**4. Final remarks**

TNFα concomitant production by Th1 and CD8<sup>+</sup>

IFNγ, IL-10 and TNFα production was analyzed by flow cytometry in CD4<sup>+</sup>

296 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

and CD8<sup>+</sup>

IFNγ<sup>+</sup>

single producers [154]. Moreover, those double producers CD4<sup>+</sup>

TNFα<sup>+</sup>

high quality CD4<sup>+</sup>

Prism 5 (GraphPad Software).

[154, 155]. As for CD8<sup>+</sup>

activity compared to IFNγ<sup>+</sup>

IL-2<sup>+</sup>

in the killing of *L. major* [151, 152] and other unrelated microorganisms (*e.g. Mycobacterium tuberculosis* [153]) than the production of IFNγ or TNFα alone. More recently, IFNγ<sup>+</sup>

cytes were stimulated *ex-vivo* with phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin A, stained for sur‐ face and intracellular molecules and analyzed in a FACSCanto flow cytometer (BD Bioscences). Cytokine single and double producers in each lymphocyte population are depicted from naïve, infected **(A, C)** or challenged **(B, D)** mice. Bars represent means ± SD of 4 to 9 animals of one experiment with statistically significant differences between naïve and infected mice indicated with \* when p<0.05, as calculated by two-tailed Mann Whitney test run in GraphPad

protocols against *L. major* and correlate with prognosis of protection much better than IFNγ

our study, we could not detect any difference in the cytotoxicity mediated by CD8<sup>+</sup>

**Figure 4.** Intracellular cytokines of CD4<sup>+</sup> and CD8<sup>+</sup> lymphocytes of HL infected and re-infected animals

, were determined to belong to the central memory subset, providing long-term protection

T cells has for long proven to be more efficient

T cells were described to be generated after several vaccination

single producer cells in HIV-infected patients [156]. However, in

T cells, they were described to have enhanced cytolytic

**(A, B)** and CD8<sup>+</sup>

TNFα<sup>+</sup>

T cells from

T cells, which can also be

**(C, D)** lymphocytes. Spleno‐

Leishmaniasis is a tropical neglected disease that urgently needs control measures, as vacci‐ nation, since nowadays the global population is at risk. As some vaccines are available for ZVL, the discovery of an effective human vaccine for VL is near. Choosing the right antigen coupled with the appropriate adjuvant for the formulation is crucial to have an effective vaccine, but immunogenicity sometimes countervail safety and complicates the scenario. Effective immunization requires the presentation of the antigen by proper APCs to mount a strong immune response and develop immunological memory, as well as it entails antigen persistence. As described previously, live vaccines produce more robust immune responses than dead parasites or defined protein or peptides but they represent an important health risk, mainly in immunosuppressed people. Furthermore, the immune response developed against live *Leishmania infantum* strains that display differences in infectivity is also unique and characteristic of each strain, being infectivity related with a stronger induction of an immune response, as showed by our experimental data.

In this chapter, we have updated the main aspects to consider when a vaccination study against *Leishmania* is planned. We aimed to show that vaccination is an effective way, and hopefully a soon reality, to prevent the spread of leishmaniasis, limiting the outcome of the disease and avoiding the parasite transmission. While successful research is close, many efforts are still needed for achieving an efficient human vaccine for leishmaniasis accessible to everyone in need.
