BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the discovery of a new human intracisternal retrovirus (HICRV) and to assays and therapy related to that virus.

Description of the Background

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily perceived as the same becomes understood by reference to the following detailed description when considered in connection with the accompany figures.

Human retroviruses are associated with several human diseases that involve disturbances of the growth of CD4 + T lymphocytes (Wong-Staal F. and R. Gallo, Nature 317: 395, 1985). Human T lymphotropic virus type I (HT V I) is associated with malignant expansion of CD4+ T cells (Poiesz B.J. et al. Proc. Natl. Acad. Sci.. USA. 77: 7415, 1980), whereas, human immunodeficiency virus (HIV-1 ) is associated with depletion of CD4+ T cells, resulting in the acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi F. et al. Science 220: 868, 1983; Popovic M. et al. Science 224: 497, 1984). HIV-1 acts by attacking lymphocytes and macrophages which display the CD4 antigen on their cellular surface. These cells are responsible for profound immunity against foreign invaders. HIV-1 defeats the hosts' ability to combat disease by killing these cell of the immune system. Retroviruses (including HIV-1 , HIV-2, HTLV I, HTLV II) act by carrying within their protein coat an RNA message that is transcribed into DNA by the viral protein reverse transcriptase. This viral DNA is then incorporated into the host DNA through homologous recombination and used as a template for future messenger RNA production. The protein sequences coded by the viral nucleotides instruct the cell to make more viral particles. And then, through means not fully understood, the virus causes lysis of the cell, releasing new viral particles into the body (Ho

D. D. et al. N. Eno. J. Med. 317: 278, 1987; Green W.C. N. Enα. J. Med. 324: 308, 1991 ).

One of the clinical hallmarks of HIV-1 is an extraordinary frequency of opportunistic infections, especially with Pneumocystis carinii. Pneumocystis carinii pneumonia (PCP) is the most common opportunistic infection in patients with AIDS (Fauci A.S. Science 239:617, 1988). Prior to the AIDS epidemic, PCP occurred primarily in immunocompromised patients, particularly patients with conditions such as lymphoma, leukemia, organ transplantation, hypogammaglobulinemia, or therapy with cytotoxic agents and corticosteroids. Rare cases of Pneumocystis carinii without predisposing conditions have been reported (Anderson CD. et at. Amer. J. Clin. Path. 71 : 156, 1961 and Lyons H.A, et al Arch. Int. Med. 108: 929. 1961 ). Using serological detection of Pneumocystis carinii by measuring circulating antibodies or antigen, PCP has also been reported in normal immunocompetent children (Pifer L.L. et al. Pediatrics. 61 (1 ): 35, 1978). More recently a number of patients with PCP and T cell deficiency, and in particular CD4+ T cell deficiency, have been described (Jacobs J.L. et al. N. Enα. J. Med. 324: 246, 1991 ; GautϊerV. et al. Clin. EXD. Allergy 21 : 63, 1991 ). All these patients were negative for HIV-1 infection.

SUMMARY OF THE INVENTION

One aspect of the invention provides cell lines in continuous culture infected with HICRV. Another aspect of the invention comprises substantially purified HICRV. Still another embodiment of the invention is monoclonal antibody against HICRV. The invention may include recombinant DNA, such as plasmid DNA, coding for HICRV viral protein.

The present invention also includes a method for determining the presence or absence of HICRV antibody or HICRV antigen in a biological sample, comprising the steps of contacting the biological sample with a reagent adapted to immunologically and preferentially bind with the antibody or antigen to form an immunological complex, and detecting the

presence or absence of the immunological complex. In one preferred embodiment of the assay method, the reagent is HICRV antigen. In another embodiment, the complex may be detected through a colorimetric change. Alternatively, the complex radioactive and the detection comprises identifying or measuring the radioactivity. Still another alternative involves detecting the complex through fluorescence. Also included within the scope of the present invention is a kit for detecting the presence or absence of HICRV antigen or anti-HICRV antibody in a sample, comprising means for forming an immunological complex with the HICRV antigen or antibody in the sample, and means for indicating the presence or absence of the complex. The indicating means can be a detectable label for labeling the complex. The means for forming the complex can be anti-HICRV antibody adapted to bind with HICRV antigen in the sample, or HICRV antigen adapted to bind with anti-HICRV antibody in the sample.

