PARATRANSGENIC STRATEGIES FOR CONTROL OF VECTOR-BORNE

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PARATRANSGENIC STRATEGIES FOR CONTROL OF
VECTOR-BORNE DISEASES

• Ravi V. Durvasula, M.D.
• Chief of Medicine, NM VA Health Care System
• Director, Center for Global Health,
• Dept of Internal Medicine
• UNM School of Medicine

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AIMS

• Provide overview of global impact of vector-borne
  diseases
• Provide overview of Chagas disease
• Discuss paratransgenic approach to Chagas disease
• Provide overview of visceral leishmaniasis
• Discuss paratransgenic approach to visceral
  leishmaniasis in India

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CONTROL OF VECTOR-BORNE DISEASES

• Vector-borne diseases remain a leading cause of
  human illness
• Over 60 billion loss annually to agriculture
• Impact on commercial livestock
• Lack of effective vaccines
• Vector eradication programs mainstay of control
• Pesticides effective over short term
• Issues of cost, environmental toxicity, adverse
  health effects and resistance

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Triatomine Vectors

• Reduviid bug aka Kissing Bug, Assassin Bug or
  La Chincha
• Multiple genera Rhodnius, Triatoma,
  Panstrongylus
• Obligate blood-feeders
• Humans are innocent bystanders in the cycle

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Reduviid Bug Habitats

• Sylvatic and peri-domestic reservoirs
• Thatch roofing and cracks of adobe walls
• Up to 10,000 bugs per house
• Highly sequestered colonies

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Transmission of T. cruzi

• Nocturnal
• Kissing bug attracted to warmth and CO2
• 30 minutes to repletion
• Release of fecal/urine droplet at end of blood
  meal
• Droplet laden with metacyclic trypomastigotes
• Entry of parasites at site of wound or mucous
  membranes

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Trypanosoma cruzi

• Flagellate protozoan
• Ubiquitous zoonotic agent
• Silent reservoir in animals
• Undergoes maturation in arthropod vector

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Acute Chagas Disease

• Regional edema, Romanas Sign
• Fever, flu-like illness
• Acute myocarditis, hepatosplenomegaly, lymph node
  enlargement
• Self-limited illness in 70-80 of cases
  indeterminate phase
• Low level of awareness

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Chagas Heart Disease

• Chronic Chagasic Cardiopathy (CCC)
• Most devastating effect of Chagas disease
• Progressive dilated cardiomyopathy
• Arrhythmias, heart blocks
• Leading cause of heart disease south of US border
• May occur years to decades after initial infection

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Gastrointestinal Manifestations of Chagas Disease

• GI tract involvement in 8-10 of infected
  individuals
• Syndromes of dysmotility megaesophagus and
  megacolon
• Dysphagia most common presentation
• Radiographic and manometric studies similar to
  idiopathic achalasia
• Megacolon often less symptomatic
• Chronic constipation

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AIDS and Chagas Disease

• Reactivation possible when CD4 count lt 200
• CNS mass lesion or acute diffuse
  meningoencephalitis
• May be confused with CNS toxoplasmosis
• Necrotizing hemorrhagic encephalitis,
  obliterative angiitis
• Amastigote forms detectable on biopsy

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TREATMENT OPTIONS

• Nifurtimox and Benznidazole
• Nifurtimox has 70 cure rates for acute Chagas
  disease less than 50 cure for Chronic Chagas
• Benznidazole shows similar efficacy
• Drugs taken for 60-120 days
• High rate of side-effects severe GI and neuro
  side-effects with Nifurtimox granulocytopenia,
  rash and neuropathies with Benznidazole
• WHO recommends treatment course for infected
  patients, regardless of stage
• No effective vaccine yet

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Control of Chagas Disease

• Vector eradication
• Blood-bank screening
• 3 highly successful programs Southern Cone,
  Central American and Andean Pact Initiatives
• Dramatic reductions in new cases
• Vector resistance and sustainability are issues

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Prospects for Chagas Control

• Insecticide use remains the mainstay
• Many success stories such as Chile
• Countries such as Bolivia and Guatemala still
  with high prevalence
• Recent resurgence of disease in Argentina

