Protective immune responses involved in host resistance to Brazilian isolates of Toxoplasma gondii: implications on vaccine development employing recombinant viral vectors

Ricardo T. Gazzinelli,*# Braulia Caetano,*# Blima Fux,*# Marianne Garcia,*# Alexandre V. Machado,* Adriana M. Ferreira,£ Ricardo W. A. Vitor, £ Maria Norma Melo,
£Oscar Bruna-Romero*#F
Departamento de Bioquímica e Imunologia,* Departamento de Microbiologia,F Departamento de Parasitologia, £ Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais; and Laboratório de Imunopatologia, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz#

Toxoplasma gondii is an intracellular protozoan parasite that is widespread in nature, with a high prevalence in a variety of warm-blooded animals, including humans. It is estimated that toxoplasmosis exists in a chronic, asymptomatic form in between five hundred million to one billion people (1). Toxoplasmosis establishes itself as a lifelong, chronic infection, with transmission occurring in two ways: (i) consumption or handling of uncooked meat bearing tissue cysts; and (ii) ingestion of food or water contaminated with oocysts shed in cat feces (1). Although infections are usually benign, high morbidity and even mortality can be seen in immunocompromised patients, particularly in cancer and transplant patients under immunosuppressive therapy or HIV infected individuals (2). In addition, infection with T. gondii is also a major cause of economical loss in veterinary medicine (3,4).
In humans, toxoplasmosis is a common cause of infectious retinitis in both immunocompetent and immunosupressed persons, accounting for 30-50% of all cases of posterior uveitis (5). Toxoplasmic retinochoroiditis is believed to result mainly from periodic reactivation of latent cysts associated with congenital infection. However, recent studies in France (6), Canada (7), Brazil (8-10) and the United States (11) indicate that ocular disease after postnatal acquired infection is also prevalent in healthy adults. Typical features of retinochoroiditis include unilateral focal retinitis at the border of a pre-existing pigmented retinochoroid lesion and an overlying vitritis. In some cases, the lesions may be atypical, consisting of large areas of retinal necrosis or retinochoroiditis without a preexisting scar (12).
Approximately, 50% of the adult population in Brazil is chronically infected with the parasite, this prevalence is similar to other countries in Latin America (8,10,13-15). However, in some areas of the country the prevalence is significantly higher, reaching up 95% of the population, e.g. Erechim, Rio Grande do Sul State (8,9). In regard to the ocular disease in the seropositive individuals, a high frequency has been described in different areas of Brazil. Studies performed in Erechim (8,9) show 17.7% of the infected population developed ocular lesions, whereas in Melquiades, Minas Gerais State, where 49% of prevalence was observed, 12.5% of the seropositive individuals presented signs of ocular disease (10). We believe that the high frequency of ocular toxoplasmosis in Brazilian populations may reflect the unique combination of infective parasite strains, host genetic background, and/or epidemiological aspects such as infection early in life as well as continuous exposure to infective parasites.
The main objectives of the studies performed in our laboratories are: (i) to define the genetic structure of T. gondii strains isolated in the Brazilian territory; (ii) to define the immunological components involved in resistance of the intermediary host to infection with Brazilian T. gondii isolates; (iii) to elaborate subunit vaccines that protect the intermediary host against infection with Brazilian T. gondii strains. Ultimately, we expect that this information may contribute to the rational design of an effective vaccine to be used in prophylaxis of T. gondii in humans and other intermediary hosts.

