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

doi:10.1126/SCITRANSLMED.AAA3043

LEISHMANIASIS

A sand fly salivary protein vaccine shows efficacy

against vector-transmitted cutaneous leishmaniasis

in nonhuman primates

Fabiano Oliveira,

1

Edgar Rowton,

2

Hamide Aslan,

1

* Regis Gomes,

1,3

Philip A. Castrovinci,

1

Patricia H. Alvarenga,

4,5

Maha Abdeladhim,

1

Clarissa Teixeira,

1,3

Claudio Meneses,

1

Lindsey T. Kleeman,

1

Anderson B. Guimarães-Costa,

1

Tobin E. Rowland,

2

Dana Gilmore,

1

Seydou Doumbia,

6

Steven G. Reed,

7

Phillip G. Lawyer,

2

John F. Andersen,

8

Shaden Kamhawi,

1†

Jesus G. Valenzuela

1†

Currently, there are no commercially available human vaccines against leishmaniasis. In rodents, cellular immunity

to salivary proteins of sand fly vectors is associated to protection against leishmaniasis, making them worthy targets

for further exploration as vaccines. We demonstrate that nonhuman primates (NHP) exposed to Phlebotomus

duboscqi uninfected sand fly bites or immunized with salivary protein PdSP15 are protected against cutaneous

leishmaniasis initiated by infected bites. Uninfected sand fly–exposed and 7 of 10 PdSP15-immunized rhesus ma-

caques displayed a significant reduction in disease and parasite burden compared to controls. Protection correlated

to the early appearance of Leishmania-specific CD4

+

IFN-g

+

lymphocytes, suggesting that immunity to saliva or

PdSP15 augments the host immune response to the parasites while maintaining minimal pathology. Notably,

the 30% unprotected PdSP15-immunized NHP developed neither immunity to PdSP15 nor an accelerated Leishmania-

specific immunity. Sera and peripheral blood mon onuclear cells from individuals naturally exposed to P. duboscqi bites

recognized PdSP15, demonstrating its immunogenicit y in humans. PdSP15 sequence and structure show no homol-

ogy to mammalian proteins, further demonstrating its potential as a component of a vaccine for human leishmaniasis.

INTRODUCTION

Leishmaniasis is a neglected tropical disease that affects the poorest of

communities and comes only second to malaria and fourth among

tropical parasitic diseases in mortality and morbidity, respectively (1).

Despite its global distribution and substantial disease burden, there

are no commercially available human leishmaniasis vaccines to date.

All forms of leishmaniasis are transmitted by the bite of infected

phlebotomine sand flies. As infected females feed on mammalian hosts,

they inject saliva, counteracting hemostasis and improving blood-

feeding success. Leishmania-infected sand flies regurgitate parasites

together with the salivary proteins into the bite wound. Exploiting the

concurrenceofsandflysalivaandparasitesinthebitesiteisanoriginal

approach to traditional Leishmania vaccines.

Experimentally, it has been shown that exposure to saliva through

bites of uninfected sand flies or immunization with an appropriate sal-

ivary protein protects rodents against cutaneous and visceral leishma-

niases (2–5). Saliva-mediated protection from leishmaniasis correlates

to the induction of a rapid sand fly saliva–specific T

H

1(Thelper1cell)

immune response at the bite site that steers the development of a faster

and more robust Leishmania-specific T

H

1 immunity with minimal pa-

thology (4, 6). Moreover, antibodies are not required for saliva-mediated

protection from leishmaniasis in murine models (3, 4).

Additionally, saliva-driven immunity protected against vector-transmitted

leishmaniasis (3, 4). This virulent mode of challenge, encompassing sand

fly saliva, promastigote secretory gel (7), and midgut-differentiated

Leishmania metacyclics, was shown to rescind the efficacy of a vaccine

established via needle challenge with Leishmania parasites (8), high-

lighting the robustness of saliva-mediated immunity to leishmaniasis.

Cutaneous leishmaniasis (CL) is the most widely distributed form of the

complex of diseases referred to as the leishmaniases. Annually, an estimated

0.7 million to 1.3 million new CL cases occur worldwide (9). Some two-

thirds of new CL cases occur in six countries including Afghanistan,

Algeria, Brazil, Colombia, Iran, and the Syrian Arab Republic (9). CL caused

by Leishmania major is prevalent in the Middle East, North Africa, and Sub-

Saharan Africa, where it is mainly transmitted by Phlebotomus papatasi or

Phlebotomus duboscqi sand flies (10). Here, we tested the capacity of ex-

posure to P. duboscqi uninfectedbitesorimmunizationwithits15-kD

salivary protein, PdSP15, in nonhuman prima te s (NHP) for prote ct io n

against vector-transmitted L. major. We uphold the concept of using

immunity to vector salivary proteins to protect humans from CL, dem-

onstrating their efficacy against vector-transmitted L. major in NHP.

