**Toxoplasmosis: Advances and Vaccine Perspectives**

Oscar Bruna-Romero1, Dulcilene Mayrink de Oliveira1 and Valter Ferreira de Andrade-Neto2,\*

*1Department of Microbiology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte-MG 2Department of Microbiology and Parasitology, Centro de Biociências, Universidade Federal do Rio Grande do Norte – UFRN, Natal-RN Brazil* 

### **1. Introduction**

168 Current Topics in Tropical Medicine

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*Toxoplasma gondii* was first identified more than 100 years ago in the tissues of birds and mammals. In 1908 Nicolle and Manceoux described it for the first time in the gundi (*Ctenodactylus gundi*), a North African rodent, in tachyzoite forms. At the same time, Splendore in Brazil, identified the parasite in rabbit tissues. Due to its bow-like shape (Greek: *Toxo =* Arc) the genus was named *Toxoplasma*. However, only in the 1970's was the complete life cycle known and the parasite recognized as a coccidian parasite (member of the phylum Apicomplexa). It is ubiquitous throughout the world and estimated to infect approximately half of the world's population. It is characterized by a polarized cell structure and two unique apical secretory organelles called micronemes and rhoptries.

*Toxoplasma* has a complex life cycle consisting of a sexual cycle in its feline definitive hosts and an asexual cycle in its intermediate hosts. The latter, including humans, can be infected by ingestion of oocysts shed in cat feces. Unlike most other Apicomplexan parasites, *Toxoplasma* can be transmitted between intermediate hosts by either vertical (via placenta) or horizontal (carnivorism) transmission.

*Toxoplasma* parasite is found in intermediate hosts in two interconvertable stages: bradyzoites and tachyzoites. Bradyzoites, a dormant form, are slow-growing, transmissible and encysted. Infections with bradyzoite-containing cysts occur upon ingestion of undercooked meat. The wall of these cysts is digested inside the host stomach and the released bradyzoites, which are resistant to gastric peptidases, subsequently invade the small intestine. There, they convert into tachyzoites, the rapidly growing, disease-causing form that can infect most nucleated cells, replicate inside a parasitophorous vacuole, egress, and then infect neighboring cells. These tachyzoites activate a potent host immune response that eliminates most of the parasites. Some tachyzoites, however, escape destruction and convert back into bradyzoites. In the absence of an adequate immune response, tachyzoites

<sup>\*</sup> Corresponding Author

Toxoplasmosis: Advances and Vaccine Perspectives 171

cells become crucial to control parasite replication, not only by serving as additional sources of IFN-but also by developing cytotoxic activity against infected cells, eliminating parasite factories and thus preventing reactivation of infection (Denkers *et al*. 1993; Denkers and Gazzinelli 1998; Bhopale 2003). Whether B cells also play a role in protection against this parasite is not clear, but studies have generated indirect evidences that IgG antibodies may be important for protection (Kang *et al*. 2000). B cell-deficient mice have shown increased susceptibility to brain inflammatory pathology in chronic infections with the parasite, despite presenting similar levels of serum and tissue pro-inflammatory cytokines, such as IFN-γ. Furthermore, adoptive transfer of polyclonal anti- *T. gondii* IgG antibodies to these

To reproduce what the immune system does naturally to protect hosts against *T. gondii* infection (and re-infection), researchers have attempted several strategies for vaccination. These include the use of whole parasites (attenuated in different ways), soluble parasite antigens, recombinant purified proteins (subunit vaccines) or recombinant live vectors that express heterologous antigen(s) within host organisms (figure 1). Currently, some of these tools are also being used in combination, as part of prime-boost immunization protocols.

Sporulated oocysts (sporozoite-containing cysts) from the environment or tissue cysts (bradyzoite-containing cysts) from infected animals are the two major sources of infection with *T. gondii* (figure 2). However, vaccine candidates that include sporozoites or sporozoite antigens have traditionally been less studied because of the ease of access to bradyzoites and tachyzoites, e.g. using animal brain cysts or acutely infected animal peritoneal lavage/cell cultures, respectively. As a result, the first *T. gondii* whole-parasite experimental vaccines were mainly based on attenuated tachyzoites/bradyzoites, in particular those generated by inactivation or irradiation. Inactive parasites were used for immunization of experimental animals from 1956 (Cutchins and Warren 1956) to 1972 (Krahenbuhl *et al*. 1972) with not much success. In contrast, gamma-irradiated *T. gondii* tachyzoites were successfully tested as experimental vaccines in 1975 (Seah and Hucal 1975), in part after taking the idea from the pioneering irradiated-sporozoite malaria vaccines, which were initially tested in the 1960s and 70s (Nussenzweig *et al*. 1967; Gwadz *et al*. 1979). In the 1975 report, all animals inoculated with highly irradiated *T. gondii* parasites survived, were free of tissue cysts and were solidly protected against a subsequent rechallenge. Later, a few reports (Dubey *et al*. 1996; Omata *et al*. 1996; Dubey *et al*. 1998) have also used irradiated sporozoites (under the form of sporulated oocysts) to vaccinate mice, cats and pigs against toxoplasmosis, but in contrast to tachyzoites, results were not very

Other attempts to induce protection against toxoplasmosis with whole-parasite vaccines included the use of live attenuated parasites (tachyzoites) such as the S-48, the cps1-1, the temperature-sensitive TS-4, the MIC1-3 knock-out or the non-replicative Δrps13 strains (McLeod *et al*. 1988; Hakim *et al*. 1991; Buxton 1993; Gigley *et al*. 2009; Lu *et al*. 2009; Hutson

mice prevented both pathology and mortality.

**3.1 Whole-parasite attenuated vaccines** 

encouraging, though some protection was also observed.

**3. Major toxoplasma vaccines and candidates studied to date** 

Below is a review of current's state of the art of most of these technologies.

will grow unabated and cause tissue destruction, which can be severe and even fatal. However, the inflammatory immune response induced by tachyzoites can cause immunemediated tissue destruction. Therefore, a subtle balance between inducing and evading the immune response is crucial for *Toxoplasma* to establish a chronic infection.

The success of *Toxoplasma* as a widespread pathogen is due to the ease in which it can be transmitted between intermediate hosts. Humans do not play a major role in transmission; consequently, pathogenesis in humans is the indirect result of adaptations to infection in other hosts and treatment of human infections is unlikely to lead to the spread of drug resistance.

Once inside a host, the parasite develops powerful tools to modulate its host cell and develop into a chronic infection that can evade the host's immune system as well as all known anti-toxoplasmatic drugs. The ability of the parasite to replicate within a host cell, evade immune responses and undergo bradyzoite development requires the parasite to effectively modulate its host.

Toxoplasmosis remains a major health concern in pregnancy, where it causes severe birth defects or miscarriage, and in immunocompromised hosts. Thus, new toxoplasmosis control strategies are needed. The development of effective human and veterinary vaccines against toxoplasmosis is a relevant goal for Public Health (Gazzinelli *et al*. 1996; Pifer and Yarovinsky 2011). Even if new therapeutic drugs, with less hypersensitivity and toxicityrelated events, are developed, not only for acute *T. gondii* infection but also for the currently untreatable latent bradyzoite form of the parasite, a prophylactic vaccine against the disease would still be the best option from the financial, epidemiological, and social points of view. A vaccine would decrease the enormous costs of diagnosis/treatment, the premature loss of lives, the extensive rates of dissemination as well as the social impact of the disease. One major fact that suggests the possibility of vaccination against toxoplasmosis is that primary infection with the *T. gondii* parasite elicits protective immunity against re-infection in most individuals.
