scispace - formally typeset
SciSpace - Your AI assistant to discover and understand research papers | Product Hunt

Journal ArticleDOI

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

03 Jun 2015-Science Translational Medicine (American Association for the Advancement of Science)-Vol. 7, Iss: 290

TL;DR: It is demonstrated that a vaccine against sand fly salivary protein can protect nonhuman primate from leishmania infection and PdSP15 sequence and structure show no homology to mammalian proteins, further demonstrating its potential as a component of a vaccine for human leishmaniasis.

AbstractCurrently, 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 macaques displayed a significant reduction in disease and parasite burden compared to controls. Protection correlated to the early appearance of Leishmania-specific CD4(+)IFN-I³(+) 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 mononuclear cells from individuals naturally exposed to P. duboscqi bites recognized PdSP15, demonstrating its immunogenicity in humans. PdSP15 sequence and structure show no homology to mammalian proteins, further demonstrating its potential as a component of a vaccine for human leishmaniasis.

Topics: Cutaneous leishmaniasis (62%), Cellular immunity (58%), Leishmaniasis (54%), Immunity (52%), Vector (epidemiology) (51%)

Summary (3 min read)

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).
  • Experimentally, it has been shown that exposure to saliva through bites of uninfected sand flies or immunization with an appropriate salivary protein protects rodents against cutaneous and visceral leishmaniases (2–5).
  • 5Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil.
  • Some twothirds of new CL cases occur in six countries including Afghanistan, Algeria, Brazil, Colombia, Iran, and the SyrianArabRepublic (9).

RESULTS

  • Exposure to uninfected sand fly bites protects NHP against sand fly–transmitted CL Next, the authors tested whether immunity generated to sand fly salivary proteins in NHP was protective against vector-transmitted CL.
  • Exact measurements and P values for all the samples tested are presented in tables S1 and S2.
  • Similar to uninfected sand fly–exposed NHP, both CD8+ and CD4+ lymphocytes produced Leishmania-specific IFN-g, but only the frequency 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+ lymphocytes.

Study design

  • This study was designed to investigate the efficacy of P. duboscqi sand fly salivary proteins as vaccine candidates against CL.
  • The authors exposed rhesus macaques to uninfected sand fly bites or immunized them with an immunogenic sand fly salivary protein (PdSP15).
  • For challenge experiments, the authors calculated their sample sizes to achieve statistically significant results if protection is at least 80% effective (a 0.835% probability of preventing or reducing the CL outcome) when considering that 90% of the naïve NHP would develop lesions after challenge.
  • Data from available frozen sera and cryopreserved PBMCs from inhabitants of an endemic area where P. duboscqi is prevalent were used to assess the immunogenicity of PdSP15 in humans.
  • The number of animals per group and per experiment is indicated in all figure legends.

Animals

  • Rhesus macaques (Macaca mulatta, Indian strain) were housed in the Walter Reed Army Institute of Research vivarium where manipulations were conducted under protocol IEO02-09 approved by the Walter Reed Army Institute of Research Institute Animal Care and Use Committee and by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee under protocol LMVR12.
  • All NHP were screened for good physical health and absence of antibodies reactive to P. duboscqi sand fly salivary proteins before enrollment into the animal protocol.
  • During experimental manipulations, they were housed individually in stainless steel cages (cubic ~2 m), kept in environmentally controlled rooms with 10 to 15 air changes per hour, temperature range 18° to 29°C, relative humidity 70%, and light/dark 12:12-hour cycle.
  • NHP were fed a staple diet of LabDiet Primate Chow #5038, supplemented by Prima Treats #F05709 (Bio-Serve) fresh fruits and vegetables with water available ad libitum from an automatic system.
  • Macaques were consistently negative for SIV (simian immunodeficiency virus) and STLV (simian T lymphotrophic virus) when serologically tested annually, and quarterly, intradermal TB tests were negative.

