Preclinical development of a molecularly-defined liposomal vaccine for cutaneous leishmaniasis

Abstract

Cutaneous leishmaniasis (CL) is a neglected tropical disease with a serious public health problem in Colombia. Clinical, epidemiological and experimental evidence suggests that an effective vaccine against CL is, in principle, possible. However, no effective vaccine for human use is currently available. Using a CL murine model by Leishmania (Viannia) panamensis, the most prevalent species in Colombia, we have found that: 1) synthetic CpG oligonucleotides work as a protective adjuvant when combined with total parasite lysate and 2) a soluble total antigen liposomal formulation was also protective against parasite challenge. Additionally, the protein trypanothione reductase (TR) was identified as a promising vaccine candidate by using a reverse vaccinology strategy, thus suggesting the possibility to develop a molecularly-defined vaccine formulation. Following this research line, our hypothesis was that the employment of a particulate delivery system could enhance the efficacy of this vaccine candidate. So, we aimed to evaluate the prophylactic efficacy of a nanostructured formulation of the recombinant TR (rTR) with/without CpG. To achieve this, the liposome preparation was optimized: different factors involved in this process were analyzed and several response variables were studied, such as size, zeta potential and encapsulation efficiency. We optimized three types of liposomes which were physically stable, cationic (+65 mV), nanometric (PBS, CpG: 900 nm) or micrometric (rTR: 1500 nm) and had encapsulation efficiency of ~70% for CpG and ~20% for rTR. For CpG liposomes, the encapsulation efficiency was performed directly, by quantifying CpG, by absorption spectrophotometry in the UV-Visible region, both in the supernatant -CpG free- and in the resuspended pellet -CpG encapsulated in the liposomes-, after centrifugation and in other stages of the preparation process. The spectrophotometric method consisted of dissolving the aliquots in chloroform:methanol 1:2 and reading absorbance at 260 nm. For the rTR liposomes, the encapsulation efficiency was also performed directly, estimating the amount of rTR in different stages of the process by SDS-PAGE coupled to densitometry. In each gel, a BSA calibration curve was employed to estimate the amount of the rTR protein. The encapsulation of CpG is relatively high, despite being a hydrophilic molecule, because it is a polyanion that interacts favorably with the internal cationic lipid layers of the liposome. The encapsulation of the rTR protein is smaller, because it is a large, hydrophilic molecule and, in addition, during the extrusion step, it suffers non-specific binding to the polycarbonate membrane. Different combinations of these liposomal formulations were evaluated in vivo in our CL murine model. We observed that vaccination with rTR liposomes plus soluble CpG induced partial protection against the infectious challenge. So, we concluded that our liposomal formulation enhanced the protective efficacy of the antigen, even at lower doses than those normally used. Hence, our main perspectives include the optimization of the vaccination scheme and dosages and the advanced characterization of these vesicles.