Research ArticleLeishmaniasis

A sand fly salivary protein vaccine shows efficacy against vector-transmitted cutaneous leishmaniasis in nonhuman primates

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Science Translational Medicine  03 Jun 2015:
Vol. 7, Issue 290, pp. 290ra90
DOI: 10.1126/scitranslmed.aaa3043
  • Fig. 1. Exposure to P. duboscqi uninfected sand flies (USFs) induces an anti-saliva immunity that protects NHP from vector-transmitted CL.

    (A to C) Immunity to USF bites 48 hours (A and B) or 2 weeks (C) after the last exposure. (A) DTH response (left panel) and a hematoxylin and eosin–stained biopsy section (right panel, ×400) from a USF bite site. (B) IFN-γ mRNA expression in biopsies of a USF bite site (Exp. Bites) or normal skin (Naïve) from the same animal. Biopsies were obtained from six randomly selected bite site–reactive NHP (P = 0.0043, Mann-Whitney test; n = 6). (C) Anti-saliva immunoglobulin G (IgG) levels before (Pre) or after (Post) exposure (P = 0.0002, t test; n = 10). Cumulative data from two independent experiments are shown. OD, optical density. (D to K) Fifteen USF-exposed NHP (Exp. Bites) and nine naïve NHP were challenged with 50 L. major–infected P. duboscqi. Cumulative data from two independent experiments are shown. (D) Disease burden (P = 0.0083, Mann-Whitney test; n = 9 to 15). (E) Maximum lesion size (P = 0.0119, Mann-Whitney test; n = 9 to 15). (F) Kaplan-Meier plot of the healing time, a cumulative measurement of lesion development from ulcer to scar [P = 0.0048, log-rank (Mantel-Cox) test; n = 9 to 15]. (G) Representative photographs 9 weeks after challenge. (H) Parasite number 12 weeks after challenge (P = 0.0237, Mann-Whitney test; n = 9 to 15). (I to K) PBMCs from seven naïve (Naïve) and nine USF-exposed NHP (Exp. Bites) were stimulated with Leishmania antigen (Leish) 2 weeks after challenge. Selection was based on cell number and viability. (I) IFN-γ SFC by enzyme-linked immunospot (ELISPOT) (P = 0.0587, Mann-Whitney test; n = 7 to 9). (J) Percent of CD4+IFN-γ+ lymphocytes by flow cytometry (P = 0.0229, Mann-Whitney test; n = 7 to 9). (K) Frequency of CD4+IFN-γ+ lymphocytes correlated to disease burden. Dashed line indicates 95% confidence interval (CI) (P = 0.0022, n = 16, Spearman test r = −0.72). Scale bar, 200 μm; lines and bars indicate the mean, and error bars indicate SEM.

  • Fig. 2. A RAS approach in NHP identifies PdSP15 as a TH1-inducing protein from saliva of the sand fly P. duboscqi.

    NHP were exposed three times to uninfected sand fly bites. Two weeks after the last exposure, animals were injected intradermally with 23 distinct DNA plasmids encoding the most abundant P. duboscqi salivary proteins or an empty plasmid as a negative control. Bites from one sand fly or the inoculation of one pair of SGH was used as positive controls. (A) Skin induration 48 hours after inoculation of plasmids measured using a Vernier caliper. Cumulative data of 14 NHP from three independent experiments are shown. (B and C) Two-millimeter skin biopsies of marked injection sites were obtained from 8 of 14 USF-exposed NHP. (B) IFN-γ and IL-4 mRNA expression by quantitative real-time fluorescence polymerase chain reaction (RT-qPCR). (C) IFN-γ/IL-4 ratio for each animal. Exact measurements and P values for all the samples tested are presented in tables S1 and S2. Lines and bars indicate the mean, and error bars indicate SEM.

  • Fig. 3. Immunization with PdSP15 protects NHP against vector-transmitted CL.

    (A to C) Immunity in PdSP15- immunized (PdSP15) or sham-immunized (CTL) NHP 48 hours (A) or 2 weeks (B and C) after last immunization. (A) Skin induration after inoculation with bovine serum albumin (CTL) or rPdSP15 (P = 0.0067, t test; n = 10). (B) IFN-γ SFC by ELISPOT (P = 0.0002, t test; n = 10). (C) Anti-saliva IgG levels before (Pre) or after (Post) immunization in controls (CTL), PdSP15-immunized NHP producing IFN-γ (PdSP15-IFN+) or not (PdSP15-IFN) (P < 0.0001, one-way ANOVA; n = 3 to 10). (D to L) Evaluation of disease (D to H) and Leishmania-specific immunity (I to L) in CTL, PdSP15-IFN+, or PdSP15-IFN NHP after challenge with 50 infected sand flies. (D) Disease burden (P = 0.0490, one-way ANOVA; n = 3 to 11). (E) Maximum lesion size (P = 0.0465, one-way ANOVA; n = 3 to 11). (F) Kaplan-Meier plot of the healing time [P = 0.1770, log-rank (Mantel-Cox) test; n = 3 to 11]. (G) Representative photographs 5 weeks after challenge. (H) Parasite number 5 weeks after challenge (P = 0.0034, one-way ANOVA; n = 3 to 8). (I to K) PBMCs stimulated with Leishmania antigen (Leish) 2 weeks after challenge in 8 to 10 NHP. Selection was based on cell number and viability. (I) IFN-γ SFC by ELISPOT (P = 0.0075, one-way ANOVA; n = 3 to 10). (J) Percent of CD4+IFN-γ+ lymphocytes by flow cytometry (P = 0.0002, one-way ANOVA; n = 3 to 10). (K) Frequency of CD4+ lymphocytes producing cytokines (P = 0.0418, one-way ANOVA; n = 4 to 6). (L) LST induration size 48 hours after the injection of Leishmania antigen at 12 weeks after challenge (P = 0.0269, one-way ANOVA; n = 3 to 10). Cumulative data for 11 CTL and 10 PdSP15 NHP from two independent experiments are shown. Lines and bars indicate the mean, and error bars indicate SEM.