I - Genetic analysis of Brazilian T. gondii isolates by multilocus PCR-RFLP
T. gondii strains can be divided in three main lineages based on various genetic markers (16). Studies in mice have shown that infection with each of the three lineages of T. gondii results in different outcomes: Type I strains are highly virulent, whereas types II and III strains are relatively avirulent (16). Type I differ genetically by 1% or less from type II and type III (17). However, the main determinants that dramatically affect the virulence of different T. gondii strains in the host and pathogenesis of toxoplasmosis are poorly understood. Type II strains of T. gondii appear to be dominant in the US territory and are frequently isolated from AIDS patients with toxoplasmic encephalitis (17). Interestingly, different studies suggest the involvement of Type I and Type I/III strains in the development of ocular disease. Consistently, Type I strain was identified as responsible for a toxoplasmosis epidemic outbreak associated with a high rate of development of acquired ocular disease (18). In addition, a recent study indicates a high frequency of Type I as well as Type I/III recombinant isolates from ocular lesions in patients from US (19).
Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) at eight independent loci was used to determine the type (I, II or III) lineage of 20 Toxoplasma gondii strains isolated from humans and animals in Brazil. RH (type I, highly virulent), ME49 (type II, avirulent) and VEG (type III, avirulent) were used as reference strains. Differently from expected frequencies, all Brazilian strains showed to have recombinant genotypes, with typical alleles of types I, II or III at almost all loci assessed. The cB21-4 locus, a microsatellite marker, showed a higher allelic polymorphism with seven alleles among strains under analysis. Data have also shown that many Brazilian T. gondii strains presented a new haplotype at the L363 locus. When results of the eight loci were combined, 14 schizodemes were characterized out of the 20 T. gondii strains isolated in Brazil. The phenogram representing PCR-RFLP data separated Brazilian strains into two distinct genetic groups associated with murine virulence phenotype, termed group I-A and group I-B. Strains from group I-A (AS28, BV and N) that were highly virulent in BALB/c mice, were clustered with RH reference strain. Only those strains presented the haplotype I at the L363 locus, suggesting that this could be a possible marker of highly virulent strains. Strains from group I-B (cystogenic strains) showed a more heterogeneous behavior regarding virulence: a few of them (EGS, RAR, SAF, D5 and D6) were virulent, others (C4, P and D8) avirulent and most of them (D1, D2, D3, D4, D7, EFP, CH1, CH2 and CH3) intermediate virulent in mice. A significant linkage disequilibrium was observed in the population surveyed. However, the role of sexual recombination in the population structure of T. gondii in Brazil seems to be more central than in Europe and North America, where most studies have been performed.
The fact that all Brazilian T. gondii strains are more closely related to the type I lineage is noteworthy. From the studied strains, 85% showed a certain degree of virulence (highly virulent, virulent and intermediate virulence) and only 15% of the strains were avirulent (20). These findings contrasts with studies performed in the US and Europe, where most strains are avirulent of type II or type III (17). Although speculative at this point, we would like to suggest that the high frequency of virulent strains closely related to type I lineage may be in part responsible for the high frequency of acquire ocular toxoplasmosis commonly found in Brazil (8-10).
It seems that sexual reproduction may plays a central role in the population structure of T. gondii in Brazil, although we have detected a significant correlation between PCR-RFLP, RAPD-PCR and SSR-PCR genetic distances, suggesting linkage disequilibrium in this population. It is also possible that the epidemiology of toxoplasmosis may be different in Brazil, with ingestion of oocysts being the main mechanism of T. gondii transmission in our country, which could explain the high percentage (100%) of recombinant strains observed in our study. Circumstantial evidence of the potential importance of oocyst transmission in Brazil was the finding that the consumption of untreated or unfiltered water was implicated as a source of T. gondii infection for human populations in north Rio de Janeiro State, a disease endemic area (15). Moreover, an outbreak of clinical toxoplasmosis in humans was epidemiologically linked to drinking water from a municipal water reservoir in Santa Isabel do Ivai, Paraná State (21). Whether asexual or sexual stage transmission is responsible for the majority of Toxoplasma infections, it should be possible to identify an oocyst-specific immunogenic peptide or whole protein that would identify the stage of the parasite responsible for causing infection. The identification of factors that contribute to the high percentage of genetic recombination in Brazilian T. gondii population would be of clinical and epidemiological importance and should be explored in the future.