Sand flies and SGH

  • P. duboscqi sand flies originally from Mali, West Africa, and reared in the insectary facilities of the Laboratory of Malaria and Vector Research, NIAID, NIH, were used for the described experiments.
  • For transmission experiments, 3- to 4-day-old sand flies were allowed to feed on blood containing L. major promastigotes as previously described (4).
  • Sand flies with mature infections (11 to 15 days after blood feeding) were used to transmit Leishmania parasites to NHP.
  • Five- to 7-day-old sand flies were used for preparation of SGH.
  • Briefly, pools of 20 salivary glands were dissected in phosphate-buffered saline.

Parasites

  • L. majorWR 2885 strain was used to infect sand flies and for preparation of specific Leishmania antigen.
  • This strain of parasites was recently isolated from a soldier deployed to Iraq as previously described (4).
  • Leishmania antigen was prepared by harvesting 1 × 109 parasites from culture flasks and repeated freeze-thaw cycles.
  • Twenty-three DNA plasmids coding to P. duboscqi salivary proteins were cloned in the VR2001-TOPO vector (Vical Inc.), and endotoxinfree DNA was purified as previously described (14).

Reverse antigen screening

  • NHP were inoculated intradermally with 30 mg of the 23 distinct DNA plasmids, once, for reverse antigen experiments.
  • Onemonth after the last uninfected sand fly bite exposure, NHP were injected intradermally using an insulin syringe with each of the DNA candidates, empty plasmid control, SGH, and bites from one uninfected sand fly in the inner thighs and/or chest.
  • Twenty-four and 48 hours after the inoculations, reactions were recorded by measurement of the induration diameter suing a Vernier caliper.
  • The structure of PdSP15 was determined by molecular replacement using a monomeric PdSP15b structure (56) as a search model in the program PHASER (57).
  • Lesion sizewasmeasuredweekly as thediameter of the skin lesionusing adigitalVernier caliper .

Leishmanin skin test

  • Animals were inoculated with 100 mg in 100 ml of the Leishmania antigen, intradermally in the left inner thigh.
  • Measurements were taken with a Vernier caliper .
  • Antibody measurements by enzyme-linked immunosorbent assay Microtiter plates were coated with P. duboscqi sand fly SGH (1 pair/ ml) or with rPdSP15 (2 mg/ml) overnight at 4 °C.
  • Alkaline phosphatase substrate was added for 30 min, and absorbance was read at 405 nm in a spectrophotometer (Molecular Devices).

Histological analysis of DTH site

  • DNA or RNA was extracted using QIAamp DNA Micro Kit or RNeasy Mini Kit, respectively, following the manufacturer’s instructions.
  • Numbers of parasites in the skin were determined by SYBR Green real-time PCR assay and primers JW11-JW12 as targets for Leishmania amplification (59).
  • Results are expressed as relative expression, where value of 1 is the normal skin baseline.

Statistical analyses

  • Lines present in the scatterplot graphs represent the mean, and bar graphs depict means ± SEM or SD as indicated.
  • Statistical differences between two groups were tested by t test (two-tailedMann-Whitney test).
  • Correlations were tested by Spearman test, and dotted lines illustrate the 95% CI.
  • Fig. S3. CD8+ lymphocytes are not critical for protection fromCL inNHPexposed to uninfected sand fly bites.
  • Fig. S4. Anti-PdSP15 antibodies and CD8+ lymphocytes are not critical for protection from CL in PdSP15-immunized NHP.

REFERENCES AND NOTES

  • R.G., C.T., andP.A.C. participated in laboratory and animal studies.
  • Leishmaniasis is transmitted by the bite of infected phlebotomine sand flies, which also transfer some of demonstrate that a vaccine against sand fly salivary protein can protect nonhuman primate from leishmaniaal.

