WO1990002335A1

WO1990002335A1 - Method of detecting human hiv

Info

Publication number: WO1990002335A1
Application number: PCT/US1989/003750
Authority: WO
Inventors: Jules V. Hallum
Original assignee: STATE OF OREGON, acting by and through THE STATE BOARD OF HIGHER EDUCATION, on behalf of OREGON HEALTH SCIENCES UNIVERSITY
Priority date: 1988-08-29
Filing date: 1989-08-29
Publication date: 1990-03-08
Also published as: AU4209689A

Abstract

An in vitro diagnostic method detects retroviruses indicative of acquired immune deficiency syndrome (AIDS) and related conditions. A sample of cells is obtained from the genital tract of a host and contacted with an immunological marker which signals the presence of the retrovirus within the cells. Cells in a cervical smear obtained during a routine Papanicolaou test (Pap smear) are contacted with a horseradish peroxidase conjugated antibody. The cells are then contacted with a colored indicator and hydrogen peroxide. Infected cells have dark colored loci that are detectable through a microscope.

Description

METHOD OF DETECTING HUMAN HIV

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates to methods for diagnosing acquired immune deficiency and related syndromes, as well as to screening techniques for indicating the presence of a retrovirus associated with those syndromes.

2. General Discussion of the Background Acquired immune deficiency syndrome (AIDS) is one of the major public health problems in the world today. It is transmitted in bodily fluids during such activities as sexual intercourse, blood transfusions, and intravenous drug use. After initial infection, a patient usually experiences a mild, transient, mononucleosis-like condition, but thereafter becomes asymptomatic. This asymptomatic stage may last for several years, during which time the virus can be unknowingly transmitted to others by engaging in high- risk activities.

After several years, patients typically develop fever, weight loss, and persistent generalized lymphadenopathy. Later stages of the disease are characterized by the presence of opportunistic infections from pathogens such as pneumocytis carinii, aspergillus, Candida, cryptococcus, cytomegalovirus, toxoplasma, myσobacteria and herpes viruses. In addition to opportunistic infections, many patients develop neoplasms such as Kaposi's sarcoma. Death usually occurs within two years of diagnosis.

As this clinical picture suggests, people with AIDS suffer from profound suppression of cell-mediated immunity. A quantitative modification of subpopulationε of T lymphocytes occurs, with an inversion of the ration of T helper/inducer cells (OKT4) to c totoxic/suppressor cells (OKT8) . This inversion results primarily from severe deficiency of T4 cells, which act as helper/inducer cells in both cell mediated and humoral immunity. It is widely believed that AIDS and its related syndromes are caused by the human immunodeficiency virus (HIV-l) , which selectively affects the human T4 cell. Donated blood in most countries is screened for the presence of antibodies to this virus using the method disclosed in U.S.

Patent No. 4,708,818, in which antisera detect blood borne antibodies to HIV. The most widely used type of antibody test is the enzyme-linked immune absorbent assay (ELISA) in which a patient's serum is added to viral antigens, and immune complexes are detected by enzyme labeled antihuma immunoglobulin. A similar approach is also used to diagnose AIDS by determining whether antibodies to the virus are present in a patient's blood. In spite of these screening and diagnostic techniques, the AIDS epidemic has continued to increase in severity. In the United States alone, there were about 50,000 AIDS cases with over 25,000 deaths from 1981 to 1987. The number of AIDS cases increased nationally from about 100 every six month period in 1981 to more than 5,000 each six month period by July 1986. Although women represent only 7- 10% of the diagnosed cases in the United States, the incidence among women has been steadily increasing since 1981. Many experts believe that the best hope for arresting the epidemic in the near future is to educate the public about high-risk behavior, and encourage modification of such behavior. An important aspect of this approach is to detect carriers of the virus and alert them to the dangers they pose to others. In contrast to the losing battle against AIDS, great reductions have been achieved in the number of deaths from cervical carcinoma. Much of this progress is due to the widespread use of Papanicolaou tests (PAP tests) which can detect 95% of early cervical neoplasia by cytologic examination of cervical smears obtained during routine annual pelvic examinations. Use of the Pap test has reduced deaths from cervical cancer by more than 50% in recent years through recognition and treatment of preinvasive neoplasia. It is estimated that 46 million Pap tests are performed in the U.S. each year, with another 46 million performed in other countries.

