Serological marker for the latent form of toxoplasmosis
In the present invention, inventors report the characterization of BCLA (Brain Cyst Load-associated Antigen), a protein exclusively expressed during the bradyzoite stage of the parasite. In cysts directly purified from the brain of mice, the protein is distributed within and at the surface of the cyst. ELISA antibody capture using a combination of serologically reactive BCLA peptides and a recombinantly expressed c-terminal domain (rBCLA) constitutes an efficient serological marker of latent infection with a high sensitivity that is clearly and exclusively correlated with the presence of cysts in the brain of mice. Antibodies directed against BCLA antigen have been detected in human patients with enriched titers in patients qualified as seropositive to Sag1 or tachyzoite related antigens. Further correlation in humans between anti-BCLA IgG synthesis and cysts is brought by significantly stronger recorded titers in pathological panels strongly related to the presence of cyst. Furthermore, newborn infants with a confirmed congenital toxoplasmosis display significantly higher anti-BCLA IgGs at birth when compared to their mother, suggesting a specific in-utero neosynthesis of such IgGs. Thus the invention relates to a new Toxoplasma gondii protein, hereafter referred as BCLA, a new serological marker whose expression is restricted to the latent form of Toxoplasmosis (bradyzoite/cyst). This specific protein and its antigenic fragments can be used to detect autoantibodies in the sera of patient for the diagnosis of the latent form of Toxoplasmosis. The invention also relates to derived antibodies, generated by BCLA immunisation that specifically binds this new protein.
1 . A method for diagnosing or confirming a diagnosis of and treating a latent form of Toxoplasmosis in a patient in need thereof, wherein the latent form of Toxoplasmosis is caused by infection with type I or type II Toxoplasma gondii , comprising:
a) obtaining a biological sample from the patient,
b) detecting via an immunoassay, in the biological sample, antibodies toward a Toxoplasma gondii polypeptide selected from the group consisting of
i) the Toxoplasma gondii polypeptide BCLA amino acid sequence (SEQ ID NO: 1);
ii) the BCLA C-terminal antigenic domain amino acid sequence (SEQ ID NO:2);
iii) a BCLA internal repeated domain amino acid sequence selected from the group consisting of:
(SEQ ID NO: 4)
TgR1,
(SEQ ID NO: 5)
TgR2,
(SEQ ID NO: 6)
TgR3,
(SEQ ID NO: 7)
TgR4,
(SEQ ID NO: 8)
TgR5,
(SEQ ID NO: 9)
TgR6,
(SEQ ID NO: 10)
TgR7,
(SEQ ID NO: 11)
TgR8,
(SEQ ID NO: 12)
TgR9,
(SEQ ID NO: 13)
tgR10,
(SEQ ID NO: 14)
TgR11,
(SEQ ID NO: 15)
TgR12
and
(SEQ ID NO: 16)
TgR13;
and
iv) a polypeptide selected from the group consisting of SEQ ID Nos 55-56, and
c) treating the patient with a folic acid antagonist and/or an antibiotic.
2 . An in vitro method for diagnosing or confirming a diagnosis of and treating congenital Toxoplasmosis in a patient in need thereof, wherein the congenital Toxoplasmosis is caused by infection with type I or type II Toxoplasma gondii , comprising:
a) obtaining a biological sample from the patient,
b) detecting via an immunoassay, in the biological sample, antibodies toward a Toxoplasma gondii polypeptide selected from the group consisting of
i) the Toxoplasma gondii polypeptide BCLA amino acid sequence (SEQ ID NO: 1);
ii) the BCLA C-terminal antigenic domain amino acid sequence (SEQ ID NO:2);
iii) a BCLA internal repeated domain amino acid sequence selected from the group consisting of:
(SEQ ID NO: 4)
TgR1,
(SEQ ID NO: 5)
TgR2,
(SEQ ID NO: 6)
TgR3,
(SEQ ID NO: 7)
TgR4,
(SEQ ID NO: 8)
TgR5,
(SEQ ID NO: 9)
TgR6,
(SEQ ID NO: 10)
TgR7,
(SEQ ID NO: 11)
TgR8,
(SEQ ID NO: 12)
TgR9,
(SEQ ID NO: 13)
TgR10,
(SEQ ID NO: 14)
TgR11,
(SEQ ID NO: 15)
TgR12 and
(SEQ ID NO: 16)
TgR13;
and
iv) a polypeptide selected from the group consisting of SEQ ID Nos 55-56, and
c) treating the patient with at least one folic acid antagonist and/or at least one antibiotic.
3 . The method according to claim 1 wherein said biological sample is a fluid sample.
4 . A method for treating a patient infected with latent form of Toxoplasmosis caused by infection with type I or type II Toxoplasma gondii , comprising:
a) detecting via an immunoassay, in a biological sample obtained from the patient, antibodies toward a Toxoplasma gondii polypeptide selected from the group consisting of
i) the Toxoplasma gondii polypeptide BCLA amino acid sequence (SEQ ID NO: 1);
ii) the BCLA C-terminal antigenic domain amino acid sequence (SEQ ID NO:2);
iii) a BCLA internal repeated domain amino acid sequence selected from the group consisting of:
(SEQ ID NO: 4)
TgR1,
(SEQ ID NO: 5)
TgR2,
(SEQ ID NO: 6)
TgR3,
(SEQ ID NO: 7)
TgR4,
(SEQ ID NO: 8)
TgR5,
(SEQ ID NO: 9)
TgR6,
(SEQ ID NO: 10)
TgR7,
(SEQ ID NO: 11)
TgR8,
(SEQ ID NO: 12)
TgR9,
(SEQ ID NO: 13)
TgR10,
(SEQ ID NO: 14)
TgR11,
(SEQ ID NO: 15)
TgR12 and
(SEQ ID NO: 16)
TgR13;
and
iv) a polypeptide selected from the group consisting of SEQ ID Nos 55-56, and
b) administering to the patient at least one folic acid antagonist and/or at least one antibiotic, or a pharmaceutical composition comprising the at least one folic acid antagonist and the at least one antibiotic compound.
5 . The method of claim 1 , wherein the at least one folic acid antagonist is pyrimethamine and the at least one antibiotic is sulfadiazine or spiramycin.
6 . The method of claim 2 , wherein the at least one folic acid antagonist is pyrimethamine and the at least one antibiotic is sulfadiazine or spiramycin.
7 . The method of claim 4 , wherein the patient is asymptomatic.