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Squirrel monkeys (Saimiri spp.), new world primates from South America, are very susceptible to toxoplasmosis. Numerous outbreaks of fatal toxoplasmosis in zoos have been identified around the world, resulting in acute respiratory distress and sudden death. To date, preventive hygiene measures or available treatments are not able to significantly reduce this mortality in zoos. Therefore, vaccination seems to be the best long-term solution to control acute toxoplasmosis. Recently, we developed a nasal vaccine composed of total extract of soluble proteins of Toxoplasma gondii associated with muco-adhesive maltodextrin-nanoparticles. The vaccine, which generated specific cellular immune responses, demonstrated efficacy against toxoplasmosis in murine and ovine experimental models. In collaboration with six French zoos, our vaccine was used as a last resort in 48 squirrel monkeys to prevent toxoplasmosis. The full protocol of vaccination includes two intranasal sprays followed by combined intranasal and s.c. administration. No local or systemic side-effects were observed irrespective of the route of administration. Blood samples were collected to study systemic humoral and cellular immune responses up to 1 year after the last vaccination. Vaccination induced a strong and lasting systemic cellular immune response mediated by specific IFN-γ secretion by peripheral blood mononuclear cells. Since the introduction of vaccination, no deaths of squirrel monkeys due to T. gondii has been observed for more than 4 years suggesting the promising usage of our vaccine. Moreover, to explain the high susceptibility of naive squirrel monkeys to toxoplasmosis, their innate immune sensors were investigated. It was observed that Toll-like and Nod-like receptors appear to be functional following T. gondii recognition suggesting that the extreme susceptibility to toxoplasmosis may not be linked to innate detection of the parasite., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Neutrophils are now recognized as major components of the response to Toxoplasma gondii by their contribution to parasite elimination by a number of mechanisms. This article focuses on recent advances in the understanding of the mechanisms of migration, cytokine release, and formation of extracellular traps by neutrophils during toxoplasmosis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The current therapeutic arsenal for toxoplasmosis is restricted to drugs non-specific to the parasite which cause important side effects. Development of more efficient and specific anti-Toxoplasma compounds is urgently needed. Imidazo[1,2-b]pyridazines designed to inhibit the calcium-dependent protein kinase 1 of Toxoplasma gondii (TgCDPK1) and effective against tachyzoite growth in vitro at submicromolar ranges were modified into hydrochloride salts to be administered in vivo in a mouse model of acute toxoplasmosis. All protonated imidazo[1,2-b]pyridazine salts (SP230, SP231 and SP232) maintained their activity on TgCDPK1 and T. gondii tachyzoites. Rat and mouse liver microsomes were used to predict half-life and intrinsic clearance, and the pharmacokinetic profile of the most rapidly degraded imidazo[1,2b]pyridazine salt (SP230) was determined in serum, brain and lungs of mice after a single administration of 50 mg/kg. Compounds were then tested in vivo in a murine model of acute toxoplasmosis. Mice infected with tachyzoites of the ME49 strain of T. gondii were treated for 4, 7 or 8 days with 25 or 50 mg/kg/day of SP230, SP231 or SP232. The parasite burdens were strongly diminished (>90% reduction under some conditions) in the spleen and the lungs of mice treated with imidazo[1,2-b]pyridazine salts compared with untreated mice, without the need for pre-treatment. Moreover, no increases in the levels of hepatic and renal toxicity markers were observed, demonstrating no significant signs of short-term toxicity. To conclude, imidazo[1,2-b]pyridazine salts have strong efficacy in vivo on acute toxoplasmosis and should be further tested in a model of mouse congenital toxoplasmosis., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Development of sub-unit mucosal vaccines requires the use of specific delivery systems or immune-modulators such as adjuvants to improve antigen immunogenicity. Nasal route for vaccine delivery by nanoparticles has attracted much interest but mechanisms triggering effective mucosal and systemic immune response are still poorly understood. Here we study the loading of porous nanoparticles (DGNP) with a total extract of Toxoplasma gondii antigens (TE), the delivery of TE by DGNP into airway epithelial, macrophage and dendritic cells, and the subsequent cellular activation. In vitro, DGNP are able to load complex antigens in a stable and quantitative manner. The outstanding amount of antigen association by DGNP is used to deliver TE in airway mucosa cells to induce a cellular maturation with an increased secretion of pro-inflammatory cytokines. Evaluation of nasal vaccine efficiency is performed in vivo on acute and chronic toxoplasmosis mouse models. A specific Th1/Th17 response is observed in vivo after vaccination with DGNP/TE. This is associated with high protection against toxoplasmosis regarding survival and parasite burden, correlated with an increased delivery of antigens by DGNP in airway mucosa cells. This study provides evidence of the potential of DGNP for the development of new