Cervical secretions and cytologic smears have been used to detect conditions other than preclinical neoplasia. Examples of conditions detectable in such secretions and smears are herpes simplex [Kapur et al., Arch Pathol Lab. Med.. 109:464-465 (1985)], chlamydia trachomatis [Kiviat et al., JAMA 253:997- 1000 (1985); Spence et al., Obstetrics and Gvnecol. 68:691-695 (1986); Weismerer et al., Obstetrics and Gvnecol. 69:347-349 (1987); Adams et al. J. of Path. 143:241-247 (1984)] neisseria gonorrhoeae [Grubon et al., Obstetrics and Gvnecol. 69:350-353 (1987)], gardnerella vaginaliε [Cano et al., Can J. Microbiol. , 29:27-32 (1983)], and group B streptocci [Castle et al., J. Clin. Pathol. 36:463-465 (1983)].

An AIDS associated retrovirus has also been isolated from vaginal and cervical secretions, as reported by ofsy et al., Lancet, March 8, 1986 at pp. 527-528. the quantity of free virus recovered from such secretions was so low, however, that identification of the virus could be achieved only by passing isolates to cultured mitogen-stimulated normal human peripheral blood mononuclear cells. SUMMARY OF THE INVENTION The present invention is an in vitro diagnostic method for detecting the presence or absence of a human retrovirus indicative of acquired immune deficiency syndrome, which virus is commonly known as the human immunodeficiency virus (HIV) . According to the method of the present invention, a sample of cells are obtained from the genital tract of a host and then directly contacted with a marker which causes a cytological change in the cell sample if virus peptides are present. The cell sample is preferably a smear of cervical and/or vaginal cells obtained during a routine Pap test, for example during a routine physical examination. In especially preferred embodiments, the virus is detected by contacting the cells with an immunocytochemical marker, such as antisera to HIV (or its peptides) which has been tagged with a fluorescent dye or other indicator for diagnosis of the disease. The cervical smear is obtained, for example, by scraping cells from the vagina, cervix, or external os of the cervix, and/or by aspirating fluids from the genital tract and placing them on a glass slide. Among the collected cells will be macrophages. The smear is then contacted with the immunological marker, which forms an immune complex with the retroviral peptides expressed on the cell surfaces. The immune complex causes a cytological change in the cells, particularly macrophages, if the retrovirus is present.

The immunological marker is preferably selected from among those which form immune complexes with peptides of HIV-1 and the related HIV-2. Semen smears containing macrophages can also be substituted for cervical smears. The present method is a relatively noninvasive procedure that does not require drawing blood from a patient. Moreover, millions of Pap smears are already performed each year, and provide a wide sampling of sexually active females who would be most likely to develop the disease. The present method will increase the incidence of HIV testing among these women, which should in turn lead to a reduction in new cases by altering the sexual activity of women who test positive for the virus.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In one disclosed embodiment of this invention, routine cytological smears, such as those obtained during a Pap test, are exposed to tagged antisera to HIV-1. The smear is made by obtaining cervical aspirate and/or scrapings from the external os or vaginal pool, placing them on a glass slide, fixing the specimen, and thereafter staining it with the antisera to form immune complexes between the antisera and viral peptides in the cell surface membrane. The slide is then washed to remove any free fluorescent antisera that has not formed an immune complex, and the slide is thereafter examined under a microscope for the presence of fluorescent immune complexes which indicate the presence of the virus in the cells which were removed from the genital tract. This method detects sufficient viral proteins on the surfaces of cells to be visible through a microscope. The genome of HIV codes for viral proteins and glycoproteins, some of which undergo post- translational change. For example, the env gene codes for the protein that makes up the outer coat or envelope of the virus. The env gene codes for a 90 kilodalton protein that is subsequently highly glycosylated to form a large glycoprotein, gp 160, which is then cleaved into an a ino terminus fragment (gp 120) and a smaller carboxy terminus fragment (gp 41) . This protein, and other viral proteins such as the spike surface membrane peptide, are expressed on the surface of cells which were collected in a cervical smear. It is the presence of such cell surface viral peptides that are detected by this method, not free virus.

