WO1994010344A1

WO1994010344A1 - Diagnostic reagents and their use in the detection of human herpesvirus 7

Info

Publication number: WO1994010344A1
Application number: PCT/US1993/010106
Authority: WO
Inventors: Zwi N. Berneman; Paolo Lusso; Marvin S. Reitz, Jr.; Robert C. Gallo
Original assignee: The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
Priority date: 1992-11-02
Filing date: 1993-10-21
Publication date: 1994-05-11
Also published as: AU5447194A

Abstract

The present invention provides probes and primers for detection of human herpesvirus 7 nucleic acids. The probes are both oligonucleotides and plasmid probes. Methods for detecting human herpesvirus 7 nucleic acids are also provided. The methods employ the probes of the present invention and may include nucleic amplification with the described primers. Also provided are human herpesvirus 7 infected continuous cell lines and methods to infect continuous cell lines with human herpesvirus 7.

Description

DIAGNOSTIC REAGENTS AND THEIR USE IN THE DETECTION OF HUMAN HERPESVIRUS

This application is a continuation-in-part application of U.S. Serial No. 07/971,102, filed November 2,

1992, and U.S. Serial No. 08/072,567, filed June 4, 1993, both of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Herpesviruses are a family of viruses which infect humans and other primates, as well as other animals. The viruses are double stranded DNA viruses having a capsid and envelope. Seven species of herpesviruses are known to infect humans and are designated human herpesvirus 1-7.

The herpesvirus family is a significant cause of human illness. Herpes simplex virus types 1 and 2, Varicella Zoster virus, human cytomegalovirus, and Epstein Barr virus are members of the herpesvirus family. The viruses cause a variety of diseases, including cutaneous herpes simplex lesions, ononucleosis, chicken pox, and disseminated multisystem infections in immunocomproroised persons.

Human herpesvirus 6 (HHV-6) is a recently described member of the herpesvirus family. The virus is T- lymphotropic. The virus is primarily associated with exanthem subitum. It is also the cause of 10% of Epstein-Barr virus- and human cytomegalovirus-negative and heterophile antibody negative cases of infectious mononucleosis. Additionally, it has been implicated as an etiologic agent of hepatitis, meningitis and meningo-encephalitis, as well as a number of acute febrile illnesses in children. It has been associated with a number of life-threatening complications in transplant recipients, including interstitial pneumonitis and graft failure after bone marrow transplantation. "Human Herpesvirus-6: Epidemiology, Molecular Biology and Clinical Pathology" in Perspectives in Medical virology. Vol. 4. Ablaεhi, Krueger, and Salahuddin (eds.), Elsevier Science Publishers, Amsterdam 1992.

The most recently described human herpesvirus species is human herpesvirus 7 (HHV-7) . The virus was isolated from activated CD4⁺ T-lymphocytes of a healthy adult. Frenkel et al., Proc. Natl. Acad. Sci. USA. 87:748-752 (1990). HHV-7 has not yet been associated with human disease, yet apparently may persistently infect humans. Wyatt and Frenkel, J. Virol.. 66:3206-3209 (1992). Apparently, exposure to HHV-7 occurs early in life, but later than HHV-6 exposure. Wyatt et al., J. Virol.. 65:6260-6265 (1991). Although a causal relationship was not advanced, HHV-7 have been isolated from a patient having chronic fatigue syndrome. Berneman et al., J. Infect. Dis.. 166:690-691 (1992).

Following the discovery of HHV-7 by Frenkel et al., supra. new independent strains have been isolated. Berneman et al..supra. One new strain (JI) was isolated from a patient having chronic fatigue syndrome (CFS) . Peripheral blood mononuclear cells infected in vitro were primarily, but not exclusively, CD4⁺.

Human herpesvirus 7 exhibits significant DNA homology to two other human herpesviruses, HHV-6 and human cytomegalovirus. Cells infected with HHV-7 also cross-react with some antibodies to cells infected with HHV-6. Wyatt et al., J. Virol.. 65:6260-6265 (1991) and Berneman et al., Proc. Natl. Acad. Sci. USA. 89:10552-10556 (1992). Both HHV-6 and human cytomegalovirus are associated with diseases with high morbidity. Because HHV-7 shares substantial homology with HHV-6 and human cytomegalovirus, and HHV-7 apparently is a constitutive organism in many humans, it is important to be able to differentiate between these viruses by rapid and reproducible means. Otherwise, HHV-7 present in a patient may cause false positive results in diagnostic testing.

It has been difficult to develop specific tests for HHV-7 because the virus has only been infected into primary peripheral blood or cord blood mononuclear cells. These cells require separation from other blood components and, as the cells cannot continuously propagate, they must be replenished. Also, infection in cord blood cells may vary considerably. Use of primary peripheral blood also may be infected with other human herpesviruses, e.g., Epstein-Barr virus, human cytomegalovirus, and HHV-6. A cell line infected with HHV-7 would provide a ready source of uncontaminated HHV-7-infected cells, virus particles and viral proteins for both diagnostic and potential therapeutic uses.

Blocking viral receptors on T-cells has been attempted to inhibit infection of different viruses. Blocking strategies to inhibit HIV infection have been attempted as AIDS and other retroviral infections have proven to be relatively resistant to other methods of treatment. Presently approved methods of HIV treatment employ synthetic pharmaceuticals, such as 3'-azido-2',3•-dideoxythy idine (AZT) and 2' ,3'-dideoxyinosine (ddl) , to block reverse transcriptase. While these medications have been shown to decrease morbidity and increase life expectancy of HIV infected individuals, neither treatment is curative. Also, these drugs have serious side-effects that are dose limiting and preclude treatment of many patients.

Many methods of blocking HIV infection have related to finding molecules which competitively inhibit HIV-1 binding to its T cell receptor. CD4 is the HIV-1 receptor on human T- cells. In one attempt by Darr et al., Proc. Natl. Acad. Sci. 87: 6574-6578 (1990) to block HIV infection in humans, soluble CD4 was given to five patients. Unfortunately, viral serum titers did not decrease during therapy. It was speculated that the lower efficacy of soluble CD4 to block HIV infection in vivo was due to lower binding affinities between the soluble CD4 molecules and the gpl20 envelope protein of primary HIV-1 isolates. It was suggested that laboratory isolates of HIV-1 had greater affinities for CD4 than primary isolates taken from individual patients. However, bioactive compounds with the ability to block HIV from binding the CD4 T cell receptor, such as isolated CD4, have been given to patients with little success.

What is needed in the art are methods of specifically and reproducibly detecting the presence of HHV-7 infected cells. Also, methods for blocking the binding of viruses to CD4 T-cell receptors could provide valuable therapeutic alternatives to presently available treatment of many diseases. A convenient source of HHV-7 viral particles, HHV-7 viral DNA, and HHV-7 viral proteins is also needed. Surprisingly, the present invention fulfills these and other related needs.

SUMMARY OF THE INVENTION

The present invention provides compositions for the detection of HHV-7 nucleic acid in a sample. The probes comprise nucleic acid sequences homologous to HHV-7 nucleic acid. The probes may be plasmids or bacteriophage. Also provided are methods for detecting the presence of human herpesvirus 7 genomic nucleic acid in a sample. The methods generally comprise (a) mixing the sample with a probe comprise nucleic acid sequences homologous to HHV-7 nucleic acid; and (b) detecting hybridization between the nucleic acid in the sample and the probe. The probes may be labelled or bound to a solid support. HHV-7 nucleic acid in the sample may be amplified prior to hybridization. The methods may be employed in clinical samples.

Kits are also provided for detecting the presence of HHV-7 in experimental and clinical samples. The kits include a probe of the present invention. The kits may also include primers for amplification of HHV-7 DNA.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1. Electron microscopy of peripheral blood CD4⁺ T cells infected with HHV-7_AL. Mature virus particles of HHV-7_JB in the extracellular space. Figs. 2A-C. Syncytia formation induced by HHV-7 infection. (A) Giant multinucleated cells in cultures of Sup-Tl cells, 5 days after infection with HHV-7^. (B) Thin sections of an infected syncytial cell, containing several nuclei and virus particles at various stages of maturation. (C) Nuclear IF staining with MAb 9A5D12 in activated peripheral blood CD4⁺ T lymphocytes.

Figs. 3A-B. PCR amplification of DNA from HHV-6- and HHV-7-infected cells. Ethidium bromide-staining to visualize: (A) the products of PCR amplification performed with two nested pairs of primers specific for HHV-6. Outer primer pair:

EX1 (S'-GCGTTTTCAGTGTGTAGTTCGGCA-S' - SEQ ID NO:16) and EX2 (5^»-TGGCCGCATTCGTACAGATACGGAGG-3' - SEQ ID NO:17) ;.inner primer pair

IN3 (S ' -GCTAGAACGTATTTGCTGCAGAACG-S ' - SEQ ID NO: 18) and

IN (S'-ATCCGAAACAACΓGTCTGACΓGGCA-S¹ - SEQ ID NO:19). (B) The products of one round of PCR amplification with primers specific for HHV-7. C «■ negative control (no-DNA sample) . M = 123-bp-ladder molecular weight markers.

Figs. 4A-B. Southern blot analysis of three HHV-7 isolates. DNA extracted from Sup-Tl cells infected with HHV-?^, HHV-7JB and HHV-7_αι was digested with (A) Hin lll or (B) BairHI , and resolved on a 0.8% agarose gel. After Southern blotting, the DNA was hybridized with different ³²P-labelled HHV-7-specific probes, as indicated.

Fig. 5. FacScan histograms illustrating the selective down-regulation of CD4 in the course of HHV-7 infection of purified normal peripheral blood CD4⁺ T cells. Im unofluorescence was assessed with a FacScan* analyzer

(Becton Dickinson Immunocytometry) , using appropriate gating to eliminate dead cells and debris.

Fig. 6. Line graph illustrating the dose-dependent inhibition of HHV-7 infection in purified normal CD4⁺ T cells by anti-CD4 monoclonal antibodies (o = Leu3a; o = 0KT4a; ♦ = OKT4) . Monoclonal antibodies to other cell surface determinants (A = HLA-DR [Chemicon]; * = CD3 (0KT3; Ortho) ; • = anti-/?₂ microglobulin) , as well as control monoclonal antibodies M26 (♦) and M90 (■) ; directed against the p24 and gpl20 proteins of HIV-1, respectively) . Data represent the mean values from at least three separate experiments.

Fig. 7. Line graph illustrating the dose-dependent inhibition of HHV-7 infection in purified normal CD4⁺ T cells by recombinant soluble CD4 (sCD4) . Three isolates of HHV-7 were tested: ■ = HHV-?^; ♦ - HHV-7_JB; A = HHV-7_αι. Data represent the mean values of two separate experiments. The virus stock (10⁵ cell culture infectious doses (CCID)) was pretreated with sCD4 for 30 min at room temperature and subsequently used to infect 1 x 10⁶ normal peripheral blood CD4⁺ T cells in 24-well plates.

Fig. 8. Bar chart illustrating the inhibition of HHV-7 binding and internalization in normal CD4⁺ T cells by anti-CD4 monoclonal antibody 0KT4a. The bar chart reports the quantitative results of densitometric scans from a Southern Blot autoradiograph. Bar No. 1 is the amount of HHV-7 probe hybridization to DNA isolated from CD4⁺ T cells exposed for 1 hour at 37°C to HHV-7_AL. Bar No. 2 is the amount of HHV-7 probe hybridization to CD4⁺ T cells pretreated with antibody OKT4a at 5μg/ml for 30 minutes at room temperature and then exposed to HHV-7_AL. Bar No. 3 is the amount of HHV-7 probe hybridization to untreated CD4⁺ T cells.

Fig. 9. Line graph illustrating the dose-dependent inhibition of HHV-7 infection by the gpl20 glycoprotein derived from three different HIV-1 isolates (■ = IIIB; ♦ = 451; A = BaL) . Native gpl20 protein was purified by immunoaffinity chromatography from infected CD4⁺ T-cell lines, as reported (Gallo, R. C. et al. Science, 224:500-504 (1984)). The native gpl20 envelope glycoprotein of HIV-1 was isolated from cell lysates of a hvunan T-cell line chronically infected by HIV using immunoaffinity chromatography columns coated with anti-gpl20 murine monoclonal antibodies, a technique that is well known to experts in the art. A higher point on the graph represents greater inhibition of HHV-7 by gpl20. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides nucleic acid sequences useful as probes for the detection of HHV-7 in samples. Generally, the samples will be biological samples and may include solid tissues, solid tissue homogenates, blood cells, physiological fluids or secretions, or cell lysates. One such probe is an isolated and purified nucleic acid sequence (SEQ ID NO:l) derived from amplification of HHV-7 DNA. The DNA sequence of SEQ ID N0:1 may be employed as a probe specific for HHV-7 or may serve as the model for the development of other probes or amplification primers. Other probes include a variety of nucleic acid sequences contained in plasmids and bacteriophage as described in more detail below.

The present invention also provides primers and probes for the detection of human herpesvirus 7 (which hereinafter may be referred to as HHV-7) infection of host cells. The primers and probes hybridize to portions of the HHV-7 genome which exhibit differing degrees of homology to human herpesvirus 6 and human cytomegalovirus. "Hybridizing" refers to the binding of two single stranded nucleic acids via complementary base pairing.

As used hereinafter, a "sequence specific to" a particular viral species is a sequence unique to the species, that is, not shared by other previously characterized species. A probe containing a subsequence complementary to a sequence specific to a species will typically not hybridize to the corresponding portion of the genome of other species under stringent conditions (e.g., washing the solid support in O.lXXSSC, 0.1% SDS at 60°C).

The oligonucleotide sequences of the hybridizing regions of the primers and probes of the invention are presented below. Those skilled in the art will realize that an oligonucleotide sequence used as the hybridizing region of a primer can also be used as the hybridizing region of a probe. Suitability of a primer sequence for use as a probe depends on the hybridization characteristics of the primer. Similarly, an oligonucleotide used as a probe can be used as a primer. The term "oligonucleotide" refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, such as primers, probes, PCR products, and nucleic acid controls. The exact size of an oligonucleotide depends on many factors and the ultimate function or use of the oligonucleotide. Oligonucleotides can be prepared by any suitable method, including, for example, direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al., 1981, Tetrahedron Lett. 22:1859-1862; and the solid support method of U.S. Patent No. 4,458,066, each of which is incorporated herein by reference.

"Bind(s) substantially" refers to complementary hybridization between an oligonucleotide and a target sequence and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired priming for the PCR polymerases or detection of hybridization signal.

"Probe" refers to an nucleic acid sequence which binds through complementary base pairing to a subsequence of a target nucleic acid. It will be understood by one of skill in the art that probes will typically substantially bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are generally either directly labelled as with isotopes or indirectly labelled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the target.

The oligonucleotides shown in Table 1 are probes for the detection of human herpesvirus 7. Probes HV9 AND HV12 specifically hybridize to HHV-7 DNA. The probes may also be employed as PCR primers. Table 1 Oljqo Seouence Listing Seouence

HV9 SEQ ID NO:2 CCTAATGAAGGCTACTTTGAAGTA-

CAAATG HV12 SEQ ID NO:3 AGAATTCTGTACCCATGGGCACATT-

TGTAC

Nucleic acid sequences which are homologous to SEQ ID NO:2 or SEQ ID NO:3 may also serve as probes of the present invention. "Nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides. The term "homologous" indicates that two or more nucleotide sequences share a majority of their sequence. Generally, this will be at least about 75% of their sequence, usually greater than about 85%, and preferably about 95% of their sequence. Another indication that sequences are homologous is if they hybridize to the same nucleotide sequence under stringent conditions (see, e.g., Sambrook et al.. Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1985, incorporated herein by reference) . Stringent conditions are sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is at least about 0.2 molar at pH 7 and the temperature is at least about 55°C.

