WO2001032206A1

WO2001032206A1 - Detection of viral pathology mediated by lymphocytes

Info

Publication number: WO2001032206A1
Application number: PCT/US2000/030575
Authority: WO
Inventors: Mark Allegretta; Richard Albertini
Original assignee: Biomosaics, Inc.
Priority date: 1999-11-01
Filing date: 2000-10-31
Publication date: 2001-05-10
Also published as: AU1470101A

Abstract

The present invention provides a method for diagnosing and/or predicting the development of pathology in a mammal, mediated by viral infection, by isolating lymphocytes bearing a selectable marker and identifying among the lymphocytes a population of infected cells. The relative frequency of the mutant cells in the population from a sample can be determined by directly plating cells in the presence of a selection medium. In one embodiment of the invention, the selectable marker is hypoxanthine-guanine phosphoribosyltransferase (hprt).

Description

Detection of Viral Pathology Mediated by Lymphocytes

Field of the Invention

The field of this invention is the area of viral pathology and the diagnosis and/or prediction of autoimmunity or cancer as a result of virally infected cells. Specifically, the present invention provides a method for the diagnosis and/or prediction of the expansion of a population of virally infected cells capable of mediating pathology, specifically adult T-cell leukemia/lymphoma.

Background of the Invention

The etiologic agent of adult T-cell leukemiallymphoma (ATL) has been identified as human T-cell lymphotropic virus type 1 (FITLy-I). The virus is endemic throughout Japan, with certain regions of the country having elevated prevalence rates. For example, in the Nagasaki Prefecture, (population: 1.5 million), prevalence of HTLV-1 carriers is approximately 10% in the age group over 40 years old. The national average is approximately 1%. Because such a large number of people are carriers of this potentially devastating virus, developing an effective measure to control the endemic cycle of HTLV-1 has been imperative. This is especially important since practical ways to prevent or control ATL are not available. Recently an HTLV-1 associated myelopathy causing paralysis called tropical spastic paraparesis (HAM/TSP) has also been associated with HTLV-1 infection. Methods for detection of HTLV- 1 infection, in general, measure exposure to the virus by detecting and quantifying antibodies to HTLV-1 antigens in blood, sera, and blood-derived products. Such assays can be used to aid diagnosis of ATL and HAM/TSP and to screen blood and blood products for previous exposure to HTLV-1, but are not useful for determining at what time a given infected individual will progress to a pathological condition.

The critical information needed to institute preventative therapy for pathology mediated by HTLV-1 is the ability to reliably identify those individuals with HTLV-1 that will develop disease, and to do so before the disease has had sufficient time to progress to a point where therapy is no longer effective. HTLV-1 carriers who have incorporated the viral genome in a specific manner in the peripheral blood cells can be identified with routine molecular tests. This condition is designated pre-ATL, and accounts for about 2% of healthy carriers of the virus. These people are at extremely high risk of developing ATL. Their ages tend to range from 32 to 80 (median 57). Many cases of pre-ATL show a long-lasting carrier state (10-year probability around 90%).

It would be useful to have a method of detecting lymphocyte amplification and viral infection. Repeated divisions of cells derived from a single parent lymphocyte result in numerous identical cells or clones. That these lymphocyte clones are derived from a common parental cell is evidenced by the commonality of their specific antigen receptors, antibodies in B-cells and T-cells receptors (TCRs) in T-cells. The amino acid sequence of a TCR protein, for example, renders it specific to a particular and limited array of antigens. In any individual there are a vast variety of TCR types estimated at 10⁶ and 10⁷. The variety of TCR specifities results from rearrangements of the regions of the nucleic acid encoding the TCR proteins. The genes encoding the TCR proteins comprise several different regions, including those termed V, C, D and J. During maturation in the thymus, each T-lymphocyte differentiates to synthesize only a single molecular species of TCR. It is believed that translocations or rearrangements of the deoxyribonucleic acid (DNA) of the V, C, D and J regions is the mechanism by which a T-lymphocyte is committed to expressing a single TCR.

Somatic gene mutations result when there is an alteration of the nucleotide sequence of a gene encoding a protein or protein fragment. Such change may include a substitution of one nucleotide for another, or the insertion or deletion of one or more nucleotides, resulting in a change in the corresponding amino acid sequence of the encoded peptide. Somatic gene mutations are distributed largely stochastically in that, with some exceptions, they are distributed at random among the genes of the genome. However, mutations are more likely to occur in cells that are undergoing mitosis, or cell division, because they often result from inaccurate DNA replication. Thus, a cell lineage that has undergone repeated divisions, as is the case in amplification of virally infected T-cells, has a higher probability of accumulating somatic gene mutations.