II – Role of cytokines and MHC haplotype in mouse resistance to infection with natural recombinant (Type I/III) strains of T. gondii.
Different studies performed in murine experimental show the important role of cytokines such as IL-12, TNF-a and IFN-y and generation of reactive nitrogen intermediates (RNI) as mediators of host resistance to early T. gondii infection (22). Thus, animals deficient in IL-12, IFN-g, inducible nitric oxide synthase (iNOS), treated with neutralizing antibodies anti-cytokines or specific inhibitors of iNOS are more susceptible to infection with T. gondii (23-27). Acquired immunity to T. gondii is associated with a Th1-type response (24). During chronic infection, neutralization of either IFN-g or TNF-a results in the reactivation of disease and the development of toxoplasmic encephalitis (TE) (28,29). Further, other host genetic factors, including MHC alleles are important determinants of host resistance and susceptibility to early infection, as well as controlling cyst numbers and encephalitis at later stages of infection with T. gondii in mice (30,31). Consistently, both CD4+ T as well as CD8+ T lymphocytes are important components in host resistance to this parasite (28,30).
Considering the immunological studies described above, we decided to characterize the importance of such immunological mechanisms in host resistance to three natural recombinant T. gondii strains (Type I/ III) isolated in Brazil, named P-Br (32), D8 and C4, which are cystogenic in mice. The IL-12/IFN-y axis and iNOS were defined as main determinants of resistance during the acute infection with the Brazilian strains. Different from the Type II strain of T. gondii (ME-49), per-oral infection with the Type I/III strains led only to a light inflammatory infiltrate and no major lesions in the intestine of the C57BL/6 mice. In addition, the BALB/c (resistant to ME-49) and C57BL/6 (susceptible to ME-49) mice were shown, respectively, to be more susceptible and resistant to cyst formation and toxoplasmic encephalitis, when infected with Type I/III strains. Consistently, a congenic BALB/c strain containing MHC haplotype “b” was highly susceptible to ME-49 (Type II strain), but not to the recombinant Type I/III strains. Together, our results indicate that MHC haplotype “b” is a major determinant of susceptibility to cyst formation and toxoplasmic encephalitis induced during infection with Type II, but not with Type I/III strains of T. gondii.

