Periodic Reporting for period 1 - HORUS (Casting light on HOst-cytomegaloviRUs interaction in Solid organ transplantation)
Periodo di rendicontazione: 2022-11-01 al 2024-04-30
Solid organ transplantation (SOT) is the treatment of choice for end-stage organ failure, but the immunosuppressive drugs used to prevent graft rejection expose to an increased risk of new symptoms or the development of post-transplant disorders such as opportunistic infections. Cytomegalovirus (CMV) is the most frequent infectious complication following solid organ transplantation. CMV is responsible for infections that usually go unnoticed, even though it remains latent in the body for life when present. In a context of organ transplant patients, who are immunocompromised people, CMV can be dangerous.
CMV may occur as a primary infection in CMV-naïve patients after SOT, or it can be reactivation of latent donor or recipient infection in CMV-seropositive SOT recipients.
"HORUS" (Casting light on HOst-cytomegaloviRUs interaction in Solid organ transplantation) study is a European research project, funded by the European Commission (Horizon Europe) involving 24 partners in 7 European countries (France, Spain, Czech Republic, Belgium, Switzerland, Germany and Italy).
The aim of HORUS study is to build the first longitudinal cohort of European SOT recipients to investigate viral, clinical and immunological characteristics of the host-virus interactions in the transplant setting in 450 solid organ transplants (heart, lung kidney and liver). Combining those parameters in the first 100 patients of the cohort will provide:
- a signature early after transplantation to predict the risk of developing CMV infection
- a signature at day 0 of infection to predict the risk of CMV disease severity
Found signatures will be validated on the whole retrospective cohort and prospectively in a “proof-of-concept" study.
Moreover, HORUS will include functional in vitro and mouse models to investigate the mechanisms of lymphocyte response to CMV and provide innovative methods to increase CMV-specific immunity.
The goal is to determine which patients are the most at risk to develop CMV infections, based on their immune response. HORUS will lead to personalized clinical prevention and treatment of CMV disease and to improvement of patient outcomes.
CMV may occur as a primary infection in CMV-naïve patients after SOT, or it can be reactivation of latent donor or recipient infection in CMV-seropositive SOT recipients.
"HORUS" (Casting light on HOst-cytomegaloviRUs interaction in Solid organ transplantation) study is a European research project, funded by the European Commission (Horizon Europe) involving 24 partners in 7 European countries (France, Spain, Czech Republic, Belgium, Switzerland, Germany and Italy).
The aim of HORUS study is to build the first longitudinal cohort of European SOT recipients to investigate viral, clinical and immunological characteristics of the host-virus interactions in the transplant setting in 450 solid organ transplants (heart, lung kidney and liver). Combining those parameters in the first 100 patients of the cohort will provide:
- a signature early after transplantation to predict the risk of developing CMV infection
- a signature at day 0 of infection to predict the risk of CMV disease severity
Found signatures will be validated on the whole retrospective cohort and prospectively in a “proof-of-concept" study.
Moreover, HORUS will include functional in vitro and mouse models to investigate the mechanisms of lymphocyte response to CMV and provide innovative methods to increase CMV-specific immunity.
The goal is to determine which patients are the most at risk to develop CMV infections, based on their immune response. HORUS will lead to personalized clinical prevention and treatment of CMV disease and to improvement of patient outcomes.
For the WP1, the teams have worked to meet the deliverables and milestones set out in the Grant Agreement. Since the KOM in January 2023, WP1 participants have been working on creating the eCRF, drafting contracts and planning FSS. FSS began in June 2023. The recruitment of 152 patients as at 30 April 2024 is below our forecasts, but has been hampered by various factors, including administrative, logistical and human factors, which we will detail in this report. Corrective measures have been implemented and the recruitment backlog should be cleared by the end of 2024/beginning of 2025. The exploratory cohort on which WP2 and WP3 depend should not be overly impacted by this delay.
Despite our recruitment delay, the report highlights our ability to absorb it and the mobilization of all the teams involved.
In the WP2, a placental model mimicking latency by stopping the viral replication using late-stages inhibitors has been set up. To perform the deep characterization of T and NK cells for WP2 and WP3, we set up cytometry panels with 2 panels for ex-vivo analysis of non-stimulated gamma-delta and alpha-beta T cells, NKT and NK cells, 1 panel for ex-vivo analysis of stimulated gamma-delta T, 1 panel for ex-vivo analysis of stimulated alpha-beta T cells and 1 panel for ex-vivo analysis of stimulated NK cells. We also set up the process for transcriptomic analysis for gamma delta and alpha beta T cells.
