FR2666657A1 - METHOD FOR DETERMINING THE RISK OF EVOLUTION OF INFECTION CAUSED BY HIV AND ITS IMPLEMENTATION - Google Patents

Abstract

The method of determining a risk of a course of an HIV infection in a subject, which is independent of other risk indices of such a course, comprises determining in a sample a fluid or a a body secretion taken from said subject with a level of DHEA which is at the lower end of, or less than the lower value of the reference range of 180-1250 ng / dl. The method can be used to determine levels of DHEA in a subject, indicating a predisposition to, or confirming a possibility of the course of HIV infection, and which can be avoided by administration of DHEA or a salt or drug. 'a conjugate of it.

Description

METHOD OF SCREENING AND ITS IMPLEMENTATION

  The present invention relates to a method for determining the risk of progression of infection caused by human immunodeficiency virus (HIV) in a subject, the method

  being based on hormone levels in the serum.

  UK-A-2,204,237 describes the use of certain 17-keto steroids, including dehydroepiandrosterone (DHEA), in the prophylaxis and treatment of retroviral infections, including HIV infection, Acquired Immunodeficiency Syndrome (AIDS). ) and the AIDS-related complex (ARC) In particular, UK-A-2,204,237 describes the inhibition of antigen production.

  HIV p 24 in macrophages infected with HIV.

  Bricaire F et al (The Medical Press, Sept 1988, 17, n 30) determined the hormonal profile (sex hormones and cortisol) in twelve people with AIDS and twenty-eight people with ARC The forty people were homosexual men whose the mean age was thirty-seven years Overall sex hormone levels, including dehydroepiandrosterone sulfate (DHEA-S), were significantly reduced in patients with AIDS and ARC;

  that the cortisol levels were unchanged.

  Jacobson MA et al (Abstracts of the 5th International Conference on AIDS, Montreal, June 1989) described fifty percent inhibition of HI Vp 24 macrophage expression at DHEA concentrations of 3-30 μg / ml. observed that seronegative homosexual men at risk for HIV have significantly higher serum DHEA-S levels than HIV-positive men of the same age or healthy HIV-negative blood donors They concluded that DHEA / DHEA-S may have a protective effect in

HIV infections.

  Wisniewski T et al (Abstracts of the 7th International Conference on AIDS, Florence, June 1991) investigated the relationship between DHEA levels and cortisol, absolute T 4 levels, and the clinical status of individual HIV disease. Ninety-eight adults with HIV infection had defined levels of cortisol and DHEA in the plasma, seventy-seven percent of whom had estimated simultaneous T4 absolute levels; Clinical status was determined by historical and physical examination The authors found an important relationship between DHEA levels and absolute levels of T 4, but not between absolute levels of T 4 and cortisol or cortisol / DHEA ratios. They concluded that

  Positive relationship exists between immune status and DHEA levels.

  Mulder JH et al (Abstracts of the 7th International Conference on AIDS, Florence, June 1991) followed a group of homosexual men, initially asymptomatic, infected with HIV-1 DHEA levels in serum were determined in forty-one subjects who have progressed to AIDS for a period of five years, the initial measurements being made on a 1985 serum sample and the final measurement, on a serum sample taken four months prior to the diagnosis of AIDS Forty-one subjects infected with HIV, of the same age, who remained asymptomatic during the follow-up period, were used as controls CD 4 + counts and antigenemia of HIV p 24 were also determined. Subjects who progressed to AIDS showed a significant decrease DHEA levels, while the controls did not exhibit this phenomenon DHEA levels, CD 4 + counts and HIV p 24 antigen status were found to be symptomatic. The authors concluded that in asymptomatic HIV-I infected subjects, a decrease in the

  DHEA is observed before the development of AIDS.

  Jacobson MA et al (Journal of Infectious Diseases, in press) described a study of serum DHEA and DHEA-S levels and subsequent AIDS progression in homosexual men with HIV infection in the US. San Francisco Men's Health Study, followed prospectively since 1984 Among one hundred and eight HIV-positive men at the beginning of the study, and having a CD4 count in the range of 200-500 cells / gl, DHEA levels below the lower limit of normal (<180 ng / dl) predict the subsequent course of AIDS, after controlling for hematocrits, age, and absolute numbers of CD4 cells. concluded that DHEA may have a protective effect

  1 i O protection in HIV infections.

  DHEA and its interconvertible DHEA-S sulfate have been described as inhibiting viral expression and have been associated with decreased cancer risk (Schwartz AG et al., Toxicologic Pathology, 1986, 14, NO 3) DHEA-S levels increased were associated with reduced occurrence of breast cancer (Schwartz A G.

  et al, Nutrition and Cancer, 1981, 3, No. 1).

  According to the present invention, there is provided a method for determining the risk of progression of an HIV infection in a subject, which is independent of other indices of the risk of such an evolution, said method comprising the determination in a sample of a body fluid or bodily secretion of said subject of the DHEA level, which is at the lower end, or lower than the lowest value, of a reference range of

  -1250 ng / dl, as defined below.