The concentration of free virus in cervical and vaginal smears is so low that it does not usually provide visually detectable fluorescent immune complexes when viewed through a microscope. As described by ofsy et al. (1986) above, detection of free virus in centrifuged and filtered cervical secretions requires amplification of the virus by culturing it with human peripheral blood mononuclear cells. The present method avoids the necessity for amplification by directly contacting fluorescent antibodies with the cells. A sufficient concentration of tagged antibodies react with viral peptide antigens on the cell surfaces to provide a detectable fluorescence when viewed through a microscope.

Viral peptides are preferably detected using polyclonal antibodies to a variety of viral peptides. Polyclonal detection of numerous viral antigens increases the concentration of fluorescent immune complexes, and results in greater visibility of the HIV infected cells under the microscope. Although the antisera is usually obtained by injecting an animal with disrupted virus particles, the polyclonal antibodies in the antisera recognize the bind to peptides expressed by the virus. Example 1 Cervical aspirates and scrapings from the external os and vaginal pool of an HIV-l seropositive female patient were placed on a glass slide and fixed with 95% methyl alcohol. Other fixatives, such as acetone, may also be used. The slide was allowed to fix for two minutes, after which time the liquid was poured off of the slide and it was allowed to dry. One hundred microliters of fluorescein conjugate of HIV 1 antisera was diluted 1:50 and an aliquot portion of this solution was then added to the slide. The fluorescein conjugate of the antisera was obtained from Viro Stat, P.O. Box 8522, Portland, Maine 04104. This product is an IgG fraction purified from whole antiserum and conjugated with high purity isomer I of fluorescein isothiocyanate in which free fluorescein has been removed from the final product. The host animal for the antisera was a goat, the virus strain was human isolate, and the specificity was for purified virions. The antisera was incubated at room - temperature for 30 minutes, then rinsed thoroughly with phosphate buffered saline-azide. After drying, the slide was examined with ultraviolet illumination under a fluorescent epi-illumination microscope. Fluorescent cells were visually detectable. The microscopic field was then illuminated with visible light, and the previously fluorescent cells were thought to be macrophages. No fluorescence was detected in endothelial cells. The same procedure was performed with cervical aspirates and scrapings from the external os and vaginal pool of an HIV-l seronegative female patient. No fluorescent cells were detected in this smear. Other Detection Methods Although antisera was used in Example 1 to detect viral antigens in macrophages and other cells of a Pap smear, other immunological or immunocytochemical methods can be used to test Pap smears for the presence of HIV infected cells.

Example 2 The same procedure would be followed as in Example 1 for obtaining and fixing the smear. Instead of using a fluorescent conjugate, however, a biotin conjugate would be used. Such a conjugate, which can be obtained from Viro-Stat of Portland, Maine, includes a purified IgG fraction of antisera covalently coupled with the N-hydroxysuccinimide ester of biotin under mild conditions' to give a high degree of substitution.

Streptavidin conjugated to fluorescein isothiocyanate is added to the slide, and binds with the viotin to become visible under a fluorescent microscope in the presence of HIV antigen.

Example 3 HIV could be detected on the cervical smear by using monoclonal antibodies to specific HIV antigens. Examples of some of these antibodies are listed in Table I:

Table I

Monoclonal Antibodies to Specific HIV Antigens Antibody Specificity

Anti-HIV pl7 Gag-encoded 17 kilodalton core protein and 55 dalton precursor protein Anti-HIV p24 Gag-encoded 24 kilodalton core protein and 55 dalton precursor protein

Anti-HIV gpl20 Env-encoded 120 kilodalton envelope ^' glycoprotein and 160 kilodalton precursor glycoprotein

Anti-HIV gp41 Env-encoded 41 kilodalton tranεmembrane glycoprotein and 160 ' kilodalton precursor glycoprotein

Anti-HIV3 'ORF Gene of HIV

Antibodies to the p24 core protein are available from E.I. du Pont de Nemours and Co., Inc. (1-800-551-2121) and Abbott Laboratories.

