Deficient Humoral Responses Underlie Susceptibility to Toxoplasma gondii in CD4-Deficient Mice

Resistance to infection with Toxoplasma gondii was studied in mice lacking CD4 expression. Such mice developed more brain cysts and survived for a shorter time than did wild-type controls after peroral infection with ME49 cysts. After immunization with the ts-4 strain of T. gondii, CD4-deficient mice exhibited impaired resistance to a challenge infection with virulent RH tachyzoites. Thus, deficient CD4 expression increases the susceptibility of mice to a primary peroral T. gondii infection with cysts and impairs their ability to be successfully vaccinated. CD8(+) T cells from blood or spleens of Toxoplasma-infected, CD4-deficient mice expressed markers of activation at frequencies similar to those of infected wild-type mice. Production of IFN-gamma in vitro was moderately depressed, and levels of Toxoplasma-specific immunoglobulin G2a in serum were substantially lower than in wild-type mice. Administration of Toxoplasma-immune serum to ts-4-vaccinated CD4-deficient mice significantly improved their resistance to RH challenge. Also, the survival of CD4-deficient mice chronically infected with ME49 was significantly prolonged by administration of immune serum. These results demonstrate that in addition to CD8(+) T cells and IFN-gamma, which are known to be critical for resistance, CD4(+) cells also contribute significantly to protection against chronic T. gondii infections and against challenge infections with highly virulent tachyzoites in immunized mice via their role as helper cells for production of isotype-switched antibodies.     

Antibody responses to Toxoplasma gondii in sera, intestinal secretions, and milk from orally infected mice and characterization of target antigens.

Toxoplasma gondii-specific antibody responses in serum, intestinal secretions, and milk were identified with an enzyme-linked immunosorbent assay following a single oral infection of mice with strain 76K cysts of T. gondii. Immunoglobulin A (IgA) production began during week 2 of infection in serum and milk and during week 3 of infection in intestinal secretions and persisted in all three throughout the experiment (17 weeks). IgG but not IgM antibodies were detected in intestinal secretions later in the infection. Serum and milk IgG and IgM production began at the same time after infection as did the IgA response. In Western blotting (immunoblotting), intestinal IgA antibodies were shown to react with antigens comigrating with the T. gondii proteins p22, p23, p30, and p43, the 28-kilodalton antigen, and the 55- and 60-kilodalton rhoptry proteins, as recognized by specific monoclonal antibodies. Milk IgA antibodies reacted with antigens comigrating with p30 and p43. Most of the antigens recognized by IgA antibodies were also detected by IgG antibodies. IgA antibodies from all three biological samples detected the same major T. gondii antigens; thus, there was apparently no specific antibody production unique to one locality.     

The MIC3 gene of Toxoplasma gondii is a novel potent vaccine candidate against toxoplasmosis

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The micronemal protein MIC3, which is a potent adhesin of T. gondii, could be a significant candidate vaccine against toxoplasmosis. In this study, all CBA/J mice intramuscularly vaccinated with a plasmid encoding the immature form of the MIC3 protein (pMIC3i) produced specific anti-MIC3 immunoglobulin G (IgG) antibodies, and their sera displayed high antibody titers. This response was increased by the coadministration of a plasmid encoding the granulocyte-macrophage colony-stimulating factor (pGM-CSF). Similarly, a specific and significant cellular immune response was obtained in mice immunized with pMIC3i, and this response was markedly enhanced by pGM-CSF coadministration. The cellular immune response was associated with the production of gamma interferon IFN-gamma and interleukin-2 (IL-2), indicating that this was a Th1-type response. This was confirmed by the production of large amounts of IgG2a. Mice immunized with pMIC3i displayed significant protection against an oral challenge with T. gondii 76K cysts, exhibiting fewer brain cysts than did the control mice. Coadministration of pGM-CSF enhanced this protection. In conclusion, this study describes the design of a potent DNA vaccine encoding the novel T. gondii target antigen, MIC3 protein, that elicits a strong specific immune response as well as providing effective protection against T. gondii infection. In the attempt to achieve complete protection against toxoplasmosis, MIC3 is a good candidate vaccine which could be combined with other relevant and previously described candidates, such as SAG1 and GRA4.     

Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection.

SAG1 protein of Toxoplasma gondii was evaluated as a protective antigen in mucosal immunization with cholera toxin as an adjuvant. CBA/J mice intranasally immunized with a combination of SAG1 and cholera toxin exhibited significantly fewer cysts in the brain after oral infection with the 76K strain of T. gondii than control mice. This acquired protection lasted at least 5 months. Protected mice developed high levels of serum anti-SAG1 immunoglobulin G antibodies as well as an enhanced systemic cellular response, as assessed by the proliferation of splenocytes in response to SAG1 restimulation in vitro. This cellular proliferation was associated with an increase of interleukin-2 and interleukin-5 synthesis and with barely detectable gamma interferon production. Splenic immune T cells were shown to convey modest protection to recipients against development of brain cysts following oral infection with T. gondii. Significant production of anti-SAG1 immunoglobulin A was induced in intestinal secretions of protected mice. These results indicate that intranasal immunization with SAG1 and cholera toxin can induce mucosal and systemic immune responses and affords partial and long-lasting resistance against the establishment of chronic toxoplasmosis.     

Toxoplasma gondii excretory secretory antigenic proteins of diagnostic potential

Infection with Toxoplasma gondii is widespread and important in humans, especially pregnant women and immunosuppressed patients. A panel of tests is usually required for diagnosis toxoplasmosis. Excretory secretory antigen (ESA) is highly immunogenic, and thus it is a good candidate for investigation into new infection markers. ESA was prepared from tachyzoites of RH strain of T. gondii by mice intraperitoneal infection. Sera were obtained from several categories of individuals who differed in their status of anti-Toxoplasma IgM, IgG and IgG avidity antibodies. The ESA was subjected to SDS-PAGE, two-dimensional gel electrophoresis and Western blot analysis. Antigenic bands of approximate molecular weights of 12, 20 and 30 kDa, when probed with anti-human IgM-HRP and IgA-HRP, showed good potential as infection markers. The highest sensitivity of the bands was 98.7% with combination of IgM and IgA blots with sera of patients with anti-Toxoplasma IgM+ IgG+. The specificities were 84% and 70% with sera from other infections and healthy controls in IgM blots and IgA blots respectively. By mass spectrometry, the 12 kDa protein was identified as thioredoxin. The two top proteins identified for 20 kDa molecule were microneme protein 10 and dense granule protein 7; whereas that for 30 kDa were phosphoglycerate mutase 1 and phosphoglycerate mutase.     

Comparative study of tissue culture and mouse inoculation methods for demonstration of Toxoplasma gondii.

Two methods for the isolation of Toxoplasma gondii were analyzed and compared. Bradyzoites or tachyzoites of three strains of T. gondii were injected into mice and introduced in parallel onto MRC5 fibroblasts cultured on cover slips. In the cultures, the parasites were more readily identified by an indirect immunofluorescence assay than by examination of unstained or Giemsa-stained cultures. With the RH strain, the tachyzoites replicated actively, and large foci of parasites were observed in 24 h. The bradyzoites or tachyzoites of the other strains could also be cultivated, but grew rather slowly; 2 days after inoculation, early stages of multiplication could be observed: from day +4, Toxoplasma clusters or foci were easily identified at a x100 magnification. The course of infection in mice was greatly dependent on the virulence of the strain and on the parasitic stage inoculated. In the chronically infected mice, evidence of Toxoplasma infection was only detected 45 days after inoculation through the demonstration of cysts in the brain or the presence of specific antibodies in the serum. The mean ratio of infected mice and positive cultures was compared in relation to the inoculum size. The tissue culture method was found to be at least as sensitive as mouse inoculation. Since Toxoplasma organisms may be isolated within a few days in tissue culture, it is proposed that this method should be used when early isolation of the parasite is crucial for the diagnosis of toxoplasmosis.     

