WO1999061909A2

WO1999061909A2 - Methods and compositions for the detection of human herpesvirus

Info

Publication number: WO1999061909A2
Application number: PCT/US1999/011407
Authority: WO
Inventors: Chou-Pong Pau
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 1998-05-26
Filing date: 1999-05-26
Publication date: 1999-12-02
Anticipated expiration: 2000-11-26
Also published as: WO1999061909A3; AU4095899A

Abstract

Compositions and methods for the detection and diagnosis of infectious diseases are provided. In particular, efficient and sensitive compositions and methods for the detection of human herpesvirus 8 are provided. The claimed diagnostic compositions and methods involve the use of peptides representative of dominant antigenic regions of human herpesvirus in detection assays. Such assays are highly specific, sensitive and accurate.

Description

METHODS AND COMPOSITIONS FOR THE DETECTION OF HUMAN HERPESVIRUS

This invention was made by the Centers for Disease Control and Prevention, an agency of the United States Government.

FIELD OF THE INVENTION

The present invention relates to the field of immunology and more particularly relates to immunoassays and novel peptides for the detection of human herpes virus 8.

BACKGROUND OF THE INVENTION

Kaposi' s Sarcoma

Prior to the advent of acquired immunodeficiency disease syndrome (AIDS), Kaposi' s sarcoma (KS) was described as a rare skin cancer found mainly in elderly Mediterranean men. AIDS-associated Kaposi' s sarcoma is an aggressive and widely disseminated neoplasm that can be life threatening and highly disabling. Generally, Kaposi' s sarcoma associated with human immunodeficiency virus (HIV) infection is characterized by lesions in the skin, mucous membrane, lymph nodes and viscera. Often the appearance of Kaposi' s sarcoma lesions may be the first notable manifestation of AIDS.

In the United States in 1996, AIDS -associated Kaposi's sarcoma occurred in approximately 17% of gay men with AIDS and 1-5% of others infected with HIV. As the most common AIDS -related tumor, the identification and diagnosis of Kaposi's sarcoma has become an important facet of the treatment of AIDS.

In some HIV-positive patients, Kaposi's sarcoma is confined to the skin and may require mild forms of therapy.

However, many AIDS-associated Kaposi's sarcoma patients suffer from an aggressive form of Kaposi's sarcoma in which lesions progress from macules to plaques and nodules, and which subsequently coalesce and develop into fungating or ulcerated masses.

Human Herpesvirus Type 8

Epidemiologic and clinical studies have that suggested the infectious cause of Kaposi's sarcoma is human herpesvirus type 8 (HHV8), a gammaherpes virus. The detection of HHV8

DNA in nearly all Kaposi's sarcoma lesions from AIDS patients provides evidence that HHV8 plays an important role in the pathogenesis of AIDS-associated Kaposi's sarcoma.

The close association and concurrent observations of Kaposi's sarcoma and AIDS has emphasized the importance of

Kaposi's sarcoma diagnosis. Early detection and treatment of Kaposi's sarcoma could diminish the severity of symptoms related to AIDS, thereby ultimately improving the myriad complications associated with AIDS. In addition, the availability of sensitive tests that accurately detect Kaposi's sarcoma would improve diagnosis and reduce erroneous characterizations of skin disorders. By correctly identifying a skin lesion as one that is the result of HHV8 infection, treatment of the disorder could be administered quickly, before the disease becomes widely disseminated and uncontrollable. There is a need, therefore, for a sensitive and reliable assay to detect the presence of HHV8 in a patient sample and thereby diagnose Kaposi's sarcoma.

Although some assays for HHV8 antibodies have been developed, including immunofluorescence assays (7, 13, 14, 16, 18), immunoblots (17), and enzyme immunoassays (EIA) (5, 22), these assays lack the sensitivity and accuracy needed for reliable diagnosis of Kaposi's sarcoma. The enzyme immunoassays of Simpson et al. utilize as antigens either a recombinant protein expressed by the HHV8 ORF 65 gene (22) or an 18-amino-acid peptide of the HHV8 capsid protein (ORF 26) conjugated to bovine serum albumin (5). However, the accuracy of these assays is suboptimal. The enzyme immunoassays of the prior art typically only detect HHV8 antibodies in only 60% to 80% of Kaposi's sarcoma patients. Therefore, discordant results have been observed when these immunoassays were compared with other assays such as infected cell-based assays.

What is needed is an assay configured for high throughput, that it is useful for routine seroepidemiologic studies of HHV8 infection, while maintaining sensitivity and accuracy. Accordingly, there remains a need for an HHV8 assay having high specificity and whose sensitivity and signal detection may be easily monitored. The need also continues in the art for a sensitive, accurate, inexpensive, and rapid assay for the detection of HHV8 in a sample.

SUMMARY OF THE INVENTION

Methods and compositions for the detection of human herpesvirus 8 (HHV8) are provided. The methods are assays for the detection of HHV8 antigens or antibodies thereof in a biological sample. Detection of an HHV8 infection can be used to diagnose AIDS-associated Kaposi's sarcoma.

The compositions are peptides corresponding to dominant epitopes of HHV8 or homologs thereof. A first preferred peptide is a dominant epitope within the protein encoded by the HHV8 ORF 65 gene. A second preferred peptide is a dominant epitope within the protein encoded by the HHV8 ORF K8.1 gene.

Antibodies to these peptides are also provided. The antibodies can be polyclonal antibodies or monoclonal antibodies or immunoreactive fragments thereof. The HHV8 peptides or antibodies to the HHV8 peptides are useful in vitro as research tools for studying Kaposi's sarcoma and HHV8 in general and as diagnostic reagents in the immunoassays described herein. The

HHV8 peptides and antibodies can be labeled with a detectable label. Detection of the label indicates the presence of an HHV8 infection. Detection of HHV8 and a determination of the concentration of HHV8 in a sample over time can be diagnostic or prognostic for the occurrence or recurrence of Kaposi's sarcoma.

Antibodies specific for HHV8 are useful therapeutically when administered to a human to passively immunize the human against HHV8 infection, thereby reducing

HHV8 related disease.

One or more of the HHV8 peptides may be combined with a pharmaceutically acceptable excipient or sustained-release compositions, such as a biodegradable polymer, to form a vaccine composition to prevent or treat HHV8 infection.

Preferably, the HHV8 peptides or antibodies thereto are labeled with a radioisotope or other detectable molecules for use with techniques, including, but not limited to, positron emission tomography, autoradiography, flow cytometry, radioreceptor binding assays, immunoprecipitation and immunohistochemistry .

Kits for detecting the presence and quantity of HHV8 peptides or antibodies thereto are also provided. The kits can be in any configuration well known to those of ordinary skill in the art and are useful for the detection or monitoring of HHV8 infection in a patient or biological sample.

Molecular probes corresponding to the ribonucleic acid and deoxyribonucleic acid molecules encoding the HHV8 peptides may also be used to detect and measure HHV8 levels in biological samples. Recombinant peptides produced from these nucleic acid molecules are also provided.

Accordingly, it is an object of the present invention to provide a sensitive method for the detection of HHV8 in a biological sample that is simple, rapid, inexpensive, easy to perform and accurate.