Certain somatic gene mutations occur and are detectable in lymphocytes. For example, 6-thioguanine resistant (TGR) T-lymphocytes result from mutation of the gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT, hprt gene). These mutations can be detected by one of two methods, a short term autoradiographic assay and a more definitive clonal assay. Both are presently used for human mutagenicity monitoring. The clonal assay involves the isolation of in vivo-derived hprt mutant cells and their in vitro propagation for full in vitro characterization. Such human somatic mutant cells have been characterized and shown to maintain the TG^R phenotype in vitro in the absence of selection, and to be deficient in HPRT activity. Furthermore, in vivo-derived mutants show hprt gene structural alterations as defined by Southern blots probed with a cDNA hprt probe, or DNA sequence analysis. Both assays are based on the fact that cells exhibiting the mutation termed hprt⁺ are able to survive in the presence of 6-thioguanine, whereas wild-type cells having the hprt⁺ genotype are killed.

TCR gene rearrangement patterns can also be defined for wild type and hprt mutant T-cell clones isolated in clonal assays. As expected on the basis of the large repertoire of possible TCR patterns most independently isolated wild-type clones show unique TCR gene rearrangement patterns when studied by Southern blots with oligonucleotide probes for the alpha, beta or gamma TCR genes. However, studies of TCR gene rearrangement patterns among spontaneously arising hprt⁺ mutant colonies show that clonal amplification of varying degrees has often occurred in vivo for clones that produce the mutants. Spontaneous somatic gene mutation in vivo in human T-lymphocytes appears to occur preferentially in those T-lymphocytes that are either actively dividing in vivo or have recently undergone division in vivo. In either case, mutation appears to mark cells derived from clones that are undergoing clonal amplification. By directing attention to only the mutant traction of lymphocytes obtained from a sample, which should be representative of the total in vivo T-lymphocyte population, the present method permits more effective identification of the small minority subpopulation of cells that are undergoing expansion in vivo. By first isolating that fraction of the sample lymphocytes which exhibit somatic mutations, the subfraction exhibiting expansion may be more readily detected. When correlated with clinical or other information such as a molecular test for the presence of integrated virus, this method serves to identify cells that are of some biologic or pathologic importance to the host, or are at greater risk of leukemic transformation.

A clonal assay to define hprt⁺ gene mutants in lymphocytes would therefore be useful in providing a method to identify, study and produce cells that are representatives of clones that are expanded in vivo and are of pathologic relevance to the host. It would be useful to identify when people with HTLV-1 will develop pathology and an assay for HTLV-1 mediated pathology would be particularly useful because the virus can be readily transferred from the peripheral blood leukocytes of antibody-positive people to leukocytes of antibody negative people when the two are cultivated together. Consequently, it appears that there is great risk of infection involved in whole blood transfusions when the transfused blood contains infected cells.

Objects and Summary of the Invention

It is therefore an object of the invention to provide a method of identifying cells that are biologically or pathologically important to the host.

It is another object of the invention to provide a method to identify, study and produce cells that are representative of clones that are expanded in vivo and represent pathologic indicators.

It is yet another object of the present invention to provide an assay for HTLV-1 mediated pathology.

It is still another object of the present invention to provide a minimally invasive method for the diagnosis and/or prediction of viral pathology in individuals infected with viruses such as the human T-lymphotropic virus type 1.

These and other objects of the invention are obtained by providing a method that includes the step of obtaining a sample of blood or tissue; culturing an aliquot of cells from the sample in a medium which is selective for cells which lack a functional hypoxanthine-20 guanine phosphoribosyltransferase; culturing a second aliquot of the cells from the sample in a medium which allows enumeration of total clonable cells; and, determining a frequency of cells lacking a functional hypoxanthine-guanine phosphoribosyltransferase, whereby a viral pathology is predicted when the frequency of cells lacking hypoxanthine-guanine phosphoribosyltransferase is greater than 6 per 106 peripheral blood mononuclear cells.

Brief Description of the Drawings

Figure 1 is a photograph of an electrophoresis gel showing HTLV-1 infection in wild type and mutant T-cells.

Figure 2(a), (b) and (c) is a photograph of electrophoresis gels showing T-cell isolates.

Figure 3 is a graphic representation of results obtained on wild type and mutant isolates from patients.