Plasmid and bacteriophage probes are also provided by the present invention. These probes may be conveniently synthesized by ligating segments of HHV-7 nucleic acid into vectors such a pBluescript plasmids or Lambda Dash II vectors. Methods for such ligation are well known to those of skill in the art as described in Sambrook et al., supra. previously incorporated herein by reference.

Briefly, supernatant from HHV-7 infected cell cultures or HHV-7 infected cell lysates is concentrated by centrifugation. The pellet is digested with a proteinase and electrophoresed. HHV-7 nucleic acid is present as a DNA band of about 155,000 basepairs. Identification of HHV-7 DNA may be confirmed by hybridization with an HHV-7-specific probe, such as p43L3 as described in Berneman et al., Proc. Natl. Acad. Sci. USA. 89:10552-10556 (1992), incorporated herein by reference.

The DNA is removed from the gel and extracted from the resulting solution. The extracted DNA may be digested with a variety of restriction enzymes. Choice of restriction enzyme will generally be guided by the vector into which the DNA will be inserted as understood by those of skill in the art. Restriction fragments are ligated into the vector. The vector is then cloned into appropriate host cells. Cloned vectors are analyzed by restriction enzyme digestion and Southern blot hybridization with purified HHV-7 DNA. Positive clones may be screened with human herpesvirus 6 and cytomegalovirus DNA for specificity. Such vector probes include pVL23, pVL23.2, pVL17, pVL17D.l, pVL17D.2, pVL17A.l, PVL17C.1, pVL18, pVLlβ.l, pVL44, pVL29, pVL3, pVL19, pVL8, pVL13, pVL13.1, XVL5, XVL10, XVL12, XVL56, XVL65, XVL73, or XVL81 as described below.

The probes of the present invention may also be nucleic acid sequences which are complementary to SEQ ID NO:2, SEQ ID NO:3, or other probes of the present invention. Further, by substituting uracil for thymine, RNA probes may also be employed in the present invention.

The probes of the present invention will generally be labeled. Probes may be labeled with several fluorophors or enzymes that generate colored products. Probes may be labeled with photoluminescents, Texas red, rhoda ine and its derivatives, red leuco dye and 3,3 ',5,5 '-tetramethylbenzidine (TMB) , fluorescein and its derivatives, dansyl, u belliferone and the like or with horse radish peroxidase, alkaline phosphatase, or the like. Alternatively, the probes may be labeled with a radioactive marker such as ³²P, ¹⁴C, ³H, ¹²⁵I, ³⁵S, or the like.

Probes can be labeled directly or indirectly. The common indirect labeling schemes covalently bind a ligand to the nucleotide and prepare labeled probe by incorporating this using random priming or nick translation. The ligand then binds an anti-ligand which is covalently bound to a fluorescent label. Ligands and anti-ligands vary widely. When a ligand has an anti-ligand, e.g., biotin, thyroxine, or cortisol, the ligand may be used in conjunction with the labelled naturally-occurring anti-ligand. Alternatively, a hapten or antigen may be used in combination with an antibody. One useful indirect DNA probe label is bio-11-dUTP (ENZO Diagnostics, New York, N.Y.). First the probes are purified by cesium chloride gradient ultracentrifugation and glass bead extraction with a Gene Clean kit (Bio 101, La Jolla, California) . Clean probe (1 microgram) is then mixed with bio-11-dUTP or other DNA probe label along with ³HdGTP tracer in the presence of 2 ng DNase per 50 microliter reaction mixture. After nick translation or random priming in the presence of polymerase, nuclease, and unlabeled nucleotides, labeled probe is separated from unincorporated nucleotides with G-50 Sephadex spin columns equilibrated in 150 mM NaCl, 15 mM sodium citrate, 0.1% sodium dodecyl sulfate (pH 7.0). Labeled probes are precipitated with a 50-fold excess of salmon sperm carrier DNA and yeast tRNA. Probes having repetitive sequences are further mixed with 100- to 500-fold excess depurinated human placental DNA. The probes are ethanol precipitated, washed twice in 70% ethanol at 0°C, and dried for 10 minutes at 23°C in a Savant speed vac. The pellets are redissolved to 8 ng/microliter in 100% deionized formamide for 30 minutes at 37°C. The solution is mixed with an equal volume of 2XSSC, 20% daxtran sulfate, lOOmM NaP04 and denatured for 5 minutes at 90°C. Denatured unique probes are chilled on ice. Probes with repetitive sequences are prehybridized to placental human DNA for 4 hours at 37°C. Fluorescent labels are bound to the probes following hybridization. The same protocol may be followed to incorporate the hapten digoxygenin-11-dUTP (Boehringer, Mannheim, Germany) into probe.

The present invention also provides primers for amplification of HHV-7 DNA sequences and subsequences.

"Amplifying" or "amplification", which typically refers to an "exponential" increase in target nucleic acid, is being used herein to describe both linear and exponential increases in the numbers of a select target sequence of nucleic acid. "Subsequence" refers to a sequence of nucleic acids that comprise a part of a longer sequence of nucleic acids.

The term "primer" refers to an oligonucleotide, whether natural or synthetic, capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization (i.e., DNA polymerase) in an appropriate buffer and at a suitable temperature. A primer is preferably a single-stranded oligodeoxyribonucleotide. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. The term "primer" may refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding one or both ends of the target region to be amplified. The term "target region" refers to a region of a nucleic acid to be analyzed and can include a polymorphic region. For instance, if a region shows significant levels of polymorphism in a population, mixtures of primers can be prepared that will amplify alternate sequences. Similar to probes as described above, a primer can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (as commonly used in an ELISA) , biotin, or haptens and proteins for which antisera or monoclonal antibodies are available. A label can also be used to "capture" the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support. The oligonucleotides shown in Table 2 are positive sense (upstream) primers or probes. Oligonucleotides HV7 and HV10 only serve as primers for HHV-7 amplification.

Table 2

Sequence 5•-GCNCCNTAYGAYATYCAYTTC-3• 5•-GCNCCNTAYGAYATYCAYTTT-3• 5^»-GCNCCNTAYGAYATACAYTTC-3• 5•-GCNCCNTAYGAYATACAYTTT-3' 5^■-TATCCCAGCTGTTTTCATATAGTAAC-3•

5^•-CAGAAATGATAGACAGATGTTGG-3• In Tables 2 and 3, N is adenine, cytosine, guanine, or thymidine; Y is either pyrimidine nucleotide (cytosine or thymidine) ; and R is either purine nucleotide (adenine or guanine) .

Table 3 lists oligonucleotides that function as negative sense (downstream) primers or as probes. The terms "sequence-specific oligonucleotide" and "SSO" refer to oligonucleotides that have a sequence, called a "hybridizing region, * exactly complementary to the sequence to be detected, typically sequences characteristic of a particular allele or variant, which under "sequence-specific, stringent hybridization conditions" will hybridize only to that exact complementary target sequence. Relaxing the stringency of the hybridizing conditions will allow sequence mismatches to be tolerated; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions. Depending on the sequences being analyzed, one or more sequence-specific oligonucleotides may be employed. The terms "probe" and "SSO probe" are used interchangeably with SSO. Oligonucleotides HV8 and HV11 are HHV-7 sequence specific oligonucleotides.

Table 3

Sequence 5'-RAANARNGTNGCRATNGGNGT-3^■ 5'-RTANARNGTNGCRATNGGNGT-3' 5^»-RAANARNGTNGCTATNGGNGT-3• 5•-RTANARNGTNGCTATNGGNGT-3' 5'-GCCTTGCGGTAGCACTAGATTTTTTG-3^■

5'-TAGATTTTTTGAAAAAGATTTAATAAC-3

The oligonucleotides may have hybridizing regions modified to include a restriction site toward the 5 '-end. The restriction site is introduced into the amplified product when one of these oligonucleotides is used as a primer. Initial hybridization conditions are chosen such that the base pair mismatches around the restriction site are tolerated. Mismatches near the 5'-end are tolerated better than those near the 3 '-end of a primer. Restriction sites may be added to the 3 '-end or 5 '-end of the primer if desired. The incorporation of such restriction sites into the amplified product facilitates cloning of the amplified product for use as probes as described below.

The desired viral sequences are conveniently inserted into a suitable vector before transformation using standard techniques for transformation host cells. Prokaryotes are preferably used for cloning, although yeast mammalian or insect cells may also be used.

The particular procedure used to introduce the altered genetic material into the host cell for expression of the viral sequences is not critical and may vary. Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasmid vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see Sambrook et al., ; ;ara, previously incorporated herein by reference). It is only necessary that the particular genetic engineering procedure utilized be capable of successfully introducing into the host cell the viral DNA in a form which provides for replication of the viral DNA sequence.

The particular vector used to transport the genetic information into the cell is also not critical. Any of the conventional vectors used for cloning DNA sequences may be used. These include pBluescript M13+ (Stratagene) , pUC18, pUC19, pUCllβ, pUC119, pSP64, PGEM-3Z, or the like.

The cloning vectors contain an appropriate origin of replication. The vectors usually comprise selectable markers such as the sodium, potassium ATPase, thymidine kinase, aminoglycoside phosphotransferase, antibiotics such as hygromycin B, ampicillin, kanamycin, and the like, phosphotransferase, xanthine-guanine phosphoribosyl transferase, CAD (carba yl phosphate synthetase, aspartate ^■ transcarba ylase, and dihydroorotase) , adenosine deaminase, DHFR, and asparagine synthetase and ouabain selection. Following ligation or recombination of the viral DNA sequence into a vector, the vector is transfected into host cells. Vector replication may be induced when appropriate. The selectable markers provide a means to identify transformed host cells. Transformed cells carrying a vector containing the viral DNA PCR product can be identified by means of hybridization with nucleic acid probes as described above. Vectors may then be isolated by well known techniques as described in Sambrook et al., previously incorporated herein by reference. The vectors may be labeled prior to or following isolation as described above.

The present invention also provides methods for detecting HHV-7 nucleic acid in a sample comprisirj_; (a) contacting the sample with a probe, which probe comprises a nucleic acid sequence homologous to human herpesvirus 7 nucleic acid; and (b) detecting hybrids formed between the subsequence and the probe. Another aspect of the present invention are methods for detecting the presence of human herpesvirus 7 genomic nucleic acid comprising: (a) amplifying a subsequence of the nucleic acid; (b) mixing the amplified nucleic acid with an oligonucleotide probe specific to the subsequence under conditions wherein the probe binds to the subsequence to form a stable hybrid duplex; and (c) detecting hybrids formed between the subsequence and the probe. Sequence-specific probe hybridization is an important step in the successful performance of the present methods. The sequence specific oligonucleotide probes of the present invention hybridize specifically with a particular segment of the HHV-7 genome and have destabilizing mismatches with the sequences from other organisms. Under sufficiently stringent hybridization conditions, the probes hybridize specifically only to exactly complementary sequences. The stringency of the hybridization conditions can be relaxed to tolerate varying amounts of sequence mismatch. Detection of the amplified product utilizes this sequence-specific hybridization to insure detection of only the correct amplified target, thereby decreasing the chance of a false positive caused by the presence of homologous sequences from related organisms.

Stringency is increased by raising temperature, lowering salt concentration, or raising formamide concentration. Adding dextran sulfate or raising its concentration can increase the effective concentration of labeled probe to increase the rate of hybridization and ultimate signal intensity. In addition, ultrasonic treatment of the reaction vessel in a commercially available sonication bath may accelerate hybridization.

In a particularly useful embodiment, the primers of the present invention are used in conjunction with a polymerase chain reaction (PCR) amplification of the target nucleic acid. "Polymerases" refers to enzymes able to catalyze the synthesis of DNA or RNA from nucleoside triphosphate precursors. In the amplification reactions of this invention, the polymerases are template-dependent and typically add nucleotides to the 3 '-end of the polymer being formed. It is most preferred that the polymerase is thermostable as described in U.S. Patent No. 4,889,819, incorporated herein by reference.

If PCR is used to test the presence of HHV-7 in blood cells, heparinized whole blood should be drawn in a sealed vacuum tube kept separated from other samples and handled with clean gloves. For best results, blood should be processed immediately after collection; if this is impossible, it should be kept in a sealed container at 4°C until use.

Other physiological fluids, such as cerebrospinal fluid, saliva, throat washings, urine and the like may be assayed. When using any of these fluids, the cells in the fluid should be separated from the fluid component by centrifugation. When testing cerebrospinal fluid, the cells tested should be from the second or third tube to avoid peripheral blood contamination.

Tissues should be roughly minced using a sterile, disposable scalpel and a sterile needle (or two scalpels) in a 5 mm Petri dish. Procedures for removing paraffin from tissue sections are described in specialized handbooks. In order to avoid the problems of PCR contamination, handling of the samples and isolation of the DNA should be performed in a hood with laminar air flow, preferably in an area not used for the preparation of DNA template-free PCR cocktails and not used for analysis and cloning of PCR products. Also, because both HHV-6 and HHV-7 can be reactivated from human peripheral blood mononuclear cells, cord blood mononuclear cells or cells from non-infected cell lines should be used as negative controls.

Although the PCR process is well known in the art (see, e.g., U.S. Patent Nos. 4,683,195; 4,683,202; and

4,965,188, each of which is incorporated herein by reference) and although commercial vendors, such as Perkin-Elmer Cetus Inc., sell PCR reagents and publish PCR protocols, some general PCR information is provided below for purposes of clarity and full understanding of the invention for those unfamiliar with the PCR process.

To amplify a target nucleic acid sequence in a sample by PCR, the sequence must be accessible to the components of the amplification system. In general, this accessibility is ensured by isolating the nucleic acids from the sample. A variety of techniques for extracting nucleic acids from biological samples are known in the art. For example, see those described in Rotbart et al., 1989, in PCR Technology (Erlich ed., Stockton Press, New York) and Han et al., 1987, Biochemistry 26:1617-1625.

Several methods are particularly well suited for use in amplifying HHV-7 DNA. The choice between these methods is typically governed by the size of the sample which is available for testing. One method is crude extraction which is useful for relatively large samples. Briefly, mononuclear cells from samples of blood, spinal fluid, throat washings, tissue or the like, are isolated by layering on sterile Ficoll-Paque gradient by standard procedures. Interphase cells are collected and washed three times in sterile phosphate buffered saline before DNA extraction. If residual red blood cells are visible (reddish pellet) , an osmotic shock (treatment of the pellet for 10 sec with distilled water) is suggested, followed by two additional washings. This will prevent the inhibitory effect of the heme group carried by hemoglobin on the PCR reaction. If PCR testing is not performed immediately after sample collection, aliquots of 10⁶ cells can be pelleted in sterile Eppendorf tubes and the dry pellet frozen at -20°C until use.

The cells are resuspended (10⁶ mononuclear cells per 100 μl) in a buffer of 50 mM Tris-HCl (pH 8.3), 50 mM KC1 1.5 mM MgCl₂, 0.5% Tween 20, 0.5% NP40 supplemented with 100 μg/ml of proteinase K. After incubating at 56^CC for 2 hr, the cells are heated to 95°C for 10 min to inactivate the proteinase K and immediately moved to wet ice (snap-cool) . If gross aggregates are present, another cycle of digestion in the same buffer should be undertaken. Ten μl of this extract will be used for amplification. When extracting DNA from tissues, the amount of the above mentioned buffer with proteinase K may vary according to the size of the tissue sample. The extract is incubated for 4-10 hrs at 50°-60°C and then at 95°C for 10 minutes to inactivate the proteinase. During longer incubations, fresh proteinase K should be added after about 4 hr at the original concentration.

