Photodynamic Vaccination of BALB/c Mice for Prophylaxis of Cutaneous Leishmaniasis Caused by Leishmania amazonensis

Abstract

Background: Photosensitizers (PS), like porphyrins and phthalocyanines (PC) are excitable by light to generate cytotoxic singlet oxygen and other reactive oxygen species in the presence of atmospheric O2. Photodynamic inactivation of Leishmania by this means renders them non-viable, but preserves their effective use as vaccines. Leishmania can be photo-inactivated after PS-sensitization by loading via their endocytic uptake of PC or endogenous induction of transgenic mutants with delta-aminolevulinate (ALA) to accumulate cytosolic uroporphyrin I (URO). Here, PS-sensitization and photo-inactivation of Leishmania amazonensis was further examined in vitro and in vivo for vaccination against cutaneous leishmaniasis (CL).Methods and Results: Leishmania amazonensis promastigotes were photodynamically inactivated in vitro by PC-loading followed by exposure to red light (1–2 J/cm2) or ALA-induction of uroporphyrinogenic transfectants to accumulate cytosolic URO followed by longwave UV exposure. When applied individually, both strategies of photodynamic inactivation were found to significantly, albeit incompletely abolish the MTT reduction activities of the promastigotes, their uptake by mouse bone marrow-derived macrophages in vitro and their infectivity to mouse ear dermis in vivo. Inactivation of Leishmania to completion by using a combination of both strategies was thus used for the sake of safety as whole-cell vaccines for immunization of BALB/c mice. Different cutaneous sites were assessed for the efficacy of such photodynamic vaccination in vivo. Each site was inoculated first with in vitro doubly PS-sensitized promastigotes and then spot-illuminated with white light (50 J/cm2) for their photo-inactivation in situ. Only in ear dermis parasites were photo-inactivated beyond detection. Mice were thus immunized once in the ear and challenged 3 weeks later at the tail base with virulent L. amazonensis. Prophylaxis was noted in mice photodynamically vaccinated with doubly photo-inactivated parasites, as indicated by a significant delay in the onset of lesion development and a substantial decrease in the parasite loads.Conclusion: Leishmania doubly PS-sensitized and in situ photo-inactivated as described proved to be safe and effective when used for one-time immunization of ear dermis, as indicated by its significant protection of the inherently very susceptible BALB/c mice against CL.

Photodynamic inactivation of Leishmania braziliensis doubly sensitized with uroporphyrin and diamino-phthalocyanine activates effector functions of macrophages in vitro

“…In the present study, Leishmania braziliensis of the Viannia subgenus was successfully photodynamically inactivated by the dual approach of genetic and chemical engineering, which has proven effective to render members of the Leishmania subgenus non-viable for safe and effective use as experimental whole-cell vaccines 12,13 . All transgenic clones produced in the present work were responsive to delta-aminolevulinate (ALA) for uroporphyrinogenesis, i.e.…”

“…Doubly photodynamic inactivation of Leishmania indeed renders them non-viable 20 , but preserves their antigenicity and adjuvanicity. The efficacy of Strategy #1 was thus demonstrated in experimental vaccination against cutaneous leishmaniasis 13 . As suggested by the in vitro work presented here with L. braziliensis, Strategy #1 may be further improved to enhance its efficacy by increasing doses for loading APC to match that of Strategy #2.…”

“…We have been exploring inactivation of Leishmania via the principle of photodynamic therapy (PDT) as a strategy to render them non-viable for safe and effective use experimentally not only as whole-cell vaccines against leishmaniasis 12 , 13 but also as carriers for vaccines against other infectious and malignant diseases 14 – 17 . Photodynamic therapy makes use of dyes as photosensitizers (PS) that are excitable by light of specific wavelength to produce biocidal radicals, like singlet oxygen ( 1 O 2 ) and reactive oxygen species (ROS).…”

“…Sensitization of L. amazonensis by a combination of endogenously accumulated URO and exogenously supplied cationic PC provided assurance of their complete photo-inactivation without exception 20 . These Leishmania are invariably non-viable, but remain structurally and immunologically intact, as their use to immunize highly susceptible BALB/c mice protected them against a homologous challenge by suppressing lesion development and significantly reducing parasite burdens 13 .…”

Effective Delivery of Cancer Vaccines with Oxidatively Photo-Inactivated Transgenic Leishmania for Tumor Immunotherapy in Mouse Models

“…Brief illumination of these photosensitized Leishmania results in their rapid oxidative inactivation initiated by the generation of extremely short-lived, albeit highly destructive singlet oxygen [14]. The safety and efficacy of such inactivated Leishmania have been demonstrated by immunization of animals, producing neither infection nor adverse effects [15], but prophylactically protect them against both cutaneous and visceral leishmaniasis [16,17], and immunotherapeutic activities clinically against drug-incurable canine leishmaniasis [18]. Moreover, Leishmania transgenically made to express ovalbumin (OVA) was shown to effectively deliver this antigen, after photodynamic inactivation, to DC for processing and presentation to activate OVA epitope-specific T cells in vitro [12].…”