Identification and characterization of a Fc receptor activity on the Toxoplasma gondii tachyzoite.

The Immunoglobulin (Ig) binding capacity of Toxoplasma gondii tachyzoites was investigated using fluorescence flow-cytometry analysis. Polyclonal mouse, human and rat immunoglobulins without specific anti-Toxoplasma activity bound to parasites in a concentration-dependent manner, saturating them at circulating serum concentrations. The immunoglobulin class and subclass specificity of binding was investigated using irrelevant monoclonal antibodies. IgM, IgA and IgG reacted with the parasite membrane. The attachment of mouse IgM to the parasite surface was hampered by mouse IgG1, IgG2a, IgG2b and IgG3. The binding of mouse IgG was proportionally reduced with increasing concentrations of mouse monoclonal IgM. The binding of murine immunoglobulin was diminished when in presence of human IgG. Purified Fc- but not Fab portions of immunoglobulins, fixed to parasites. Using labelled calibrated beads, the Ig binding capacity of parasites was estimated to be 6900 +/- 500 sites per tachyzoite. The Kd of the T. gondii Fc Receptor (FcR) activity was determined at 1.4 +/- 0.1 microM (mean +/- SEM). Such FcR activity was reduced by phospholipase C, trypsin and pronase treatment of the parasites. These data show a low affinity FcR activity on T. gondii tachyzoites which recognizes Ig of different species and isotypes and is likely supported by a glycosyl-phosphatidylinositol (GPI)-anchored surface protein of the parasite.

Serodiagnosis of toxoplasmosis by using a recombinant form of the 54-kilodalton rhoptry antigen expressed in Escherichia coli.

A 330-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii 54-kDa rhoptry protein (ROP2) was expressed in Escherichia coli as a fusion polypeptide containing a 48-amino-acid sequence derived from phage lambda protein Cro and E. coli protein LacI followed by six consecutive histidyl residues. Metal chelate affinity chromatography provided an easy way to isolate the recombinant product in a highly purified form (> 95%). When this material was used to develop an immunoglobulin G (IgG) enzyme-linked immunosorbent assay for diagnosis of toxoplasmosis, the test reached a sensitivity of 89%. The sensitivity of the assay was similar whether the sera contained T. gondii-specific IgM or were devoid of such IgM. It was also found that ROP2 is a conserved antigen since antibodies against the recombinant antigen could be detected in mice experimentally infected with 11 independent T. gondii isolates originating from infected human tissues tested. Thus, the 54-kDa rhoptry antigen could advantageously complement other previously described T. gondii antigens for the development of more-sensitive and more-informative recombinant antigen-based tests for toxoplasmosis diagnosis.

Human T cell clone identifies a potentially protective 54-kDa protein antigen of Toxoplasma gondii cloned and expressed in Escherichia coli.

Protective immunity against Toxoplasma gondii is recognized to be cell mediated and IFN-gamma is considered to be the major mediator of resistance. Thus, protective Ag of the parasite must induce IFN-gamma-producing T cells. In order to identify such Ag, we have constructed a T. gondii cDNA library in the cloning/expression vector lambda gt11, screened this library with a pool of sera of immune donors, and further screened the set of selected recombinant Ag using, as probe, a T. gondii-reactive T-cell clone (TCC) derived from an infected/immune individual and producing a high level of IFN-gamma. One recombinant Ag was shown to induce TCC proliferation and was characterized. The corresponding mature T. gondii Ag has an apparent molecular mass of 54 kDa and the sequence of the cDNA clone suggests that it is membrane associated. The epitope defined by the TCC on this Ag was found to be present in three Toxoplasma strains independently of their phenotype (virulent or cyst forming). Recognition of this Ag by the TCC was shown to be restricted by HLA-DPw4, the most frequent allele in the Caucasian population (approximately 40%). The use of this Ag as a vaccine component is proposed.

Prenatal diagnosis of congenital toxoplasmosis.

Prenatal diagnosis of congenital toxoplasmosis was attempted in 50 pregnant women at risk for giving birth to an affected child. Fifteen of these patients seroconverted during pregnancy and 35 had a high initial antibody level in their first serum sample. Prenatal diagnosis consisted of a combination of ultrasound screening, amniocentesis, and funipuncture at about 20 weeks' gestation. Diagnosis of congenital toxoplasmosis was based on a positive toxoplasma culture of amniotic fluid or fetal blood and on the presence of specific immunoglobulin M antibodies in fetal blood. In addition, alterations in fetal hematology, cellular immunology, and fetal liver tests were indicative of infection. Fetal infection was detected in six fetuses; two died in utero as a consequence of the infection and four were born after 37 weeks' gestation. Despite antibiotic treatment with pyrimethamine and sulfadiazine, one child has internal hydrocephalus and chorioretinitis and another has unilateral chorioretinitis. In the two other children, the disease is still subclinical. Of the 44 children born after a negative prenatal diagnosis, 35 have reached the age of 1 year; toxoplasma antibodies have disappeared in all of them. Investigation of the remaining nine children showed a decrease in toxoplasma antibodies, suggesting that none of them are affected. Prenatal diagnosis was never associated with fetal loss, and premature delivery occurred in only two cases. We conclude that prenatal diagnosis of congenital toxoplasmosis is safe and reliable.

