Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates

Abstract

Summary Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP‐HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients’ urine. Specifically, these proteins were the following: Li‐isd1 (), Li‐txn1 () and Li‐ntf2 (). Initial vaccine validation studies were performed with the rLi‐ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA‐SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li‐ntf2+ BpMPLA‐SE had a marked parasite burden reduction in spleens at 40 days post‐challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

Immunogenicity in dogs and protection against visceral leishmaniasis induced by a 14kDa Leishmania infantum recombinant polypeptide

“…In our former studies in mice we vaccinated the animals with Li-ntf2 formulated with the adjuvant Bp MPLA-SE and showed that this formulation induced IgG2a antibody response and production of IFN-γ by Li-ntf2 specific T cells thus a Th1 biased response [18]. To confirm that this immunization protocol was also effective in dogs, two groups of six month old, male Beagles were immunized three times (one month apart) with either Li-ntf2 mixed with Bp MPLA-SE or with Li-ntf2 in saline.…”

“…Of note, it is important to highlight that despite the reduced MW of Li-ntf2 (13.9 kDa) and consequent limited antigenic epitope repertoire, this molecule induced partial protection in mice [18] and dogs (this study). In contrast, the successful commercially available dog vaccines [14;15;41-50], which induce better protection than Li-ntf2 , are composed of multiple high MW proteins ranging from 40kDa to 100kDa.…”

“…To confirm in dogs the immunogenicity of the vaccine formulation that we have previously tested in mice [18], 2 groups of 3 dogs (6 month old, male, Beagle) were immunized subcutaneously three times (three weeks apart) with either Li ntf2 (50μg) + Bp MPLA-SE (50μg), group 1, or with Li ntf2 (50μg) and no adjuvant, group 2. Bp MPLA-SE (Institute Butantan, São Paulo, Brazil) is an oil-in water emulsion containing monophosphoryl lipid A (MPLA) derived from Bordetella pertussis [18].…”

“…Bp MPLA-SE (Institute Butantan, São Paulo, Brazil) is an oil-in water emulsion containing monophosphoryl lipid A (MPLA) derived from Bordetella pertussis [18]. …”

“…This approach led to the identification of several polypeptides of L. infantum. When tested as vaccine candidates formulated with the adjuvant monophosphoryl lipid A (MPLA) from Bordetella pertussi s emulsified with squalene (Butantan Institute, São Paulo, SP, Brazil) one of the polypeptides, L. infantum nuclear transport factor-2 ( Li-ntf 2) induced marked parasite burden reduction in the vaccinated animals compared to control mice [18]. …”