Elsevier

Vaccine

Volume 27, Issue 11, 10 March 2009, Pages 1750-1757
Vaccine

Exosomes are an effective vaccine against congenital toxoplasmosis in mice

https://doi.org/10.1016/j.vaccine.2009.01.022Get rights and content

Abstract

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.

Introduction

Toxoplasmosis is caused by Toxoplasma gondii, a protozoan found worldwide. T. gondii infection may be asymptomatic in immunocompetent individuals, but may cause severe, life-threatening illness in immunocompromised individuals and foetal complications if the mother experiences a primary infection during pregnancy. The parasite may cause infection via three main routes: (1) the ingestion of tissue cysts in undercooked infected meat, (2) the ingestion of sporulated oocysts shed in the faeces of an infected cat and spreading to food or water and (3) vertical transmission, across the placenta, from mother to foetus after infection of the mother during pregnancy. Congenital toxoplasmosis is a serious public health problem not only for humans, but also for livestock, with significant economic implications for farmers and importers. It has been estimated that 1.2–2.2% of the 16 million ewes in the UK suffer from the abortion of foetuses due to T. gondii infection [1]. The incidence of human congenital toxoplasmosis in the United States has been estimated at between 1 in 10,000 and 1 in 1000 births [2]. In up to 4% of cases, congenital toxoplasmosis may result in abortion, a stillborn child, or a child with serious physical and/or mental retardation, hydrocephalus, intracranial calcifications and retinochoroiditis during the first few years of life [3], [4]. Serious clinical signs may develop during childhood and early adulthood. By the age of 20, up to 85% of affected subjects, including many free of symptoms at birth, have chorioretinitis [2], [5]. Various factors have been implicated in the development of congenital toxoplasmosis: the immunological status of the mother, the virulence of the parasite and the time of gestation at which infection occurs. About one third of infected mothers give birth to an infant with toxoplasmosis [6], [7].

As previously reported, T. gondii infection is predominantly controlled by cell-mediated immunity [8]. The interferon gamma (IFN-γ) produced by natural killer cells, CD4+ and CD8+ lymphocytes plays a major role in controlling infection [9]. Interleukin (IL)-12 is also crucial for the induction of an efficient immune response to T. gondii, resulting in the differentiation and clonal expansion of Th1-type T cells and this cytokine is produced by macrophages, neutrophils and dendritic cells (DCs), in particular [10], [11], [12]. IL-12 is a key cytokine, rapidly triggering IFN-γ secretion after T. gondii infection. IL-10 cytokine also plays an important role in down-regulating systemic IFN-γ responses in C57BL/6 mice and immunoregulating IL-12 levels [13]. Immunity induced by infections contracted before pregnancy prevents the parasite from crossing the placenta and completely protects the foetus, suggesting that it may be possible to block vertical transmission of the parasite by appropriate vaccination before pregnancy, although no tools have yet been developed for the effective prevention of congenital transmission or abortion.

DCs are professional antigen-presenting cells that control the outcome of the immune response. The central role of DCs in controlling immunity makes these cells ideal tools for priming functional immune responses [14]. Many studies have proposed the use of DCs as vaccine vectors. However, expensive treatments of this type, based on living cells, could be envisaged only for severe diseases, such as cancers, which are specific to the individual due to MHC restriction. For ethical reasons, it is not possible to use live cell lines in an immunisation protocol in humans. New approaches, involving the development of non-live and DNA-free vaccines, must therefore be pursued. Moreover, the use of DCs is limited by the difficulty of obtaining large numbers of cells suitable for vaccination purposes. DCs secrete exosomes cell-free membranous vesicles expressing functional MHC class I and class II and T-cell costimulatory molecules on their surface [15]. The properties of these structures are compatible with their use as an alternative to DC-based vaccines.

A previous study showed that DC-derived exosomes pulsed with TAg induced a protective immune response against T. gondii in both syngeneic CBA/J mice and allogeneic C57BL/6 mice [16], [17]. Immune protection was associated with the induction of humoral and cellular TAg-specific responses. In this study, we evaluated whether DC-derived exosomes could induce a protective immune response in pregnant CBA/J mice and protect young mice from congenital toxoplasmosis in an experimental model.

We provide the first demonstration that the vaccination of CBA/J mice, before pregnancy, with exosomes released from T. gondii-pulsed DCs, strongly protects pups against parasite infection during gestation. This protection was associated with a specific and protective T-cell response. These data suggest that exosomes, which have both antigenic and adjuvant characteristics, may be a suitable alternative candidate anti-toxoplasmosis vaccine.

Section snippets

Animals

Six- to 10-week-old female and male CBA/J mice (H-2k) were purchased from Janvier (France) and maintained in a pathogen-free environment. Female Swiss (OF1) and CBA/J mice were used to maintain the T. gondii cycle. All experiments were performed in accordance with the Tours University guidelines for animal experimentation.

Parasites

Tachyzoites of the RH strain (Type I) of T. gondii were harvested from the peritoneal fluids of Swiss OF1 mice 3–4 days after intraperitoneal infection with 1 × 106 tachyzoites.

The fertility rate was lower in TAg-vaccinated mice than in Exo-TAg- and DC-TAg-vaccinated mice

The fertility rate for this protocol was about 65–70% and the length of the gestation period of the mice was 19–20 days (Table 1A). However, a much smaller number of litters was obtained for the group of mice vaccinated with TAg than for the other groups of mice (Table 1A). Indeed, only 27.3% of mice vaccinated with TAg became pregnant. Moreover, fertility rates were slightly lower for the Exo-NP vaccinated group than for controls, with only 45.5% of mice becoming pregnant. By contrast, the

Discussion

Toxoplasmosis is a serious disease in humans that may cause abortion or congenital infection if a woman is exposed to the parasite, T. gondii, for the first time during pregnancy. Infection before pregnancy generally results in immune responses that protect the foetus. Similar observations have been reported in mice, suggesting that mice are a suitable model animal for studying congenital disease. Only one vaccine against toxoplasmosis has been licensed, Ovilis Toxovax® (Intervet), which

Acknowledgements

C.B. holds a fellowship from the Ministère de la Recherche et de l’Enseignement Français. We thank T. Papin for skilful technical assistance and S. Bigot for secretarial assistance.

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    Céline Beauvillain and Matthieu Juste contributed equally to this work.

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