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Paratransgenesis Using Gut Symbionts of
Triatomines

• Triatomines harbor bacterial symbionts that have
  nutritional role
• The symbionts can be genetically transformed to
  express a gene product that interferes with T.
  cruzi transmission
• Insect symbionts can be replaced with genetically
  modified symbionts

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Paratransgenesis
A Novel Approach to Preventing Insect-Borne
Disease. New England Journal of Medicine. Conte
JE Jr. Sept 1997 337(11) 785-6.
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PARATRANSGENIC EXPRESSION IN R. PROLIXUS

• shuttle plasmid pRrThioCec with cDNA for
  L-cecropin A
• clearance of T. cruzi in 65 of experimental
  group (n100) and log 2-3 reduction in parasites
  in 35 (Durvasula et al. PNAS 1997)
• shuttle plasmid pRrMDWK6 with gene encoding rDB3
• Expression of functional VH-Kappa in R. prolixus
  (Durvasula et al. Med. Vet . Entomology 1998 )
• Shuttle plasmid pBAP5 with L1 mycobacteriophage
  integrase (Dotson et al. Inf Gen Imm 2003)
• Rhodococcal expression plasmids with genes
  encoding AMPs
• (Fieck et al 2009)

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T.cruzi coated with mAb WIC29.26

• Glycan epitope of gp72
• Related glycans of T. cruzi surface
• Targets for single chain antibodies expressed via
  recombinant R. rhodnii

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Red Fluorescent Protein- sFv

• Engineered single chain antibody
• VH-RFP-VL structure
• Recognizes sialic acid residues
• Photoactivation by white light

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Surface of T.cruzi

• Arthrobacter-derived lyticase
• Alpha mannosidase
• Genes encoding endoglucanases expressed via R.
  rhodnii

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Activity of recombinant lyticase against
Trypanosoma cruzi

• cDNA encoding lyticase of Arthrobacter cloned
  into Rhodococcal-E. coli shuttle plasmid
• Extracts of E. coli transformed to express
  lyticase
• Marked decline in OD 600nm of T. cruzi culture
  exposed to cell extracts
• Lyticase-treated T. cruzi failed to propagate in
  fresh LIT
• Transformation of R. rhodnii underway

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Table 1 Minimal Inhibitory and Bactericidal
Peptide Concentration Determinations 24
Hour Single AMP Treatment All
samples plated in triplicate
Values averaged from three independent trials
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SYMBIONT SPREAD
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Greenhouse Study of Transgenic Insects
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Greenhouse Study Summary
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Potential Modes of Environmental Spread of GM
Bacteria

• Transfer of GM bacteria to non-target arthropods
• Ants, cockroaches, or flies ingesting GM bacteria
• Horizontal Gene Transfer (HGT) amongst compatible
  host bacteria

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A Paratransgenic Approach to Visceral
Leishmaniasis

• Heidi Hillesland
• University of New Mexico School of Medicine
• Howard Hughes Medical Fellow
• Mentors Ravi Durvasula, M.D. and Ivy Hurwitz,
  Ph.D.

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Visceral Leishmaniasis (VL) Endemic Regions
Visceral leishmaniasis what are the needs for
diagnosis, treatment and control? François
Chappuis, Shyam Sundar, Asrat Hailu, Hashim
Ghalib, Suman Rijal, Rosanna W. Peeling, Jorge
Alvar Marleen Boelaert. Nature Reviews
Microbiology 5, S7-S16 (November 2007)
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40 species of Phlebotomus Old world
30 species of Lutzomyia- Americas
Lutzomyia longipalpis Lutzomyia migonei Lutzomyia
ovalesi Lutzomyia verrucarum Lutzomyia peruensis
Phlebotomus argentipes Phlebotomus
papatasi Phlebotomus ariasi Phlebotomus duboscqi
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VL Clinical Manifestations

