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

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.


Introduction
Leishmaniasis is a parasitic disease caused by protozoans of the genus Leishmania that are transmitted by sand fly insects.There are several clinical presentations of leishmaniasis which vary according to the parasite species.The leishmaniases cause high morbidity and mortality worldwide and are an important public health problem [1].In 2021, Brazil reported 15,023 cases of cutaneous/mucosal leishmaniasis [2] caused mainly by Leishmania braziliensis.Although different vaccine formulations have conferred protection against Leishmania spp. in animal models [3], to this date, there are no leishmaniasis vaccines approved for human use.
Leishmania infection can stimulate a protective response that depends upon the generation of IFN-γ-producing CD4 + T cells, leading to macrophage activation and subsequent parasite killing [4].On the other hand, disease development is mainly driven by the production of non-protective Th2-associated cytokines IL-4, IL-10, IL-13, and TGF-β [5,6].Durable protective immunity acquired following leishmanization led to the development of several live attenuated Leishmania strains as prophylactic vaccines [7].The possibility of using genetically modified Leishmania spp. as candidate vaccines has expanded given the feasibility of large-scale and cost-effective production [8].Adaptation of CRISPR/Cas9 methods to Leishmania genome editing enabled the development of marker-free Leishmania strains [9].Research towards the deletion of genes related to parasite survival and/or virulence is promising, as these modifications maintain the antigenic repertoire of wildtype counterparts and thus the ability to induce a broad-spectrum immune response while eliminating the pathology associated with infection [10].Centrin 1 is a calcium-binding protein that plays a fundamental role in centrosome duplication [11].Centrin 1 was initially characterized in L. donovani as a homolog of human centrin 2, and infection of mice with centrin-deficient L. donovani (LdCen −/− ) induces a multifunctional T-cell response that significantly reduces the parasite load upon virulent challenge, conferring protection against disease [12].
L. braziliensis has been little explored in terms of vaccine development despite its importance as the causative agent of mucosal and disseminated leishmaniasis, two severe clinical manifestations of leishmaniasis [13].Given the promising results obtained with centrin-deficient Leishmania spp., we developed a centrin-deficient L. braziliensis cell line (Lb-Cen −/− ) [14].We previously showed that centrin deletion did not cause off-target mutations and that LbCen −/− axenic amastigotes become multinucleated cells and display impaired survival in macrophages similar to other centrin-deleted Leishmania species [12, 15,16].In vivo, LbCen −/− failed to induce cutaneous lesions in BALB/c mice [14].Herein, we explored LbCen −/− as a vaccine candidate against cutaneous leishmaniasis caused by L. braziliensis.Although LbCen −/− is safe and non-pathogenic, it did not induce a protective immune response, differently from what was observed in mice that healed from a primary infection with Leishmania braziliensis wild type (LbWT) or mice inoculated with centrindeficient L. donovani (LdonCen −/− ).Our results point out the differences in the ability of attenuated Leishmania spp. to induce protective immune responses, as well as the divergent mechanisms of protection that may be necessary, collectively highlighting the hurdles of vaccine development against some species causing American Tegumentary Leishmaniasis.

Bone Marrow-Derived Macrophage (BMDM) Culture and In Vitro Exposure to LbCen −/−
Bone marrow cells were isolated from femurs and tibias of BALB/c mice and cultured with RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 2.5% HEPES (all from Invitrogen) and further complemented with conditioned media from the mouse fibroblast cell line L929 (ThermoFisher).After differentiation, bone marrow-derived macrophages (BMDMs) were seeded at 3 × 10 5 cells/500 µL/coverslip per well in 24-well plates.BMDM monolayers were washed to remove non-adherent cells and infected with stationary-phase promastigotes LbWT or LbCen −/− at a 10:1 parasite/host cell ratio in supplemented RPMI 1640/well.Plates were incubated at 37 • C under 5% CO 2 for 6 h or 24 h.Coverslips were extensively washed to remove any non-internalized Leishmania and were methanol-fixed and stained with hematoxylin and eosin (H&E).Leishmania uptake and internalized parasites were assessed by observing 200 macrophages, and the number of cells with and without Leishmania as well as the total number of intracellular parasites were counted by optical microscopy (magnification ×1000).Alternatively, coverslips were washed after 24 h of infection and further cultured in 500 µL of supplemented RPMI 1640/well at 37 • C and 5% CO 2 for a total of 72 h.Then, cells were washed and cultured in supplemented Schneider's insect medium for 5 days at 26 • C. Promastigote numbers were determined at days 3 and 5 by counting in a hemocytometer.

