Noroviruses

Duncan Steele

Bill & Melinda Gates Foundation

Acknowledgements:

Ben Lopman and Umesh Parashar, CDC

Megan Carey and Julia Bosch, BMGF

1

Global norovirus burden

• Globally, norovirus is associated with 18% (95% CI: 17-20%) of diarrhoeal disease

• Estimated to cause approximately:

– 1 billion episodes annually

– 200,000 deaths annually, about >70,000 of which are among children in developing countries.

• Disease occurs across the age range in all settings, but incidence is highest in young children.

• Noroviruses are transmitted by multiple routes

– Person-to-person spread predominates

– Foodborne transmission is estimated to account for approximately 15% of disease

2

0

1

2

3

4

5

1980 1985 1990 1995 2000 2005 2010 2015

877,000 25%

1980

440,000 22%

1994

527,000 29%

2004

453,000 37%

2008

ROTAVIRUS DEATH ESTIMATES

90,000 13%

2010

NOROVIRUS DEATH ESTIMATES

212,000 12%

2004

71,000 9.9%

2011

197,000 28%

2010

173,000 26%

2011

To

tal

dia

rrh

ea d

eath

s (M

illi

on

s)

Mid-point of Study Period

Deaths % of diarrheal deaths

Year

Deaths % of diarrheal deaths

Year

3

Estimates of norovirus mortality

Norovirus detection is common amongst

healthy controls

1) True asymptomatic infection

2) Shedding of virus in stool continues long after the resolution of symptoms

3) Ingested virus may even transit the gut without replicating

• For this combination of reasons, norovirus can frequently be detected using RT-qPCR in stools from healthy individuals.

• However, norovirus is clearly a pathogen

• Complicates interpretation of etiological studies including MAL-ED or GEMS

4

Norovirus illness and infection rates in children

in community based cohort studies

5

Sapovirus (not previously tested)

Shigella (PCR vs. culture)

LT-ETEC (PCR vs. culture)

tEPEC (PCR vs. culture)

Adenovirus 40/41 (PCR vs. EIA)

ST-ETEC (PCR vs. culture)

Cryptosporidium (PCR vs. EIA)

Astrovirus (PCR vs. EIA)

C. jejuni/coli (PCR vs. EIA)

Rotavirus (PCR vs. EIA)

Norovirus GII (both PCR) MAL-ED WorkupTAC

0% 2% 4% 6% 8% 10% 12% 14% 16% 18%

Sapovirus (not previously tested)Salmonella (PCR vs. culture)

E. histolytica(PCR vs. EIA)EIEC (PCR vs. culture)

Adenovirus 40/41 (PCR vs. EIA)LT-ETEC (PCR vs. culture)

Cryptosporidium(PCR vs. EIA)ST-ETEC (PCR vs. culture)Shigella (PCR vs. culture)

Astrovirus (PCR vs. EIA)Rotavirus (PCR vs. EIA)

Norovirus GII (both PCR)C. jejuni/coli (PCR vs. EIA)

Attributable Fraction (%)

1-2

yea

rs o

f ag

e

0-1

yea

rs o

f ag

e

0% 2% 4% 6% 8% 10% 12% 14% 16% 18%

Sapovirus (not previously tested)Salmonella (PCR vs. culture)

E. histolytica(PCR vs. EIA)EIEC (PCR vs. culture)

Adenovirus 40/41 (PCR vs. EIA)LT-ETEC (PCR vs. culture)

Cryptosporidium(PCR vs. EIA)ST-ETEC (PCR vs. culture)Shigella (PCR vs. culture)

Astrovirus (PCR vs. EIA)Rotavirus (PCR vs. EIA)

Norovirus GII (both PCR)C. jejuni/coli (PCR vs. EIA)

Attributable Fraction (%)

POSTER TEMPL ATE BY:

www.PosterPresentations.com

▪ Studies of childhood diarrhea in

developing countries have traditionally

employed a wide range of diagnostic

modalities including culture, microscopy,

enzyme immunoassay, and polymerase chain

reaction.