RESULTS

Exposure to uninfected sand fly bites protects NHP against

sand fly–transmitted CL

To induce immunity to sand fly saliva in NHP, we exposed naïve rhesus

macaques to 20 P. duboscqi uninfected sand fly bites four times every

1

Vector Molecular Biology Section , Laboratory of Malaria and Vector Research, National

Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD

20852, USA.

2

Department of Entomology, Walter Reed Army Institute of Research, Silver

Spring, MD 20910, USA.

3

Centro de Pesquisas Gonçalo Moniz (CPqGM)–Fundação

Oswaldo Cruz (FIOCRUZ), Salvador, Bahia 40296-710, Brazil.

4

Laboratório de Bioquímica de

Resposta ao Estresse, Instituto de Bioquímica Médica, Universidade Federal do Rio de

Janeiro, Rio de Janeiro 21941-902, Brazil.

5

Instituto Nacional de Ciência e Tecnologia em

Entomologia Molecular (INCT-EM), Universidade Federal do Rio de Janeiro, Rio de Janeiro

21941-902, Brazil.

6

Faculty of Medicine, Pharmacy and Odontostomatology, University of

Bamako, Bamako 1805, Mali.

7

Infectious Disease Research Institute, Seattle, WA 98102,

USA.

8

Vector Biology Section, Laboratory of Malaria and Vector Research, National Institute

of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.

*Present address: Nursing Department, Faculty of Health Science, Selahaddin Eyyubi

University, Diyarbakir, Turkey.

†Corresponding author. E-mail: jvalenzuela@niaid.nih.gov (J.G.V.); skamhawi@niaid.nih.gov (S.K.)

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21 days. Most NHP (63%) developed a

delayed-type hypersensitivity (DTH) re-

sponse showing a marked recruitment

of mononuclear cells to the dermis 48 hours

after the last exposure (Fig. 1A). The re-

active bite site showed a relative abun-

dance of interferon-g (IFN-g)compared

to controls ascribing a T

H

1-like environ-

ment to the observed cell infiltrate (P =

0.0043, Mann-Whitney test; n =6;Fig.1B).

Exposure to uninfected sand flies also

generated a humoral response that was

more pronounced in DTH-positive reac-

tive animals (P = 0.0002, t test; n =10;

Fig. 1C). To test the efficacy of the observed

immunity to uninfected sand fly bites in

protection fro m Leishmania parasites,

we developed a natural model of vector-

transmitted CL in NHP using L. major–

infected P. duboscqi sand flies. Two groups

of NHP challenged with either 20 or 50

infected sand flies presented with 50 and

90% of diseased animals, respectively.

Lesion clusters evolved from papules to

nodules to ulcerated lesions, which pro-

gressed to healed scars (fig. S1), charac-

terist ics that mirror those observed in

human CL caused by L. major (11). Next,

we tested whether immunity generated

tosandflysalivaryproteinsinNHPwas

protective against vector-transmitted CL.

Naïve and uninfected sand fly–exposed

NHP were challenged with 50 Leishmania-

infected sand flies. Compared to naïve

animals, uninfected sand fly–exposed NHP

controlled the infection with a significant

reduction in disease burden, as defined

by the computation of the cumulative

measurement of the largest diameter of

each lesion on a weekly basis (P = 0.0083,

Mann-Whitney test; n =9to15;Fig.1D),

maximum lesion size (P = 0.0119, Mann-

Whitney test; n = 9 to 15; Fig. 1E), and

time to heal [P = 0.0048, log-rank (Mantel-

Cox) test; n =9to15;Fig.1F].Atthe

9-week post-challenge time point, 70%

of naïve animals displayed ulcerated le-

sions compare d to only 30% of uninfec ted

sand fly–exposedNHP(Fig.1G).Thisre-

duction in disease severity correlated to a

significantly lower number of parasites in

lesion biopsies (P = 0.0237, Mann-Whitney

test; n = 9 to 15;Fig. 1H). Notably, in order

not to disrupt the course of lesion develop-

ment, the parasite burden was measured

at 12 weeks after infection when les io n s

of both naïv e and uninfected sand fly–

ex p os e d NH P we r e he a l i n g . Furthermore,

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-g 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).Cumulativedatafrom

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 =9to15).

