The role of TLR9 on Leishmania amazonensis infection and its influence on intranasal LaAg vaccine efficacy.

Leishmania (L.) amazonensis is one of the etiological agents of cutaneous leishmaniasis (CL) in Brazil. Currently, there is no vaccine approved for human use against leishmaniasis, although several vaccine preparations are in experimental stages. One of them is Leishvacin, or LaAg, a first-generation vaccine composed of total L. amazonensis antigens that has consistently shown an increase of mouse resistance against CL when administered intranasally (i.n.). Since Toll-like receptor 9 (TLR9) is highly expressed in the nasal mucosa and LaAg is composed of TLR9-binding DNA CpG motifs, in this study we proposed to investigate the role of TLR9 in both L. amazonensis infection and in LaAg vaccine efficacy in C57BL/6 (WT) mice and TLR9(-/-) mice. First, we evaluated, the infection of macrophages by L. amazonensis in vitro, showing no significant difference between macrophages from WT and TLR9(-/-) mice in terms of both infection percentage and total number of intracellular amastigotes, as well as NO production. In addition, neutrophils from WT and TLR9(-/-) mice had similar capacity to produce neutrophil extracellular traps (NETs) in response to L. amazonesis. L. amazonensis was did not activate dendritic cells from WT and TLR9(-/-) mice, analysed by MHCII and CD86 expression. However, in vivo, TLR9(-/-) mice were slightly more susceptible to L. amazonensis infection than WT mice, presenting a larger lesion and an increased parasite load at the peak of infection and in the chronic phase. The increased TLR9(-/-) mice susceptibility was accompanied by an increased IgG and IgG1 production; a decrease of IFN- in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN- producing CD8(+) T cells, but not CD4(+) T cells in the lesion-draining lymph nodes. Also, TLR9(-/-) mice could not control parasite growth following i.n. LaAg vaccination unlike the WT mice. This protection failure was associated with a reduction of the hypersensitivity response induced by immunization. The TLR9(-/-) vaccinated mice failed to respond to antigen stimulation and to produce IFN- by lymph node cells. Together, these results suggest that TLR9 contributes to C57BL/6 mouse resistance against L. amazonensis, and that the TLR9-binding LaAg comprising CpG motifs may be important for intranasal vaccine efficacy against CL. Author summary Leishmaniasis is a major neglected tropical disease, being responsible for more than 20 million deaths per year. The high mortality rate highlights the difficulties and ineffectiveness of the current prophylactic approaches and treatments currently available. Therefore, the development of an effective vaccine would be highly advantageous to circumvent these problems. Despite the many vaccines preparations that have been studied in the last few years, none have shown satisfactory efficacy to be approved for human use. Immune receptors, including the TLR family, are known to be important for host defense during parasitic infections, such as leishmaniasis, and also for vaccine efficacy. In this work, we investigate the role of TLR9 during Leishmania amazonensis infection in vaccinated and non-vaccinated mice. We used a C57BL/6 TLR9-/- mouse model and a first-generation vaccine preparation (LaAg) composed of a total lysate of L. amazonesis. We demonstrate that TLR9 is important for controlling leishmaniasis infection caused by L. amazonensis and is involved in the efficacy of the LaAg vaccine. These findings will certainly help in the development of a better vaccine against leishmaniasis.