**4. Conclusion**

178 Current Topics in Tropical Medicine

Viral vectors exhibit many advantages for the development of a vaccine against toxoplasmosis. In summary, viral vectors express foreign antigens directly inside host cells very efficiently; as a result they present antigen fragments in association with MHC molecules more proficiently and, subsequently, they better stimulate the required antitoxoplasma T cell responses (Th1 and CTL) because they act as natural adjuvants and stimulate intracellular innate immunity receptors. In addition, they can be administrated through the natural route of infection, such as via nasal mucosa, and they are able to induce

Our group has tested adenoviruses and influenza viruses as feasible vaccine vectors against toxoplasmosis (Caetano *et al*. 2006; Machado *et al*. 2010; Mendes *et al*. 2011) and they have shown significant improvement in comparison with naked plasmid vaccines. For those studies we have focused on possible formulations and immunization protocols using *T. gondii* surface antigens (SAGs). These molecules are involved in host cell attachment and invasion, and their sequences are conserved among different strains of *T. gondii*, sharing a high degree of homology even between type I (pathogenic and lethal in mice) and type II/III strains (cystogenic). However, these favorable traits do not ensure that these antigens will end up displaying sufficient protective capacities, but the proofs-of-principle obtained with their use will surely be maintained for any other antigens that display better protective properties. In Caetano *et al*. (Caetano *et al*. 2006), we generated three recombinant adenoviruses encoding genetically modified SAG1, SAG2 and SAG3, without the 3'-end GPI anchoring motifs to ensure secretion and subsequent induction of combined Th/CTL immune responses. BALB/c mice received rAd/SAG1, rAd/SAG2, rAd/SAG3, or a combination of the three viruses (rAdMIX) and were challenged with 100 live tachyzoites of the *T. gondii* RH strain or with 20 cysts of the P-Br strain. Adenovirus immunization elicited potent antibody responses against each protein and displayed a significant bias toward a Th1 profile. When comparing the three recombinant viruses, rAd/SAG2 was the most efficient in eliciting antigen-specific antibodies. A significant reduction in cysts loads in the brain was observed in animals challenged with the P-Br strain. Vaccination with a mixture of all viruses promoted the highest level of inhibition of cyst formation, about 80%. However, no protection was observed against tachyzoites of the

In the study by Machado *et al*. (2010), we generated a recombinant Influenza A vector encoding SAG2 of *T. gondii* and explored an original heterologous prime-boost immunization protocol using influenza virus (rFLU/SAG2) and a recombinant adenovirus (rAd/SAG2). Influenza A viruses are promising but currently under-explored vectors, which display some advantageous features to be used as live recombinant vaccines, such as the ability to infect and activate antigen presenting cells as well as to present high immunogenicity at mucosal and systemic levels (Rocha *et al*. 2004; Machado *et al*. 2010). BALB/c mice primed with an intranasal rFLU/SAG2 dose and boosted with a subcutaneous rAd/SAG2 dose elicited both humoral and cellular immune responses specific for SAG2. Moreover, when immunized animals were challenged with the cystogenic P-Br strain of *T. gondii*, they displayed up to 85% of reduction in parasite burden. These results demonstrate the potential use of recombinant influenza and adenoviruses in vaccination protocols to protect against oral challenge with *T.* 

Literature shows that, for other diseases, there is a good reproducibility of results when transferring experimental results obtained by immunization with some viral vectors (in particular adenoviruses) from small experimental animals to larger animal models or

effective and long lasting immune responses.

highly virulent RH strain (Caetano *et al*. 2006).

*gondii* (Machado *et al*. 2010), although there is room for improvement.

Recombinant subunit vaccines (proteins in adjuvants, DNA vaccines and recombinant live vectors) are the present trends for the development of a vaccine against Toxoplasmosis. A myriad of parasite antigens have been described and researchers are testing them in many animal models of the disease. It is our belief that, more than the description of new parasite antigens that could be used in a final vaccine formulation, one of the major issues for the next future is to develop and test highly antigenic formulations using currently known antigens. Developing this type of formulations requires a deep knowledge of the immune system's antigen processing and presentation pathways, proficiency in the use of molecular biology techniques to adapt the parasite antigen sequences to enter those pathways, and using the new generation of adjuvants and delivery vectors in a manner that can best stimulate the pretended anti-parasite Th1 cellular (and probably humoral) immune responses. The options and combinations are so broad, and yet untested, that several years of research will be needed before we can decide which combination will be more adequate [antigen(s) + adjuvant(s) + vector(s)] or what will be the most efficient immunization protocol (single dose, multiple dose, homologous or heterologous prime-boost, etc.).

Finally, we would like to call attention to the fact that a possible result of the immunization/protection tests may actually be the achievement of a cost-effective vaccine that may be suitable for large-scale production and use. Then, one key question will arise for the future, regarding the correct use of that vaccine. In principle, the vaccine should be applied to animals, because preventing oocyst shedding by cats and tissue cyst formation in meat-producing animals should have great impact on both environmental contamination and public health. But this intervention could pose a risk because of the loss of herd immunity against the parasite and the resurgence of a different profile of *Toxoplasma*-related pathologies because of the primo-infection of non-vaccinated adults traveling to other countries or regions instead of kids or young adults being infected at its home places. To solve this, a possibility would be the universal vaccination of all children against toxoplasmosis, although this might end up being not feasible in practice or even might not be considered as a priority. Researchers should include these topics amongst those to be discussed in the forthcoming years within the field of vaccine development against toxoplasmosis.