Protective Role for Interleukin-5 during Chronic Toxoplasma gondii Infection

To investigate the role of interleukin-5 (IL-5) during Toxoplasma gondii infection, IL-5 knockout (KO) mice and C57BL/6 control mice were infected intraperitoneally with ME49 cysts and the course of infection was monitored. The mortality rate during chronic infection was significantly greater in IL-5-deficient animals, and consistent with this finding, the KO mice harbored a greater number of brain cysts and tachyzoites than did their wild-type counterparts. Although the IL-5 KO animals did not succumb until late during infection, increased susceptibility, as measured by accelerated weight loss, was detectable during the acute stages of infection. The amounts of total immunoglobulin (Ig), IgM, and IgG2b were comparable in both strains, while the amount of IgG1 was much smaller in IL-5 KO mice. Spleen cell production of IL-12 in response to T. gondii antigen was approximately threefold lower in the KO strain, and this decrease correlated with a selective loss of B lymphocytes during culture. A link between the presence of B cells and augmented IL-12 production was established by the finding that after removal of B cells with monoclonal antibody and complement, wild-type- and KO-derived cells produced equivalent levels of IL-12 in response to T. gondii antigen. These results demonstrate a protective role of IL-5 against T. gondii infection and suggest that IL-5 may play a role in the production of IL-12.     

Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene

Infection with the protozoan parasite Toxoplasma gondii is transmitted to humans from infected animals by tissue cysts and oocysts excreted by cats. Immunization with inactivated parasites or recombinant proteins has at best shown partial protection. We constructed a plasmid expressing the SAG1 surface antigen of T. gondii, p1tPASAG1, and showed that animals immunized with the plasmid produce anti-SAG1 antibodies which recognize the native SAG1. Mice immunized with p1tPASAG1 showed 80 to 100% protection against challenge with the non-cyst-producing, virulent RH isolate, compared to an 80% mortality in mice immunized with empty plasmid, which is the greatest efficacy of any vaccine against T. gondii produced so far. The SAG1 molecule was analyzed for potential cytotoxic T-lymphocyte (CTL) epitopes, and four peptides with the best fit were synthesized. The ability of the peptides to stimulate gamma interferon production by CD8(+) T cells from p1tPASAG1-immunized mice was tested in an ELISPOT assay, and one new CTL epitope was identified. Adoptive transfer of CD8(+) T cells from p1tPASAG1-immunized to na?ve mice showed partial protection. In conclusion, DNA vaccination with p1tPASAG1 gave effective protection in mice against T. gondii infection and the protection could be adoptively transferred by purified CD8(+) T cells.     

Detection of specific immunoglobulin E in patients with toxoplasmosis.

An immunocapture assay was developed to detect Toxoplasma gondii-specific immunoglobulin E (IgE) in sera from adults with acute acquired infection or reactivation and from babies with congenital toxoplasmosis. The components of this assay were monoclonal antibody to human IgE, samples from patients, and T. gondii tachyzoites treated with Formalin. When T. gondii-specific IgE antibodies were present, visually detectable agglutination occurred. Sera, umbilical cord blood, fetal blood, cerebrospinal fluid, and amniotic fluid were tested by this method. Specific IgE antibodies were detected in sera from 25 (86%) of 29 adults who developed specific IgG antibody during pregnancy or had specific IgA and IgM antibodies. Specific IgE was present early during infection, at the time that IgM antibodies were present, and slightly preceding the presence of specific IgA antibodies. In 23 patients tested serially, IgE antibodies never persisted for longer than 4 months. No nonspecific anti-T. gondii IgE was detected in sera from uninfected individuals. Maternal IgE antibodies did not cross the placenta. In sera of patients with congenital toxoplasmosis, specific IgE antibodies were found at birth, during the first year of life, and during immunologic recrudescence following discontinuation of pyrimethamine-sulfonamide therapy. The IgE immunocapture assay is simple to perform. It is especially useful for determining when T. gondii was acquired by recently infected pregnant women.     

Immune responses and resistance to Toxoplasma gondii in mice immunized with antigens of the parasite incorporated into immunostimulating complexes.

