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

Zika Virus Infection Clinical Trial

— ZITOPEx  

Official title:

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

Clinical Trial Details — Status: Not yet recruiting

Administrative data

• NCT number: NCT05589012
• Other study ID #: 2020-A01318-31
• Status: Not yet recruiting
• Start date: November 1, 2023
• Est. completion date: April 1, 2024

Study information

• Verified date: August 2022
• Source: Assistance Publique - Hôpitaux de Paris
• Contact: PICONE Olivier
• Phone: 01 47 60 66 36
• Email: olivier.picone[@]aphp.fr
• Is FDA regulated: No
• Study type: Observational

Clinical Trial Summary

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

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.

Recruitment information / eligibility

• Status: Not yet recruiting
• Enrollment: 8
• Est. completion date: April 1, 2024
• Est. primary completion date: April 1, 2024
• Accepts healthy volunteers: No
• Gender: Female
• Age group: 18 Years and older

Eligibility

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)

Related Conditions & MeSH terms

• Zika Virus Infection

Intervention

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.
• 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.
• 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.

Locations

Country	Name	City	State
n/a

Sponsors (1)

Lead Sponsor	Collaborator
Assistance Publique - Hôpitaux de Paris

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	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	Validate the ex vivo placental model for TONV.	Analysis of viral load in placental explants for TONV in RNA copies/mL	Inclusion
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	Compare the placental infection of TONV versus ZIKV.	Analysis of viral load depending on the virus (ZIKV vs TONV) in RNA copies/mL	inclusion
Secondary	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
Secondary	Compare the placental infection of TONV versus ZIKV.	Analysis of histological lesions depending on the virus (ZIKV vs TONV) in RNA copies/mL	inclusion
Secondary	Highlight the cellular targets of TONV.	Histological analysis and labeling of TONV cells in RNA copies/mL	inclusion
Secondary	Highlight the cellular targets of TONV.	Histological analysis and labeling of placental cells in RNA copies/mL	inclusion