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

Juliana Elena Silveira Pratti,Herbert Leonel De Matos Guedes,Diogo Oliveira-Maciel,Andre Macedo Vale,Tadeu Diniz Ramos,Thiago Soares De Souza Vieira,Daniel C Oliveira Gomes,Joyce Carvalho Pereira,Celio G Freire-De-Lima,Bartira Rossi-Bergmann,Leandra Linhares Lacerda,Luan Firmino-Cruz,Elvira M Saraiva,Juliana Paiva Da Silva,Alessandra Marcia Da Fonseca Martins

doi:10.1371/journal.pntd.0007146

Juliana Elena Silveira Pratti, Herbert Leonel De Matos Guedes + Show 13 more

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https://doi.org/10.1371/journal.pntd.0007146

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Abstract

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. amazonensis. L. amazonensis 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-γ in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN-γ 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-γ 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.

Highlights

Leishmaniasis is a group of chronic, non-contagious diseases caused by flagellate protozoa of the genus Leishmania [1]
We investigated the role of Toll-like receptor 9 (TLR9) during L. amazonensis infection in C57BL/6 (WT) mice and C57BL/6 TLR9-/- mice, the adjuvant effect of the DNA containing CpG motifs present in Leishmania amazonensis promastigote antigens (LaAg) vaccine and their efficacy when administered by the mucosal route, which presents high expression of TLR9 [17]
The release of dsDNA using the DNA picogreen assay of neutrophils from WT and TLR9-/- neutrophils was measured (Fig 2C), and, again, no difference was observed. These results suggest that TLR9 does not participate in neutrophil extracellular traps (NETs) induction by L. amazonensis infection in vitro

Summary

Introduction

Leishmaniasis is a group of chronic, non-contagious diseases caused by flagellate protozoa of the genus Leishmania [1]. Leishmania (L.) amazonensis is an etiological agent for a broad spectrum of leishmaniases in South American countries [2], including Brazil, where it is a causative agent of localized cutaneous leishmaniasis, diffuse cutaneous leishmaniasis and, rarely, visceral leishmaniasis [2]. The Leishvacin (or LaAg) vaccine is a first-generation vaccine composed of total proteins, lipids, carbohydrates, RNA and DNA of L. amazonensis and has been studied for years [3,4]. The efficacy of the mucosa as administration route of LaAg vaccine has already been tested in both oral and intranasal routes [5,6]. Studies show that intranasal administration of LaAg provides greater protection to BALB/c mice challenged with L. amazonensis and is more advantageous due to the easier application, and lower doses of antigen required as compared to the oral route. The protection achieved by intranasal immunization was accompanied by the development of a long-term immune memory and adaptive immunity [5,7]

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