Brief Description of the Drawings

Other objects, advantages and features of the present invention will become apparent to those skilled in the art from the following discussion .

Figure 1 is a series of transmission electron micrographs of mononuclear cells from the patient (photographs A and B) and the daughter (C). Viral particles are indicated by arrows.

Figure 2 is a bar graph illustrating the results of a reverse transcriptase assay from supernatants of PHA stimulated, IL-2 activated patient mononuclear cells. Figure 3 illustrates the results from a PCR amplification of genomic

DNA using primers for HIV and HTLV tax gene regions. Amplified nucleic acid was separated by agarose gel eiectrophoresis and stained with ethidium bromide.

Figure 4 illustrates the results from a PCR amplification of genomic DNA using primers for HTLV I or HTLV II pol gene regions. Amplified nucleic acid was separated by agarose gel eiectrophoresis and stained

with ethidium bromide.

Figure 5 is a histogram illustrating the presence of anti-HICRV in the patients serum as compared with normal control serum.

Figure 6 is a Western Blot analysis of HICRV infected H9 and control cultures separated by one-dimensional SDS polyacrylamide gel eiectrophoresis. Arrows illustrate protein bands not found in control cultures.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to the discovery of a novel human intracisternal retrovirus (HICRV). The virus was initially isolated from a patient suffering with PCP and had no risk factors for HIV-1 infection.

Ultrastructurally, enzymatically, serologically and by polymerase chain reaction (PCR), HICRV is distinct from HIV-1 , HIV-2, HTLV I, HTLV II and the recently described A type intracisternal retroviral particle (Garry R.F. et al. Science 250: 1127, 1990. As with HI V-1 , CD4 + cells appear to be the primary target for HICRV infection. This observation is based on the severe quantitative and qualitative deficiency of CD4+ cells in the patient, along with in vitro transmissibility of the virus to CD4+ H9 cells

(Popovic M. et al. Science 224: 497, 1984). However, it is possible that cells lacking CD4 might also be the target of HICRV infection.

The patient reported here is different from any previously reported. This patient was asymptomatic at the time of diagnosis and has remained asymptomatic for 18 months following the diagnosis of PCP. No evidence of infection with HIV-1, HIV-2, HTLV I or HTLV II was observed as determined by serological, enzymatic, and PCR techniques. Furthermore, it has been demonstrated that the patient's cells are infected with HICRV. The patient had a blood transfusion in 1949-50 following bleeding from a spontaneous abortion. It is probable that the patient was infected with the virus early in life via blood transfusion and developed immunodeficiency over several decades.

HICRV poses a potential long term threat to the safety of the blood supply. It is apparently communicated between individuals through sexual or other close contact, by blood transfusions, and through placenta from the mother to the child. The extent of HICRV infection in the population remains to be determined; however, HICRV infection has been detected in the patient's own immediate family (the daughter, Example 5) and in the general population (as demonstrated by positive immunofluorescence antibody test for HICRV in samples obtained from the U.S. Centers for Disease Control, Example 9). All these patients had low CD4+ T cells and were negative for HIV-1 , HIV-2, HTLV I and HTLV

II infection. Interestingly, 3 of 4 patients tested positive had a history of blood transfusion.

The virus has been grown in continuous culture in H9 cells, as reported in Example 4. Cell lines in continuous culture infected with the virus comprise one aspect of the invention. Such cell lines are infected as in Example 4, and are maintained and expanded using techniques well known to those or ordinary skill in the HIV and other retrovirus arts. One particular HICRV-infected H9 cell line, identified as H9/200, (H9/HICRV) is maintained by the inventor, Dr. Gupta, in his laboratory at the University of California, Irvine. This cell line is available to the U.S.

Patent and Trademark Office during the pendency of this application and will be publicly available after issuance of the patent. If properly required by the Patent and Trademark Office, it will be deposited with a commercial depository contractually obligated to maintain the deposit for a length of time and under such conditions of public availability as are acceptable to the Patent and Trademark Office. A number of other cell lines are currently been used to propagate HICRV. These cell lines include: AA-2 (Chaffee S. et al. J. EXP. Med. 168: 605, 1988; CEM-SS (Foley G.E. et al. Cancer 18: 522, 1965; Nara P.L. et al. AIDS Res. Hum. Retroviruses 3: 283, 1987), HUT78 (Gazdar et al. Blood 55: 409,

1990), Sup-T1 (Smith S.D. et al. Cancer Res. 44: 5657, 1984), U937 (Ralph P. et al. J. Exp. Med. 143: 1528, 1976).

