WO2016168741A2 - Detection of human cytomegalovirus - Google Patents

Abstract

Provided herein, inter alia, are methods for detection of human cytomegalovirus (HCMV) infection in biological samples provided by individuals who are seronegative for antibodies to HCMV via assaying for the presence of the HCMV-encoded viral interleukin-10 (cmvIL-10) mRNA or protein.

Description

DETECTION OF HUMAN CYTOMEGALOVIRUS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/148,629, filed April 16, 2015, the disclosure of which is incorporated by reference herein in its entirety,

FIELD OF INVENTION

[0002] The invention relates generally to the field of methods of screening biological samples, such as donated blood, serum, or other blood-derived products, for the presence of human cytomegalovirus.

BACKGROUND

[0003] Human cytomegalovirus (HCMV) is a widespread pathogen that infects more than 70% of the general population (de la Hoz et aL (2002) ,/ Clin Virol., 25 Suppl 2S 1-12). In most individuals, primary infection with HCMV is asymptomatic; however, serious symptoms can occur in patients with compromised immune systems. HCMV pneumonitis greatly impacts the morbidity and mortality of organ transplant recipients, and HIV patients are frequently diagnosed with severe HCMV retinitis (de la Hoz et al., (2002) J Clin Virol., 25 Suppl 2S 1-12). HCMV can be transmitted from mother to child during pregnancy, and infection can result in serious congenital defects, including deafness, mental retardation, and other neurological deficiencies (Damato & Wirmern (2002) / Obstet Gynecol Neonatal Nurs 31: 86-92). The CDC reports that about 1 in 150 children is born with congenital HCMV infections, and about 1 in every 5 children born with the virus will suffer permanent physical problems; such as hearing loss or developmental disabilities.

[0004] Assays for determining if HCMV infection has occurred in individuals are widely available from commercial sources. The enzyme-linked immunosorbent assay (or ELISA) is the most commonly available serologic test for measuring and/or detecting antibody to HCMV. However, assays like ELISA require the production of antibodies by the infected individual in order to achieve an accurate result. As antibody production occurs sometime after infection, these commonly-used assays can miss detecting the virus in recently-infected individuals by registering a false negative result. As such, there is a particular need for improved methods and kits for the detection of HCMV infections, particularly for screening biological samples derived from individuals such as newborns, organ donors, blood donors, and the immunosuppressed for HCMV infections.

[0005] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.

SUMMARY

[0006] The invention provided herein discloses, inter alia, improved methods for detection of human cytomegalovirus (HCMV) infection in individuals who are seronegative for antibodies to HCMV via assaying for the presence of the HCMV-encoded viral interleukin-10 (cmvIL-10) mRNA or protein.

[0007] Accordingly, in some aspects, provided herein are methods for detecting human cytomegalovirus (HCMV) in a biological sample provided by an individual, the method comprising: detecting the presence of viral interleukin 10 (cmvIL-10) in the sample, wherein the individual is seronegative for HCMV. In some embodiments, the sample is selected from the group consisting of a blood sample, a tissue sample, a urine sample, a saliva sample, a semen sample, a tear sample, or a breast milk sample. In some embodiments of any of the embodiments described herein, cmvIL-10 is detected by detecting a cmvIL-10 nucleic acid in the sample. In some embodiments, the cmvIL-10 nucleic acid is DNA. In some embodiments, the cmvIL-10 DNA is detected by PCR or Southern Blotting. In some embodiments, the cmvIL-10 nucleic acid is RNA. In some embodiments, the cmvIL-10 RNA is detected by RT-PCR, Northern Blotting, in situ hybridization, microarray, or RNase protection assay. In some embodiments of any of the embodiments described herein, the cmvIL-10 is detected by detecting a cmvIL-10 protein in the sample. In some embodiments, the cmvIL-10 protein is detected by Western Blotting, immunoprecipitation,

immunocytochemistry, immunohistochemistry, immunoelectron microscopy,

radioimmunoassay, Enzyme-Linked ImmunoSpot (ELISPOT) assay, 2D gel electrophoresis, or enzyme-linked immunosorbent assay (ELISA). In some embodiments, the cmvIL-10 protein is detected by ELISA. In some embodiments, the antibody used in the ELISA is a polyclonal antibody. In some embodiments, the antibody used in the ELISA assay is a monoclonal antibody. In some embodiments of any of the embodiments described herein, the individual is pregnant. In some embodiments of any of the embodiments described herein, the individual is immunocompromised. In some embodiments, the individual is infected with human immunodeficiency virus (HIV). In some embodiments, the individual is an organ transplant recipient. In some embodiments, the individual is between 0 days and 2 years of age. In some embodiments of any of the embodiments described herein, the individual is immunocompetent. In some embodiments, the individual has one or more conditions selected from the group consisting of cytomegalovirus (CMV) mononucleosis, posttransfusion CMV, arterial hypertension, and Guillain-Barre syndrome. In some

embodiments of any of the embodiments described herein, the individual is asymptomatic. In some embodiments of any of the embodiments described herein, the individual is a pre-natal infant. In some embodiments of any of the embodiments described herein, the individual is undergoing lytic infection with HCMV. In some embodiments of any of the embodiments described herein, the individual is undergoing latent infection with HCMV. In some embodiments of any of the embodiments described herein, the individual is an organ donor. In some embodiments of any of the embodiments described herein, the cmvIL-10 is full length cmvIL-10. In some embodiments of any of the embodiments described herein, the cmvIL-10 is truncated cmvIL-10 (LAcmvILlO). In some embodiments of any of the embodiments described herein, the biological sample provided by the individual is donated blood. In some embodiments of any of the embodiments described herein, the individual is a blood donor.

[0008] In other aspects, provided herein is a kit for detecting human cytomegalovirus

(HCMV) in a biological sample provided by an individual comprising: a) a probe for detecting the presence of viral interleukin 10 (cmvIL-10) in the sample; and b) one or more buffers and/or reagents, wherein the individual is seronegative for HCMV. In some embodiments, the probe is selected from the group consisting of a nucleic acid probe or an antibody. In some embodiments of any of the embodiments described herein, the kit further comprises c) a secondary antibody. In some embodiments of any of the embodiments described herein, the antibody or the secondary antibody is conjugated to an enzyme. In some embodiments of any of the embodiments described herein, the kit further comprises d) a substrate. In some embodiments of any of the embodiments described herein, the biological sample provided by the individual is donated blood. In some embodiments of any of the embodiments described herein, the individual is a blood donor. In some embodiments of any of the embodiments described herein, the kit comprises reagents necessary for performing an ELISA.

[0009] In yet other aspects, provided herein are methods for detecting human cytomegalovirus (HCMV) in a biological sample provided by an individual, the method comprising: (a) contacting the biological sample comprising viral interleukin 10 (cmvIL-10) with a probe that specifically binds to a cmvIL-10 polypeptide or nucleic acid; and (b) detecting the presence of cmvIL-10 when a complex is formed between the probe and cmvIL- 10 polypeptide or nucleic acid, wherein the individual is seronegative for HCMV. In some embodiments, the probe comprises one or more nucleic acids. In some embodiments, the one or more nucleic acids specifically hybridize to a nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments of any of the embodiments disclosed herein, said one or more nucleic acids are PCR primers and PCR is performed subsequent to the complex forming between the PCR primers and the cmvIL-10 nucleic acid. In some embodiments of any of the embodiments disclosed herein, the one or more nucleic acids are detectably labeled. In some embodiments, the probe comprises an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, polyclonal antibody is produced using a recombinantly-produced cmvIL-10 polypeptide immunogen comprising A26 to K176 of SEQ ID NO:3. In some embodiments of any of the embodiments disclosed herein, the polyclonal antibody is derived from goat. In some embodiments of any of the embodiments disclosed herein, the individual is pregnant. In some embodiments of any of the embodiments disclosed herein, the individual is immunocompromised. In some embodiments, the individual is infected with human immunodeficiency virus (HIV). In some embodiments, the individual is an organ transplant recipient. In some embodiments of any of the embodiments disclosed herein, the individual is between 0 days and 2 years of age. In some embodiments of any of the embodiments disclosed herein, the individual is

immunocompetent. In some embodiments, the individual has one or more conditions selected from the group consisting of cytomegalovirus (CMV) mononucleosis, posttransfusion CMV, arterial hypertension, and Guillain-Barre syndrome. In some

embodiments of any of the embodiments disclosed herein, the individual is asymptomatic. In some embodiments of any of the embodiments disclosed herein, the individual is a pre-natal infant. In some embodiments of any of the embodiments disclosed herein, the individual is undergoing lytic infection with HCMV. In some embodiments of any of the embodiments disclosed herein, the individual is undergoing latent infection with HCMV. In some embodiments of any of the embodiments disclosed herein, the individual is an organ donor. In some embodiments of any of the embodiments disclosed herein, the cmvIL-10 comprises full length cmvIL-10. In some embodiments of any of the embodiments disclosed herein, the cmvIL-10 comprises truncated cmvIL-10 (LAcmvILlO). In some embodiments of any of the embodiments disclosed herein, the biological sample provided by the individual comprises donated blood. In some embodiments of any of the embodiments disclosed herein, the individual is a blood donor.

