LmCen-/- based vaccine is protective against canine visceral leishmaniasis following three natural exposures in Tunisia

Abstract

Dogs are the main reservoir host of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis (ZVL). An effective vaccine against Canine Visceral Leishmaniasis (CVL) will help the control and elimination of ZVL. In this study, we evaluated in dogs the safety, immunogenicity, and efficacy of a live attenuated Leishmania major Centrin gene-deleted (LmCen^-/-) as a vaccine. Two doses (10⁶ or 10⁷) of LmCen^-/- vaccine were administered intradermally in a prime-boost regimen. Both vaccine doses induced equally high level of IgG anti-Leishmania and exhibited strong antigen-specific cellular responses with IFN-γ production by CD4 + T cells one-month post-immunization. A second cohort of dogs was vaccinated with 10⁶ LmCen^-/- parasites one month prior to their transfer to a CVL endemic focus in Northern Tunisia for exposure to sand fly bites during three successive transmission seasons. Dogs were exposed to bite from naturally infected sandflies for 3-5 months per year. Our results showed that only 1/11 vaccinated dogs became PCR positive for Leishmania and developed clinical signs of CVL. In contrast, 4/11 unvaccinated dogs were tested PCR positive for Leishmania and displayed oligosymptomatic CVL, demonstrating that immunization with LmCen^-/- vaccine confers long-term protection with an efficacy of 82.5% against CVL in natural transmission settings.

Fig. 1. Humoral immune response in LmCen^−/− vaccinated dogs.

a Protocol for analysis of the Immunogenicity of LmCen^−/−. Three groups of dogs were included in this step: dogs receiving 10⁶ or 10⁷ live LmCen^−/− parasites and as negative control dogs receiving 100 µl of medium. Immunization was done by intradermal injection of 100 µl of vaccine in the ear of dog. Boost with vaccine was done at the same conditions as the first immunization at an interval of 2 months. b–d Histograms represent the mean of total IgG anti-SLA at different kinetics. Vertical bars indicate the standard deviation (SD). Total IgG titers are measured using SLA from L.major. Statistical analyses are done using GraphPad Prism, *p < 0.05: difference is statistically significant.

Fig. 2. IFN-γ production in LmCen^−/− vaccinated dogs.

One month post-Immunization, PBMCs from immunized dogs (10⁶/10⁷ LmCen^−/−) or control groups (medium) were incubated with soluble Leishmania antigens (SLA, 10 µg/ml). a IFN-γ levels measured in cell culture supernatants collected at 72 h using sandwich ELISA. b Percentage of CD4⁺T cells producing IFN-γ in response to stimulation with SLA. Statistical analyses are done using GraphPad Prism, *p < 0.05: statistically significant difference.

Fig. 3. Natural Challenge of dog in the endemic area for transmission of L. infantum.

a Dogs receiving 10⁶ LmCen^−/− (n = 11) and those receiving 100 µl of PBS (Negative control, n = 11) are transported to an endemic area for the transmission of L. infantum (North of Tunisia) during three successive seasons 2019, 2020 and 2021. b Phenology of P. perniciosus and P. perfiliewi in the exposure site. One-month post-prime, dogs were placed in a rural area (36°58’N, 10°03’E) during September-October 2019, June-November 2020, and September-November 2021. The densities of P. perniciosus and P. perfiliewi as well as their infection prevalence with L. infantum were assessed during August-October 2019 (1^st exp), June-November 2020 (2^nd exp), and August-November 2021 (3^rd exp). The phenology of collected sand fly species by sticky traps was studied during the same periods in the same site. Density of sandflies was recorded as the number of sand fly species per m² of sticky trap. The infection prevalence of collected sandflies by CDC light traps was assessed during the same periods. The minimum infection rate (MIR) of sandflies by Leishmania parasites defined as the number of positive pools divided by the total number of tested specimen × 100.

Fig. 4. IgG antibodies specific for sand fly saliva and L. infantum in dogs before and after natural exposure to L. infantum.

IgG anti- SGH and anti-SLA are measured in sera from dogs immunized with LmCen^−/− at a dose of 10⁶ and those receiving 100 µl of PBS (Negative control). Titers of IgG anti-SGH measured in dogs before (a) or at return from the field after successive expositions 2019 (b), 2020 (c) and 2021 (d). Titers of IgG anti-SLA before (e) and after the exposure in the endemic area (f–h) measured using SLA from L.infantum. Histograms represent the mean of IgG anti-SGH or anti-SLA for each group. Bars represent the standard deviation for each group. Pie charts represent number of dog positive and/or negative for IgG anti-SLA and IgG anti-SGH at return from the field in 2019 (i–l), 2020 (j–m) and 2021 (k–n). Cutoff of IgG anti-SLA is fixed at OD = 0.23; cutoff for IgG anti-SGH fixed at OD = 0.08.

Fig. 5. Impact of the vaccination of dog with LmCen^−/− after three exposures in the endemic area for transmission of L. infantum in Tunisia.

a Results of biochemical, hemogram and (b) clinical score after the 3^rd exposure in the endemic area. c The PCR parasite load in the whole blood/Bl and spleen aspirate/SA every year during, six months after the successive seasons 2019, 2020, and 2021.