One aspect of the invention relates to immunoassays for detection of HICRV. One assay using serum from infected individuals is reported in Example 8. A Western Blot assay is also reported in Example 8. Monoclonal antibody and purified antigen based assays are also contemplated, as illustrated in Examples 11, 12, and 20. Of course, the exemplified assays are only illustrative. Polyclonal or monoclonal antibody against HICRV antigen, and purified viral antigen, can be used in radioimmunoassays (RIA), enzyme linked immunosorbent assays (ELISA), and immunofiuorescence assays, using those reagents in direct and indirect competitive assays, sandwich assays, flow cytometry or other conventional assays. These assays can be used to determine the presence of viral antigen or to detect antibodies against HICRV antigen. Also contemplated are DNA probes directed against the proviral DNA in infected cells, and reverse transcriptase assays. The cell free virus in the supernatants of HICRV infected cell lines activated with phorbol myristate acetate (to amplify virus production) may be purified, the nucleic acid can be isolated, and cloned and then be used in well known manners to determine the amino acid sequence of specific viral antigens, or to make nucleic acid probes to identify and/or isolate the RNA and proviral DNA of HICRV, and ultimately to use those viral genes to make cDNA piasmid constructs which can express viral proteins in vitro.

The following Examples are illustrative of certain embodiments of the invention; however, the invention is not to be limited only to these specific embodiments. Instead, it will be recognized that numerous other related aspects of the invention can be practiced by those skilled in the art based on the disclosure herein. It is intended that all such aspects of the invention be covered by this patent. It should be particularly noted that modern biotechnology provides conventional techniques which, although time consuming, can with confidence provide monoclonal antibodies against HICRV, isolate whole viral particles, purified viral proteins or antigens, recombinant DNA or RNA coding for viral proteins,

organisms expressing those recombinant genes, assays using antigen, antibody, and DNA or RNA probes to determine the presence of HICRV or HICRV infection. It is to those already skilled in these modern immunology and molecular genetics techniques that the present disclosure is directed.

Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein for purposes of illustration only.

Example 1 : Lymphocyte Subsets Peripheral blood lymphocyte subsets were analyzed with monoclonal antibodies, anti-CD2 (Leu 5), anti-CD3 (Leu 4), anti-CD4 (Leu 3), anti-CD8 (Leu 2), anti-CD25 (IL-2 receptor), and anti-CD20 (Leu 16) using FACScan (Becton Dickinson, San Jose, CA). Analysis was done on whole blood samples using lysing solution. Mononuclear cells were mixed with appropriate dilutions of monoclonal antibodies for 30 minutes at 4°C, washed three times with phosphate buffer saline (PBS) and resuspended in PBS. Mouse Ig of the same isotypes as that of monoclonal antibodies were used as controls. Ten thousand cells were analyzed using FACScan. Data are presented for both percentage and the absolute number of subsets in Table 1 below.

Table 1 : Immunological Analysis of the Patient

TESTS PATIENT CONTROLS % (Numbers) % (Numbers)

Lymphocyte Subsets:

CD2 + T cells 61 (1041 ) 65-85 (1467-3003) CD3 + T cells 52 (857) 59-76 (1277-2929) CD4+ T cells 10 (1 71 ) 32-47 (782-1 626) CD8+ T cells 50 (700) 24-36 (463-1369) CD4 + /CD8 + ratio 0.2 0.9-2.1 CD20+ B cells 17 (290) 4.6-16.8 (163-41 1 ) CD25 + T cells 72 54-78

The data presented show a marked decrease in proportions and numbers of CD4+ and increase in the proportions and numbers of CD8+ T cells in the patient as compared with the controls. These findings demonstrate severe CD4+ T cell deficiency in this patient. CD25 antigen expression was measured on mononuclear cells activated with PHA for three days and the cells expressing CD25 antigen in the patient were comparable to controls. Unstimulated cells served as controls.

