Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection

Análise da reatividade de anticorpos anti-Toxoplasma gondii em amostras de soros de cães doentes e aparentes sadios pelos métodos de IFI, ELISA e "WESTERN-BLOT".

CNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoTrabalho de Conclusão de Curso (Graduação)Este estudo teve como objetivo analisar o perfil imunoquímico e a reatividade de anticorpos anti-Toxoplasma gondii em amostras de soros de cães doentes e aparentemente saudáveis, procedentes do Hospital Veterinário da Universidade Federal de Uberlândia e Centro de Controle de Zoonoses de Uberlândia - MG

Strongyloides venezuelensis: the antigenic identity of eight strains for the immunodiagnosis of human strongyloidiasis

The antigens of eight strains of Strongyloides venezuelensis were identified by means of the indirect immunofluorescence antibody (IFAT), enzyme-linked immunosorbent assay (ELISA) and immunoblot (IB) tests. Infective larvae (L3) from these strains were obtained from Rattus norvegicus feces cultures. For IFAT, sections of L3 were used while the ELISA and IB, tests were conducted with alkaline extract. Ninety serum samples were tested: 30 from patients with S. stercoralis, 30 from patients with other parasitic diseases, and 30 from healthy subjects (free of parasites). Average sensitivity and specificity among all eight strains, both for IFAT and ELISA, were, respectively, 93% and 100%. In the IB, anti-S. stercoralis IgG recognized a single antigenic fraction with 45 kDa. Serum samples from patients with S. stercoralis revealed antigens from different strains of S. venezuelensis, indicating antigenic identity for possible use in the synthesis of recombinant antigen that could be useful in immunodiagnosis and vaccine against this parasite1191714COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão tem95/9691/

Strongyloides Venezuelensis: The Antigenic Identity Of Eight Strains For The Immunodiagnosis Of Human Strongyloidiasis.

The antigens of eight strains of Strongyloides venezuelensis were identified by means of the indirect immunofluorescence antibody (IFAT), enzyme-linked immunosorbent assay (ELISA) and immunoblot (IB) tests. Infective larvae (L3) from these strains were obtained from Rattus norvegicus feces cultures. For IFAT, sections of L3 were used while the ELISA and IB, tests were conducted with alkaline extract. Ninety serum samples were tested: 30 from patients with S. stercoralis, 30 from patients with other parasitic diseases, and 30 from healthy subjects (free of parasites). Average sensitivity and specificity among all eight strains, both for IFAT and ELISA, were, respectively, 93% and 100%. In the IB, anti-S. stercoralis IgG recognized a single antigenic fraction with 45 kDa. Serum samples from patients with S. stercoralis revealed antigens from different strains of S. venezuelensis, indicating antigenic identity for possible use in the synthesis of recombinant antigen that could be useful in immunodiagnosis and vaccine against this parasite.1197-1

Infectivity Of Strongyloides Venezuelensis Is Influenced By Variations In Temperature And Time Of Culture.

The present research investigated the influence of temperature and time of larvae culture on the infectivity of Strongyloides venezuelensis. Mice were infected s.c. with 1500 larvae of S. venezuelensis maintained at 28 °C for three days of culture (dc), 28 °C for seven dc or 18 °C for seven dc. On days 1, 3, 5, 7, 14 and 21 post-infection the animals were sacrificed and cell numbers in the blood, peritoneal cavity fluid (PCF), broncoalveolar fluid (BALF), cytokines, immunoglobulins, number of parasites and eggs/g of feces were quantified. Results demonstrated an increase in eosinophils and mononuclear cells in the blood, PCF and BALF of infected mice. Larvae at 28 °C/3dc induced earlier eosinophils in the PCF and BALF as opposed to larvae at 28 °C/7dc and 18 °C/7dc. Larvae at 28 °C/7dc induced higher synthesis of IL-4, IL-5 and IL-10 on days 5 and 7 post-infection. Larvae at 28 °C/3dc in culture induced higher synthesis of IL-12 than larvae of seven dc, but time in culture induced better synthesis of IFN-γ after larval migration had ceased and only adult worms were present. Larvae at 28 °C/3dc in culture induced higher synthesis of IgG and IgG1 and expelled less female parasites than larvae cultivated for seven days. In conclusion, it was observed that the infectivity of S. venezuelensis is influenced by variations in temperature and time of culture.12772-