It is another object of the present invention to provide a sensitive method for the diagnosis and prognosis of Kaposi's sarcoma. It is another object of the present invention to identify the dominant epitopes of the HHV8 virus and the amino acid sequences of these epitopes.

It is another object of the present invention to provide a sensitive immunological diagnostic method for the detection of HHV8 using peptides from the dominant epitope of the virus.

It is yet another object of the present invention to provide a sensitive diagnostic method for the detection of human herpes virus 8 wherein the peptides used are derived from the dominant epitope within the HHV8 ORF 65 encoded protein.

It is yet another object of the present invention to provide a sensitive diagnostic method for the detection of human herpes virus 8 wherein the peptides used are derived from the dominant epitope within the HHV8 ORF K8.1 encoded protein. An additional object of the invention is to provide a method of detection of HHV8 for monitoring the effectiveness of therapeutic treatments of Kaposi's sarcoma.

An advantage of the invention is that it is highly specific for HHV8. Another advantage is that the assay yields reproducible results.

Yet another advantage is that the methods are particularly suited for rapid throughput and screening of samples, without the need for extensive training or expensive laboratory equipment. These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Seroreactivities of specimens from 20 KS+/HIV+ (I), 20 KS-/HIV+ (II) and 20 KS-/HIV- (III) persons with overlapping peptide PI, P2, P3 and P4 derived from the C- terminal half of HHV8 ORF 65 gene product. Dotted lines indicate cutoff values as defined by the mean baseline-corrected OD₄₅₀ of the 20 KS-/HIV- serum specimens plus 5 standard deviations.

Figure 2. Seroreactivities of three HHV8 antibody positive serum specimens with P4 peptide analogs which differ from P4 by one residue. Substitutions and residue number corresponding to HHV8 ORF 65 protein are shown in the X-axis.

Relative reactivity of 1.0 was assigned to peptide P4.

Figure 3. Seroreactivity of P4 with serum specimens (n=6) preincubated with 0-10 μg/100 μl of competing peptides (a)

P4 itself, and (b) P4 homolog derived from the C-terminus of BFRF3-encoded Epstein-Barr virus protein.

Figure 4. Graph showing results of experiment for mapping immunodominant region of K8.1a. PCA and PCB are HHV8-positive sera and NC is a negative control.

Figure 5. Graph showing results of experiment for fine mapping immunodominant region of K8.1a.

DETAILED DESCRIPTION Compositions and methods for the detection of human herpesvirus 8 (HHV8) are described herein. Detection of an HHV8 infection can be used to diagnose or monitor AIDS- associated Kaposi's sarcoma. The compositions are isolated, synthetic, or recombinant peptides corresponding to dominant epitopes of HHV8 or analogs thereof. A first preferred peptide is a dominant epitope within the protein encoded by the HHV8 open reading frame (ORF) 65 gene. The HHV8 ORF 65 gene product is expressed during the lytic cycle. A second preferred peptide is a dominant epitope within the protein encoded by the HHV8 ORF K8.1 gene. The HHV8 ORF K8.1 gene product is also expressed during infection.

The HHV8 peptides or antibodies to the HHV8 peptides are useful in vitro as research tools for studying Kaposi's sarcoma and HHV8 in general and as diagnostic reagents in the immunoassays described herein. The HHV8 peptides and antibodies can be labeled with a detectable label so that detection of the label indicates the presence of an HHV8 infection. Detection of HHV8 and a determination of the concentration of HHV8 in a sample over time can be diagnostic or prognostic for the occurrence or recurrence of Kaposi's sarcoma.

The methods are assays for the detection of HHV8 antigens or antibodies thereof in a biological sample in which the HHV8 peptides, nucleic acid probes encoding the HHV8 peptides, or HHV8 peptide-specific antibodies are used as reagents.

Definitions

The terms "a", "an" and "the" as used herein are defined to mean "one or more" and include the plural unless the context is inappropriate. As used herein the term "biological fluid" or "biological sample" refers to any source in which HHV8 peptides may exist. For example, the sample or fluid may include, but is not limited to, wounds, blood, tissues, saliva, semen, tears, urine, bone, muscle, cartilage, or skin.

HHV8 Peptides

The dominant epitopes of the proteins encoded by the HHV8 ORF 65 and HHV8 ORF K8.1 genes are identified herein. Dominant epitopes, also referred to as the immunodominant regions of the antigen, are highly immunogenic areas of an antigen to which a majority of the antibodies specific for the antigen bind. The amino acid sequences of the full length proteins encoded by ORF 65 and ORF K8.1 are provided in SEQ ID NOS: 1 and 2, respectively. A first preferred dominant epitope of HHV8 is a peptide containing an amino acid sequence corresponding to the last 31 amino acid residues of the C-terminal (amino acids 140-170) of the protein encoded by the HHV8 ORF 65 gene (SEQ ID NO: 5). More preferably, the dominant HHV8 epitope is a peptide containing an amino acid sequence corresponding to the last 14 amino acid residues of the C-terminal of the protein encoded by the HHV8 ORF 65 gene (SEQ ID NO: 6).

It will be understood by those skilled in the art that the preferred dominant epitope includes peptide analogs, which are defined herein as antigenic peptides containing amino acid sequences differing from SEQ ID NOS: 5 or 6 by an amino acid substitution at any position or having other molecules attached to amino acid functional groups. Exemplary peptide analogs, their amino acid sequences (SEQ ID NOS: 7-17), and their experimental abbreviation are set forth in Table 1 below. Most preferably, the dominant HHV8 epitope is a peptide containing an amino acid sequence corresponding to the penultimate 8 amino acid residues of the C-terminal of the protein encoded by the HHV8 ORF 65 gene having the amino acid sequence RKPPSGKK (SEQ ID NO: 18).

A second preferred dominant epitope of HHV8 is a peptide comprising an amino acid sequence contained within the protein encoded by the HHV8 ORF K8.1 gene (SEQ ID NO: 2). HHV8 ORF K8.1 encodes immunogenic glycoproteins (K8.1a and K8.1b) generated by spliced transcripts (Chandran et al. Virology

249:140-149 (1998)). Peptide K8.1a (SEQ ID NO: 22) is composed of 228 amino acids with the first 142 identical to the deduced amino acids sequence of the ORF K8.1 gene. Peptide K8.1b (SEQ ID NO: 23) is similar to K8.1a but with a 61 amino acid deletion beginning at codon 82 and an S to R substitution at codon 81. A preferred dominant epitope of HHV8 ORF K8.1 is a peptide containing an amino acid sequence corresponding to the first 31 amino acid residues of the N-terminal portion of the protein encoded by the HHV8 ORF K8.1 gene (SEQ ID NO: 19). As further described in the Examples, twelve overlapping peptides (20 to 22 amino acids long) encompassing codon 25 to 197 of K8.1a were synthesized and probed with HHV8 positive sera. Peptide K2 (SEQ ID NO: 24) was discovered to be the most immunogenic (see Figure 4). Most preferably therefore, the dominant HHV8 epitope is a peptide containing an amino acid sequence corresponding to peptide K2 (SEQ ID NO: 24).

