Toxoplasmosis: Advances and Vaccine Perspectives (Tropical Diseases Due to Protozoa and Helminths) Part 1

Introduction

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 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 toxicity-related 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.

Mechanisms of protective immunity against toxoplasmosis

Immune responses during the early stages of T. gondii infection are characterized by activation of innate mechanisms mediated by macrophages and dendritic cells (DC) (Gazzinelli et al. 1996; Pifer and Yarovinsky 2011). These cells are activated in mice (not yet known how in humans) after parasite internalization, by engagement of endosomal toll-like receptor 11 (and probably others) with tachyzoite products, which drives subsequent production of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α). In turn, IL-12 activates natural killer (NK) cells (Denkers et al. 1993) to secrete gamma interferon (IFN-γ) (Gazzinelli et al. 1994), which then acts as stimulus for T-cell activation and, in synergy with TNF-α, mediates killing of tachyzoites by macrophages through enhanced production of free oxygen radicals and nitric oxide (NO).

Acquired immunity against T. gondii develops afterward, and is characterized by strong CD4+ and CD8+ T cell activity (Gazzinelli et al. 1992). The cytokine IFN-γ continues to be central in resistance to the parasite during the successive acute and chronic stages of infection, driving the differentiation of CD4+ T lymphocytes specific for parasite antigens to a helper T cell type (Th1) cytokine profile. More important, the newly generated CD8+ T cells become crucial to control parasite replication, not only by serving as additional sources of IFN-γ 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 mice prevented both pathology and mortality.

Major toxoplasma vaccines and candidates studied to date

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

Whole-parasite attenuated vaccines

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 encouraging, though some protection was also observed.

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 et al. 2010; Mevelec et al. 2010).

Potential advantages (+) and concerns (-) of the major vaccination strategies used to immunize hosts against T. gondii infection. Abbreviations: STAg, Soluble Tachyzoite Antigen; TSo, Tachyzoites Sonicate.

Fig. 1. Potential advantages (+) and concerns (-) of the major vaccination strategies used to immunize hosts against T. gondii infection. Abbreviations: STAg, Soluble Tachyzoite Antigen; TSo, Tachyzoites Sonicate.

Major T. gondii antigens identified to date in the different stages of the parasite's life cycle and major routes of parasite transmission. Thin black arrow = horizontal transmission via oocysts; thick black arrow = horizontal transmission via tissue cysts; dotted arrows = vertical transmission via tachyzoites. Abbreviations: SAG, surface antigen; ROP, rhoptry protein; GRA, dense granules; MIC, microneme protein; SRS, SAG-related sequences; BSR, bradyzoite-specific recombinant; MAG, matrix antigen; LDH, lactate dehydrogenase; ENO, enolase; TgERP0, T. gondii embryogenesis-related protein.

Fig. 2. Major T. gondii antigens identified to date in the different stages of the parasite’s life cycle and major routes of parasite transmission. Thin black arrow = horizontal transmission via oocysts; thick black arrow = horizontal transmission via tissue cysts; dotted arrows = vertical transmission via tachyzoites. Abbreviations: SAG, surface antigen; ROP, rhoptry protein; GRA, dense granules; MIC, microneme protein; SRS, SAG-related sequences; BSR, bradyzoite-specific recombinant; MAG, matrix antigen; LDH, lactate dehydrogenase; ENO, enolase; TgERP0, T. gondii embryogenesis-related protein.

The only vaccine commercialized for veterinary purposes, Ovilis®Toxovax (Intervet/Schering-Plough Animal Health, UK), based on the incomplete parasite S-48 strain (not able to generate either tissue cysts or oocysts), began to be marketed in New Zealand and the United Kingdom in 1988 to control miscarriages provoked by T. gondii in sheep. Reduction in fetal loss and in formation of cysts in the meat used for consumption has been reported. This vaccine seems to reduce infection in sheep, which in free-range grazing are constantly exposed to oocyst contamination.

Interestingly, up to date, the most recent and technologically advanced recombinant vaccine formulations have reached, at best, the same levels of protective immunity induced by whole-parasite vaccines. Three main reasons may be responsible for that difference: (i) true protective antigens (or more plausibly antigen combinations) of the parasite have not yet been identified, (ii) while the whole organism and the recombinant vaccines contain the same antigenic sequences, the process by which the recombinant products are generated result in the loss of crucial features that are key for protein’s immunogenicity (Crampton and Vanniasinkam 2007) or, finally, (iii) gamma-irradiated or otherwise attenuated parasites maintain metabolic functions, retain the capacity to invade mammalian cells, present antigens to the host’s immune system and elicit cellular immunity and cytokine responses in a highly similar way to natural infection (Hiramoto et al. 2002), and exogenous recombinant antigens do not.

However, even though protection has been repeatedly demonstrated after immunization with whole-parasite vaccines, real concerns also exist regarding the use of this type of vaccines, in particular for uses other than veterinary immunization. The major fear is that attenuated parasites could revert to the pathogenic phenotype. For this reason, studies towards developing a human vaccine have focused on parasite extracts or recombinant technologies that use defined immunodominant antigens and delivery strategies.

Immunogenic parasite extracts

Identification and functional characterization of proteins of the tachyzoite stage of T. gondii has been the focus of extensive research, because antigens within this stage are presented to the immune system effectively during natural infection, forcing the parasite to enter (in less than two weeks) into the latent bradyzoite stage seeking for protection. This strategy results in physical parasite shielding by encystation and different, and much lower, antigen availability for the immune system.

The soluble tachyzoite antigen extract (STAg) was the first protein blend identified as source of protective products, before wide-scale proteomic analyses were available (Denkers et al. 1993; Yap et al. 1998). Protection with STAg is only partial, even when the very efficient CpG oligodeoxynucleotides are used as adjuvants (Yin et al. 2007). Similar partial protection was also induced by the T. gondii sonicate of tachyzoites (TSo), even when mixed with cholera toxin (CT, a mucosal adjuvant) for oral administration (Bourguin et al. 1991; Bourguin et al. 1993). One of the reasons why immunogenic parasite extracts render non-protective immunity may be the diversification of immune responses amongst all the different antigens (immunodominant or not) present in those extracts. Additionally, the extraction process (in the case of STAg) may have eliminated some of the innate immunity activators, namely TLR agonists, present in the whole parasite. Current proteomic analyses (high-throughput 2-dimensional electrophoresis combined with mass spectrometry) have identified nine novel vaccine candidates within STAg (Ma et al. 2009) and we should see some of these promising antigens being tested in vivo as recombinant subunit or vectorised vaccines in the near future.

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