Functional Transcriptomics of Wild-Caught <i>Lutzomyia intermedia</i> Salivary Glands: Identification of a Protective Salivary Protein against <i>Leishmania braziliensis</i> Infection

<div><p>Background</p><p><i>Leishmania</i> parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with <i>Phlebotomus papatasi</i> salivary gland (SG) homogenate are protected against <i>Leishmania major</i> infection, while immunity to <i>Lutzomyia intermedia</i> SG homogenate exacerbated experimental <i>Leishmania braziliensis</i> infection. In humans, antibodies to <i>Lu. intermedia</i> saliva are associated with risk of acquiring <i>L. braziliensis</i> infection. Despite these important findings, there is no information regarding the repertoire of <i>Lu. intermedia</i> salivary proteins.</p><p>Methods and Findings</p><p>A cDNA library from the Salivary Glands (SGs) of wild-caught <i>Lu. intermedia</i> was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11—coding for a 4.5-kDa protein—induced a cellular immune response and conferred protection against <i>L. braziliensis</i> infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells.</p><p>Conclusions</p><p>We identified the most abundant and novel proteins present in the SGs of <i>Lu. intermedia</i>, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from <i>Lu. intermedia</i> can protect against cutaneous leishmaniasis caused by <i>L. braziliensis</i>.</p></div

Immune response to <i>Lutzomyia whitmani</i> salivary molecules.

<p>BALB/c mice were immunized three times with <i>Lu</i>. <i>whitmani</i> SGS (equivalent to 1 pair of salivary glands) (red circles) or were inoculated with saline (control) (black circles), in the right ear, at two week intervals. (A) IgG respose to <i>Lu</i>. <i>whitmani</i> SGS determined by ELISA at different time points. Data are shown individually, from one representative experiment (bar at mean ± SEM). (B) Western blot analysis of <i>Lu</i>. <i>whitmani</i> salivary proteins using sera from immunized mice and SDS-PAGE depicting <i>Lu</i>. <i>whitmani</i> salivary proteins. (C) Two weeks after the last immunization, mice were challenged in the opposite ear with <i>Lu</i>. <i>whitmani</i> SGS and DTH response was measure 48h later. Data, shown as (median ± SD), are from two experiments performed with five mice in each group (D) Ear sections were obtained 48h later and stained with H&E. Sections were analyzed by optical microscopy under (100X). ** p<0.01.</p

Frequency and absolute number of cytokine-producing cells in mice immunized with <i>Lutzomyia whitmani</i> SGS.

<p>BALB/c mice were immunized three times with <i>Lu</i>. <i>whitmani</i> SGS (equivalent to 1 pair of salivary glands) or were inoculated with saline (control), in the right ear, at two week intervals. Forty eight hours after SGS inoculation in the left ear dermis, mice were euthanized and draining lymph node cells were stimulated with anti-CD3 and anti-CD28 for 16h. Cells were subsequently stained for determination of frequency and absolute numbers of (<b>A</b>) CD4⁺TCR⁺IFN-⁺ and CD8⁺TCR⁺IFN-⁺ and (<b>B</b>) CD4⁺TCR⁺IL-10⁺ and CD8⁺TCR⁺IL-10⁺ T cells. The numbers shown represent mean ± SEM from three independent experiments, each performed with three mice. Frequency plots are representative from one experiment.</p

<i>Lutzomyia whitmani</i> saliva immunization induces protection against experimental <i>Leishmania braziliensis</i> infection.

<p>BALB/c mice were immunized three times with <i>Lu</i>. <i>whitmani</i> SGS (equivalent to 1 pair of salivary glands) (red circles) or were inoculated with saline (control) (black circles), in the right ear, at two week intervals. Two weeks after the last immunization mice challenged in the opposite ear with 10⁵ <i>L</i>. <i>braziliensis</i> plus SGS (equivalent to one pair of salivary glands). (<b>A</b>) Lesion development was monitored weekly. Data, shown as mean ± SEM, are from one representative experiment, performed with five mice in each group. (<b>B</b>) Parasite load was determined ten weeks after challenge via a limiting dilution assay. Data (bar at mean ± SEM) are shown individually. (<b>C</b>) Ten weeks after challenge, spleen cells were stimulated with Soluble <i>Leishmania</i> Antigen (SLA) and IFN- levels were measured in culture supernatants by ELISA, 72h later. Data, shown as mean ± SEM, are from one representative experiment, performed with five mice in each group. * p<0.05; **, p<0.01; ***, p< 0.001.</p

Human serum IgG response against <i>Lutzomyia whitmani</i> SGS.

<p>(<b>A</b>) ELISA was performed against <i>Lu</i>. <i>whitmani</i> SGS using human sera were from individuals from a Cutaneous Leishmaniasis (CL) endemic area (n = 264) or from control individuals (n = 13). (<b>B</b>) ELISA was performed against <i>Lu</i>. <i>whitmani</i> SGS with human sera from CL individuals (n = 30), sera from healthy individuals with either a negative (n = 231) or a positive (n = 33) <i>Leishmania</i> skin test (LST). The dotted line represents the cut-off value for the assay. Data are shown individually, bar at mean. p<0.01; ***, p< 0.001.</p

Immunity to <i>Lutzomyia whitmani</i> Saliva Protects against Experimental <i>Leishmania braziliensis</i> Infection