It will be understood by those skilled in the art that the preferred dominant epitope includes peptide analogs, which are defined herein as antigenic peptides containing amino acid sequences differing from SEQ ID NOS: 19 or 24 by an amino acid substitution at any position or having other molecules attached to amino acid functional groups. Exemplary peptide analogs, their amino acid sequences (SEQ ID NOS: 26-53), and their experimental abbreviations are set forth in Table 1 below.

Other relevant epitopes include peptides encoded by the HHV8 ORF K12 gene, (SEQ ID NO: 20) and the HHV8 ORF 73 gene, (SEQ ID NO: 21) including fragments and analogs thereof. A preferred embodiment of the present invention comprises a bivalent peptide, P2833 (SEQ ID NO: 25) incorporating the dominant epitopes of both ORF 65 and of ORF K8.1a.

Table 1 HHV8 Peptides

The HHV8 peptides and peptide analogs have a variety of uses. Peptides that bind to HHV8 antibodies with high specificity and avidity may be radiolabeled and employed for visualization and quantitation using autoradiographic and membrane binding techniques. Such applications provide important diagnostic and research tools. HHV8 peptides may be employed to develop affinity columns for isolation of HHV8 antibodies.

The HHV8 peptides are also useful in diagnostic methods and kits to detect and quantify antibodies capable of binding HHV8. The HHV8 detection methods and kits permit detection of circulating HHV8 antibodies in order to indicate the presence or level of HHV8 infection. Patients having circulating anti-HHV8 antibodies may be more likely to develop skin lesions and other complications, and those that have HIV infection may be able to take precautions or receive treatment to prevent, decrease or delay serious manifestation of symptoms such as Kaposi's sarcoma.

HHV8 peptides may be chemically coupled to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules, chemiluminescent, bioluminescent and other compounds for a variety of applications. The efficiency of the coupling reaction is determined using different techniques appropriate for the specific reaction. For example, radiolabeling of an HHV8 peptide with I is accomplished using chloramine T and Na¹²⁵I of high specific activity. The reaction is terminated with sodium metabisulfite and the mixture is desalted on disposable columns. The labeled peptide is eluted from the column and fractions are collected. Aliquots are removed from each fraction and radioactivity measured in a gamma counter. In this manner, the unreacted Na I is separated from the labeled HHV8 peptide. The peptide fractions with the highest specific radioactivity are stored for subsequent use such as analysis of the ability to bind to HHV8 antisera. The HHV8 peptides can be isolated by standard protein purification and enzymatic cleavage or synthesized by chemical or biological methods (e.g., cell culture, recombinant gene expression, peptide synthesis, and in vitro enzymatic catalysis of larger encompassing peptides to yield active peptides).

Recombinant techniques include gene amplification from DNA sources using the polymerase chain reaction (PCR), and gene amplification from RNA sources using reverse transcriptase/PCR. The present invention also encompasses compositions comprising vectors containing a DNA sequence encoding HHV8 peptides, wherein the vectors are capable of expressing HHV8 peptides when present in a cell, compositions comprising cells containing vectors, wherein the vector contains a DNA sequence encoding HHV8 peptides or fragments or analogs thereof, and wherein the vectors are capable of expressing HHV8 peptides when present in the cell. The invention further comprises methods comprising implanting into a cell a vector, wherein the vector contains a DNA sequence encoding HHV8 peptides, and wherein the vector is capable of expressing HHV8 peptides when present in a cell.

Antibodies to HHV8 Peptides

The isolated, recombinant or synthetic HHV8 peptides described herein are useful for generating antibodies specific for HHV8. The antibodies can be either polyclonal antibodies or monoclonal antibodies. Antibodies that specifically bind to HHV8 can then be used in diagnostic methods and kits, as described below, to detect or quantify HHV8 in a biological sample such as a human fluid or tissue sample. Results from these tests can be used to diagnose or predict the occurrence or recurrence of HHV8 mediated diseases such as Kaposi's sarcoma. Antibodies to the HHV8 peptides may also be used in production facilities or laboratories to isolate additional quantities of the peptides, such as by affinity chromatography. The isolated, recombinant or synthetic peptides can be administered to animals as immunogens or antigens, alone or in combination with an adjuvant, for the production of antisera reactive with HHV8 proteins. In addition, the peptides can be used to screen antisera for hyperimmune patients from whom can be derived antibodies having a very high affinity for the proteins. Antibodies isolated from the antisera are useful for the specific detection of HHV8 infection or as research tools.

The term "antibodies" as used herein includes monoclonal antibodies, polyclonal, chimeric, single chain, bispecific, simianized, and humanized antibodies as well as Fab fragments, including the products of an Fab immunoglobulin expression library.

Monoclonal antibodies are generated by methods well known to those skilled in the art. The preferred method is a modified version of the method of Kearney, et al., J. Immunol. 123:1548-1558 (1979), which is incorporated by reference herein. Briefly, animals such as mice or rabbits are inoculated with the immunogen in adjuvant, and spleen cells are harvested and mixed with a myeloma cell line, such as P3X63Ag8,653. The cells are induced to fuse by the addition of polyethylene glycol. Hybridomas are chemically selected by plating the cells in a selection medium containing hypoxanthine, aminopterin and thymidine (HAT). Hybridomas are subsequently screened for the ability to produce anti-trichloroethylene monoclonal antibodies.

Hybridomas producing antibodies are cloned, expanded and stored frozen for future production.

Polyclonal antisera are also raised using established techniques known to those skilled in the art. For example, polyclonal antisera may be raised in rabbits, sheep, goats or other animals. Isolated, recombinant or synthetic HHV8 peptides conjugated to a carrier molecule such as bovine serum albumin, may be combined with an adjuvant mixture, emulsified and injected subcutaneously at multiple sites on the back, neck, flanks, and sometimes in the footpads of the animals. Booster injections are made at regular intervals, such as every 2 to 4 weeks. Blood samples are obtained by venipuncture, for example using the marginal ear veins after dilation, approximately 7 to 10 days after each injection. The blood samples are allowed to clot and are centrifuged, and the serum removed, aliquoted, and stored under refrigeration for immediate use or frozen for subsequent analysis.

Techniques for the production of single chain antibodies are known to those skilled in the art and described in U.S. Patent No. 4,946,778 and can be used to produce single chain antibodies to the proteins described herein. Phage display technology may be used to select antibody genes having binding activities for HHV8 proteins from PCR-amplified genes of lymphocytes from humans screened for having antibodies to the HHV8 proteins or libraries. Bispecific antibodies have two antigen binding domains wherein each domain is directed against a different epitope.

The antibody may be labeled directly with a detectable label for identification and quantitation of HHV8. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances including colored particles such as colloidal gold and latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).

Alternatively, the antibody may be labeled indirectly by reaction with labeled substances that have an affinity for immunoglobulin, such as protein A or G or second antibodies. The antibody may be conjugated with a second substance and detected with a labeled third substance having an affinity for the second substance conjugated to the antibody. For example, the antibody may be conjugated to biotin and the antibody-biotin conjugate detected using labeled avidin or streptavidin. Similarly, the antibody may be conjugated to a hapten and the antibody-_N hapten conjugate detected using labeled anti-hapten antibody. These and other methods of labeling antibodies and assay conjugates are well known to those skilled in the art.