▪ Molecular diagnostics offer more consistent

diagnostic performance, however they

substantially increase the background rate of

pathogen detection.

▪ Quantitative analysis of molecular testing

can help identify the subset of clinically

significant molecular detections.

Molecular re-analysis of community burdens of diarrhea in children

in developing countries in the multisite MAL-ED cohort study

James A. Platts-Mills1*, Jie Liu1*, Jean Gratz1,2, Darwin Operario1, Amidou Samie3, Ira Praharaj4, Gagandeep Kang4, Carl Mason5, Ladaporn Bodhidatta5, Sadia Shakoor6,

Furqan Kabir6, Shahida Qureshi6, Rashidul Haque7, Mami Taniuchi1,7, Margaret Kosek8.9, Mery S. Salas9, Alexandre Havt10, Aldo A. Lima10, Eric R. Houpt1

1Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, USA, 2Haydom Lutheran Hospital, Haydom, Tanzania, 3University of Venda, Thohoyandou, South Africa, 4Christian Medical College, Vellore, India, 5Armed Forces Research Institute of Medical

Sciences, Bangkok, Thailand, 6Aga Khan University, Karachi, Pakistan, 7International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh, 8Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA, 9Asociación Benéfica PRISMA, Iquitos, Peru, 10Clinical

Research Unit and Institute of Biomedicine, Federal University of Ceara, Fortaleza, Brazil, *Contributed equally

BACKGROUND

STUDY DEISGN AND STOOL TESTING

RESULTS ANALYSIS METHODS

CONCLUSIONS

CONTACT INFORMATION

James A. Platts-Mills, Assistant Professor

Infectious Diseases and International Health, University of Virginia

jp5t@virginia.edu

MAL-ED Study: http://mal-ed.fnih.org/

REFERENCES

1. Platts-Mills JA, Babji S, Bodhidatta L, Gratz J, Haque R, Havt A,

et al. Pathogen-specific burdens of community diarrhoea in

developing countries (MAL-ED): a multisite birth cohort study.

Lancet Global Health 2015, in press.

2. Liu J, Kabir F, Manneh J, Lertsethtakarn P, Begum S, Gratz J,

Becker SM, Operario DJ, Taniuchi M, Janaki L, Platts-Mills JA,

et al. Development and assessment of molecular diagnostic

tests for 15 enteropathogens causing childhood diarrhoea: a

multicentre study. Lancet Infect Dis 2014;8:716-24.

▪ We re-analyzed stool specimens from a

community-based multisite birth cohort study,

involving a total of 2086 children from 8 sites.1

▪ Diarrhea was defined as 3 or more loose

stools in 24 hours or presence of blood in stool.

Asymptomatic control specimens were

collected monthly; specimens collected within 7

days of an episode of diarrhea were excluded

from the analysis

▪ We re-tested diarrheal as well as quarterly

surveillance stools from the first two years of

life from a subset of approximately 60 children

per site with singleplex, quantitative PCR for a

broad range of enteropathogens using TaqMan

Array Cards (TAC).

▪ A total of 8508 diarrheal and 24238 control

stools were tested for a broad range of

enteropathogens in the primary analysis; 2182

diarrheal and 3244 control stools from

approximately 60 children per site were re-

analyzed by TAC.

▪ The total AF for all pathogens increased from

24.7% to 34.9% in the first year of life and 38.0%

to 57.6% in the second year of life.

▪ In comparison to conventional detection

methods, the AF for Shigella, ST-ETEC, and

adenovirus 40/41 increased substantially.

Sapovirus was not previously assayed for in the

study and is revealed to have a substantial

burden of disease in these settings.

▪ This re-analysis substantially revises prior

estimates of pathogen-specific burdens of

diarrhea in children in developing countries and

can help refine priorities for development of

pathogen-specific interventions.