Nitric Oxide and Cytokine Quantification
BMDMs were primed or not with lipopolysaccharide (LPS) (100 ng/mL) (Invitrogen) and infected with LbWT or LbCen −/− as described above for 24 h.Cell culture supernatants were collected after 24 h for nitric oxide quantification using a Griess reagent [17].Cytokine levels (IL-10, TNF-a, IL-12p70, and IL-1B) were determined by sandwich ELISA, following the manufacturer's instructions (Invitrogen).(3 × 10 6 stationary-phase promastigotes), using a 27G needle.Ear thickness was measured weekly using a digital caliper (Thermo Scientific).Parasite load was evaluated at the inoculation site (ear) and in draining lymph nodes (dLNs), two and five weeks later (five mice per time point), by limiting dilution analysis, as previously described [18].

Safety and Efficacy of LbCen
To evaluate protection, mice (n = 12) were inoculated with LbCen −/− , as described above.Five weeks later, mice were challenged in the left ear dermis with 10 5 LbWT.Controls consisted of age-matched naïve mice (n = 10) infected with 10 5 LbWT.Parasite load was determined five weeks after challenge (6 mice per time point), as described above.

Leishmanization
BALB/c mice (n = 12) were inoculated in the right ear dermis with LbWT (10 5 stationary-phase promastigotes), using a 27G needle.Ear thickness was measured weekly using a digital caliper (Thermo Scientific).Ten weeks later, after lesion healing, we evaluated the cellular and humoral response.Delayed-Type Hypersensitivity (DTH) was evaluated (n = 6 mice) by inoculation of L. braziliensis SLA (10 ug/mL), prepared as described elsewhere [19], in the left ear dermis.Ear induration was measured 24, 48, and 72 h later, using a digital caliper (Thermo Scientific).For the humoral response (n = 6 mice), MaxiSorp 96-plates were coated with Soluble Leishmania Antigen (SLA) (10 ug/mL) and incubated overnight at 4 • C. Plates were washed with PBS 1X +0.05% Tween 20 (PBS-T) and blocked with 5% non-fat milk in PBS-T for 2 h at room temperature.Sera were diluted 1/100 in PBS-T +5% non-fat milk and were incubated overnight at 4 • C. The plates were washed, incubated with IgG1 or IgG2a HRP-conjugated antibodies (Invitrogen) (1/1000 in PBS-T +5% non-fat milk) for 2 h at room temperature.The plates were washed, TMB solution (Invitrogen) was added, and reactions were stopped with H 2 SO 4 2N.The plates were read at 450 nm using a FilterMax F3 Multi-mode Microplate Reader (Molecular Devices).SoftMax Pro Software (Version 6.3) was used to obtain an indirect measure of antibody titers in serum samples.Sera from naïve mice were used as controls.
Ten weeks after the primary LbWT infection, healed (leishmanized) mice (n = 6) were challenged with 10 5 stationary-phase LbWT promastigotes, inoculated in the left ear dermis.Controls consisted of age-matched naïve mice (n = 6) inoculated with stationary-phase LbWT promastigotes.Parasite load was determined five weeks after challenge, as described above, with 6 mice per time point.To evaluate the cellular response, dLN cells (1 × 10 6 ) were seeded on 24 well-plates and stimulated with SLA (10 ug/mL) or ConA (1 ug/mL) or left unstimulated, in supplemented RPMI.Cells were incubated at 37 • C and 5% CO 2 for 48 h and cytokine production was assayed in culture supernatants by a Luminex Assay (MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel, Merck Millipore, Cat#MCYTOMAG-70K), following the manufacturer's instructions.Data acquisition was performed with the Luminex LX200 (Merck Millipore).Mean Fluorescent Intensity was calculated against standards using the Milliplex Analyst software Version 5.1.
2.6.Immunization with LbCen −/− and Challenge with LbWT BALB/c mice (n = 6) were inoculated in the right ear dermis with LbCen −/− (3 × 10 6 stationary-phase promastigotes) or LbWT (10 5 stationary-phase promastigotes) using a 27G needle.Ear thickness was measured weekly using a digital caliper (Thermo Scientific).The cellular response (DTH, n = 3 mice; re-stimulation of dLN cells with SLA, n = 3 mice) and humoral immune response (IgG1 and IgG2a, n = 6 mice) were evaluated as described above at the following time points: ten weeks after the healing of the primary infection with LbWT (leishmanization) and four weeks after inoculation with LbCen −/− .2.7.Efficacy of Immunization with LbCen −/− versus Leishmanization BALB/c mice (n = 6 per group) were either leishmanized or immunized with LbCen −/− as described above.Ten weeks after leishmanization and six weeks after immunization, mice were challenged with LbWT (10 5 stationary-phase promastigotes).Controls consisted of age-matched naïve mice (n = 6) inoculated with stationary-phase LbWT promastigotes.Six weeks post challenge, mice were euthanized for the determination of parasite load and cytokine production in dLN cells.Challenged ears were excised and fixed in 10% formalin.Tissues were embedded in paraffin, cut, and stained with hematoxylin and eosin (H&E).Slides were examined under a light microscope and images were obtained using a Slide scanner Olympus VS110 system and photographs were obtained using OlyVIA Software (version 3.8).