[00010] In further aspects, provided herein is a complex comprising (a) a probe and (b) a cmvILlO protein or nucleic acid, wherein the cmvILlO protein or nucleic acid is derived from a biological sample from an individual infected with human cytomegalovirus (HCMV) but has not undergone seroconversion. In some embodiments, the probe comprises one or more nucleic acids. In some embodiments, the one or more nucleic acids specifically hybridize to a nucleic acid of SEQ ID NO: l or SEQ ID NO: 2. In some embodiments of any of the embodiments disclosed herein, said one or more nucleic acids are PCR primers and PCR is performed subsequent to the complex forming between the PCR primers and the cmvIL-10 nucleic acid. In some embodiments of any of the embodiments disclosed herein, the one or more nucleic acids are detectably labeled. In some embodiments, the probe comprises an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a monoclonal antibody. In some embodiments, the antibody comprises a polyclonal antibody. In some embodiments, the polyclonal antibody is produced using a recombinantly-produced cmvIL-10 polypeptide immunogen comprising A26 to K176 of SEQ ID NO:3. In some embodiments of any of the embodiments disclosed herein, the polyclonal antibody is derived from goat. In some embodiments of any of the embodiments disclosed herein, the individual is pregnant. In some embodiments of any of the embodiments disclosed herein, the individual is immunocompromised. In some embodiments, the individual is infected with human immunodeficiency virus (HIV). In some embodiments, the individual is an organ transplant recipient. In some embodiments of any of the embodiments disclosed herein, the individual is between 0 days and 2 years of age. In some embodiments of any of the embodiments disclosed herein, the individual is immunocompetent. In some embodiments, the individual has one or more conditions selected from the group consisting of cytomegalovirus (CMV) mononucleosis, post-transfusion CMV, arterial hypertension, and Guillain-Barre syndrome. In some embodiments of any of the embodiments disclosed herein, the individual is asymptomatic. In some embodiments of any of the embodiments disclosed herein, the individual is a pre-natal infant. In some embodiments of any of the embodiments disclosed herein, the individual is undergoing lytic infection with HCMV. In some embodiments of any of the embodiments disclosed herein, the individual is undergoing latent infection with HCMV. In some embodiments of any of the embodiments disclosed herein, the individual is an organ donor. In some embodiments of any of the embodiments disclosed herein, the cmvIL- 10 comprises full length cmvIL- 10. In some embodiments of any of the embodiments disclosed herein, the cmvIL- 10 comprises truncated cmvIL- 10 (LAcmvILlO). In some embodiments of any of the embodiments disclosed herein, the biological sample provided by the individual comprises donated blood. In some

embodiments of any of the embodiments disclosed herein, the individual is a blood donor

[00011] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

[00012] FIG. 1 depicts detection of vIL- 10 proteins by ELISA (enzyme linked immune sorbent assay). A) Newborn human foreskin fibroblasts (NuFF- 1) were infected with HCMV strain AD 169 at an MOI of 0.1. Supernatants were collected and analyzed daily using both our vIL- 10 ELISA and a commercial human IL- 10 (ML- 10) ELISA kit (R&D Systems). vIL-10 was evident from day 3-9 post infection, while hIL-10 was not detected at any point during the infection. The vIL-10 ELISA uses a commercial preparation of goat polyclonal antiserum directed against vIL- 10 purchased from R&D systems (detailed protocol provided on following pages). B) Purified recombinant cytokines (R&D Systems) were used to test the assay for cross-reactivity. In addition to the cellular cytokine hIL- 10, a cytokine from a related herpesvirus Epstein-Barr virus, ebvIL- 10, and an unrelated cytokine, IFN-γ, were also tested. PBS served as a negative control. Error bars represent standard error. These results are each representative of 3 independent experiments.

[00013] FIG. 2 depicts detection of two distinct isofora s of v IL- 10. cmvIL- 10 and LAcmv IL- 10. A) Schematic illustration of the two viral gene products derived from alternative splicing of HCMV UL11 1 A. B-D) Human embryonic kidney (HEK)-293 cells were transfected with pcDNA plasmid DNA encoding either cmvIL-lQ or LAcmvIL-10 only. Lysates from day 2 were separated using a Western blot to visualize the two different isoforms, blotted with anti-cmvIL-10 polyclonal antibody (R&D Systems, 1: 1000 dilution) or anti-MAPK antibody as a control (B). Supernatants from each day were collected and analyzed via the vIL-10 ELISA (C-D). Error bars represent standard error. The results in B- D are each representative of 3 independent experiments.

[00014] FIG. 3 depicts demographics and standard curve. A) A cartoon depiction of the process of blood collection beginning from blood donation and collection of whole blood to the harvest of plasma. B) The blood donors are mapped by gender, age, ethnicity, and HCMV status to provide a better background of the donor population. C) A standard curve was created using 10% plasma in PBS as a diluent to show that plasma proteins did not interfere with the sensitivity and specificity of the assay. D) Plasma was harvested for quantification of viral cytokine levels using the vIL-10 and results are shown as a comparison between HCMV status. E) Plasma vIL-10 levels are depicted based on HCMV status and gender. F) Plasma vIL-10 detection is supported by the presence of the viral IE1 gene by PCR of whole blood gDNA. Also, IE1 is detected in both HCMV + and - donors.

[00015] FIG. 4 depicts detection of vIL-10 in a representative set of healthy blood donors. Four samples were HCMV seronegative (S9-S 12) and four samples were HCMV seropositive (S 13-16). Bar graph shows vIL-10 levels as determined by ELISA, and vIL-10 was detected in two seronegative specimens. The seronegative donors with vIL- 10 lacked any measurable IgG or IgM response to HCMV (Trinity Bioscience ELISA), but viral DNA could be detected by PCR. A nested PCR procedure was used to detect exon 4 of HCMV IE1 (detailed protocol on next pages) on genomic DNA isolated from the whole blood sample, and top panel represents first round of PCR. Middle panel, second round of PCR shows that two seronegative donors have viral DNA present in their blood, which correlates with detection of vIL-10 protein. Lower panel, detection of β-actin as a control.

[00016] FIG. 5 depicts detection of human cytokines in human blood samples. The same specimens tested for vIL-10 levels were also tested for levels of three human cytokines: hIL-10, IL-12, and TNFoc via commercial ELISA kits (R&D Systems). Top panels show each levels of each cytokine in HCMV+ and HCMV- samples. Bottom panel shows relationship between levels of each cytokine with vIL-10 levels. DETAILED DESCRIPTION

[00017] This invention provides, inter alia, methods, compositions, and kits for the detection of Human Cytomegalovirus (HCMV) in biological samples provided by individuals who are seronegative for HCMV infection. The invention is based, in part, on the inventors' surprising discovery that the HCMV-encoded cytokine crnvIL-10, which is a potent immunosuppressive viral ortholog of human IL-IO, is detectable in biological samples derived from individuals who do not possess antibodies to HCM V {i.e. who are seronegative for HCMV) and thus would not be considered to be infected with this virus by the most commonly used laboratory tests employed to screen for the presence of HCMV. As such, the methods and kits disclosed herein have the potential to greatly improve the accuracy and reliability of assays designed to identify HCMV infection in biological samples provided by and to individuals such as expectant mothers, newborn children, organ donors, those who are immunocompromised, or blood donors.

I. General Techniques

[00018] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A

Laboratory Manual, fourth edition (Sambrook et a!., 2012) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (jointly referred to herein as "Sambrook"); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, including supplements through 2014); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Antibodies: A Laboratory Manual, Second edition, Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY (Greenfield, ed., 2014), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000, (including supplements through 2014), Gene Transfer and. Expression in Mammalian Cells (Makrides, ed., Elsevier Sciences B.V., Amsterdam, 2003), and Current Protocols in

Immunology (Horgan K and S. Shaw (1994) (including supplements through 2014).

II. Definitions

[00019] "Seroconversion," as used herein, refers to the point in time when a specific antibody (such as an antibody to HCMV, such as an HCMV-expressed protein) becomes detectable in a biological sample provided by an individual. During an infection or immunization, antigens enter the blood and the immune system begins to produce antibodies in response. Seroconversion is the point in time when the antibody becomes detectable. Before seroconversion, the antigen may be detectable, but the antibody is not.

[00020] As used herein, the phrase "seronegative for HCMV," means that an individual does not produce any detectible antibodies against HCMV or an HCMV-expressed protein (e.g. antibodies detectable by currently available, standard, or routine diagnostic tests). In some embodiments, the phrase refers to a biological sample provided by an individual that lacks any detectable antibodies against HCMV.

[00021] "Viral interleukin- 10," or "cmvIL-10," or "vIL- 10" or "cytomegalovirus interleukin- 10" can be used interchangeably to refer to the full length mRNA or protein product of the HCMV UL111A gene. In some embodiments, cmvIL-10 also refers to truncated cmvIL- 10 (i.e., latency associated cmvIL-10 or LAcmvIL- 10).

[00022] "Latency Associated cmvIL- 10" or "LAcmvIL-10," as used herein, refers to a truncated mRNA or protein product of the HCMV UL111A gene. The LAcmvIL-10 protein is co-linear with full length cmvIL- 10 for the first 127 residues and then diverges in sequence at the truncated C-terminal domain (139 amino acids total compared to 175 for full length cmvIL-10).