Polyclonal antibodies are preferably used to provide a greater degree of fluorescence than can be obtained with monoclonal antibodies. Such polyclonal antibodies could include, for example, any combination of the monoclonal antibodies listed in Table I.

Example 4 At least one antibody (either mono- or polyclonal), such as those of Table I, could be conjugated with horseradish peroxidase, as described in Laboratorv Techniques in Biochemistrv and Molecular Biology, 2nd edition, Clausen, J. (1981) , publisher - Elsevier/North Holland Biomedical Press. , New York, New York 10017, p. 197 et seq.

The resulting conjugated antibody (marker antibody) could then, according to a procedure discussed in Selected Methods in Immunology. Editors - B. Mishell and S. Shiigi (1980) , publishers - .H. Freeman and Co. , San Francisco, California, p. 292 et seq. , be brought into contact with cells that were collected and fixed by the method of Example 1. The antibody could then be treated with an indicator compound and hydrogen perioxide. The indicator could comprise one of a large number of known compounds, such as aminoethyl carbazpl or 3,3' dia inobenzidine. After incubation for about thirty minutes and rinsing with distilled water to remove unreacted antibodies, the slides could be observed under low power magnification. Dark-colored loci would indicate infected cells.

Example 5

At least one antibody (either mono- or polyclonal) , such as those of Table I, could also be used according to the double antibody method described in Selected Methods in Immunology, Editors - B. Mishell and S. Shiigi (1980) , publishers - W.H.

Freeman and Co., San Francisco, California, p. 292 et seq.

By this procedure, slides prepared according to Example 1 would be treated with at least one antibody that is specific for an HIV antigen, incubated, and rinsed to remove unreactant antibodies. A second antibody, specific for the immunoglobulin species of the first antibody, could be conjugated with horseradish peroxidase as described in Laboratory Techniques in Biochemistrv and Molecular Biology. 2nd edition₇ Clausen, J. (1981) , publisher Elsevier/North Holland Biomedical Press., New York, New York 10017, p. 197 et seq. The resulting horseradish peroxidase- conjugated antibody (marker antibody) would then be added to the smear and allowed to react with the first, bound antibody. The smear would then be rinsed with distilled water to remove any excess of the second antibody. An indicator, such as aminoethylcarbozol or 3,3' diaminobenzidine, would then be added along with hydrogen peroxide. After about 30 minutes, the slide would be rinsed and observed under low power magnification. Dark colored loci would indicate infected cells.

Example 6 Antibodies labeled using alkaline phosphatase could also be employed, with either the single or the double antibody technique described above in Examples 4 and 5.

In the case of the single antibody method, poly- or mono-clonal antibodies would be conjugated with alkaline phosphatase. The conjugated antibodies (marker antibodies) are then added in sufficient volume to cover the cells in a smear formed according to the procedure of Example l. After incubation for about 30 minutes, excess antibodies are removed by rinsing with distilled water. Next, the smear is developed by covering the cells with a solution which is reactive with the alkaline phosphatase to cause a color change. The reactant solution contains nitro blue tetrozolium in dimethyl-formanide (NTB) and 5- bromo-4-chloro-3-indolylphosphate in dimethyl- formanide (BC1P) at a ratio of 2:1, u:v. After 15 minutes, the cell smear is rinsed and viewed under a light microscope. Infected cells could be identified by purple colored loci.

With the double antibody method, HIV specific antibody serum would be applied to the cells in a smear. After incubation, excess antibodies would be removed by rinsing. A second antibody (marker antibody) , that contains the label and that is specific to the species of the HIV specific antibody, would then be added. After rinsing off the excess second antibody, the color reaction is developed and read as above.

Specific procedures for -labeling antibodies with alkaline phosphatase are found in the Promega Catalog, 1988, and Laboratorv Techniques in Biochemistry and Molecular Biology, p. 157 et seq. (1981) , published by Elsevier/North Holland Biomedical Press, New York, New York.

Example 7 The cells of the cervical smear could also be tested for retroviruseε related to HIV-l. The presence of HIV-2 is asεociated with AIDS in Senegal and other countries in Western Africa, and at leaεt one AIDS case in the United States has been associated with HIV-2. Both HIV-l and HIV-2 share core proteins. Brun-Vezinet et al., Lancet 1:28 (1987). The DuPont ELISA test for HIV-l, for example, cross reacts with HIV-2 antibody.