Figure 2. Overall adjusted attributable fractions derived for conventional diagnostic work-up (red) and TAC re-analysis (blue)

▪ Generalized estimating equations were used to

fit logistic regression models to calculate odds

ratios for the association between the detection

of each pathogen and diarrhea.

▪ Attributable fractions (AF) were calculated as

where N = total number of cases of diarrhea,

ORi = odds ratio from the fitted model for each

case i in which the pathogen was detected.

Sapovirus*(not*previously*tested)*

Shigella*(PCR*vs.*culture)*

LT<ETEC*(PCR*vs.*culture)*

tEPEC*(PCR*vs.*culture)*

Adenovirus*40/41*(PCR*vs.*EIA)*

ST<ETEC*(PCR*vs.*culture)*

Cryptosporidium*(PCR*vs.*EIA)*

Astrovirus*(PCR*vs.*EIA)*

C.*jejuni/coli*(PCR*vs.*EIA)*

Rotavirus*(PCR*vs.*EIA)*

Norovirus*GII*(both*PCR)* MAL<ED*Workup*TAC*

0%* 2%* 4%* 6%* 8%* 10%* 12%* 14%* 16%* 18%*

Sapovirus*(not*previously*tested)*

Salmonella*(PCR*vs.*culture)*

E.*histolyQca(PCR*vs.*EIA)*

EIEC*(PCR*vs.*culture)*

Adenovirus*40/41*(PCR*vs.*EIA)*

LT<ETEC*(PCR*vs.*culture)*

Cryptosporidium(PCR*vs.*EIA)*

ST<ETEC*(PCR*vs.*culture)*

Shigella*(PCR*vs.*culture)*

Astrovirus*(PCR*vs.*EIA)*

Rotavirus*(PCR*vs.*EIA)*

Norovirus*GII*(both*PCR)*

C.*jejuni/coli*(PCR*vs.*EIA)*

A" ributable+Frac. on+(%)+

1<2*years*of*age

**********************************0<1*years*of*a

ge*

Figure 1. Sensitivity (blue) and specificity (red) of comparator methods vs. TAC

! " =( 1/& )∑*↑▒( - . ↓* −1)/ - . ↓*  

A total of 8,508 diarrhoeal and 24,238 control stools were tested for a broad range of enteropathogens in the primary analysis; 2,182 diarrhoeal and 3,244 control stools from approximately 60 children per site were re-analyzed by qPCR assays in TAC

6

Molecular re-analysis of MAL-ED

Challenges to Estimating Global

Norovirus Burden

• Little routine testing performed in ongoing surveillance platforms; or in developing countries

• Sensitive assays have only recently been widely available, and are not available for most settings

• Community-based studies are expensive and challenging

• Some norovirus acute gastroenteritis cases do not present with diarrhoea, only vomiting

• Challenges in interpretation of diagnostic results

Birth cohort study in Vellore, India

Leveraging the global rotavirus networks with TAC assays

8

Norovirus: basic virology

• Noroviruses are a highly diverse group of ssRNA viruses.

• GII.4 norovirus: – is the most common genotype causing cases

and outbreaks across the age range – evolves in a boom-and-bust cycling of

epochal evolution and escape population immunity with new variants emerge every 2-4 years

– cause more severe disease and affect both young and elderly vulnerable populations.

• Real time RT-PCR is – the gold standard for norovirus diagnostics – exquisitely sensitive and frequently detects

virus in the stool of healthy individuals • Uncultivable, but there has been important

recent progress in in vitro cell culture for norovirus.

9

Norovirus vaccines

• A number of noroviruses vaccines being developed.

• Based on the expression of VP1 leading to the production of VLPs or P particle subunit in various expression systems.

• Preclinical and early human studies of various concentrations of monovalent or bivalent norovirus antigens, with and without adjuvants, and various routes of immunization have shown safety and immunogenicity.