[00023] As used herein, a "nucleic acid" or "oligonucleotide" refers to two or more deoxyribonueleotides and/or ribonucleotides covalently joined together in either single or double-stranded form.

[00024] As used herein, the term "protein" includes polypeptides, peptides, fragments of polypeptides, and fusion polypeptides.

[00025] The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase

"consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel

characteristic(s)" of the claimed invention. [00026] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[00027] As used herein, the singular terms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise.

III. Human Cytomegalovirus (HCMV)

[00028] Human Cytomegalovirus (HCMV) belongs to the β-Herpesvirinae subfamily of the family Herpesviridae, including human herpes virus 6 (HHV-6) and human herpes virus 7 (HHV-7). HCMV is a double- stranded DNA virus, comprising 230 kbp coding more than 200 genes with the diameter of about 180 nm. HCMV is the biggest virus among the family Herpesviridae. HCMV exhibits strong species specificity as no other animals are known to be vulnerable to HCMV infection. The specific mode of HCMV transmission from person to person is entirely unknown but is presumed to occur through bodily fluids.

[00029] After infection, HCMV remains latent in lymphocytes in the body for the rest of the person's life. Overt disease rarely occurs unless immunity is suppressed either by drugs, infection or old age. Initial HCMV infection, which often is asymptomatic, is followed by a prolonged, latent infection during which the virus resides in mononuclear cells without causing detectable damage or clinical illness. However, in immunocompromised patients such as hemodialysis patients, cancer patients, patients who take immunosuppressants, HIV- carriers, bone-marrow transplant patients, and organ transplant patients with

immunocompromised status, HCMV can be reactivated into an active, lytic infection. Life- threatening diseases such as interstitial pneumonia, retinitis, gastroenteritis, and encephalitis can subsequently develop in these individuals.

[00030] Persons who have been infected with HCMV develop antibodies to the virus and these antibodies persist in the body for the lifetime of that individual. A number of laboratory tests that detect these antibodies to HCMV have been developed to determine if infection has occurred and are widely available from commercial laboratories. The enzyme- linked immunosorbent assay (or ELISA) is the most commonly available serologic test for measuring and/or detecting antibody to HCMV. For example, the result of a currently available ELISA test can be used to determine if acute infection, prior infection, or passively acquired maternal antibody in a newborn infant is present. [00031] Aside from ELISA, another method that can be used to determine if an individual is infected with HCMV is assaying for the presence of a HCMV-derived nucleic acids (such as an RNA or genomic DNA) in sample obtained from the individual. In some embodiments, PCR or RT-PCR is used to determine if an individual is infected with HCMV. In one embodiment, the PCR is performed to detect the presence of HCMV genomic DNA (such as the DNA sequence encoded by Genbank Accession no. X17403 or a portion thereof). In another embodiment, the PCR assays for the presence of the HCMV IE I gene. In a further embodiment, the PCR assay for the HCMV IE1 gene uses a forward primer haying the sequence AAGTGAGTTCTGTCGGGTGCT and a reverse primer having the sequence GTGACACCAGAGAATCAGAGGA.

[00032] HCMV assays are also part of the standard screening for non-directed blood donation (i.e., donations not specified for a particular patient) in the U.S., the UK and many other countries throughout the world. HCMV-negative donations are earmarked for transfusion to infants or immunocompromised patients, who are at risk for the potentially severe complications known to be associated with HCMV infection. Some blood donation centers maintain lists of donors whose blood is CMV negative due to special demands.

IV. Viral interleukin-10 (cmvIL-10)

[00033] The cmv IL-10 protein is a homolog of human IL-10 encoded by the UL111A gene product of HCMV (Kotenko et al., (2000) Proc Nad Acad Sci U S A 97: 1695--- 1700; Genbank Accession no. X17403 (HCMV AD 169 Whole Genome)). Despite having only 27% sequence identity to human IL-10, cmv IL-10 binds to the cellular IL-10 receptor (IL-10R) and displays many of the immune suppressive functions of human IL-10 (Slobedman et al., (2009) ,/ Virol 83: 961.8-9629; Spencer et al., (2002 ) J Virol 76: 1285-1292 ). These include regulation of IFN-γ, IL-la, GM-CSF, IL-6 and TNF-a, which are all pro-inflammatory cytokines. CmvIL-10 has also been shown to play a role in downregulating MHC I and MHC II and up regulating HLA-G (non-classical MHC I). These two events allow for immune evasion by suppressing the cell-mediated immune response and natural killer cell response, respectively.

[00034] CmvIL-10 is encoded as a discontinuous open reading frame containing two introns (nucleotides 159678 to 160364 of HCMV AD169 Whole Genome). atg ctgtcggtga tggtctcttc ctctctggtc ctgatcgtct tttttctagg cgcttccgag gaggcgaagc cggcgacgac gacgataaag aatacaaagc cgcagtgtcg tccagaggat tacgcgacca gattgcaaga tctccgcgtc acctttcatc gagtaaaacc tacgttggta ggtcacgtag gtacggttta ttgcgacggt ctttcttttc cgcgtgtcgg gtgacgtagt tttcctcttg tagcaacgtg aggacgacta ctccgtgtgg ctcgacggta cggtggtcaa aggctgttgg ggatgcagcg tcatggactg gttgttgagg cggtatctgg agatcgtgtt tcccgcaggc gaccacgtct atcccggact caagacggaa ttgcatagta tgcgctcgac gctagaatcc atctacaaag acatgcggca atgtgtaagt gtctctgtgg cggcgctgtc cgcacagagg taacaacgtg ttcatagcac gctgttttac ttttgtcggg ctcccagcct ctgttaggtt gcggagataa gtccgtgatt agtcggctgt ctcaggaggc ggaaaggaaa tcggataacg gcacgcggaa aggtctcagc gagttggaca cgttgtttag ccgtctcgaa gagtatctgc actcgagaaa gtag (SEQ ID N0: 1) (introns shown in bold)

[00035] The presence of introns in the UL111 A gene encoding the cmvIL-10 mRNA allows for the possibility of alternative splicing, and this has been documented to occur in latently infected granulocyte-macrophage progenitor cells (Jenkins et al., (2004) Virol. 78(3): 1440-7). The truncated UL111A region latency-associated (LAcmvIL-10) transcript differs from full length cmvIL-10 transcripts in that it contains only one intron (nucleotides 159678 to 160173 of HCMV AD 169 Whole Genome). tgcggcgatg ctgtcggtga tggtctcttc ctctctggtc ctgatcgtct tttttctagg cgcttccgag gaggcgaagc cggcgacgac gacgataaag aatacaaagc cgcagtgtcg tccagaggat tacgcgacca gattgcaaga tctccgcgtc acctttcatc gagtaaaacc tacgttggta ggtcacgtag gtacggttta ttgcgacggt ctttcttttc cgcgtgtcgg gtgacgtagt tttcctcttg tagcaacgtg aggacgacta ctccgtgtgg ctcgacggta cggtggtcaa aggctgttgg ggatgcagcg tcatggactg gttgttgagg cggtatctgg agatcgtgtt tcccgcaggc gaccacgtct atcccggact caagacggaa ttgcatagta tgcgctcgac gctagaatcc atctacaaag acatgcggca atgtgtaagt gtctctgtgg cggcgctgtc cgcacagagg taa (SEQ ID NO:2) (intron shown in bold)

[00036] The LAcmvIL- 10 protein product (SEQ ID NO: 4) is co-linear with cmvIL- 10 for the first 127 residues and then diverges in sequence at the truncated C-terminal domain (139 amino acids total compared to 175 for full length cmvIL-10 (SEQ ID NO:3)).

MLSVMVSSSL VLIVFFLGAS EEAKPATTTT I NT PQCRP EDYATRLQDL RVTFHRVKPT LQREDDYSVW LDGTVVKGCW GCSVMDWLLR RYLEIVFPAG DHVYPGL TE LHS MRS TLBS IYKDMRQCPL LGCGDKSVIS RLSQEAERKS

DNGTRKGLSE LDTLFSRLEE YLHSRK (SEQ ID NO:3)

MLSVMVSSSL VLIVFFLGAS EEAKPATTTT IKNTKPQCRP EDYATRLQDL RVTFHRVKPT LQREDDYSVW LDGTVVKGCW GCSVMDWLLR RYLEIVFPAG DHVYPGLKTE LHSMRSTLES IYKDMRQCVS VSVAALSAQR (SEQ ID NO: 4)

[00037] Whereas full length cmvIL-10 exhibits a broad range of inhibitory functions associated with human IL-10, including inhibition of PBMC proliferation, impairment of dendritic cell maturation expression, suppression of inflammatory cytokine synthesis, and reduction of class II MHC expression, the immunosuppressive activities of LAcmvIL-10 appear to be more attenuated.

[00038] The UL111 A gene is expressed in both lytic and latent HCMV infection. Both cmvIL-10 and LAcmv IL-10 are expressed during lytic HCMV infection. However, only LAcmv IL-10 has been shown to be produced during latent infection.