(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-Whi tney 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-g SFC by enzyme-linked immuno-

spot (ELISPOT) (P = 0.0587, Mann-Whitney test; n = 7 to 9). (J) Percent of CD4

+

IFN-g

+

lymphocytes by flow

cytometry (P = 0.0229, Mann-Whitney test; n = 7 to 9). (K) Frequency of CD4

+

IFN-g

+

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 mm; lines and bars indicate the mean, and error bars indicate SEM.

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protection in uninfected sand fly–exposed NHP correlated to the induc-

tion of an early Leishmania-specific immune response 2 weeks after

infection. After stimulation with Leishmania antigen (Leish), pe-

ripheral blood mononuclear cells (PBMCs) from uninfected sand fly–

exposed NHP produced a high er numbe r of IFN-g spot-forming cells

(SFC) compared to controls (P = 0.0587, Mann-Whitney test; n =7

to 9; Fig. 1I). We corroborated these data by flow cytometric analysis

demonstrating that CD3

+

cells were the main source of specific anti-

Leishmania IFN-g (fig.S2).AlthoughCD4

+

(Fig. 1J) and CD8

+

(fig.

S3A) lymphocytes produced Leishmania-specific IFN-g,onlythefre-

quency of CD4

+

IFN-g

+

cells was statistically higher between uninfected

sand fly–exposed and naïve animals (P = 0.0229, Mann-Whitney test;

n = 7 to 9; Fig. 1J). Moreover, the frequency of CD4

+

IFN-g

+

cells

inversely correlated to dise as e burd en in uni nfected sa n d fl y –exposed

NHP (P < 0.0022, n = 16, Spearman rank correlation test r = −0 .7211;

Fig. 1K and fig. S3B), suggesting that Leishmania-specific CD4 T cells are

participating in parasite clearance.

Reverse antigen screening of P. duboscqi sand fly salivary

molecules in saliva-exposed NHP identifies PdSP15 as a

vaccine candidate against CL

Having established that exposure to uninfected sand fly bites protects

NHP against CL, our next objective was to identify the salivary protein

responsible for the protective effect. Therefore, we screen about 23 se-

creted salivary proteins of P. duboscqi in NHP. To identify protective

sand fly salivary proteins while minimizing the number of NHP, we

developed an approach named “reverse antigen screening” (RAS). The

approach is based on exploiting the adaptive immunity generated against

salivary proteins in uninfected sand fly–exposed NHP. Uninfected

sand fly–exposed and bite site–reactive NHP were injected intrader-

mally with DNA plasmids coding for the most abundant secreted

P. duboscqi salivary proteins. Using the host machinery as a natural pro-

tein expression system, we selected salivary molecules that induced a

T

H

1-DTH 48 hours after inoculation. Salivary gland homogenate

(SGH) and bites from one uninfected sand fly, and empty plasmid were

used as positive and negative controls, respectively. From the 23 tested

DNA plasmids, we selected the top five molecules based on their in-

duction of the largest skin induration as measured by the diameter of

the skin reaction. These included PdMu54 (2.4 mm), PdSP15 (1.83 mm),

PdMu29 (1.79 mm), PdMu49 (1.69 mm), and PdMu35 (1.66 mm). We

also selected a negative control, empty DNA plasmid (1.01 mm), and

two positive controls, SGH (3.63 mm) and a bite site (2.84 mm) (Fig. 2A

and table S1). Of the five, PdSP15 was the only molecule displaying a

significant increase in IFN-g mRNA message compared to the ne gat i v e

control [P = 0.0109, one-way analysis of variance (ANOVA); n =8],and

the one exhibiting the lowest level of interleukin-4 (IL-4) (Fig. 2B);

this translated to a high IFN-g /IL-4 ratio indicative of a T

H

1-biased im-

mune response (P = 0.0470, one-way ANOVA; n =8;Fig.2C).Exact

P values for the five tested samples are presented in table S2.

Immunization with PdSP15 protects NHP against sand

fly–transmitted CL

NHP were immunized intradermally with PdSP15 DNA two times 21

days apart and boosted 21 days later with recombinant PdSP15 (rPdSP15)

and glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE). Con-

trol animals were inoculated with empty plasmid followed by a boost

with bovine serum albumin and GLA-SE. In contrast to controls, 70%

of PdSP15-immunized NHP displayed a distinct skin induration at the

injection site 48 hours after the rPdSP15 boost (P = 0.0067, Mann-

Whitney test; n = 10; Fig. 3A). Two weeks later, PBMCs of skin-reactive

PdSP15-immunized NHP produced significantly higher IFN-g SFC

after stimulation with rPdSP15 compared to controls (P = 0.0002, Mann-

Whitney test; n =7;Fig.3B,solidsquares).ThenumberofIFN-g SFC

in the 30% nonreactive PdSP15-immunized animals was similar to

controls (Fig. 3B, empty squares) but produced significantly high levels

of specific anti-rPdSP15 IgG antibodies (Fig. 3C, empty squares; P <

0.0001, one-way ANOVA; n = 3), with antibody levels showing a

negative cor relation to IFN-g production (P = 0.0037; r = –0.84,

Fig. 2. A RAS approach in NHP

identifies PdSP15 as a T

H

1-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 ex-

posure, animals were injected in-

tradermally with 23 distinct DNA

plasmids encoding the most abun-

dant P. duboscqi salivary proteins or an empty plasmid as a negative con-

trol. 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 plas-

mids 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-g and IL-4 mRNA expression by quantitative real-time fluores-

cence polymerase chain reaction (RT-qPCR). (C) IFN-g/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.

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Spearman correlation; n = 10; fig. S4A).

Because of the dichotomy of responses

to Pd SP 1 5 im mu niz atio n, we split PdSP15

vaccinated NHP to those that produced

IFN-g (PdSP15 –IFN-g

+

)orthosewhere

PdSP15 immunization induced a strong

antibody and a poor IFN-g resp onse

(PdSP15–IFN-g

−

). Control (CTL)- and

PdSP15-immunized NHP were challenged

with 50 L. major–infected sa n d fl i e s

1 month after the last immunization.

Compared to controls, PdSP15–IFN-g

+

(solid squares) NHP had significantly re-

d uced disease burden (P = 0.0490, one-

way ANOVA; n = 3 to 11; Fig. 3D) and reduced maximum lesion

size (P = 0.0465, one-way ANOVA; n = 3 to 11; Fig. 3E). PdSP15–IFN-g

−

NHP (empty squares) were not protected and had a disease burden

comparable to controls (Fig. 3, D and E). We did not observe a reduc-

tioninthetimetohealwhencomparingPdSP15–IFN-g

+

and controls

(Fig. 3F). Representative photographs illustrate the reduction in lesion

severity at 5 weeks after infection in PdSP15–IFN-g

+

NHP compared

to controls (Fig. 3G). Disease amelioration was further echoed by a

Fi g. 3. Immunization with PdSP15 pro-

tects 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 inoc-

ulation with bovine serum albumin (CTL) or

rPdSP15 (P = 0.0067, t test; n =10).(B)IFN-g

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-g (PdSP15-

IFN

+

) or not (PdSP15-IFN

−

)(P < 0.0001, one-way

ANOV A; n =3to10).(D to L) Evaluation of

disease (D to H) and Leishmania-specific im-

munity (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) Max-

imum 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 =3to8).(ItoK)

PBMCs stimulated with Leishmania antigen

(Leish) 2 weeks after challenge in 8 to 10 NHP.

Selection was based on cell number and vi-

ability. (I) IFN-g SFC by ELISPOT (P = 0.0075,

one-way ANOVA; n = 3 to 10). (J) Percent of

CD4

+

IFN-g

+

lymphocytes by flow cytometry

(P = 0.0002, one-way ANOVA; n =3to10).

(K) Frequency of CD4

+

lymphocytes produc-

ing 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.

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significant reduction in the number of parasites in PdSP15–IFN-g

+

NHP compared to controls (P = 0.0034, one-way ANOVA; n =3to

8; Fig. 3H). PdSP15–IFN-g

−

NHP harbored parasite numbers compa-

rable to controls (Fig. 3H).

To understand how cellular immunity to PdSP15 protects against

vector-transmitted CL, we explored the early immune response to

Leishmania in PdSP15-immunized NHP 2 weeks after challenge with

infected sand flies. Similar to uninfected sand fly–exposed NHP, CD3

+

cells were the main source of specific anti-Leishmania IFN-g (fig.

S4B). Compared to controls, PdSP15–IFN-g

+

NHP (solid squares) de-

veloped a stronger anti-Leishmania immune response after challenge,

showing a significant increase in the number of IFN-g SFC (P = 0.0075,

one-way ANOVA; n = 3 to 8; Fig. 3I) and in the frequency of CD4

+

IFN-g

+

lymphocytes (P = 0.0002, one-way ANOVA; n = 3 to 10; Fig. 3J). No-

tably, a significant increase in the proportion of Leishmania-specific

CD4

+

IFN-g

+

IL-2

+

cells was also observed in PdSP15–IFN- g

+

NHP com-

pared to controls (P = 0.0418, one-way ANOVA; n = 4 to 6; Fig. 3K).

