EP2558486A1 - Antigenic structure and uses thereof for screening trypanosomiases in humans and animals - Google Patents

Abstract

The invention relates to an antigenic structure that contains a tryptophan epitope, characterised in that said structure is made up of a tryptophan pattern W or a peptide of 3 or 4 amino acids comprising a pattern W, coupled with glutaraldehyde. The invention can be used for screening trypanosomiasis in humans or animals.

Description

 Antigenic construct and its applications for trypanosomosis screening in humans and animals

The subject of the invention is an antigenic construct and its use for developing a kit and implementing a method for trypanosomosis screening in humans and animals.

 Human African trypanosomiasis (HAT) and animal trypanosomiasis (AAT) mainly affect poor rural communities and are often neglected diseases, which are a serious public health problem and a hindrance to the economic development of many countries. The greatest difficulty for the control of these diseases results from the great variability of the clinical symptomatology associated with the lack of sensitivity and specificity or the heaviness of carrying out the available screening tests.

 Active screening of the population at risk is therefore essential to identify infected individuals early and reduce transmission by reducing the parasite reservoir. In addition, screening for HAT should include a diagnosis of stage 1 (lymphatic-blood) or 2 (neurological), because patients in stage 2 are treated with melarsoprol whose toxic side effects are important (overall mortality of 5-10% ). The diagnosis of stage still depends today on the examination of the cerebrospinal fluid after lumbar puncture and the positivity threshold of the cytorachia is still debated (5, 10 or 20 cells / μΐ).

Currently, the CATT (Card Agglutination Test For Trypanosomiasis) /! ¹ . b. gambiense is the most commonly used serological test. The assay includes fixed trypanosomes of LiTat variant 1.3. A variant of CATT consists of semi-purified antigens (LiTat 1.3, 1.5 and 1.6) coupled to latex beads. The main problem related to CATT is a lack of specificity because the antigens used are responsible for many cross-reactions and thus false positives. Moreover, blood, diluted blood or plasma have variable antibody titers against these antigens. Thus parasito logically positive patients may be CATT negative. Finally, the CATT does not allow the diagnosis of stage and a satisfactory serological follow-up of the patients after treatment, in particular because of the cross reactions with other infections.

It is therefore important to have new antigens available to improve screening, staging and monitoring of HATs and TAAs. Although some antigens have proved interesting from a pathophysiological point of view, their manipulation in field conditions (Semballa et al., 2004 (ref.1)) has proved difficult. Tryptophan epitopes (WE), which represent constant antigens of trypanosome, induce specific antibodies in the host (human or animal). These epitopes comprise the amino acid L-tryptophan and are located in constant domains of the C-terminal portion of the variable surface glycoproteins (VSG). High titers of anti-WE antibodies of IgM isotype were measured by ELISA in the serum of patients with human African trypanosomiasis (HAT), and increased titers were found in stage 2 patients (Okomo-Assoumou et al. al., 1995 (ref 2)).

 These pioneering results were obtained by the inventors' laboratory using synthetic epitopes proposed to study their recognition by anti-WE antibodies, in particular WE-glutaraldehyde-BSA constructs. In these constructs, glutaraldehyde acts as a fixative between the synthetic antigen WE and the carrier protein (here BSA bovine albumin). These constructs have demonstrated the potential of the WE epitope as a diagnostic target.

 The work of the inventors in this field has shown an unexpected effect of glutaraldehyde on the optimal orientation that it confers on synthetic antigen. Indeed, it has been found that glutaraldehyde serves as a spacer and orienting arm of the synthetic antigen WE when it is coupled to an inert support such as latex beads or ELISA plates. As illustrated in FIGS. 1A and 1B, to which the following example refers, the antigenic construct of the invention constitutes a mimotope of the natural antigen, when it is coupled only to the latter, which allows better recognition by anti-WE antibodies.

 The object of the invention is therefore to provide a new antigenic construct. It also aims to take advantage of the properties of this construction in a kit and a screening method for human and animal trypanosomoses.