V. Methods of the Invention

[00039] The present invention is directed to methods for detecting human

cytomegalovirus (HCMV) infection in a biological sample provided by an individual who is seronegative for HCMV. The method encompasses detecting the presence of viral interleukin-10 (cmvIL-10) in the sample. In some embodiments, the methods of the present invention optionally include the step of assaying the biological sample from the individual for the presence of, for example, antibodies to HCMV to ascertain the individual's HCMV serostatus. In another embodiment, the method encompasses contacting a sample comprising cmvIL-10 or LAcmvIL-10 with a probe that specifically binds to a cmv IL-10 or LAcmv IL-10 polypeptide or nucleic acid; and detecting the presence of cmv IL-10 or LAcmv IL-10 when a complex is formed between the probe and cmvIL-10 or LAcmv IL-10 polypeptide or nucleic acid.

[00040] Biological samples can be obtained from individuals by any means known in the art and can include, without limitation, blood (including, e.g., products derived from whole blood such as serum or platelets), tissue, urine, saliva, semen, tears, cerebrospinal fluid, or breast milk.

A Detection of cmvIL-10 expression

[00041] Viral interleukin-10 expression or activity can be detected in samples using any means known in the art. CmvIL-10 expression encompasses the existence of the full and intact UL111A viral DNA sequence (including, e.g., promoter elements, enhancer sequences, introns, and exons), the conversion of the UL111 A gene sequence into transcribed mRNA (including, e.g., the initial unspliced mRNA transcript or the mature processed mRNA), or the translated cmvIL-10 (or LAcmvIL-10) protein product (including, e.g., any

posttranslational modifications such as, but not limited to, ubiquitination, sumoylation, acetylation, methylation, glycosylation, and/or hydroxylation).

L cmvIL-10 gene and mRNA expression

[00042] The assessment of cmvIL-10 expression or activity in a sample can be at the levels of mRNA or DNA. Assessment of mRNA expression levels of gene transcripts is routine and well known in the art. For example, one flexible and sensitive quantitative method for assessing mRNA expression levels in a biological sample is by quantitative RT- PCR (qRT-PCR) or by any other comparable quantitative PCR-based method. Additional methods for assessing cmvIL-10 mRNA expression include, but are not limited to, Northern blotting, microarrays, in situ hybridization, and serial analysis of gene expression (SAGE).

[00043] Techniques such as Northern blotting or RT-PCR rely on the use of a probe or primers complementary to the cmv-ILlO mRNA. The term "complementary" and

"complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5'-A-T-G-C-3' is complementary to the sequence 5'-G-C-A-T-3'. Complementarity may be "partial," in which case only some of the bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in PCR-based amplification reactions, as well as detection methods that depend upon binding between nucleic acids. In some embodiments, the nucleic acid probes such as oligonucleotides, oligonucleotide arrays, and/or primers for use in the methods of the present invention are complementary to a nucleic acid of SEQ ID NO: l or SEQ ID NO:2. In other embodiments, nucleic acid probes such as oligonucleotides, oligonucleotide arrays, and/or primers for use in the methods of the present invention are any of about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a nucleic acid of SEQ ID NO: l or SEQ ID NO:2. In further embodiments, nucleic acid probes such as oligonucleotides, oligonucleotide arrays, and/or primers for use in the methods of the present invention are at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a nucleic acid of SEQ ID NO: l or SEQ ID NO:2. In some embodiments, nucleic acid probes such as oligonucleotides, oligonucleotide arrays, and/or primers for use in the methods of the present invention are at most about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a nucleic acid of SEQ ID NO: l or SEQ ID NO:2. All individual values for numbers and percentages as used herein can be combined to create upper and lower limits for ranges. For example, in additional embodiments, nucleic acid probes such as oligonucleotides, oligonucleotide arrays, and/or primers for use in the methods of the present invention can be about 65%- 100%, 75%- 95%, 80%-90%, 75%-100%, 80%-100%, 85%-100%, 85%-95%, 90%-100%, or 95%-100% complementary to a nucleic acid of SEQ ID NO: l or SEQ ID NO:2.

[00044] Nucleic acid binding molecules such as probes, oligonucleotides,

oligonucleotide arrays, and primers can be used in assays to detect cmvIL-10 or LAcmvIL-10 RNA expression in biological samples from individuals. In one embodiment, RT-PCR is used according to standard methods known in the art. In another embodiment, PCR assays such as Taqman® assays available from, e.g., Applied Biosystems, can be used to detect nucleic acids and variants thereof. In another embodiment, a two stage nested PCT assay (such as that performed in Example 5, infra) is performed to assess the existence of the cmvIL-10 mRNA or genomic DNA sequence. In other embodiments, qPCR and nucleic acid microarrays can be used to detect nucleic acids. Reagents that bind to cmvIL-10 or

LAcmvIL-10 can be prepared according to methods known to those of skill in the art or purchased commercially.

[00045] Analysis of cmvIL-10 or LAcmvIL-10 nucleic acids can be achieved using routine techniques such as Southern blot analysis, PCR, Northern blot analysis, RT-PCR, or any other methods based on hybridization to a nucleic acid sequence that is complementary to a portion of the cmvIL-10 or LAcmvIL-10 coding sequence (e.g., slot blot hybridization) are also within the scope of the present invention. General nucleic acid hybridization methods are described in Anderson, "Nucleic Acid Hybridization," BIOS Scientific Publishers, 1999. Amplification or hybridization of a plurality of nucleic acid sequences (e.g. , genomic DNA, mRNA or cDNA) can also be performed from mRNA or cDNA sequences arranged in a microarray. Microarray methods are generally described in Hardiman, "Microarrays Methods and Applications: Nuts & Bolts," DNA Press, 2003; and Baldi et al., "DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling," Cambridge University Press, 2002.

[00046] Analysis of the gene encoding cmvIL-10 (UL1 11 A) can be performed using techniques known in the art including, without limitation, microarrays, polymerase chain reaction (PCR)-based analysis, sequence analysis, and electrophoretic analysis. A non- limiting example of a PCR-based analysis includes a Taqman® allelic discrimination assay available from Applied Biosystems. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al., Nat. Biotechnol., 16:381-384 (1998)), and sequencing by hybridization. Chee et al., Science, 274:610-614 (1996); Drmanac et al., Science, 260: 1649-1652 (1993); Drmanac et al., Nat. Biotechnol., 16:54-58 (1998). Non- limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Other methods for detecting nucleic acids include, e.g., the INVADER^® assay from Third Wave Technologies, Inc., restriction fragment length polymorphism

(RFLP) analysis, allele- specific oligonucleotide hybridization, a heteroduplex mobility assay, single strand conformational polymorphism (SSCP) analysis, single-nucleotide primer extension (SNUPE) and pyrosequencing.

[00047] A detectable moiety or detectable label can be used in the assays described herein for detection of cmvIL-10 nucleic acids. A wide variety of detectable moieties can be used, with the choice of label depending on the sensitivity required, ease of conjugation, stability requirements, and available instrumentation and disposal provisions. Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, and the like.

[00048] Detection of cmvIL-10 nucleic acids can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of biological samples. Alternatively, single sample formats could be developed to facilitate diagnosis or prognosis in a timely fashion.

[00049] Alternatively, the nucleic acid probes of the invention can be applied to sections of biological sample biopsies immobilized on microscope slides. The resulting staining or in situ hybridization pattern can be visualized using any one of a variety of light or fluorescent microscopic methods known in the art.

[00050] Also provided herein are reagents for in vivo imaging of cmvIL-10 (or LAcmvIL-10) such as, for instance, the imaging of labeled regents that detect cmvIL-10 nucleic acids. For in vivo imaging purposes, reagents that detect the presence of cmvIL-10 (or LAcmvIL-10) nucleic acids, may be labeled using an appropriate marker, such as a fluorescent marker.

2. Protein expression

[00051] Similarly, assessment of protein expression levels is routine in the art. For example, one method of measuring protein levels is via Western blotting or

immunohistochemistry using antibodies to cmvIL-10 (or LAcmvIL-10). Other well-known and reliable methods for assaying for the existence of cmvIL-10 protein in a sample include, without limitation, radioimmunoassay (RIA), ELISA (such as the ELISA described in Example 5, infra), flow cytometry, immunohistochemistry, immunocytochemistry, or any other antibody-mediated technique. In one embodiment, the method for assaying the existence of cmvIL-10 protein is an ELISA. In another embodiment, the ELISA can detect at least about 5 pg/mL of cmvIL-10 protein in the sample.

[00052] Antibody reagents can be used in assays to detect expression of cmvIL-10 (or LAcmvIL-10) in patient samples using any of a number of immunoassays known to those skilled in the art. "Antibody" as used herein is meant to include intact molecules as well as fragments which retain the ability to bind antigen, such as cmvIL-10 (e.g., Fab and F(ab') fragments). These fragments are typically produced by proteolytically cleaving intact antibodies using enzymes such as a papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The term "antibody" also refers to both monoclonal antibodies and polyclonal antibodies. Polyclonal antibodies are derived from the sera of animals immunized with the antigen.

[00053] Antibodies having specificity for a specific protein, such as a protein product of the UL1 1 1 A gene (such as the proteins encoded by SEQ ID NOs:3-4), rnRNA, or a fragment thereof, may be prepared by conventional methods. A mammal, (e.g. a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the peptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.