Immunostimulating complexes were prepared with antigens extracted from tachyzoites of Toxoplasma gondii and were used to immunize mice. The major antigens incorporated into the immunostimulating complexes were the P30 and P22 antigens and an antigen with an approximate molecular weight of 6,000. Other antigens of molecular weights above 30,000 were also present. High antibody titers to T. gondii antigens and a delayed-type hypersensitivity reaction were noted for the immunized mice. Challenge of these mice with tachyzoites injected interperitoneally or with oocysts administered orally resulted in a statistically significant (P < 0.001) conditional probability of survival compared with that of controls. In contrast, the differences between immunized mice and controls challenged with tissue cysts did not attain statistical significance.     

Vaccination against murine toxoplasmosis using recombinant Toxoplasma gondii SAG3 antigen alone or in combination with Quil A

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.     

Intranasal immunization with SAG1 and nontoxic mutant heat-labile enterotoxins protects mice against Toxoplasma gondii

Effective protection against intestinal pathogens requires both mucosal and systemic immune responses. Intranasal administration of antigens induces these responses but generally fails to trigger a strong protective immunity. Mucosal adjuvants can significantly enhance the immunogenicities of intranasally administered antigens. Cholera toxin (CT) and heat-labile enterotoxin (LT) are strong mucosal adjuvants with a variety of antigens. Moreover, the toxicities of CT and LT do not permit their use in humans. Two nontoxic mutant LTs, LTR72 and LTK63, were tested with Toxoplasma gondii SAG1 protein in intranasal vaccination of CBA/J mice. Vaccination with SAG1 plus LTR72 or LTK63 induced strong systemic (immunoglobulin G [IgG]) and mucosal (IgA) humoral responses. Splenocytes and mesenteric lymph node cells from mice immunized with LTR72 plus SAG1, but not those from mice immunized with LTK63 plus SAG1, responded to restimulation with a T. gondii lysate antigen in vitro. Gamma interferon and interleukin 2 (IL-2) production by splenocytes and IL-2 production by mesenteric lymph node cells were observed in vitro after antigen restimulation, underlying a Th1-like response. High-level protection as assessed by the decreased load of cerebral cysts after a challenge with the 76K strain of T. gondii was obtained in the group immunized with LTR72 plus SAG1 and LTK63 plus SAG1. They were as well protected as the mice immunized with the antigen plus native toxins. This is the first report showing protection against a parasite by using combinations of nontoxic mutant LTs and SAG1 antigen. These nontoxic mutant LTs are now attractive candidates for the development of mucosally delivered vaccines.     

STAg和霍乱毒素不同程序滴鼻免疫小鼠诱导抗弓形虫作用

目的观察弓形虫可溶性速殖子抗原（soluble tachyzoite antigen，STAg）和霍乱毒素（cholera toxin，CT）佐剂不同程序滴鼻免疫小鼠诱导的抗弓形虫感染能力，确定STAg和CT滴鼻免疫的最佳程序。方法BALB／c小鼠随机分为3组：1次、2次和3次免疫组，用20μgSTAg＋1μgCT／只分别滴鼻免疫1次，2次或3次，前2次间隔2周，末次间隔1周。末次免疫后第14天，用4×10^4个速殖子／只灌胃攻击所有小鼠，观察小鼠健康及死亡情况，攻击后第30天处死，ELISA法检测血清IgG和粪便IgA，计数肝、脑组织内弓形虫速殖子，分离并计数派伊尔结（Peyer＇s patches，PP）和脾淋巴细胞数。结果2次和3次免疫组小鼠存活率明显高于1次免疫组（P〈0．05），肝、脑组织内虫荷显著低于1次免疫组（P〈0．001），血清IgG和粪便IgA高于1次免疫组，PP和脾淋巴细胞数无显著性变化。结论STAg和CT佐剂滴鼻免疫2次或3次能有效诱导小鼠抗弓形虫感染。     

Antigen(s) responsible for immunoglobulin G responses specific for the acute stage of Toxoplasma infection in humans.