  The reference range used below for DHEA is the reference range of Nichol's Institute, San Francisco, US A. Low DHEA levels, that is, <180 ng / dl, have been determined at HIV-positive individuals appear to be associated with disease progression regardless of factors such as age, CD4 counts and hematocrits (Jacobson MA et al (Journal of Infectious Diseases, in press)).

  is included here for reference).

  In addition, preferably, the sample of fluid or body secretion is taken from a subject having a number of lymphocytes

CD 4 of 200-500 cells / il.

  In addition, preferably, the sample used for the

  Determination of the DHEA according to the invention is a body fluid.

  The body fluid is preferably blood or a fraction of the blood.

  The blood fraction is preferably plasma or serum.

  The amount of DHEA can be measured in different ways secundem artem Thus, DHEA can be measured by liquid gas chromatography (GLC). However, clinical laboratories or surgical practitioners are generally not equipped with the apparatus necessary to perform a GLC test. Thus, preferably, the determination of DHEA according to the present invention is carried out by

an immunoassay.

  Consequently, DHEA can be determined by a radioimmunoassay or enzymoimmunoassay. As for the GLC, clinical laboratories may not be equipped and it is unlikely that surgeon practitioners will have the equipment and means to perform radioimmunoassays. Thus, the immunoassay for the determination of the DHEA according to the invention is preferably carried out by an assay.

enzymoimmunologic.

  Thus, according to one embodiment of the present invention, there is provided an immunoassay method for determining the DHEA antigen component of the DHEA-antibody antigen reaction which comprises: a) adding a sample of a fluid or body secretion, containing DHEA, to an amount of anti-DHEA in an insolubilized form; b) the course of the immunochemical reaction; and c) estimating the amount of DHEA bound to said insolubilized anti-DHEA. The amount of bound DHEA can be determined by Western

  Blotting in a manner known per se.

  However, the amount of bound DHEA can also be

  determined enzymatically in a manner known per se.

  Thus, an immunoassay method according to the invention may comprise: a) adding a sample of a fluid or body secretion containing DHEA to an amount of anti-DHEA in an insolubilized form; b) the course of the immunochemical reaction, while simultaneously or subsequently adding a predetermined amount of labeled anti-DHEA; and c) estimating the amount of DHEA by determining the amount of free or bound labeled anti-DHEA, in a manner

known in itself.

  The bound anti-DHEA can be contacted with anti-mammalian immunoglobulin prior to enzymatic determination. The insolubilized antibody may be a polyclonal antibody or

a monoclonal antibody.

  When the monoclonal antibody is used, it may preferably be a human monoclonal antibody, mouse or rat, prepared by conventional methods including the methods generally available for the production of monoclonal antibodies on an industrial scale, antibodies or fragments of monoclonal antibodies prepared by genetic engineering or antibodies produced

  by in vitro immunization of appropriate cells.

  Preferably, the monoclonal antibody is of the IgG class. The immunoassay methods according to the present invention can be carried out using any known format, for example beads, rods, membranes, particles, plates, rods, strips, etc. For example, the insolubilized or solid phase antibody as used according to the present invention is preferably bound to a bead, a rod, a membrane, a particle, a plate, a stick, a tape, tube, cell, or the like, of plastics material or glass, in a manner known per se Latex, nylon or other suitable material beads may be used in accordance with

known per se.

  In particular, the insolubilized form of the antibody comprises said antibody adsorbed on the surface adapted for the adsorption of proteins. Said surface may be a bead, a liposome, a membrane, a particle, a plate, a stick, a tube, a

  cell, or the like, and of a material as specified above.

  Under the appropriate laboratory conditions, the surface comprises a plate or microtiter strip of plastic material, suitable for protein adsorption, in which the immunochemical reaction and the estimation of DHEA can take place. The microtiter plates are preferably polystyrene microtiter plates comprising flat cells, marketed by Dynatech under the trade name MICROELISA. Examples of strips are strips marketed by Dynatech under the trade name of

Removawell.

  The surface concerned can be coated directly with an optimal dilution of the polyclonal antibody prepared by separation of the immunoglobulin fraction concerned from the antiserum or according to

  a variant of the monoclonal antibody.

  The estimation of the bound DHEA in the sample can be carried out by enzymatic, fluorometric, luminometric or radiometric assay, using enzymes, fluorochromes, light emitting samples or radioactive markers, respectively in the qualitative and semiquantitative assays. The estimation can be performed visually, for example when the assay involves the use of colored or other beads, in a manner known per se, as the method

described in EP-A 0 154 749.

  The labeled agents for use in the assays according to the invention are conventionally prepared or purchased from the appropriate suppliers. These labeled agents are generally in conjugated form such as enzyme-labeled antibodies for use in Competitive binding assays The labeled agent may be an antibody covalently bound to a radiolabel usable in a radiometric assay, when assays are performed under laboratory conditions providing the necessary equipment and know-how. Under laboratory conditions, the estimation of bound DHEA can be carried out by enzymoimmunoassay, for example by using a suitable peroxidase-labeled antibody or other suitable peroxidase conjugate. A suitable peroxidase is horseradish peroxidase Another conjugate of appropriate peroxidase is a peroxidase complex

  avidin-biotin, which can be used with an antibody-conjugate

  biotin to enhance the enzyme immunoassay in a conventional manner In an enzyme immunoassay of this type, the antigen insolubilized on the solid phase antibody binds to the antibody-biotin complex which in turn binds to the avidin / streptavidin peroxidase complex. -biotin, which allows

measure peroxidase activity.