In the present invention, the cervical smear could be prepared in accordance with the procedure of Example 1, and then exposed to 100 ml of fluorescein conjugate of HIV-2 antisera in a 1:50 dilution. The slide would be incubated and rinsed aε deεcribed in Example 1, and then examined with ultraviolet illumination under a fluorescent microscope. Fluorescent cells, especially virally infected macrophages, would be visually detectable.

Example 8 The procedure of Example 1 could be modified to include genital secretions other than cervical smears. Semen from male subjects could be placed on a slide to make a smear for microscopic examination. The semen smear would then be exposed to the fluorescein conjugate of antisera of Example 1, incubated, and examined with ultraviolet illumination under a fluorescent microscope. Cells in the smear, particularly macrophages which are infected with HIV-l, would be illuminated. It is believed that the macrophages would express viral peptides on their cell surfaces, which would form the fluorescent immune complexes detected by ultraviolet illumination.

The present invention, as embodied in the examples, is a substantial improvement over prior methods which relied on detection of free virions in bodily secretions. In the present method, cells from a subject's genital tract are directly contacted with an indicator which detects the presence of a retrovirus in the cell. This method contrasts with the prior art in which an indicator is not directly contacted with cells taken from the genital tract. These prior methods instead cultured the virions with blood cells to amplify the virus, and then expose the virus to an im unofluorescence assay. No direct exposure occurs between the cells of the genital tract and the indicator in such prior art methods. The indicator is instead indirectly exposed to the virus after amplification of the virus in culturing cells. Having illustrated and described the principles of my invention with reference to several preferred embodiments,- it should be apparent to those persons skilled in the art that such invention may be modified in arrangement and detail without departing from such principles. I claim as my invention all such modifications aε come within the true spirit and scope of the following claims.

Claims

I CLAIM:

1. An in vitro diagnostic method for detecting the presence or absence of a human retrovirus indicative of acquired immune deficiency syndrome in a host, which method comprises the steps of: obtaining a sample of cells of said host from the genital tract of said host; and contacting the sample with a means for signalling the presence of the retrovirus for a time and under conditions sufficient to signal the presence of the retrovirus if it is preεent in the cellε of the sample.

2. The method of claim 1 further comprising the step of placing the sample of cells on a slide, and then contacting the sample with the signalling means on the slide.

3. The method of claim 1 wherein the contacting step includes contacting the sample with an immunological marker which signals the presence of the retrovirus within the cells.

4. The method of claim 3 wherein the contacting step includes contacting the sample with an immunological marker which formε an immune complex with peptides produced by the retrovirus.

5. The method of claim 3 wherein the contacting step includes contacting the sample with an immunological marker on a slide to form an immune complex to peptide products of the retrovirus in the plasma membranes of cells* infected with the retrovirus.

6. The method of claim 3 wherein the contacting step includes contacting the sample with an immunological marker which formε an immune complex with antibodies to the retrovirus.

7. The method of claim 3 wherein the contacting step includes contacting the sample with an IgG fraction of antisera to the retrovirus.

8. The method of claim l wherein the contacting step includeε contacting the sample with an immunocytochemical marker which causes a characteristic detectable cytological change in the sample which indicates the presence of the retrovirus.

9. The method of claim 8 wherein the immunocytochemical marker is a conjugate of fluorescent material and antisera to the retrovirus.

10. The method of claim 8 further comprising the step of examining the sample for the characteristic cytological change.

11. The method of claim 10 wherein the step of obtaining the sample includes obtaining macrophages from the genital tract.

12. The method of claim 11 wherein the examining step includeε examining the macrophages for the characteristic cytological change.

13. The method of claim 1 wherein said sample is semen.

14. The method of claim 1 wherein said means for signaling the presence of the retrovirus iε εuitable to signal the presence of HIV-l.

15. The method of claim 1 wherein said means for signaling the presence of the retrovirus is suitable to signal the presence of HIV-2.

16. The method of claim 1 wherein the host is female and the step of obtaining a sample includes obtaining cells from the cervix of the female.