Example 2: Lymphocyte DNA Synthesis Mononuclear cells isolated on a Ficoll-Hypaque density gradient were stimulated with soluble antigens and mitogens in 96 well microtiter plates. One hundred thousand cells/well (in triplicates) were incubated with optimum concentrations of soluble antigens (mumps, Candid albicans, tetanus toxoid, and PPD) and mitogens (phytohemagglutinin, concanavalin A and pokeweek mitogen) for 5 and 3 days respectively. Cells were pulsed with 1 mCi/well of ³ H thymidine and cultured for the final 18 hours. Cells were harvested and ³ H thymidine incorporation was measured using a scintillation counter. Data shown in Table 2 below are expressed as mean net counts per minute of triplicate cultures. Table 2: DNA S n he is C un s Per Minute

These data show an inability of the patient's immune system to respond to antigen and mitogen stimulation, further demonstrating the ineffectiveness of this patient's T-cells.

Example 3: Serological Tests Serum immunoglobulins were measured by nephelometry. Serum

HIV-1 antibodies were measured by ELISA (DuPont) and Western blot, and P24 antigen by antigen capture ELISA technique (DuPont). Antibodies against HTLV I and HTLV II were examined by immunofluorescence assay (Dr. Jay Levy, University of California, San Francisco) and no activity was observed. All of these test showed no cross activity between HICRV and HIV-1 , HTLV I or HTLV II, indicating that HICRV is distinct from these viruses.

Example 4: Co-Culture of Patient's Cells With H9 Cell

Mononuclear cells (2 X 10°7ml) in 12 X 75 mm falcon test tubes were suspended in medium containing 10% fetal calf serum and antibodies, and cultured with 10mg/ml of PHA for 72 hours. Medium was changed and cells were cultured in the presence of 10 unit/ml of recombinant interleukin 2 (AMGEM, Thousand Oaks, CA). At day 7, cells were co-cultured with H9 cells at a ratio of 1 :3 (patient : H9 cells), and culture medium (RPMI-1640 and 10% fetal calf serum) was changed every 3 days. After 3 weeks in culture, cells were analyzed for virus particles by electron microscopy. Supernatants of donor cells activated with PHA and IL-2 and of co-cultures with H9 cells were analyzed for precipitable reverse transcriptase (RT) activity in the presence of divalent cations Mn ^{+ +} or Mg ^{+ +} , using dTrA and dGrC as synthetic primers (Salahuddin, S.Z. et al, Proc. Natl. Acad. Sci. USA 82: 5530, 1985). In addition, infected cells were used for screening of patient's serum for the presence of antibody against the virus, using immunofluorescence and Western blot techniques.

Example 5: Electron Microscopic Studies

Co-cultures of H9 cells and the patient's cells were pelleted, fixed in cold 2.5% glutaraldehyde in 0.12 M phosphate buffer (pH. 7.4) for 30 min. and rinsed in buffer priorto postfixation in 1 % osmium tetroxide for 30 minutes. The pellets were embedded in 4% bact-agar solution to hold them together during processing. Blocks were made using a razor blade and dehydrated with ethyl alcohol and propylene oxide before being embedded in Medcast. Ultrathin sections of these embedded blocks were cut at a thickness of 50-60 nm and examined with a Phillips CM 10 electron microscope. AH the intact cells showed virus particles as seen in Figures 1A - 1C. Intracisternal viral particles were regularly seen in the cytoplasmic vesicles of the H9 cells (Figure 1A and IB [higher magnification]. The HICRV particles appeared uniform, approximately 80 nm in diameter with two concentric rings of electron dense material and a clear center. Twenty to thirty particles in each cell were seen per this section. In addition, similar viral particles were seen H9 cells co-cultured with the mononuclear cells taken from the patient's daughter (Figure 1C).