• Incubation period varying from weeks to months to
  years
• Prolonged fever
• Cachexia
• Hepatosplenomegaly with predominance of
  splenomegaly
• Pancytopenia
• Hypergammaglobulinemia , mostly IgG
• Hypoalbuminemia
• Death most commonly due to secondary infection

www.sfgate.com/.../a/2002/08/19/MN33767.DTLo1
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VL in Bihar, India
National Vector Borne Disease Control Programme,
22, Shamnath Marg, Delhi - 110054.
www.nvbdcp.gov.in/kal9.html
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Life cycle of sandfly
Larvae-4 instar
Egg
Pupae
Adult fly
34-70 days, 28ºC

• Terrestrial lifecycle
• Breeding sites characterized at periphery of
• cattle sheds in Bihar for P. argentipes
• Moist microhabitats-rock crevices, animal
• burrows, termite mounds, cavities of tree,
• domestic animal shelters, organic debris

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Paratransgenesis

• Transform commensal bacteria to express
  anti-leishmania compound

4. Larvae undergo pupation

• Introduce transformed commensal bacteria to soil
  breeding sites

5. Sandfly emerges with transformed bacteria
within its gut

• Larvae feed on soil containing transformed
  bacteria

• Blocked parasite development in adult sandfly

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P. argentipes Samples From Four Regions in Bihar
Identification of Aerobic Gut Bacteria from the
Kala Azar Vector Phlebotomus argentipes A
Platform for Potential Paratransgeneic
Manipulation of Sand flies. American Journal of
Tropical Medicine and Hygiene. Hillesland H,
Read A, Subhadra B, Hurwitz I, McKelvey R, Ghosh
K, Durvasula R, Das P. 2008 76 (6) 881-6.
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P. argentipes Gut Bacteria Isolates
Candidates for paratransgenesis non-pathogenic
soil bacteria that are amenable to transformation
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Transformation of Candidate Bacteria B.
megaterium, B. subtilis, and B. pumilus
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Development of Paratransgenic Sand Fly
No bacteria added
Bacillus subtilis
Bacillus subtilis pAD43-25 GFP
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Development of Paratransgenic Sand Fly
Bright field and fluorescent microscopy of gut
dissection

• and b. Bacillus subtilis fly at 10x
• c. and d. Bacillus subtilis pAD43-25 at 10x

a
b
c
d
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Development of Paratransgenic Sand Fly
Fluorescent microscopy of gut dissection Bacillus
subtilis pAD43-25 fly at 40x
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NEXT STEPS

• Evaluation of antimicrobial peptide activity
  against strains of L. donovani
• Cloning of AMP-encoding genes into Bacillus
  expression plasmids
• Testing of RFP activity clone VH-RFP-VL encoding
  gene
• Potential killing of sandflies with light
  exposure

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THE FUTURE

• Laying down blankets of insecticides over cities
  does not appear to have a bright future. And only
  50 years after penicillin seemed a cure-all, the
  efficacy of nearly all of the roughly 150
  antibiotics in the doctors satchel is waning. If
  the arsenal for disease prevention continues
  emptying, the decision will become more focused
  how do we weigh the fear of knowingly disrupting
  our own natural habitat with transgenics versus
  the fear of taking no action in face of a new
  epidemic? Jack Hitt, The New York Times
  Magazine May 6, 2001

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ACKNOWLEDGEMENTS

• NIH/NIAID
• HHMI
• USDA
• Burroughs Wellcome Foundation
• The Yale/ UNM Team
• Dr. Ranjini Sundaram
• Dr. Sahar Usmani
• Dr. D. V. Subba Rao
• Scott Matthews
• Bobban Subhadra
• Dr. Ivy Hurwitz
• Annabeth Fieck
• Heidi Hillesland
• Sarah Weiss
• Dr. V.Sree Hari Rao
• Dr. Gabriel Lopez
• Dr. Amber Read
• Dr. Robin McKelvey

• MERTU/G , UDV, Guatemala
• Celia Cordon-Rosales
• Dr. Pamela Pennington
• Walter Reed Army Institute of Research, Bethesda
• Dr. Ed Rowton
• Dr. Kashinath Ghosh
• APTIV Inc., Portland, Oregon
• Philipp Kirch
• Dr. John McLaughlin
• Univ. Buenos Aires, Argentina
• Dr. Ricardo Gurtler