10

Transgenic plant-based

norovirus vaccine

Norovirus P particle and

combination vaccines

Trivalent norovirus/ rotavirus

combination vaccine

Bivalent norovirus VLP

vaccine

Principal

inventor

Charles Arntzen, PhD

Arizona State University

Xi Jason Jiang, PhD

Cincinnati Children's Hospital

Timo Vesikari, MD PhD

University of Tampere, Finland

Mary K. Estes, Ph.D.

Baylor College of Medicine

Norovirus

antigen(s)

(all based on

VP1)

Norwalk virus (GI.1) VLP Two to three noroviruses P

domains representing

different GI and GII strains

GII-4 and GI-3 VLP GI.1 and GII.4 consensus

VLP

Other

antigen(s)

None Chimeric norovirus P-

rotavirus VP8* particle,

experimental formulations

include influenza, Hepatitis E

Rotavirus VP6 None

Adjuvant(s) gardiquimod or none when

delivered with GelVac

5 mg chitosan, 50 μg MPL,

and TNC buffer 24920797

None, but some evidence that the

RV VP6 component adjuvant effect

alum and MPL (3-O-

desacyl-4’ monophosphoryl

lipid A)

Route of

administrati

on

Intranasal by GelVac dry

powder and oral by ingestion

of raw potato

Intranasal Intramuscular and intranasal Intramuscular, previously

intranasal and oral

Commercial

partner

UMN Pharma (Japan). Takeda Pharmaceuticals

Norovirus vaccine candidates

11

Human efficacy data from challenge studies

• The only products with human efficacy data are being developed by Takeda Pharmaceuticals. – An intranasal monovalent formulation was shown to be effective against

infection and disease following GI.1 challenge. • 47% (95% CI, 15%–67%) against AGE

• Norwalk virus infection by 26% (95% CI, 1%–45%)

– An IM bivalent formulation showed a degree of protection against disease

following GII.4 challenge sufficient to warrant further clinical development. • Non-significant reduction in AGE (26.0% among vaccinees ; 33.3% among

placebo recipients)

• Reductions of more severe disease and diarrhoea and vomiting in vaccine recipients following GII.4 challenge

12

Critical questions for a norovirus vaccine

• Can a vaccine elicit broad protection against multiple genotypes?

• What will be the duration of protection from vaccination?

• Will a norovirus vaccine have to be regularly updated in order to keep up with natural evolution of the virus?

• How will prior norovirus infection history affect vaccine immunogenicity and effectiveness?

• Will the same vaccine formulation be effective in all population groups, including in infants or low-income settings?

• How will the variation in human genetic susceptibility affect vaccine outcomes?

13

Target groups for vaccination strategies

Inci

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aliz

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De

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Soci

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He

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care

cost

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Ro

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isk

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Ch

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vacc

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imm

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Ch

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s in

vacc

inat

ing:

pro

gram

mat

ic

Children

(<5 years)

High

High High

Med

.

High High High Naïve: may

need multiple

doses

Interaction with

other routine

immunizations

Older children

(5-14 years)

Med Low Low Low Med Low Med History of

exposure

Younger

adults

(15-64 years)

Med Low Low Low Med Low Med History of

exposure

Generally low

coverage

Older adults

(≥65 years)

Low Med High High

Low High Low History of

exposure

immune

senescence

Generally low

coverage

Top priority?

14

Specific sub-population target groups for

vaccination

• Healthcare workers

• Travelers

• Military personnel

• Immunocompromised patients

• Food service workers

Each group has unique epidemiological, economic and programmatic considerations

15

Specific roles for WHO in 2016-2017

• Study design to help quantify incidence and burden , particularly in developing countries

• Call for better epidemiology data on norovirus – Birth cohort studies

– Regional norovirus surveillance networks with standardized protocols

– Development / optimization of diagnostics for use in etiology and clinical studies

• Development of preferred product characteristics; TPPs – Different population targets will likely have different TPPs

• Confirmation of currently proposed immune correlates of protection; validation in different populations

16

THE WORK IS COMPLICATED. WHY WE DO IT, IS NOT