[00054] In some embodiments, a polyclonal antibody that binds to cmvIL-10 and LAcmvIL- 10 for use in any of the antibody-based detection methods disclosed herein (e.g., ELISA) is produced using an immunogenic peptide encoding SEQ ID NOs:3 or 4 or a portion of the proteins encoded by SEQ ID NOs:3 or 4. In other embodiments, the polyclonal antibody is produced using an immunogenic peptide having any of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the proteins encoded by SEQ ID NOs:3 or 4. In other embodiments, the polyclonal antibody is produced using an immunogenic peptide at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the proteins encoded by SEQ ID NOs:3 or 4. In other embodiments, the polyclonal antibody is produced using an immunogenic peptide having at most about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the proteins encoded by SEQ ID NOs:3 or 4. In further

embodiments, the polyclonal antibody is produced using an immunogenic peptide about 50%- 100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 55%- 100%, 55%-90%, 55%-80%, 55%-70%, 55%-65%, 60%- 100%, 60%-90%, 65%-80%, 65%-75%, 65%- 100%, 75%-95%, 80%-90%, 75%- 100%, 80%- 100%, 85%- 100%, 85%-95%, 90%- 100%, or 95%- 100% identity to the proteins encoded by SEQ ID NOs:3 or 4.

[00055] In a further embodiment, the polyclonal antibody is produced using an immunogenic peptide comprising A26 to K176 of SEQ ID NO:3. In another embodiment, the polyclonal antibody is produced in goat. In one embodiment, the immunogenic peptide is recombinantly produced in a bacterial species (such as, but not limited to E. coli). In yet another embodiment, the Neutralization Dose (ND₅₀) of the polyclonal antibody is about 0.15-0.9 μg/mL (for example, about 0.15 μg/mL-0.8 μg/mL, 0.15 μg/mL-0.7 μg/mL, 0.15 μg/mL-0.6 μg/mL, 0.15 μg/mL-0.5 μg/mL, 0.15 μg/mL-0.4 μg/mL, 0.2 μg/mL-0.8 μg/mL, 0.3 μg/mL-0.7 μg/mL, 0.4 μg/mL-0.6 μg/mL, 0.5 μg/mL-0.9 μg/mL, 0.6 μg/mL-0.9 μg/mL, or 0.7 μg/mL-0.9 μg/mL), such as any of about 0.15 μg/mL, 0.2 μg/mL, 0.25 μg/mL, 0.3 μg/mL, 0.35 μg/mL, 0.4 μg/mL, 0.45 μg/mL, 0.5 μg/mL, 0.55 μg/mL, 0.6 μg/mL, 0.65 μg/mL, 0.7 μg/mL, 0.75 μg/mL, 0.8 μg/mL, 0.85 μg/mL, or 0.9 μg/mL, inclusive of all values falling in between these concentrations) in the presence of 2 ng/mL recombinant cmvIL-10. In other embodiments, the Neutralization Dose (ND₅₀) of the polyclonal antibody is at least about 0.15 μg/mL, 0.2 μg/mL, 0.25 μg/mL, 0.3 μg/mL, 0.35 μg/mL, 0.4 μg/mL, 0.45 μg/mL, 0.5 μg/mL, 0.55 μg/mL, 0.6 μg/mL, 0.65 μg/mL, 0.7 μg/mL, 0.75 μg/mL, 0.8 μg/mL, 0.85 μg/mL, or 0.9 μg/mL, inclusive of all values falling in between these concentrations) in the presence of 2 ng/mL recombinant cmvIL-10. In some embodiments, the Neutralization Dose (ND₅₀) of the polyclonal antibody is at most about 0.15 μg/mL, 0.2 μg/mL, 0.25 μg/mL, 0.3 μg/mL, 0.35 μg/mL, 0.4 μg/mL, 0.45 μg/mL, 0.5 μg/mL, 0.55 μg/mL, 0.6 μg/mL, 0.65 μg/mL, 0.7 μg/mL, 0.75 μg/mL, 0.8 μg/mL, 0.85 μg/mL, or 0.9 μg/mL, inclusive of all values falling in between these concentrations) in the presence of 2 ng/mL recombinant cmvIL-10.

[00056] Monoclonal antibodies can be prepared using hybridoma technology (Kohler, et al., Nature 256:495 (1975)). In general, this technology involves immunizing an animal, usually a mouse. The splenocytes of the immunized animals are extracted and fused with suitable myeloma cells, e.g., SP20 cells. After fusion, the resulting hybridoma cells are selectively maintained in a culture medium and then cloned by limiting dilution (Wands, et al., Gastroenterology 80:225-232 (1981)). The cells obtained through such selection are then assayed to identify clones which secrete antibodies capable of binding to septin family member proteins or fragments thereof. Such techniques are well known in the art, {e.g. the hybridoma technique originally developed by Kohler and Milstein {Nature 256:495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Methods Enzymol, 121 : 140-67 (1986)), and screening of combinatorial antibody libraries (Huse et al., Science 246: 1275 (1989)). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated.

[00057] Immunoassay techniques and protocols using antibodies or fragments thereof to cmvIL-10 or LAcmvIL-10 are generally described in Price and Newman, "Principles and Practice of Immunoassay," 2nd Edition, Grove's Dictionaries, 1997; and Gosling,

"Immunoassays: A Practical Approach " Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used. See, e.g., Self et al., Curr. Opin. Biotechnol., 7:60-65 (1996). The term immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme

immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated.

Immunoassays can also be used in conjunction with laser induced fluorescence. See, e.g., Schmalzing et al., Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. Sci., 699:463-80 (1997). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. See, e.g., Rongen et al., . Immunol. Methods, 204: 105-133 (1997). In addition, nephelometry assays, in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention. Nephelometry assays are commercially available from Beckman Coulter (Brea, Calif.; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biochem., 27:261-276 (1989)). [00058] Specific immunological binding of the antibody to cmvIL- 10 or LAcmvIL-10 can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. An antibody labeled with

125

iodine- 125 (^{^J}I) can be used. A chemiluminescence assay using a chemiluminescent secondary antibody specific for the antibody to cmvIL- 10 or LAcmvIL-10 is suitable for sensitive, non-radioactive detection of protein levels. A secondary antibody labeled with a fluorochrome is also suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, urease, and the like. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β- galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β- D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm. A urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals; St. Louis, Mo.).

[00059] A signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect

125

radiation such as a gamma counter for detection of I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, Calif.) in accordance with the manufacturer's instructions. If desired, the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected

simultaneously.

[00060] In some embodiments, the antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g. , microtiter wells), pieces of a solid substrate material or membrane (e.g. , plastic, nylon, paper, nitrocellulose), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.

B. Target populations

[00061] The methods for detecting HCMV in biological samples disclosed herein can be used to test biological samples derived from members of at-risk target populations for the presence of HCMV. As such, the methods disclosed herein can be used to differentiate individuals who are infected with HCMV but who do not produce detectable levels of antibodies to the virus. The ability to differentiate between individuals who are infected with HCMV from those who are not is of particular relevance to the medical treatment and care of individuals who are members of certain target populations. These target populations include, without limitation, expectant mothers, newborn children, organ donors, those who are immunocompromised (such as those diagnosed with or suspected of having HIV or AIDS or organ donor recipients on immnosuppressive therapy), or blood donors. As such, the methods disclosed herein are particularly applicable with respect to ensuring individuals who are members of these target populations do not receive medical treatments (e.g., blood transfusions) or biologically-derived products (donor organs) from HCMV-infected individuals.

V. Compositions of the Invention

[00062] Also provided herein are complexes comprising a probe and a cmvILlO (or LAcmvIL-10) protein or nucleic acid, wherein the cmvILlO protein or nucleic acid is derived from a biological sample from an individual infected with human cytomegalovirus (HCMV) but has not undergone seroconversion. The probe can be any of the nucleic acid or polypeptide probes described herein (such as an antibody or functional fragment thereof). Additionally, the biological sample (such as, blood) from which the cmvILlO protein or nucleic acid is derived can be processed, prior to formation of the complex, such as by isolation of serum, total protein, or nucleic acids.

VI. Kits

[00063] In addition, the present invention includes a kit for carrying out the subject cmvIL-10 (or LAcmvIL- 10) assays. The kit can include one or more probes specific for identification of cmvIL-10 (and/or LAcmvIL-10) mRNA or protein in a biological sample from an individual. Such probes include can include antibodies (either polyclonal, monoclonal, or fragments thereof) or oligonucleotide probes. The probes can optionally

3 1 2 12^ include a signal such as a radioactive isotope (such as, but not limited to, H, ^' C, H, ^"I,

32 33

^' P, or ^~'"P), a signal-producing enzyme (such as, for example, horseradish peroxidase, luciferase), or a signal-producing protein (e.g., but not limited to, green fluorescent protein). Where the probe is an antibody or fragment thereof, the kit can also include a secondary antibody conjugated to a signal. In other embodiments, the probe can be an aptamer, photoaptamer, protein, peptide, peptidomimetic or a small molecule chemical compound.

[00064] Other materials useful for performing the subject method can also be included as part of the kit. For example, the kit can include buffers or iabware necessary to obtain or store a biological sample from an individual, or isolate or purify target mRNA or protein from the biological sample. Further, the kit can include materials (e.g. , chemicals or buffers or substrates for eliciting signals from a signal-producing enzyme) or Iabware for performing hybridization and detection procedures. The kit can also include labeling materials for labeling the probes. Written materials describing the steps involved in the subject method can be included for instructing the user how to use the article of manufacture or kit.

