Immunization with centrin-Deficient Leishmania braziliensis Does Not Protect against Homologous Challenge

Abstract

Immunization with various Leishmania species lacking centrin induces robust immunity against a homologous and heterologous virulent challenge, making centrin mutants a putative candidate for a leishmaniasis vaccine. Centrin is a calcium-binding cytoskeletal protein involved in centrosome duplication in higher eukaryotes and Leishmania spp. lacking centrin are unable to replicate in vivo and are non-pathogenic. We developed a centrin-deficient Leishmania braziliensis (LbCen^-/-) cell line and confirmed its impaired survival following phagocytosis by macrophages. Upon experimental inoculation into BALB/c mice, LbCen^-/- failed to induce lesions and parasites were rapidly eliminated. The immune response following inoculation with LbCen^-/- was characterized by a mixed IFN-γ, IL-4, and IL-10 response and did not confer protection against L. braziliensis infection, distinct from L. major, L. donovani, and L mexicana centrin-deficient mutants. A prime-boost strategy also did not lead to a protective immune response against homologous challenge. On the contrary, immunization with centrin-deficient L. donovani (LdonCen^-/-) cross-protected against L. braziliensis challenge, illustrating the ability of LdonCen^-/- to induce the Th1-dominant protective immunity needed for leishmaniasis control. In conclusion, while centrin deficiency in L. braziliensis causes attenuation of virulence, and disrupts the ability to cause disease, it fails to stimulate a protective immune response.

Keywords: cutaneous leishmaniasis; immunization; live attenuated vaccine; vaccination.

Figure 1

Impaired survival of LbCen^−/− in murine macrophages. Bone marrow-derived macrophages (BMDMs) were infected with LbWT or LbCen^−/− promastigotes. After 24 h, cells were washed and stained with H&E (at 24 h or 72 h). The percentage of infection (A) and the number of amastigotes per 200 macrophages (B) were determined by optical microscopy. Alternatively, infected macrophages were washed (72 h) and cultured in Schneider medium. Three or five days later, the number of promastigotes was evaluated (C). Results are presented as means ± SEM and are pooled from two independent experiments, each performed in quadruplicate. * p < 0.05.

Figure 2

Innate immune response in LbCen^−/−-infected macrophages. Bone marrow-derived macrophages (BMDMs) were primed with LPS (100 ng/mL) and were later infected with LbWT or LbCen^−/− promastigotes. Controls (Mo) were left unprimed. After 24 h, culture supernatants were collected and the presence of TNF (A), IL-10 (B), IL-1B (C), and IL-10 (D) was determined by ELISA. (E) Nitrite concentration was evaluated by Griess reaction. Results are presented as mean ± SEM and are from a representative experiment, performed in quadruplicate. * p < 0.05.

Figure 3

Safety of LbCen^−/− upon experimental infection. BALB/c mice were inoculated with LbWT or LbCen^−/− promastigotes. (A) Lesion development was measured weekly. Parasite load was measured in dLNs two (B) and five (C) weeks post challenge. Results are expressed as means ± SEM and are from one representative experiment, performed with five mice. ** p < 0.01. ND, not detected.

Figure 4

Efficacy of LbCen^−/− against L. braziliensis. BALB/c mice were inoculated with LbCen^−/− stationary promastigotes in the ear dermis. Five weeks later, mice were challenged with LbWT and (A) lesion development was measured weekly. (B) Parasite load was measured five weeks after challenge in the ear (B) and dLNs (C). Results are expressed as means ± SEM and are from one representative experiment performed with six mice.

Figure 5

Immune response induced by LbCen^−/− versus a primary L. braziliensis infection. BALB/c mice were primary infected with LbWT and left to heal or were inoculated with LbCen^−/− promastigotes. (A) Lesion development was measured weekly. (B) DTH response to SLA, serology (C) IgG1 and IgG2a, and (D) cytokine production in dLNs were measured ten weeks after mice healed from the primary infection (LbWT) or four weeks after inoculation with LbCen^−/−. Results are expressed as means ± SEM and are from one representative experiment, performed with five mice. ** p < 0.01.

Figure 6

Efficacy of leishmanization vs. immunization with LbCen^−/−. BALB/c mice were inoculated with LbWT or LbCen^−/− promastigotes. Healed (LbWT) or immunized (LbCen^−/−) mice were challenged with LbWT. Controls consisted of age-matched naïve mice. (A) Lesion development was measured weekly and histopathological analysis of ears was performed six weeks post challenge. (B) Areas under the curves (AUCs) of ear thicknesses shown in (A). (C) Parasite load in ears and draining lymph nodes six weeks post challenge. (D) Cytokine response in draining lymph nodes six weeks post challenge. Results are expressed as means ± SEM and are from one representative experiment, performed with six mice. * p < 0.05; ** p < 0.01. ns, not significant.

Figure 7

Prime boost with LbCen^−/− cannot improve immunity against L. braziliensis. BALB/c mice were inoculated with 3 × 10⁶ LbCen^−/− promastigotes and, two weeks later, mice received a booster inoculation. (A) DTH response and (B) cytokine response in dLNs cells following re-stimulation with SLA. (C) Frequency of CD4⁺ T central memory cells (CD4⁺CD44⁺CD62L⁺). Evaluations were performed two weeks after the boost inoculation. Mice were later challenged with 10⁵ LbWT promastigotes and lesion development was measured weekly (D). (E) Parasite load, (F) cytokine production in DLN cells, and frequency of CD4⁺ T central memory cells (G) were determined five weeks after challenge. Results are expressed as means ± SEM and are from one representative experiment, performed with eight mice. ns, not significant, dotted line represents baseline ear thickness.

Figure 8

Protective immunity of LdCen^−/− against L. braziliensis. Mice were immunized with LdCen^−/− promastigotes and, five weeks later, mice were challenged with LbWT promastigotes. Lesion development was monitored weekly (A). Parasite load in ears and draining lymph nodes (B) was determined by limiting dilution five weeks post challenge. Results are expressed as means ± SEM and are from one experiment, performed with six mice. * p < 0.05; ** p < 0.01. ns, not significant.