Primary and Booster Immunization
BALB/c mice (n = 8) were inoculated in the right ear dermis with LbCen −/− (3 × 10 6 stationary-phase promastigotes), and two weeks later, the mice were boosted at the same site.Two weeks after the booster inoculation, the cellular response (DTH and re-stimulation of dLN cells with SLA) was evaluated as described above.The frequency of central memory T cells (TCMs) was determined by flow cytometry.Briefly, dLN cells (5 × 10 5 ) were incubated with Fc Block (BD Biosciences, clone 2.4G2) for 5 min at 4 • C and then stained with CD4 (eBioscience, clone GK1.5), CD44 (BD Pharmingen), and CD62L (BD Pharmingen) antibodies in the dark for 20 min at 4 • C. The cells were washed in PBS and resuspended in PBS containing Hoechst 33258 at 5 ug/mL.Acquisition was performed using an LSRFortessa™ flow cytometer (BD Biosciences) and analyzed using FlowJo Software version 10. Isotype controls for each antibody used under similar conditions indicated specific binding of the test antibody.Electronic compensation was performed with single-stained cells with individual mAbs used in the test samples.
To evaluate the efficacy of the prime-boost strategy, mice (n = 8) were immunized as described and, five weeks later, were challenged with LbWT (10 5 stationary-phase promastigotes).Controls consisted of age-matched naïve mice (n = 8) inoculated with stationary-phase LbWT promastigotes.Lesion development was measured weekly.Five weeks after the challenge, parasite load, cytokine response in dLN cells stimulated with SLA, and the frequency of TCM were determined, as described above.

Statistical Analysis
The course of disease was plotted individually for mice in all experimental and control groups.Comparisons between two groups were performed using Mann-Whitney nonparametric t-tests and comparisons among more than two groups were performed using the Kruskal-Wallis method.Data are presented as the mean ± error of the mean.Analyses were conducted using Prism (V.8.0, GraphPad) and a p-value ≤ 0.05 was considered significant.

LbCen −/− Parasites Exhibit Impaired Growth and Survival in BMDMs
BMDMs were infected with LbCen −/− or LbWT and, after 24 h and 72 h, the number of infected macrophages and intracellular parasites was determined.Exposure to LbCen −/− led to significantly reduced survival (p < 0.05) compared to LbWT, both at 24 h and 72 h (Figure 1A,B).After 72 h, the BMDMs were washed and cultured in a supplemented Schneider medium to determine the number of recovered parasites.At both time points, we observed a significantly decreased (p < 0.05) recovery of LbCen −/− compared to LbWT (Figure 1C).This outcome was not due to impaired uptake by the host cell, as the number of BMDMs harboring LbCen −/− or LbWT was similar after 6 h of exposure (Supplemental Figure S1A,B) as was the number of intracellular amastigotes.These results confirm that lack of centrin in L. braziliensis impairs survival and replication within the host cell.
phase LbCen −/− or with metacyclic LdonCen −/− via the tail vein.Five weeks later, mice were challenged with LbWT (10 5 stationary-phase promastigotes) in the left ear dermis.Controls consisted of age-matched naïve mice (n = 6).Ear thickness was measured weekly.Six weeks post challenge, parasite load was determined by limiting-dilution analysis, as described above.