Example 6: Reverse Transcriptase activity Culture supernatants from the patient's mononuclear cells stimulated for 3 days with 10mg/ml PHA plus 10U/ml rL-2 were examined for precipitable reverse transcriptase (RT) activity in the presence or absence of divalent cations Mg ⁺⁺ or Mn ⁺⁺ , using dTrA and dGrC as synthetic primers. Supernatants were centrifuged at 10,000 x g for 15 minutes and treated with polyethylene glycol 8000 (30% W/V with 0.4 M NaCl). The mixture was maintained overnight at 0°C. All specimen were centrifuged at 40,000 x g for 45 minutes at 4 ^β C. Supernatants were discarded and pellets were resuspended in 100ml of buffer containing Triton 100. Specimens were frozen at -70°C and analyzed simultaneously. Data are expressed as counts per minute. Background counts were <1 ,000 cpm. Figure 2 shows a significant RT

activity that was predominantly Mn ^{+ +} -dependent. Columns 1- and 2 with Mn ^{+ +} and Mg ^{+ +} respectively, and dGrC and dTrA as template primers (columns 3 and 4 respectively).

Example 7: DNA Primer Analysis Although patient's serum was negative for antibodies against

HIV-1 , HTLV-I, and HTLV-II, and for P24 antigens, in order to definitively demonstrate that there was not HIV or HTLV infection, genomic DNA was isolated from (the patient's) PHA stimulated and IL-2 activated mononuclear cells, HIV-1 infected and HTLV I infected MT-2 cell lines, and examined for HIV and HTLV proviruses by polymerase chain reaction, using S 145 and SK 431 primers to amplify both HIV-1 and HIV-2 provirus (gag), SK 1 10 and SK 1 1 1 primers to amplify HTLV-I and HTLV-II provirus (pol), and SK 43 and SK 44 to amplify HTLV I and HTLV II provirus (tax). Figure 3A shows lack of HIV-1 or HIV-2 (gag) proviruses in mononuclear cells from patient (200), healthy normal control or with MT-2/HTLV-1 but a positive band of with U1 /HIV-1 . Figure 3B shows a lack of HTLV I and HTLV II (tax) and Figure 4 shows a lack of HTLV I or HTLV II (pol) proviruses in control mononuclear cells, patient's cells (200) or U1 /HIV-1 . A positive reaction was however, seen with MT-2/HTLV 1. Polymerase chain reaction data and serological data strongly suggest that CD4+ T cell deficiency in the present patient is not due to infection with HIV-1 , HIV-2, HTLV-I or HTLV-II. Lack of HIV-1 provirus in the patient's mononuclear cells and negative serology for HIV-1 distinguishes HICRV from A-type intracisternal retroviral particle.

Example 8:

(a) Detection of serum antibodies against HICRV Serum antibodies against the virus were detected by immunofluorescence and Western Blot assays.

(b) Immunofluorescence Assay

A modification of the technique described for HIV-1 P24 antigen was used (Ohlsson-Wilheϊm, B.M. et at. J. Infect. Pis.. 162: 1018, 1990). In brief, 2 X 10 ⁶ HICRV-infected H9 cells (H9/HICRV, as determined by electron microscopy and reverse transcriptase activity) were fixed in medium containing 1.5% formaldehyde for 10 minutes at room temperature. Cells were centrifuged and pellets were resuspended in PBS containing 0.5% Triton X-100 (Sigma Chemicals, St. Louis, MO) and incubated for 3 minutes at 4°C. Cells were washed with cold PBS three times and incubated with pooled human AB serum for 30 minutes at 4°C to block all sites for non-specific binding to human serum immunoglobulins or to FITC-conjugated goat IgG anti-human immunoglobulin used for counterstaining. After incubation cells were washed and incubated with normal serum (as control) and patient's serum (1 :2 dilution) for 30 minutes at 4°C. Cells were washed with PBS and counterstained with FITC-conjugated goat IgG (Fab') ² anti-human immunoglobulin for 30 minutes at 4°C. Cells were washed three times with PBS, resuspended, and examined for positive fluorescence, using FACScan (Becton-Dickinson, San Jose, CA). Figure 5 show a positive reaction with the patient serum. No reaction was observed with normal serum. Now we have made further modification in this technique. Instead fixation of cells with formaldehyde and treatment with Triton X 100, the cells are fixed with 70% methanol at -20°C for 10 minutes. The remaining protocol remains the same.

(b) Western Blot Analysis

In addition to the immunofluorescence assay, we assayed for the presence of antibodies to HICRV by Western blot analysis. Five million H9 and H9/HICRV cells were lysed in 500ml of a lysing buffer and nuclei were removed by microcentrifugation for 1 hour at 4°C. One hundred microliters of supernatants were mixed with equal volume of sample loading buffer, and 20ml of the sample was applied to 10% SDS-PAGE.