Did you find this useful? Give us your feedback

...read more

Content maybe subject to copyright    Report

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 flyexposed 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 (25). Saliva-mediated protection from leishmaniasis correlates
to the induction of a rapid sand fly salivaspecific 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 flytransmitted 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 dica, 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.)
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 3 June 2015 Vol 7 Issue 290 290ra90 1
on July 7, 2015Downloaded from

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 flyexposed
NHP were challenged with 50 Leishmania-
infected sand flies. Compared to naïve
animals, uninfected sand flyexposed 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 flyexposedNHP(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 eosinstained 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 sitereactive 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. majorinfected 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.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 3 June 2015 Vol 7 Issue 290 290ra90 2
on July 7, 2015Downloaded from

protection in uninfected sand flyexposed 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 flyexposed 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 flyexposed NHP. Uninfected
sand flyexposed and bite sitereactive 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
flytransmitted 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.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 3 June 2015 Vol 7 Issue 290 290ra90 3
on July 7, 2015Downloaded from

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
(PdSP15IFN-g
). Control (CTL)- and
PdSP15-immunized NHP were challenged
with 50 L. majorinfected sa n d fl i e s
1 month after the last immunization.
Compared to controls, PdSP15IFN-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). PdSP15IFN-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-
tioninthetimetohealwhencomparingPdSP15IFN-g
+
and controls
(Fig. 3F). Representative photographs illustrate the reduction in lesion
severity at 5 weeks after infection in PdSP15IFN-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.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 3 June 2015 Vol 7 Issue 290 290ra90 4
on July 7, 2015Downloaded from

significant reduction in the number of parasites in PdSP15IFN-g
+
NHP compared to controls (P = 0.0034, one-way ANOVA; n =3to
8; Fig. 3H). PdSP15IFN-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 flyexposed NHP, CD3
+
cells were the main source of specific anti-Leishmania IFN-g (fig.
S4B). Compared to controls, PdSP15IFN-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 PdSP15IFN- g
+
NHP com-
pared to controls (P = 0.0418, one-way ANOVA; n = 4 to 6; Fig. 3K).
Similar to uninfected sand flyexposed NHP, both CD8
+
and CD4
+
lymphocytes produced Leishmania-specific IFN-g, but only the fre-
quency of the latter was significantly higher in PdSP15IFN-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 PdSP15IFN-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,
PdSP15IFN-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
PdSP15IFN-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 PdSP15IFN-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 flyexposed 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.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 3 June 2015 Vol 7 Issue 290 290ra90 5
on July 7, 2015Downloaded from

Figures (3)
Citations
More filters

Journal ArticleDOI
TL;DR: The early events associated with infection, the CD4- T cells that mediate protective immunity and the pathological role that CD8+ T cells can have in cutaneous leishmaniasis are discussed.
Abstract: Cutaneous leishmaniasis is a major public health problem and causes a range of diseases from self-healing infections to chronic disfiguring disease. Currently, there is no vaccine for leishmaniasis, and drug therapy is often ineffective. Since the discovery of CD4(+) T helper 1 (TH1) cells and TH2 cells 30 years ago, studies of cutaneous leishmaniasis in mice have answered basic immunological questions concerning the development and maintenance of CD4(+) T cell subsets. However, new strategies for controlling the human disease have not been forthcoming. Nevertheless, advances in our knowledge of the cells that participate in protection against Leishmania infection and the cells that mediate increased pathology have highlighted new approaches for vaccine development and immunotherapy. In this Review, we discuss the early events associated with infection, the CD4(+) T cells that mediate protective immunity and the pathological role that CD8(+) T cells can have in cutaneous leishmaniasis.

289 citations


Journal ArticleDOI
03 Jun 2016-Vaccine
TL;DR: Leishmaniasis is a vector-borne neglected tropical disease caused by a protozoan parasite of the genus Leishmania and transmitted to humans by the bite of a sand fly, and both VL and CL vaccines have been shown to be cost-effective in economic modeling studies.
Abstract: A number of leishmaniasis vaccine candidates are at various stages of pre-clinical and clinical development. Leishmaniasis is a vector-borne neglected tropical disease (NTD) caused by a protozoan parasite of the genus Leishmania and transmitted to humans by the bite of a sand fly. Visceral leishmaniasis (VL, kala-azar) is a high mortality NTD found mostly in South Asia and East Africa, while cutaneous leishmaniasis (CL) is a disfiguring NTD highly endemic in the Middle East, Central Asia, North Africa, and the Americas. Estimates attribute 50,000 annual deaths and 3.3 million disability-adjusted life years to leishmaniasis. There are only a few approved drug treatments, no prophylactic drug and no vaccine. Ideally, an effective vaccine against leishmaniasis will elicit long-lasting immunity and protect broadly against VL and CL. Vaccines such as Leish-F1, F2 and F3, developed at IDRI and designed based on selected Leishmania antigen epitopes, have been in clinical trials. Other groups, including the Sabin Vaccine Institute in collaboration with the National Institutes of Health are investigating recombinant Leishmania antigens in combination with selected sand fly salivary gland antigens in order to augment host immunity. To date, both VL and CL vaccines have been shown to be cost-effective in economic modeling studies.

