Download Animal Models of Leishmaniasis Relevant to

Animal Models of Leishmaniasis Relevant to
Vaccine Development
David Sacks
Laboratory of Parasitic Diseases
NIAID, NIH
Leishmaniasis: A spectrum of diseases associated with a
diversity of parasite species, vectors, and reservoir hosts
zoonotic
cutaneous
leishmanaisis
Le. major
Le. amazonensis
Le. mexicana
Le. venezuelensis
Le. braziliensis
Le. panamensis
Le. peruviana
anthroponotic
mucocutaneous
cutaneous
leishmanaisis leishmanaisis
Le. tropica
Le. braziliensis
diffuse
zoonotic
cutaneous
visceral
leishmanaisis leishmanaisis
Le. amazonensis
Le. aethiopica
anthroponotic
visceral
leishmanaisis
Le. infantum
Le. chagasi
Le. donovani
Proven Sand fly Vector Species
P. (La.) longipes P. (La.) pedifer P. (Pa.) alexandri P. (E.) argentipes P. (S.) celiae P. (S.) martini
P.(La) orientalis P. (Pa.) alexandri P. (La.) ariasiP. (A.) brevis P. (La.) langeroni P. (La.) pernisciosus
P. (La.) perfiliewi P. (Ph.) duboscqi P. (Ph.) papatasi P. (La.) aculeatus P. (Pa.) sergenti P. (La.) kandelakii
Lu. (N.) flaviscutellata Lu. (Ps.) carrerrai Lu. (Ps.) complexa Lu. (N.) intermedia Lu. (Ps.) wellcomei Lu. (N.) whitmani
Lu. (Lu.) longipalpis Lu. (H.) hartmanni Lu. (N.) umbratilis Lu. (D.) anthophora Lu. (H.) ayacuchensis Lu. (C.) christophei
Lu. (Lu.) diabolica Lu. (N.) olmeca Lu. (V.) ovallesi Lu. (N.) trapidoi Lu. (N.) ylephiletor Lu. (H.) peruensis Lu. (V.) verrucarum
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Leishmania Life Cycle
Zoonotic Cutaneous Leishmaniasis
Caused by L. major
P. duboscqi
P. papatasi
C57Bl/6 mice inoculated in the ear dermis with a
low dose (100-1000) of L. major metacyclic
promastigotes develop self-healing lesions that
resemble the human disease
C57Bl/6 mice, ear
Human, arm
C57BL/6 Resistant Mouse model of Leishmania major Cutaneous Infection
Parasite Growth
Priming Phase
Parasite Killing
Effector Phase
Parasite Maintenance
Chronic Phase
Th1
IFN-
LESION
# PARASITES
2°
Weeks-Post Needle Infection
Healed primary infection “vaccinates”
against secondary infection
Effective Vaccination AgainstCutaneous Leishmaniasis
Using Live Challenge (Leishmanization)
Ref erence
No. Subjects
Primary Inf ection
Secondary Challenge
Sokolova (1940) 1000
Soviet Union
1000
Experimental L. major
Natural exposure
None
Natural Exposure
Naggan(1972)
Israel
Experimental L. major
None
100
400
Natural Exposure
Natural Exposure
Result
6%
64%
0%
30%
Chronic lesions associated with leishmanization
day 75
day 160
day 190
day 250
Defined subunit vaccines against L. major
In mice,
A whole cell, killed vaccine, Autoclaved Leishmania major (ALM)+CpG, provides strong
protection following needle challenge (Rhee et. al. JEM 2002; Darrah et al., Nat Med 2007).
In contrast
Autoclaved Leishmania major (ALM)+BCG used extensively in human trials does not
provide protection following natural exposure.
Efficacy of Killed Whole Parasite Vaccine – A Meta Analysis
Iran
Iran
Iran
Iran
Iran
Noazim et al, Vaccine J., 2009
Is there a problem with the mouse models?
• Variability in challenge systems; dose and stage of
parasite, site and route of inoculation, duration
between vaccination and challenge.
• Variability in definitions of protection; pathology,
parasitic load in inoculation site or sites of
dissemination, time points examined
• Variability in immune parameters examined; cytokine
profiles, frequencies of memory and effector subsets,
tissues examined
At the time the human trials were carried out,
no experimental Leishmania vaccine had ever been
evaluated under laboratory conditions
using infected sand fly challenge.