  The methods described above can be used to determine the levels of DHEA in a subject, indicating a predisposition to, or confirming a probability of progression of HIV infection that can be avoided by administration of

  DHEA or a salt or conjugate thereof.

  The invention also provides different kits or test boxes containing the components / ingredients necessary to perform the diagnostic methods according to the invention. Such a kit or test box may contain antibody-coated tubes containing all the components necessary for carrying out the test. diagnostic methods according to the invention when a sample of one

body fluid.

  It is also possible to provide a strip containing all the components / ingredients necessary to perform a rapid one-step assay according to the present invention by depositing

  above a body fluid in a manner known per se.

  The theoretical basis of the invention is the following study, described by Jacobson et al (Journal of Infectious Diseases, In Press)

mentioned above.

  Subjects participating in a San Francisco Health Study that began in 1984 were evaluated at six-month intervals, called cycles, by taking serum samples that were frozen at -70. Serum samples from 108 male, homosexual, HIV-seropositive individuals participating in this study were tested to determine the association between DHEA levels and the course of AIDS in the fifth cycle of study, when each of the subjects had a CD4 count in the range of 200-500 cells / g 11, DHEA levels were determined by radioimmunoassay. DHEA levels were thus measured at each follow-up cycle and subjects were divided in two groups according to whether their DHEA levels in the plasma were (i) <180 ng / dl or (ii) 2 180 ng / dl Both groups were then subdivided according to whether the subjects completely developed AIDS or not Pen During an intermediate follow-up period of 37 months, 16 out of 27 individuals with lower than normal DHEA levels (<180 ng / dl) evolved to the AIDS stage, compared with 22 out of 81

normal DHEA levels.

  The follow-up study demonstrated that there is a relationship between low DHEA levels in subjects involved in the study and progression to fully developed AIDS, regardless of age, number of CD4's and hematocrit

  of the subject, as shown below.

  In the evaluation of the data, a series of Cox proportional hazard regression models were fitted to estimate the relative risk of AIDS development associated with lower than normal DHEA, both alone and adjusted to the rates of the others.

covariate factors.

  The relative risks of AIDS development associated with below-normal DIHEA levels, alone and adjusted for the influence of other covariate factors, measured during the fifth cycle,

are summarized in Table 1.

TABLE 1

  RISKS RELATED TO AIDS DEVELOPMENT

  Prediction agent Risk p-value relative DHEA <180 (single) 2.38 <0.01 DHEA <180 (adjusted) 2.34 0.02 Age / 10 2.18 <0.01 Hematocrits <40 4.57 < 0.01 CD cells 4/10 0.99 0.71 The present invention is therefore based on the above analyzes which confirm a significant association between DHEA levels <180 ng / dl and an increased risk of evolution. This association has been shown to persist even after adjusting for the number of hematocrits and CD4 cells, which are known as independent predictors of evolution.

  diseases caused by HIV.

Claims (9)

  1 Method for determining the risk of progression of an HIV-induced infection in a subject, independent of other indices of the risk of such an evolution, said method being characterized in that it comprises the determination in a sample of body fluid or body secretion taken from said subject, the level of DHEA which is at the lower end, or lower than the lower value, of a range of reference of 180-1250 ng / dl.   2 Method according to claim 1, characterized in that the sample of fluid or body secretion is taken from a subject having a CD4 lymphocyte count of -500 cells / 4 l.   Method according to claim 1 or 2, characterized in   that the rate of DHEA is <180 ng / dl.   4 Method according to any one of claims 1   at 3, characterized in that the body fluid is plasma or serum.   Method according to any one of the claims   previous ones, characterized in that the DHEA is determined by gaseous liquid chromatography.   6 Method according to any one of claims 1   at 4, characterized in that the determination of the DHEA is carried out by immunoassay.   7 Immunoassay method for the   determination of the DIEA antigen of the DHEA antigen reaction   antibody, which comprises: a) adding a sample of a fluid or body secretion containing DHEA to an amount of anti-DHEA in an insolubilized form; b) the course of the immunochemical reaction; and c) estimating the amount of DHEA bound to said insolubilized anti-DHEA. The method of claim 7, which comprises: a) adding a sample of a fluid or body secretion containing DHEA to an amount of anti-DHEA in insolubilized form; b) performing the immunochemical reaction, while simultaneously or subsequently adding a predetermined amount of labeled anti-DHEA; and c) estimating the amount of DHEA by determining the amount of free or bound labeled anti-DHEA, in a manner known in soil.   9 Test kit containing one or more component (s), for carrying out the method according to any one of Claims 1 to 8.