An agglutination test for immunoglobulin G (IgG) Toxoplasma antibodies with acetone-fixed tachyzoites (AC antigens; AC agglutination test) was positive only with sera from patients during the acute stage of their infection. In contrast, when the test was performed with Formalin-fixed tachyzoites (HS antigens; HS agglutination test), positive results were obtained during both the acute and chronic (latent) stages of the infection. Studies were performed to define the antigen(s) of T. gondii which are detected by IgG antibodies present only during the acute stage of the infection. Sera of mice immunized with AC antigens recognized predominantly 10 antigens of tachyzoites by immunoblot analysis. Sera from individuals with the acute but not chronic infection reacted strongly with these same 10 antigens in immunoblots. Sera of mice immunized with HS antigens recognized more Toxoplasma antigens on immunoblots than did mouse AC antibodies. Absorption of the latter with HS antigens removed detectable reactivity of AC antibodies in immunoblots and in the AC and HS agglutination tests, suggesting that AC antigens are a selected portion of HS antigens. AC antigens were specific for tachyzoites in that AC antibodies reacted with the cell membranes of both forms of the organism in an indirect fluorescent-antibody test. Tachyzoite-specific antigen(s) appear to be useful in differentiating between the acute and chronic stages of Toxoplasma infection through their detection by IgG antibodies.     

Antigenaemia and antibody response to Toxoplasma gondii in human immunodeficiency virus-infected patients

Toxoplasma encephalitis in immunocompromised patients results from reactivation of previously acquired (latent) infection. The aim of the study is to assess the antigenaemia and antibody response to Toxoplasma gondii in human immunodeficiency virus (HIV)-infected patients to determine the best marker for early diagnosis of toxoplasmosis in such patients. Indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG, IgM and IgA anti-toxoplasma antibodies and double-sandwich ELISA for toxoplasma antigen is carried out in serum samples collected from 100 HIV seropositive patients and 75 controls. Toxoplasma-specific IgG, IgM and IgA antibody response and antigenaemia were detected in 12%, 6%, 7% and 14% of HIV-infected patients, respectively. On retrospective analysis of 14 patients with antigenaemia only one had central nervous system (CNS) symptoms attributable to toxoplasma infection. In this patient, the CD4+ cell count was below 50/microL and none of the specific immunoglobulin isotype responses could be detected. The patient showed clinical improvement following specific chemotherapy for toxoplasmosis. In 25 HIV-negative and anti-toxoplasma IgG antibody-positive controls, IgM was detected in two (8%), IgA in five (20%) and antigenaemia in 10 (40%), while 50 HIV seronegative healthy controls were negative for both antigen and antibody responses. The study indicates that detection of toxoplasma antigen in addition to IgG antibody response may prove to be a useful indicator in the early diagnosis of reactivated toxoplasmosis in HIV/AIDS patients.     

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.     

Diagnosis of congenital toxoplasmosis by two-dimensional immunoblot differentiation of mother and child immunoglobulin g profiles

Differentiation between the specific immunoglobulin G (IgG) response to Toxoplasma gondii by a mother and her newborn child is helpful in the diagnosis of congenital infection with T. gondii in newborns without T. gondii-specific IgM and/or IgA antibodies at birth. Previous methods include immunoblotting and complexing T. gondii antigen with the sera from the mother and child and comparing the bands after electrophoresis. We developed a two-dimensional immunoblotting (2DIB) method with T. gondii RH strain tachyzoite antigen and validated the method with sera from 11 children identified through the neonatal screening program for congenital toxoplasmosis in Denmark. The children were identified by using Toxoplasma-specific IgM antibodies at the screening test, but the presence of T. gondii-specific IgM and/or IgA antibodies could not be confirmed at the subsequent serum sample tested. The children were monitored for at least 12 months, and in seven of eight patients monitored for 12 months the results of the 2DIB-predicted congenital infection were confirmed by the presence of persistent Toxoplasma-specific IgG antibodies. 2DIB is a sensitive technique that allows early differentiation between passively transferred maternal T. gondii-specific IgG antibodies and antibodies synthesized by the newborn child.     