When the sample contains a small number of cells, extraction may be accomplished by methods as described in

Higuchi, "Simple and Rapid Preparation of Samples for PCR", in PCR Technolog^y. Ehrlich, H.A. (ed.), Stockton Press, New York, which is incorporated herein by reference. PCR may be employed to detect human viruses in very small numbers of cells (1000-5000) derived from individual colonies from bone marrow and peripheral blood cultures. The cells in the sample are suspended in 20 μl of PCR lysis buffer (10 mM Tris-HCl (pH 8.3), 50 mM KC1, 2.5 mM MgCl₂, 0.1 mg/ml gelatin, 0.45% NP40, 0.45% Tween 20) and frozen until use. When PCR is to be performed, 0.6μl of proteinase K (2 mg/ml) is added to the cells in the PCR lysis buffer. The sample is then heated to about 60°C and incubated for 1 hr. Digestion is stopped through inactivation of the proteinase K by heating the samples to 95°C for 10 min and then cooling on ice. A relatively easy procedure for extracting DNA for

PCR is salting out procedure adapted from the method described by Miller et al.. Nucleic Acids Res.. 16:1215 (1988), which is incorporated herein by reference. Mononuclear cells are separated on a Ficoll-Hypaque gradient. The cells are resuspended in 3 ml of lysis buffer (10 mM Tris-HCl, 400 mM

NaCl, 2 mM Na₂EDTA, pH 8.2). Fifty μl of a 20 mg/ml solution of proteinase K and 150 μl of a 20% SDS solution are added to the cells and then incubated at 37°C overnight. Rocking the tubes during incubation will improve the digestion of the sample. If the proteinase K digestion is incomplete after overnight incubation (fragments are still visible) , an additional 50 μl of the 20 ϋg/ml pr -ainase K solution is mixed in the solution and incubated zor another night at 37°C on a gently rocking or rotating platform. Following adequate digestion, one ml of a 6M NaCl solution is added to the sample and vigorously mixed. The resulting solution is centrifuged for 15 minutes at 3000 rpm. The pellet contains the precipitated cellular proteins, while the supernatant contains the DNA. The supernatant is removed to a 15 ml tube that contains 4 ml of isopropanol. The contents of the tube are mixed gently until the water and the alcohol phases have mixed and a white DNA precipitate has formed. The DNA precipitate is removed and dipped in a solution of 70% ethanol and gently mixed. The DNA precipitate is removed from the ethanol and air dried. The precipitate is placed in distilled water and dissolved.

Kits are also commercially available for the extraction of high-molecular weight DNA for PCR. These kits include Genomic Isolation Kit A.S.A.P. (Boehringer Mannheim, Indianapolis, IN), Genomic DNA Isolation System (GIBCO BRL, Gaithersburg, MD) , Elu-Quik DNA Purification Kit (Schleicher & Schuell, Keene, NH) , DNA Extraction Kit (Stratagene, La Jolla, CA) , TurboGen Isolation Kit (Invitrogen, San Diego, CA) , and the like. Use of these kits according to the manufacturer's instructions is generally acceptable for purification of DNA prior to practicing the methods of the present invention.

The concentration and purity of the extracted DNA may be determined by εpectrophotometric analysis of the absorbance of a diluted aliquot at 260 nm and 280 nm. Either 10 μl of crude extract, or 1 μg of purified DNA by the alternate methods are used for PCR amplification.

After extraction of the DNA, PCR amplification may proceed. The first step of each cycle of the PCR involves the separation of the nucleic acid duplex. Of course, if the target nucleic acid is single- stranded, i.e., single-stranded RNA, no initial separation step is required. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complementary copies of the target strands. For successful PCR amplification, the primers are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement) , serves as a template for the extension of the other primer. The cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.

In the preferred embodiment of the PCR process, strand separation is achieved by heating the reaction to a sufficiently high temperature for an sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase (see U.S. Patent No. 4,965,188, incorporated herein by reference). Typical heat denaturation involves temperatures ranging from about 80°C to 105°C for times ranging from seconds to minutes. Strand separation, however, can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic means. Strand separation may be induced by a helicase, for example, or an enzyme capable of exhibiting helicase activity. For example, the enzyme RecA has helicase activity in the presence of ATP. The reaction conditions suitable for strand separation by helicases are known in the art (see Kuhn Hoffman-Berling, 1978, CSH-Ouantitative Biology 43:63-67; and Radding, 1982, Ann. Rev. Genetics 16:405-436, each of which is incorporated herein by reference) .

Template-dependent extension of primers in PCR is catalyzed by a polymerizing agent in the presence of adequate amounts of four deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and dTTP) in a reaction medium comprised of the appropriate salts, metal cations, and pH buffering system. Suitable polymerizing agents are enzymes known to catalyze template-dependent DNA synthesis. Because some primers of the present invention are apparently subsequences of a potential protein encoding HHV-7 gene, it may be possible to perform PCR to identify mRNA encoding the protein. When PCR is performed to generate a cDNA library from RNA for further amplification, the initial template for primer extension is RNA. Polymerizing agents suitable for synthesizing a complementary, copy-DNA (cDNA) sequence from the RNA template are reverse transcriptase (RT) , such as avian myeloblastosis virus RT, Moloney murine leukemia virus RT, or Ther us thermophilus (Tth) DNA polymerase, a thermostable DNA polymerase with reverse transcriptase activity marketed by Perkin Elmer Cetus, Inc. Typically, the genomic RNA template is heat degraded during the first denaturation step after the initial reverse transcription step leaving only DNA template. Suitable polymerases for use with a DNA template include, for example, £. coli DNA polymerase I or its Klenow fragment, T₄ DNA polymerase, Tth polymerase, and Tag polymerase, a heat-stable DNA polymerase isolated from Thermus acruaticus and commercially available from Perkin Elmer Cetus, Inc. The latter enzyme is widely used in the amplification and sequencing of nucleic acids. The reaction conditions for using Tag polymerase are known in the art and are described in Gelfand, 1989, PCR Technology, supra.

The PCR method can be performed in a step-wise fashion, where after each step new reagents are added, or in a fashion where all of the reagents are added simultaneously, or in a partial step-wise fashion, where fresh or different reagents are added after a given number of steps. For example, if strand separation is induced by heat, and the polymerase is heat-sensitive, then the polymerase must be replenished following each round of strand separation.

However, if, for example, a helicase is used for denaturation, or if a thermostable polymerase is used for extension, then all of the reagents may be added initially, or, alternatively, if molar ratios of reagents are of consequence to the reaction, the reagents may be replenished periodically as they are depleted by the synthetic reaction.

Those skilled in the art will know that the PCR process is most usually carried out as an automated process with a thermostable enzyme. In this process, the temperature of the reaction mixture is cycled through a denaturing region, a primer annealing region, and an extension reaction region. Alternatively, the annealing and extension temperature can be the same. A machine specifically adapted for use with a thermostable enzyme is commercially available from Perkin Elmer Cetus, Inc.

Those skilled in the art will also be aware of the problems of contamination of a PCR by the amplified nucleic acid from previous reactions and nonspecific amplification. Methods to reduce these problems are pre ided in PCT patent application Serial No. 91/05210, filed July 23, 1991, incorporated herein by reference. The method allows the enzymatic degradation of any amplified DNA from previous reactions and reduces nonspecific amplification. The PCR amplification is carried out in the presence of dUTP instead of dTTP. The resulting double-stranded, uracil-containing product is subject to degradation by uracil N-glycosylase (UNG) , whereas normal thymidine-containing DNA is not degraded by UNG. Adding UNG to the amplification reaction mixture before the amplification is started degrades all uracil- containing DNA that might serve as target. Because the only source of uracil-containing DNA is the amplified product of a previous reaction, this method effectively sterilizes the reaction mixture, eliminating the problem of contamination from previous reactions (carry-over) . UNG itself is rendered temporarily inactive by heat, so the denaturation steps in the amplification procedure also serve to inactivate the UNG. New amplification products, therefore, though incorporating uracil, are formed in an effectively UNG-free environment and are not degraded. Alternatively, other methods to reduce PCR contamination, such as ultraviolet irradiation of PCR reagents, may be employed.

"Amplification reaction mixture" refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid. These include: enzymes, aqueous buffers, salts, target nucleic acid, and deoxynuσleoside triphosphates. Depending upon the context, the mixture can be either a complete or incomplete amplification reaction mixture.

A particularly useful method for amplifying HHV-7 DNA by PCR employs primer pairs HV7 and HV8 or HV10 and HV11. These primers amplify HHV-7 DNA, but have not been shown to amplify DNA from other human herpesviruses, including 12 strains of HHV-6. Briefly, the amplification reaction mixture is prepared as follows. Distilled water is added to 10 μl of the 10X PCR reaction buffer (100 mM Tris-HCl (pH 8.3), 500 mM KC1, 15 mM MgCl₂, 1 mg/ml gelatin), 16 μl of the 1.25 mM dNTP mix (1.25 mM mix of dATP, dCTP, dGTP and dTTP (made from 100 mM pre-made solutions, US Biochemical, Cleveland, OH)), 2.5 μl of 20 μM primers HV7 and HV8 or HV10 and HVll, and 0.5 μl of 5 U/μl Taq DNA polymerase to make a final volume of 100 μl. One μg of the DNA sample is added to the

Amplification reaction mixture described above. For primer pairs HV7/HV8 the following thermal cycler program is used: 30-40 cycles of 1 min at 94^βC, 2 min at 60°C, 2 min at 72°C with an increase of 2 sec per cycle; followed by a final extension at 72°C for 7 min. For primer pairs HV10/HV11 the following alternate program is used: 30-40 cycles of 1 min at 94°C, 2 min at 55°C, 2 min at 72°C with an increase of 2 sec per cycle; followed by a final extension at 72°C for 7 min. Following thermal cycling, 10-15 μl of the amplification reaction mixture is mixed with gel-loading solution, and run on 6% polyacrylamide gels or on 2% agarose gels. The gels are stained in IX TBE solution (10X TBE = 0.89 M Tris, 0.89 M boric acid, 0.02 M Na₂EDTA, pH 8.3) containing ethidium bromide, and visualized on a UV transilluminator. The PCR products are transferred to a membrane, such as a nitrocellulose membrane or a nylon membrane (e.g., Nytran membranes, Schleicher & Schuell, Keene NH) and baked or UV cross-linked, as appropriate.

Those practicing the present invention should note that, although the preferred embodiment incorporates PCR amplification, amplification of target sequences in a sample may be accomplished by any known method, such as ligase chain reaction (LCR) , transcription amplification, and self- sustained sequence replication, each of which provides sufficient amplification so that the target sequence can be detected by nucleic acid hybridization to an SSO probe. Alternatively, methods that amplify the probe to detectable levels can be used, such as Q/3-replicase amplification. The term "probe" encompasses the sequence specific oligonucleotides used in the above procedures; for instance, the two or more oligonucleotides used in LCR are "probes" for purposes of the present invention, even though some embodiments of LCR only require ligation of the probes to indicate the presence of an allele.

The assay methods for detecting hybrids formed between SSO probes and nucleic acid sequences can require that the probes contain additional features in addition to the hybridizing region. For example, if the probe is first immobilized, as in the "reverse" dot blot format described below, the probe can also contain long stretches of poly-dT that can be fixed to a nylon support by irradiation, a technique described in more detail in PCT Patent Publication No. 89/11548, incorporated herein by reference. In the dot blot format, immobilized target is hybridized with probes containing a compound used in the detection process, as discussed below. The probes of the present invention can be synthesized and labeled using the techniques described above for synthesizing oligonucleotides. The probe can be labeled at the 5'-end with ³²P by incubating the probe with ³²P-dCTP T4 polynucleotide kinase. A suitable nonradioactive label for SSO probes is horseradish peroxidase (HRP) or a chromagen as described above. Methods for preparing and detecting probes containing this label are described in the Examples below and in U.S. Patent Nos. 4,914,210 and 4,962,029; the latter patents are incorporated herein by reference. For additional information on the use of such labeled probes, see U.S. Patent No. 4,789,630; Seiki et al., 1988, N.Enq.J.Med. 319:537-541; and Bugawan et al., 1988, Bio/Technology 6:943-947, each of which is incorporated herein by reference.

Plasmid and bacteriophage probes may be labeled by a variety of methods, either directly or indirectly. The common indirect labeling schemes covalently bind a ligand to the nucleotide and prepare labeled probe by incorporating this using random priming, primer extension, or nick translation. The ligand then binds an anti-ligand which is covalently bound to a fluorescent label. Ligands and anti-ligands vary widely. When a ligand has an anti-ligand, e.g., biotin, thyroxine, or cortisol, the ligand may be used in conjunction with the labelled naturally-occurring anti-ligand. Alternatively, a hapten or antigen may be used in combination with an antibody.

One preferred indirect DNA probe label is bio-11- dUTP (ENZO Diagnostics, New York, N.Y.). First the probes are purified by cesium chloride gradient ultracentrifugation and glass bead extraction with a Gene Clean kit (Bio 101, La Jolla, California). Clean probe (1 microgram) is then mixed with bio-li-dUTP or other DNA probe label along with ³HdGTP tracer in the presence of 2 ng DNase per 50 microliter reaction mixture. After nick translation or random priming in the presence of polymerase, nuclease, and unlabeled nucleotides, labeled probe is separated from unincorporated nucleotides with G-50 Sephadex spin columns equilibrated in 150 mM NaCl, 15 mM sodium citrate, 0.1% sodium dodecyl sulfate (pH 7.0). Labeled probes are precipitated with a 50-fold excess of salmon sperm carrier DNA and yeast tRNA. Probes having repetitive sequences are further mixed with 100- to 500-fold excess depurinated human placental DNA. The probes are ethanol precipitated, washed twice in 70% ethanol at 0°C, and dried for 10 minutes at 23°C in a Savant speed vac. The pellets are redissolved to 8ng/microliter in 100% deionized formamide for 30 minutes at 37°C. The solution is mixed with an equal volume of 2XSSC, 20% dextran sulfate, lOOmM NaP04 and denatured for 5 minutes at 90°C. Denatured unique probes are chilled on ice. Probes with repetitive sequences are prehybridized to placental human DNA for 4 hours at 37°C. Fluorescent labels are bound to the probes following hybridization. The same protocol may be followed to incorporate the hapten digoxygenin-11-dUTP (Boehringer, Mannheim, Germany) into probe.

The probes of the invention can be used to determine if viral sequences are present in a sample by determining if the SSO probes bind to the viral sequences present in the sample. Suitable assay methods for purposes of the present invention to detect hybrids formed between SSO probes and nucleic acid sequences in a sample are known in the art. For example, the detection can be accomplished using a dot blot format, as described in Example 2. In the dot blot format, the unlabeled amplified sample is bound to a solid support, such as a membrane, the membrane incubated with labeled probe under suitable hybridization conditions, the unhybridized probe removed by washing, and the filter monitored for the presence of bound probe. When multiple samples are analyzed with a single probe, the dot blot format is quite useful. Many samples can be immobilized at discrete locations on a single membrane and hybridized simultaneously by immersing the membrane in a solution of probe. An alternate method that is quite useful when large numbers of different probes are to be used is a "reverse" dot blot format, in which the amplified sequence contains a label, and the probe is bound to the solid support. This format would be useful if the test of the present invention were used as one of a battery of tests to be performed simultaneously, such as might be useful for simultaneously detecting HHV-6 and HHV-7 nucleic acid in a sample. In this format, the unlabeled SSO probes are bound to the membrane and exposed to the labeled sample under appropriately stringent hybridization conditions. Unhybridized labeled sample is then removed by washing under suitably stringent conditions, and the filter is then monitored for the presence of bound sequences.

Both the forward and reverse dot blot assays can be carried out conveniently in a microtiter plate. The probes can be attached to bovine serum albumen (BSA) , for example, which adheres to the microliter plate, thereby immobilizing the probe.

Another suitable assay system employs a procedure in which a labeled probe is added during the PCR amplification process. Any SSO probe that hybridizes to target DNA during each synthesis step is degraded by the 5 ' to 3 ' exonuclease activity of a polymerase, e.g.. Tag polymerase. The degradation product from the probe is then detected. Thus, the presence of the breakdown product indicates that the hybridization between the SSO probe and the target DNA occurred.