 The antigenic construct of the invention is based on the coupling of an epitope of interest to glutaraldehyde and their attachment to an inert carrier. The invention thus relates to an antigenic construct containing a tryptophan epitope, characterized in that it is formed of a tryptophan motif W, or a peptide of 3 or 4 amino acids comprising a motif W, coupled with glutaraldehyde.

 Preferably, said peptide corresponds to the sequence SEQ ID No. 1 CxWy, in which "x" represents K, A, S, V, T, R, I, E, D, N or G, and "y" represents D, S, T, E, N, K, G, Q, R or I, either of "x" or "y" may be absent.

A representative sequence, SEQ ID No. 2, is of the CKWD type. According to an advantageous arrangement, said coupling product is grafted onto latex beads. This formulation makes it possible to develop a rapid agglutination technique on the sera of patients or animals affected, respectively, with THA or TAA.

 According to another provision, advantageously used with the previous one, the coupling product is grafted onto ELISA plates, making it possible to develop a quantitative technique of "anti-trypanosome" antibody levels in patients or animals suffering from HAT or TAA.

 According to yet another arrangement, the coupling product as defined above is grafted onto poly-L-lysine deposited at the bottom of ELISA plates.

 This arrangement makes it possible to have a completely synthetic system, whether for the development in the form of beads or ELISA test.

 The invention also relates to a screening kit for THA or TAA, characterized in that it comprises

 at least one antigenic construct as defined above, with possibly

 one or more reagents for the antigen / antibody reaction and / or buffer solutions and / or reagents for the detection, quantification or visualization of antigen / antibody complexes when present.

 The kit above advantageously comprises, according to a further feature of the invention, the reagents for carrying out an immunoassay, especially an indirect ELISA or an ELISA inhibition test, making an anti-monoclonal antibody compete with one another. -WE with anti-WE antibodies infected host sera.

 In an alternative embodiment, the antigenic construct is attached to a support.

The invention is further directed to a method for screening for THA or TAA, characterized in that it comprises

 . contacting a serum sample of a patient or an animal with an antigenic construct as defined above, advantageously using a kit as described above,

. revealing an immunological reaction of the antigen-antibody type. In the variant where the antigenic construct is attached to latex beads, contacting with the serum sample leads to agglutination of the serum if the patient or animal is respectively HAT or TAA. This serological diagnostic method allows for mass screening, staging and monitoring of HAT and TAA. It has the advantage of high sensitivity and specificity by avoiding cross-reactions with other infections (false positives) and false negatives (seronegative at CATT in parasita logy).

 This new serological diagnostic test, which is cheap, stable and easy to handle, combined with an indirect ELISA or an ELISA-based inhibition test based on anti-WE antibodies and which has proved its effectiveness, can be used for a diagnosis of mass and stage (determination of antibody titers of patients in stages 1 and 2) of HAT or TAA in the field, with high specificity. This test is particularly suitable for a specific screening of human or animal African trypanosomiasis.

 Other characteristics and advantages of the invention are given by way of illustration in the following examples of embodiment of the invention. In these examples, reference is made to FIGS. 1 to 5, which represent, respectively,

 Figures 1A-1C, the native steric hindrance of the CKWD sequence (Figure 1A), an inert carrier-type sequence with amine-glutaraldehyde-W function (Figure 1B), and a sequence attached to an ELISA plate (Fig. . 1 C)

 Figure 2 and 3 the diagnostic test results, respectively, on human serum and blood,

 Figure 4, a graph of results obtained with animal sera infected with different species of Trypanosomes;

 Figure 5, the levels of anti-W antibodies in the serum of patients with HAT (stages 1 and 2).

Example 1: synthesis of coupling products W-glutaraldehyde or C-x-W-y-glutaraldehyde Production of coupled latex beads

 At 100 μl of hapten (tryptophan or peptide) at a concentration of 5 mg / ml in a 1.5 M acetate buffer, pH 8.3, vial 100 μl of 5% glutaraldehyde are added under vortex for approximately 10 seconds.

 Without waiting, 50 μΐ of amino latex beads (Sigma) are added and allowed to react for at least 10 minutes with slow stirring.

The reaction is stopped by addition of 100 μl of 1M NaBH ₄ and leave the mixture for 10 minutes while stirring slowly. The mixture is dialyzed against distilled water supplemented with NaBH ₄ (lspatula / 51) all day long by changing the dialysis water 2 to 3 times, then about 14h (all night) with water, remove the excess of NaBH ₄ .