Statistical Analysis
The course of disease was plotted individually for mice in all experimental and control groups.Comparisons between two groups were performed using Mann-Whitney non-parametric t-tests and comparisons among more than two groups were performed using the Kruskal-Wallis method.Data are presented as the mean ± error of the mean.Analyses were conducted using Prism (V.8.0, GraphPad) and a p-value ≤ 0.05 was considered significant.

LbCen −/− Parasites Exhibit Impaired Growth and Survival in BMDMs
BMDMs were infected with LbCen −/− or LbWT and, after 24 h and 72 h, the number of infected macrophages and intracellular parasites was determined.Exposure to LbCen −/− led to significantly reduced survival (p < 0.05) compared to LbWT, both at 24 h and 72 h (Figure 1A,B).After 72 h, the BMDMs were washed and cultured in a supplemented Schneider medium to determine the number of recovered parasites.At both time points, we observed a significantly decreased (p < 0.05) recovery of LbCen −/− compared to LbWT (Figure 1C).This outcome was not due to impaired uptake by the host cell, as the number of BMDMs harboring LbCen −/− or LbWT was similar after 6 h of exposure (Supplemental Figure S1A,B) as was the number of intracellular amastigotes.These results confirm that lack of centrin in L. braziliensis impairs survival and replication within the host cell.

Macrophage Exposure to LbCen −/− or LbWT Induces Similar Cytokine Response
BMDMs were exposed to LbCen −/− or LbWT in the presence or absence of LPS, and cytokine production was evaluated after 24 h.Levels of TNF were similar in both LbCen −/− and LbWT infections and significantly higher (p < 0.05) compared to controls (Figure 2A).Levels of IL-12p70 were also similar for LbCen −/− and LbWT and significantly lower (p < 0.05) compared to controls stimulated with LPS (Figure 2B).Exposure to LbWT impaired IL-1β secretion, whereas LbCen −/− did not (Figure 2C).IL-10 levels (Figure 2D) and NO production were not changed in comparison to controls (Figure 2E).Taken together, these results show that the macrophages exposed to LbWT or LbCen −/− parasites produce a similar innate immune response, except for IL-1β.
Levels of IL-12p70 were also similar for LbCen −/− and LbWT and significantly lower (p < 0.05) compared to controls stimulated with LPS (Figure 2B).Exposure to LbWT impaired IL-1β secretion, whereas LbCen −/− did not (Figure 2C).IL-10 levels (Figure 2D) and NO production were not changed in comparison to controls (Figure 2E).Taken together, these results show that the macrophages exposed to LbWT or LbCen −/− parasites produce a similar innate immune response, except for IL-1β.

Immunization with LbCen −/− Does Not Cause Lesions and Is Safe
The deletion of centrin affects amastigote growth, and mice inoculated with centrindeficient Leishmania do not develop disease [15,16].Deletion of the centrin in L. braziliensis reproduced this avirulent phenotype, as BALB/c inoculated with LbCen −/− parasites failed to develop cutaneous lesions (Figure 3A), whereas inoculation of LbWT leads to lesions that develop and heal spontaneously [13].Two weeks after inoculation, the parasite load was significantly lower (p < 0.05) in the ears of mice inoculated with LbCen −/− compared to LbWT (Figure 3B).Viable parasites were recovered from the inoculation site for both LbWT and LbCen −/− groups at 2 weeks p.i., but only from the LbWT group and not from the LbCen −/− group from the dLNs 2 weeks p.i. (Figure 3B).Five weeks post inoculation, LbCen −/− parasites were not detected at either the inoculation site or in dLNs (Figure 3C), whereas in LbWT-infected mice, parasites were detected at both sites.

Immunization with LbCen −/− Does Not Cause Lesions and Is Safe
The deletion of centrin affects amastigote growth, and mice inoculated with centrindeficient Leishmania do not develop disease [15,16].Deletion of the centrin in L. braziliensis reproduced this avirulent phenotype, as BALB/c inoculated with LbCen −/− parasites failed to develop cutaneous lesions (Figure 3A), whereas inoculation of LbWT leads to lesions that develop and heal spontaneously [13].Two weeks after inoculation, the parasite load was significantly lower (p < 0.05) in the ears of mice inoculated with LbCen −/− compared to LbWT (Figure 3B).Viable parasites were recovered from the inoculation site for both LbWT and LbCen −/− groups at 2 weeks p.i., but only from the LbWT group and not from the LbCen −/− group from the dLNs 2 weeks p.i. (Figure 3B).Five weeks post inoculation, LbCen −/− parasites were not detected at either the inoculation site or in dLNs (Figure 3C), whereas in LbWT-infected mice, parasites were detected at both sites.