A particularly useful means of hybridization and detection of HHV-7 DNA following PCR is as follows. The nylon filter to which the sample or amplification products have been blotted is soaked in a closed container (on a rocker or rotating platform) with hybridization solution (see above) or in Hybrisol I (Oncor, Gaithersburg, MD) solution at 37°C for at least 10 min. When a segment of HHV-7 nucleic acid has been amplified in the sample, a probe is chosen which is homologous to the a segment of the amplified sequence. Depending on the primer pairs used for amplification, an oligonucleotide probe, e.g., HV9 or HV12, is chosen (see Table 1) , end-labelled with ³²P-dCTP using T4 polynucleotide kinase and added to the hybridization solution (10⁶ cpm/ml) . Hybridization is performed overnight at 37°C on a rocking or rotating platform.

The filter is rinsed twice at room temperature with 6X SSC-0.5% SDS and then washed twice for 10 min at 55°C or 60°C with 6X SSC-0.5% SDS. Following washing, the filter is rinsed with 2X SSC at room temperature, blotted to dampness with 3mm Whatman paper, and wrapped in plastic wrap. The filter is then autoradiographed by standard methods as described in Sambrook et al., supra.

The nucleotide sequences provided above are an important aspect of the present invention. Although only one strand of the sequence is shown, those of skill in the art recognize that the other strand of the sequence can be inferred from the information depicted above. This information enables the construction of other probes and primers of the invention.

The nucleotide sequence presented in SEQ ID N0:1 is the product of PCR amplification of HHV-7 DNA. SEQ ID N0:1 may be employed as a probe or to develop other probes or amplification primers. The sequence may be compared to DNA sequences of HHV-6, human cytomegalovirus, or other human herpesviruses to determine subsequences which are specific HHV-7. Generally the oligonucleotide probes or primers will be between about 15 and 50 nucleotides in length, although longer or shorter subsequences may be employed, depending on the use.

The present invention also relates to kits and multicontainer units comprising useful components for practicing the present method. A useful kit can contain SSO probes for detecting HHV-7 nucleic acid. In some cases, the SSO probes may be fixed to an appropriate support membrane. The kit may also contain primers for PCR, as such primers are useful in the preferred embodiment of the invention. Other optional components of the kit include, for example, reverse- transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin- enzyme conjugate and enzyme substrate and chromogen if the label is biotin) , and the appropriate buffers for PCR or hybridization reactions. In addition to the above components, the kit can also contain instructions for carrying out the present method.

The present invention also provides HHV-7 infected continuous cell lines and methods for infecting continuous cell lines with HHV-7. Immortalized cell lines may be infected with HHV-7 to provide a source of HHV-7 virions, nucleic acid sequences and proteins for diagnostic and therapeutic uses. The cell lines are incubated with HHV-7 containing supernatant under suitable conditions, typically for 0.5-6 hours at 30°-40°C. Corticosteroids like hydrocortisone are generally added to the cell culture medium after incubation with the HHV-7 supernatant. The corticosteroids may also be present during infection. Typical hydrocortisone concentrations are 0.5 μg/ml to 50 μ/ml, although the hydrocortisone concentration may vary. While cells in culture may be infected in the absence of hydrocortisone, the frequency of cellular infectivity is greater and more reproducible in the presence of hydrocortisone. Infectivity may be determined, e.g., by the appearance of very large cells, decrease in the size of cellular clusters, the appearance of large adherent cells, the methods of the present invention, electron microscopy, or positive immunofluorescence with anti-HHV-7 antibodies such as HHV-7 seropositive human serum and/or cross-reacting HHV-6 mAbs as described in Wyatt et al., J. Virol.. 65:6260-6265 (1991) and Berneman et al., Proc. Natl. Acad. Sci USA, 89:10552-10556 (1992). One particularly useful cell line which may be infected with HHV-7 is SUP-T1, an immature T- lymphocyte cell line (Smith et al.. Cancer Res.. 44:5657-5660 (1984).

The HHV-7 infected continuous cell lines provide a means for raising immunoglobulins specific for HHV-7. This is typically accomplished either by harvesting viral particles from cell culture or lysing infected cells and immunizing animals with the viral particles or purified or crude viral proteins from the lysates. A number of standard procedures for purifying proteins can be used, such as, for example, ammonium sulfate precipitation, gel electrophoresis, ion exchange chromatography, size exclusion chromatography or affinity chromatography. See, generally_f Scopes, R. , Protein . Purification, Springer-Verlag, N.Y. (1982). In addition to the use of proteins encoded by the desired viral genome, peptides that mimic epitopes specific to the virus can also be used to raise antibodies specific to the virus. Such technology is well known to those of skill in the art and is described, for instance, in Geysen et al., Multipin Peptide Synthesis - A Review. Coselco Mimotypes, PTY Ltd., which is incorporated herein by reference.

The immunoglobulins may be used for a variety of applications. For instance, they can be used to assay for the presence of the virus in a biological sample or to purify the viral antigens, for example, using affinity chromatography.

They may also be used to neutralize the corresponding protein, including inhibition of infectiousness of the entire virus. The multitude of techniques available to those skilled in the art for production and manipulation of various immunoglobulin molecules can thus be readily applied to produce molecules useful for diagnosis and detection of the particular species. The vector probes of the present invention may also be used for recombinant synthesis of HHV-7 proteins or polypeptides. HHV-7 nucleic acid sequences may be excised from the probes and ligated into expression vectors. The expression vectors may be transformed into eukaryotic or prokaryotic host cells. See, e.g., Sambrook et al., supra. Procaryotes may be employed for cloning and expressing DNA sequences to produce HHV-7 proteins or polypeptides for use in the present invention. Several methods may be employed to produce the desired polypeptides such as those described in Sambrook et al., supra. Several different procaryotic hosts are suitable for cloning the desired DNA sequences. For example, £j. coli K12 strain 294 (ATCC No. 31446) is particularly useful. Other microbial strains which may be used include E__s_ coli strains such as Ej_ coli B, and E__s_ coli X1776 (ATCC No. 31537) , and E^. coli c600 and cβOOhfl, E^. coli W3110 (F^",λ^", prototrophic,

ATCC No. 27325) , bacilli such as Bacillus subtilus. and other enterobacteriaceae such as Salmonella tvphimurium or Serratia marcescens _f and various Pseudomonas species. When expressed in procaryotes the polypeptides used in the present invention typically contain an N-terminal methionine or a formyl methionine, and are not glycosylated. These examples are, of course, intended to be illustrative rather than limiting.

The present invention also provides DNA constructs encoding potential HHV-7 proteins or fragments thereof. The DNA constructs generally comprise a transcriptional promoter, a DNA sequence encoding an HHV-7 protein or a fragment thereof, and a transcriptional terminator.

In general, plasmid vectors containing replication and control sequences which are derived from species compatible with the recombinant host cells are used in connection with these hosts. Other vectors, such as λ-phage, cosmids, or yeast artificial chromosomes may be employed. The vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells. For example, E. coli may be transformed using pBR322, a plasmid derived from an Ej. coli species. Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying and selecting transformed cells. The pBR322 plasmid, or microbial plasmid must also contain, or be modified to contain, promoters which can be used by the microbial organism for an expression of its own proteins. Those promoters most commonly used in recombinant DNA construction include /3-lactamase (penicillinaεe) and lactose promoter systems and a tryptophan (trp) promoter system. One suitable promoter is contained in the in vitro transcription vector pGEM-1. The promoter is a T7 and S06 polymerase promoter. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors. The promoters are operably linked to a nucleic acid sequence encoding an HHV-7 protein or a homolog or fragment thereof. The promoters may be inducible or constitutive and provide a means to express the encoded HHV-7 protein in the procaryotic host. Following expression, the polypeptide may be purified by standard methods such as described below.

Alternatively, a DNA sequence encoding an HHV-7 protein or fragment thereof may be inserted into a suitable eukaryotic expression vector, which in turn is used to transfeet eukaryotic cells. A eukaryotic expression vector, as used herein, is meant to indicate a DNA construct containing elements which direct the transcription and translation of DNA sequences encoding polypeptides of interest. Such elements include promoters, enhancers, transcription terminators and polyadenylation signals. By virtue of the inclusion of these elements operably linked within the DNA constructs, the resulting eukaryotic expression vectors contain the information necessary for expression of the polypeptides of interest.

Host cells for use in expressing recombinant HHV-7 proteins of interest include mammalian, avian, insect and fungal cells. Fungal cells, including species of yeast (a.g., Saccharomyces spp. , Schizosaccharomyces spp.) or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.) may be used as host cells for producing polypeptides useful in the present invention. Suitable vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected. Preferred selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources. The expression units may also include a transcriptional terminator. Choice of a particular host and selectable marker is well within the level of ordinary skill in the art.

Cultured mammalian cells may be used as host cells within the present invention. Cultured mammalian cells for use in the present invention may include human monocytoid, lymphocytoid, and fibroblastoid cell lines. A particularly useful mammalian cell line is the HeLa-tat cells that are HeLa derived cells which produce constitutively HIV-1 Tat (Schwartz et al., supra) . Mammalian expression vectors for use in carrying out the present invention will include a promoter capable of directing the transcription of a cloned gene or cDNA. Useful promoters include viral promoters and cellular promoters. Viral promoters include the immediate early cytomegalovirus promoter (Boshart et al.. Cell 41:521-530, 1985) and the SV40 promoter (Subramani et al., Mol♦ Cell. Biol. 1:854-864, 1981). Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., U.S. Patent No. 4,579,821), a mouse V_κ promoter (Bergman et al., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983); Grant et al., Nuc. Acids Res. 15:5496, 1987) and a mouse V_H promoter (Loh et al.. Cell 33:85-93, 1983).

A particularly preferred promoter is the HIV LTR promoter from HIV-1. Such expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence encoding the polypeptide or protein of interest. Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes. Also contained in the expression vectors is a polyadenylation signal located downstream ,of the coding sequence of interest. Polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the Adenovirus 5 E1B region and the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9:3719-3730, 1981). The expression vectors may include a noncoding viral leader sequence, such as the Adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites. Vectors may also include enhancer sequences, such as the SV40 enhancer and the mouse μ enhancer (Gillies, Cell 33: 717-728, 1983). Expression vectors may also include sequences encoding the adenovirus VA RNAs.

Cloned DNA sequences may be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al.. Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973). Other techniques for introducing cloned DNA sequences into mammalian cells, such as electroporation (Neumann et al., EMBO J. 1:841-845, 1982), may also be used. In order to identify cells that have included or integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker such as the DHFR gene. Selectable markers are reviewed by Thilly (Mammalian Cell Technology. Butterworth Publishers, Stoneham, MA, which is incorporated herein by reference) . The choice of selectable markers is well within the level of ordinary skill in the art.

Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequence(s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels. The HHV-7 proteins and fragments thereof produced according to the present invention, either authentic (native) or recombinant polypeptides, may be purified by a variety of means, including via affinity chromatography, e.g., on an antibody column using antibodies directed against the HHV-7 proteins or using HHV-7 binding substances, such as purified CD4 receptor. Additional purification may be achieved by conventional chemical purification means, such as liquid chromatography, gradient centrifugation, and gel electrophoresis, among others. Methods of protein purification are known in the art (see generally. Scopes, R. , supra) and may be applied to the purification of the recombinant HHV-7 proteins described herein; see also a purification protocol described in U.S. 4,929,604, incorporated herein by reference.

Substantially pure HHV-7 proteins or polypeptides of at least about 50% is preferred, at least about 70-80% more preferred, and 95-99% or more homogeneity most preferred, particularly for many diagnostic and pharmaceutical uses.

Once purified, partially or to homogeneity, as desired, the HHV-7 protein or fragment thereof may then be used therapeutically, diagnoεtically, or in screening asεayε as described herein. The purified HHV-7 proteins may then be combined in pharmaceutical compositions for a variety of therapeutic useε. The compositions may be administered to persons or animals infected, or at risk for infection, by HIV. Pharmaceutical compositions may be produced that comprise CD4 receptor binding proteins of HHV-7. The HHV-7 CD4 receptors will competitively inhibit binding of HIV to CD4 receptors on the surface of T cells. The concentration of the purified HHV-7 proteinε, or fragments thereof, in these formulationε can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to aε much as 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Actual methods for preparing parenterally administrable compounds will be known or apparent to thoεe skilled in the art and are described in detail in, for example. Remington's Pharmaceutical Science, 17th ed. , Mack Publishing Company, Easton, PA (1985) , which is incorporated herein by reference. Preferably, the pharmaceutical compositionε are administered parenterally, i.e., intravenouεly, subcutaneously, or intramuscularly. Thus, this invention provides compositions for parenteral administration which comprise a solution of an inhibitory peptide of the present invention or dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water," buffered water, 0.9% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. In some instances such as treating neurological disease or lesions, it can be desirable to package the proteins in liposomeε for administration. These compositions can be sterilized by conventional, well known sterilization techniques. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositionε can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such an pH adjusting and buffering agentε, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potasεium chloride, calcium chloride, etc. Appropriate dosage ranges and routes of administration can be determined by standard techniques as described in Remington'ε Pharmaceutical Science, 17th ed. , Mack Publishing Company, Easton, PA (1985), previously incorporated herein by reference.

Alternatively, attenuated live virus can be given to a patient suffering from HIV infection to inhibit the progression of AIDS. The attenuated virus may replicate harmlessly in the patient and may interfere with the ability of HIV-1 to infect CD4+ cells by blocking binding of CD4 receptor sites on T cells. Another aspect of the present invention is the addition of genes encoding expression products of HHV-7 genes, or fragments thereof, to host cells. In this manner, the expression product of HHV-7 protein-encoding genes may be produced by the host cells in vivo or in vitro. The host cells then serve to self-administer the HHV-7 protein encoded by the transfected HHV-7 gene. For example, transfection of human hematological stem cells (CD34⁺⁾ with genes encoding the HHV-7 protein that binds the CD4 receptor could provide a means to competitively inhibit binding of HIV virions to CD4 molecules in T cells. In some instances the DNA constructs of the present invention, e.g., pVL23, pVL23.2, pVL17, pVL17D.l, PVL17D.2, pVL17A.l, pVL17C.l, pVLlδ, pVLlβ.l, pVL44, pVL29, pVL3, pVL19, pVL8, pVL13, or pVL13.1 may be employed as vectorε for gene therapy.

Addition of the genes to host cells may be accomplished by several methods including those described in International Patent Publication No. WO88/08450, incorporated herein by reference. A particularly useful method is gene complementation as described in Donehower et al. , Prog. Med. Virol.. 34:1-32 (1987) or McLachlin et al. Prog. Nuc. Acids Res. Mol. Biol.. 38:91-135 (1990), both herein incorporated by reference. Briefly, nucleic acid sequences encoding the desired HHV-7 protein or polypeptide may be introduced into retroviral vectors, such aε Mo-MLV-baεed defective expreεεion vectorε. The vector may be packaged as virions and used to transfect host cells. Preferably, a retroviral promoter sequence controls the expression of the gene. The gene may also be under the control of a conεtitutive promoter. Once transfection of the host cells haε been achieved, expreεsion of the gene may be accomplished by appropriate means. See Drumm et al.. Cell. 62:1227-1233 (1990), Hoeben et al., J. Biol. Chem.. 265:7318-7323 (1990), Kasidet al., Proc. Natl. Acad. Sci. USA. 86:8927-8931 (1989), and Morgan et al. AIDS Res. Hum. Retroviruses. 6:183-191 (1990), all of which are incorporated herein by reference.

Another aspect of the present invention is the use of HHV-7 and molecular clones thereof as vectorε for gene therapy. As latent and persistent HHV-7 infection is commonly found in T cells of healthy individuals, employing infectiouε HHV-7 particles or HHV-7 DNA could provide a safe and effective means for introducing protein or peptide encoding nucleic acids into host cellε, especially CD4 expressing cells. The viral particles or DNA may carry a wide range of different protein- or polypeptide-encoding nucleic acid sequences. Following infection or transfection of the host cells, the introduced nucleic acid sequences may be induced to express the desired protein or polypeptide.