Separated proteins were transferred to nitrocellulose paper, using Novex transfer apparatus (Encinitas, CA). A Western blot was probed with normal serum or the patient's serum (1 :10 dilution) preabsorbed with mononuclear cells from healthy donor. Protein bound to serum antibodies was visualized with alkaline phosphatase-conjugated goat anti-human Ig and a Blot detection kit (Amersham, Arlington Heights, IL.). Figure 6A shows two (2) distinct bands of approximately 24kd in H9/200 (H9/HICRV) and not in H9 cells. Normal serum showed no bands at 24kd either in H9 or H9/200 cells (Figure 6B). This further confirmed the presence of antibodies against HICRV.

Example 9: HICRV Infection exists in the general population

Fifteen blind serum samples were obtained from the United Stated Centers for Disease Control. Five of these samples were from individuals who were HIV negative and who exhibited low CD4 values. Serum from three of those five individuals tested positive for HICRV in the assay of

Example 6, as did serum from the wife of one of those three individuals (Table 1 ). The HIV-1 negative heterosexual and homosexual controls tested negative, as did the HIV-1 and HTLV-1 positive controls. When analyzed by Fisher's exact test for significance, the p value is .027 (after excluding the wife of the HICRV positive individual).

These results indicated that HICRV is indigenous in the general population and that it can be transmitted from infected individuals to other family members. Furthermore, HICRV can produce CD4 + T cell dysfunction/deficiency.

Example 10: Monoclonal antibodies against HICRV

The lysates of HICRV infected cell lines and cell free virus are used to immunize mice. Subsequently, the mice are sacrificed and homogenized spleens from those animals are used to prepare hybridomas

(Coligan J.E. et al. Current Protocols in Immunology, John Wiley Press, London, 1991 ). Primary hybridoma supernatants are screened to

examine which wells contains hybridomas that secrete antibodies against HICRV, using ELISA or immunofluorescence assays. After candidate hybridomas are identified, they are expended and fed, then cells are both frozen and cloned by limiting dilution. Large scale monoclonal antibodies are produced in culture supernatants and as ascites fluid (Coligan J.E. et al. Current Protocols in Immunology, pp. 2.5, 1991 ). The monoclonal antibodies are purified by ammonium sulfate precipitation and size exclusion chromatography followed by DE52 ion exchange chromatography (Colligan et al. Current Protocols in Immunology pp. 2.71 , 1991 ). All column fractions are retained until SDS-PAGE and antibody activity with ELISA and immunofluorescence assays have been done. Antibodies are also conjugated with various fluorochromes for direct immunofluorescence assay.

Example 11: (a) ELISA for HICRV

Monoclonal antibodies produced in accordance with Example 10 is adsorbed to a nylon membrane. Bove serum albumin (0.1 % in phosphate buffer saline [PBS]) is then added to block nonspecific binding sites on the membrane. After washing and drying, the membrane is contacted with a sample suspected of containing HICRV antigen. The membrane is washed with PBS and contacted with monoclonal antibody from Example 10 which has been conjugated with horseradish peroxϊdase or alkaline phosphatase enzyme. Following another washing step using PBS, the membrane is then incubated with a substrate solution, 0.4 mM 2,2 ^r -azo-di(3-ethyl- benzothiazoline sulfonate)ϊn 0.05 M citric acid (pH 4.0) containing 0.3% H ₂ O ₂ (ABTS, Kirkegaard and Perry, Gaithesburg, MD). The color produced is read by a ELISA plate reader.

(b) POLYMERASE CHAIN REACTION (PCR) HICRV provirus in the mononuclear cells is detected by PCR.

Genomic DNA from the mononuclear cells from subject in question and HICRV infected cell lines is isolated by the proteinase K/phenol extraction method (Strauss W.M. in: Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York pp. 2.2.1 , 1989). DNA from normal cells is used as a negative control. 0.5mg of each DNA is used as template for PCR. Primers are designed to amplify the pol, the gag and the tax regions of HICRV are used. PCR is done with a thermal cycler at 35-45 cycles for 1 to 3 minutes. Amplified DNA is confirmed by Southern hybridization analysis with probes.