Alternatively, the HHV8 antibodies can be labeled with short lived isotopes to enable visualization of HHV8 peptides in vivo using positron emission tomography or other modern radiographic techniques to locate emerging infectious sites.

All serum samples from generation of polyclonal antisera, or media samples from production of monoclonal antisera, are analyzed for determination of antibody titer. Titer is established through several means, for example, using dot blots and density analysis, and also with precipitation of radiolabeled peptide-antibody complexes using protein A, secondary antisera, cold ethanol or charcoal-dextran followed by activity measurement with a gamma counter. The highest titer antisera are also purified on affinity columns which are commercially available. HHV8 peptides are coupled to the gel in the affinity column. Antiserum samples are passed through the column and anti-HHV8 antibodies remain bound to the column. These antibodies are subsequently eluted, collected and evaluated for determination of titer and specificity.

The highest titer HHV8 antisera is tested to establish the following: (a) optimal antiserum dilution for highest specific binding of the antigen and lowest non-specific binding, (b) the ability to bind increasing amounts of HHV8 peptide in a standard displacement curve, (c) potential cross-reactivity with related peptides and proteins, such as those of other viral species, and (d) ability to detect HHV8 peptides in extracts of plasma, urine, tissues, and in cell culture media. The peptides and antibodies to the proteins are useful for the treatment and diagnosis of HHV8 infections as described below with regard to diagnosis method and for the development of anti-HHV8 vaccines for active or passive immunization.

Nucleic Acid Sequences

Nucleic acid sequences encoding the HHV8 peptides are useful for the production of recombinant peptides or as nucleic acid probes for the detection of HHV8 infection in a sample or specimen with high sensitivity and specificity. The probes can be used to detect the presence of HHV8 in the sample, diagnose infection with the disease, quantify the amount of HHV8 virus in the sample, or monitor the progress of therapies used to treat the infection. The nucleic acid and amino acid sequences are also useful as laboratory research tools to study the organism and the disease and to develop therapies and treatments for the disease.

Nucleic acid probes selectively hybridize with nucleic acid molecules encoding the peptides described herein or complementary sequences thereof. By "selective" or "selectively" is meant a sequence which does not hybridize with other nucleic acids to prevent adequate detection of the HHV8 DNA. Therefore, in the design of hybridizing nucleic acids, selectivity will depend upon the other components present in a sample. The hybridizing nucleic acid should have at least 70% complementarity with the segment of the nucleic acid to which it hybridizes. As used herein to describe nucleic acids, the term "selectively hybridizes" excludes the occasional randomly hybridizing nucleic acids, and thus, has the same meaning as "specifically hybridizing". The selectively hybridizing nucleic acids of the invention can have at least 70%, 80%, 85%, 90%,

95%, 97%, 98%, and 99% complementarity with the segment of the sequence to which it hybridizes.

The invention contemplates sequences, probes and primers which selectively hybridize to the encoding DNA or the complementary, or opposite, strand of DNA. Specific hybridization with nucleic acid can occur with minor modifications or substitutions in the nucleic acid, so long as functional species-specific hybridization capability is maintained. By "probe" is meant nucleic acid sequences that can be used as probes or primers for selective hybridization with complementary nucleic acid sequences for their detection or amplification, which probes can vary in length from about 5 to 100 nucleotides, or preferably from about 10 to 50 nucleotides, or most preferably about 18-24 nucleotides. Therefore, the terms "probe" or "probes" as used herein are defined to include "primers". Isolated nucleic acids are provided herein that selectively hybridize with the species-specific nucleic acids under stringent conditions and should have at least five nucleotides complementary to the sequence of interest as described by Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. MOLECULAR CLONING: A LABORATORY

MANUAL, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

If used as primers, the composition preferably includes at least two nucleic acid molecules which hybridize to different regions of the target molecule so as to amplify a desired region. Depending on the length of the probe or primer, the target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions. For example, for the purpose of diagnosing the presence of the HHV8, the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes is at least enough to distinguish hybridization with a nucleic acid from other organisms.

The nucleic acid sequences encoding the HHV8 peptides can be inserted into a vector, such as a plasmid, and recombinantly expressed in a living organism to produce recombinant HHV8 peptides. HHV8 Detection Methods

Methods for the detection of HHV8 peptides in biological samples, such as fluids and tissues are provided for the diagnosis or prognosis of Kaposi's sarcoma. The methods involve obtaining a sample suspected of containing HHV8. The sample may be taken from an individual, such as a wound, blood, saliva, tissues, bone, muscle, cartilage, or skin sample. The cells can then be lysed, and the DNA or RNA extracted, precipitated and amplified. Detection of HHV8 DNA is achieved by hybridizing the amplified DNA with an HHV8 probe that selectively hybridizes with the DNA as described above.

Detection of hybridization is indicative of the presence of HHV8. Preferably, detection of nucleic acid (e.g., probes or primers) hybridization can be facilitated by the use of detectable moieties. For example, the probes can be labeled with biotin and used in a streptavidin-coated microtiter plate assay. Other detectable moieties include radioactive labeling, enzyme labeling, and fluorescent labeling, for example.

DNA may be detected directly or may be amplified enzymatically using polymerase chain reaction (PCR) or other amplification techniques prior to analysis. RNA or cDNA can be similarly detected. Increased or decreased expression of HHV8 peptides can be measured using any of the methods well known in the art for the quantitation of nucleic acid molecules, such as, for example, amplification, PCR, RT-PCR, RNase protection,

Northern blotting, and other hybridization methods.

Diagnostic assay for HHV8 peptides or HHV8 antibodies may also be used to detect the presence of an infection.

Assay techniques for determining protein or antibody levels in a sample are well known to those skilled in the art and include methods such as radioimmunoasssay, Western blot analysis and

ELISA assays.

Despite the high correlation of P4 antibodies in patients' serum with KS, this assay may not be able to detect latent HHV8 infections since the ORF 65 gene product may not be highly expressed during latent infection. A highly sensitive assay for HHV8 infection may require a cocktail of peptides derived from latent and lytic-cycle proteins. Therefore, identification of dominant epitopes within the latent antigens is critical for the development of better diagnostics for HHV8 infection. In addition, independent methods, such as a virus culture or polymerase chain reaction, are needed to validate serological methods.

Treatment of Kaposi's Sarcoma

The present invention further includes methods of treating or preventing viral diseases such as Kaposi's sarcoma, by stimulating the production of antibodies specific for HHV8 and HHV8 peptides, and/or by administering substantially purified HHV8 peptide antibodies, or HHV8 peptide agonists or antagonists, and/or HHV8 peptide antisera or antisera directed against HHV8 peptide antisera to a patient. Additional treatment methods include administration of HHV8 peptide antibodies, HHV8 peptide fragments, HHV8 peptide analogs, HHV8 peptide antisera, or HHV8 peptide receptor agonists and antagonists linked to cytotoxic agents. HHV8 peptides can be produced synthetically by chemical reaction or by recombinant techniques in conjunction with expression systems. HHV8 peptides can also be produced by enzymatically cleaving isolated ORF 65, ORF K8.1 or other HHV8 proteins to generate antigenic peptides having antibody producing activity.

Antibodies specific for HHV8 are useful therapeutically when administered to a patient to passively immunize the patient against HHV8 infection, thereby reducing HHV8 related disease.