141 citations


Journal ArticleDOI
TL;DR: Although Old World CL is generally not fatal, clinical symptoms can lead to disfiguring scars that result in social stigmatization and psychological consequences, and the World Health Organization has estimated that around 2.4 million disability-adjusted life years (DALYs) are lost due to CL and visceral leishmaniasis globally.
Abstract: The Syrian refugee crisis has precipitated a catastrophic outbreak of Old World cutaneous leishmaniasis now affecting hundreds of thousands of people living in refugee camps or trapped in conflict zones. A similar situation may also be unfolding in eastern Libya and Yemen. Leishmaniasis has been endemic in Syria for over two centuries, with the first case ever reported being as early as 1745, when it was known as the “Aleppo boil” [1,2]. Old World cutaneous leishmaniasis (CL) is characterized most notably by disfiguring skin lesions, nodules, or papules, and in the Middle East and North Africa (MENA) region it is primarily caused either by Leishmania tropica (anthroponotic) or L. major (zoonotic), with some sporadic cases also caused by L. infantum (Box 1) [3–5]. In North Africa, a chronic form of CL also can be caused by L. killicki [6–7]. Box 1. Old World Cutaneous Leishmaniasis (CL) in the MENA Region Anthroponotic CL Major etiologic agent: Leishmania tropica [4,5,7] Major vector: Phlebotomus sergenti [4,5] Zoonotic CL Major etiologic agent: L. major [4,5,7] Minor etiologic agent: L. infantum [4,5] Vectors: Ph. papatasi for L. major; Ph. perfiliewi, Ph. perniciosus, Ph. longicuspis, and Ph. ariasi for L. infantum [5] Major animal reservoirs: Rodents (L. major) and dogs (L. infantum) [4,7] Although Old World CL is generally not fatal, clinical symptoms can lead to disfiguring scars that result in social stigmatization and psychological consequences. The World Health Organization (WHO) has estimated that around 2.4 million disability-adjusted life years (DALYs) are lost due to CL and visceral leishmaniasis (VL) globally [8]; however, the number of DALYs attributed to CL is still under evaluation. The 2013 Global Burden of Disease Study determined that CL causes only 41,700 DALYs [9], while other studies have found that these figures may represent profound underestimates [10,11]. Studies observing the impact of marring CL facial scars have found that the social stigmatization involved leads to anxiety, depression, and decreased quality of life for patients [12]. The scars can lead to a changed perception of self and can limit individuals’ abilities to participate in society, further decreasing their social, psychological, and economic well-being, as employment opportunities become scarce. Women, adolescents, and children are particularly susceptible to the social stigmatization of disfiguring scars [13]. The hardships caused by CL extend beyond physical symptoms and manifest most prominently in patients’ social, psychological, and economic well-being. Like many neglected tropical diseases (NTDs), CL not only occurs in settings of poverty but the disease also has the ability to perpetuate and reinforce poverty, catalyzing a positive feedback loop between disease and poverty [14]. For many of these reasons, the WHO classifies leishmaniasis as one of 17 NTDs [15], although the cutaneous form is often not prioritized in major global health initiatives, unlike the NTDs now targeted by integrated preventive chemotherapy [11].

91 citations


Cites background from "A sand fly salivary protein vaccine..."

  • ...Development of a commercially available vaccine for Old World CL should also be made a priority, as one does not currently exist even though it would enhance efficacy of disease and vector control programs [71,72]....

