Development of Novel Prime-Boost Strategies Based on a Tri-Gene Fusion Recombinant L. tarentolae Vaccine against Experimental Murine Visceral Leishmaniasis

Abstract

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL.

Assessment of Protection Induced by DNA and Live Vaccine Encoding Leishmania MHC Class I Restricted Epitopes against L. major Challenge in Balb/c Mice Model

“…Previously L. tarentolae has proven promising by long lasting protection while expressing multiple candidate antigens (all among potential vaccine candidates as A2) [24,48] especially along with sandfly salivary immune-stimulatory proteins [49]. This is a confirmatory proof that peptides included must be among high avidity ones previously evaluated in in vivo infectious challenge.…”

“…Real-time PCR was used to quantify parasite burden in draining lymph nodes at both early and late phase of infection as described before [24]. Genomic DNA was extracted from homogenized lymph nodes of 4 individual mice.…”

“…Reaction was stopped by 1% SDS solution and absorbance was measured at 405 nm with reference filter. Antibody ELISA assay was followed by previously described instructions [24]. Briefly maxisorb ELISA plate was coated with either P1+P4 or P2+P3 mixture (5 μg/mL each peptide) then loaded with 100 µl of 1:10 diluted pooled sera of each group.…”

Current treatment of visceral leishmaniasis (Kala-azar): an overview

“…49 The vaccine has already completed phase 1 clinical trials in the U.S. to demonstrate safety and the ability to evoke a robust immune response. [46][47][48] The IDRI vaccine, called LEISH-F3 + GLA-SE, is a highly purified, recombinant vaccine which incorporates two fused leishmania parasite proteins, given along with an additive to boost the immune response. It works by activating the immune system to kill the parasite that causes VL, the first of its kind till now developed.…”

Recent updates and perspectives on approaches for the development of vaccines against visceral leishmaniasis

“…The use of vaccines combining different proteins could provide the benefi ts of increased simplicity and reduced production costs, since it would only be necessary to produce a single vaccine to protect against different Leishmania species (56) . However, few studies have evaluated chimeric vaccines aimed at protection against VL (25) (57) , since the majority of reports have been of investigations of single antigens (14) (58) (59) (60) . The development of a multi-antigenic vaccine requires an appropriate choice of the biological targets for use in its composition.…”