Immunological and Pharmaceutical Compositions

Immunological compositions, including vaccine, and other pharmaceutical compositions containing the HHV8 peptides or antibodies thereof are included within the scope of the present invention. One or more of the peptides can be formulated and packaged, alone or in combination with other antigens, using methods and materials known to those skilled in the art for vaccines. The immunological response may be used therapeutically or prophylactically and may provide antibody immunity or cellular immunity such as that produced by T lymphocytes such as cytotoxic T lymphocytes or CD4+ T lymphocytes.

To enhance immunogenicity, the peptides may be conjugated to a carrier molecule. Suitable immunogenic carriers include proteins, polypeptides or peptides such as albumin, hemocyanin, thyroglobulin and derivatives thereof, particularly bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), polysaccharides, carbohydrates, polymers, and solid phases. Other protein derived or non-protein derived substances are known to those skilled in the art. An immunogenic carrier typically has a molecular weight of at least 1,000 daltons, preferably greater than 10,000 daltons. Carrier molecules often contain a reactive group to facilitate covalent conjugation to the hapten. The carboxylic acid group or amine group of amino acids or the sugar groups of glycoproteins are often used in this manner. Carriers lacking such groups can often be reacted with an appropriate chemical to produce them. Preferably, an immune response is produced when the immunogen is injected into animals such as mice, rabbits, rats, goats, sheep, guinea pigs, chickens, and other animals, most preferably mice and rabbits. Alternatively, a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide may be sufficiently antigenic to improve immunogenicity without the use of a carrier. The HHV8 peptides may be administered with an adjuvant in an amount effective to enhance the immunogenic response against the conjugate. At this time, the only adjuvant widely used in humans has been alum (aluminum phosphate or aluminum hydroxide). Saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have toxicities which limit their potential use in human vaccines. However, chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman-Snitkoff et al. J. Immunol. 147:410-415 (1991) and incorporated by reference herein, encapsulation of the conjugate within a proteoliposome as described by Miller et al., J. Exp. Med. 176: 1739-1744 (1992) and incorporated by reference herein, and encapsulation of the protein in lipid vesicles such as Novasome™ lipid vesicles (Micro Vescular Systems, Inc., Nashua,

NH) may also be useful.

The term "vaccine" as used herein includes DNA vaccines in which the nucleic acid molecule encoding HHV8 peptides in a pharmaceutical composition is administered to a patient. For genetic immunization, suitable delivery methods known to those skilled in the art include direct injection of plasmid DNA into muscles (Wolff et al, Hum. Mol. Genet. 1:363 (1992)), delivery of DNA complexed with specific protein carriers (Wu et al, J. Biol. Chem. 264:16985 (1989), coprecipitation of DNA with calcium phosphate (Benvenisty and

Reshef, Proc. Natl. Acad. Sci. 83:9551 (1986)), encapsulation of DNA in liposomes (Kaneda et al., Science 243:375 (1989)), particle bombardment (Tang et al., Nature 356:152 (1992) and Eisenbraun et al, DNA Cell Biol. 12:791 (1993)), and in vivo infection using cloned retroviral vectors (Seeger et al., Proc.

Natl. Acad. Sci. 81:5849 (1984)).

In a preferred embodiment, a vaccine is packaged in a single dosage for immunization by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. The vaccine is most preferably injected intramuscularly into the deltoid muscle. The vaccine is preferably combined with a pharmaceutically acceptable carrier to facilitate administration. The carrier is usually water or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution.

The carrier to which the protein may be conjugated may also be a polymeric delayed release system. Synthetic polymers are particularly useful in the formulation of a vaccine to effect the controlled release of antigens. For example, the polymerization of methyl methacrylate into spheres having diameters less than one micron has been reported by Kreuter, J., MICROCAPSULES AND NANOPARTICLES IN MEDICINE AND PHARMACOLOGY, M. Donbrow (Ed). CRC Press, p. 125-148

(1991).

Microencapsulation of the peptide will also give a controlled release. A number of factors contribute to the selection of a particular polymer for microencapsulation. The reproducibility of polymer synthesis and the microencapsulation process, the cost of the microencapsulation materials and process, the toxicological profile, the requirements for variable release kinetics and the physicochemical compatibility of the polymer and the antigens are all factors that must be considered. Examples of useful polymers are polycarbonates, polyesters, polyurethanes, polyorthoesters polyamides, poly (d,l-lactide-co-glycolide) (PLGA) and other biodegradable polymers. The use of PLGA for the controlled release of antigen is reviewed by Eldridge, J.H., et al. CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY, 146:59-66 (1989).

The preferred dose for human administration is from 0.01 mg/kg to 10 mg/kg, preferably approximately 1 mg/kg. Based on this range, equivalent dosages for heavier body weights can be determined. The dose should be adjusted to suit the individual to whom the composition is administered and will vary with age, weight and metabolism of the individual. The vaccine may additionally contain stabilizers such as thimerosal (ethyl(2- mercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Company, St. Louis, MO) or physiologically acceptable preservatives. Labeled Conjugates

When labeled with a detectable biomolecule or chemical, the HHV8 peptides and antibodies described herein are useful for purposes such as in vivo and in vitro diagnostics and laboratory research. Various types of labels and methods of conjugating the labels to the peptides and antibodies are well known to those skilled in the art. Several specific labels are set forth below. The labels are particularly useful when conjugated to a protein such as an antibody or receptor.

For example, the peptides and antibodies can be conjugated to a radiolabel such as, but not restricted to, 32p, 3H, 1⁴C, ³⁵S, 125i_{s or} 13 li. Detection of a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography.

Bioluminescent labels, such as derivatives of firefly luciferin. are also useful. The bioluminescent substance is covalently bound to the peptide or antibody by conventional methods, and the labeled peptide or antibody is detected when an enzyme, such as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light.

Fluorogens may also be used as labels. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, and Texas Red. The fluorogens are generally detected by a fluorescence detector.

The peptides and antibodies can alternatively be labeled with a chromogen to provide an enzyme or affinity label. For example, the peptide or antibody can be biotinylated so that it can be utilized in a biotin-avidin reaction, which may also be coupled to a label such as an enzyme or fluorogen. For example, the peptide or antibody can be labeled with peroxidase, alkaline phosphatase or other enzymes giving a chromogenic or fluorogenic reaction upon addition of substrate. Additives such as 5-amino-2,3-dihydro-l,4-phthalazinedione (also known as Luminol™) (Sigma Chemical Company, St. Louis, MO) and rate enhancers such as p-hydroxybiphenyl (also known as p-phenylphenol) (Sigma Chemical Company, St. Louis, MO) can be used to amplify enzymes such as horseradish peroxidase through a luminescent reaction; and luminogeneic or fluorogenic dioxetane derivatives of enzyme substrates can also be used. Such labels can be detected using enzyme-linked immunoassays (ELISA) or by detecting a color change with the aid of a spectrophotometer. In addition, peptides or antibodies may be labeled with colloidal gold for use in immunoelectron microscopy in accordance with methods well known to those skilled in the art.

