Insights from experimentally infecting humans with hookworm

Emeritus Professor Rick SpeareSchool of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University

&Director, Tropical Health Solutions Townsville, Queensland, Australia

17 October 2012

Presentation to Medical Research Council (Gambia)Available at: http

//www.tropicalhealthsolutions.com/hookworm

Orientation - map

Townsville

Anton Breinl Centre for Public Health and Tropical

Medicine

Dr John Croese & Castle Hill

Hygiene Hypothesis: STH style

• Although soil transmitted helminths (STH) cause significant morbidity in developing countries, the lack of STH in developed countries is hypothesised to increase the incidence of allergic and autoimmune disease.

• STH will induce a Th2 response against the parasite and this will be down-regulated by regulatory T cells which will also non-specifically down-regulate the destructive immune response (Th1) of the host that is responsible for allergic and autoimmune diseases.

Relationship between prevalence of multiple sclerosis and STH

Fleming & Cook. Neurology 2006;67:2085

• Trichuris trichiura as a marker of STH & low levels of community sanitation

• Prevalence of MS & Tt in 35 countries

• Correlational study

Multiple Sclerosis vs Whipworm

• Prevalence of MS falls steeply once threshold of 10% Tt is reached

• This applied for marginalised populations sharing same country (Jews – Arabs in Israel)

Fleming & Cook. Neurology 2006;67:2085

10%Is the protective effect

due to Tt or gut exposure to

pathogens?

Some worms are protective; some aren’t: Care is needed!

• Meta-analysis of asthma and association with STH

• STH in general no significant effect

• Ascaris is associated with asthma

• Hookworm is protective

Leonardi-Bee et al Am J Resp Crit Care Med 2006;174:514

Effect of anthelmintics on allergic reactions

• Van den Biggelaar et al J Inf Dis 2004;189:892

Skin reactivity to dust mite antigen increased after STH controlled

• Flohr et al. J Aller Clin Immunol 2006;118:1305

How do parasites exert an effect?

• Brown & Jackson Para Immunol 2004;26:429

Immune response

Trichuris suis for IBD

• Whipworm eggs collected from worms in experimentally infected pigs

• Incubated, made aseptic• 2500 eggs orally every 3 weeks

• All open trials• Significant percent of

remissions for both Crohn’s Disease and Ulcerative Colitis

• No significant side effects

Summers et al. Am J Gastrolenterol 2003;98:2034

Summers et al. Gut 2005;54:87

80% of patients benefited

Necator americanus

Life cycle

• Skin penetration• Blood-lung

migration• Occurs within

48 hrs PI• Juvenile worms

arrive in sm int in 4 weeks PI

• Adults mature at 7-8 weeks PI

N. americanus in gut

• Attaches to mucosa of small intestine

• Sucks blood• Little known about

behaviour of NA in human gut since can not be directly observed

Infective larvae (L3i)

• 25°C• In dark• 7 days

Harada-Mori Technique

12 hr post-infection

1 day post-infection

2 days post-infection

Infection worked!

• We inoculated 9 immunosuppressed Crohn’s disease patients and 3 normal donors with 15 to 101 L3i

• All 9 Crohn’s patients and 2/3 donors developed a patent infection

Insight: Imunosuppressed and normal hosts can be infected with Necator with no

or minimal negative effects.

What did the hosts think?

• Human hosts loved their worms!

Insight: Finding subjects to be inoculated with hookworms is not difficult.

Some problems!

• Penetration site

• Small intestine

2 days post-infection

4 days post-infection

6 days post-infection

7 days post-infection

8 days post-infection

9 days post-infection

10 days post-infection

11 days post-infection

12 days post-infection

15 days post-infection

20 days post-infection

Severe local response on reinfection

Insight: Penetration of L3i is uneventful in most, worrying in some

Why the severe local reaction?

L3i are “dirty”

• L3i are heavily contaminated with faecal bacteria.

• Standard techniques of multiple washing do not reduce the contamination.

• Is the hookworm sheath left on skin or does it remain in epidermis?

• Washing in providone iodine for 15 min was added after harvesting.

• Could L3i be made sterile?

Better Quality L3i

• Melissa Logan – BMedLabSc honours thesis

Bacteria are associated with L3i

In vitro tests

In vivo tests: penetration• 15 L3i; 3 groups – 5 subjects per group

Patency of infection• 100% patency for both techniques

Antibiotic treated L3i were equivalent to standard L3i

Conclusion

• Treatment of L3i with bleach and antibiotics gives viable larvae of comparable potency.

• These L3i are sterile.• Bleach and antibiotic technique should be the

gold standard for L3i for experimental infection.

Recommended Protocol

• Harvest L3i and centrifuge• Place in 0.25% sodium hypochlorite for 10 mins• Wash twice using centrifugation in sterile distilled

water to remove any residual sodium hypochlorite• Place in a solution of benzylepenicillin (180 mg/L) and

ceftazadime (1.0 mg/L) for 60 mins• Centrifuge to remove excess solution• Wash L3i twice using centrifugation in sterile distilled

water to remove any residual antibiotic solution.