Similar to uninfected sand fly–exposed NHP, both CD8

+

and CD4

+

lymphocytes produced Leishmania-specific IFN-g, but only the fre-

quency of the latter was significantly higher in PdSP15–IFN-g

+

NHP

compared to controls (Fig. 3J and fig. S4C), reinforcing the conclusion

that the protective immune response is mostly driven by CD4

+

lym-

phocytes. This rapidly developing robust immunity against Leishmania

parasites was not observed in PdSP15–IFN-g

−

NHP (Fig. 3, I and J,

empty squares). Our findings suggest that PdSP15-specific IFN-g pro-

motes a microenvironment that facilitates priming of an early Leishmania-

specific protective CD4

+

T cell response.

In humans, the presence of a DTH after intradermal inocula-

tion with killed Leishmania, known as a positive Leishmanin skin

test (LST), is considered a signature of lifelong protective immunity

against CL. Twelve weeks after infection,

PdSP15–IFN-g

+

NHP had a significantly

larger LST induration size (Fig. 3L, solid

squares) compared to controls (P=0.0269,

one-way ANOVA; n = 3 to 11) and to

PdSP15–IFN-g

−

animals (Fig. 3L, empty

squares). This suggests that infected con-

trols and PdSP15 –IFN-g

−

NHP developed

a weaker immunity to Leishmania com-

pared to PdSP15–IFN-g

+

NHP after reso-

lution of the infection.

PdSP15 is a member of the

insect family of odorant-binding

proteins with no sequence or

structure homology to known

human proteins

The protective salivary antigen PdSP15

shares sequence homology only to the

small odorant-binding protein family

found exclusively in the salivary glands

of sand flies (Fig. 4A), with 67 and 54%

identity to the P. papatasi and Phlebotomus

sergenti salivary proteins PpSP15 and

PsSP15, respectively (Fig. 4B). To exclude

any structural similarities to human pro-

teins, the crystal structure of PdPS15 was

solved to a 2.95-nm resolution (Fig. 4C,

table S3). The structure is available at the Research Collaboratory

for Structural Bioinformatics Protein Data Bank (RCSB PDB) with

PDB code 4OZD. PdSP15 contains six a-helical elements designated

a, c, d, e, f, and g that match the homologous secondary structures of

insect odorant-binding proteins. Helix e is elongated relative to other

described insect proteins and contains a number of basic (arginine

and lysine) residues. Structural search with the program DALI (12)

showed a distant similarity to insect odorant-binding protein family

members including the D7 proteins found in the saliva of mosquitoes

and did not identify structural similarities to mammalian proteins

(fig. S5).

PdSP15 is immunogenic in humans naturally exposed to

P. duboscqi bites

Having established that PdSP15 is an antigen foreign to humans, we

investigated the immunogenicity of rPdSP15 in individuals naturally

exposed to P. duboscqi bites (13). Sand fly–exposed individuals with

antibodies to whole saliva produced significant levels of antibodies

to rPdSP15 (P < 0.0001, Mann-Whitney test; n = 12 to 30; Fig. 5A) or

the SGH (P < 0.0001, Mann-Whitney test; n =12to30;Fig.5A).PBMCs

from 14 individuals naturally exposed to P. duboscqi bites (18 to 65 years

old) were stimulated with SGH or rPdSP15 in vitro, and supernatants

were collected 96 hours after stimulation. Levels of IFN-g, IL-10, IL-17,

IL-5, and IL-13 were detected by a Luminex assay (Fig. 5B). Levels of

IL-2, IL-4, and IL-9 in these samples were below the limit of detection

of the assay. Compared to controls, stimulation with SGH induced sig-

nificant levels of IFN-g (mean, 294.6 pg/ml; P = 0.0354, one-way

ANOV A; n = 14; Fig. 5B), IL-10 (mean, 32.47 pg/ml; P = 0.0112, one-

way ANOVA; n = 14; Fig. 5B), IL-17 (mean, 245.4 pg/ml; P = 0.0004, one-

way ANOVA; n = 14; Fig. 5B), and IL-5 (mean, 65.27 pg/ml; P = 0.0344,

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 a-helical

elements designated as a, c, d, e, f, and g.

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

Summary (3 min read)

INTRODUCTION

RESULTS

Study design

Animals

Sand flies and SGH

Parasites

Reverse antigen screening

Leishmanin skin test

Histological analysis of DTH site

Statistical analyses

REFERENCES AND NOTES

Figures (3)

Citations

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References

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