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Genetically Modified Live Attenuated Parasites as Leishmania vaccines : Evaluation of Safety and Efficacy Hira Nakhasi, Ph.D. Director DETTD/OBRR/CBER/USFDA Bethesda, MD, USA July 13, 2006. Impact of Visceral Leishmaniasis. Drug Treatment unsatisfactory Painful, long (30 day) - PowerPoint PPT Presentation
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Genetically Modified Live Attenuated Parasites as Leishmania vaccines : Evaluation of Safety and Efficacy
Hira Nakhasi, Ph.D.Director
DETTD/OBRR/CBER/USFDABethesda, MD, USA
July 13, 2006
•350 million are at risk worldwide•1-2 million new cases each year•Estimated death toll of ~50,000/yr
•Post Kala-azar Dermal L.
•Emergence of Drug resistance
•Re-emergence w/ HIV•Drug Treatment unsatisfactory Painful, long (30 day) Expensive
Impact of Visceral Leishmaniasis
•T-T Transmission
•Mother to Child Transmission
Relevance to US Public HealthRelevance to US Public Health
• Millions of American travelers and thousands of US Millions of American travelers and thousands of US troops are visitors or deployed in malaria, Chagas troops are visitors or deployed in malaria, Chagas and and LeishmaniaLeishmania endemic areas (World is a global endemic areas (World is a global village)village)
• Increasing rates of immigration raises concern about Increasing rates of immigration raises concern about the potential for transmissionthe potential for transmission
• Known cases of Visceral Leishmania transmission Known cases of Visceral Leishmania transmission through transfusionthrough transfusion
• Significant number of potential donors are deferred Significant number of potential donors are deferred based on potential exposurebased on potential exposure
• No donor screening assays are availableNo donor screening assays are available• No vaccines are availableNo vaccines are available
Adapted from Paulo Pimenta
Life cycle of Leishmania
Promastigote
AmastigoteSandfly vector
Human host
Differentiation
Differentiation
Growth
Macrophage
Differentiation
Dwyer, D.M.
Dwyer, D.M.
Debrabant, A
.
Out come of previous studies on Out come of previous studies on LeishmaniaLeishmania vaccine development vaccine development
Various strategies of vaccine developmentVarious strategies of vaccine development• Whole cell lysates/enriched fractionsWhole cell lysates/enriched fractions• Attenuation through long term cultureAttenuation through long term culture• Chemical MutagenesisChemical Mutagenesis• Irradiated parasitesIrradiated parasites• Killed parasitesKilled parasites• Recombinant and Synthetic antigens + AdjuvantRecombinant and Synthetic antigens + Adjuvant• DNA vaccines and CpG ODNsDNA vaccines and CpG ODNs• All the above studies lead to the conclusion that All the above studies lead to the conclusion that
parasite persistenceparasite persistence may be required to maintain the may be required to maintain the immunological memory to prevent reinfectionimmunological memory to prevent reinfection
Could be achieved by live-attenuated parasites immunization Could be achieved by live-attenuated parasites immunization that could persist indefinitely w/o inducing diseasethat could persist indefinitely w/o inducing disease
Selvapandiyan, et. al. 2006, Ind. J. Med. Res. 123: 455-466
Specific AimsSpecific Aims
1.1. Develop Develop attenuated linesattenuated lines of of Leishmania Leishmania donovanidonovani by defined genetic alteration by defined genetic alteration
2.2. Assess the Assess the immunogenecityimmunogenecity of genetically of genetically defined parasitesdefined parasites
3.3. Identify Identify biomarkersbiomarkers of genetic stability to of genetic stability to monitor vaccine safety of live attenuated monitor vaccine safety of live attenuated parasitesparasites
Ld genome
Development of Development of L. donovaniL. donovani parasite with deletion of growth parasite with deletion of growth controlling centrin genecontrolling centrin gene
LdCen
Cen 3’UTRCen 5’UTR
Cen 3’UTRCen 5’UTR
BamHI KpnISalI SpeI
Neor
DoubleDouble KO KO
Selvapandiyan et. al. (2004) J. Biol. Chem. 279: 25702-10
Hygr
SummarySummary• The Leishmania centrin null
mutants show differential growth arrest
• Axenic amastigote centrin null mutants are growth arrested in the G2/M phase and multi-nucleated.
• Duplication of Basal bodies is affected in the axenic amastigotes
• Centrin KO amastigotes do not survive in the macrophages
• Leishmania centrin null mutants are potential attenuated vaccine candidates
Selvapandiyan et. al. JBC (2004) 276:43253-61
Hours postinfection
% I
nfe
cted
mac
rop
hag
esP
aras
ites
/in
fect
edm
acro
ph
age
∗
∗ **
-/-
LdCEN3+/+
120 hr
240 hr
Am
M M
+/+
LdCEN3-/-
LdCEN3+/+
ProLdCEN3-/-
ProLdCEN3-/-
AmLdCEN3+/+
Am
LdCEN3+/+
LdCEN3-/-
2 M
012345678
0 24 48 72 96 120
Ce l
ls X
107 /
ml
Time (hr)
012345678
0 24 48 72 96 120
● -/- ◆ +/+ ▲ +/-
AmastigotePromastigote
Immunogenecity and pathogenicity of centrin null mutants of L. donovani in BALB/c mice
WT KO
Saha et al., (unpublished data)
IL-4 IFN-g
KO
1. Reduced Survival of KO parasites2. Reduced Pathogenicity of KO parasites3. Infection with KO parasites resulted in a Th1 (IFN-g)
predominated antileishmanial T cell response
Identification of Biomarkers to monitor vaccine safety of live attenuated centrin
null mutant Leishmania•A microarray of 4224 clones printed in triplicate.
•Axenic Amastigote samples of Ldcen+/+ RNA and of Ldcen-/- RNA were used for hybridization to the microarray
•Differentially expressed genes were identified
A B C A B C A B C
Stat
Am
KO
Am
KO
AB A
m
1.1. Expression is significantly reduced in KO vs wild type parasiteExpression is significantly reduced in KO vs wild type parasite2.2. Expression is directly related to centrin modificationExpression is directly related to centrin modification3.3. Expression of Calpain-like cysteine peptidase in CentrinExpression of Calpain-like cysteine peptidase in Centrin
KO parasites in vaccinated animals can be used to monitor KO parasites in vaccinated animals can be used to monitor for its genetic stabilityfor its genetic stability
Expression Pattern of Calpain-like Cysteine Expression Pattern of Calpain-like Cysteine Peptidase as a BiomarkerPeptidase as a Biomarker
calpain-like cysteine peptidase
Conclusions and Future DirectionsConclusions and Future Directions • Centrin deleted parasites can be exploited in the
development of an attenuated Leishmania parasite• Disruption of centrin gene results in growth arrest in vitro and in
vivo• Reduced survival and pathogenicity in animal model• Induction of antileishmanial immune response
• Assess the immunogenecity of centrin deleted attenuated parasites in small and large animal models
• Characterize genes identified through microarray analysis as biomarkers to monitor the genetic stability of the centrin-deleted live attenuated vaccine.
• Develop additional genetically defined live attenuated L. donovani parasites to be tested as vaccine candidates
AcknowledgmentsAcknowledgmentsFDA UCSFAngamuthu Selvapandiyan CC WangRobert Duncan Parveen Kumar
Alain Debrabant National Center for CellGannavaram Sreenivas Science, Pune, IndiaNancy Lee Bhaskar Saha
ICMR, New Delhi, IndiaPoonam Salotra
CDRI, Lucknow, IndiaNeena Goyal
Mayo Clinic, Rochester, MinnesotaJeff Salisbury