    [...]


Journal ArticleDOI
TL;DR: The results obtained shall pave the way for the development of field-application tools that could contribute to the management of leishmaniasis in endemic areas as well as address the impact of such molecules on the biology of the host–sand fly–parasite interaction.
Abstract: Background Leishmaniases are parasitic diseases present worldwide that are transmitted to the vertebrate host by the bite of an infected sand fly during a blood feeding. Phlebotomine sand flies inoculate into the mammalian host Leishmania parasites embedded in promastigote secretory gel (PSG) with saliva, which is composed of a diverse group of molecules with pharmacological and immunomodulatory properties. Methods and findings In this review, we focus on 3 main aspects of sand fly salivary molecules: (1) structure and composition of salivary glands, including the properties of salivary molecules related to hemostasis and blood feeding, (2) immunomodulatory properties of salivary molecules and the diverse impacts of these molecules on leishmaniasis, ranging from disease exacerbation to vaccine development, and (3) use of salivary molecules for field applications, including monitoring host exposure to sand flies and the risk of Leishmania transmission. Studies showed interesting differences between salivary proteins of Phlebotomus and Lutzomyia species, however, no data were ever published on salivary proteins of Sergentomyia species. Conclusions In the last 15 years, numerous studies have characterized sand fly salivary proteins and, in parallel, have addressed the impact of such molecules on the biology of the host–sand fly–parasite interaction. The results obtained shall pave the way for the development of field-application tools that could contribute to the management of leishmaniasis in endemic areas.

87 citations


Journal ArticleDOI
TL;DR: It is shown that ingestion of a second uninfected blood meal by Leishmania-infected sand flies triggers dedifferentiation of metacyclic promastigotes to a replicative form, termed the retroleptomonad promastsigote, which amplifies parasite numbers in the flies.
Abstract: Sand flies, similar to most vectors, take multiple blood meals during their lifetime1-4. The effect of subsequent blood meals on pathogens developing in the vector and their impact on disease transmission have never been examined. Here, we show that ingestion of a second uninfected blood meal by Leishmania-infected sand flies triggers dedifferentiation of metacyclic promastigotes, considered a terminally differentiated stage inside the vector 5 , to a leptomonad-like stage, the retroleptomonad promastigote. Reverse metacyclogenesis occurs after every subsequent blood meal where retroleptomonad promastigotes rapidly multiply and differentiate to metacyclic promastigotes enhancing sand fly infectiousness. Importantly, a subsequent blood meal amplifies the few Leishmania parasites acquired by feeding on infected hosts by 125-fold, and increases lesion frequency by fourfold, in twice-fed compared with single-fed flies. These findings place readily available blood sources as a critical element in transmission and propagation of vector-borne pathogens.

76 citations


References
More filters

Journal ArticleDOI
TL;DR: A description is given of Phaser-2.1: software for phasing macromolecular crystal structures by molecular replacement and single-wavelength anomalous dispersion phasing.
Abstract: Phaser is a program for phasing macromolecular crystal structures by both molecular replacement and experimental phasing methods. The novel phasing algorithms implemented in Phaser have been developed using maximum likelihood and multivariate statistics. For molecular replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solutions from noise, and for single-wavelength anomalous dispersion experimental phasing, the new algorithms, which account for correlations between F+ and F−, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallographic community.

15,505 citations


Journal ArticleDOI
TL;DR: The key purpose of interactive analysis is to check whether conserved residues line up in multiple structural alignments and how Conserved residues and ligands cluster together in multiple structure superimpositions.
Abstract: Our web site (http://ekhidna.biocenter.helsinki.fi/dali_server) runs the Dali program for protein structure comparison. The web site consists of three parts: (i) the Dali server compares newly solved structures against structures in the Protein Data Bank (PDB), (ii) the Dali database allows browsing precomputed structural neighbourhoods and (iii) the pairwise comparison generates suboptimal alignments for a pair of structures. Each part has its own query form and a common format for the results page. The inputs are either PDB identifiers or novel structures uploaded by the user. The results pages are hyperlinked to aid interactive analysis. The web interface is simple and easy to use. The key purpose of interactive analysis is to check whether conserved residues line up in multiple structural alignments and how conserved residues and ligands cluster together in multiple structure superimpositions. In favourable cases, protein structure comparison can lead to evolutionary discoveries not detected by sequence analysis.