The location of an HHV8 infection can be determined by labeling an antibody as described above and detecting the label in accordance with methods well known to those skilled in the art, such as immunofluorescence microscopy using procedures such as those described by Warren and Nelson, Mol. Cell. Biol. 7:

1326-1337 (1987).

HHV8 Immunoassays

There are many techniques known in the art for detecting a component such as a peptide in a mixture and/or measuring its amount. Immunoassays, which employ antibodies that bind specifically to the peptide of interest, are one of the better known measurement techniques. Classical methods involve reacting a sample containing the peptide with a known excess amount of antibody specific for the peptide, separating bound from free antibody, and determining the amount of one or the other. Often the antibody is labeled with a reporter group to aid in the determination of the amount of bound analyte. The reporter group or "label" is commonly a fluorescent or radioactive group or an enzyme.

An immunoassay is performed for the detection of HHV8 virus in a sample as follows:

A sample is collected or obtained using methods well known to those skilled in the art. The sample containing the HHV8 proteins or antibodies to be detected may be obtained from any biological source. For example, the sample may be a biological fluid, such as blood serum, blood plasma, urine, spinal fluid, fermentation fluid, lymph fluid, tissue culture fluid and ascites fluid. The sample may be diluted, purified, concentrated, filtered, dissolved, suspended or otherwise manipulated prior to immunoassay to optimize the immunoassay results.

To detect HHV8 protein, the sample is incubated with an HHV8 antibody, produced as described herein. The antibody may be labeled or conjugated to a solid phase bead or particle as also described herein. The labeled antibody is then detected using known methods well known to those skilled in the art. The term "detecting" or "detected" as used herein means using known techniques of detection of biologic molecules such as immunochemical or histological methods. Such methods include immunological techniques employing monoclonal or polyclonal antibodies to the peptides, such as enzyme linked immunosorbant assays, radioimmunoassay, chemiluminescent assays, or other types of assays involving antibodies known to those skilled in the art. Current binding assay technology benefits from the diversity of detection systems developed that use enzyme- catalyzed chromogenic reactions, radionuclides, chemiluminescence, bioluminescence, fluorescence, fluorescence polarization and a variety of potentiometric and optical biosensor techniques.

Binding assays rely on the binding of analyte by analyte receptors to determine the concentrations of analyte in a sample. Analyte-receptor assays can be described as either competitive or non-competitive. Non-competitive assays generally utilize analyte receptors in substantial excess over the concentration of analyte to be determined in the assay. Sandwich assays, in which the analyte is detected by binding to two analyte receptors, one analyte receptor labeled to permit detection and a second analyte receptor, frequently bound to a solid phase, to facilitate separation from unbound reagents, such as unbound labeled first analyte receptor, are examples of non-competitive assays.

Competitive assays generally involve a sample suspected of containing analyte, an analyte-analogue conjugate, and the competition of these species for a limited number of binding sites provided by the analyte receptor. Competitive assays can be further described as being either homogeneous or heterogeneous. In homogeneous assays all of the reactants participating in the competition are mixed together and the quantity of analyte is determined by its effect on the extent of binding between analyte receptor and analyte-conjugate or analyte analogue-conjugate. The signal observed is modulated by the extent of this binding and can be related to the amount of analyte in the sample. U.S. Patent No. 3,817,837 describes such a homogeneous, competitive assay in which the analyte analogue conjugate is a analyte analogue-enzyme conjugate and the analyte receptor, in this case an antibody, is capable of binding to either the analyte or the analyte analogue. The binding of the antibody to the analyte analogue-enzyme conjugate decreases the activity of the enzyme relative to the activity observed when the enzyme is in the unbound state. Due to competition between unbound analyte and analyte analogue-enzyme conjugate for analyte-receptor binding sites, as the analyte concentration increases the amount of unbound analyte analogue-enzyme conjugate increases and thereby increases the observed signal. The product of the enzyme reaction may then be measured kinetically using a spectrophotometer.

Heterogeneous, competitive assays require a separation of analyte analogue conjugate bound to analyte receptor from the free analyte analogue conjugate and measurements of either the bound or the free fractions. Separation of the bound from the free may be accomplished by removal of the analyte receptor and anything bound to it from the free analyte analogue conjugate by immobilization of the analyte receptor on a solid phase or precipitation. The amount of the analyte analogue conjugate in the bound or the free fraction can then be determined and related to the concentration of the analyte in the sample. Normally the bound fraction is in a convenient form, for example, on a solid phase, so that it can be washed, if necessary, to remove remaining unbound analyte analogue conjugate and the measurement of the bound analyte analogue conjugate or related products is facilitated. The free fraction is normally in a liquid form that is generally inconvenient for measurements. If multiple analytes are being determined in a single assay, the determination of the free fraction of analyte analogue conjugate for each analyte is made impossible if all are mixed in a single liquid unless the responses of the individual analyte analogue conjugates can be distinguished in some manner. However, detecting the free fraction of analyte analogue conjugate in assays that are visually interpreted is a distinct advantage because the density of the color developed in such assays is generally proportional to the analyte concentration over much of the range of analyte concentration.

A preferred method of HHV8 detection involves the use of an enzyme immunoassay (EIA). Because EIAs are well suited and configured for high throughput, they are particulary useful for routine seroepidemiologic studies of HHV8 infection. In addition, EIA techniques and methods are well known and neither extensive training nor expensive materials would be required to implement such diagnostic systems.

Kits for Detecting HHV8 Infections

Kits for detecting the presence and quantity of HHV8 peptides or antibodies thereto are also provided. The kits can be in any configuration well known to those of ordinary skill in the art and are useful for the detection or monitoring of HHV8 infection in a patient.

The kits preferably contain one or more HHV8 peptides or antibodies which can be used for the detection of HHV8 antibodies or proteins in a biological sample. Such a kit can additionally contain the appropriate reagents for binding the peptides or antibodies described herein to the corresponding HHV8 antibodies or proteins in the sample and detecting bound peptides or antibodies. Preferably, the HHV8 peptides or antibodies are labeled with a radioisotope or other detectable molecules for use with techniques, including, but not limited to, positron emission tomography, autoradiography, flow cytometry, radioreceptor binding assays, and immunohistochemistry. The kits described herein may additionally contain equipment for safely obtaining the sample, a vessel for containing the reagents, a timing means, a buffer for diluting the sample, and a colorimeter, reflectometer, or standard against which a color change may be measured. In a preferred embodiment, the reagents, including the protein or antibody, are lyophilized, most preferably in a single vessel. Addition of aqueous sample to the vessel results in solubilization of the lyophilized reagents, causing them to react. Most preferably, the reagents are sequentially lyophilized in a single container, in accordance with methods well known to those skilled in the art that minimize reaction by the reagents prior to addition of the sample.

The assay kits include but are not limited to the following techniques: competitive and non-competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry. For each kit, the range, sensitivity, precision, reliability, specificity and reproducibility of the assay are established. Intraassay and interassay variation is established at 20%, 50% and 80% points on the standard curves of displacement or activity.