Detailed Description of the Invention

In a preferred embodiment, the method of the invention requires a sample of lymphocytes from the individual, for example obtained from a sample of body fluid or tissue. Preferably, this body fluid is whole blood obtained by venipuncture. An anticoagulant, such as heparin, is combined with the blood sample to prevent clotting. Alternatively, other appropriate body fluids, tissues or samples containing lymphocytes such as lymph node biopsies, synovial fluid, cerebrospinal fluid, pleural or peritoneal fluid or others may be used. Lymphocytes are separated from the body fluid, tissue or sample, preferably by Ficoll Hypaque density gradient centrifugation, although other appropriate methods may be employed such as methods utilizing antibody-conjugated magnetic particles, fluoresence-5 activated cell sorting, or other density gradient separation methods utilizing media other than Ficoll Hypaque. The lymphocytes so obtained are washed, preferably with isotonic saline, and transferred to tissue culture medium, such as RPMI-1640, or other appropriate medium. Preferably, the medium contains a nutrient source, such as serum. In addition, an agent such as a mitogen may be added to activate or "prime^"' the cells. Preferably, phytohemagglutinin (PHA) is the priming agent. Other appropriate priming agents include lectins such as concanavalin-A (con-A), Poke Weed Mitogen (PWM), or antibody-mediated ligation of lymphocyte surface molecules such as CD3 and/or CD28m, or by use of phorbol myristic acetate and a calcium ionophore. Lymphocytes are preferably primed or activated for 24 to 48 hours. This interval is chosen so that new cell division does not occur in vitro. Cell division at this stage is undesirable in that it provides a propitious opportunity for new mutation events to occur. Alternatively, when working with continuously dividing cell populations, the plating in the selecting agent may begin without priming.

Lymphocytes are removed from the culture medium, washed and replated in appropriate medium at an appropriate dilution. The primed cells are then inoculated into the wells of microtiter plates in very small volumes (approximately 10 to 200 microliters) in limiting dilutions. The total inoculum in each well includes appropriate medium such as RPMI 1640 or Dulbecco's Minimal Essential Medium, a nutrient source, and, preferably, inactivated feeder cells, a growth factor, such as interleukin 2 (1L2), interleukins 4, 7, 10, 15, or others for T-cells or a B-cell growth factor (BCGF) for B-cells. These limiting dilutions inocula are selected to contain 0.5, 1 or 2 cells per well, in those wells that are in plates to be used to determine the sample cloning efficiency. Wells in other plates used for determining the presence of mutation at a gene locus are inoculated with approximately 1 x 10⁴ cells per well in an inoculum medium as above which also includes an indicator agent. For example, 6-thioguanine can be used to determine whether cells have the wild-type hprt⁺ allele or a mutant hprt⁺ allele. Cells with the wild-type allele do not survive in the presence of 6- thioguanine, while those cells with the mutant genotype can survive. The hprt⁺ allele is extremely rare in natural cell populations. Mutation in the gene encoding hprt has been shown to occur preferentially in dividing T cells. The mutations can arise through errors in DNA replication, through inadequate DNA repair or by fixation of mutation. Thus, the frequency of mutant cells is enriched in cells that have recently divided, or have divided extensively in vivo. The frequency of hprt-deficient cells in normal adults is 5.4.+-A8 X 10°. Growth of cell colonies in the presence of this indicator agent therefore indicates that a prior mutation event has occurred within the cell line. Other indicators can be used, however, including 8-azaguanine which can detect mutations at the hprt locus, and 6- mercaptopurine which can also detect mutations at the hprt locus. However, 8-azaguanine provides less stringent selection than 6-thioguanine, thus altering the frequency and profile of mutant cells pbtained. Other possible indicators include diphtheria toxin, Ouabain, diamino-purine, anti-HLA antibodies and complement, which detect mutations in the diphtheria-resistance, Oubain-resistance, adenine phosphoribosyltransferase and HLA genes, respectively.

The indicator agent need not necessarily kill cells of a particular genotype so long as it provides a mechanism to distinguish between wild-type and mutant genotypes. For example, the indicator could potentially bind to cells of a certain genotype, thereby allowing separation by use of a fluorescence activated cell sorter, or magnetic separation methods. Other mechanisms of distinguishing genotypes will be evident to those skilled in the art. Therefore, two sets of plates are prepared: those that include very low numbers of cells and no indicator agent, and those that include larger number of cells in the presence of an indicator agent. The latter plates are used to determine the cloning efficiency under selection conditions (mutant fraction). The technical details of this limiting dilution inoculation can be varied. For example, although preferably round bottom microtiter wells are used, flat bottom wells may allow better outgrowth of cells. Although a variety of feeder cells such as autologous or isologous peripheral blood mononuclear cells can be used, X-irradiated B-lyrnphoblastoid cells are preferred. The number of cells inoculated into the wells containing the indicator agent to determine mutant fractions is varied depending upon the anticipated mutant frequency. For example, if a high or elevated mutant frequency is expected, less that 10⁴ cells per well are inoculated, for example 10^3' Replicate plates, such as three or more each containing 96 microtiter wells are inoculated with cells at low density to determine cloning efficiency. As large a number of plates as is practical is inoculated with a higher density of cells in the presence of the indicator agent.

