Evaluation of Viral Replication by Tonate Virus (TONV) and Zika Virus (ZIKV), Within an ex Vivo Trophoblast and Placental Model (ZITOPEx)

Prospective, non-interventional study carried out after culturing placental trophoblastic tissue ex vivo and infection with Zika and Tonate

Study Overview

• Status: Not yet recruiting
• Conditions: Zika Virus Infection
• Intervention / Treatment: Biological: Cell line and virus; Biological: Infection of human placental culture explants; Biological: Determination of viral load in tissues by qRT-PCR

Detailed Description

The analysis will relate for each virus studied + control + favipiravir to 1 placenta in the 1st trimester and 1 placenta at term.

2 experiments per virus will be planned.

• Study Type: Observational
• Enrollment (Anticipated): 8

Contacts and Locations

• Study Contact: Name: PICONE Olivier; Phone Number: 01 47 60 66 36; Email: olivier.picone@aphp.fr

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years and older (ADULT, OLDER_ADULT)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: Female

• Sampling Method: Non-Probability Sample

Study Population

Pregnant women

Description

Inclusion Criteria:

• Patient over 18 years old
• Singleton pregnancy of normal course, at term, birth by caesarean section OR
• Trophoblast from Voluntary Termination of Pregnancy (IVG), after endo-uterine aspiration

Exclusion Criteria:

• Need for pathological, genetic or bacteriological examination of the placenta
• Multiple pregnancy
• HBV+ (Hepatitis B positive), HCV+ (Hepatitis C positive) , known CMV seroconversion during pregnancy (CytoMégaloVirus)
• Immunosuppression (drugs, corticosteroids, etc.)
• Diabetes
• Pre eclampsia
• Intrauterine growth retardation (IUGR),
• Vascular or placental pathology.
• Refusal to participate
• Patient under guardianship / curatorship / security measure
• Patient under AME (state medical aid)

Study Plan

How is the study designed?

Number of groups / cohorts

1

Cohorts and Interventions

Group / Cohort

Intervention / Treatment

Human placentas and trophoblasts (biological waste) from uncomplicated pregnancies.; The placentas will be collected following a birth by caesarean section after oral and written information by delivery of the information note for women who have had a normal singleton pregnancy at term. Similarly, trophoblasts are collected following endo-uterine aspiration in the context of voluntary termination of pregnancy.; Cell line and virus; Infection of human placental culture explants; Determination of viral load in tissues by qRT-PCR

Biological: Cell line and virus; C6/36 cells will be cultured in L15 culture medium (Leibovitz's L-15 medium), supplemented with 5% FBS (Fetal Bovine Serum) and 1% penicillin/streptomycin at 28°C in a humidified atmosphere containing 5% C02. ZIKV and TONV will be expanded and titrated using C6/36 cells. The viral stock will be aliquoted in 100 µL aliquots and stored at -80°C.; Biological: Infection of human placental culture explants; Placental tissues will be handled within one hour of delivery. The chorionic villi will be dissected into 5mm sections and the tissues washed abundantly, minimum 3 times with a standard culture medium (RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% L-Glutamine and 1% Penicillin/Streptomycin) to remove maternal blood, membranes and blood clots. Collagen gel sponges will be placed in 6-well plate wells containing 3mL of culture medium (RPMI-1640 supplemented with 15% heat-inactivated fetal bovine serum (FCS), 1% Penicillin/streptomycin, 0 1% Gentamycin, 1% Amphotericin B, 1% L-Glutamine, 1% non-essential amino acid, 1% sodium pyruvate) per well. Chorionic villi will be dissected into 5mm sections and placed on top of collagen gel sponges at the interface between culture medium and air.; Biological: Determination of viral load in tissues by qRT-PCR; The tissues will be lysed by mechanical disruption in a lysis buffer + 4%TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) (Machery-Nagel ref: 740395.107) with the Precellys system. The lysed tissues will be diluted in 100 mg/mL of Macherey-Nagel lysis buffer, aliquoted and stored at -80°C until extraction. Total RNA will be extracted in duplicate using the Nucleospin 96 RNA Core kit (Macherey Nagel ref: 740466.4), following the supplier's instructions. Standard samples, controls and ZIKV and TONV viral RNA will be extracted and tested in parallel under the same conditions. The extracted RNA will be subjected to reverse transcriptase, using the Superscript One-Step RT-qPCR kit (Invitrogen ref: 11732088) according to the manufacturer's recommendations, with a probe and primer specific for ZIKV and TONV.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
To assess the replication potential of TONV and ZIKV after placenta infection ex vivo.	Determine the viral load (RT-qtPCR) of TONV and ZIKV after placenta infection, ex vivo in RNA copies/mL	inclusion

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Validate the ex vivo placental model for TONV.	Analysis of viral load in placental explants for TONV in RNA copies/mL	Inclusion
Compare viral infection of the placenta at different terms of pregnancy (first trimester and term).	Analysis of the viral load according to the term in RNA copies/mL	first trimester
Compare viral infection of the placenta at different terms of pregnancy (first trimester and term).	Analysis of gene expression of the different cytokines according to the term in RNA copies/mL	first trimester
Compare viral infection of the placenta at different terms of pregnancy (first trimester and term).	Analysis of the histological lesions according to the term in RNA copies/mL	first trimester
Compare viral infection of the placenta at different terms of pregnancy (first trimester and term).	Analysis of the viral load according to the term in RNA copies/mL	term
Compare the placental infection of TONV versus ZIKV.	Analysis of viral load depending on the virus (ZIKV vs TONV) in RNA copies/mL	inclusion
Compare the placental infection of TONV versus ZIKV.	Analysis of gene expression of different cytokines depending on the virus (ZIKV vs TONV) in RNA copies/mL	inclusion
Compare the placental infection of TONV versus ZIKV.	Analysis of histological lesions depending on the virus (ZIKV vs TONV) in RNA copies/mL	inclusion
Highlight the cellular targets of TONV.	Histological analysis and labeling of TONV cells in RNA copies/mL	inclusion
Highlight the cellular targets of TONV.	Histological analysis and labeling of placental cells in RNA copies/mL	inclusion

Collaborators and Investigators

• Sponsor: Assistance Publique - Hôpitaux de Paris

Study record dates

Study Major Dates

• Study Start (ANTICIPATED): November 1, 2023
• Primary Completion (ANTICIPATED): April 1, 2024
• Study Completion (ANTICIPATED): April 1, 2024

Study Registration Dates

• First Submitted: August 22, 2022
• First Submitted That Met QC Criteria: October 17, 2022
• First Posted (ACTUAL): October 21, 2022

Study Record Updates

• Last Update Posted (ACTUAL): October 21, 2022
• Last Update Submitted That Met QC Criteria: October 17, 2022
• Last Verified: August 1, 2022

More Information

Terms related to this study

• Keywords: placenta; trophoblast
• Additional Relevant MeSH Terms: RNA Virus Infections; Virus Diseases; Infections; Arbovirus Infections; Vector Borne Diseases; Flavivirus Infections; Flaviviridae Infections; Zika Virus Infection
• Other Study ID Numbers: 2020-A01318-31

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No