vaccines against a range of pathogens., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The development of an effective vaccine against Toxoplasma gondii infection is an important issue due to the seriousness of the related public health problems, and the economic importance of this parasitic disease worldwide. Rhoptry neck proteins (RONs) are components of the moving junction macromolecular complex formed during invasion. The aim of this study was to evaluate the vaccine potential of RON4 using two vaccination strategies: DNA vaccination by the intramuscular route, and recombinant protein vaccination by the nasal route. We produced recombinant RON4 protein (RON4S2) using the Schneider insect cells expression system, and validated its antigenicity and immunogenicity. We also constructed optimized plasmids encoding full length RON4 (pRON4), or only the N-terminal (pNRON4), or the C-terminal part (pCRON4) of RON4. CBA/J mice immunized with pRON4, pNRON4 or pCRON4 plus a plasmid encoding the granulocyte-macrophage-colony-stimulating factor showed high IgG titers against rRON4S2. Mice immunized by the nasal route with rRON4S2 plus cholera toxin exhibited low levels of anti-RON4S2 IgG antibodies, and no intestinal IgA antibodies specific to RON4 were detected. Both DNA and protein vaccination generated a mixed Th1/Th2 response polarized towards the IgG1 antibody isotype. Both DNA and protein vaccination primed CD4+ T cells in vivo. In addition to the production of IFN-γ, and IL-2, Il-10 and IL-5 were also produced by the spleen cells of the immunized mice stimulated with RON4S2, suggesting that a mixed Th1/Th2 type immune response occurred in all the immunized groups. No cytokine was detectable in stimulated mesenteric lymph nodes from mice immunized by the nasal route. Immune responses were induced by both DNA and protein vaccination, but failed to protect the mice against a subsequent oral challenge with T. gondii cysts. In conclusion, strategies designed to enhance the immunogenicity and to redirect the cellular response towards a Th1 type response against RON4 could lead to more encouraging results., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Neospora caninum is an intracellular protozoan pathogen that causes abortion in cattle. We studied how the interaction between murine conventional dendritic cells or macrophages and N. caninum influences the generation of cell-mediated immunity against the parasite. We first explored the invasion and survival ability of N. caninum in dendritic cells and macrophages. We observed that protozoa rapidly invaded and proliferated into these two cell populations. We then investigated how Neospora-exposed macrophages or dendritic cells distinguish between viable and non-viable (heat-killed tachyzoites and antigenic extract) parasites. Viable tachyzoites and antigenic extract, but not killed parasites, altered the phenotype of immature dendritic cells. Dendritic cells infected with viable parasites down-regulated the expression of MHC-II, CD40, CD80 and CD86 whereas dendritic cells exposed to N. caninum antigenic extract up-regulated the expression of MHC-II and CD40 and down-regulated CD80 and CD86 expression. Moreover, only viable tachyzoites and antigenic extract induced IL-12 synthesis by dendritic cells. MHC-II expression was up-regulated and CD86 expression was down-regulated at the surface of macrophages, regardless of the parasitic form was encountered. However, IL-12 secretion by macrophages was only observed under conditions using viable and heat-killed parasite. We then analysed how macrophages and dendritic cells were involved in inducing T-cell responses. T lymphocyte IFN-γ-secretion in correlation with IL-12 production occurred after interactions between T cells and dendritic cells exposed to viable tachyzoites or antigenic extract. By contrast, for macrophages IFN-γ production was IL-12-independent and only occurred after interactions between T cells and macrophages exposed to antigenic extract. Thus, N. caninum-induced activation of murine dendritic cells, but not that of macrophages, was associated with T cell IFN-γ production after IL-12 secretion., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Biodegradable nanoparticles with surface adsorbed antigens represent a promising method for in vivo delivery of vaccines targeting a wide range of infectious diseases or cancers. We investigated the feasibility of loading dendritic cells with a vaccine antigen, HIV p24 protein, on the surface of surfactant-free anionic (d,l-lactic acid, PLA) nanoparticles. The p24 protein had a high affinity for the nanoparticles and the antigenicity and immunogenicity of the p24 protein on the nanoparticle was well preserved after immunization. p24-coated nanoparticles were efficiently taken up by mouse dendritic cells (DCs), inducing DC maturation by increasing MHC-I, MHC-II, CD40, CD80 and CD86 surface expression and secreting IL-12 (p70) and IL-4. We evaluated the ability of DCs pulsed with p24-coated nanoparticles to elicit an optimal humoral and cellular immune response in the blood and intestine. DCs pulsed with p24-nanoparticles induced high seric and mucosal antibody production and elicited strong systemic and local lymproliferative responses, correlated with a Th1/Th2-type response, and systemic CTL responses in mice. Thus, DCs pulsed with antigen-loaded PLA nanoparticles may provide a novel delivery tool for cell therapy vaccination against chronic infectious diseases.