[00065] In one embodiment, cmvTL- 10 (and/or LAcmvIL- 10) proteins derived from a sample can be immobilized on a solid phase or support. The kits may therefore also include reagents and means for measuring the quantity of cmvIL-10 proteins, or fragments thereof. For example, the kits can employ immunoassays, mass spectrometry analysis technology, or chromatographic technology, or a combination of said technologies.

[00066] In one embodiment, the kit comprises antibodies, antigen-binding, or complementary nucleic acids for cmvIL- 10 (and/or LAcmvIL-10). The kit may comprise probes or assays for detecting expression of mRNA, cDNA or protein corresponding cmvIL- 10 (and/or LAcmvIL-10). Suitable probes or assays may include complementary nucleic acids (including cDNA or oligonucleotides, for example) or antibodies, fragments thereof, or antigen-binding polypeptides directed against (i.e. capable of binding) the corresponding cmvIL-10 (and/or LAcmvIL- 10) proteins.

[00067] The kit may include instructions for use in detecting breast cancer, determining risk of metastasis, determining tumor grade, and determining tumor sub-type. In a specific example, the kit may be useful in predicting metastatic potential of a breast cancer tumor.

[00068] In another embodiment, the kit contains reagents necessary for performing an ELISA in accordance with the methods of the present invention (such as the ELISA performed in Example 1, infra). Such kits can include, without limitation, polyclonal or monoclonal antibodies to cmvIL-10 (or LAcmvIL-10), purified cmvIL-10 protein, a secondary antibody (e.g., a biotinylated secondary antibody such as a Biotinylated Affinity Purified Polyclonal Goat IgG antibody), was buffer, blocking buffer, a signal molecule (such as, but not limited to Streptavidin-HRP), substrate solution, and/or stop solution (such as sulfuric acid). In some embodiments, the ELISA is sensitive to only cmvIL-10 (or LAcmvIL-10) and does not cross react with other human cytokines (such as, without limitation, ML- 10, ebvIL-10, or IFN-γ). In another embodiment, the ELISA reagents contained in the kit can detect at least about 5 pg/mL of cmvIL-10 protein in the sample.

[00069] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[00070] The invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting.

EXAMPLES

Example 1: cmvIL-10 can be detected in supernatants of HCMV-infected cells

[00071] This example describes the development and validation of a sensitive ELISA test for assaying for the presence of cmvIL-10 in HCMV-infected ceils.

Materials and Methods:

[00072] Cell culture and viral infection: Newborn human foreskin fibroblasts (NuFF-1) were infected at 0.1 MOI with HCMV AD169-strain for a period of 9 days. Supernatants from each day were collected for ELISA.

[00073] cmvIL-10 ELISA: Viral HCMV IL-10 Affinity Purified Polyclonal Ab, Goat IgG (R&D #AF117) was reconstituted in 500 μΐ of sterile PBS for a final concentration of 200 ig/ml. This was aliquoted into 20 tubes with 25 μΐ each and store at -20°C.

Recombinant viral HCMV IL-10 (R&D #117-VL-025) was reconstituted in 250 id of sterile PBS containing 0.1% bovine serum albumin (BS A) for a final concentration at 100 μg ml and aliquoted into 25 tubes with 10 μΐ each and store at -20°C. Viral HCMV IL-10 Biotinvlated Affinity Purified Pab, Goat IgG (R&D #BAF117) was reconstituted in 250 μΐ of sterile PBS for a final concentration of 200 ^g/ml and aliquoted into 25 tubes with 10 μΐ each and store at -20°C.

[00074] Plates were coated with 50μ1Λνε11 of 2 μ^ηιΐ viral HCM V IL-10 Affinity Purified Polyclonal Ab, Goat IgG (R&D #AF1 I7) diluted in PBS and sealed with adhesive plate cover. The plate was incubated overnight at 4°C. The plate was washed 3x with wash buffer (PBS + 0.05% Tween) followed by addition of blocking buffer (PBS + 1% BSA), sealing with adhesive, and incubation for 1 hour at room temperature (RT) on a shaker.

Following incubation, plates were washed 3x with wash buffer. Samples and standards were then added to the plate and sealed with adhesive cover for a two hour incubation at RT on a shaker. For standards, an 8-poini standard curve stalling at 1000 pg/ml in PBS containing 10% seronegative human serum was constructed using Recombinant Viral HCMV IL-10 (R&D #117-VL-025) with 2 fold dilution, and PBS with 10% seronegative human serum as blank for an 8th point. Blood samples were tested at 10%: 16 μΐ plasma + 144 μΐ PBS.

[00075] Following incubation, plates were washed 3x with wash buffer. Detection antibody (Viral HCMV IL-10 Biotinylated Affinity Purified Pab, Goat IgG (R&D

#BAF117)) was then added at 0.2 μ^πιΐ in PBS, seal with adhesive cover, and incubated for 2 hours at RT on a shaker followed by washing 3x with wash buffer. The samples were then incubated with 1:200 Streptavidin-HRP (R&D #DY998; or as indicated by vendor instructions) in PBS for 20 minutes at RT on a shaker (cover with foil to avoid exposure to light) followed by washing 3x with wash buffer. Substrate Solution (R&D #DY999) was then incubated for 20 minutes max at RT (avoid direct light exposure) followed by addition of stop solution (1M H₂S0₄). Plates were read at 450 nm within 30 minutes. Results:

[00076] As shown in Figure 1A, newborn human foreskin fibroblasts (NuFF-1) were infected at 0, 1 MOI with HCMV AD169-strain for a period of 9 days. Supernatants from each day were collected and analyzed via the cmvIL-10 ELISA described above and a commercial ML- 10 ELISA kit. As detailed in Figure IB, various cytokines were used to test the assay for cross-reactivity. In addition to the cellular cytokine ML- 10, a cytokine from a related herpesvirus, Epstein-Barr virus (ebvIL-10), and an unrelated cytokine, IFN-γ, were also tested. PBS served as a negative control. These results demonstrate that cmvIL-10 can be detected using ELISA in supernatants of HCMV-infected cells. The cmvIL-10 ELISA used in this Example is sensitive and specific for cmvIL-10, as none of the other cytokines tested (ML- 10, ebvIL-10, and IFN-γ) showed any cross-reactivity with our antibodies despite similarity between the IL-10 homologs.

Example 2: Both vIL-10 isoforms can be detected by ELISA in transfected cells

[00077] This Example shows that the ELISA described in Example 1 can be used to detect both cmvIL-10 and LAcmvIL-10 in transfected cells.

Materials and Methods:

[00078] Cell culture and trarasfectioB: Human embryonic kidney cells (HEK293) were cultured in DMEM media supplemented with 10% FBS. Cells were seeded at 2 x 10⁵ cells/well into 6-well dishes and after 24 hours the medium was removed and replaced with DMEM containing 1% FBS. Cells were transfected with a mixture of plasmid DNA and Fugene Transfection Reagent (Roche) in a ratio of 9:3 (ul Fugene: μg DNA) as recommended by the supplier. Cells were either mock transfected (no DNA), or transfected with pcDNA- cmvIL-10 or pcDNA-LA-cmvIL-10 as indicated. Aliquots (400 ul) of supernatants from transfected cells were collected daily for 5 days post-transfection for ELISA. Cells from duplicate transfected were lysed and collected for Western blotting as described below.

[00079] Western Blots: For western blotting, transfected cells were harvested into cell lysis buffer (150 mM NaCl, 20 mM HEPES, 0.5% Triton-X-100, 1 mM NaOV₄, 1 mM EDTA, 0.1% NaN₃). Lysates were clarified, proteins were separated via SDS-PAGE, and then transferred to a nitrocellulose membrane. Then membrane was incubated in blocking solution (5% milk + IX TBS-T) for 1 hr, then probed with primary antibody to cmvIL-10 or α-ΜΑΡΚ (control) at a 1 : 1000 in blocking solution overnight at 4°C. After washing, the membranes were incubated with a 1 :2000 dilution of appropriate AP-conjugated secondary antibody and bands were detected using Western Blue stabilized AP substrate (Promega, Madison, WI).

[00080] cmvIL-10 ELISA was performed as described above. Results:

[00081] Figure 2A depicts a schematic illustration of the two viral gene products of HCMV UL111 A. Figure 2B shows the results of a Western Blot for cmvIL-10

demonstrating that the antibody used is specific for both the full length cmvIL-10 protein as well as the truncated LAcmvIL-10 transcript. Figures 2C and 21) demonstrate that the ELISA described in Example 1 can accurately detect the presence of both cmvIL-10 and LAcmvIL-10.

Example 3: Use of vIL-10 ELISA with human blood specimens

[00082] This Example demonstrates that the cmvIL-10 ELISA described in Example 1, supra, can be used to detect cnivIL-lG in human blood specimens.

