Identification of Host Factors of Norovirus Infections in Mini-Gut Model

NCT ID: NCT03342547

Last Updated: 2018-09-20

Basic Information

• Recruitment Status: UNKNOWN
• Clinical Phase: NA
• Total Enrollment: 20 participants
• Study Classification: INTERVENTIONAL
• Study Start Date: 2018-04-18
• Study Completion Date: 2020-12-31

Brief Summary

The primary objective in this study is to establish a list of host cellular proteins that mediate norovirus infection.

Norovirus is one of the most common pathogens attributed to diarrheal diseases from unsafe food. It is also the primary cause of mortality among young children and adults in foodborne infections. Norovirus is not just a foodborne burden. In a recent meta-analysis, norovirus accounts for nearly one-fifth of all causes of (including person-to-person transmission) acute gastroenteritis in both sporadic and outbreak settings and affects all age groups. Undoubtedly, norovirus is of paramount public health concern in both developed and developing countries. Research efforts to better understand norovirus pathobiology will be necessary for targeted intervention.

From Middle East respiratory syndrome coronavirus to Zika virus, efforts to identify host factors important for mediating virus infection has always been a research priority. Such information will shed light on potential therapeutic targets in antiviral intervention. Norovirus virus-host interaction studies have been hampered by the lack of a robust cell culture model in the past 20 years. In 2016, norovirus has finally been successfully cultivated in a stem cell-derived three-dimensional human gut-like structure called enteroid or mini-gut.

In this study, intestinal stem cells will be isolated from duodenal biopsies collected from participants, followed by differentiation into mini-guts. Genome-wide genetic screening for host essential and restrictive factors will be performed on infected mini-guts by knockout CRISPR and gain-of-function CRISPR SAM, respectively. Shortlisted candidates will undergo preliminary functional validation in cell lines. These data will provide insights into potential therapeutic targets against norovirus infection.

Detailed Description

Study design and overview - This is a laboratory-based study. The investigators plan to establish a list of host cellular proteins regulating norovirus infection. Methods will include establishment of patient-specific stem cell-derived human intestinal enteroids as an infection model and genome-wide CRISPR and CRISPR SAM genetic screens upon infections of two norovirus genotypes.

***Establishment of human intestinal enteroid model***

1. Duodenal biopsies and saliva collection - After obtaining IRB-approved informed consent, two duodenal biopsy samples will be obtained from each participant undergoing upper gastrointestinal endoscopy without contraindications for biopsy sampling, such as participants on anti-coagulation or with other causes of bleeding diathesis. Biopsy samples will be immersed in ice-cold 1xPBS and transported immediately to the laboratory within 30 minutes for further processing. In addition, saliva (1-2 mL) will be collected from each participant for secretor status testing by ELISA.

2. Isolation of crypt stem cells and differentiation - Upon arrival, biopsy samples will first be chopped into smaller pieces. Intestinal crypt cells will be isolated by repeated washing and incubation with 1x Complete Chelating solution (1xCCS) and EDTA buffer. Supernatant containing crypt cells will then be grown in Matrigel containing complete medium with growth factors (CMGF+) (Wnt 3 and Rspo-1 conditioned media, Noggin, A83, B27, EGF, N2, n-acetylcysteine, gastrin, nicotinamide, and SB202190). Budding crypts will then be incubated in differentiation medium (CMGF+, excluding Wnt 3 conditioned medium, SB202190 and nicotinamide as well as having reduced amount of Rspo-1 conditioned medium and Noggin). Formation of three-dimensional gut-like enteroids will be monitored daily.

Conditions

Gastrointestinal Infection

Study Design

• Allocation Method: NA
• Intervention Model: SINGLE_GROUP
• Primary Study Purpose: BASIC_SCIENCE
• Blinding Strategy: NONE

Study Groups

Intestinal stem cell-derived enteroids

Duodenal biopsy samples and saliva will be collected from participants and then processed in laboratory to generate intestinal stem cell-derived enteroids.

Group Type: EXPERIMENTAL

Duodenal biopsy

Intervention Type: PROCEDURE

Two duodenal biopsy samples will be collected from each participant

Saliva

Intervention Type: PROCEDURE

Saliva (1-2 mL) will be collected from each participant for secretor status testing

Interventions

Duodenal biopsy

Two duodenal biopsy samples will be collected from each participant

Intervention Type: PROCEDURE

Saliva

Saliva (1-2 mL) will be collected from each participant for secretor status testing

Intervention Type: PROCEDURE

Eligibility Criteria

Inclusion Criteria

• Adults aged 18 years or above
• Prospective outpatients undergoing gastrointestinal endoscopy for symptoms of dyspepsia in the Endoscopy Unit of the Prince of Wales Hospital, Shatin, Hong Kong, China
• Able and willing to provide informed written consent

Exclusion Criteria

• Use of anti-coagulants and/or aspirin that may have increased risk of bleeding
• History of bleeding diathesis
• Contraindications for biopsy sampling

• Minimum Eligible Age: 18 Years
• Eligible Sex: ALL
• Accepts Healthy Volunteers: Yes

Sponsors

Chinese University of Hong Kong

OTHER

Sponsor Role: lead

Responsible Party

CHAN CHI WAI

Assistant Professor

Responsibility Role: PRINCIPAL_INVESTIGATOR

Principal Investigators

Chi-Wai Chan, PhD

Role: PRINCIPAL_INVESTIGATOR

Chinese University of Hong Kong

Locations

Endoscopy Centre, Prince of Wales Hospital

Hong Kong, , China

Site Status: RECRUITING

Countries

China

Central Contacts

Chi-Wai Chan, PhD

Role: CONTACT

martin.chan@cuhk.edu.hk

+852 35051523

Facility Contacts

Chi-Wai Chan, PhD

Role: primary

martin.chan@cuhk.edu.hk

+852 35051523

Other Identifiers

MIC_CHAN_GRF_17_18_v1_20160930

Identifier Type: -

Identifier Source: org_study_id