Possible differences between needle and
sand fly challenge:
• Dose and fidelity of metacyclic promastigotes
• Tissue damage associated with fly bites
• Sand flies co-inject SALIVA and midgut flora
with known inflammatory and immune
modulatory properties
Infected Sand Fly Challenge Model
Phlebotomus duboscqi
14 Days
L. major Amastigotes
+
Mouse Blood
P. duboscqi infected with RFP- L. major
Stable transfected line carrying the [pKS NEO DsRed] plasmid
Intravital visualization of peripheral inflammation
using Two-Photon Microscopy
femtosecond-pulsed IR
laser ~800-1060 nm
Galvos
PMT-based
detection
Dichroic
(700sp)
2nd harmonic / collagen
L. major dsRed
LysM-EGFPbright / neutrophils
LysM-EGFPdim/ monocytes/macs
z
(Faust et al., Blood, 2002)
focal plane
Exposed ear of an
anesthetized mouse
Maximum intensity projection images across X, Y, and Z dimensions derived from 2P-IVM
40 min post-bite
y
y
x
z
2nd harmonic / collagen
L. major dsRed
LysM-EGFP
z
x
pPeters et al., Science 2008
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y
x
z
x
y
z
Analysis of protective immunity generated by “Live” versus antigen vaccination
following needle or infected sand fly challenge
Needle Challenge (103 L.major)
Infected Sandfly Challenge
Chronic Phase 18 weeks
2° Site (ear)
Analysis
Live-Vaccinated (Live-V)
Antigen Vaccine (Ag-V)
104 L.major (s.c. footpad)
Autoclaved L. major (ALM)+CpG
Age Matched Control (AMC)
Non-living vaccines do not work as well as Leishmanization “live-vaccination”
Only healed
mice are protected against sand fly challenge
# L.m. /Each bitten ear
4 Weeks post needle inoculation or L.m.-Infected Sand Fly Bite
***
AMC ALM+CpG Healed
Needle Challenge
AMC ALM+CpG Healed
Infected Sand Fly Challenge
Peters et. al. PLoS Path. 2009
Clinical development of Leish-111f–MPL-SE
(monophosphoryl lipid A in a stable emulsion)
Prophylactic and Therapeutic trials in Columbia, Brazil, and India are
ongoing to evaluate safety and/or efficacy of the candidate vaccine.
Coler and Reed, Trends in Parasitology, 2005
Leish-110f–MPL-SE protects against needle but not
infected sand fly challenge
(21 wks post vaccination: 6 wks post challenge)
Sand Fly
Rapid recruitment of TH1 effectors to site of infected sand fly challenge
in live but not Ag- vaccinated mice
72 Hrs.
No Antigen
Antigen-Restimulation
0.7% (5)
Age Matched
Control
24 Hrs.
No Antigen
Antigen-Restimulation
0.7% (17)
0.7%
CD4
Ag-Vaccination
5.0% (123)
3.4%
Live-Vaccination
IFN
Rapid recruitment of multi-functional effector cells to the site of sand fly challenge
in mice with healed primary lesion (24 hr)
BMDC + L.m. Ag-restimulation CD3+CD4+ gated
L.m. Infected Sand Fly
AMC
Uninfected Sand Fly
AMC
Healed
Healed
2.1%
TNF-a
0.9%
1.0%
2.0%
IFN-
Rapidly-recruited, Tissue-seeking, Cytokine-producing CD4+ T Cells are the best immune correlate
for protection against cutaneous Leishmaniasis. These cells likely require Ag to be maintained,
and are UNLIKELY to be memory cells
Determining the Contribution of CD4 T Cell Subsets to Protective Immunity in the Skin
CD44 and CD62L define three CD4+ T cell subsets
Cm (9%)
CD44hi
CD62L
Naïve (33%)
IFN
CD44hi
CD62L
CD4+
CD4+
Effector
Central Memory
T Cell
T Cell
IL-7R
Eff (46%)
CD44
Recently Activated
In the Re-circulating Pool
Prior Activation
In Lymphoid Organs
Tissue
Homing
Division
in DLN (2d)
Immediate
IFN-
Tissue
Homing
Ag
IFN-
Why does ALM+CpG Ag-Vaccine protect against needle
but not sand fly challenge?
Recruitment of Th1 effectors to challenge sites is comparable
1 Week
638
309
77
53
Nature of Sand Fly
Transmission
Is the Neutrophil Response to Sand Fly Bite Different than Needle Inoculation?
2 Hours
Day 4-5
Day 8-12
Sand Fly
Sand Fly
Needle
Needle
y
x
z
GFPhi = Neutrophils
RFP = L. major
Neutrophil depletion promotes the efficacy of the ALM+CpG vaccine against sand fly
challenge
*** p<0.0001 vs. AMC(R14)
** p=0.0059 vs. ALM+CpG
Dermal cells in bite site 1d
(21349)
(25632)
F4/80
# L.m. / Each Bitten Ear
GL113 CD11b+ RB6-8C5 CD11b+
***
**
Summary
• Only mice with healed primary lesions (live vaccinated
mice) were significantly protected against infected
sand fly challenge.
• Rapidly-recruited, tissue-seeking, multifunctional
cytokine-producing CD4+ T cells are the best immune
correlates for protection.
• These cells likely require persistent Ag to be
maintained.
• The acute neutrophilic, wound healing response at the
site of vector transmission is immune modulatory
Conclusions
Our mouse models may be fine at predicting
vaccination success against L. major, so long as more
stringent evaluation criteria are used:
- Protection against ‘natural’ sand fly challenge
- Potency and durability of protection comparable to
naturally acquired resistance (healed infection)
- Immune correlates of protection comparable to
those associated with naturally acquired resistance
Acknowledgements
Nathan Peters
Flavia Ribeiro-Gomes
Charles Anderson
Susanne Nylen
Kim Beacht
Sand Flies
Phil Lawyer
Dia Elnaiem
Rachel Patrick
Lisa Stamper
Naglia Secundino
Shaden Kamhawi
Nikki Kimblin
IDRI
Steve Reed
Sylvie Bertholet