Immunization with LbCen −/− Does Not Protect against LbWT Infection
To evaluate the protective capacity of LbCen −/− , we immunized BALB/c mice with LbCen −/− and, 5 weeks later, challenged them with LbWT.Surprisingly, immunization with LbCen −/− did not protect against LbWT infection (Figure 4A).Mice developed cutaneous lesions that were comparable to those observed in naïve controls upon a challenge infection with LbWT at week 10 (Figure 4A).In a separate experiment, after 5 weeks of immunization followed by five weeks of WT challenge, there was no difference in the parasite load in the two groups, both at the inoculation site and in dLNs (Figure 4B and Figure 4C, respectively).Thus, a lack of centrin in L. braziliensis renders parasites unable to cause pathology but incapable of conferring protection against infection with wild-type L. braziliensis, differently from the centrin deletion mutation of other Leishmania spp.[12,15,16].

Immunization with LbCen −/− Does Not Protect against LbWT Infection
To evaluate the protective capacity of LbCen −/− , we immunized BALB/c mice with LbCen −/− and, 5 weeks later, challenged them with LbWT.Surprisingly, immunization with LbCen −/− did not protect against LbWT infection (Figure 4A).Mice developed cutaneous lesions that were comparable to those observed in naïve controls upon a challenge infection with LbWT at week 10 (Figure 4A).In a separate experiment, after 5 weeks of immunization followed by five weeks of WT challenge, there was no difference in the parasite load in the two groups, both at the inoculation site and in dLNs (Figure 4B and Figure 4C, respectively).Thus, a lack of centrin in L. braziliensis renders parasites unable to cause pathology but incapable of conferring protection against infection with wild-type L. braziliensis, differently from the centrin deletion mutation of other Leishmania spp.Leishmanization, known as the deliberate inoculation of Leishmania parasites to provide protective immunity to re-infection, is effective and has been successfully tested in pre-clinical animal models [20,21] and in humans [22].We thus evaluated the immune response in mice that healed from a primary infection with LbWT.Following inoculation of LbWT, BALB/c mice developed lesions that peaked at around week 5 and spontaneously healed by week 10 (Supplemental Figure S2A).These primary infected and healed mice mounted a DTH response against SLA (Supplemental Figure S2B) and produced both IgG1 and IgG2a (Supplemental Figure S2C).Upon re-infection of healed mice with LbWT, lesions did not develop (Supplemental Figure S2D), differently from the control (naïve) mice.This protective outcome is accompanied by a significantly lower (p < 0.01) parasite load at the inoculation site but not in dLNs (Supplemental Figure S2E).Mice that healed from a primary infection also produced significantly (p < 0.01) lower levels of IFN-γ, IL-4, and IL-10 (p < 0.01) (Supplemental Figure S2F) compared to control (naïve) mice.Thus, experimental infection with LbWT induces disease development that is spontaneously cured in BALB/c mice, and healed mice are immune to challenge.
Next, we wanted to compare the immune response of leishmanized versus immunized animals.As observed before, LbCen −/− does not cause lesion development, differently from LbWT (Figure 5A).Although the DTH responses in the two groups were not significantly different (Figure 5B), IgG1 and IgG2a levels (Figure 5C) were significantly higher (p < 0.01) in leishmanized mice compared to immunized animals.Moreover, while IFN-γ production was similar in the two groups, IL-4, IL-10 and IL-17 levels were higher in mice immunized with LbCen −/− (Figure 5D).These results showed that the immune responses that developed upon inoculation with LbCen −/− were not Th1-dominant, contrary to that developed by mice that healed from a primary infection with LbWT.