Plates are placed in the incubator under standard conditions for cell growth, and the wells observed for growing clones. Usually, wells are inspected after 7, 10 and 14 days. The number of wells in each plate that contain growing cells is determined, and the fraction of positive wells per plate calculated. This fraction is simply the number of positive wells containing growing clones divided by the total number of wells. Cloning efficiencies are calculated for the low density cloning efficiency plates and the high density mutant fraction plates containing the indicator agent, such as 6-thioguanine. The cloning efficiencies are determined by the P class of the Poisson distribution which is defined as P₀=e^" where P₀ is the proportion of wells with no growing colonies and x is the average number of cells that were capable of growth originally plated into the wells, divided by inoculum size. In essence, two cloning efficiencies are calculated for each experiment. A cloning efficiency can be determined both for cells growing without an indicator agent and for cells inoculated and growing in the presence of an indicator agent. The former is termed the cloning efficiency (non-selection) and the latter the cloning efficiency (selection). The mutant frequency is defined as the cloning efficiency (selection) divided by the cloning efficiency (non-selection). By example, determining the cloning efficiency of cells derived from a blood sample and incorporating this into the mutant frequency calculation allows the mutant frequency to be equilibrated for the total number of peripheral blood mononuclear cells (the population of white blood cells with a monomoφhic nucleus) present in the sample. This in turn allows the mutant frequency to be equilibrated for comparison between blood samples from different individuals, or blood samples form the same individual taken at different points in time. Growing cells are transferred to progressively larger culture vessels to develop large populations of cloned cells. Once large cloned populations are developed, these can be characterized as desired. For example, cells can be phenotyped for cell surface markers, e.g., CD4, CD8, CD1 1 and CD3, etc. by standard and well-known techniques. The enzyme activity of HPRT can be determined in wild type and mutant clones to define the true loss of enzyme activity expected in the mutants. Chromosomal analysis can be performed on these clones. The cells can be tested for viral integration by polymerase chain reaction and/or _g

southern blot techniques, or other molecular analysis methods. TCR gene usage can be determined by Southern blot analysis or by reverse transcriptase-polymerase chain reaction and sequencing of the genes. Similarly, the isolates can be characterized by functional assays that determine the nature and specificity of subsequent immune responses they may generate. For example, cytokine production, proliferation, activation of transcription, up-regulation of cell surface molecules and other indicators of immune responses can be determined by standard assay methods described in the literature and practiced by those skilled in the art. Other methods are available for detecting and characterizing mutations, such as nucleic acid sequencing, or RNAase cleavage. All of these permit definition of the changes and spectrum of changes that have occurred in the gene, and to define the functional characteristics of the mutant populations.

The present method may be modified for use with B-cells. Again, lymphocyte samples are obtained and separated as described. In this instance, however, priming or activation will require an anti-lgM or anti-C'3 B-receptor in order to activate or stimulate the cells prior to cloning. Cloning is performed preferably in the presence of a B-cell growth factor. The assay for hprt gene mutation for B-cells is performed as described herein for T-cells. The rearrangement patterns analyzed are the rearrangements of the regions of nucleic acid encoding the immunoglobulin molecules. Clonal amplifications are recognized in a manner similar to that described above.

