The accomplishments of the section are: 1) Immunity to Sand Fly Salivary Protein LJM11 Modulates Host Response to Vector-Transmitted Leishmania Conferring pathology-Free Protection. Leishmania vaccines that protect against needle challenge fail against the potency of a Leishmania-infected sand fly transmission. Here, we demonstrate that intradermal immunization of mice with 500ng of the sand fly salivary recombinant protein LJM11 (rLJM11) from Lutzomyia longipalpis, in the absence of adjuvant, induces long-lasting immunity that results in ulcer-free protection against Leishmania major delivered by vector bites. This protection is antibody independent and abrogated by depletion of CD4(+) T cells. Two weeks after challenge, early induction of IFN-gamma;specifically to rLJM11 correlates to diminished parasite replication in protected animals. At this time point, Leishmania-specific induction of IFN-gamma;in these mice is low in comparison with its high level in non-protected controls. We hypothesize that early control of parasites in a T-cell helper type 1 environment induced by immunity to LJM11 permits the slow development of Leishmania-specific immunity in the absence of open ulcers. Leishmania-specific immunity observed 5 weeks after infection in rLJM11-immunized mice shows a twofold increase over controls in the percentage of IFN-gamma;-producing CD4(+) T cells. We propose LJM11 as an immunomodulator that drives an efficient and controlled protective immune response to a sand fly-transmitted Leishmania somewhat mimicking """"""""leishmanization""""""""-induced protective immunity but without its associated lesions 2) KSAC, a defined Leishmania antigen, plus adjuvant protects against the virulence of L. major transmitted by its natural vector Phlebotomus duboscqi. Recombinant KSAC and L110f are promising Leishmania vaccine candidates. Both antigens protected BALB/c mice against L. major infection following needle challenge. Considering the virulence of vector-transmitted Leishmania infections, we vaccinated BALB/c mice with either KSAC+GLA-SE or L110f+GLA-SE to assess protection against L. major transmitted via its vector Phlebotomus duboscqi. Mice receiving the KSAC or L110f vaccines were challenged by needle or L. major-infected sand flies. Following sand fly challenge, KSAC-vaccinated mice were protected while L110f-vaccinated animals showed partial protection. Protection correlated with the ability of SLA to induce IFN-gamma;-producing CD4(+)CD62L(low)CCR7(low) effector memory T cells pre- and post-sand fly challenge. This study demonstrates the protective efficacy of KSAC+GLA-SE against sand fly challenge;the importance of vector-transmitted challenge in evaluating vaccine candidates against Leishmania infection;and the necessity of a rapid potent Th1 response against Leishmania to attain true protection. 3) Salivary gland transcriptomes and proteomes of Phlebotomus tobbi and Phlebotomus sergenti, vectors of leishmaniasis. Phlebotomus tobbi is a vector of Leishmania infantum, and P. sergenti is a vector of Leishmania tropica. To better understand the components and possible implications of sand fly saliva in leishmaniasis, the transcriptomes of the salivary glands (SGs) of these two sand fly species were sequenced, characterized and compared. The most abundant, secreted putative proteins were categorized as antigen 5-related proteins, apyrases, hyaluronidases, D7-related and PpSP15-like proteins, ParSP25-like proteins, PpSP32-like proteins, yellow-related proteins, the 33-kDa salivary proteins, and the 41.9-kDa superfamily of proteins. This analysis of P. sergenti is the first description of the subgenus Paraphlebotomus salivary components. The investigation of the subgenus Larroussius sand fly P. tobbi expands the repertoire of salivary proteins in vectors of Le. infantum. Although P. tobbi transmits a cutaneous form of leishmaniasis, its salivary proteins are most similar to other Larroussius subgenus species transmitting visceral leishmaniasis. These transcriptomic and proteomic analyses provide a better understanding of sand fly salivary proteins across species and subgenera that will be vital in vector-pathogen and vector-host research. 4) Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis, Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo.
The aim of the study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Several L. longipalpis salivary proteins were expressed in mammalian cells (293 F) and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant of 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. 5) The protein LJM 111 from Lutzomyia longipalpis salivary gland extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model. 5) The protein LJM 111 from Lutzomyia longipalpis salivary gland extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model. Several studies have pointed out the immunomodulatory properties of the Salivary Gland Extract (SGE) from Lutzomyia longipalpis.
We aim ed to identify the SGE component (s) responsible for its effect on ovalbumin (OVA)-induced neutrophil migration (NM) and to evaluate the effect of SGE and components in the antigen-induced arthritis (AIA) model. We tested the anti-arthritic activities of SGE and the recombinant LJM111 salivary protein (rLJM111) by measuring the mechanical hypernociception and the NM into synovial cavity. Furthermore, we measured IL-17, TNF- and IFN- released by lymph nodes cells stimulated with mBSA or anti-CD3 using enzyme-linked immunosorbent assay (ELISA). Additionally, we tested the effect of SGE and rLJM111 on co-stimulatory molecules expression (MHC-II and CD-86) by flow cytometry, TNF- and IL-10 production (ELISA) of bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, chemotaxis and actin polymerization from neutrophils. Besides, the effect of SGE on CXCR2 and GRK-2 expression on neutrophils was investigated. We identified one plasmid expressing the protein LJM111 that prevented NM in OVA-challenged immunized mice. Furthermore, both SGE and rLJM111 inhibited NM and pain sensitivity in AIA and reduced IL-17, TNF- and IFN-. SGE and rLJM111 also reduced MHC-II and CD-86 expression and TNF- whereas increased IL-10 release by LPS-stimulated BMDCs. SGE, but not LJM 111, inhibited neutrophils chemotaxis and actin polymerization. Additionally, SGE reduced neutrophil CXCR2 expression and increased GRK-2. Thus, rLJM111 is partially responsible for SGE mechanisms by diminishing DC function and maturation but not chemoattraction of neutrophils.

Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2012
Total Cost
$1,222,735
Indirect Cost
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