Immunization with LbCen −/− Cannot Recapitulate the Immune Response Induced by Healed Primary Infection with LbWT
Leishmanization, known as the deliberate inoculation of Leishmania para provide protective immunity to re-infection, is effective and has been successfull in pre-clinical animal models [20,21] and in humans [22].We thus evaluated the i response in mice that healed from a primary infection with LbWT.Following inoc of LbWT, BALB/c mice developed lesions that peaked at around week spontaneously healed by week 10 (Supplemental Figure S2A).These primary infec healed mice mounted a DTH response against SLA (Supplemental Figure S2 produced both IgG1 and IgG2a (Supplemental Figure S2C).Upon re-infection of mice with LbWT, lesions did not develop (Supplemental Figure S2D), differently f control (naïve) mice.This protective outcome is accompanied by a significantly lo 0.01) parasite load at the inoculation site but not in dLNs (Supplemental Figure S2 Given the immune responses developed by mice immunized with LbCen −/− versus leishmanized mice, we compared the outcome of a challenge with LbWT in these two settings.At the peak of lesion development, tissue sections from the ears of LbCen −/−immunized mice showed signs of chronic inflammation, features not observed in leishmanized mice (Figure 6A), yet not as prominent as those observed in the naïve mice.On the contrary, leishmanized mice showed only mild signs of inflammation.Leishmanized mice presented a significantly lower (p < 0.01) disease burden (AUC) compared to mice inoculated with LbCen −/− or control (naïve) mice (Figure 6B) and significantly lower (p < 0.01) parasite load at the inoculation site, compared to LbCen −/− -inoculated or control (naïve) Vaccines 2024, 12, 310 9 of 15 mice (Figure 7C).While the three experimental groups displayed similar levels of IFN-γ, detected upon stimulation of dLN cells (Figure 6D), the production of IL-4, IL-10, and IL-17 was significantly higher in mice inoculated with LbCen −/− (Figure 6D).Thus, we can speculate that the cellular recruitment induced by immunization with LbCen −/− may have favored parasite establishment.Taken together, these results demonstrate that inoculation with LbCen −/− cannot recapitulate the immune profile induced by leishmanization and, as such, is incapable of conferring protection against challenge with L. braziliensis.
immunized mice showed signs of chronic inflammation, features not observed in leishmanized mice (Figure 6A), yet not as prominent as those observed in the naïve mice.On the contrary, leishmanized mice showed only mild signs of inflammation.Leishmanized mice presented a significantly lower (p < 0.01) disease burden (AUC) compared to mice inoculated with LbCen −/− or control (naïve) mice (Figure 6B) and significantly lower (p < 0.01) parasite load at the inoculation site, compared to LbCen −/−inoculated or control (naïve) mice (Figure 7C).While the three experimental groups displayed similar levels of IFN-γ, detected upon stimulation of dLN cells (Figure 6D), the production of IL-4, IL-10, and IL-17 was significantly higher in mice inoculated with LbCen −/− (Figure 6D).Thus, we can speculate that the cellular recruitment induced by immunization with LbCen −/− may have favored parasite establishment.Taken together, these results demonstrate that inoculation with LbCen −/− cannot recapitulate the immune profile induced by leishmanization and, as such, is incapable of conferring protection against challenge with L. braziliensis.

Priming/Boosting Induces Prolonged Parasite Persistence
For most vaccine strategies, multiple immunizations are required to induce efficient protection [23].Although this has not been necessary in studies performed with Leishmania spp.lacking centrin [15,16], we investigated whether a second immunization with LbCen −/− could boost the primary immune response and, hence, alter the outcome of infection after challenge.Priming and boosting with LbCen −/− parasites continued to be safe as mice remained lesion-free (data not shown).The cellular response, measured by DTH (Figure 7A), was similar to that observed with a single immunization (Figure 5B).The recall response also showed that while IFN-γ was produced, IL-10 was detected at comparatively higher levels (Figure 7B).Two immunizations with LbCen −/− were also not capable of generating a significant increase in CD4 + T central memory cells (CD4 + CD44 + CD62L + ) (Figure 7C), as seen upon a single immunization with LmexCen −/− [16].Given that a booster inoculation with LbCen −/− did not alter the immune response observed earlier, we expected that the outcome of a challenge infection would also be the same.Indeed, mice receiving two inoculations with LbCen −/− continued to be susceptible to a challenge infection with LbWT (Figure 7D), with lesions developing similarly to the control, naïve mice.The parasite load in the two groups was similar (Figure 7E).Lastly, no significant differences were observed in the production of IFN-γ (Figure 7F) or IL-10 (Figure 7G), comparing mice primed and boosted with LbCen −/− and control, naïve mice.Collectively, our results show that a booster LbCen −/− immunization does not alter the lack of protection observed upon a single immunization.

