F. Santoro

Induction of a protective antibody-dependent response against toxoplasmosis by in vitro excreted/secreted antigens from tachyzoites of Toxoplasma gondii

Summary Toxoplasma gondii is a worldwide protozoan parasite which causes severe disease in congenitally infected children and in immunocompromised patients. Besides the well‐defined cytoplasmic and membrane antigens of tachyzoites, we felt that excreted/secreted antigens could play a major role in the immune response. We first report the development of a well‐controlled procedure for obtaining tachyzoite excreted/secreted antigens (E/SA) in cell‐free incubation media. The E/SA immunogenic in human, rat and mouse toxoplasmosis were then characterized. The major E/SA recognized by human sera from the chronic phase of toxoplasmosis had molecular weights of 108, 97, 86, 69,60, 57, 42, 39, 28.5, 27 and 26 kD. When injected into +/+ Fischer rats, E/SA elicited high antibody titres. In addition, passive transfer of these sera to highly susceptible nu/nu littermates induced a significant degree of protection towards the virulent RH strain of T. gondii. This work, which demonstrates the key role played by E/SA in the protective immune response, suggests that these antigens should be of value both for diagnostic purposes and for the development of new strategies for immunization against toxoplasmosis.

Development of metacyclic Leishmania promastigotes is associated with the increasing expression of GP65, the major surface antigen

Summary Using immunofluorescence techniques and flow microfluorometry analysis, we have demonstrated that the binding of a monoclonal antibody (VD5/ 25) produced against GP65, the major surface antigen of Leishmania braziliensis, increased on the surface of stationary‐phase promastigotes from all the New World Leishmania species causing mucocutaneous or cutaneous disease as compared with the log‐phase parasites. In addition, a sequential development of Leishmania amazonensis promastigotes from a non‐infective to an infective stage was demonstrated. Indeed, promastigotes in the stationary phase (days 6–7) were found to be far more infective than those in the logarithmic phase of growth (day 3) both in vitro for mouse peritoneal macrophages and in vivo for BALB/c mice. The intracellular survival and multiplication of L. amazonensis were significantly inhibited when infective promastigotes were treated with the VD5/25 monoclonal antibody. The increasing expression of GP65 on the promastigote surface may thus contribute to Leishmania infectivity. This seems to represent a characteristic mechanism applicable to all New World Leishmania species studied.

The immunodominant epitope of the major membrane tachyzoite protein (P30) of Toxoplasma gondii

Summary Several physicochemical characteristics of the repeated epitope of the major surface protein (P30) of Toxoplasma gondii were investigated with an anti‐P30 mAb by two different methods: (a) a one‐site/inhibition assay that detects molecules containing single or multiple epitopes and (b) a two‐site/one antibody radiometric assay that is only effective with antigenic molecules containing two or more identical epitopes. Using both techniques, the repeated epitope within purified P30 was stable after 1 h at 63°C, but labile at 100°C. It was also resistant to successive freezing and thawing, and not affected after one year at – 70°C. Lyophilization and acidic or basic treatment had no effect. This epitope was also resistant to 20% trichloroacetic acid precipitation (activity recovered in the pellet) and to precipitation with cold acetone.

Use of a monoclonal antibody in a double-sandwich ELISA for detection of IgM antibodies to Toxoplasma gondii major surface protein (P30)

A double-sandwich ELISA, developed for detection of IgM antibodies to the major surface protein of Toxoplasma gondii (P30), is proposed for the diagnosis of acute acquired toxoplasmosis. The method is based on the capture of serum IgM antibodies, which are revealed indirectly by the sequential addition of a Toxoplasma extract and a beta-galactosidase-conjugated anti-P30 monoclonal antibody. All 57 patients tested with serological characteristics of recently acquired toxoplasmosis showed high levels of IgM anti-P30 antibodies. In addition, 5 out of the 24 patients with chronic toxoplasmosis and all 7 patients with a clinical acute infection in which the classical IgM serology was negative, also presented significant anti-P30 IgM antibodies. Patients with either rheumatoid factor or antinuclear antibodies were all negative. In view of its simplicity, specificity and sensitivity, this method is recommended for the current diagnosis of T. gondii infection.