As used herein, the term "lymphocyte-mediated pathology" refers to any condition in which an inappropriate lymphocyte response is a component of the pathology. While the normal immune system is closely regulated, aberrations in immune response are not uncommon. In some cases, the immune system functions inappropriately and reacts to a component of the host as if it were foreign. Such a response results in an autoimmune disease, in which the host's immune system attacks the host's own tissue. In other instances, lymphocytes replicate inappropriately and without control. Such replication results in a cancerous condition known as a lymphoma or leukemia. T cells, being the primary regulators of the immune system, directly or indirectly effect such autoimmune pathologies. The term is intended to include both diseases directly mediated by T cells and those, such as myasthenia gravis, which are characterized primarily by damage resulting from antibody binding, but also reflect an inappropriate T cell response which contributes to the production of those antibodies, and cancerous cellular transformation. Once mutated cloned cell populations are isolated they can be tested for reactivity to the antigen in question, or for the presence of viral integration. Alternatively, viral infection can be determined on a clone by clone basis, to enumerate the frequency of such cells within the mutant fraction. The presence of viral infection in a clone of cells may be determined by molecular techniques such as polymerase chain reaction, and is routine to those skilled in the art. By enriching for in vivo expanded clones with the methods described herein, the sensitivity of detection of a viral infection may be increased relative to that of the unselected population of lymphocytes. This enrichment may allow for the identification of a pathologic process with greater sensitivity than previously attainable, or may allow the identification of a pathologic process at a point in time prior to its ability to be detected by current methods.

Examples

The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof. Other features and advantages of the present invention will become apparent from the following, more detailed Examples which illustrate, by way of example, the principles of the invention.

Example I

Cloning of I-Lymphocytes:

T-cell lymphocytes were cloned according to the method of O'Neill, et at. (1987), Mutagenesis 2:87, which is incoφorated herein by reference. Human peripheral blood samples were obtained by venipuncture and heparinized within 10 units/mi whole blood beef lung heparin (Upjohn, Washington, D.C.). The samples were overlaid into sterile 50 ml centrifuge tubes (Corning Glass Co., Corning, N.Y.) Containing Ficoll (m. w. 400,000)- Hypaque-M, 90% (specific gravity 1.077) (Ficoll obtained from Sigma Chemical Co., St. Louis, Mo.; Hypaque obtained from Sterling Drug [Winthrop], New York, N.Y.) at a ratio of 2.1 whole blood to Ficoll-Hypaque. The sample-containing tubes were centrifuged at 600 x G for 30 minutes at 20° C. The mo no nuclear cell fractions, represented by the white band at the plasma-Ficoll-Hypaque interface, were transferred to fresh tubes, and washed twice with phosphate buffered saline (PBS). The "basic medium" employed was RPMI 1640 containing 25 MM HEPES buffer, 2 mM L-glutamine, 100 units/ml penicillin and lOO μg/ml streptomycin sulfate which was adjusted to pH 7.2 before the addition of 2 g/1 sodium bicarbonate. All lymphocyte cultures were in basic medium containing 20% nutrient medium HL-1 (Ventrex Laboratories, Portland, Me.). I-lymphocytes were cloned as follows: fresh, single donor mononuclear cells were initially primed by culturing 1 X 10⁶ mononuclear cells/mi in growth medium containing 15% FBS and 1 m/ml phytohemagglutinin-M (PHA-M; Welicome Diagnostics, Greensville, S.C.) for 36 hours. The cell suspensions were then centrifuged at 500 X g for 10 minutes. Cells were inoculated into microtiter plates in two sets. In order to determine cloning efficiency, a sample volume of cell suspension equivalent to 1 to 2 cells/well was placed in individual wells of 96-well microtiter plates. To one set of wells was added growth medium containing 15% FBS and an optimal amount of T-cell growth factor (TCGF), determined as described below and 5 X 10 feeder cells, to a final volume of 0.2 ml/well. The feeder cells used were mycoplasma-free, hprt- derivatives of WI- L2 lymphoblastoid cells designated TK6 and grown in medium RPMI 1640 containing 10% horse serum and no antibiotics. The TK6 cells had been previously irradiated with a cesium 137 source at 150 rad/minute, for a total irradiation of 10 krad. Parallel sets of plates were inoculated with 10⁴ cells/wells in 0.2 ml growth medium to which 10 μM 6-thioguanine (Sigma Chemical Co., St. Louis, Mo.) and 5 X 10³ feeder cells were added. Cells that survive in the selection medium have had a mutation event at the hprt locus, and are termed TG^R mutants. The plates were then incubated, without change of medium, for 14 days to permit colony growth. The wells were monitored for colony growth by use of an inverted phase contrast microscope. Each plate was scored for cell growth by two individuals on days 7, 10 and 14. Sources of growth factor include recombinant or natural IL-2 or other cytokines, or the supernatant of ex-vivo activated lymphokine-activated killer cells that contain recombinant IL-2 (LAK-Sup). Three different assays were employed for testing The TCGF activity to define the optimal amount for T-cell growth. The first assay employed was a short- term culture that measures tritiated thymidine (³H-dT) incoφoration with growth factor dependent T-lymphocytes. The T-lymphocytes employed in this assay had been grown in vitro for at least 14 days and were no longer responsive to Pl-IA. The cells were plated at 2 X 10⁴ cells per microtiter well (96-well, flat bottom) in 200 μ\ of medium containing the designated amount of TCGF, incubated for 24 hours and I Ci³H-thymidine added (spec, activ. 6 Ci/mmol) and incubated for an additional 18 hours. The incoφoration of radioactivity was determined by the use of a cell harvester and a liquid scintillation spectrometer. The second assay was performed as described below with cells incubated with 1 μg/ml PHA for 40 house and then plated at I or 2 cells/round-bottom well in different amount of TCGF. The third assay was a mass culture growth with the same cells plated in a cloning assay. The cells