17. The method of claim 16 wherein the step of obtaining a sample of cells includes scraping the external os of the cervix.

18. The method of claim 1 wherein the host is female and the step of obtaining a sample includes obtaining cells from the vagina of the female.

19. The method of claim' 18 wherein the step of obtaining a sample of cells includes obtaining a specimen from the vaginal pool.

20. The method of claim 1 wherein the step of obtaining a sample of cells includes aspirating fluids from the genital tract.

21. The method of claim 1 wherein the step of obtaining a sample includes obtaining macrophages from the genital tract.

22. An in vitro diagnostic method for detecting the presence or absence of a human retrovirus indicative of acquired immune deficiency syndrome, which method comprises the steps of: obtaining a Pap smear of cells from the genital tract of a female; and contacting the cells of said smear with a cytochemical detector means for signalling the presence of said retroviruε in εaid cellε, for a time and under conditionε sufficient for the detector means to signal the presence of said retrovirus if it is present.

23. The method of claim 22 wherein said contacting step includes contacting said smear with an immunocytochemical detector which causes a characteristic cytological change in cellε of said smear if said retrovirus is present in said cells.

24. The changed cells of claim 23.

25. The method of claim 23 further comprising the step of examining said sample for said characteristic cytological change.

26. An in vitro diagnostic method for detecting the presence or absence of HIV-l or HIV-2 in a host, which method comprises the steps of: obtaining a sample of cells from the vagina or cervix of a human female host; placing the sample of cells on a microscope slide; contacting the sample of cells with sufficient amounts of antisera to form a fluorescent immune complex with viral peptides in the cell surface membranes of the cells, which immune complexes are visible under ultraviolet illumination through a microscope; and then examining the sample of cellε to determine if any immune complexeε are viεible.

27. An in vitro diagnostic method for detecting the presence or absence of a human retrovirus indicative of acquired immune deficiency syndrome in a εubject, which method co priεes the εteps of: obtaining a sample of cells from the genital tract of a human εubject; contacting the sample with a marker antibody that is specific for antigens present on a retroviruε that is indicative of acquired immune deficiency syndrome, the marker antibody having been conjugated with horseradiεh peroxidaεe; contacting the εa ple with amountε of an indicator and hydrogen peroxide εufficient to bind the indicator with the marker antibody; rinsing the sample to remove any indicator not bound to the marker antibody; and observing the extent to which the indicator is retained in the rinsed εample to determine whether the retrovirus is present in the cells of the sample.

28. The method of claim 27 wherein the marker antibody is εpecific for HIV antigens.

29. The method of claim 27 wherein: the marker antibody is specific for the immunoglobulin species of an other antibody, the other antibody being specific for HIV antigens; and the method further comprises the step of contacting the sample with the other antibody before the contacting of the sample with the marker antibody.

30. The method.of claim 27 wherein the indicator is selected from the group consisting of aminoethyl carbazol and 3,3' diaminobenzidine.

31. An in vitro diagnostic method for detecting the presence or absence of a human retrovirus indicative of acquired immune deficiency syndrome in a subject, which method comprises the steps of: obtaining a sample of cells from the genital tract of a human subject: contacting the sample with a marker antibody that is specific for antigens present on a retroviruε_. that is indicative of acquired immune deficiency syndrome, the marker antibody having been conjugated with alkaline phoεphataεe; contacting the εample with a reactant which reacts with the alkaline phoεphataεe to bind an indicator with the marker antibody; rinεing the sample to remove any reactant and indicator not bound to the marker antibody; and observing the extent to which the indicator is retained in the rinsed sample to determine whether the retrovirus is present in the cells of the εample.

32. The method of claim 31 wherein the marker antibody is specific for HIV antigens.

33. The method of claim 31 wherein: the marker antibody is specific for the immunoglobulin species of an other antibody, the other antibody being specific for HIV antigens; and the method further comprises the step of contacting the sample with the other antibody before the contacting of the. sample with the marker antibody.

34. The method of claim 31 wherein: the reactant is 5-bromo-4-chloro-3- indoly/phoεphate; the indicator iε nitro blue tetrozolium; and the observing comprises viewing the sample under a light microεcope to detect cellε containing purple loci.