Molecular Approaches to Develop Therapies Against Exotic Pathogens

Fondation Mérieux: Healthy and unhealthy health markets

21st January 2015

Rob Harrison Alistair Reid Venom Research Unit Liverpool School of Tropical Medicine The global neglect of snakebite victims and their treatment: an overview

Rob Harrison Alistair Reid Venom Research Unit Liverpool School of Tropical Medicine Antivenom is an effective treatment

Elapids (cobras) Neurotoxic

Vipers Haemotoxic Antivenom is an effective treatment

Elapids (cobras) Neurotoxic

Vipers Haemotoxic 45 antivenom manufacturers globally

http://apps.who.int/bloodproducts/snakeantivenoms/database/

Sub-Saharan Africa Manufacturer in: Middle East/N Africa South Africa Europe Manufacturers in: Manufacturers in: Iran France Saudi Arabia Poland Israel UK Egypt Russia Tunisia India N America Serbia Algeria 5 manufacturers Manufacturers in: USA Mexico Asia Manufacturers in: Thailand C America Japan Manufacturers in: China Costa Rica Indonesia Phillipines Korea S America Myanmar Manufacturers in: Pakistan Brazil Colombia Argentina Australia Peru CSL Venezuela Uruguay Global Poverty Ecuador 45 antivenom manufacturers globally Yet snakebite mortality is high Snakebite Mortality

Global Poverty 45 antivenom manufacturers globally How many victims die from snakebite? Snakebite Mortality

Envenomings Deaths Europe 10,000 128 Middle East 3,000 78 USA 2,500 7 C & S America 129,000 2,300 Africa 420,000 32,000 Asia 2,000,000 57,500 Australasia 1,250 4

Global mortality from snakebite is estimated at 95,000 deaths per year Kasturiratne et al, PLoS Med, 2009 The need for more accurate snakebite disease-burden data

45,000 deaths pa

Envenomings Deaths Europe 10,000 128 Middle East 3,000 78 USA 2,500 7 C & S America 129,000 2,300 Africa 420,000 32,000 Asia 2,000,000 57,500 Australasia 1,250 4

Global mortality from snakebite is estimated at 95,000 deaths per year Kasturiratne et al, PLoS Med, 2009 The need for more accurate snakebite disease-burden data

45,000 deaths pa

Envenomings Deaths Europe 10,000 128 Recommendation: Middle East 3,000 78 Region-wide surveys of USA 2,500 7 • Incidence, Mortality, Morbidity C & S America 129,000 2,300 • Socioeconomic impact Africa 420,000 32,000 • Availability of effective AV Asia 2,000,000 57,500 Sponsored by Gov’ts & IHAs Australasia 1,250 4

Global mortality from snakebite is estimated at 95,000 deaths per year Kasturiratne et al, PLoS Med, 2009 45 antivenom manufacturers globally Who is dying from snakebite, and why? Snakebite Mortality

Envenomings Deaths % Europe 10,000 128 1.3 Middle East 3,000 78 2.6 USA 2,500 7 0.3 C & S America 129,000 2,300 1.7 Africa 420,000 32,000 7.6 Asia 2,000,000 57,500 4.8 Australasia 1,250 4 0.3 Total: 95,000 Most snakebite deaths occur in Asia and Africa Case fatality is highest in African snakebite victims Snakebite mortality and gov’t expenditure on health

Snakebite Mortality

Government Per Capita Expenditure on Health (US$) Snakebite Mortality (log) Mortality Snakebite

Those countries than can least afford antivenom have the highest snakebite deaths Harrison et al, PLoS NTD, 2009 Snakebite mortality: low quality of life and poverty

Snakebite Mortality

Human Development Index – an index of quality of life education literacy income life expectancy

Countries with lowest HDI ranking have highest snakebite mortality rates Harrison et al, PLoS NTD, 2009 Snakebite mortality: low quality of life and poverty

Snakebite Mortality

Recommendation: Human Development Index IHA sponsorship of antivenom: • that is effective – an index of quality of life • and affordable education literacy IHA support for regional AV income manufacture in Africa life expectancy

Countries with lowest HDI ranking have highest snakebite mortality rates Harrison et al, PLoS NTD, 2009 Snakebite – a disease of rural poverty causing high mortality and permanent disability

Snakebite Mortality

Snakebite is an another Neglected Tropical Disease (WHO, 20010) Harrison et al, PLoS NTD, 2009 Snakebite – a disease of rural poverty causing high mortality and permanent disability

In some areas of W Africa, snakebite is in the top 10 causes of hospital admission (N. Ghana) and over 70% beds (NE Nigeria) can be occupied by snakebite victims Snakebite – a disease of rural poverty causing high mortality and permanent disability

In some areas of W Africa, snakebite is in the top 10 causes of hospital admission (N. Ghana) and over 70% beds (NE Nigeria) can be occupied by snakebite victims.