Materials and Methods:

[00083] Blood sample processing: Whole blood stored in EDTA tubes was obtained from a blood bank. Tubes were rotated for 10 minutes at room temperature (RT) on a 360° rotator/rotisserie to ensure proper mixing. After mixing, 400 μΐ of whole blood for 2 preps of genomic DNA preparation and PGR was collected. The remainder of the blood in the tube was then centrifuged at 1300 x g for 10 minutes at 4°C to obtain plasma (Figure 3A). As much plasma as possible was removed without disturbing buffy coat layer and red blood cells. Plasma was aliquoted into Eppendorf tubes (500 μΐ/tube) and assayed immediately for vIL-10 by ELISA and the remainder stored at -20°C for later use. The remainder of plasma + buffy coat + cells (2 ml.,) was collected and stored for isolation of mononuclear cells.

[00084] Mononuclear cell isolation and RNA extraction: Mononuclear cells were isolated using the Ficoll-Paque Premium protocol (GE Healthcare Bio-Sciences AB). RNA was extracted from cells immediately after isolation using the Qiagen RNeasy Midi Kit. RNA was stored at -80°C. [00085] Extracting genomic DNA (gDNA) from whole Mood; Genomic DNA was isolated from whole blood for PGR using the Promega ReliaPrep Blood gDNA Miniprep System (Part #TM330) based on the manufacturer's protocol. Two separate preps were typically performed for each blood sample. The blood sample was thoroughly mixed for 10 minutes (min) in a rotisserie shaker at room temperature (RT). About 20 μΐ of Proteinase (PK) was dispensed into a 1.5ml centrifuge tube followed by addition of 200 μΐ of whole blood which was briefly mixed. 200 μΐ of Cell Lysis Buffer (CLD) was added to the tube. The tube was then capped and mixed by vortexing for 10 seconds. The tube was then incubated at 56°C for 10 min. The contents of the tube were then added to the ReliaPrep Binding Column and placed in a microcentrifuge for lmin at max speed. The binding column was placed into a fresh collection tube and 500 ul of Column Wash Solution (CWD) was added to the column 3x followed by centrifugation for 3min at maximum speed. 50 μΐ of nuclease-free water was added to the column followed by centrifugation for 1 min at maximum speed to eiute the gDNA.

[00086] PCR OH genomic DNA (gDNA): HCMV viral load was determined by detecting IE1 in gDNA of donor blood. This protocol was based on the TaKaRa Ex Taq recommended reaction mixture (Cat# RR001A).

[00087] Primers utilized for PCR experiments in Examples 3 and 4 are shown in Table 1.

Table 1: Primer sequences

[00088] PGR used the inner IE1 primers (IE! F2 and IE1 R2) shown in Table 1 for each sample. The PGR reaction mixture included 0.50 uL ofTaKaRa ExTaq (5 units/ul), 5 μΐ ΙΟχ Ex Taq Buffer, 4 μΐ dNTP Mixture (2.5mM each), < 500 ng gDNA, 1 μ.Μ of each primer, and molecular biology grade water up to 50 ul. PGR reaction tubes were placed in a BioRad MyCycler Thermal Cycler and samples were run under the following PGR conditions: 95°C 5min; 35 cycles of 94°C 30s, 58°C 30s, 72°C 60s; 72°C 5min. PGR products were run on a 1 % agarose gel and visualized using ethidium bromide, a BioRad ChemiDic MP Imaging

System, and Image Lab 4.0 Software.

Results:

[00089] Figure 3B shows blood donors mapped by gender, age, ethnicity, and HCMV status. Figure 3C shows a standard curve created using 10% plasma in PBS as a diluent to show that plasma proteins did not interfere with the sensitivity and specificity of the assay . Plasma was harvested for quantification of viral cytokine levels using the cmvIL-10 ELLS A and results are shown as a comparison between HCMV status in Figure 3D while Figure 3E depicts plasma cmvIL-10 levels based on HCMV status and gender. As shown in Figure 3F, plasma cmvIL-10 detection is supported by the presence of the viral IE1 gene by PCR of whole blood gDNA. Also, IE1 is detected in both HCMV + and - donors. These results indicate that the ELISA test described in Example 1 can be used to accurately detect cmvIL- 10 in the blood of donors and that the gene encoding cmvIL-10 is found in individuals who are seronegative for HCMV infection based on the standard screening techniques used by blood banks.

Example 4: Detection of vIL-10 in a representative set of healthy blood donors.

[00090] This Example demonstrates that the ELISA test described in Example 1 can be used to detect cmvIL-10 in the blood of individuals who are seronegative for HCMV.

Materials and Methods:

[00091] cmvIL-10 ELISA is performed as described in Example 1.

[00092] Nested PCR on genomic DNA (gDNA) using IE! primers: HCMV viral load was determined by detecting IE1 in gDNA of donor blood. This protocol was based on the TaKaRa Ex Taq recommended reaction mixture (Cat# RR001 A). Primers utilized for nested PCR experiments are shown in Table 1, supra.

[00093] Two rounds of PCR were performed. The first round of PCR used the outer IE1 primers (IE1 Fl and IE1 Rl) and beta actin positive control primers for each sample. The second round of PCR used the first round of PCR as DNA template and the inner IE1 primers (IE1 F2 and IE1 R2). For the first round of PCR, the reaction mixture included 0.50 Ε ofTaKaRa ExTaq (5 units/ul), 5 μΐ ΙΟχ Ex Taq Buffer, 4 ul dNTP Mixture (2.5mM each), < 500 ng gDNA, 1 μΜ of each primer, and molecular biology grade water up to 50 μΐ. PCR reaction tubes were placed in a BioRad MyCycler Thermal Cycler and samples were run under the following PCR conditions: 95°C 5min; 35 cycles of 94°C 30s, 58°C 30s, 72°C 60s; 72°C 5min. PCR Round 1 products may be visualized on 1% agarose gel electrophoresis. For the second round of PCR, the reaction mixture included 0.25 μΕ ofTaKaRa ExTaq (5 units/ul), 2.5 μΐ ΙΟχ Ex Taq Buffer, 2 μΐ dNTP Mixture (2.5mM each), < 500 ng gDNA, 1 μΜ of each primer, and molecular biology grade water up to 25 μΐ. PCR reaction tubes were placed in a BioRad MyCycler Thermal Cycler and samples were run under the following PCR conditions: 95°C 5 mm; 30 cycles of 94°C 30s, 58°C 30s, 72°C 50s; 72°C 5min. PCR products were run on a 1% agarose gel and visualized using ethidium bromide, a BioRad ChemiDic MP Imaging System, and Image Lab 4.0 Software.

Results:

[00094] Forty eight (48) samples were used in the present Example. Representative results are depicted in Figure 4. Four blood samples were HCMV seronegative (S9-S 12) and four samples were HCMV seropositive (S 13-16). vIL-10 levels as determined by ELISA are shown in the bar graph in Figure 4 (top). vIL-10 was detected in two seronegative specimens. The seronegative donors with vIL-10 lacked any measurable IgG or IgM response to HCMV (Trinity Bioscience ELISA), but viral DNA could be detected by PCR. A nested PCR procedure was used to detect exon 4 of HCMV IE1 on genomic DNA isolated from the whole blood sample, and the top panel of Figure 4 (bottom) represents first round of PCR. The second round of PCR shown in the middle panel (bottom) of Figure 4 shows that two seronegative donors have viral DNA present in their blood, which correlates with detection of vIL-10 protein, β-actin as a control (Figure 4 (bottom, lower panel)). These results suggest that vIL-10 is produced at measurable levels in healthy adults and that seronegative donors may still harbor HCMV. [00095] In total, the results of the blood study described in this Example show that though only 54% (26/48) of the donors were HCMV positive, 71% (34/48) of donors had detectable levels of vIL-10. Nine seronegative donors showed high levels of vIL-10, six had low levels, and seven had no detectable vIL-10 levels. The seronegative donors lacked any measurable IgG or IgM response to HCMV. The results suggest that vIL-10 is produced at measurable levels in healthy adults and that seronegative donors may still harbor HCMV.

Example 5: Host cytokine levels in human blood

[00096] This example shows the correlation between cmvIL-10 and other human cytokines in donated blood.

Materials and Methods:

[00097] ELISA: cmvIL-10 ELISA was performed as described in Example 1, supra. ELISAs for ML- 10, IL-12, and TNFa were performed via commercially ELISA kits (R&D Systems) according to manufacturer's instructions.

Results:

[00098] The same specimens tested for vIL-10 levels in Example 4 were also tested for levels of three human cytokines: hIL-10, IL-12, and TNFa via commercially available ELISA kits. The top panels of Figure 5 show each levels of each cytokine in HCMV+ and HCMV- samples. The bottom panel shows relationship between levels of each cytokine with vIL-10 levels. Although there was a correlation between some samples with higher vIL- 10 levels and higher levels of the other cytokines, overall there was not strong linear correlation.

Claims

CLAIMS We claim:

1. A method for detecting human cytomegalovirus (HCMV) in a biological sample provided by an individual, the method comprising: detecting the presence of viral interleukin 10 (cmvIL-10) in the sample, wherein the individual is seronegative for HCMV.

2. The method of claim 1, wherein the sample is selected from the group consisting of a blood sample, a tissue sample, a urine sample, a saliva sample, a semen sample, a tear sample, or a breast milk sample.