Preparation and antitubercular activity of lipophilic diamines and amino alcohols

A series of diamines and amino alcohols derived from 1-dodecanol, 1-tetradecanol, 1,2-dodecanediol and 1,2-tetradecanediol were synthesized and tested for their antitubercular activity. Compounds 3, 8 and 9 were found to be the most active (MIC of 6.25 ¼g/mL). Nine other compounds displayed activity against Mycobacterium tuberculosis, with a MIC of 12.5 ¼g/mL

Specific cell-mediated immune responses elicited by immunization with microneme proteins.

<p>Spleen cells were harvested from immunized (indicated as TgMICs) and control mice (PBS) on day 15 post the last antigen injection and cultured in the presence of medium only, STAg (10 μg/ml), or Concanavalin A (2 μg/ml) for 72 h. (A) Proliferation of spleen cells was measured by [3H]-thymidine incorporation assay. Each bar represents the average of four mice per group and is representative of three independent experiments. Statistical significance is denoted as *p < 0.05 compared to the control group. (B–D) Cytokine concentration was measured by ELISA in the supernatant of spleen cell cultures. Panels show the IL-12 (B), IFNγ(C), and IL-10 (D) concentrations. Each bar represents the mean ± SD of triplicate samples and the results are representative of three independent experiments. Statistical significance is denoted as *p < 0.05 compared to PBS-inoculated mice; # p < 0.05 compared to non-stimulated cells of the same group.</p

Number of brain cysts and survival of mice immunized with microneme proteins and infected with <i>T</i>. <i>gondii</i>.

<p>Mice immunized on days 0, 15, and 30 with the indicated preparations of TgMICs or with the vehicle (PBS) were challenged after one month (day 60) with <i>T</i>. <i>gondii</i> infection, provoked by gavage with cysts of the ME49 strain. (A) Cyst numbers were counted from whole brain homogenates of mice, harvested one month after challenge with 40 cysts. The results are expressed as means ± SD for each group. Significance is denoted as *p < 0.05, compared to the PBS group. (B) Survival curves of mice that were challenged with 80 cysts of the ME49 strain and observed daily for mortality. Data are representative of six experiments with similar results.</p

Cladogram analysis of TgMIC1, TgMIC4, and TgMIC6 of the <i>T</i>. <i>gondii</i> isolates.

<p>Dendrograms grouping microneme proteins of <i>T</i>. <i>gondii</i> isolates showing similarity of approximately 99% according to their sequences. (A) TgMIC1. (B) TgMIC4. (C) TgMIC6. Amino acids sequences were obtained from available sequence database at NCBI, and the alignment was performed using MEGA 6.06 software.</p

SDS-PAGE and western blot analysis of native and recombinant microneme proteins.

<p>SDS-PAGE of recombinant proteins (panels A, B, and C, Coomassie Blue stained) or native (panel D, silver-stained) proteins. Heterologous expression was noted in <i>E</i>. <i>coli</i> (DE3) and recombinant proteins were detected in inclusion bodies. Lane 1: protein expression before induction. Lane 2: protein expression after induction. Lane 3: purified and refolded histidine-tagged recombinant proteins, displayed apparent molecular masses of 70-kDa (TgMIC1, panel A), 80-kDa (TgMIC4, panel B), and 30-kDa (TgMIC6, panel C). Panel E shows the electrophoretical profile of the Lac+ fraction, composed of the native proteins TgMIC1 (53-kDa) and TgMIC4 (68-kDa). Lane M: Molecular mass markers. Reactivity of recombinant and native microneme proteins with anti-Lac+ mouse serum was examined by western blot (Panel E), developed with peroxidase-conjugated goat anti-mouse IgG.</p