Monoclonal antibodies identify new Toxoplasma gondii soluble antigens.

In order to characterize Toxoplasma gondii antigens, we have produced a panel of monoclonal antibodies specific for the parasite. A total of 22 hybridomas were derived from the spleen cells of mice immunized either with a 100,000 g supernatant of a sonicate from the RH strain (called F3), or chronically infected with the Wiktor or the 76K strain. Except for one hybridoma producing an IgM, all the hybridomas derived from mice immunized with F3 produced IgG1 antibodies while those obtained from chronically infected mice produced antibodies belonging to the IgG2b, IgG2a and IgM subclasses. Western-blot analysis showed that the panel of monoclonal antibodies defines at least 7 distinct antigens or antigen families. An antigen of apparent Mw 25 kD present exclusively in the 100,000 g supernatant of the T. gondii sonicate was recognized by the majority of monoclonal antibodies derived from mice immunized with the F3 fraction. Two other antigens of apparent Mw 27 kD and 29 kD present in the soluble and insoluble fractions of the sonicate were also identified. Monoclonal antibodies against the previously described 21 kD and 31 kD surface antigens and belonging to the IgG2a but also to the IgG1 subclasses were able to mediate lysis of the parasite in the presence of human non immune serum. The 22 monoclonal antibodies did not identify antigenic differences between the two independently isolated RH and Wiktor strains.

[Efficacy of treatments in experimental toxoplasmosis]. / Etude de l'efficacité des traitements dans la toxoplasmose expérimentale.

Experimental study of the intraocular penetration of six drugs with therapeutic effect on toxoplasma. A direct and an indirect method are used. The penetration of six drugs administered sub-conjunctival, retrobulbar and intramuscular in one shot is measured in the anterior chamber, the vitreous and the retina-choroid of a healthy rabbit eye. The best results are obtained for: spiramycin, trimethoprim-sulfamethoxazole and clindamycin. The therapeutic efficiency of four drugs on infected rabbit eyes with toxoplasma are studied using an indirect method: pyrimethamine and especially doxycycline have a positive effect.

Impact of primary prevention on the incidence of toxoplasmosis during pregnancy.

Until now, it was assumed that primary prevention of congenital toxoplasmosis was possible by means of specific hygienic measures. A prospective survey of pregnant women was made at a hospital in Brussels over the period 1979-1986 to assess the impact of such a prevention program. In the first study period (1979-1982), when no prophylactic measures were taught, 2986 consecutive women demonstrated a seroconversion rate of 1.43% among the nonimmunized subjects; 1.07% of the seropositive patients had high antibody levels in their first serum sample. In the second study period (1983-1986), all 3563 patients were instructed to adopt prophylactic measures. The seroconversion rate in seronegative patients and the percentage of patients with high initial antibody level were 0.95 and 1.26%, respectively. Although the percentage of seroconversion was reduced by 34% in the second study period, this difference did not attain significance. These results indicate that the impact of a primary prevention program aimed at reducing congenital toxoplasmosis is limited.

A new cryopathic syndrome.

One case of a syndrome simulating a systemic disease is reported. An HLA-B, patient presented with apparent toxoplasmosis, retinal vein occlusion, autoantibodies against cephalin, contact factors and lipoproteins, immune complexes, rheumatoid factor, and cryoprecipitate. A representation of the immunological mechanisms involved is proposed.

[Urinary infections in children]. / Infection urinaire chez l'enfant.

The E. Coli antibody response was studied in 81 infants and children with urinary tract infection. An haemagglutination technique was used. The antigen used was the bacteria present in urine. The correlation between the site of infection and response of haemagglutinating antibodies was statistically demonstrated. In some cases, the antibody titers were followed for several weeks, allowing an appreciation on the effectiveness of treatment. On the other hand, the correlation between serology and some of the results emphasize the interest of these tests in the general investigation of urinary tract infection in childhood.

[Indirect flourescent antibody test in toxoplasmosis. Advantage of the use of fluorescent anti-IgG conjugate (author's transl)]. / La réaction d'immuno-fluorescence indirecte dans la toxoplasmose. L'avantage de l'emploi du conjugué fluorescent anti-IgG

The indirect fluorescent antibody (IFA) test effected with anti-human conjugate gives appreciably equivalent results to those obtained with the dye (SABIN-FELDMAN) test, but weak serum dilutions may raise non specific reactions, due to the presence of substances possessing the IgM antibody properties. Those substances are not present in the serum of children during the first months of their life. The application of an anti-IgG conjugate allows to prevent those unspecific reactions. The application of the modified IFA test for detecting the toxoplasmic infection during pregnancy and the use of the REMINGTON test for the early diagnosis of the congenital toxoplasmosis are discussed.