3,347 citations


Journal ArticleDOI
15 Jun 2006-Blood
TL;DR: The quality of the HIV-specific CD8(+) T-cell functional response serves as an immune correlate of HIV disease progression and a potential qualifying factor for evaluation of HIV vaccine efficacy.
Abstract: Establishing a CD8+ T cell–mediated immune correlate of protection in HIV disease is crucial to the development of vaccines designed to generate cell-mediated immunity. Historically, neither the quantity nor breadth of the HIV-specific CD8+ T-cell response has correlated conclusively with protection. Here, we assess the quality of the HIV-specific CD8+ T-cell response by measuring 5 CD8+ T-cell functions (degranulation, IFN-γ, MIP-1β, TNF-α, and IL-2) simultaneously in chronically HIV-infected individuals and elite nonprogressors. We find that the functional profile of HIV-specific CD8+ T cells in progressors is limited compared to that of nonprogressors, who consistently maintain highly functional CD8+ T cells. This limited functionality is independent of HLA type and T-cell memory phenotype, is HIV-specific rather than generalized, and is not effectively restored by therapeutic intervention. Whereas the total HIV-specific CD8+ T-cell frequency did not correlate with viral load, the frequency and proportion of the HIV-specific T-cell response with highest functionality inversely correlated with viral load in the progressors. Thus, rather than quantity or phenotype, the quality of the CD8+ T-cell functional response serves as an immune correlate of HIV disease progression and a potential qualifying factor for evaluation of HIV vaccine efficacy.

1,738 citations


Journal ArticleDOI
TL;DR: The importance of using multiparameter flow cytometry to better understand the functional capacity of effector and memory T-cell responses, thereby enabling the development of preventative and therapeutic vaccine strategies for infections is highlighted.
Abstract: T cells mediate effector functions through a variety of mechanisms. Recently, multiparameter flow cytometry has allowed a simultaneous assessment of the phenotype and multiple effector functions of single T cells; the delineation of T cells into distinct functional populations defines the quality of the response. New evidence suggests that the quality of T-cell responses is crucial for determining the disease outcome to various infections. This Review highlights the importance of using multiparameter flow cytometry to better understand the functional capacity of effector and memory T-cell responses, thereby enabling the development of preventative and therapeutic vaccine strategies for infections.

1,362 citations


Journal ArticleDOI
TL;DR: The quality of a CD4+ T-cell cytokine response can be a crucial determinant in whether a vaccine is protective, and may provide a new and useful prospective immune correlate of protection for vaccines based on T-helper type 1 (TH1) cells.
Abstract: CD4+ T cells have a crucial role in mediating protection against a variety of pathogens through production of specific cytokines. However, substantial heterogeneity in CD4+ T-cell cytokine responses has limited the ability to define an immune correlate of protection after vaccination. Here, using multiparameter flow cytometry to assess the immune responses after immunization, we show that the degree of protection against Leishmania major infection in mice is predicted by the frequency of CD4+ T cells simultaneously producing interferon-gamma, interleukin-2 and tumor necrosis factor. Notably, multifunctional effector cells generated by all vaccines tested are unique in their capacity to produce high amounts of interferon-gamma. These data show that the quality of a CD4+ T-cell cytokine response can be a crucial determinant in whether a vaccine is protective, and may provide a new and useful prospective immune correlate of protection for vaccines based on T-helper type 1 (TH1) cells.

1,227 citations


"A sand fly salivary protein vaccine..." refers background in this paper

  • ...The presence of polyfunctional T cells has been associated with effectiveness of the protective immune response in several vaccine candidates for diverse infectious diseases such as leishmaniasis (36), smallpox (37), hepatitis C virus (38), and tuberculosis (TB) (39, 40)....

    [...]


Related Papers (5)