The present study was conducted mainly to evaluate the contribution of the cellular and the humoral responses in protection conferred by the MIC3 DNA vaccine (pMIC3i) that was proved as a potent vaccine against toxoplasmosis. We performed the adoptive transfer of CD4(+) and CD8(+) T lymphocytes from pMIC3i immunized mice to naive ones and the role of humoral immunity was evaluated by in vitro invasion assays. We also constructed plasmids encoding the EGF-like domains and the Lectin-like domain of MIC3, to define which domains of MIC3 are involved in the protection. Furthermore, the adjuvant effect of the GM-CSF-expressing vector (granulocyte-macrophage colony-stimulating factor) required the precise temporal and spatial codelivery of GM-CSF with antigen, thus, we constructed a bicistronic plasmid expressing MIC3 and GM-CSF. In conclusion, the protection induced by pMIC3i was mainly mediated by CD4(+) and CD8(+) T lymphocytes and both EGF and Lectin domains of MIC3 conferred protection. Furthermore, the codelivery of GM-CSF by a bicistronic plasmid appeared to be a most effective way for enhancing the adjuvant properties of GM-CSF.

Toll-like receptor (TLR) ligands are attractive adjuvant candidates in vaccine development. Eimeria tenella profilin-like protein has recently been shown to be a potent agonist of the innate immune system through its recognition by Toll-like receptor-11. In this report, we studied the systemic and mucosal adjuvant activity of Eimeria profilin-like protein within a vaccinal strategy against Toxoplasma gondii in mice. Using intraperitoneal (i.p.) immunization, we observed that coadministration of the recombinant Eimeria antigen (rEA) with T. gondii antigen (TAg) effectively elevates plasma levels of IL-12p70 and consequently induced both enhanced specific humoral and Th1 cellular immune responses. The co-administration of TAg plus rEA by i.p route significantly enhanced the protection against T. gondii infection (62% brain cyst reduction) in comparison with control mice and with mice immunized with TAg alone (only 36% brain cyst reduction). After intranasal immunization, humoral and cellular responses were weak. However mice immunized nasally with TAg plus rEA were significantly protected with 50% of brain cyst reduction, conversely TAg immunized mice did not present any brain cyst reduction.These results indicate that Eimeria profilin-like protein would serve as an efficacious systemic and mucosal adjuvant inducing protective immune response against chronical stage of T. gondii infection through TLR11 activation.

Toxoplasmosis is a serious disease in humans and may cause abortion or congenital infection if a woman is exposed to the disease for the first time during pregnancy. Infection before pregnancy normally results in immunity protecting the foetus, suggesting that it may be possible to block vertical transmission of the parasite by appropriate vaccination before pregnancy. We found that the vaccination of CBA/J mice, before pregnancy, with exosomes secreted by SRDCs pulsed in vitro with Toxoplasma gondii-derived antigens (TAg) induced a protective response in the pups. Indeed, vaccination resulted in the presence of significantly fewer cysts in pup brains. This protection was associated with strong humoral responses in the serum in vivo. We also observed cellular responses in vivo, with cell proliferation associated with the production of cytokines by the splenocytes. Thus, exosomes are nucleic acid-free vesicles able to induce immune responses correlated with protection against T. gondii infection in a congenital model. They are therefore a potentially useful tool for vaccination against infectious disease.

Toxoplasma gondii enters the mucosal surfaces of the host, and so immunity at these sites is of major interest. Due to the compartmentalization of the immune response, systemic immunization does not induce high levels of immunity at mucosal surfaces. Intranasal immunization has been shown to be very effective in inducing both systemic and mucosal immune responses. Immunization with mRNA can induce both humoral and cell-mediated immune responses, both of which are important in conferring immunity to T. gondii. The efficacy of RNA vaccination by the nasal route with T. gondii RNA was evaluated. We assessed the percentage of cumulative survival after an oral challenge with a lethal dose of T. gondii cysts (40 cysts), and the number of brain cysts following a challenge with a sublethal dose of T. gondii 76 K cysts (15 cysts). Vaccinated mice were found to be significantly better protected than non-immunized mice after a challenge with a lethal dose of cysts; and a challenge with a sublethal dose also resulted in fewer brain cysts than in non-immunized mice. Sera and intestinal secretions of immunized mice recognized T. gondii antigens, suggesting that a specific humoral immune response may occur. Moreover, a specific lymphoproliferative response observed in cervical lymph nodes may confer protection. These preliminary findings suggest that RNA vaccination by a mucosal route could be feasible.