<div><p>Background</p><p>Previous works showed that immunization with saliva from <i>Lutzomyia intermedia</i>, a vector of <i>Leishmania braziliensis</i>, does not protect against experimental infection. However, <i>L</i>. <i>braziliensis</i> is also transmitted by <i>Lutzomyia whitmani</i>, a sand fly species closely related to <i>Lu</i>. <i>intermedia</i>. Herein we describe the immune response following immunization with <i>Lu</i>. <i>whitmani</i> saliva and the outcome of this response after <i>L</i>. <i>braziliensis</i> infection.</p><p>Methods and findings</p><p>BALB/c mice immunized with <i>Lu</i>. <i>whitmani</i> saliva developed robust humoral and cellular immune responses, the latter characterized by an intense cellular infiltrate and production of IFN-γ and IL-10, by both CD4⁺ and CD8⁺ cells. Mice immunized as above and challenged with <i>L</i>. <i>braziliensis</i> plus <i>Lu</i>. <i>whitmani</i> saliva displayed significantly smaller lesions and parasite load at the challenge site. This protection was associated with a higher (p<0.05) IFN-γ production in response to SLA stimulation. Long-term persisting immunity was also detected in mice immunized with <i>Lu</i>. <i>whitmani</i> saliva. Furthermore, individuals residing in an endemic area for cutaneous leishmaniasis (CL) presented antibody responses to <i>Lu</i>. <i>whitmani</i> saliva. However CL patients, with active lesions, displayed a lower humoral response to <i>Lu</i>. <i>whitmani</i> saliva compared to individuals with subclinical <i>Leishmania</i> infection.</p><p>Conclusion</p><p>Pre-exposure to <i>Lu</i>. <i>whitmani</i> saliva induces protection against <i>L</i>. <i>braziliensis</i> in a murine model. We also show that <i>Lu</i>. <i>whitmani</i> salivary proteins are immunogenic in naturally exposed individuals. Our results reinforce the importance of investigating the immunomodulatory effect of saliva from different species of closely related sand flies.</p></div

Persistent immunity induced by <i>Lutzomyia whitmani</i> saliva protects against experimental <i>Leishmania braziliensis</i> infection.

<p>BALB/c mice were immunized three times with <i>Lu</i>. <i>whitmani</i> SGS (equivalent to 1 pair of salivary glands) (red circles) or were inoculated with saline (control) (black circles), in the right ear, at two week intervals. Twelve weeks after the last immunization mice challenged in the opposite ear with 10⁵ <i>L</i>. <i>braziliensis</i> plus SGS (equivalent to one pair of salivary glands). (<b>A</b>) Lesion development was monitored weekly. Data, shown as mean ± SEM, are from one representative experiment, performed with five mice in each group. (<b>B</b>) Parasite load was determined eight weeks after challenge via a limiting dilution assay. Data (bar at mean ± SEM) are shown individually. (<b>C</b>) IgG response to <i>Lu</i>. <i>whitmani</i> SGS determined by ELISA at twelve weeks before challenge. Data are shown individually, from one representative experiment (bar at mean ± SEM). * p<0.05; **, p<0.01; ***, p< 0.001.</p

Leukocyte recruitment in air pouch exudates in response to <i>L. braziliensis</i> parasites and <i>L. intermedia</i> saliva.

<p>BALB/c mice (five to six per group) received three immunizations with <i>L. intermedia</i> SGS. Fifteen days after the last immunization, air pouches were raised in naïve mice and in mice immunized with <i>L. intermedia</i> SGS. Air pouches were inoculated with <i>L. braziliensis</i> alone (Lb) or with <i>L. braziliensis</i>+<i>L. intermedia</i> saliva (Lb+SGS). Exudates were collected twelve hours later. Leukocytes were enumerated microscopically. (A) Total number of leukocytes accumulated in air pouches and (B) total number of neutrophils, monocytes, eosinophils, and lymphocytes accumulated in air pouches of naïve mice. (C) Total number of leukocytes accumulated in air pouches and (D) total number of neutrophils, monocytes, eosinophils, and lymphocytes accumulated in air pouches of SGS-immunized mice The data shown are from a single experiment representative of three independent experiments. (* P<0.05).</p

Chemokine and cytokine expression in air pouch exudates in response to <i>L. braziliensis</i> parasites and <i>L. intermedia</i> saliva.

<p>BALB/c mice (five to six per group) received three immunizations with <i>L. intermedia</i> SGS. Fifteen days after the last immunization, air pouches were raised in naïve mice and mice immunized with <i>L. intermedia</i> SGS. Air pouches were inoculated with <i>L. braziliensis</i> alone (Lb) or with <i>L. braziliensis</i>+<i>L. intermedia</i> saliva (Lb+SGS). Air pouch lining tissue was submitted to real-time PCR for relative quantification of chemokines and cytokines. Chemokine expression in (A) naïve and (B) SGS-immunized mice. Cytokine expression in (C) naïve and (D) SGS-immunized mice. Bars represent the means and standard errors of the means of five mice per group. The data shown are from a single experiment representative of two independent experiments. (* P<0.05; ** P<0.01).</p

Modulation in leukocyte recruitment and gene expression in mice pre-exposed to <i>L. intermedia</i> saliva.

<p>BALB/c mice (five to six per group) received three inoculations with endotoxin-free PBS or <i>L. intermedia</i> SGS. Fifteen days after the last immunization, air pouches were raised and inoculated with <i>L. intermedia</i> SGS. Exudates and air pouch lining tissue were collected twelve hours later. Leukocytes were enumerated microscopically. (A) Total number of recruited leukocyte and (B) total number of neutrophils, monocytes, eosinophils, and lymphocytes accumulated in air pouches. Relative expression of chemokines (C) and cytokines (D) in the air pouch lining tissue was determined by real-time PCR. The data shown are from a single experiment representative of two independent experiments. (* P<0.05; ** P<0.01).</p