III – Protection against challenge with a natural recombinant (Type I/III) strain of T. gondii in mice vaccinated with recombinant viruses coding the main tachyzoite surface antigens.
Vaccine development is based on the observation that exposure to T. gondii can elicit a life-long immune response, capable of protecting the host against a secondary challenge with the parasite (22). In early stages of infection, this response is characterized by activation of innate mechanisms mediated by macrophages, that internalize free tachyzoites and produce IL-12, and by NK cells, which secrete IFN-g (22). This last cytokine controls the replication of the parasite and, in conjunction with IL-12, drives the differentiation of CD4+ T lymphocytes specific for parasite antigens to a Th1 cytokine profile (24). In addition, CD8+ T cells are crucial to control parasite replication in the chronic phase of the disease, preventing reactivation of infection. Both CD4+ and CD8+ T cells secrete IFN-g, and CD8+ T cells develop cytotoxic activity against infected cells (30,31).
Initial attempts to induce protection against toxoplasmosis involved the use of live attenuated tachyzoites from mutant strains of T. gondii, like the heat sensitive ts-4 (33). Despite the high efficiency in terms of activation of CD4+ and CD8+ T cells and the efficacy of protection, vaccines based on live tachyzoites are not applicable to humans, due to the risk of pathogenic side effects. Thus, most recent protocols focus on development of recombinant vaccines. As target antigens, there is a great interest in tachyzoite surface proteins, particularly SAG1 (34-38). In addition, a few studies have also been performed with SAG2 and SAG3 (39). The SAG proteins, the most abundant on the tachyzoite surface, are anchored to the parasite membrane by glicosylphosphatidylinositol (GPI) structures and are believed to be involved in the process of host cell invasion. Further, their sequences are highly conserved among different strains of T. gondii, sharing high degree of homology between type I (pathogenic and lethal to mice) and type II/III (cystogenic) strains (32,40).
Genes coding for T. gondii surface antigens SAG1 and SAG2 have been cloned and expressed in recombinant procariotic and eucaryotic systems, and both recombinant protein and plasmid have been used to immunize mice and other species. It was observed that recombinant proteins generally induce high titers of IgG1 antibodies, which have poor effect against challenge with pathogenic strains of T. gondii (38), and that improvement of the response depends on combination with proper adjuvants. On the other hand, vaccines based on plasmids coding for SAG1 and SAG2 were able in many cases, depending on administration route and formulation, to induce cellular immune response with Th1 profile, inducing better protection (34).
In this context, viral vectors such as adenoviruses can improve the immune responses in comparison to naked plasmid vaccines, since they are more efficient in transferring the genetic sequences and inducing protein expression (41). Adenoviruses infect a great variety of cells, including key immune cells, like dendritic cells, leading to an efficient activation of CD4+ and CD8+ T cells (42) and are capable of driving the immune response to a desired Th1 type (43). We generated recombinant adenoviruses encoding three genetically modified surface antigens (SAG) of the parasite Toxoplasma gondii, i.e. AdSAG1, AdSAG2 and AdSAG3 (44). Modifications included the removal of their GPI-anchoring motifs and, in some cases, the exchange of the native signal peptide by influenza virus haemaglutinin signal sequence (HASS). Adenovirus immunization of BALB/c mice elicited potent antibody responses against each protein, displaying a characteristic bias to a Th1 profile when the IgG2a/IgG1 antibody ratio was determined. Furthermore, the presence of parasite-specific IFN-g producing CD8+ T cells was analyzed in the same animals by ELISPOT. Splenocytes from immunized mice secreted IFN-g after recognition of antigen-presenting cells infected with each recombinant adenovirus. However, only AdSAG2 was able to induce in vivo sufficient numbers of antigen-specific lymphocytes to be readily distinguished from the control animals vaccinated with an irrelevant virus. We finally tested the capacity of the immune responses detected to protect mice against a challenge with live T. gondii parasites. Although no major protection was observed against tachyzoites of the highly virulent RH strain, a significant reduction in brain cyst loads was observed in animals challenged with cysts of the P-Br strain. Thus, up to 80% of the parasitic forms were eliminated from animals vaccinated with a mix of the three recombinant viruses. Since adenoviruses seemed capable of inducing broad Th1-biased protective immune responses against T. gondii antigens, other parasite antigens should be tested alone or in combination with the ones described here to further develop a protective vaccine against toxoplasmosis.
Considering that the regular route of infection with T. gondii is through the mucosa of the digestive tract, we decided to direct our studies towards a vaccine that induces a strong mucosal immunity. For that we are considering the use a heterologous prime-boost protocol with SAG2 encoding adenovirus and influenza virus. It is noteworthy that both viruses infect cells from the digestive tract and can be use to elicit mucosal immunity. Thus, we have also constructed influenza vector (45) expressing the T. gondii surface antigens, and the experiments with heterologous prime-boost with recombinant virus are being performed.

IV – Conclusions
We conclude that the great majority of the T. gondii Brazilian isolates are natural recombinants generated from lineages Type I and III. Importantly, the majority of these isolates are virulent in mice. Whereas the IL-12/IFN-g and iNOS were critical for host resistance to these isolates, we found that the mouse MHC haplotype “b” is not a determinant of susceptibility to infection, as previously determined by parasites of Type II lineage. Finally, adenovirus coding modified genes of SAG1, SAG2 and SAG3 led to induction of protective immunity, as indicated by 80% reduction of cyst numbers in mice challenged with a natural recombinant Type I/III Brazilian isolate.

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