2 ₄ were plated in 2 cm wells at 1 X 10 cells/well in 2 ml of medium containing 2 X 10 irradiated TK6 cells and different amounts of TCGF. Cell number was determined by the use of a Coulter Counter. The maximum cell number (usually attained after 7-9 days incubation) was used as the measure of TCGF activity. TCGF was used to produce maximum cell growth and cloning as determined by testing, usually 20% TCGF. Alternatively, commercial TCGF (human T-cell polyclone; Collaborative Research Inc., Cambridge, Mass.) Can be used at 5 or 10% TCGF. The cloning efficiency in non-selection medium and the cloning efficiency in selection medium are calculated by the Poisson relationship P₀=e^" , which defines x as the average number of clonable cells/well. The value of x divided by the number of cells added to each well defines the cloning efficiency (non-selection) and the cloning efficiency (selection), respectively. The cloning efficiency (selection) divided by the cloning efficiency (non- selection) yields the measured mutant frequency. Cells from the clones were grown and expanded in vitro to characterize the T- lymphocyte colonies. The modified RPMI 1640 medium containing optimal amounts of TCGF and 2.5 X 10⁵ irradiated TKo cells per cm² or surface area were used. The colonies in positive wells in microtiter dishes contained I X 10⁴ to 2 X 10⁵ cells after 10-14 days incubation. These cells were transferred to 2 cm² wells containing 2 ml of medium (and lO μM 6-thioguanine for selected colonies) and incubated until the surface of the well is 70 to 80% confluent, usually in 3 to 6 days. The cells were then removed and centrifuged at 500 X 6 for 10 minutes to remove the depleted medium and transferred to three 4 cm² wells in 4 ml of medium at about 1 X 10⁵ cells/mi were attained. Four cryopreserved samples of peripheral blood mononuclear cells from patients with HAM/TSP infected with HTLV-1 were tested as above. The results are shown below. Table 1. Cloning efficiency and mutant frequency in 4 individuals infected with HTLV-1.

Example II

Identification of HTLV-1 Infection:

In order to demonstrate that wild type and TG T-cells are infected with HTLV-1, primers were developed for polymerase chain reaction analysis and used on DNA samples from the cell line MJ. This cell line is constitutively infected with The HTLV-1 virus, and is used as a positive control for molecular studies. To isolate the DNA, 50,000 cells for each isolate were washed and snap-frozen in liquid nitrogen. 50 ul TENS buffer (25 mM Tris-HCI pH 8, 100 mM NaCI, 10 mM EDTA, 0.6% SDS) and Proteinase K (0.5 mg) was added, and the solution was heated to 56°C for 1 hour (Yang, J.L., Maher, V.M., and McCormick, J.J. Geme 83:347, 1989). Primary polymerase chain reaction reactions were sued to amplify HTLV-1 sequences from The HTLV-1 tax region using 1-3 μg of DNA sample. The procedure was performed using a 50 μ\ volume that contained the DNA sample, lOXBuffer (lOOmM Tris-HCL, 15mM MgCl₂, 500mMKCL, pH 8.3), 0.5 μM each primer (Gibco-BRL) 0.2 mM dNTP, 2.5 units Taq polymerase, and an overlay of mineral oil (Perkin Elmer). PCR reactions were completed in a Perkin Elmer thermal cycler as follows: 25 cycles of denaturation for 1 min. at 94°C, annealing for 1 min. at 54°c extension for 1 min. at 72° followed by a 10 min. extension at 72°C after the final cycle. The primer pairs used in polymerase chain reaction analysis were as follows:

Tax Primary SK44 GAGGGCATAACGCGTCCATCG (SEQ ID NO: l) HTLV-Outer GCTCTTCCTGCTTTCTCCGGG (SEQ ID NO:2)

