Production of potent polyvalent antivenom against

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Production of potent polyvalent antivenom against
elapid snakes of Thailand..A long winding road.
Kavi Ratanabanangkoon Laboratory of
Immunology Chulabhorn Research Institute
Global Issues in Clinical Toxicology 08, Melbourne
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ELAPIDS of THAILAND
Naja kaouthia (Thai cobra) Naja siamensis
(Splitting cobra) Bungarus fasciatus
(Yellow-banded krait) Bungarus candidus
(White-banded Krait) Ophiophagus hannah (King
cobra)
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Postsynaptic toxins - the cause of
death, by respiratory paralysis -
species diagnosis - difficult - LD50 20
ug/kg.? Presynaptic toxins? Membrane active
peptides (cardiotoxins, cytotoxins)? Enzymes
Elapids Cobra, King cobra, Krait
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0.2 0.1 0 0.2 0.1 0
3.0 2.0 1.0
0.10 0.05 0 0.04 0.02 0 4.0 2.0
0
0 50 100 150 200
250 300 350 400
450
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PROBLEMS WITH THERAPEUTIC ANTIVENOMS

• (1) DIFFICULT TO PRODUCE
• (2) LOW POTENCY
• (3) MONOVALENCY
• (4) IMPURITY

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PROBLEMS WITH THERAPEUTIC ANTIVENOMS

• (1) DIFFICULT TO PRODUCE
• - low responder horses
• - long immunization period
• - use large quantity of venom
• in the immunization
• high cost
• short supply

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PROBLEMS WITH THERAPEUTIC ANTIVENOMS

• (2) LOW POTENCY
• - Large volume of horse sera administered
• high of adverse reactions
• e.g.
  hypersensitivity
• high cost of treatment

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PROBLEMS WITH THERAPEUTIC ANTIVENOMS

• (3) MONOVALENCY
• - one AV for one venom
• - no significant cross-neutralization
• species diagnosis essential
• - diagnosis from signs and symptoms
  difficult or impossible
• lethal toxins are chemically
  pharmacologically similar but
  immunochemically distinct
• use of immunodiagnostic kit
• production of polyvalent AV

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PRODUCTION OF ANTIVENOM AGAINST N.kaouthia

• - Postsynaptic toxin 3, 71 a.a., MW 7821
• - 25-30 of venom protein
• - highest among terrestrial snakes
• - Irreversible binding to ?-subunits of nAchR
• - Toxicity limit immunization dose
• - Low MW low immunogenicity
• Only 1/6 horses responded to
  immunization

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AIMS OF RESEARCH PROJECT

• To improve the production of monovalent AV esp.
  
• AV against N. kaouthia (Thai cobra)
• responder horse
• potency of AV
• (2) To produce potent polyvalent AV's against
• Elapids (neurotoxic venoms)
• Viperids (hematotoxic venoms)
• To develop effective large scale fractionation
  of AV
• To develop in vitro assays for toxins and AVs

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STRATEGY
(1) Development of in vitro assays of AV
potency (2) Improvement of immunogenicities of
snake venoms (toxins) 2.1 use of purified toxins
(to avoid antigenic competition) 2.2 use of
various polymers and conjugates of toxins 2.3
use of potent adjuvants
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In vitro assay of AV potency - essential
for large scale experiments requiring
statistical analysis - to reduce the use
of live animals Rationale Since venoms
also contain non-toxic, immunogenic proteins
ELISA should quantitate only Ab against lethal
component(s)
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0.2 0.1 0 0.2 0.1 0
3.0 2.0 1.0
0.10 0.05 0 0.04 0.02 0 4.0 2.0
0
0 50 100 150 200
250 300 350 400
450
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ELISA Ag crude venom
Ag toxin fraction
Ag pure toxin 3
J. Rungsiwongse and K.Ratanabanangkoon J.
Immunol. Methods. 136 37-43 (1991)
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CHOICES OF IMMUNOGENS
Native venoms - Readily available -
'Antigenic competition' ... Approx. 100
proteins/venom - Elapid neurotoxin ... MW 7821
Low immunogenicity AV with low neutralizing
potency Purified toxins - A major hurdle for
some labs - Lethal component(s) not yet
identified AV with no Ab against less toxic
components Chemically modified toxins/venoms
- Treated with aldehydes toxoid / venoid -
Simple reaction but difficult to control /
reproduce AV with low neutralizing potency
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P. Sunthornandh, et. al. Molecular Immunol. 29
501-10 (1992).
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P. Sunthornandh, et. al. Molecular Immunol. 29
501-10 (1992).
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P. Sunthornandh, et. al. Molecular Immunol. 29
501-10 (1992).
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EFFECTS OF ADJUVANTS

• Toxin polymers/conjugates showed high Ab
  response
• when CFA was used in rats
• - With IFA, bentonite or squalene/Arlacel A,
  these
• polymers / conjugates gave low Ab response
• Therefore adjuvant was the determining factor

P.Sunthornandh and K.Ratanabanangkoon Toxicon,
32 561-71 (1994).
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USE OF CFA IN HORSES