  • Fig. 4. PdSP15 is an odorant-binding protein in saliva of phlebotomine sand flies.

    (A) Phylogenetic tree analysis shows the similarity of odorant-binding proteins in New and Old World sand fly species and their divergence from odorant-binding proteins (OBP) of other dipterans and humans. Bootstrap value, 10,000. PdSP15 location is underlined in red. (B) Sequence alignment between PdSP15 from P. duboscqi (accession number 112361953) and its orthologs in P. papatasi (PpSP15, accession number 449060564) and P. sergenti (PsSP15, accession number 299829414). Black shading and gray shading represent identical and similar amino acids, respectively. (C) Crystal structure of PdSP15 (4OZD) containing six α-helical elements designated as a, c, d, e, f, and g.

  • Fig. 5. PdSP15 is immunogenic in humans.

    (A and B) Sera and PBMCs were obtained from individuals living in central Mali where P. duboscqi sand flies are prevalent. (A) Anti-PdSP15 IgG levels (P < 0.0001, Mann-Whitney test; n = 12 to 30) and anti-saliva (Anti-SGH) (P < 0.0001, Mann-Whitney test; n = 12 to 30) in 30 Malians (Endemic). National Institutes of Health (NIH) blood bank healthy donors (n = 12) were used as controls (Non-endemic). Dashed lines indicate the cutoff (calculated as the mean in the non-exposed group ± 2.5 SD). (B) Cytokine levels produced by PBMCs from 14 Malian villagers after stimulation with SGH or PdSP15: IFN-γ (mean, 294.6 pg/ml; P = 0.0354, one-way ANOVA; n = 14), IL-10 (mean, 32.47 pg/ml; P = 0.0112, one-way ANOVA; n = 14), IL-17 (mean, 245.4 pg/ml; P = 0.0004, one-way ANOVA; n = 14), and IL-5 (mean, 65.27 pg/ml; P = 0.0344, one-way ANOVA; n = 14), or stimulation with rPdSP15 yielded IFN-γ (mean, 342 pg/ml; P = 0.0082, one-way ANOVA; n = 14), IL-10 (mean, 6.757 pg/ml; P = 0.0145, one-way ANOVA; n = 14), and IL-17 (mean, 198.7 pg/ml; P = 0.0153, one-way ANOVA; n = 14), but not IL-5 (mean, 14.68 pg/ml; P = 0.7409, one-way ANOVA; n = 14). PBMCs were selected on the basis of cell viability. Lines indicate the mean.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/290/290ra90/DC1

    Fig. S1. Disease progression of CL in NHP after transmission of L. major by bites of 50 infected P. duboscqi sand flies.

    Fig. S2. CD3+ lymphocytes are the main source of Leishmania-specific IFN-γ in NHP exposed to uninfected P. duboscqi sand flies.

    Fig. S3. CD8+ lymphocytes are not critical for protection from CL in NHP exposed to uninfected sand fly bites.

    Fig. S4. Anti-PdSP15 antibodies and CD8+ lymphocytes are not critical for protection from CL in PdSP15-immunized NHP.

    Fig. S5. The crystal structure of PdSP15.

    Table S1. Measurements (mm) of skin indurations 48 hours after inoculation with plasmids coding for selected sand fly salivary proteins.

    Table S2. IFN-γ and IL-4 mRNA expression by RT-qPCR.

    Table S3. Data collection and refinement statistics for PdSP15.

    Source data. Excel file

  • Supplementary Material for:

    A sand fly salivary protein vaccine shows efficacy against vectortransmitted cutaneous leishmaniasis in nonhuman primates

    Fabiano Oliveira, Edgar Rowton, Hamide Aslan, Regis Gomes, Philip A. Castrovinci, Patricia H. Alvarenga, Maha Abdeladhim, Clarissa Teixeira, Claudio Meneses, Lindsey T. Kleeman, Anderson B. Guimarães-Costa, Tobin E. Rowland, Dana Gilmore, Seydou Doumbia, Steven G. Reed, Phillip G. Lawyer, John F. Andersen, Shaden Kamhawi,* Jesus G. Valenzuela*

    *Corresponding author. E-mail: jvalenzuela{at}niaid.nih.gov (J.G.V.); skamhawi{at}niaid.nih.gov (S.K.)

    Published 3 June 2015, Sci. Transl. Med. 7, 290ra90 (2015)
    DOI: 10.1126/scitranslmed.aaa3043

    This PDF file includes:

    • Fig. S1. Disease progression of CL in NHP after transmission of L. major by bites of 50 infected P. duboscqi sand flies.
    • Fig. S2. CD3+ lymphocytes are the main source of Leishmania-specific IFN-γ in NHP exposed to uninfected P. duboscqi sand flies.
    • Fig. S3. CD8+ lymphocytes are not critical for protection from CL in NHP exposed to uninfected sand fly bites.
    • Fig. S4. Anti-PdSP15 antibodies and CD8+ lymphocytes are not critical for protection from CL in PdSP15-immunized NHP.
    • Fig. S5. The crystal structure of PdSP15.
    • Table S1. Measurements (mm) of skin indurations 48 hours after inoculation with plasmids coding for selected sand fly salivary proteins.
    • Table S2. IFN-γ and IL-4 mRNA expression by RT-qPCR.
    • Table S3. Data collection and refinement statistics for PdSP15.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Source data. Excel file

    [Download Source Data File]

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