The examples of the present invention presented below are provided only for illustrative purposes and not to limit the scope of the invention. Numerous embodiments of the invention within the scope of the claims that follow the examples will be apparent to those of ordinary skill in the art from reading the foregoing text and following examples.

EXAMPLES Example

This example describeε infection of a continuous cell line with HHV-7. Cellular infection was enhanced by treatment of the cellε with hydrocortiεone. Growing HHV-7 in a cell line obviates the need to use primary cells which are not as readily available and which do not grow continuously and may also be latently infected with herpes and other viruses.

A pellet of SUP-T1 cells (about 1.4 x 10⁶) waε incubated with 1 to 2 ml of virus-containing supernatant for 2 hr. at 37°C. Following incubation, RPMI 1640 medium supplemented with 20% fetal calf serum, L-glutamine, penicillin and streptomycin and 5 microgram/ml of hydrocortisone was added, so that the final cell concentration was 100,000/ml. The culture was carried at 37°C in an incubator gaεεed with 5% C0₂. The same culture medium was added to the culture twice during the firεt 10 dayε (the final volume waε 4X that of the original volume) . The culture medium waε replaced approximately twice weekly, depending on the acidity level of the culture. The culture was assayed for the appearance of very large cells, the decrease in size of the SUP-T1 clusters, and in culture flasks which were kept flat, for the appearance of large adherent cells. The latter correlated well with HHV-7 viral protein expression, as assayed by immunofluorescence with HHV-7-seropositive antiserum obtained from HHV-7 seropoεitive individualε and with HHV-6 monoclonal antibodieε which cross-react with HHV-7 infected cells. The level of positive immunofluorescence in cultures carried with hydrocortiεone was as high as 80% of the cells in culture. During the peak of infection, the level of positivity varied between 10 and 80%. Carrying the infected culture in medium without hydrocortisone reεulted in more variable rateε of successful HHV-7 infection.

Example 2

This example describes the preparation of an HHV- εpecific probe, HV9 and detection of amplified HHV-7 DNA with the probe. Viral DNA waε obtained from HHV-7 infected cellε.

SUP-T1 cell line or phytohemagglutinin A-stimulated cord blood mononuclear cells (CBMC) were incubated for 1-2 hr at 37°C with virus-containing culture supernatant. The cell line was cultured in RPMI 1640 medium supplemented with 2%-10% fetal calf serum (FCS) , while the CBMC were grown in the same medium containing 5%-20% FCS with or without 10% (volume/volume) of interleukin 2 (ABI, Columbia, MD) . Infection was asεeεεed by the appearance of large cellε, immunofluorescence, and electron microεcopy. Indirect immunofluorescence with human polyclonal and murine monoclonal antisera was carried out on infected and control uninfected cells on 8- or 10-well-slideε. The following anti-HHV-6 monoclonal antibodieε were used to detect HHV-7 infection (the HHV-6 viral protein antigenic determinants are in parentheses) : 9A5D12 (p41) , 12B3G4

(pl35) , 6A5G3 (gpll6/64/54) , 2D6 (gp82/105) , 7A2 (gpl02) Bala Chandran et al., J. Virol.. 63:2835-2840 (1989); and C-5 (p38/41) Iyengar et al.. Int. J. Cancer. 49:551-557 (1991). Only antibodies 9A5D12, 12B3G4, and C-5 provided positive immunofluorescence with cells shown to infected with HHV-7 by other meanε.

Amplification by PCR was performed on HHV-7 nucleic acid obtained from infected cells. The oligonucleotides used for PCR analysis are listed in Tables 2 and 3. Since hybridization studieε εhowed that HHV-7 waε related to HHV-6 (see, Frenkel et al., Proc. Natl. Acad. Sci. USA. 87:748-752 (1990) and Berneman et al., Proc. Natl. Acad. Sci. USA. 89:10552-10556 (1992)), HHV-7 DNA was amplified using primers designed after comparing sequences from HHV-6 and HCMV. Based on homology between homologous putative proteins of HHV-6, and human cytomegalovirus, degenerate oligonucleotide primers covering the two regions of amino acid identity were syntheεized (Tableε 2 and 3): HV3.1, HV3.2, HV3.3 and HV3.4 for the APYDIHF peptide motif, in a εenεe orientation; and HV4.1, HV4.2, HV4.3 and HV4.4 for the TPIATLF/Y motif, in an antiεenεe direction.

The HHV-7 sequence and clone were obtained by PCR as follows: Initial PCR amplification was in a 100 μl reaction, containing 2 μg of HHV-7-infected SUP-T1 cell DNA, 10 mM Tris- HCl (pH 8.3), 50 mM KC1, 1.5, 2.0 or 2.5 mM MgCl₂, 0.01% gelatin, 0.2 mM dATP, dCTP, dGTP and dTTP, 4μM of primers HV3.1, HV3.2, HV3.3, HV3.4, HV4.1, HV4.2, HV4.3 and HV4.4, and 5 units of Taq DNA polymerase. Forty cycles of PCR amplification were performed according to the following program: 94°C for 1 min, 45°C for 2 min, 72°C for 2 min with an increase of 2 sec per cycle, and an additional 7 min at the end at 72°C. The PCR products were analyzed as described Berneman et al, Proc. Natl. Acad. Sci. USA. 89:3005-3009

(1992) . The polyacrylamide gel piece containing the 228-bp PCR product waε cut out and the amplified DNA eluted out of the gel piece in 0.5 M ammonium acetate and 1 mM EDTA at 37°C overnight. After phenol-chloroform extraction and ethanol precipitation, the amplified fragment waε redissolved in distilled water, treated with T4 polynucleotide kinase, extracted with phenol-chloroform, ethanol precipitated, rediεsolved in distilled water, and blunt-end ligated overnight at room temperature into Eco RV-digested and phoεphatased pBluescript plasmid (Stratagene, La Jolla, CA) . Recombinant bacterial colonies were screened with the ³²p-labelled amplified 228bp HHV-7 DNA fragment. The insert of one selected recombinant plasmid (designated "3L25") was sequenced using the Sequenase Version 2.0 DNA Sequencing kit (US Biochemical, Cleveland, OH) . Based on the DNA εequence located internally to the regions covered by the degenerate primers, the HV7 and HV8 primers were synthesized (Tables 2 and 3) . Thirty cycles of PCR amplification were carried out in a final volume of 100 μl containing 1 μg of HHV-7-infected CBMC DNA, 10 mM Triε-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 0.2 mM of each dNTP, 0.5 μM of the HV7 and HV8 primers and 2.5 units of Taq DNA polymerase, with the following program: 1 min at 94^βC, 2 min at 60°C, 2 min at 72°C with an increaεe of 2 εec per cycle, and an additional final extenεion at 72°C for 7 min.

The HHV-7 εequences were determined on the 3L25 clone. Only the sequence located internally to the regions covered by the degenerate primers is indicated in SEQ ID N0:1. Localization of the oligonucleotides (Tables 2 and 3) on the sequence is as follows: HV7, 1-26 (εenεe) ; HV8, 186-161 (antiεenεe); HV9, 82-111 (sense); HV10, 48-70 (sense); HVll, 171-145 (antiεenεe); and HV12, 132-103 (antiεenεe). Sequence alignment between thiε HHV-7 εequence

(originating from the 3L25 clone) and HHV-6 and human cytomegaloviruε DNA sequences showed a nucleotide homology of 57.5% between HHV-7 and HHV-6, of 36% between HHV-7 and HCMV, and of 39.8% between HHV-6 and HCMV. At the amino acid level there is, respectively, an identity and a similarity of 51.6% and 19\4% between HHV-7 and HHV-6, of 25.8% and 17.7% between HHV-7 and HCMV, and of 27.4% and 19.4% between HHV-6 and HCMV. Comparison and alignment of DNA and protein sequenceε waε performed uεing CLUSTAL and PALIGN programs in PC/Gene Software (Intelligenetics. Mountain View, CA) .

PCR amplification of KHV-7 DNA seguences, using primers HV10 and HVll waε alεo performed. The amplification waε carried out using the same protocol as described for primer pair HV7/HV8 as above, except that annealing occurred at 55°C instead of 60°C. The PCR products were transferred by electroblotting from gels to nylon filters and hybridized to probe HV12 (washing conditions: 6X SSC-0.5% SDS at 60°C, twice for 10 min; autoradiography after 4 hr at -80^CC) . The hybridization showed a positive signal on the PCR products amplified from HHV-7-infected cell DNA only.

Example 3 A plasmid probe was prepared which incorporated the

DNA sequences produced by PCR with primers HV7 and HV8. The plasmid was denoted p43L3 and demonstrated εpecific hybridization to HHV-7 viral DNA.

The amplified DNA εequence resulting from PCR performed with HV7 and HV8 primers as described above was cloned using the TA Cloning System Version 1.0 (Invitrogen, San Diego, CA) . The manufacturer's instructions were followed for the dilution of the PCR product, ligation into the vector and transformation into bacterial cells. Transformed bacterial colonies were screened with the ³²P-end-labelled HV9 oligonucleotide probe (Table 1) . The probe was prepared by end labelling with T4 polynucleotide kinaεe followed by hybridization, waεhing, and autoradiography procedures. Some plasmid probes demonεtrated hybridization to the HV9 probe. Plaεmid p43L3 waε then employed as probes in Southern blot hybridizations with different human herpeεviruεeε.

High-molecular-weight DNA from cellε infected with HHV-7, HHV-6, herpes εimplex virus type 1, Epstein Barr virus, varicella zoster virus and human cytomegalovirus was examined. Following digestion with Hind III or Bam HI, Southern blot analysis was performed using p43L3 and several HHV-6 probes. Five μg of DNA of Hind Ill-digested DNA from each viral strain was electrophoresed in a 0.8% agarose gel and transferred to a nylon filters by Southern blotting. The nylon filterε were prehybridized for at leaεt 2 hr at 37°C in Hybriεol I solution (Oncor, Gaitherεburg, MD) . Following prehybridization, 10⁶ cpm/ml of each DNA probe that was ³²P-labelled by nick translation was contacted to different filters. Hybridization was overnight at 37°C. The filters were washed with 6x SSC-0.5% SDS, twice for 15 min at 60°C, followed by further washing with increasing stringency. The most stringent conditions were O.lx SSC-0.5% SDS at 60°C. The filters were then autoradiographed.

While all the HHV-6 probes uεed hybridized to DNA of all HHV-6 εtrainε tested so far, only 3 of them hybridized to HHV-7 DNA. The p43L3 plasmid probe, containing the amplified HHV-7 DNA fragment without the region covered by the degenerate primerε, only hybridized to HHV-7 DNA and not to any other human herpesvirus DNA tested (HSV-1, EBV, VZV, HCMV, HHV-6) , including 12 different HHV-6 strains.

Example 4

This example describes isolation and characterization of different HHV-7 isolates.

Three isolates of HHV-7 were characterized in this study. Two of the isolateε, JI (Berneman, Z. N. et al., J. Infect. Dis.. 166:690-691 (1992)) and JB, were derived from the peripheral blood of patients affected by the chronic fatigue syndrome (CFS) , while one isolate, denoted AL, was obtained from the peripheral blood of a healthy adult individual. All three isolateε were characterized aε HHV-7 on the basis of immunologic and genetic analyses as discuεεed below. See Berneman, Z. N. et al., Proc. Natl. Acad. Sci. USA. 89:10552-10556 (1992) and Berneman, Z. N. et al., ^ Infect. Diε._f 166:690-691 (1992) in regard to iεolate JI. Peripheral Blood Mononuclear Cellε (PBMC) from either CFS or normal patientε were cultured in RPMI 1640 supplemented with 20% fetal calf serum, phytohemagglutinin (PH, 5 μg/mL) , and interleukin 2 (IL-2, 10% vol/vol) . After 9 days of culture, the primary cells were treated with 25 μg/mL mitomycin C for 30 min at 37°C, washed with RPMI 1640, and cocultured in IL-2-containing medium with cord blood mononuclear cells. Due to extensive cell death, the cells were again cocultured with new, PHA-prestimulated cord blood mononuclear cells about 4 weeks later. At 10 days after the second coculture, large cells εtated to appear, reminiscent of the cytopathic effect seen after infection with HHV-6. This cytopathic effect peaked after 13 days, and most of the cells were lysed after 17 dayε. Culture of new PHA-stimulated cord blood mononuclear cells with supernatant of the infected culture resulted in the appearance of new large cells, with a peak after 8 days. Those cells (approximately 10% of the whole culture) reacted by immunofluorescence with the patient's serum, indicating foreign antigen expression. The serum had an IgG titer against strain JI of 1:200 and against HHV-6 strain GS of 1:20.

There was no reactivity of the infected cells with specific antibodies directed against herpes simplex virus (HSV) types 1 and 2, Epεtein-Barr viruε (EBV) , human cytomegaloviruε (HCMV) , or varicella-zoεter viruε (VZV) . Southern blot hybridization with probes recognizing HSV-1, EBV, HCMV, VZV and herpesviruε εaimiri DNA or with the HHV-6 probe pZVH14 (Josephs et al.. Science. 234:601-603 (1986)) did not reveal any hybridization signal.

Transmission electron microscopy was performed on glutaraldehyde-fixed thin sections from cells infected with HHV-7 isolateε. Thiε analyεis revealed the presence of viral particles with the characteristic morphological features of herpesviruses (Fig. 1) . Both intranuclear and cytoplasmic virions at different stages of maturation were visible. Mature, enveloped virions, approximately 200 nm in diameter, were released into the extracellular space. They contained an electron-denεe core εurrounded by the capsid membrane, a tegument and an external envelope, sometimes exhibiting typical spike-shaped projections on the external εurface.

To characterize the JB and AL herpesvirus isolates, acetone-fixed infected cells were prepared for analysiε by indirect immunofluorescence (IF) with murine monoclonal antibodies (MAbs), aε described in Lusso et al., J. Exp. Med.. 167:1659-1670 (1988). Fluoreεcein iεothiocyanate (FITC)- conjugated goat-anti-mouse-immunoglobulin-G (IgG) antiserum was used as a second reagent. The two isolates (JB and AL) exhibited the typical reactivity pattern of HHV-7. Infected cells were recognized by two MAbε, 9A5D12 (p41) and 12B3G4 (pl35) , originally developed against HHV-6, but known to cross-react with HHV-7. In contrast, no reactivity was seen with other MAbs specific for HHV-6, (2D6 and 13D6 [gp82/105], 6A5G3 [gpll6/65/54]) , or with MAbε εpecific for hCMV (9220 [early nuclear protein], 9221 [late nuclear protein]) (Dupont) .

To better inveεtigate the in vitro propertieε of HHV-7, optimized conditions for the growth of the new iεolateε in primary human lymphocyte cultureε were developed. Becauεe the predominant target cellε for HHV-7 appear to be T lymphocytes of the CD4⁺ phenotype, enriched, PHA-activated populations of CD4⁺ T lymphocytes derived from the peripheral blood of healthy blood donors were used. The cells were purified by negative immunomagnetic selection, uεing magnetic beads coated with goat-anti mouse-IgG antiεerum (Dynal) , after the cellε were labelled with a cocktail of MAbε to CD8 (Leu-2a) , CD14 (Leu-M3), CD16 (Leu-llb) , CD19 (Leu-12) , and CD56 (Leu-19) (Becton Dickinεon) . In εuch cultureε, infection by both new iεolateε, as well as by another HHV-7 isolate (JI) previously obtained from a CFS patient was rapid and generalized. Virus titers of up to 10⁵-10⁶ infectious particles/ml were detected in the culture supernatantε 7-10 dayε after infection, aε determined by infecting normal CD4⁺ T lymphocyteε with serial ten-fold dilutions of the virus stock.