• - Low dose, low volumn, multi-site injections
• (2 mg venom in 0.05 0.1 ml x 10-20 sites)
• Inflammation
• total surface area of dropletes
• Involvement of more immune cells in
• more extensive lymphatic systems

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Neutralization activities of pooled sera against
N. kaouthia from various groups of horses

R. Pratanaphon, et. al. Vaccine 15, 1523-8 (1997)
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Production of polyvalent AV against
elapids Anti-NK antibody
C. Chotwiwatthanakun, et. al. Toxicon, 39,
1487-1494 (2001)
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Median effective dose (ED50) of various
preparations of antivenoms
3LD50
C. Chotwiwatthanakun, et. al. Toxicon, 39,
1487-1494 (2001)
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Polyvalent antivenom against 3 vipers
Numbers in parentheses are 95 fiducial limits.
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Amount of snake venoms used in the production of
antivenoms
X2.597
X0.276
serum

S. Sriprapat, et. al. Toxicon 41, 57-64 (2003)
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Comparisons on the responses of horses immunized
under two different immunization procedures
Statistically significant (plt0.05)
S. Sriprapat, et. al. Toxicon 41, 57-64 (2003)
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ADVANTAGES OF THE LOW DOSE, LOW VOLUME,
MULTI-SITE IMMUNIZATION PROTOCOL

• Potent polyvalent / monovalent antivenoms
  produced
• No serious health problem on horses
• Venom economy
• No fractionation / detoxification of venom needed
• Implications on immunization / vaccination

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A LONG WINDING ROAD..
highly potent AV ? Thai cobra
low dose, low volume, multi-site CFA
highly immunogenic toxin polymers/conjugates
weak adjuvants
in vitro assays of AV potency
- highly toxic venoms - low MW toxin -
bentonite/AlPO4
antivenoms - low
potency - monovalency - low
responder horses - high venom cost - long
immunization period - highly impure
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RESEARCH TEAM

• Jarusporn Rungsiwongse Ronachai Pratanaphon
• Leera Kittigul Charoonroj Chotwiwattanakun
• Patcharee Sunthornandh Somporng Sapsuthipas
• Supod Sriprapat (QSMI) Rutai Raweerith
• Surasak Akesowan (QSMI) Pornchai
  Matangkasombut
• Orawan Khow (QSMI) Kavi Ratanabanangkoon

RESEARCH GRANTS
USAID Grant no. 936-5546-G-00-5079 STDB
Grant no. CPT 89-1-05-143.
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P. Sunthornandh, et. al. Molecular Immunol. 29
501-10 (1992).
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Immunization Regimens and Use of Adjuvants

• Preparation of venom immunogens
• Venom solution preparation
• Detoxification of venom
• Immunological adjuvants and
• preparation of immunogens

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Preparation of venom immunogensVenom solution
preparation

• - venom allergenic, hygroscopic
• - refrigerated venom condensation
• weighing error proteolytic digestion

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Preparation of venom immunogensVenom solution
preparation

• - venom solution foaming
• protein denaturation

• each venom solution should be prepared
  separately,
• not as a mixture

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PRODUCTION OF ANTIVENOMS AGAINST SNAKES
1985 Refinement of AV at QSMI 1955 P.
Christensen..Bentonite 1937 J.
Freund...Oil adjuvants 1926 A.T.
GlennyAlum as adjuvant 1924 G.
Ramon.."Adjuvants" discovered 1923 QSMI
established 1894 A. CalmetteAV against
N.kaouthia
QSMI Queen Saovabha Memorial Institute
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PRODUCTION OF THERAPEUTIC ANTIVENOMS
in vitro / in vivo assay of potency
Pepsin digestion
Plasmapheresis
Standardization
Plasma serum
Therapeutic antivenom
Animal
Blood
F(ab')2

• horse
• sheep
• hen

• Fractionation
• (NH4)2S04 precipitation
• caprylic acid precipitation
• affinity chromatography
• - ion-exchange, etc.

• Immunogen
• native venom
• purified toxins
• - chemically
• modified toxins
• (toxoid, venoid)

• Adjuvants
• Bentonite
• Al(OH)3
• AIP04
• Freund's

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Schematic drawing of rabbit shaved of fur on its
back and proximal limbs. The black dots over the
shaved area represent intradermal injection sites
of 30-50 ul
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C. Chotwiwatthanakun, R. Pratanaphon, S.
Akesowan, S. Sriprapat and K.Ratanabanangkoon
Production of potent polyvalent antivenom
against three elapid venoms using a low dose, low
volume, multi-site immunization protocol.
Toxicon, 39, 1487-1494 (2001).
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C. Chotwiwatthanakun, R. Pratanaphon, S.
Akesowan, S. Sriprapat and K.Ratanabanangkoon
Production of potent polyvalent antivenom
against three elapid venoms using a low dose, low
volume, multi-site immunization protocol.
Toxicon, 39, 1487-1494 (2001).
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NH4OAC conc. (M)
OD 280
Fraction number