Example 12: Immunofluorescence assay for HICRV

This method is utilized for the detection of HICRV in the biological sample of cells by direct or indirect immunofluorescence assay. Cells are incubated with unconjugated or direct fluorochrome conjugated (Example 10) monoclonal antibodies against various viral antigens. Cells are washed with PBS and in the case of unconjugated antibodies, are counterstained with FITC/PE/TR-conjugated anti-mouse Ig. After 30 minutes of incubation, cells are washed with PBS and examined for positive fluorescence, using microscope to flow cytometry. Cells from healthy subjects and HICRV infected cells serve as negative and positive controls. Mouse Ig of the same isotypes as that of the monoclonal antibodies are used as negative controls.

Example 13: Purification of HICRV

Virus-containing supernatants from HICRV infected cell lines are passed through 0.45m filter. Supernatants are centrifuged at 17,000 rpm for 3 hours at 4°C to pellet virions. Viral pellets are resuspended in a total volume of 1 ml TNE buffer and kept overnight at 4°C. Virus is further purified by column chromatography, using 1 10 ml column of sepharose 4B at room temperature with TNE as the column buffer. The eluted virus is placed in microcentrifuge tubes and pelleted at full speed for 1 hour and 15 minutes in a microcentrifuge at 4°C. Supernatants are

removed and virus pellets are resuspended in 150ml of buffer, such as JD.5% Triton X-100 in CMF-PBS. The virus is approximately x 10,000 concentrated and can be used for ELISA or Western blotting.

Example 14: Immunochromatographic isolation of viral antigens:

Monoclonal antibodies from Example 10 are bound to immunochromatography column. Virus from supernatants of HICRV infected cell lines (Example 4) are purified by sepharose 4B column chromatography, are directed through monoclonal antibody affinity column. Viral antigens are eluted with Glycine/HCI buffer (0.1 M adjusted to pH 2.5 with 0.2M HCI).

Example 15: Sequencing of Purified Viral Antigens

The amino acid sequence of viral antigen protein is determined using repeated cycles of the Edman degradation reactors as described (G. Allen. In "Seouencing of Protein and Peptides" [T.S. Work and R.H. Burdon, edsJ, Amsterdam, New York: Elsevier, 1981 ). Each degradation cycle consists of three steps. The N-terminal amino acid is modified with phenyl isothiocyanate, cleaved from protein, and finally modified amino acid will be determined by an automatic amino acid sequencer.

Example 16: HICRV probe preparation

HICRV probe is prepared for screening a genomic library by three different methods. [1] Genomic RNA is isolated from purified ^' viral particles and used as a template to synthesize cDNA probe for RT. It is possible that purified RNA is lost during cDNA synthesis, this method may not be practical for repeated preparation of cDNA probe. [2] Alternatively, double stranded cDNA is synthesized, using purified viral RNA as template and commercially available cDNA kit (Invitrogen, San Diego, CA). The cDNA is digested with restriction endonucleases. The

restricted DNA fragments are cloned in E. coli, using pUC plasmids and will be used as probes. [3] If Southern hybridization analysis shows any restriction fragment(s) of proviral DNA homologous to HIV or HTLV probes, DNA fragments are cloned in E. coli, using plasmid pUC plasmid. [4] The amino acid sequence of viral antigen protein is used as a basis of oligonucleotide probe synthesis.

Example 17: Preparation and screening of provirus libraries

To clone viral antigen gene, a proviral DNA library and a genomic library of virus infected cells are constructed. Unintegrated proviral DNA is isolated from the cytoplasm of virus-infected cells and chromosomal

DNA is isolated from virus-infected cells (Hirt B. J. Mol. Biol. 26: 365, 1967) . Expression libraries are made with lambda bacteriophage vectors (Maniatis T. et al. Molecular Cloning. A Laboratory Manual, pp.270, 1987. Cold Spring Harbor Lab. New York), including lambda gt 1 1 . The libraries are screened with monoclonal antibodies raised against viral antigens and viral DNA probes. The clones expressing viral antigens will be isolated and viral antigen genes will be subcloned in M13-derivative plasmids for DNA sequencing.