Infection of normal CD4⁺ T cells by all three iεolates of HHV-7 was progressive and cytopathic, and characterized by the appearance of large, refractile cells and increaεing loεε of cell viability. A variable proportion pf cells exhibited the typical morphological features of giant multinucleated cells (Fig. 2A) , as also revealed by electron microscopy (Fig. 2B) and nuclear IF staining with MAb 9A5D12 (Fig. 2C) . These εyncytia were indistinguishable from those induced by human immunodeficiency virus, the cauεative agent of AIDS.

Aε previously described in Berneman et al. supra. the neoplastic CD4⁺ T-cell line Sup-Tl, originally derived from a pediatric patient with non-Hodgkin*s T-cell lymphoma, was found to be suεceptible to HHV-7 infection. When inoculated with high-titered, cell-free supernatant from infected CD4⁺ T cells, Sup-Tl cells showed generalized and progressive cytopathic effect, with more than 80% antigen- positive cells by day 8-10 poεt-infection. However, cocultivation with infected primary lymphocyteε waε consistently more rapid and efficient than cell-free viral transmission. Low-level HHV-7 replication was also detected in two additional neoplastic CD4⁺ T-cell lines, Jurkat and Molt-3.

PCR amplification was carried out using primerε εpecific for HHV-6 or HHV-7, aε reported in Berneman et al., εupra. Primerε derived from a highly conserved region of the HHV-6 genome did not amplify DNA extracted from cells infected with HHV-7_AL or HHV-7_JB (Fig. 3A) . However, the expected 186 bp PCR product was amplified from both iεolateε after a εingle round of amplification using a pair of HHV-7-specific primers, HV7 and HV8 (Fig. 3B) . The specificity of the reaction was then confirmed by Southern blot hybridization of the PCR product with the internal, ³²P-labelled oligodeoxynucleotide probe HV12.

To definitively establiεh the identity of the new HHV-7 iεolateε, we performed Southern blot hybridizations using a panel of recombinant DNA probes cloned in pBluescript plaεmid (Stratagene) from HHV-7_jj. High-molecular-weight DNA, extracted from Sup-Tl cellε infected with iεolateε AL, JB and JI, waε digeεted with different restriction endonucleases and analysed by Southern blot. Genomic DNA from cells uninfected and infected with HHV-6_GS was also included as control. Under stringent conditions, all the HHV-7 probes showed reactivity with HHV-7_AL, HHV-7_JB and HHV-7J_J DNA, but none cross- hybridized with HHV-6 DNA (Figs. 4A-B) . As expected, none of the probes used reacted with cellular DNA from uninfected cultureε. Moreover, no hybridization was observed using an HHV-6-specific probe, pZVH14.

While most of the HHV-7 probeε used (pVL44, pVL23, pVL18, pVL17) yielded the same restriction patterns for all three viral iεolates, genetic polymorphism was observed using one probe, pVL8 (Figs. 4A-B) . After Hind III digestion,

HHV-7_jB and HHV-7_jj yielded a single large fragment of about 11 Kb. In contrast, a band of lower molecular weight (approximately 5.5 Kb) was seen with HHV-7_AL (Fig. 4A) . Similarly, after Sam HI digeεtion, the reεtriction pattern of HHV-7_JB waε the εame aε that of HHV-7_JJ, but different from that of HHV-7_AL (Fig. 4B) . It iε noteworthy that the isolates displaying a similar restriction pattern were both independently derived from patients with CFS.

The availability of optimized culture conditionε for HHV-7 growth permitted eεtabliεhment of a teεt for in vitro neutralization of HHV-7 infectivity or syncytia formation. The neutralization teεt employed purified populations of CD4⁺ T lymphocytes from normal peripheral blood. An HHV-7 inoculum containing approximately 2 x 10⁴ cell culture infectious_, doses/ml waε pretreated with serial dilutions of the test serum for 30 min at room temperature and subsequently used to infect 10⁵ purified CD4⁺ T cellε, previouεly activated with PHA. Incubation waε continued for 5 days in 24-well plates (Coεtar) in a total volume of 1 ml of complete medium. The infection waε evaluated at day 5 poεt-infection by indirect IF on acetone-fixed cells with mAb 9A5D12. For neutralization of HHV-7-induced syncytia formation, 4 x 10⁴ HHV-7-infected normal CD4⁺ T lymphocytes were pretreated for 30 min at room temperature with serial dilutions of the test serum and then cocultured with 2 x 10⁵ uninfected Sup-Tl cells in 24-well plates in a total volume of 1 ml. Syncytia formation waε scored at 20 and 40 h after cocultivation. Serum from the JB patient contained antibodies to

HHV-7 antigens expressed on the surface membrane of HHV-7- infected cells. Infected Sup-Tl cellε were exposed to serum from patient JB at 1:80 dilution and analyεed by fluorocytometry after εtaining with FITC-conjugated goat-anti- human IgG antiεerum. A εtrong εurface membrane fluorescence was obεerved in the majority of the infected cellε, but not in uninfected control Sup-Tl. To test for the presence of neutralizing antibodies, serial two-fold dilutionε of the JB serum, previously heat-inactivated by incubation at 56°C for 40 min, were used to pretreat the HHV-7 inoculum. The neutralizing antibody titer was determined as the reciprocal of the highest dilution of serum that completely prevented large cell formation and IF signs of productive HHV-7 infection. The neutralizing titer observed in the JB serum was 1280. A similar result were obtained in the εyncytia inhibition teεt.

Example 5

Thiε example demonstrates synthesis of vector probes of the present invention. The probes hybridize to HHV-7 nucleic acid.

Supernatant from HHV-7-infected SUP-T1 cells was concentrated by ultracentrifugation. The pellet was embedded into a plug of low melting point agarose gel, digested with proteinase K and electrophoreεed using field inversion gel electrophoresis. A DNA band of approximately 155,000 base pairs was demonεtrated to hybridize with p43L3, an HHV-7 specific probe described in Berneman et al. , Proc. Natl. Acad. Sci. USA. 89:10552-10556 (1992), previously incorporated herein by reference. The band was cut from the gel and digested overnight with beta-agarase I (New England Biolabs) . Following phenol/chloroform extractions, the DNA was ethanol precipitated, washed in 70% ethanol, air-dried and redisεolved in distilled water. The DNA was digested with BamHI and ligated into BamHI-digeεted phoεphataεed pBlueεcript plaεmids (Stratagehe) . Recombinant plasmids were extracted from transformed bacterial colonies, analyzed by restriction enzyme digeεtion and Southern blot hybridization with ³ P-labelled purified viral DNA. HHV-7 specificity was aεεayed by Southern blot hybridization of DNA from cellε either infected or not infected with different human herpesviruseε, including HHV-7. Plasmid clones were selected on the basis of different sized BamHI inεertε. Redundancy was analyzed by crosε-hybridization analysis.

The following HHV-7 plasmid clones have been obtained: pVL23: contains a 13 kilobasepair (kbp) insert; hybridizes to HHV-7 only. pVL23.2 iε an identical (sub)clone. pVL17: originally isolated as a clone with a 4 kbp insert; similar clones include pVL17D.l and pVL17D.2. Other related clones (for instance PVL17A.1, pVLI7C.l) contain a 6kbp insert. The 4 and 6 kbp insertε croεε-hybridize, and most probably are identical over a 4 kbp region. The kbp insert hybridizes to HHV-7 only. pVL18: contains 5.5 kbp insert; hybridizeε to HHV-7 and HHV-6. Hybridization to HHV-6 iε seen after low-stringency washes only (6X SSC at 60°C) . There is an identical (sub) clone with the name XPVL18.1. pVL44: contains a 3.5 kbp insert; hybridizes to HHV-7 only. pVL29: contains a 2.2 kbp insert; hybridizes to HHV-7 only. pVL3: contains a 2.1 kbp insert; hybridizes to HHV-7 only. pVL19: contains a 1.3 kbp insert; hybridizes to HHV-7 only. pVL13: this clone originally contained 2 BamHI insertε, of 0.6 kbp and 0.3 kbp; only the former ("pVL13-0.6K or pVL13.1") hybridizeε to viral DNA, and then to HHV-7 only.

Alternatively, BamHI inεertε were ligated into BamHI lambda bacteriophage (Lambda Daεh II, Stratagene) to form a BamHI lambda bacteriophage library. Plaques were screened as above. The following lambda bacteriophage clones have been developed: XVL5: containε a 15 kbp fragment; hybridizeε to both

HHV-7 and HHV-6, even after high-εtringency washes. Contains the HHV-7 εequence of the inεert of plaεmid p43L3 aε described in Berneman et al., Proc. Natl. Acad. Sci. USA. 89:10552-10556 (1992), as well aε the GGGTTA repeat aε deεcribed in Berneman et al., εupra and Kiεhi et al., J. Virol.. 62:4824-4627 (1988) .

XVL12: contains a 16 kbp fragment; hybridizes to HHV-7 only.

XVL56: contains an 11 kbp fragment; hybridizes to HHV-7 and HHV-6. Clone λVLlO contains the same 11 kbp insert as XVL56, in addition to the insert of plasmid clone pVL29.

XVL10: this clone contains the same 11 kbp insert as XVL56, in addition to the insert of plasmid clone pVL29. XVL65: this clone contains 2 HHV-7 insertε, a 15 kbp inεert identical to the insert of XVL5 (that contains the GGGTTA repeat), aε well aε a 8.5-9 kbp inεert that hybridizeε with the inεert of pVL8 but doeε not εeem to contain the GGGTTA repeat.

XVL73: thiε clone containε 2 HHV-7 inserts, a 13 kbp insert identical to the insert of pVL23, as well as a 8.5-9 kbp insert that hybridizes with the insert of pVL8 and alεo seems to contain the GGGTTA repeat. XVL81: this clone containε 2 HHV-7 inεertε, a 5.5 kbp inεert identical to the inεert of PVL18, aε well aε a 8.5-9 kbp insert that hybridizes with the insert of PVL8 and also seems to contain the GGGTTA repeat.

Example 6

This example demonstrateε lymphocyte expression of CD4 receptors during HHV-7 infection. Receptor expresεion iε downregulated during infection.

The expression of several cell membrane antigens was meaεured by fluorocytometry of normal peripheral blood CD4* T lymphocyteε during the course of HHV-7 infection. In parallel, HHV-7 expresεion waε monitored by indirect immunofluoreεcence on acetone-fixed cellε using monoclonal antibody 9A5D12. Thiε monoclonal antibody recognizeε viral antigenε present inside HHV-7 infected T lymphocyteε.

The cells were purified by a_βdouble rosetting procedure using immunomagnetic beads (Dynal) , as described previously (Lusso, P. et al., J. EXP. Med.. 167:1659-1670 (1988)) and briefly below. Cells were first incubated with a cocktail of monoclonal antibodieε (e.g., anti-CD8, CD16, CD19, CD20, CD56) . After 30 minutes in ice, they were washed and incubated with the magnetic spheres coated with anti-mouse IgG antiserum developed in goats. It was expected that cells with a bound monoclonal antibody would be attached to the magnetic beads. The reaction was prolonged for 30 minuteε under continuouε rotation after which a magnet was applied to the plastic tube, so as to attract all the cells with bound microspheres to the wall. The unbound cells were free to float and were collected by decanting the liquid phase into a new tube. Subsequently cellε were activated with 1 μg/ml of purified PHA (Wellcome) .

The cellε were infected with HHV-7_AL grown in enriched normal CD4⁺ T cells. Viral antigen expresεion waε evaluated at three day intervals by indirect immunofluorescence on acetone-fixed cells with mAb 9A5D12 (Lusεo et al., J. EXP. Med.. 167:1659-1670 (1988), incorporated herein by reference) . The cellε were firεt washed, counted and an aliquot dried on a εerology εlide. After thorough desiccation, they were fixed with pure cold acetone for 5 minutes. Subsequently, the monoclonal antibody was applied at 5 μg/ml in a volume of 20 μl. After 20 min at room temperature, the slide was rinsed repeatedly in saline buffer. The cells were then treated with the secondary antibody (goat-anti-mouse IgG) for 20 minuteε at room temperature and again rinsed in saline buffer. Finally, a coverslide waε mounted with glycerol and the cellε were εcored under a fluorescence microscope (Leitz) . The mAbs used for fluorocytometric analysis were fluorescein-isothiocyanate (FITC)-labelled Leu-5a (anti-CD2) , phycoerythrin (PE)-labelled Leu4 (anti-CD3) and Leu3a (anti- CD4) (Becton Dickinεon), PE-labelled OKT4 (anti-CD4) (ortho Diagnoεticε) and FITC-labelled anti-CD44 (AMAC) . Irrelevant iεotype-matched mouse immunoglobulins conjugated with FITC or PE (Becton Dickinson) were used as controls for each test and are depicted in the histograms of Fig. 5 as empty profiles. At leaεt 5,000 eventε were accumulated for each teεt. While the expression of CD2, CD3 and CD44 was substantially unaltered even at the late stages of HHV-7 infection, a progressive downregulation of CD4 was observed in cultures infected by HHV-7_AL (Fig. 5).

Virtually no CD4 remained detectable at day 9 post- infection. At this time, HHV-7 antigens were detectable in more than 80% of the cells. Expression of CD4 was assayed using two different mAbs (Leu3a and 0KT4) which recognize two distinct domains of the glycoprotein (Sattentau, Q. J., et al.. Science. 234:1120-1123 (1986)). Analogous results were obtained with HHV-7_JB and HHV-7JJ isolates grown in normal CD4⁺ T cells, as well aε with HHV-7_AL and HHV-7_JB grown in a neoplaεtic CD4⁺ T-cell line, SupTl, derived from a pediatric non-Hodgkin•ε lymphoma and previouεly shown to be susceptible to HHV-7 (Berneman, Z. N. et al., Proc. Natl. Acad. Sci. USA. 89:10552-10556 (1992)).

As receptor downregulation iε a common biological sequela of receptor-ligand interaction, CD4 could be involved in the receptor εtructure for HHV-7 on the cellular membrane. Infection inhibition experimentε were performed in enriched normal CD4⁺ T lymphocyteε using different mAbs directed to CD4. Antibodies against CD4 could inhibit HHV-7 infection if the viral particleε used thiε glycoprotein aε part of itε receptor complex.

Example 7

Thiε example demonstrates inhibition of HHV-7 infection by anti-CD4 antibodies.

Enriched, normal peripheral blood CD4⁺ T cells (1 x 10⁶/test) were treated with monoclonal antibodieε OKT4a, Leu3a or OKT4 for 30 minutes at room temperature (RT) in 24-well plates (Costar*) and then expoεed to HHV-7 at an approximate Multiplicity of Infection (MOI) of 0.1 (i.e., 0.1 CCID/cell) . The monoclonal antibodieε were maintained at the εame original concentration throughout the culture period. After 24 hours human IL-2 was added to maintain the cultures at 10 U/ml. Infection was monitored by indirect immunofluorescence with mAb 9A5D12, as described above.