This situation exacerbated by cessation of antivenom supply by European manufacturers in 2000 Antivenom is lifesaving – so why what caused antivenom supply crisis to Africa? Vipers Haemotoxic Partly because: • AV is expensive (>$200/treatment) • AV dose-efficacy is poor (2-20 vials) • AV causes adverse effects (50% patients) Elapids Neurotoxic • AV is ineffective against local effects Antivenom is lifesaving – so what caused cessation in antivenom supply to Africa? Vipers Haemotoxic Partly because: • AV is expensive (>$200/treatment) • AV dose-efficacy is poor (2-20 vials) • AV causes adverse effects (50% patients) Elapids Neurotoxic • AV is ineffective against local effects Also - compounding economic factors: • In Africa – AVs are manufactured only by commercial companies • AV is expensive to manufacture • Demand poor • Very poor commercial profits • AV Manufacture (Europe) ceased • AV Supply to W Africa near to nil The antivenom market failure in Africa resulted in a dangerous therapeutic vacuum

Antivenom manufactured with venom from Indian snakes was 1/10th price of FAV-Afrique but ineffective – increasing case fatality from 1.8% to 12.1% The African antivenom market failure can be reversed – with support from governments, international health agencies and pharmaceutical companies

Antivenom manufactured with venom from Indian snakes was 1/10th price of FAV-Afrique but ineffective – increasing case fatality from 1.8% to 12.1%

Recommendation: • Improve regional recognition of snakebite disease burden – advocacy

• Improve regional antivenom regulatory control • Establish regional pre-clinical testing labs • Establish regional clinical snakebite-treatment training centres

• Support pharmaceutical companies to develop effective & affordable antivenoms • Gov’t & IHA support for aggressive advocacy/marketing of effective, cheap antivenoms

• Establish funding for research to develop next-generation snakebite therapies The African antivenom market failure can be sustainably reversed – with research to develop next-generation snakebite therapies Vipers Haemotoxic Research priorities to improve antivenom: • Dose efficacy

• Cross-species clinical efficacy

Elapids Neurotoxic • Affordability

• Safety

AND • Develop treatment of venom-induced tissue destruction The African antivenom market failure can be sustainably reversed – with research to develop next-generation snakebite therapies Vipers Haemotoxic Research priorities to improve antivenom: • Dose efficacy

• Cross-species clinical efficacy

Elapids Neurotoxic • Affordability

• Safety

AND • Develop treatment of venom-induced tissue destruction

- Research to revise current antivenom manufacture protocols The African antivenom market failure can be sustainably reversed – with research to develop next-generation snakebite therapies Research priorities to improve antivenom: • Dose efficacy

• Cross-species clinical efficacy

• Affordability

• Safety

AND • Develop treatment of venom-induced tissue destruction

- Research to revise current antivenom manufacture protocols Research to resolve the African antivenom market failure

 Dose Efficacy of current antivenom

10 ml

IgG Antivenom

Treatment of victim with 1 vial of EchiTabG antivenom

Most vials of antivenom are 10 ml Research to resolve the African antivenom market failure

 Dose Efficacy of current antivenom is poor

10 ml

IgG Antivenom % anti-E.ocellatus IgG bound to venom column 1ml E. ocellatus venom 10.2% immobilised on sepharose IgG specific to venom proteins

Only 10% of antivenom IgG is specific to venom proteins – 90% is redundant Research to resolve the African antivenom market failure

 Dose Efficacy of current antivenom is very poor

10 ml

IgG Antivenom Many venom proteins are weakly or non-pathogenic 1ml 0.5 ml IgG specific to pathogenic toxins IgG specific to venom proteins

Only about 5% of antivenom IgG targets pathogenic venom proteins Research to resolve the African antivenom market failure

