Dados do Trabalho

Título

IMMUNIZATION WITH CENTRIN-DEFICIENT LEISHMANIA BRAZILIENSIS DOES NOT CONFER PROTECTION AGAINST SUBSEQUENT INFECTION

Introdução

Leishmaniasis affects millions of people in major areas of the globe. Despite this disease burden, a vaccine remains unavailable. Previous infection by Leishmania induces robust immunity against subsequent disease, indicating that vaccination is an attainable goal. Recent advances in this field have shown that immunization with Leishmania lacking Centrin confers robust protection against challenge. Centrin is a calcium-binding cytoskeletal protein involved in centrosome duplication. Centrin deficiency in Leishmania causes arrested division at the amastigote stage, resulting in an attenuated cell line, unable to cause disease upon experimental infection.

Objetivo (s)

We aimed to employ the LeishGEdit toolbox to generate a Centrin-deficient Leishmania braziliensis, the causative agent of mucosal and disseminated disease, as immunoprophylaxis tool.

Material e Métodos

We generated a transgenic cell line expressing Cas9 and T7 RNA polymerase, which was later employed for the targeted deletion of Centrin. LbCen-/- was evaluated for in vitro growth and murine macrophage infectivity. Safety and protective capacity of LbCen-/- was assessed in vivo in a murine model of cut leish.

Resultados e Conclusão

Whole-genome sequencing of centrin-deficient L. braziliensis (LbCen-/-) did not indicate the presence of off-target mutations. LbCen-/- promastigotes displayed normal in vitro growth whereas axenic and intracellular LbCen-/- amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon experimental infection of BALB/c mice with LbCen-/- promastigotes, lesions failed to develop whereas parasites were detected two weeks later at the inoculation site. Parasites became undetectable after five weeks of infection, indicating impaired survival of LbCen-/- in vivo. Surprisingly, mice immunized with LbCen-/- and challenged with LbWT parasites were not protected as lesion development and parasite multiplication were observed. On the contrary, mice that healed a primary infection with wild-type L. braziliensis were successfully protected, failing to develop lesions, and displaying a significantly lower parasite load, compared to control mice. The complexity of the immune response against Leishmania sp. highlights differences regarding protective immune responses and indicates that investigating these discrepancies shall contribute to advances in the field of vaccine development.

Palavras-chave

LeishGEedit; leishmaniasis; genetic manipulation; attenuation; vaccine development

Agradecimentos

Financial support: FIOCRUZ, CAPES