A dose-dependent inhibition of HHV-7_AL infection was observed when the cellε were pretreated with OKT4a, Leu3a or OKT4, as documented by indirect immunofluorescence analysis with mAb 9A5D12 (Fig. 6) . Data represent the mean values from at least three separate experiments. As discussed above, mAb 9A5D12 binds to viral antigens present in infected cells. A lower amount of 9A5D12 binding indicates fewer infected cells. Dose-dependent inhibition of HHV-7 infection was shown in purified normal CD4⁺ T cells by anti-CD4 mAbs (o = Leu3a; □ = OKT4a; ♦ = 0KT4). The 50% inhibitory dose (ID₅₀) for 0KT4 was similar to that obεerved for OKT4A (between 0.01 and 0.02 μg/ml) . Analogous results were obtained with HHV-7_JJ and HHV-

⁷JB*

MAbs to other cell surface determinants (A = HLA-DR (Chemicon) ; * = CD3 (OKT3; Ortho) ; • - anti-S₂ microglobulin, as well as irrelevant mAbs (♦ = M26; ■ = M90; directed against the p24 and gpl20 proteins of HIV-1, reεpectively) were used as controls. Several mAbs used aε controls failed to induce significant inhibition of HHV-7 infection, including mAbs directed to class II human leukocyte antigen (HLA)-DR, β₂ microglobulin and CD3, as well as two irrelevant mAbs directed to the p24 core and gpl20 envelope proteins of HIV-1. We also performed experiments in which treatment with anti-CD4 mAbs was started after binding and internalization of HHV-7. When OKT4 or OKT4A (used at 10 μg/ml) were added 4 hr after exposure of normal CD4⁺ T cells to HHV-7_AL at 37°C, significant reduction, but not complete abrogation of HHV-7 infection was observed.

Following these experiments the ability of soluble CD4 to block infection of HHV-7 was tested. It would be expected that if CD4 iε a part of the HHV-7 receptor complex, it would bind to viral particles and inhibit infection.

Example 8

This example demonstrates inhibition of HHV-7 infection of lymphocyteε by εoluble CD4.

A doεe-dependent inhibition of HHV-7 infection was observed when the HHV-7 stock waε pretreated with the εoluble form of CD4 (Fig. 7) . Three strains of HHV-7 (isolated as discussed above) were tested: ■ = HHV-7_AL; ♦ = HHV-7_JB; A = HHV-7_JJ. Data in Fig. 7 represent the mean values of two separate experiments. The viruε εtock (10⁵ CCID) waε pretreated with soluble CD4 (sCD4) for 30 min at room temperature and subsequently used to infect 1 x 10⁶ normal peripheral blood CD4⁺ T cells in 24-well plates by standard well known means. Complete abrogation of infection, aε meaεured by binding with antibody 9A5D12, was demonstrated by all three strainε of HHV-7 in normal peripheral blood CD4⁺ T cellε uεing 500 μg/ml of εCD4 (for HHV-?^, ID₅₀ = 63.7 μg/ml). Theεe values are comparable to those reported for εome primary iεolateε of HIV-1 (Daar, E. S., et al., Proc. Natl. Acad. Sci. USA. 87:6574-6578 (1990)).

To concluεively demonεtrate that CD4 iε involved in binding and internalization of HHV-7, the preεence of HHV-7 DNA in normal CD4⁺ T cellε expoεed to HHV-7 in the preεence or absence of anti-CD4 antibody 0KT4a waε determined by Southern blot analyεiε. Aε demonstrated previously, antibody 0KT4a inhibits HHV-7 infection by competitively binding the CD4 receptor.

Example 9

CD4⁺ T cells were exposed to HHV-7 for 1 hour at 37?C in the preεence or abεence of 5 μg/ml of OKT4a. DNA run on the gel waε extracted by the following well known εalting out procedure.

Isolated T-lymphocytes were resuspended in 15 ml centrifugation tubes with 3 mlε of lysis buffer (10 mM Tris- HCL, 400 M NaCl and 2mM Na₂EDTA, pH 8.2). The cell lyεates were digested overnight at 37^βC with 0.15 ml of 20% SDS and 0.05 ml of a protease K εolution (20 mg/ml proteaεe K) . After digestion was complete, 1 ml of saturated NaCl (approximately 6M) was added to each tube and shaken vigorously for 15 seconds, followed by centrifugation at 2500 rpm for 15 minutes. The precipitated protein pellet was left at the bottom of the tube and the εupernatant containing the DNA waε transferred to another 15 ml tube. One volume of room temperature isopropanol was added and the tubes inverted several times until the DNA precipitated. The precipitated DNA strands were removed with a plastic spatula or pipette, washed in 70% ETOH, air dried, and transferred to a 1.5 ml microcentrifuge tube containing 100-200 μl TE buffer (10 mM Tris-HCL, 0.2 mM Na₂EDTA, pH 7.5). Following isolation, the DNA was digested with Hind III, electrophoresed in a 0.8% agarose gel (10 μg of DNA per lane) and transferred to a nylon membrane by well known Southern blotting techniques. Prehybridization of the nylon filter was carried out for 2 hours at 37°C in Hybriεol I^# εolution (Oncor) , followed by addition of 10⁶ cpm/ml of the HHV-7 DNA probe pVL44 labelled with [³²P] α-dATP by nick tranεlation. Hybridization continued overnight at 37°C. The filter waε washed with 6X standard saline/citrate and 0.5% SDS twice for 15 minutes at 60°C. The gel was autoradiographed for 72 hours at -70°C.

On the Southern Blot lane 1 was DNA isolated by standard methods from CD4⁺ T cells exposed for 1 hr at 37°C to HHV-7_AL. In lane 2 was DNA from CD4⁺ T cells pretreated with OKT4a at 5 μg/ml for 30 min at room temperature and then exposed to HHV-7; and lane 3 contained DNA from uninfected CD4⁺ T cellε. Hybridization with a 3.5 Kb DNA probe specific for HHV-7 yielded a distinct signal of the expected molecular weight in the absence of OKT4a, but pretreatment of the cells with OKT4a dramatically reduced the binding and/or internalization of HHV-7.

The pVL44 probe was derived aε follows. Supernatant from HHV-7_jj-infected SUP-T1 cells was concentrated by ultracentrifugation. The pellet was embedded into a plug of low melting point agarose, digeεted with proteinaεe K and electrophoreεed using field inversion gel electrophoresis in a low melting point agarose gel. A DNA band of approximately 155,000 basepairs - and previously shown to hybridize to the HHV-7-εpecific probe p43L3 waε cut out and the low melting point agaroεe digested overnight with beta-agarose I (New

England Biolabs) . After phenol/chloroform extractions, the DNA was ethanol precipitated, washed with 70% ethanol, air- dried and redissolved in distilled water. DNA was then digested with BamHI and ligated into BamHI-digested and phosphatased pBluescript plasmid (Stratagene, San Diego) .

Recombinant plasmids were extracted from transformed bacterial colonies, analyzed by restriction digestion with BamHI and Southern blot hybridization with ³²P-labelled purified viral DNA. HHV-7 εpecificity waε aεεayed by Southern blot hybridization of DNA from cellε infected or not infected with different human herpes viruses, including HHV-7_JJ. Plaεmid clones were also selected based on the different size of their BamHI inεert, and redundancy examined by cross-hybridization analysis of all plasmid clones collected.

The extent of DNA hybridization to the HHV-7 specific probe pVL44 was quantitated using a video densitometry εyεtem (Phosphorimager^β, Molecular Dynamics). The values indicated on the y axiε of Fig. 8 repreεent arbitrary unitε of peak intenεity of the hybridization εignal. Aε indicated in Fig. 8, the quantity of HHV-7 DNA isolated from cells pretreated with 0KT4a was about 80% less than the quantity of HHV-7 DNA isolated from infected, non-OKT4a pretreated cells. This demonstrates a specific reduction in the amount of HHV-7 DNA in cells that have been pretreated with an anti-CD4 antibody. For this reason CD4 appears to be intimately involved in the infectious pathway for HHV-7.

In addition to the above experiments, we tested the effect of anti-CD4 mAbs on the formation of giant multinucleated cellε induced by HHV-7.

Example 10 Thiε example demonstrates inhibition of giant cell formation by treatment of cellε with anti-CD4 antibody prior to exposure to infectious HHV-7 particleε.

Both 0KT4a and OKT4 uεed at 2 μg/ml completely abrogated the formation of εyncytia following overnight ^• cocultivation of uninfected and infected εupTl cellε. SupTl iε a neoplastic CD4⁺ T-cell line derived from a pediatric non- Hodgkin'ε lymphoma and previouεly εhown to be εusceptible to HHV-7 infection (Berneman, Z. N. et al., Proc. Natl. Acad. Sci. USA. 89:10552-10556 (1992)). In this experiment SupTl cells were cocultured overnight with HHV-7_AL-infected SupTl at 5:1 ratio between uninfected and infected cells. Monoclonal antibodies 0KT4a and 0KT4 were added at 2 μg/ml to uninfected cells 30 minutes prior to cocultivation with infected cells. SupTl cells pretreated with either OKT4a or OKT4 exhibited virtually no syncytia formation whereas cells that were not pretreated formed a large number of multi-nucleated cells. Altogether, the above examples strongly indicate that CD4 is critically involved in the receptor mechanism for HHV-7 in CD4⁺ T cellε. Since CD4 alεo represents the major receptor for HIV (Dalgleish, A. G. et al.. Nature. 312:763-767 (1984); Klatzman, D. et al.. Nature. 312:767-769 (1984)), the causative agent of the acquired immunodeficiency εyndrome (Barre-εinouεεi, F. et al.. Science, 220:868-871 (1983); Gallo, R. C. et al.. Science. 224:500-504 (1984)), it was investigated whether receptor interference could occur between HHV-7 and HIV-1.

Example 11

Thiε example demonstrates inhibition of HIV-1 infection of cells infected with HHV-7.

The effect of sequential or simultaneous HHV-7 coinfection on the ability of HIV-1 to infect CD4⁺ T lymphocytes was evaluated. In studies of sequential coinfection, different strainε of HIV-1 were added to enriched normal peripheral blood CD4⁺ T cells which had been infected with HHV-7_A 48 hours earlier.

Enriched populations of CD4⁺ T cells from normal peripheral blood were infected with HHV-7_AL, at the approximate MOI of 1. Forty-eight hours later, HHV-7-infected cellε and uninfected controlε (10⁶ cellε/teεt) were exposed to different HIV-1 isolateε. All the HIV-1 inocula contained 100,000 cpm of reverεe transcriptase per 10⁶ cells. HIV-1₅₇₁, HIV-1₅₇₃ and HIV-1_B0 were iεolated from PBMCε of different individualε with HIV-1 infection, and had been paεsaged only once in normal, PHA-activated peripheral blood mononuclear cells. HIV-1_RF and HIV-1_IIIB were obtained from persistently infected SupTl cells. After exposure to HIV-1, the T cells were extensively washed and recultured in 24-well microtiter plateε (Costar ) in the presence of purified human interleukin 2 (IL-2) (10 U/ml; Boehringer Mannheim). At day 4 post- infection, the cell viability was greater than 80% in all the cultures. The release of HIV-1 p24 antigen waε meaεured uεing a commercially available ELISA kit (Dupont) . The data preεented in TABLE 1 indicate the amount of HIV-1 p24 antigen (pg/ml) releaεed into the εupernatant fluid of the cultureε at day 4 poεt-infection.

In εeparate experiments with CD4⁺ T cells from two different donors, HHV-7 pre-incubation totally abrogated superinfection by five εtrainε of HIV-1 (TABLE 4) , including two well characterized laboratory iεolates (RF, IIIB) (Gallo, R. C. et al.. Science. 224:500-504 (1984); Popovic, M. , et al.. Science. 224:497-500 (1984)) and three field isolateε (571, 573, BO) passaged only once in vi tro in unfractionated, PHA-activated normal peripheral blood mononuclear cells.

TABLE 4 HIV-1 Replication (pg/ml of p24Y in:

HIV-1 isolate CD4⁺ T cells HHV-7-infected

CD4⁺ T Cells

571 (exp. 1) 15,895 0 (exp. 2) 799 0

(exp. 3) 3,310 0

573 (exp. 1) 5,500 0

(exp. 2) 403 0

(exp. 3) 2,672 0 BO (exp. 1) 1,154 0

(exp. 2) 17,700 0

(exp. 3) 1,360 0

RF (exp. 1) 22 0

(exp. 2) 3,900 0 (exp. 3) 5,520 0

IIIB (exp. 1) 1,868 32

(exp. 2) 5,300 0

(exp. 3) 547 0

It waε also studied whether HIV-l infection could be affected by simultaneous coinfection with HHV-7. Example 12

Thiε example demonstrates suppreεεion of expression of p24 gag protein in cells co-infected with HIV-l and HHV-7. Normal CD4⁺ T lymphocytes (10⁶/test) were exposed to either HHV-7_AL at the approximate MOI of 5 for 30 minutes at 4°C, or to uninfected culture medium as a control. Ten-fold dilutions of an HIV-1₅₇₁ inoculum containing 10⁶ CCID/ml were then added for 1 hour at 37°C, after which the cells were extensively washed and recultured in the presence of human IL- 2 (10 U/ml) in 24-well plateε. At the time of HIV-l p24 testing, the cell viability was greater than to 80% in all the cultures. The data in TABLE 5 indicate the amount of HIV-l p24 antigen (pg/ml) released into the culture supernatant at day 5 post-infection as measured by a commercially available ELISA kit (Dupont) .

Simultaneous coinfection with HHV-7 reduced the extracellular release of HIV-l p24 antigen by more than 99% (Table 5). At low MOI (0.01), HIV-l infection was totally suppressed by HHV-7 coinfection. These same results were found with Ultraviolet light treated HHV-7 virus. These data indicate that HHV-7 significantly interferes with HIV-l infection in vitro.

Table 5 HIV-l replication (pg/ml of p24)

MOI used for HIV-l infection

Culture 1.0 0.1 0.01

CD4⁺ T cells

(exp. 1) (exp. 2) (exp. 3)

CD4⁺ T cells + HHV-7

(exp. 1) (exp. 2) (exp. 3)

CD4⁺ T cells + UV-tr<

(exp. 1) (exp. 2) (exp. 3)

To elucidate whether HIV-l could in turn interfere with HHV-7 infection, the ability of the gpl20 major envelope glycoprotein of HIV-l to affect HHV-7 infection was εtudied.

Example 13

This example demonstrateε inhibition of HHV-7 infectivity by treatment with εoluble HIV-l gpl20 antigen. A εample of CD4⁺ T cellε were pretreated with εoluble gpl20 for 30 min at room temperature. Native gpl20 protein waε purified by immunoaffinity chromatography from HIV-l infected CD4⁺ T-cell lineε, aε reported (Gallo, R. C. et al.. Science. 224:500-504 (1984)). HHV-7 waε εubεequently added to the pretreated T cellε in 24-well plateε by εtandard well known meanε. Fig. 9 illuεtrates the dose-dependent inhibition of HHV-7 infection by pretreating CD4⁺ cellε with gpl20. In thiε example, gpl20 waε derived from three different HIV-l isolates (■ = IIIB; ♦ = 451; = BaL) . As seen in Fig. 9, purified, native gpl20 from three different isolates of HIV-l (IIIB, 451, BaL) caused a dose- dependent inhibition of HHV-7_A infection. It was also analyzed whether chronic infection by HIV-l could induce CD4⁺ T cellε to become resistant to superinfection by HHV-7.

Example 14 This example εhowε inhibition of HHV-7 infectivity in cellε chronically infected by HIV-l.

The SUPT1 cell line waε persistently infected with two different strainε of HIV-l, IIIB and RF, and εubεeguently expoεed to HHV-7_AIi. SupTl cellε were infected with HIV-1_IIIB or HIV-1_RF. After the peak of the cytopathic effect had faded, a population of persistently and productively infected cells were recovered, which were negative for surface CD4 antigen, as assessed with monoclonal antibody Leu3a. SupTl cells uninfected or persistently infected with HIV-l were exposed to HHV-7^ at the approximate MOI of 1. HHV-7 antigen expresεion was monitored at three day intervals by indirect immunofluorescence with mAb 9A5D12.

HHV-7 infection was monitored by indirect immunofluorescence on acetone-fixed cells for up to 20 days post-infection. While previously uninfected SupTl were readily infected by HHV-7, no signs of HHV-7 antigen expresεion were detected in SupTl persistently infected by either strain of HIV-l.