 Efficacy of current antivenom is restricted to the snake whose venom was used in its manufacture

10 ml

IgG Antivenom

1ml 0.5 ml IgG specific to venom proteins IgG specific to pathogenic toxins Antivenom efficacy is snake species and therefore geographically restricted Research to resolve the African antivenom market failure

The saw-scaled viper  The need for polyspecific antivenom in Africa

The puff adder The spitting cobra

The green mamba The Egyptian cobra

1ml 0.5 ml

The boomslang Research to resolve the African antivenom market failure

The spitting cobra

 Efficacy of current antivenom is further compromised by adding additional venoms to increase species cover because: The puff adder

10 ml The more venoms used in manufacture: – the more immunogens - the more distinct IgGs generated - the less IgG to each snake venom

- the more vials required for cure IgG - the greater the adverse effects - the greater the cost

Research needed to increase amount of Antivenom effective IgG in a vial of antivenom

0.5 ml

IgG specific to pathogenic toxins Antivenom efficacy is snake species and therefore geographically restricted Research to resolve the African antivenom market failure

Antivenom that is toxin-specific • Immunisation with few, rationally selected immunogens

Improve dose efficacy - reduce adverse effects - increase affordability Patient MoH Increase demand Increase commercial manufacturing incentives Improve delivery of antivenom to rural poor communities

We are pursuing a gene-based approach to developing toxin-specific antivenom Molecular construction of a toxin-specific snake antivenom for all African Echis saw-scaled vipers

1.00 Echis ocellatus E. ocellatus. E. jogeri 1.00 Echis pyramidum leakeyi E. pyramidum, E. leucogaster, E. khosatzkii 0.92 Echis coloratus E. coloratus and E. omanensis Casewell et al, PLoS NTD 2010 Molecular construction of a toxin-specific snake antivenom • Isolate all genes from snake venom glands Molecular construction of a toxin-specific snake antivenom • Isolate all genes from snake venom glands • Transcriptomics to define and distinguish toxins from non-toxic proteins

Construct an epitope-string – a synthetic protein/DNA sequence used for immunisation to generate toxin-specific antibodies Molecular construction of a toxin-specific snake antivenom • Neutralisation of venom-induced haemorrhage by anti-SVMP epitope string IgG

(PBS) (pVS) (MP)

MHD assay E ocellatus venom

(DC) (MP+DC) (Eo string)

Wagstaff et al, 2006 PLOS Medicine, e184 HIS-Tag SUMO-Tag MBP-Tag 100# 225# 100# 150# 75# 75# 100# 50# 75# 50# 35# 50# 35# 25# 35# 25# 25#

15#

15# 10# Serological responses15# of antivenom vs mice immunised with recombinant Echis toxin-specific epitope-strings: African viper venoms 10# 10#

A# B# C# D# E# F# G# H# I# A# B# C# D# E# F# G# H# I# >200A# B #venomC# D #proteinE# F# immunogensG# H# I# Pre-immune Mixed monospecific AEchiTAbGn venom antivenom - Echitab G

100# 225# A: E ocellatus 75# 150# 225# 100# 150# B: E p leakeyi 100# 50# C: E leucogaster 75# 75# D: E coloratus 35# E: E c sochureki 50# 50# F: C cerastes 25# G: B arietans (Ghana) 35# 35# H: B arietans (Zimbabwe) I: B gabonica (West Africa)

15# 7 mg/lane 15# 15# 10# 10# 10#

A# B# C# D# E# F# G# H# I# A# B# C# D# E# F# G# H# I# A# B# C# D# E# F# G# H# I# HIS-Tag SUMO-Tag MBP-Tag 100# 225# 100# 150# 75# 75# 100# 50# 75# 50# 35# 50# 35# 25# 35# 25# 25#

15#

15# 10# Serological responses15# of antivenom vs mice immunised with recombinant Echis toxin-specific epitope-strings: African viper venoms 10# 10#

A# B# C# D# E# F# G# H# I# 13A# epitopeB# C# D-string# E# immunogensF# G# H# I# >200A# B venom# C# D #proteinE# F# immunogensG# H# I# Pre-immune MMixedixed mtoxinono-sspecificpecific IgGs AEchiTAbGn venom antivenom - Echitab G