Infectivity of Leishmania promastigotes is associated with surface antigenic expression

Differentiation between a non-infective and an infective Leishmania promastigote population was demonstrated. Promastigotes in the stationary phase (day 5) were found to be highly infective in vitro to BALB/c mouse peritoneal macrophages, compared with those of the logarithmic phase (day 3). The infective promastigotes showed surface antigenic determinants different from non-infective ones. Polyclonal anti-3 day and anti-5 day antibodies were bound specifically to the surface of corresponding promastigotes in both SRIA and IFAT; no strong cross-reactions were observed otherwise. Also, polyclonal anti-5 day but not anti-3 day antibodies recognized efficiently the antigenic molecules on the surface of late stage (day 7) sandfly promastigotes. This clearly indicates the appearance of new antigenic molecules on the surface of infective promastigote forms. Intracellular multiplication of Leishmania was significantly inhibited by anti-5 day antibodies compared with anti-3 day antibodies. The presence of new surface molecules on late stage promastigotes may contribute to Leishmania infectivity.

Killer cells in human cutaneous leishmaniasis

In French Guiana, American cutaneous leishmaniasis is localized in the skin. The host response appears to be effective since few extra- or intracellular organisms can be found in tissue lesions, and we never observed any cutaneous dissemination or visceral involvement. However, this response is not fully effective since lesions may last for months. By using immunoperoxidase techniques and monoclonal antibodies directed against various cell populations, we examined the local immune response in skin biopsies. We found a high percentage of cells with the K/NK phenotype, a variable but usually high percentage of cells with the T cell phenotype bearing TAC receptors, and moderate numbers of monocytes and B cells. These results suggest that K/NK cells could play a role in the local control of parasite dissemination.

Identification of a major 72 kilodalton surface antigen in twelve isolates of Leishmania braziliensis braziliensis

The study of the surface antigens of Leishmania braziliensis braziliensis revealed a great homogeneity among ten strains isolated from Bolivia and two reference strains from Brazil and Belize. A 72 kDa major protein, present in all L. b. braziliensis strains, was recognized by both cutaneous and mucocutaneous human sera, but was not recognized by Kala-azar and chagasic sera. No cross-reactive antigens were found among strains of Leishmania braziliensis guyanensis, Leishmania braziliensis panamensis, Leishmania mexicana amazonensis and Leishmania donovani chagasi testing these strains with hamster and human anti-L. b. braziliensis sera. Moreover, these strains possessed major antigens with molecular weights different from those of L. b. braziliensis strains. A microheterogeneity of L. b. braziliensis surface antigens was detected for the high molecular weight antigens and seemed to be related to the isoenzymic microheterogeneity.

Schistosoma mansoni: ultrastructural damages due to complement on schistosomula in vitro.

Abstract The ultrastructural damage induced by complement in vitro on the schistosomula of Schistosoma mansoni was studied using transmission and scanning electron microscopy. The sequence of events which leads to the killing of the schistosomula is as follows: (a) the lytic activity starts at the anterior end of the schistosomula; (b) lesions progress simultaneously in two distinct directions: from the anterior to the posterior end, and from the outer membrane to the muscle layer; (c) “bubbles” appear around parasite which might correspond to increased membrane permeability; (d) the lytic activity of the late complement components occur in the syncytium; (e) the schistosomula lose their tegument completely, exposing the muscle layer. These findings and our previous work suggest that the activation of the alternate pathway and late complement components is sufficient, without antibodies or cells, to kill schistosomula in vitro.

Schistosoma mansoni: role in vivo of complement in primary infection of mice.

Abstract The role of complement in the control of the primary Schistosoma mansoni infection in mice was investigated in vivo . The number of recovered adult schistosomes 6–7 weeks postinfection was used as a parasitological criterion of immunity. No significant difference in the worm burden was observed between C5-sufficient and C5-deficient mice. In contrast, when cobra venom factor (CVF) was injected into normal or C5-deficient mice 24 hr before challenge, a significant increase of the worm burden was noticed in comparison to the untreated mice. These results indicated that, although C5 and probably the late complement components are not essential for the control of the primary infection, the alternative pathway and some of its components are involved. In fact, the injection of C3 2 hr before infection of CVF-treated mice completely restored the immunity. A role for C3, in association with effector cells, in the nonspecific immunity occurring in the first hours after a primary S. mansoni infection is suggested.

Schistosoma mansoni anticomplementary antigens (SACA). Detection in schistosomula and adult worms

In previous studies we have shown that schistosomula of Schistosoma mansoni are able to activate complement (C), in the absence of antibodies, by both the classical (CP) and the alternative C pathway (AP). In the present work we have demonstrated that certain factors present in the antigen extract of schistosomula and adult worms also showed an anticomplementary activity. These schistosome anticomplementary antigens (SACA) were found in the low molecular weight fraction (< 35,000 daltons) of the whole extract of adult schistosomes and were able to deplete C through both the CP and the AP.