Similar data were obtained with HHV-7_JB or HHV-7 _j. Thus, reciprocal interference can occur between HIV-l and HHV- 7. The results described in this report demonstrate that HHV- 7, like both the human and simian immunodeficiency retroviruses, uses CD4 aε a critical component of its membrane receptor. It is anticipated that this discovery could lead to treatments for persons suffering from HIV infection in vivo.

Example 15

This example demonstrates in vivo suppression of HIV-l infectivity with HHV-7. A patient with documented HIV-l infection is identified by standard well-known means. At day 0, and once a day for 14 days thereafter, a pharmaceutical composition containing HHV-7 or HHV-7-derived product(s) is intravenously injected into the patient. On the 14th day, blood εerum is isolated from the patient and tested for levels of HIV-l. In addition, CD4⁺ T cells are isolated and counted. A lower level of HIV-l virions or HIV-l genetic material iε found in the patient'ε blood εerum due to competitive inhibition of HIV-l by the HHV-7 viral particleε. After εeveral 14 day cycleε of treatment, a higher number of CD4⁺ T cellε iε found due to inhibition of the HIV-l infection.

It iε alεo anticipated that modified verεionε of HHV-7, εuch as attenuated viruses may also function in a εimilar manner. Attenuated viral particleε would ideally be able to replicate themselves without promoting T cell death. . These attenuated viruses would then replicate at a high rate in CD4⁺ cells and be secreted into the bloodstream. Once the attenuated HHV-7 particles were in the bloodstream they would competitively inhibit further HIV-l infection.

Attenuated viruεeε, genetically altered viruεes, or inactivated viruseε are uεed to eliminate εome of the unwanted effectε of diεseminating a virulent infectious agent. Attenuation can be achieved by εeveral means, well known to personε experts in the art. For example, long-term, high multiplicity passage in vitro has been used to obtain less pathogenic natural variants. Ultraviolet light produces virions which are unable to replicate but can εtill tranεduce viral or foreign genes into the cells. Introduction of selective mutations, by well known methods, into genes which are essential for viral pathogenicity can be performed. Finally, the entire viral genome, or portions of it, can be cloned and engineered to obtain molecular variants with diminished or absent pathogenic activity.

Many methods for introducing wild-type or attenuated HHV-7 to a mammal are well known to those of ordinary skill in the art. For instance HHV-7 could be mixed with a pharmaceutically acceptable buffer at physiological pH. In addition, HHV-7 could be given intravenously, orally, topically or by any other means known to those in the art. Advantageous carriers or adjuvants admixed with HHV-7 are also within the scope of the present invention. It may be deεirable to introduce an infectiouε amount of HHV-7 for treatment in order to provide an in vivo therapeutic effect. An infectiouε amount will vary among individuals and strain of HHV-7. However, an infectiouε amount can be determined by titration of the amount of HHV-7 required for infection uεing standard methods known to those having ordinary skill in the art.

In addition to introducing wild-type, attenuated, or inactivated HHV-7, it is anticipated that the HHV-7 envelope protein responsible for binding to HHV-7 to CD4 will provide a treatment for HIV-l infection in a mammal. The envelope protein, or receptor binding regions thereof, may be administered to a patient. The envelope protein or receptor binding region may block binding of HIV-l to CD4 receptors on T cell membranes, thus blocking cellular infection.

Example 16

This example demonεtrateε jLn vivo treatment of HIV-l infection with εoluble envelope protein from HHV-7. It is anticipated that the HHV-7 envelope protein reεponεible for binding HHV-7 to the CD4 receptor will competitively inhibit HIV-l infection in vivo. By way of definition, when discussing the HHV-7 envelope protein we are referring to any and all proteins reεponεible for HHV-7 binding to the CD4 receptor. In addition, HHV-7 protein fragmentε with binding affinity for CD4 are also within the scope of the present invention. Similarly, HHV-7 protein fragments with the ability to interfere with HIV-l binding to the CD4 receptor are also anticipated by the present invention.

Further, the native HHV-7 protein(ε) can be obtained by purification on affinity chromatography columnε from large- scale lysates of infected cell cultures. In addition, modified forms of the original protein can be genetically engineered, which may add other desired properties to the natural competitive function on HIV. For example, chimeric proteins produced by fusion of soluble CD4 with the Fc moiety of the immunoglobulin G have been obtained (the so-called "immunoadheεinε") . Such chimeric conεtructε have a marked pharmacokinetic advantage, aε they can εurvive in the bloodεtream much longer than εoluble CD4 itself.

One method of isolating the HHV-7 gene responεible for encoding the protein that binds to CD4 would be to clone the viral genes into expression vectors. DNA is isolated from a supernatant having HHV-7 viral particles or cDNA is iεolated from infected cellε. The DNA iε digested with Eco Rl and subcloned into the lambda gtll expresεion vector by well known methodε. Labelled CD4 proteins are used aε probe to εelect the lambda vector carrying a gene for the desired CD4-binding protein.

Once the recombinant protein haε been produced it can be introduced into the εerum of a patient having an HIV-l infection. A pharmaceutical composition containing between lμg and lmg of envelope protein is intravenously injected into a person having a HIV-l infection. Following injection, the HHV-7 envelope protein can competitively inhibit the HIV-l infection in vivo. Many other well known methods are available for cloning and expressing the desired genes from HHV-7.

Example 17

This example demonstrates tranεduction of HHV-7 envelope protein encoding geneε into host cells.

Methods for producing the envelope protein encoded by HHV-7 are also within the purview of the present invention. For instance, the lambda gtll vector carrying the gene encoding the envelope protein from the previouε example can be subcloned into a high level expresεion vector, εuch aε baculoviruε. Thiε type of εubcloning iε well known in the art. Poεitive baculovirus vectors could be identified by screening with labeled CD4 receptor. Those baculovirus vectors exhibiting secretion of CD4 binding molecules are selected for further study. Other high level expression systems known to those of skill in the art can also be used to express high quantities of the envelope protein. Following this cloning, the baculovirus clone iε used to transfect εelected cellε in vitro. Uεing other vectorε for cloning, e.g., retroviral-baεed, AAV-baεed vectorε, the HHV-7 proteins can be expresεed in mammals also in vivo. Other techniqueε, εuch aε direct DNA injection or DNA "bombardment" can be employed for in vivo delivery.

For the in vivo methodε, the adminiεtration route of the expression clone can be intradermal, oral, intravenous, intramuscular, subcutaneous, or intrathecal. In these methodε, the DNA εolution iε introduced in a pharmaceutically effective εolution. The transduced cell will then begin to produce high levels of the HHV-7 envelope protein. This high level of expresεion will then block the available CD4 receptorε on other cellε, effectively limiting the number of sites for HIV-l infection. This method provides an additional protocol for inhibiting HIV-l infection either in vitro or in vivo.

In addition to treating a patient having an HIV-l infection with either wildtype virus, attenuated virus, inactivated virus, or purified recombinant envelope protein(s), HHV-7 can be uεed to transduce CD4+ cells with genes possessing intrinsic anti-HIV activity, by the following methods.

Example 18

Thiε example demonεtrateε transduction of CD4 cells with genes carried by HHV-7 or HHV-7-based vectors.

A foreign gene with intrinsic anti-HIV activity can be inserted by well known techniqueε into infectiouε wild-type HHV-7 viruε, molecularly cloned HHV-7, or partε of the HHV-7 genome molecularly cloned. In addition, other feature geneε, εuch aε a selectable marker (e.g., antibiotic resiεtance genes) , can also be inserted along with the anti-HIV protective gene(s). These novel constructs cart be transduced by the HHV-7 vector into CD4 positive cells (T cells and monocyte/macrophages) in vitro and in vivo.

Novel genes or antisense oligonucleotides could be specifically introduced in CD4⁺ cells, monocytes or macrophages in vitro or in vivo by using HHV-7 as a vector. Following internalization, the viral DNA could persiεt in the cell in episomal form and/or integrate into the host cell genome. This procesε of viral mediated genomic integration and epiεomal persistence are well known properties of some members of the Herpesviradae family.

Following HHV-7 mediated transduction, and during the active viral life cycle, HHV-7 can transcribe the inserted gene. For instance, a gene encoding an antiεenεe message against the HIV-l TAT protein could inhibit HIV-l reproduction in that cell. Other antisenεe messages, εuch aε those against the HIV-l genes REV and GAG are also provided. By this procesε, cellε harboring HIV-l can be inhibited from producing HIV-l particleε due to the antisense blockage of the TAT protein. For instance, a patient having an HIV-l infection is intravenously injected with a solution containing a pharmaceutically active amount of HHV-7. In this inεtance, the HHV-7 viral particles are genetically engineered to contain antisense mesεage againεt the HIV-l TAT protein. Following injection, HHV-7 binds and iε incorporated into CD4⁺ T cellε, monocytes and macrophageε. Following internalization and possible genomic integration or persiεtence in epiεomal form, the engineered HHV-7 viruε directε tranεcription of the antiεenεe message against the TAT protein. Theεe anti-TAT RNA'ε inhibit production of HIV-l viral particleε harbored in the CD4⁺ cellε of the patient.

Other geneε can also anticipated to be incorporated in the HHV-7 genome to inhibit HIV-l progression. For instance, geneε coding for proteinε which inhibit the HIV-l viral life cycle, εuch aε transdominant mutants of GAG or REV could be introduced by well known methods into the HHV-7 genome. Following binding to CD4⁺ T cells, the HHV-7 viral particleε will be internalized into the target cells and express their genes. During the HHV-7 life cycle, the inserted inhibitory protein will also be expressed inside the cell. This protein may be a regulatory protein which binds to the HIV-l DNA, or in some other method disrupts the HIV-l viral life cycle preventing it from producing progeny viral particleε. For inεtance, geneε compriεing polymeric decoyε of naturally occurring HIV regulatory εequenceε, εuch aε poly-TAR or poly-RRE could be effective. Methodε of inεerting new genes into viruεeε are well known in the art. Similar to HIV, HHV-7 has a tropiε for CD4⁺ T lymphocytes and, in vitro, exerts on them a cytopathic effect. Unlike HIV, however, HHV-7 does not seem to be etiologically linked to immunodeficiency, aε suggested by its fairly ubiquitous distribution (Berneman et al., Proc. Natl. Acad. Sci. USA. 89:10552-10556 (1992) and Wyatt, L. S., et al., £j_ Virol.. 65:6250-6265 (1992)) and its recovery from human peripheral blood (Frenkel, N. et al., Proc. Natl. Acad. Sci. USA., 87:748-752 (1990)) and saliva (Wyatt, L. S. et al., £.. Virol.. 66:3206-3209 (1992)) in the absence of any pathological findings. For this reason, it is possible that introduction of HHV-7 may not be harmful and provide an advantageous treatment for HIV-l infection.

It was found that previous or simultaneous coinfection by HHV-7 interferes with HIV-l, markedly suppreεεing infection by several HIV-l isolates, including field isolates passaged only once in vitro. This suggests that HHV-7 can play an important role as a negative cofactor in the course of HIV infection both in vitro and in vivo.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. Although the fox-.going invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obviouε that certain changes and modifications may be practiced within the εcope of the appended claims. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Berneman, Zwi N. Lusso, Paolo Reitz, Jr., Marvin S. Gallo, Robert C.

(ii) TITLE OF INVENTION: MOLECULAR CLONES OF HUMAN HERPES 7 AND METHODS OF USE THEREOF

(iii) NUMBER OF SEQUENCES: 19

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Townsend and Townsend Khourie and Crew

(B) STREET: One Market Plaza, Steuart Street Tower

(C) CITY: San Francisco

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 94105-1492

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/971,102

(B) FILING DATE: 02-NOV-1992

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Parmelee, Steven W.

(B) REGISTRATION NUMBER: 31,990

(C) REFERENCE/DOCKET NUMBER: 15280-90-1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 206-467-9600

(B) TELEFAX: 415-543-5043

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GGGTTAGGGT TAGGGTTAGG GTTAGGGTTA GGGTTA 36

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CCTAATGAAG GCTACTTTGA AGTACAAATG 30

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AGAATTCTGT ACCCATGGGC ACATTTGTAC 30

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GCNCCNTAYG AYATYCAYTT C 21

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GCNCCNTAYG AYATYCAYTT T 21

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCNCCNTAYG AYATACAYTT C 21

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GCNCCNTAYG AYATACAYTT T 21

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: RAANARNGTN GCRATNGGNG T 21

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: RTANARNGTN GCRATNGGNG T 21

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: RAANARNGTN GCTATNGGNG T 21 (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: RTANARNGTN GCTATNGGNG T 21

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TATCCCAGCT GTTTTCATAT AGTAAC 26

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GCCTTGCGGT AGCACTAGAT TTTTTG 26

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CAGAAATGAT AGACAGATGT TGG 23

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: TAGATTTTTT GAAAAAGATT TAATAAC 27

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCGTTTTCAG TGTGTAGTTC GGCA 24

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TGGCCGCATT CGTACAGATA CGGAGG 26

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GCTAGAACGT ATTTGCTGCA GAACG 25

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: ATCCGAAACA ACTGTCTGAC TGGCA 25

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a probe for detecting the presence of human herpesvirus 7 nucleic acid in a sample, which probe compriseε a plaεmid containing a nucleic acid εequence homologouε to human herpeε viruε 7 nucleic acid or a fragment thereof.

2. A probe aε in claim 1, wherein the plaεmid iε pVL23, pVL23.2, pVL17, pVL17D.l, pVL17D.2, pVL17A.l, pVL17C.l, pVL18, pVLlβ.l, pVL44, pVL29, pVL3, pVL19, pVL8, pVL13, or pVL13.1.

3. A compoεition compriεing a probe for detecting the preεence of human herpesvirus 7 nucleic acid in a sample, which probe compriεes a bacteriophage containing a nucleic acid εequence homologouε to human herpeε viruε 7 nucleic acid or a fragment thereof.

4. A compoεition aε in claim 3, wherein the bacteriophage iε a λ bacteriophage.

5. A compoεition aε in claim 4, wherein the λ bacteriophage iε XVL5, XVL10, XVL12, XVL56, XVL65, XVL73, or XVL81.

6. A process for detecting the presence of human herpesvirus 7 genomic nucleic acid in a sample comprising:

(a) contacting the sample with a probe, which probe compriεeε a nucleic acid εequence homologous to human herpesvirus 7 nucleic acid; and

(b) detecting hybrids formed between the εubsequence and the probe.

7. A process as in claim 6, wherein the probe is a plasmid.

8. A proceεs as in claim 7, wherein the plasmid iε pVL23, pVL23.2, pVL17, pVL17D.l, pVL17D.2, pVL17A.l, pVL17C.l, pVL18, pVLlβ.l, pVL44, pVL29, pVL3, pVL19, pVL8, pVL13, or PVL13.1.

9. A proceεε as in claim 6, wherein the probe is a bacteriophage.

10. A process as in claim 9, wherein the bacteriophage is XVL5, XVL10, XVL12, XVL56, XVL65, XVL73, or XVL81.

11. A proceεs as in claim 6, further comprising amplifying human herpesviruε 7 nucleic acid in the εample.

12. A proceεε aε in claim 11, wherein the human herpesvirus 7 nucleic acid is amplified by polymerase chain reaction.

13. A proceεε aε in claim 7, wherein the probe iε bound to a εolid surface.

14. A process as in claim 13, wherein the εolid εurface iε a well of a microtiter plate.

15. A process as in claim 7, wherein the probe is labelled.

16. A kit useful for detecting the presence of human herpesvirus 7 nucleic acid comprising a probe, which probe comprises a nucleic acid sequence homologous to human herpesvirus 7 nucleic acid.

17. A kit as in claim 16, wherein the probe is pVL23, pVL23.2, pVL17, pVL17D.l, pVL17D.2, pVL17A.l, pVL17C.l, pVL18, pVLlβ.l, pVL4 , pVL29, pVL3, pVL19, pVL8, pVL13, pVL13.1, XVL5, XVL10, XVL12, XVL56, XVL65, XVL73, or XVL81.