For WP2, in vitro and in vivo models have started to understand T cell dysfunction. γδ Vδ2neg T cells either PD1+CD85j+, PD1+CD85-, PD1-CD85j+ cells and double negative are put in culture to compare IFN- γ production in medium alone, with non-infected fibroblasts and CMV-infected fibroblast with a reduction of IFN-γ production by the T cells expressing PD-1 and/or CD85j when co-cultured with hCMV-infected fibroblasts. We used the mouse model owing consecutive CMV infections and tested whether 1 versus 4 MCMV boosts would affect γδ T cell protective antiviral function upon adoptive transfer in T cell deficient hosts.
For WP2 and 3, to determine the functional activity of NK cells against hCMV-infected target cells, an hCMV intestinal colitis model has been set up, in which human intestinal organoids (HIO) are infected with hCMV (TB40 strain). We have also shown that hCMV infection results in reproducible changes in the expression of surface molecules on infected epithelial cells within HIO. Several of these surface molecules can serve as ligands for NK cells, and we are currently in the process of validating the changes in surface expression (upregulation and downregulation) of these putative NK cell receptor ligands.
Parean encompasses the development and implementation of comprehensive single-omics and multi-omics analysis workflows.
For WP3, longitudinal quantitative CMV monitoring will be done by CMV PCR capture using « SureSelectXT HS Target Enrichment System” and has been set up. Also, functions, phenotype, molecular pathways and transcriptome of naïve T cells responding to CMV are ongoing and attest their fast capacities of activation compared to alpha-beta T cells.
Deep understanding of naïve immunity activation in mouse models to recapitulate the physiology of CMV-immune response of a naïve individual in the context of immunosuppression is setting-up. The drafting of the protocol to obtain authorization to recover fetal tissues from the “Agence de la Biomedecine” is in progress. NSG mice are already in our possession. We are currently investigating alternatives to fibroblast injection to deliver the virus in a way that more closely resembles transplantation. For WP4, VHIR, USAAR and UBx, that will run the ELISPOT and flow-cytometry assays are conducting a pre-validation study of common SOPs for the two assays. For WP5, the Pproof-of-concept study of signature-based improvement of dysfunctional T-cells during CMV infection is under drafting.
Despite our recruitment delay, the report highlights our ability to absorb it and the mobilization of all the teams involved.
In the WP2, a placental model mimicking latency by stopping the viral replication using late-stages inhibitors has been set up. To perform the deep characterization of T and NK cells for WP2 and WP3, we set up cytometry panels with 2 panels for ex-vivo analysis of non-stimulated gamma-delta and alpha-beta T cells, NKT and NK cells, 1 panel for ex-vivo analysis of stimulated gamma-delta T, 1 panel for ex-vivo analysis of stimulated alpha-beta T cells and 1 panel for ex-vivo analysis of stimulated NK cells. We also set up the process for transcriptomic analysis for gamma delta and alpha beta T cells.
For WP2, in vitro and in vivo models have started to understand T cell dysfunction. γδ Vδ2neg T cells either PD1+CD85j+, PD1+CD85-, PD1-CD85j+ cells and double negative are put in culture to compare IFN- γ production in medium alone, with non-infected fibroblasts and CMV-infected fibroblast with a reduction of IFN-γ production by the T cells expressing PD-1 and/or CD85j when co-cultured with hCMV-infected fibroblasts. We used the mouse model owing consecutive CMV infections and tested whether 1 versus 4 MCMV boosts would affect γδ T cell protective antiviral function upon adoptive transfer in T cell deficient hosts.
For WP2 and 3, to determine the functional activity of NK cells against hCMV-infected target cells, an hCMV intestinal colitis model has been set up, in which human intestinal organoids (HIO) are infected with hCMV (TB40 strain). We have also shown that hCMV infection results in reproducible changes in the expression of surface molecules on infected epithelial cells within HIO. Several of these surface molecules can serve as ligands for NK cells, and we are currently in the process of validating the changes in surface expression (upregulation and downregulation) of these putative NK cell receptor ligands.
Parean encompasses the development and implementation of comprehensive single-omics and multi-omics analysis workflows.
For WP3, longitudinal quantitative CMV monitoring will be done by CMV PCR capture using « SureSelectXT HS Target Enrichment System” and has been set up. Also, functions, phenotype, molecular pathways and transcriptome of naïve T cells responding to CMV are ongoing and attest their fast capacities of activation compared to alpha-beta T cells.
Deep understanding of naïve immunity activation in mouse models to recapitulate the physiology of CMV-immune response of a naïve individual in the context of immunosuppression is setting-up. The drafting of the protocol to obtain authorization to recover fetal tissues from the “Agence de la Biomedecine” is in progress. NSG mice are already in our possession. We are currently investigating alternatives to fibroblast injection to deliver the virus in a way that more closely resembles transplantation. For WP4, VHIR, USAAR and UBx, that will run the ELISPOT and flow-cytometry assays are conducting a pre-validation study of common SOPs for the two assays. For WP5, the Pproof-of-concept study of signature-based improvement of dysfunctional T-cells during CMV infection is under drafting.