Example 18: Expression of viral antigen genes The genes encoding viral antigens are cloned in both prokaryotic and eukaryotic expression plasmids, and expressed in E. coli and eukaryotic cell lines. We will stably transfect DNA including viral gene expression system into eukaryotic cell lines (Ausubel F.M. et al. (eds.) in: Current Protocols in Molecular Biology, pp. 16.0.1 , 1989. John Wiley & Sons, Inc. New York). Viral antigen gene is fused to a strong partner of bacteriophages (e.g. T3, T7, lambda etc.) and E.coli (LacZ, trpE etc.) and transformed into E. coli strains for expression. In addition, viral antigen gene is expressed using a eukaryotic expression system, such as the Baculoviral expression system and the vaccinia virus expression system. Alternatively, fusion plasmids of constitutive eukaryotic

promotor and viral antigen gene are made and transfected to mammalian cell lines, including COS and CHO, for viral antigen expression.

Example 19: Purification of recombinant viral antigens

Expressed viral antigens are purified by an affinity chromatography, using CNBR-activated sepharose 4B column conjugated with monoclonal antibodies against viral antigens. Cell extracts containing expressed viral antigens are applied to the affinity column. Viral antigens are bound to the column and the remaining cell components are washed with PBS. Viral antigens are eluted from the column by glycine/HCI buffer (0.1M adjusted to pH 2.5 with 0.2M HCI).

Example 20:

(a) Assays for anti-HICRV antibodies

Several assays are employed for the measurements of anti-HICRV antibodies in biological fluid samples including the following.

(b) Immunofluorescence assays

Purified viral antigens from Example 19 are immobilized on a solid styrene support. The antigen-labeled support is incubated with test sample from subject suspected of HICRV infection, washed with PBS and incubated with FITC-conjugated goat-anti-human IgG. Complexes are washed and anti-HICRV antibody is measured by fluorescence of the labeled antibody remaining on the support.

(c) Western blot analysis

Supernatants of cell lysates of normal and HICRV infected cells or purified antigen are mixed with sample loading buffer, and 20ml sample is applied to 10% SDS-PAGE. Separated proteins are transferred to nitrocellulose paper. A Western blot is probed with normal serum or patient's serum preabsorbed with normal mononuclear cells. Proteins

bound to serum antibodies are visualized with alkaline phosphate-conjugated goat-anti-human Ig and a Blot detection kit.

(d) Radioimmunoprecipitation assay (RIPA)

The RIPA is carried out in five steps (Aldovini A. and Walker B.D. in: Techniques in HIV Research (Stockton Press), New York, pp. 1 1 ,

1990). These are [1 ] metabolic labelling of the virus infected cell lines with ³⁵ S-Iabeled cysteine, ³⁵ S-labeled methionine or ³ H-labeled leucine, [2] lysis of the cells and solubilizing the proteins, [3] addition of lysate to test sample antibodies bound to protein A and subsequent formation of antigen-antibody complexes, [4] washing of the complexes to remove unbound antigen, and [5] resolution of the radioactively labeled antigen recognized by serum antibodies through polyacrylamide gel eiectrophoresis.

(e) ELISA Assay HICRV purified virus from Example 13 is lysed in PBS with 10% non-idet P-4 (Sigma, St. Louis, MO.) Dilutions of virus are prepared in PBS. Dilutions of antigen (1 :200 to 1 :2000) are bound to microtiter wells by incubation at 4°C overnight. After washing with PBS, nonspecific binding sites are blocked with 1 % Bovine Serum Albumen (BSA) and 2% human control serum in PBS for 1 hour at 37°C. Blocking is followed by successive washes of PBS Tween 20. Samples of patient serum, H9/HICRV supernatant (positive control), and uninfected H9 supernatant (negative control) are added to the wells. After incubation at 4°C overnight the wells are washed and horseradish peroxidase conjugated goat anti-human antibody is added to each sample. Orthophenylenedimine (OPD) and peroxide is used to illuminate the conjugates. The absorbance of each well is measured at 490nm with a microplate reader. Alternatively, purified viral antigen from Example 19 is used in place of the viral proteins explained above.

Deposit of Cells

The cell line containing HICRV was deposited under the Budapest Treaty on September 11 , 1992 with the ATCC and it was ad warded ATCC Accession CRL No. 11121.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.