3. The method of claim 1 or 2, wherein cmvIL-10 is detected by detecting a cmvIL - 10 nucleic acid in the sample.

4. The method of claim 3, wherein the cmvIL-10 nucleic acid is DNA.

5. The method of claim 4, wherein the cmvIL-10 DNA is detected by PCR or Southern Blotting.

6. The method of claim 3, wherein the cmvIL-10 nucleic acid is RNA.

7. The method of claim 6, wherein the cmvIL-10 RNA is detected by RT-PCR, Northern Blotting, in situ hybridization, microarray, or RNase protection assay.

8. The method of claim 1 or 2, wherein the cmvIL-10 is detected by detecting a cmvIL-10 protein in the sample.

9. The method of claim 8, wherein the cmvIL-10 protein is detected by Western Blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry,

immunoelectron microscopy, radioimmunoassay, Enzyme-Linked ImmunoSpot (ELISPOT) assay, 2D gel electrophoresis, or enzyme-linked immunosorbent assay (ELISA).

10. The method of claim 9, wherein the cmvIL-10 protein is detected by ELISA.

11. The method of claim 10, wherein the antibody used in the ELISA is a polyclonal antibody.

12. The method of claim 10, wherein the antibody used in the ELISA assay is a monoclonal antibody.

13. The method of any one of claims 1-13, wherein the individual is pregnant.

14. The method of any one of claims 1-13, wherein the individual is

immunocompromised.

15. The method of claim 14, wherein the individual is infected with human immunodeficiency virus (HIV).

16. The method of claim 14, wherein the individual is an organ transplant recipient.

17. The method of claim 14, wherein the individual is between 0 days and 2 years of age.

18. The method of any one of claims 1-13, wherein the individual is

immunocompetent.

19. The method of claim 18, wherein the individual has one or more conditions selected from the group consisting of cytomegalovirus (CMV) mononucleosis, posttransfusion CMV, arterial hypertension, and Guillain-Barre syndrome.

20. The method of any one of claims 1-13, wherein the individual is asymptomatic.

21. The method of any one of claim 1-13, wherein the individual is a pre-natal infant.

22. The method of any one of claims 1-21, wherein the individual is undergoing lytic infection with HCMV.

23. The method of any one of claims 1-21, wherein the individual is undergoing latent infection with HCMV.

24. The method of any one of claims 17-23, wherein the individual is an organ donor.

25. The method of any one of claims 1-24, wherein the cmvIL-10 is full length cmvIL-10.

26. The method of any one of claims 1-24, wherein the cmvIL-10 is truncated cmvIL-10 (LAcmvILlO).

27. The method of any one of claims 1-26, wherein the biological sample provided by the individual is donated blood.

28. The method of any one of claims 1-26, wherein the individual is a blood donor.

29. A kit for detecting human cytomegalovirus (HCMV) in a biological sample provided by an individual comprising: a) a probe for detecting the presence of viral interleukin 10 (cmvIL-10) in the sample; and b) one or more buffers and/or reagents, wherein the individual is seronegative for HCMV.

30. The kit of claim 29, wherein the probe is selected from the group consisting of a nucleic acid probe or an antibody.

31. The kit of claim 29 or 30, further comprising c) a secondary antibody.

32. The kit of any one of claims 29-31, wherein the antibody or the secondary antibody is conjugated to an enzyme.

33. The kit of any one of claims 29-32, further comprising d) a substrate.

34. The kit of any one of claims 29-33, the biological sample provided by the individual is donated blood.

35. The kit of any one of claims 29-33, wherein the individual is a blood donor.

36. The kit of any one of claims 29-35, wherein the kit comprises reagents necessary for performing an ELISA.

37. A method for detecting human cytomegalovirus (HCMV) in a biological sample provided by an individual, the method comprising: (a) contacting the biological sample comprising viral interleukin 10 (cmvIL-10) with a probe that specifically binds to a cmvIL-10 polypeptide or nucleic acid; and (b) detecting the presence of cmvIL-10 when a complex is formed between the probe and cmvIL-10 polypeptide or nucleic acid, wherein the individual is seronegative for HCMV.

38. The method of claim 37, wherein the probe comprises one or more nucleic acids.

39. The method of claim 38, wherein the one or more nucleic acids specifically hybridize to a nucleic acid of SEQ ID NO: l or SEQ ID NO: 2.

40. The method of any one of claims 37-39, wherein said one or more nucleic acids are PCR primers and PCR is performed subsequent to the complex forming between the PCR primers and the cmvIL-10 nucleic acid.

40. The method of any one of claims 37-39, wherein the one or more nucleic acids is detectably labeled.

41. The method of claim 37, wherein the probe comprises an antibody or fragment thereof.

42. The method of claim 41, wherein the antibody or fragment thereof is a monoclonal antibody.

43. The method of claim 41, wherein the antibody is a polyclonal antibody.

44. The method of claim 43, wherein the polyclonal antibody is produced using a recombinantly-produced cmvIL-10 polypeptide immunogen comprising A26 to K176 of SEQ ID NO:3.

45. The method of claim 43 or 44, wherein the polyclonal antibody is derived from goat.

46. The method of any one of claims 37-45, wherein the individual is pregnant.

47. The method of any one of claims 37-45, wherein the individual is

immunocompromised.

48. The method of claim 47, wherein the individual is infected with human immunodeficiency virus (HIV).

49. The method of claim 47, wherein the individual is an organ transplant recipient.

50. The method of any one of claims 37-45, wherein the individual is between 0 days and 2 years of age.

51. The method of any one of claims 37-45, wherein the individual is immunocompetent.

52. The method of claim 51, wherein the individual has one or more conditions selected from the group consisting of cytomegalovirus (CMV) mononucleosis, posttransfusion CMV, arterial hypertension, and Guillain-Barre syndrome.

53. The method of any one of claims 37-45, wherein the individual is asymptomatic.

54. The method of any one of claim 37-45, wherein the individual is a pre-natal infant.

55. The method of any one of claims 37-54, wherein the individual is undergoing lytic infection with HCMV.

56. The method of any one of claims 37-54, wherein the individual is undergoing latent infection with HCMV.

57. The method of any one of claims 37-45, wherein the individual is an organ donor.

58. The method of any one of claims 37-57, wherein the cmvIL-10 comprises full length cmvIL-10.

59. The method of any one of claims 37-57, wherein the cmvIL-10 comprises truncated cmvIL-10 (LAcmvILlO).

60. The method of any one of claims 37-59, wherein the biological sample provided by the individual comprises donated blood.

61. The method of any one of claims 37-59, wherein the individual is a blood donor.

62. A complex comprising (a) a probe and (b) a cmvILlO protein or nucleic acid, wherein the cmvILlO protein or nucleic acid is derived from a biological sample from an individual infected with human cytomegalovirus (HCMV) but has not undergone

seroconversion.

63. The complex of claim 62, wherein the probe comprises one or more nucleic acids.

64. The complex of claim 63, wherein the one or more nucleic acids specifically hybridize to a nucleic acid of SEQ ID NO: l or SEQ ID NO: 2.

65. The complex of any one of claims 62-64, wherein said one or more nucleic acids are PCR primers and PCR is performed subsequent to the complex forming between the PCR primers and the cmvIL-10 nucleic acid.

66. The complex of any one of claims 62-64, wherein the one or more nucleic acids is detectably labeled.

67. The complex of claim 62, wherein the probe comprises an antibody or fragment thereof.

68. The complex of claim 67, wherein the antibody or fragment thereof comprises a monoclonal antibody.

69. The complex of claim 67, wherein the antibody comprises a polyclonal antibody.

70. The complex of claim 69, wherein the polyclonal antibody is produced using a recombinantly-produced cmvIL-10 polypeptide immunogen comprising A26 to K176 of SEQ ID NO:3.

71. The complex of claim 69 or 70, wherein the polyclonal antibody is derived from goat.

72. The complex of any one of claims 62-70, wherein the individual is pregnant.

73. The complex of any one of claims 62-70, wherein the individual is

immunocompromised.

74. The complex of claim 73, wherein the individual is infected with human immunodeficiency virus (HIV).

75. The complex of claim 73, wherein the individual is an organ transplant recipient.

76. The complex of any one of claims 62-70, wherein the individual is between 0 days and 2 years of age.

77. The complex of any one of claims 62-70, wherein the individual is

immunocompetent.

78. The complex of claim 51, wherein the individual has one or more conditions selected from the group consisting of cytomegalovirus (CMV) mononucleosis, posttransfusion CMV, arterial hypertension, and Guillain-Barre syndrome.

79. The complex of any one of claims 62-70, wherein the individual is asymptomatic.

80. The complex of any one of claim 62-70, wherein the individual is a pre-natal infant.

81. The complex of any one of claims 62-70, wherein the individual is undergoing lytic infection with HCMV.

82. The complex of any one of claims 62-70, wherein the individual is undergoing latent infection with HCMV.

83. The complex of any one of claims 62-70, wherein the individual is an organ donor.

84. The complex of any one of claims 62-83, wherein the cmvIL-10 comprises full length cmvIL-10.

85. The complex of any one of claims 62-83, wherein the cmvIL-10 comprises truncated cmvIL-10 (LAcmvILlO).

86. The complex of any one of claims 62-85 wherein the biological sample provided by the individual comprises donated blood.

87. The complex of any one of claims 62-85, wherein the individual is a blood donor.