15# 7 mg/lane 15# 15# 10# 10# 10#

A# B# C# D# E# F# G# H# I# A# B# C# D# E# F# G# H# I# A# B# C# D# E# F# G# H# I# HIS-Tag SUMO-Tag MBP-Tag 100# 225# 100# 150# 75# 75# 100# 50# 75# 50# 35# 50# 35# 25# 35# 25# 25#

15#

15# 10# Serological responses15# of antivenom vs mice immunised with

10# recombinant Echis toxin-specific10# epitope-strings: African viper venoms

15# 7 mg/lane 15# 15# 10# 10# 10#

A# B# C# D# E# F# G# H# I# A# B# C# D# E# F# G# H# I# A# B# C# D# E# F# G# H# I# Next: - Expand this approach to all medically-important African snakes – continent wide efficacy - Convert to toxin-specific Mcabs - to increase dose efficacy by 90% and reduce costs Research to resolve the African antivenom market failure

Research priorities to improve antivenom: • Dose efficacy

• Cross-species clinical efficacy

• Affordability

• Safety

AND • Develop treatment of venom-induced tissue destruction – no medicinal treatment

• Permanent disability in ~100,000 victims pa • 8,000 amputations in sub-Saharan Africa Research to resolve the African antivenom market failure - by exploiting the unique VHH domain of heavy-chain only camelid IgG Heavy-chain only IgG

VH VH VHH VHH VL VL CH1 CH1 CL CL VHH V H 15 kDa H

CH2 CH2 CH2 CH2 CH2 CH2

CH3 CH3 CH3

150 kDa Conventional IgG1 IgG2 IgG3

The papain-cleaved 15 kDa VHH has same tissue distribution dynamics as venom toxins – therefore anti-venom VHH is a promising candidate to reduce venom-induced tissue necrosis. Research to resolve the African antivenom market failure - preclinical efficacy of anti-venom camelid IgG VHH

Neutralisation of venom induced: Camel IgG type: Lethality (µg) Haemorrhage (µg) Coagulation (µg) Total IgG 1250 600 200 IgG2 (HcIgG) ND 150 50 VHH 250 75 100 Horse

SAVP - F(ab')2 720 300 480

Heavy-chain only IgG

V V H H Heavy-chain only IgG VHH VHH VL VL VH V Heavy-chain only IgG H CH1 CH1 VHH VHH CL CL VL VL VHH VHH CH1 CH1 CL CL VHH VHH VH VH VHH VHH C 2 C 2 V V H H V L L H CH2 C 2 VC 2 C 2 H HH H CH1 CH1 CH2 CH2 C C C 2 V H VHH L L H CH2 CH2 CH2 H VHH VHH VL VL C 3 C 3 C 3 H C 1 H H CH3 CH3 CH3 H CH1 CH2 CH2 C 2 C 2 C C H H CH2 CH2 Conventional IgG L L 1 IgG2 IgG V H Conventional IgG 3 H VHH 1 IgG2 IgG3 CH3 CH3 CH3

Conventional IgG 1 IgG2 IgG3 CH2 CH2 CH2 CH2 CH2 CH2

CH3 CH3 CH3

Conventional IgG 1 IgG2 IgG3 Research to resolve the African antivenom market failure: feasible but requires more support from research-funding agencies

Vipers Haemotoxic Research priorities to improve antivenom: • Dose efficacy

• Cross-species clinical efficacy

Elapids Neurotoxic • Affordability

• Safety

AND • Develop treatment of venom-induced tissue destruction MRC, Wellcome Trust, Rob Harrison Leverhulme Trust, BBSRC, CVRL Alistair Reid Venom Research Unit Topic 1 Liverpool School of Tropical Medicine

Key Points: • Snakebite is an important, neglected disease of the rural poor in Africa and Asia • more accurate data is needed on mortality, morbidity and socioeconomic impact – raise awareness • need Gov’ts and IHAs to recognise and act to reduce snakebite deaths and morbidity

• Current antivenom therapy can be effective but requires urgent improvement • better regulatory control needed • need to establish regional training on clinical management of snakebite • need Gov’ts and IHAs to support development and delivery of effective, affordable antivenom

• Need for funding agencies to support science devising more effective, affordable snakebite therapy • Gov’ts and IHAs to liaise with Pharma to support delivery of these new effective, affordable therapies

References Photographs: Warrell, Wuster