Are skin reactions less?

Safety when working with L3i

• For any item in the laboratory a Material Safety Data Sheet (MSDS) is needed.

• MSDS important when L3i of N. americanus are being used in laboratory.

• The MSDS for hookworm said kill spills of L3i with bleach (Health Canada 2001).

• Did this actually work?

What kills L3i rapidly?

• Bleach did not kill L3i• Ethanol 70-100% did• Dettol killed L3i

Insight: Current MSDS was inaccurate! A new one based on

evidence was needed.

New Hookworm MSDS• Speare et al. Australian Journal of Medical Science

2008;29:91-96.

• Online at http://www.tropicalhealthsolutions.com/MSDSNecatoramericanus

Abdominal pain

• Hosts developed central abdominal pain about 3 weeks post-inoculation

Healthy Volunteers:

• 1 of 3: developed vigorous local reaction• 3 of 3: Central, non-colicky abdominal pain

started about 3.5 weeks and persisted for about 5 weeks

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Abdominal pain - Post Infection with Necator americanus

0 = none; 1 = mild; 2 = moderate; 3 = severe

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Reinfection

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Abdominal pain - Post Re-Infection with 50 L3i Necator amer-icanus on day 502

0 = none; 1 = mild; 2 = moderate; 3 = severe

Day Post-Infection

What’s happening?

• Allergic reaction develops at the attachment site of the worm.

• Local eosinophilic response.• Each hookworm secretes chemicals to

suppress this reaction.• If this is unsuccessful, the worm lets go and is

carried down the gut.• It attaches to a new site.

Red spots / white spots

Look at the hookworms with capsule endoscopy

• In the past worms in the small intestine could not be studied since they were not accessible to gastroscope or colonoscope

• Now we have capsule endoscopy (CE)• CE was used in this study to count worms,

determine where they were attached, and the host reaction to feeding

The capsule: camera in a pill!

The camera

• Takes 2 images per second

• Transmits these to sensors on abdomen

• Stored on computer• Images read like a

video

Data capture

Data capture

Worms in situ

Male and female

Red spots / white spots

Distribution along small intestine

Yellow bar = NA; Red = red spot; White = white spot

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Transit

RD2: CE #5

Value of capsule endoscopy

• Numbers of hookworm can be counted.• Their relative position along small intestine

can be plotted.• Host response to feeding can be quantified.

Insight: We now have a tool to study hookworms in the small intestine.

What happens when a person is reinfected?

• Two controls reinfected with 50 L3i.• One had originally received 31 L3i and had 6

hookworms in small intestine.• Another had originally received 101 L3i and

had 16 hookworms in small intestine.• Reinfection at 400 and 500 days post-

inoculation.

Rejection started when Necator arrived in small intestine

Croese & Speare. Trends in Parasitology 2006;22(12):547-550.

• Immature worms rejected in small intestine• Each individual host seems to have a “pre-set”

number of worms that will be allowedCroese et al. Gastroenterology 2006;131(2):402-409.

Insight: Repeat infections will be safe unless the host has a high level of

permissiveness.

Can hookworms be used to treat Crohn’s Disease?

• Crohn’s Disease (CD) is an autoimmune disease of the small intestine.

• Severe inflammation, usually segmental.• Typically starts in young adults.• Requires immunosuppressive agents to

control.

Methods

• Single unblinded trial• 9 Crohn’s disease (CD) patients

– 25 L3i of NA for 3; 50 L3i for 5, 100 L3i for 1• 3 healthy volunteers

– 30, 50, 100 L3i • Monitored by Crohn’s Disease Activity Index

(CDAI), Inflammatory Disease Bowel Questionnaire, FBC, clinical biochemistry

• CD patients managed individually by gastroenterologist

Inflammatory Bowel Disease Questionnaire scores

• The IBDQ scores were increased after 20 weeks compared to the scores at the outset (week-20 mean 179, CI 20, vs week-1 mean 151, CI 14: P=0.0544).

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Time from inoculation

IBD

QRemission:IDBQ>170

Crohn’s Disease Activity Index

• The initial CDAI score of each Crohn’s subject vs the scores at week-20 and at week-45 (mean 165, CI 145 vs 64, CI 25, P=0.132; mean 165 vs 75, CI 29; P=0.246).

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• The 4-week CDAI mean for the all subjects () at the end of the study was lower compared to lead-in score (week 17-20 mean 87, CI 15 vs week 1-4 mean 141, CI 31; P=0.001);

• This improvement reflected 4 of the 5 subjects with active disease at the outset () achieving a remission (CDAI>150; mean 112, CI 21 vs 211, CI 33; P=0.0001).

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Conclusions

• Crohn’s disease subjects, including those on immunosuppressive drugs, acquired a patent N. americanus infection and appeared to benefit as a result.