Referring now to Figure 1 below, there is shown a confirmatory polymerase chain reaction analysis of HTLV-1 tax region in an infected positive control cell line (MJ). A band at 608 base pairs corresponding to a portion of the viral tax region can be seen indicating presence of integrated virus in this cell line. DNA from this cell line was used as a positive control for testing T-cell isolates from HAM/TSP patients. The band was isolated and the nucleotides sequenced to confirm the specificity of the primers for the tax region. As shown in Figure 1, repeated samples of The MJ cell line are positive for amplification of a 608 base pair fragment of The HTLV-1 tax region. Representative bands were cut from the electrophoresis gel, processed with Gene-Clean (Bio- 101), and the nucleotides sequenced to confirm the specificity of the primers for the HTLV-1 tax region. Identical conditions were used to screen wild type and mutant T-cell clones from individuals with HAM/TSP known to be infected with HTLV-1.

Example III

Identification of HTLV-1 Infection in Wild Type and Mutant T-Cells

The frequency of HTLV-1 infection in wild type and hprt mutant T-cells from HAM/TSP 20 patients was determined to identify enrichment in the mutant fraction of cells. The methods described above for the cloning and isolation of wild type and hprt mutant T-cells, and the subsequent polymerase chain reaction analysis for determination of I-ITLV-1 infection was applied to patients with HAM/TSP. Figure 2 a-c, shows representative polymerase chain reaction analysis of a panel of T-cells isolates from experiments LS31B, LS31C, LS32A and LS32B. Several of the T-cell isolates are positive for the 608 base pair fragment of the tax gene, while others are negative. This analysis was performed on 136 isolates from 4 individuals with HAM/TSP. The cumulative results are shown in Figure 3. The percentage of wild type of mutant isolates positive for viral integration is shown for each patient. In each case, there is enrichment for virally-infected cells in The hprt mutant reaction of isolates. In Experiment LS3 I B, the wild type T-cell isolates similarly analyzed showed a high degree of Vital integration. Referring to Figure 2, polymerase chain reaction analysis of HTLV-1 infection in T-cell clones from I-JAM TSP patients is depicted. Representative polymerase chain reaction analysis of wild type and hprt mutant T-cell isolates from HAM/TSP patients (experiments LS31B, LS31C, LS32A, LS32B) are clearly indicated. In positive samples, a clear band of 608 base pairs corresponding to a portion the HTLV-1 tax region can be seen, as was shown for the positive control sample (MJ). Figure 3 illustrates cumulative results from HTLV-1 analysis on wild type and mutant isolates from I-JAM/TSP patients (experiments LS31B, LS31C, LS32A, and LS32B).

Claims

What is Claimed is:

1. A method for prediction of a viral pathology mediated by virally-infected cells in an individual, said method comprising the steps of:

(a) obtaining a fluid sample from said individual;

(b) culturing an first aliquot of cells from said fluid sample in a first medium which is selective for cells which lack a functional hypoxanthine-guanine phosphoribosyltransferase;

(c) culturing a second aliquot of cells from said fluid sample in a second medium which allows enumeration of total clonable cells;

(d) determining a frequency of cells lacking a functional hypoxanthine-guanine phosphoribosyltransferase by comparing said first aliquot to said second aliquot, whereby a viral pathology is predicted when said frequency is greater than 6 per 10⁶ peripheral blood mononuclear cells.

2. The method of claim 1 wherein said first medium includes an indicator selected from the group consisting of 6-thioguanine, 8-alaguanine, dipthena toxin, Ouabain, diaminopurine, or anti-HLA antibodies with complements.

3. The method of claim 1 wherein said virus is human T-lymphotropic virus type 1.

4. The method of claim 1 wherein said fluid sample is a blood sample.

5. The method of claim 1 wherein said peripheral blood mononuclear cells are separated from said blood sample prior to culturing steps (b) and (c).

6. A method for prediction of a viral pathology mediated by virally-infected cells in an individual, said method comprising the steps of:

(a) obtaining a plurality of time-sequential fluid samples from said individual;

(b) for each said time-sequential sample, culturing an first aliquot of cells from said fluid sample in a first medium which is selective for cells which lack a functional hypoxanthine-guanine phosphoribosyltransferase; (c) for each said time-sequential sample, culturing a second aliquot of cells from said fluid sample in a second medium which allows enumeration of total clonable cells;

(d) determining a frequency of cells lacking a functional hypoxanthine-guanine phosphoribosyltransferase by comparing said first aliquot to said second aliquot, whereby a viral pathology is predicted when there is an increase in the frequency of said cells lacking functional hypoxanthine-guanine phosphoribosyltransferase over time as reflected in said sequential samples.