 By way of illustration, FIGS. 1A and 1B show the planarized chemical formulas of the native epitopic sequence and the chemical sequences used according to the invention which clearly show that the construction of the invention reproduces the molecular configuration of the epitope of the invention. 'interest.

Manufacture of ELISA plates

By well of ELISA plate (Nunc, PolySorp or MaxiSorp), 200 μl of a mixture of ΙΟΟμΙ of hapten (tryptophan or peptide, 5 mg / ml in a 1.5 M acetate buffer pH 8.3) are added. 100 μl of 5% glutaraldehyde added under vortex for about 10 seconds. Allowed to react for at least 30 minutes with slow stirring.

The reaction is stopped by the addition of 100 μl of 1M NaBH ₄ and the mixture is left for 10 minutes while stirring slowly.

The plates are washed (5 times 1 hour) with distilled water containing NaBH ₄ (lspatule / 51), then about 14 hours (overnight) with water only, to remove excess NaBH ₄ .

 A construction with a Poly-L-lysine motif attached to a LISA plate is given for illustrative purposes in Figure 1C.

Example 2: Diagnostic test on human serum (or animal) with latex beads

For rapid detection, 20 μl of conjugated latex beads are mixed with 20 μl of diluted or non-diluted sera or 20 μl of whole blood. After 5 to 10 minutes, as shown in Figures 2 (with serum) and 3 (with blood), a ring is observed characteristic of the positivity of the reaction. Otherwise the reaction is considered negative.

Example 3: Diagnostic Test Associating an ELISA Test for the Diagnosis of Stage

The results obtained are illustrated by Fig. The use of ELISA plates with sera from patients with HAT shows an increase in levels of anti-WE antibodies during the passage in stage 2 (neurological damage).

Bibliographical references

- Semballa et al, experimental Parasitology 115 (2007) 173-180

- Okomo-Assoumou et al, Ann. J. Too much. Med. Hyg., 1995, pp. 461-467 tracking of human or animal trypanosomosis.

Claims

1 - An antigenic construct containing a tryptophan epitope, characterized in that it is formed of a tryptophan motif W, or a peptide of 3 or 4 amino acids comprising a motif W, coupled with glutaraldehyde.  2 - antigenic construct according to claim 1, characterized in that said peptide corresponds to the sequence SEQ ID No. 1 CxW (-y), in which "x" represents K, A, S, V, T, R, I, E, D, N or G, and "y" represents D, S, T, E, N, K, G, Q, R or I, one of "x" or "y" being absent.  3 - antigenic construct according to claim 1 or 2, characterized in that said peptide corresponds to the sequence SEQ ID No. 2 CKWD.  4 - antigenic construct according to any one of claims 1 to 3, characterized in that said coupling product is grafted to latex beads or poly-L-lysine.  5 - antigenic construct according to any one of claims 1 to 4, characterized in that said coupling product is grafted onto ELISA plates.  6 - Construction according to claim 5, characterized in that the ELISA plates comprise poly-L-lysine.  7 - Screening kit for human or animal trypanosomosis, characterized in that it comprises  at least one antigenic construct according to any one of claims 1 to 5, optionally  one or more reagents for the antigen / antibody reaction and / or buffer solutions and / or reagents for the detection, quantification or visualization of antigen / antibody complexes when present.  8 - Kit according to claim 7, characterized in that it further comprises the reagents for performing an enzyme immunoassay, including an indirect ELISA or an ELISA inhibition test, in competition with an anti-monoclonal antibody. - WE with anti-WE antibodies infected host sera.  9 - Kit according to claim 7 or 8, characterized in that the antigenic construction is fixed on a support.  10- Kit according to any one of claims 7 to 9, characterized in that it is used for the screening of African trypanosomiasis human or animal. 11 - Method for screening for HAT or TAA, characterized in that it comprises . contacting a serum sample of a patient or an animal with an antigenic construct as defined above, advantageously using a kit according to any one of claims 7 to 10,  . revealing an immunological reaction of the antigen-antibody type.