Structure of SALO, a leishmaniasis vaccine candidate from the sand fly Lutzomyia longipalpis

Abstract

BackgroundImmunity to the sand fly salivary protein SALO (Salivary Anticomplement of Lutzomyia longipalpis) protected hamsters against Leishmania infantum and L. braziliensis infection and, more recently, a vaccine combination of a genetically modified Leishmania with SALO conferred strong protection against L. donovani infection. Because of the importance of SALO as a potential component of a leishmaniasis vaccine, a plan to produce this recombinant protein for future scale manufacturing as well as knowledge of its structural characteristics are needed to move SALO forward for the clinical path.Methodology/Principal findingsRecombinant SALO was expressed as a soluble secreted protein using Pichia pastoris, rSALO(P), with yields of 1g/L and >99% purity as assessed by SEC-MALS and SDS-PAGE. Unlike its native counterpart, rSALO(P) does not inhibit the classical pathway of complement; however, antibodies to rSALO(P) inhibit the anti-complement activity of sand fly salivary gland homogenate. Immunization with rSALO(P) produces a delayed type hypersensitivity response in C57BL/6 mice, suggesting rSALO(P) lacked anti-complement activity but retained its immunogenicity. The structure of rSALO(P) was solved by S-SAD at Cu-Kalpha to 1.94 Å and refined to Rfactor 17%. SALO is ~80% helical, has no appreciable structural similarities to any human protein, and has limited structural similarity in the C-terminus to members of insect odorant binding proteins. SALO has three predicted human CD4+ T cell epitopes on surface exposed helices.Conclusions/SignificanceThe results indicate that SALO as expressed and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing. SALO has a novel structure, is not similar to any human proteins, is immunogenic in rodents, and does not have the anti-complement activity observed in the native salivary protein which are all important attributes to move this vaccine candidate forward to the clinical path.

Functional and structural similarities of D7 proteins in the independently-evolved salivary secretions of sand flies and mosquitoes

“…duboscqi is a 15 kDa OBP that binds polyphosphate and heparin and inhibits the contact pathway of coagulation, whose activation results in production of the proinflammatory effector bradykinin 19 . SALO, a protein closely related to PdSP15 from the new world species Lutzomyia longipalpis inhibits the classical pathway of complement activation by binding protein complexes involved in the initiation of complement activation 20,21 . The structures of these proteins show a typical single-domain OBP fold, but do not contain an internal binding pocket, and exhibit little detailed similarity to the short-form D7 proteins of mosquitoes.…”

A defined subunit vaccine that protects against vector-borne visceral leishmaniasis

“…In this work, we show that the Leishmania -derived LEISH-F3+ fusion protein can protect against VL initiated by a sand fly challenge. A combination of LEISH-F3+ with a salivary protein, possibly LJM19, 15 may produce an even more robust vaccine for use in Leishmania -endemic regions.…”

Lutzomyia longipalpis: an update on this sand fly vector

“…The first is a Salivary Anti-complement from Lu. longipalpis (SALO) (Ferreira et al 2016), considered a leishmaniasis vaccine candidate (Asojo et al 2017) and the second, a soluble intestinal inhibitor (Saab et al 2020).…”