• Patients with more severe disease benefited the most.

• Reinoculation did not appear to impact negatively on CD subjects.

• These results justify progression to more sophisticated clinical trials.

Hookworm to treat Crohn’s disease?

Insight: Crohn’s disease patients improved, particularly those with more severe

disease, but additional research is needed to clarify role of hookworms in therapy.

How do hookworms modify host immune responses?

• Brown & Jackson Para Immunol 2004;26:429

Immune response

• Since CD patients were immunosuppressed, we were unable to study effect of N. americanus on immune response.

• Needed people with an autoimmune problem, but not on immunosuppressives.

• Inoculated people with coeliac disease (gluten autoimmunity).

• Test group (received Necator); control group (sham)

Daveson et al. PLoS One 2011;6(3):e17366

Results• All 10 people in test group became infected• Abdominal pain increased wk 3; baseline wk 16• Hookworm group less lethargic

Results (cont)

• Gluten challenge (5 days) – hookworm did not protect.

• Marsh score deteriorated in both groups.• Trend to be less in hookworm group, but not

significant.• Specific markers of systemic immune response

to gluten (EPLISpot) increased significantly after challenge only in control group.

Necator for Treating Coeliac Disease?

Insight: Necator did not protect coeliac disease patients against severe gluten

challenge.However, there was a subjective

impression that Necator “dulled” response to small amounts of gluten.

• Hookworm induced a specific Th2 response

Gaze et al PLoS Pathogens 2012;8(2):e1002520

Systemic immune response

Response in duodenum

• IL-5 increased adjacent to hookworm attachment site

• Plus other changes (read paper – too complex for me!)

Gaze et al PLoS Pathogens 2012;8(2):e1002520

Conclusions

• HW infection induces a systemic, Necator antigen-specific, Th2 response, and a mucosal Th2 response.

• Evidence also of a systemic, but not mucosal HW-specific Th1 response.

• HW dramatically suppressed pro-inflammatory cytokine production (IL-23) in duodenum and upregulated anti-inflammatory and wound healing cytokines (eg, IL-22).

Insights: This is a valuable experimental model for

studying the human immunological response in the gut to hookworm.

Hookworms secrete chemicals with powerful anti-inflammatory effects.

Pharmaceuticals versus Worms?

• Hookworms secrete potent chemicals that have promise for treating human inflammatory diseases.

• Are these best isolated and sold as pills? or• Infect people with hookworms and let the worms

do the immune regulation?• More profit in the former approach, and it may be

more effective and controllable.• Latter approach is simple and cheaper, but

probably less effective.• More evidence needed; so jury is out!

NA

juice

Publications• Croese J, O’Neil J, Masson J, Cooke S, Melrose W, Pritchard D, Speare R. A proof of concept study

establishing Necator americanus in Crohn’s patients and reservoir donors. Gut 2006;55:136-137.• Croese J, Speare R, Wood M, Melrose W. Allergy controls the population density of Necator americanus in

the small intestine. Gastroenterology 2006;131(2):402-409.• Croese J, Speare R. Intestinal allergy expels hookworms: seeing is believing. Trends in Parasitology

2006;22(12):547-550.• Daveson AJ, Jones DM, Gaze S, McSorley H, Clouston A, Pascoe A, Cooke S, Speare R, Macdonald GA,

Anderson R, McCarthy JS, Loukas A, Croese J. Effect of hookworm infection on wheat challenge in celiac disease - a randomised double-blinded placebo controlled trial. PLoS One 2011;6(3):e17366.

• Gaze S, McSorley HJ, Daveson J, Jones D, Bethony JM, Oliviera LM, Speare R, Mccarthy JS, Engwerda CR, Croese J, Loukas A. Characterising the mucosal and systemic immune responses to experimental human hookworm infection. PLoS pathogens 2012;8(2):e1002520.

• Logan M. Methods in improving the quality of Nector americanus larvae for use in therapeutic applications. BMedLabSc honours thesis, James Cook University. 2009.

• McSorley HJ, Gaze S, Daveson J, Jones D, Anderson RP, Clouston A, Ruyssers NE, Speare R, McCarthy JS, Engwerda CR, Croese J, Loukas A. Suppression of inflammatory immune responses in celiac disease by experimental hookworm infection. PLoS One 2011;6(9):e24092.

• Speare R, Melrose W, Cooke S, Croese J. Techniques to kill infective larvae of human hookworm, Necator americanus, in the laboratory and a new Material Safety Data Sheet. Australian Journal of Medical Science 2008;29:91-96.

Source of Images(if not original)

• N. americanus en face view: http://www.columbia.edu/itc/hs/medical/pathophys/parasitology/2004/lecture02Slides_files/slide0005.html

• Hookworm life cycle: CDC Diagnostic Parasitology web site• Trichuris and hookworm eggs: CDC Diagnostic Parasitology web site