The assay kit provides instructions, antiserum, HHV8 or HHV8 peptides, and possibly radiolabeled HHV8 and/or reagents for precipitation of bound HHV8 antibody-HHV8 complexes. The kit is useful for the measurement of HHV8 in biological fluids and tissue extracts of animals and humans with and without Kaposi's sarcoma. One example of a diagnostic kit is used for localization of HHV8 in tissues and cells. This HHV8 immunohistochemistry kit provides instructions, HHV8 antiserum, and possibly blocking serum and secondary antiserum linked to a fluorescent molecule such as fluorescein isothiocyanate, or to some other reagent used to visualize the primary antiserum.

Immunohistochemistry techniques are well known to those skilled in the art. This HHV8 immunohistochemistry kit permits localization of HHV8 in tissue sections and cultured cells using both light and electron microscopy. It is used for both research and clinical purposes. For example, samples from skin lesions are prepared to examine HHV8 infection. Such information is useful for diagnostic and possibly therapeutic purposes in the detection and treatment of Kaposi's sarcoma.

This invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention.

EXAMPLES Example 1 Identification ofHHV8 Dominant Epitope in Protein

Encoded by ORF 65 Materials and Methods Serum Panel

All serum specimens (n=61) were collected from the Atlanta metropolitan area as part of past Centers for Disease Control and Prevention studies, and were unlinked from personal identifiers prior to testing. One specimen was from a patient with classical KS (i.e., an elderly individual who was HTV seronegative) and the remaining 60 specimens were from three different groups of 20 individuals each. The first group

(KS+/HIV+) consisted of HIV-infected homosexual men who had biopsy-confirmed KS (CD4⁺ T-cell counts ranged from 10- 660/μL; mean, 269/μL). The second group (KS-/HIV+) consisted of HIV-infected homosexual men who did not have KS (CD4⁺ T- cell counts ranged from 7-1246/μL; mean, 255/μL). The third group (KS-/HIV-) consisted of healthy HIV-negative blood donors (10 men and 10 women).

Synthetic Peptides Peptides were synthesized according to manufacturer's protocol on an automatic synthesizer (Model 432A, Applied Biosystems, Foster City, CA), partially purified by reverse-phase high-performance liquid chromatography (BioRad, Richmond, CA), lyophilized, and stored desiccated at room temperature until use.

Overlapping peptides of 31 to 34 residues (PI, aa91- 124 SEQ ID NO: 3; P2, aal 17-147 SEQ ID NO: 4; and P3, aal40-170 SEQ ID NO: 5) encompassing the C-terminal 80- residues HHV8 ORF 65 protein were synthesized for initial antibody screening. A shorter version of P3 (P4, aal57-170 SEQ

ID NO: 6) corresponding to the last 14 amino acids of the protein was also used. For epitope mapping, P4 and 11 of its analogs (P4.l-P4.l l SEQ ID NOS: 7-17) which differ from P4 by one amino acid were used (Table 2). For the competition study, the Epstein-Barr virus (EBV) homolog (QPHDTAPRGARKKQ) derived from the corresponding segment of the EBV BFRF3- encoded protein (2) was used as the competing peptide. Peptide EIA

Published procedures for peptide EIA were followed (20). Briefly, peptides were dissolved in a carbonate-bicarbonate buffer (0.1M, pH 9.4) to a final concentration of 5 μg/ml, and 100 μl of this solution was used to coat microtiter wells by overnight incubation at 4°C. Peptide-coated wells were washed once in pH 7.4 phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-Tw), air-dried, and stored desiccated at -20°C until use. Nonspecific binding sites of the peptide-coated wells were blocked with 5% non-fat dry milk (Nestle Food Co.,

Glendale. CA) in PBS containing 0.3% Tween-20 (milk buffer) for 30 minutes at 37°C just prior to assay. Sera were diluted 1: 100 in a milk buffer and allowed to react with peptide-coated wells for 1 hour at 37°C. Bound antibodies were detected with goat anti-human IgG (H+L) - peroxidate conjugate (BioRad) and tetramethyl-benzidine/hydrogen peroxide substrates (Kirkegaard and Perry Laboratories, Gaithersburg, MD) after the plates were washed 5 times with PBS-Tw. Baseline-corrected optical density (OD₄₅o) was calculated as A₄₅₀ - A₆3o- The mean corrected OD₄₅₀ of the 20 KS-/HIV- specimens plus 5 standard deviations was arbitrarily chosen as the assay cutoff for each peptide.

Epitope Mapping

Three serum specimens highly reactive to peptide P4 were tested on all 12 peptides listed in Table 2 on a single microtiter plate. Antibody reactivity of each peptide analog was then compared with that of P4. Table 2

C-Terminal Sequence of HHV8 ORF 65 Protein (P4) and its Peptide Analogs for Fine Mapping of the Dominant Epitope

Competition Assay

Six P4-reactive serum specimens (3 highly reactive and 3 moderately reactive) were chosen for this study. Diluted serum samples (1 : 100 in milk buffer) were first incubated with the competing EBV peptide or P4 itself, ranging from 0.01 to 10.0 μg/100 μl at room temperature for 15 minutes. The peptide- serum mixtures were then added to the P4-coated plate, and the peptide EIA procedure as described was then followed.

Detection ofHHV8 Antibodies by Immunofluorescence Assay

A mouse monoclonal antibody enhanced immunofluorescence assay (MIFA) as described by Lennette et al. (16) with a slight modification in slide preparation (3) was followed. Briefly, tetradecanoyl phorbol ester acetate (Sigma)- induced BCBL-1 cells were harvested on day 6 post induction, washed once in PBS, and suspended in PBS to give a final concentration of 10⁶ cells/ml. One drop of this suspension was applied to the slide, air-dried, and fixed in cold acetone for 5 minutes at -20°C. Fixed cells were incubated with 1:10 diluted serum specimens for 30 minutes, followed by mouse monoclonal anti-human IgG (ATCC HF6508) and then fluorescein conjugated anti-mouse IgG (Cappel, Durham, NC) at 1: 100 dilution, and examined by using a fluorescence microscope.

Results

Antigenicity of Overlapping Peptides

The seroreactivity patterns of the 60 human serum specimens determined by the peptide EIA with overlapping peptides PI, P2, P3, and P4 are shown in Figure 1. Eighteen

(90%) of the 20 KS+/HIV+; four (20%) of the 20 KS-/HIV+; and none of the KS-/HIV- specimens reacted with P4. A virtually identical reactivity pattern was observed with P3. However, no KS+/HIV+ and only one KS-/HIV+ (5%) reacted with PI. Five KS+/HIV+ (25%) specimens and one KS-/HIV+ (5%) specimen reacted with P2. The specimen from the patient with classical KS was tested only on P3 and P4, and was positive for both peptides (data not shown).

Fine Epitope Mapping

Figure 2 depicts the reactivity patterns of three HHV8-positive serum specimens with P4 analogs. Although the effect of amino-acid substitutions varied from specimen to specimen, residues 165 (P) and 169 (K) appeared to be most important for antibody recognition in all three specimens examined. Based on the overall lower reactivities of these three specimens with peptide analogs having substitutions at residue 162-169, it was deduced that the sequence RKPPSGKK comprises the immunodominant domain of the C-terminal region of the ORF 65 gene product. Competition Study with EBV Peptide Analog

No inhibition of P4 reactivity by the homologous

EBV peptide (up to 10 μg/100 μl) was observed in peptide EIA (Figure 3b), while the autologous peptide greatly diminished the assay signals at 0.1 to 1.0 μg/100 μl with the six serum specimens examined (Figure 3 a).