7. The method of claim 6 wherein said viral pathology is mediated by human T-2 lymphotropic virus type 1.

8. A method for prediction of a pathology mediated by a virally-infected cell in an individual, said method comprising the steps of:

(a) obtaining a fluid sample from individual;

(b) culturing a first aliquot of cells from said fluid sample in a first medium which is selective for those cells which lack a functional hypoxanthine-guanine phosphoribosyltransferase, to produce a first set of cloned cell populations;

(c) culturing a second aliquot of the cells from said fluid sample in a second medium which allows expansion of total clonable cells, to produce a second set of cloned cell populations;

(d) determining the presence of virus in said cloned cell populations obtained in (b) and (c);

(e) identifying selected fluid samples of said individual where a percentage of cloned cell populations obtained in (b) determined to contain a virus is greater than a percentage of cloned cell populations obtained in (c) determined to contain a virus.

9. The method of claim 8 wherein the determination of the presence of virus comprises the steps of:

(a) isolating deoxyribonucleic acid;

(b) performing polymerase chain reaction, using oligonucleotide primers having nucleotide sequences such that an identifiable region of a virus can be amplified and detected from deoxyribonucleic acid obtained in step (a), on cultures derived from claim 8, (b) and (c); (c) analyzing the product of the polymerase chain reaction of step (e) to determine The presence of integrated virus,

(d) identifying those tissue or fluid samples of said individual where the percentage of cloned cell populations obtained in claim 8 (b) determined to contain a virus is greater than those derived from cloned cell populations obtained in claim 8 (c) determined to contain a virus.

10. The method of claim 9 wherein the medium that is selective for cells lacking a functional hypoxanthine-guanine phosphoribosyltransferase comprises 6-thioguanine.

11. The method of claim 9 wherein said oligonucleotide primers constitute the sequences GAGGGCATAACGCGTCCATCG (SEQ ID NO:l) and GCTCTTCCTGCTTTCTCCGGG (SEQ ED NO:2) to amplify the tax region of the human T-lymphotropic virus type 1.

12. The method of claim 8 wherein said fluid sample is a blood sample.

13. The method of claim 8 wherein said peripheral blood mononuclear cells are separated from said blood sample prior to culturing steps (b) and (c).

14. A method for diagnosis or prediction of a pathology mediated by a virally-infected cell in an individual infected with a virus, said method comprising the steps of:

(a) obtaining a tissue or fluid of said individual;

(b) culturing an aliquot of cells from the tissue or fluid sample obtained in step (a) in a medium which is selective for those cells which lack a functional hypoxanthine-guanine phosphoribosyltransferase, to produce an aggregate cell population;

(c) culturing a second aliquot of the cells from the tissue or fluid sample obtained in step (a) in a medium which allows expansion of total clonable cells, to produce an aggregate cell population;

(e) identifying those tissue or fluid samples of said individual where the amount of virus in the aggregate cell population obtained in (b) is greater that the amount of virus in the aggregate cell population obtained in (c);

15. The method of claim 14 where determining the presence of virus constitutes the method comprising the steps of:

(a) isolating deoxyribonucleic acid;

(b) performing polymerase chain reaction, using oligonucleotide primers having nucleotide sequences such that an identifiable region of a virus can be amplified and detected from deoxyribonucleic acid obtained in step (a), on cultures derived from claim 15, (b) and

(c);

(c) analyzing the product of the polymerase chain reaction of step (b) to determine the presence of integrated virus,

(d) identifying those tissue or fluid samples of said individual where the amount of virus in the aggregate cell population obtained in (b) is greater that the amount of virus in the aggregate cell population obtained in (c).

16. The method of claim 14 wherein the medium that is selective for cells lacking a functional hypoxanthine-guanine phosphoribosyltransferase comprise 6-thioguanine.

17. The method of claim 14 wherein said virus is human T-lymphotropic virus type 1.

18. The method of claim 17 wherein said oligonucleotide primers constitutes the sequences GAGGGCATAACGCGTCCATCG (SEQ ID NO: l) and GCTCTTCCTGCTTTCTCCGGG (SEQ ID NO:2) to amplify the tax region of the human T-4 lymphotropic virus type 1.

19. The method of claim 15 wherein said tissue or fluid sample is a blood sample.

20. The method of claim 15 wherein said peripheral blood mononuclear cells are separated from said blood sample prior to culturing steps (b) and (c).