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

NCT ID: NCT05589012

Last Updated: 2022-10-21

Basic Information

• Recruitment Status: UNKNOWN
• Total Enrollment: 8 participants
• Study Classification: OBSERVATIONAL
• Study Start Date: 2023-11-01
• Study Completion Date: 2024-04-01

Brief Summary

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

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.

Conditions

Zika Virus Infection

Study Design

• Observational Model Type: COHORT
• Study Time Perspective: PROSPECTIVE

Study Groups

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

Cell line and virus

Intervention Type: BIOLOGICAL

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.

Infection of human placental culture explants

Intervention Type: BIOLOGICAL

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.

Determination of viral load in tissues by qRT-PCR

Intervention Type: BIOLOGICAL

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.

Interventions

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.

Intervention Type: 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.

Intervention Type: 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.

Intervention Type: BIOLOGICAL

Eligibility Criteria

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)

• Minimum Eligible Age: 18 Years
• Eligible Sex: FEMALE
• Accepts Healthy Volunteers: No

Sponsors

Assistance Publique - Hôpitaux de Paris

OTHER

Sponsor Role: lead

Responsible Party

Responsibility Role: SPONSOR

Central Contacts

PICONE Olivier

Role: CONTACT

olivier.picone@aphp.fr

01 47 60 66 36

DECHANET Aline

Role: CONTACT

aline.dechanet@aphp.fr

Other Identifiers

2020-A01318-31

Identifier Type: -

Identifier Source: org_study_id