Comparison between Peptide EIA. and MIFA MIFA identified 19 of 20 (95%) KS+/HIV+; 9 of 20

(45%) KS-/HIV+; and 5 of 20 (25%) KS-/HIV- specimens positive for HHV8 antibodies. The results were consistent with previously published results in these categories (16). The specimen from the patient with classical KS was also tested positive by the MIFA. Nearly all KS+/HIV+ specimens (18 of 19) which scored positive by the MIFA were also positive by the peptide EIA. Only one of the 20 KS+/HIV+ specimens was negative by both assays. For both assays, an intermediate number of positives were found in the KS-/HIV+ set, and the fewest in the KS-/HIV- set. Less agreement between the two assays was also observed in these two sets of specimens. For the KS-/HIV+ set, three of the nine MIFA-positive specimens were also positive for the peptide EIA, and only one of the 11 MIFA-negative specimens was positive by the peptide EIA.

Example 2

Identification ofHHV8 Dominant Epitope in Protein

Encoded by ORF K8.1

Another antigenic peptide, P2641, isolated according to the methods described above in Example 1 was identifed as a 31 amino acid peptide derived from the N-terminal half (codons 32-62) of the HHV8 ORF K8.1 gene product SEQ ID NO: 19. The sensitivity and specificity of the assay using this peptide as antigen was similar to that of peptide P4 (SEQ ID NO: 6) derived from the ORF 65 gene product. Both assays identified 26 of 30 specimens (87%) positive for HHV8 antibodies from patients with biopsy-confirmed Kaposi's sarcoma. However, four specimens gave discordant results; two were postive by P2461 and negative by P4 and the other two were positive by P4 and negative by

P2461. Combining the results of the two assays, the overall sensitivity would increase from 87% to 93%.

Example 3 Identification ofHHV8 Dominant Epitope in Protein

Encoded by ORF K8. la

Twelve overlapping peptides (20 to 22 amino acids long) encompassing codon 25 to 197 of K8.1a were synthesized (Table 3) and probed with HHV8 positive sera according the methods and protocols known to those skilled in the art and as described above.

Table 3

Overlapping Peptides for Mapping of the Immunodominant Region in K8.1a

The results are shown in Figure 4. Peptide K2 (SEQ ID NO: 24) was identified as most antigenic.

Example 4

Fine Mapping ofK8.1 Epitope

Seventeen K2 peptide analogs (Table 4), which differ from K2 by one amino acid, were synthesized and probed with HHV8 positive sera again to identify the most critical amino acids for antibody binding. A typical reactivity profile is shown in Figure 5. From this figure, the epitope as GQVYQDWXXXXXC (peptides K2.4-K2.16, codon 44-56) was identified. This epitope corresponds to peptide P2641 (SEQ ID NO: 19). Table 4

K2 Peptide Analogs for Fine Mapping of the

Dominant E ito e o K8.1a

Example 5 Development of Bivalent Peptide for HHV8 Antibody Detection A bivalent peptide, P2833 (SEQ ID NO: 25) incorporating the dominant epitope of ORF 65 (SEQ ID NO: 18) and the dominant epitope of K8.1a (SEQ ID NO: 24) was synthesized. Forty serum specimens were tested from individuals who were HIV+ and had biopsy-confirmed KS. Of the specimens tested, 92.5% tested positive by our assay. Two of the negative specimens also tested negative by a published immunofluorescence assay (MIFA)(Table 5, specimen numbers 1 and 3). A fourth specimen (specimen number 6) was positive by the peptide assay but negative by MIFA. Overall, the two assays were 95% concordant.

Methods

Forty KS-- specimens were tested by peptide EIA using P2833 as the antigen. Twenty KS- normal human sera were used as the negative control. An assay cutoff of 0.161 was established using the average OD of the negative controls plus 3 standard deviations. Results of a published immunofluorescence assay (MIFA) were included for comparison.

Table 5 Pe tide EIA Results

Modifications and variations of the present method will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims. All references cited herein are incorporated by reference in their entirety.

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Claims

1. A method of detecting the presence human herpesvirus 8 in a biological sample, said method comprising: (a) contacting one or more isolated, immunogenic human herpesvirus 8 peptides with an antibody-containing biological sample, and

(b) detecting the formation of a complex between the immunogenic peptide and the antibody wherein the presence of a peptide-antibody complex indicates the presence of human herpesvirus 8.

2. The method of Claim 1, wherein the immunogenic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-53, and conservative variations thereof.

3. The method of Claim 2, wherein the immunogenic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 6, 19, 22, 23, 24 and 25 and conservative variations thereof.

4. The method of Claim 1, wherein the peptide is bound to a solid phase.

5. The method of Claim 1, wherein the peptide is labeled.

6. The method of Claim 5, wherein the label is selected from the group consisting of an electrochemiluminescent label, a chemiluminescent label, an enzymatic label, a bioluminescent label, and a fluorescent label.

7. The method of Claim 1, further comprising incubating the peptide-antibody complex with a second antibody specific for the peptide, wherein the second antibody is labeled with a detectable label and binds to the peptide-antibody complex.

8. The method of Claim 7, wherein the label is selected from the group consisting of an electrochemiluminescent label, a chemiluminescent label, an enzymatic label, a bioluminescent label, and a fluorescent label.

9. The method of Claim 1, wherein the biological sample comprises wounds, blood, tissues, saliva, semen, tears, urine, bone, muscle, cartilage, or skin.

10. An immunogenic composition comprising a pharmaceutically acceptable carrier and an isolated, immunogenic human herpesvirus 8 peptide in an amount sufficient to induce a protective immune response to human herpesvirus 8 in a mammal, said immunogenic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-

53, and conservative variations thereof.

11. The composition of Claim 10, wherein the immunogenic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 6, 19, 22, 23, 24 and 25 and conservative variations thereof.

12. The composition of Claim 10, wherein the immunogenic peptide is conjugated to a carrier protein.

13. An isolated, immunogenic human herpesvirus 8 peptide, said immunogenic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1- 53, and conservative variations thereof.

14. The immunogenic peptide in accordance with claim 11, wherein said immunogenic peptide binds to an antibody specifically immunoreactive with a peptide selected from the group consisting of SEQ ID NOS: 1-53, and conservative variations thereof.

15. The immunogenic peptide in accordance with claim 11, wherein said immunogenic peptide is used to detect the presence of human herpesvirus 8 antibodies in a biological sample comprising wounds, blood, tissues, saliva, semen, tears, urine, bone, muscle, cartilage, or skin.

16. An isolated antibody capable of binding to a human herpesvirus 8 immunogenic peptide.

17. The isolated antibody of Claim 16, wherein the immunogenic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-53, and conservative variations thereof.

18. The isolated antibody of Claim 16, wherein the immunogenic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 6, 19, 22, 23, 24 and 25 and conservative variations thereof.

19. The isolated antibody of Claim 16, wherein the antibody is isolated from a biological sample comprising wounds, blood, tissues, saliva, semen, tears, urine, bone, muscle, cartilage, or skin.

20. The isolated antibody of Claim 16, wherein the antibody is a monoclonal antibody.