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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 QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. 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 QuickTime™ and a MPEG-4 Video decompressor are needed to see this picture. QuickTime™ and a MPEG-4 Video decompressor are needed to see this picture. QuickTime™ and a MPEG-4 Video decompressor are needed to see this picture. 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