined as
 described for genetically normal mice.
 The qualitative and quantitative analysis of AT plaques, as well as the
 kinetics of development, indicate how MCMV infection alters the number and
 complexity of these plaques in the genetically hypercholesterolemic mice,
 which show strong similarities to genetic deficiencies in humans. Since
 the genetically hypercholesterolemic mice develop mature AT plaques after
 MCMV infection, the ratio of apoptotic cells is determined by
 terminal-deoxynucleotidyl transferase mediated DNA-end labeling (TUNEL).
 Preliminary results show that MCMV replicates in the aortic wall of BALB/c
 and C57/BL mice
 TABLE 1
 Treatment protocol for normal mice
 Days of virus
 reactivation
 by .gamma.-
 Immediate treatment irradiation
 Groups I* D* G* 100 200
 1 + -- -- -- --
 2 + -- -- + +
 3 + -- + -- --
 4 + -- + + +
 5 + + -- -- --
 6 + + -- + +
 7 + + + -- --
 8 + + + + +
 9 -- -- + -- --
 10 -- -- + + +
 11 -- + -- -- --
 12 -- + -- + +
 13 -- + + -- --
 14 -- + + + +
 15 -- -- -- -- --
 16 -- -- -- + +
 *I - intraperitoneal infection with MCMV; D = cholesterol diet; G
 = .gamma.-irradiated
 EXAMPLE 10
 Contribution of Coinfection with MCMV and C. pneumoniae to the Development
 of AT Lesions
 Eleven groups of C57/BL6 mice (20/group) (Charles River Laboratory) are
 infected and treated as outlined in Table 2. Inoculation with the second
 pathogen is carried out on day 25 after inoculation with the first
 pathogen to avoid possible interference between the susceptibility of
 cells to these two pathogens, and to allow sufficient time for the
 development of an inflammatory response to the first pathogen.
 Ten mice are sacrificed 25 days after inoculation with the second pathogen.
 As controls, mice infected with C. pneumoniae are tested with appropriate
 antibiotics against C. pneumoniae to establish C. pneumoniae-specificity
 of the lesions in mice infected with the bacterium, but not treated with
 antibiotics.
 Hearts are processed for qualitative and quantitative evaluation of AT
 lesions; presence of MCMV and C. pneumoniae antigens in the aortic wall by
 immunofluorescence assay; presence of cellular Hsp 70 family, vascular
 endothelial growth factor, Le.sup.x, ICAM-1, VCAM-1, NO, IL-12, IL-8,
 IL-10, and IFN-.gamma., and presence of apoptotic cells in the lesions by
 TUNEL.
 Sera are processed for determination of TC and LDL-C levels; and serum
 ICAM-1, serum VCAM-1 levels and Hsp 70 antibodies. Lungs and livers are
 processed for infectious MCMV and C. pneumoniae.
 These analyses show whether the injury initiated by MCMV infection
 progresses to accelerated and/or more complicated lesions as a result of
 C. pneumoniae superinfection, and whether primary C. pneumoniae infection
 initiates aortic wall injury that develops into AT upon secondary MCMV
 infection. Also, these analyses show whether MCMV or C. pneumoniae-induced
 apoptosis is involved in the development of mature plaques, and whether
 cellular molecules are induced that are involved in the development of AT.
 A decrease of susceptibility of aortic wall (or the whole mouse) to the
 second pathogen due to induction of some cellular factors (e.g.
 IFN-.alpha.) by the first pathogen is expected to be only transient.
 Scheduling the second infection at different times after the first
 infection overcomes this difficulty.
 TABLE 2
 Treatment protocol for MCMV- and C. pneumoniae-
 coinfected mice
 Immediate treatment Later Treatment
 Groups Infection .gamma.-irradiation Infection
 .gamma.-irradiation
 1 MCMV + C. pneumoniae --
 2 MCMV + -- --
 3 -- + C. pneumoniae --
 4 -- -- C. pneumoniae --
 5 -- + -- --
 6 C. pneumoniae -- MCMV +
 7 C. pneumoniae -- -- --
 8 -- -- MCMV +
 9 -- -- MCMV --
 10 -- -- -- +
 11 -- -- -- --
 EXAMPLE 11
 Coinfection of Human Arterial Endothelial and Smooth Muscle Cells in vitro
 with HCMV and C. pneumoniae Influences Their Replication
 Both HCMV and C. pneumoniae replicate in human arterial endothelial and
 smooth muscle cells. The interaction between the replication of these two
 pathogens and the effects of the coinfecting pathogens on the infected
 endothelial, smooth muscle, or any other cells are addressed as follows.
 A. Coinfection Experiments
 The following cells cultures: (a) human aortic endothelial cells (obtained
 from Dr. Jay Nelson, Oregon Health Sciences University, Portland, Oreg.);
 (b) human iliac arterial endothelial cells; and (c) human iliac arterial
 smooth muscle cells (both obtained from Dr. Eliott Levine, The Wistar
 Institute), are treated as follows.
 Cell cultures are infected with HCMV and C. pneumoniae simultaneously.
 Other cell cultures are infected consecutively (with HCMV followed by C.
 pneumoniae, with C. pneumoniae followed by HCMV. Still another set of cell
 cultures is infected with only one of these pathogens. Control cultures
 are uninfected.
 Co-infected cultures are tested for replication kinetics of the two
 pathogens, by quantitating the expression of C. pneumoniae and
 CMV-immediate antigens (IE) in immunofluorescence tests using monoclonal
 antibodies specific to these antigens, and by quantitating the production
 of infectious CMV and C. pneumoniae in susceptible cell cultures. The
 quantitative expression of various adhesion molecules are tested by
 immunochemical staining. Production of various cellular factors, including
 the Hsp60 family, NO synthetase, and vascular endothelial growth factor
 are tested by immunological methods. The ratio of apoptotic cells in
 infected cultures is tested by sodium-iodide staining with subsequent
 flow-cytometric determination of the percentage of hypodiploid-nuclei.
 For assessment of colocalization of apoptotic cells with infected cells, a
 double-staining process including TUNEL and pathogen-specific
 immunostaining is performed. Adhesion molecules, as well as Hsp60, are
 thought to be involved in the development of AT. The presence of apoptotic
 cells in human AT lesions has been demonstrated and MCMV is known to
 induce apoptosis of T cells.
 These experiments reveal: (a) how these two pathogens influence the
 replication of each other; (b) how the expression of adhesion molecules
 and various cellular factors is influenced by these co-infecting
 pathogens, as compared with that of the single pathogen-infected cultures;
 and (c) how apoptosis is induced by the two pathogens in cells infected
 with one pathogen or with two pathways sequentially.
 B. Transfection and Infection
 Because the human CMV-IE proteins, especially the IE2 protein, are potent
 transactivators of heterologous promoters, C. pneumoniae replication and
 the production of adhesion molecules and various growth factors in cell
 lines stably transfected with eukaryotic expression plasmids expressing
 the IE1 and 2 (pRL43a) or only the IE2 protein of HCMV (pMC18) [both
 obtained from Dr. Gary Hayward of Johns Hopkins University, Baltimore,
 Md.)] are analyzed. A stably transfected rhabdomyoma cell line was
 transfected with these plasmids. The parental cell lines are tested for
 susceptibility to C. pneumoniae infection. Stably transfected Hep2 or
 McCoy cells, which are highly susceptible for C. pneumoniae are also
 transfected.
 Transfected cultures are tested for replication of C. pneumoniae as well as
 for the expression of adhesion molecules and various cellular factors and
 mechanism(s) as described for the co-infection experiments.
 All published documents are incorporated by reference herein. 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.