Comparative immunoglobulin G antibody profiles between mother and child (CGMC test) for early diagnosis of congenital toxoplasmosis

Early diagnosis of congenital toxoplasmosis is rendered difficult when specific immunoglobulin M (IgM) and/or IgA antibodies are absent in the blood of the newborn infant. Since maternal IgG antibodies can cross the placenta, determination of IgG antibodies in newborn infants has hitherto not been used routinely for the diagnosis of congenital infection. The aim of this study was to assess the diagnostic usefulness of an immunoblot assay which compares the early IgG profiles between the mother and her child (comparative IgG profile between mother and child; CGMC test) directed against a total cell lysate of Toxoplasma gondii tachyzoites. Serum samples from 97 newborn infants at risk of toxoplasma infection were obtained from umbilical cord blood at birth or postnatally until 3 months of life and were directly compared with serum samples from the respective mothers. Congenital toxoplasmosis was diagnosed only when IgG-reactive protein bands that were present in any newborn serum samples were absent in the corresponding maternal serum sample. Congenital infection was defined by conventional serological assays when IgM and/or IgA antibodies were present in newborn infant blood or when IgG titers rose within the first 12 months or were persistently stable for more than 8 months. Using these criteria, congenital infection was definitely confirmed in 11 cases. Three additional cases were diagnosed based on indicative data. The CGMC test, which was performed without knowledge of the results of conventional serologal assays, had sensitivity and specificity of 82.4 and 93.0%, respectively, and positive and negative predictive values of 73.7 and 95.7%, respectively. When true positives and true negatives were considered, the comparative IgG profile had a ratio of 90.9% true results. The CGMC test thus is useful as an additional assay for the rapid diagnosis of congenital toxoplasmosis when paired serum samples from mother and child are available.     

Detection of Specific Immunoglobulin E during Maternal, Fetal, and Congenital Toxoplasmosis

Toxoplasma immunoglobulin E (IgE) antibodies in 664 serum samples were evaluated by using an immunocapture method with a suspension of tachyzoites prepared in the laboratory in order to evaluate its usefulness in the diagnosis of acute Toxoplasma gondii infection during pregnancy, congenital infection, and progressive toxoplasmosis. IgE antibodies were never detected in sera from seronegative women, from patients with chronic toxoplasma infection, or from infants without congenital toxoplasmosis. In contrast, they were detected in 86.6% of patients with toxoplasmic seroconversion, and compared with IgA and IgM, the short kinetics of IgE was useful to date the infection precisely. For the diagnosis of congenital toxoplasmosis, specific IgE detected was less frequently than IgM or IgA (25 versus 67.3%), but its detection during follow-up of children may be interesting, reflecting an immunological rebound. Finally, IgE was detected early and persisted longer in progressive toxoplasmosis with cervical adenopathies, so it was also a good marker of the evolution of toxoplasma infection.     

Analysis of IgG subclasses (IgG1 and IgG3) to recombinant SAG2A protein from Toxoplasma gondii in sequential serum samples from patients with toxoplasmosis

The kinetics of the humoral immune response was evaluated using the recombinant SAG2A protein comparatively to soluble Toxoplasma antigen (STAg) by ELISA in sequential serum samples of patients with toxoplasmosis up to 12 months of illness onset. The follow up of IgM and IgA levels to STAg showed a gradual decrease, with the majority of patients (88%) seropositive for IgM up to 12 months of infection, whereas IgA seropositivity was relatively low (78%) compared to IgM (100%) in the first 3 months of infection. The follow up of IgG and IgG1 antibodies showed a similar increasing profile for both SAG2A and STAg, with slightly higher seropositivity for STAg. The kinetics of IgG3 to STAg was similar to that of IgG1, contrasting with the kinetics of IgG3 to SAG2A that showed high levels up to 6 months of infection, with continuous decreasing over the time. Higher IgG3 seropositivity to SAG2A than STAg was also observed in the initial phases of infection. A higher IgG3/IgG1 ratio for SAG2A than STAg was detected in the first 3 months of infection, with decreasing profile over the time. The associations of IgG3/IgG1 ratio>1.0 with positive IgM or IgA antibodies were predominantly found in the first 3 months of infection, whereas associations of IgG3/IgG1 ratio<1.0 with positive IgM or negative IgA antibodies were mostly observed from 3 to 12 months of infection. In conclusion, our results demonstrate a differential kinetics of IgG3 antibodies to SAG2A and STAg in patients with toxoplasmosis up to 12 months of infection. Also, the IgG3/IgG1 ratio to SAG2A in association with classical serological markers of acute phase could be potential tools to distinguish early acute from convalescent phases of Toxoplasma gondii infection.