Priming/Boosting Induces Prolonged Parasite Persistence
For most vaccine strategies, multiple immunizations are required to induce efficient protection [23].Although this has not been necessary in studies performed with Leishmania spp.lacking centrin [15,16], we investigated whether a second immunization with LbCen −/− could boost the primary immune response and, hence, alter the outcome of infection after challenge.Priming and boosting with LbCen −/− parasites continued to be safe as mice remained lesion-free (data not shown).The cellular response, measured by DTH (Figure 7A), was similar to that observed with a single immunization (Figure 5B).The recall response also showed that while IFN-γ was produced, IL-10 was detected at comparatively higher levels (Figure 7B).Two immunizations with LbCen −/− were also not capable of generating a significant increase in CD4 + T central memory cells (CD4 + CD44 + CD62L + ) (Figure 7C), as seen upon a single immunization with LmexCen −/− [16].Given that a booster inoculation with LbCen −/− did not alter the immune response observed earlier, we expected that the outcome of a challenge infection would also be the same.Indeed, mice receiving two inoculations with LbCen −/− continued to be susceptible to a challenge infection with LbWT (Figure 7D), with lesions developing similarly to the control, naïve mice.The parasite load in the two groups was similar (Figure 7E).Lastly, no significant differences were observed in the production of IFN-γ (Figure 7F) or IL-10 (Figure 7G), comparing mice primed and boosted with LbCen −/− and control, naïve mice.Previously, it was shown that immunization with LdonCen −/− induces cross-protection against L. braziliensis injected subcutaneously [12].We sought to investigate whether a similar outcome would be observed using the L. braziliensis strain employed in the generation of our centrin-deleted mutant, using an intradermal model of infection.We thus compared the outcome of immunization with LdonCen −/− versus LbCen −/− followed by a challenge with LbWT in the ear dermis.We confirmed that immunization with LdonCen −/− protects against intradermal inoculation of LbWT, differently from LbCen −/− (Figure 8A).Protection was paralleled by a significantly lower parasite (p < 0.01) load at the inoculation site and in dLNs (Figure 8B), suggesting that the immune response induced by LdonCen −/− controls parasite load even at distal sites.Collectively, our results show that a booster LbCen −/− immunization does not alter the lack of protection observed upon a single immunization.

Immunization with LdonCen −/− Cross-Protects against LbWT
Previously, it was shown that immunization with LdonCen −/− induces cross-protection against L. braziliensis injected subcutaneously [12].We sought to investigate whether a similar outcome would be observed using the L. braziliensis strain employed in the generation of our centrin-deleted mutant, using an intradermal model of infection.We thus compared the outcome of immunization with LdonCen −/− versus LbCen −/− followed by a challenge with LbWT in the ear dermis.We confirmed that immunization with LdonCen −/− protects against intradermal inoculation of LbWT, differently from LbCen −/− (Figure 8A).Protection was paralleled by a significantly lower parasite (p < 0.01) load at the inoculation site and in dLNs (Figure 8B), suggesting that the immune response induced by LdonCen −/− controls parasite load even at distal sites.

Discussion
The current therapeutic options for leishmaniasis remain limited because of toxicity, drug administration challenges, treatment failure, and drug resistance [24].Protection against leishmaniasis can be achieved through leishmanization [25], but this method presents safety concerns.To this end, attenuated Leishmania spp.cell lines are an attractive alternative, as safety issues can be overcome while the delivery of the near-complete −/− in BALB/c Mice BALB/c mice (n = 10) were inoculated in the right ear dermis with LbCen −/−

Figure 1 .
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 1 .
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 3 .
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.

3. 5 .
Immunization with LbCen −/− Cannot Recapitulate the Immune Response Induced by a Healed Primary Infection with LbWT

Vaccines 2024 ,
12, x FOR PEER REVIEW

Figure 4 .
Figure 4. Efficacy of LbCen −/− against L. braziliensis.BALB/c mice were inoculated with stationary promastigotes in the ear dermis.Five weeks later, mice were challenged with L (A) lesion development was measured weekly.(B) Parasite load was measured five we challenge in the ear (B) and dLNs (C).Results are expressed as means ± SEM and are f representative experiment performed with six mice.

Figure 4 .
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 .
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 5 .
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.
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 .
Figure 7. Prime boost with LbCen −/− cannot improve immunity against L. braziliensis.BALB/c mice were inoculated with 3 × 10 6 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 10 5 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 7 .
Figure 7. Prime boost with LbCen −/− cannot improve immunity against L. braziliensis.BALB/c mice were inoculated with 3 × 10 6 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 10 5 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 .
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.

Figure 8 .
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.