Valentin Leducq

Kaposi's sarcoma‐associated herpesvirus (KSHV) has been associated with a variety of diseases and is endemic in Sub‐Saharan Africa. We performed KSHV‐whole‐genome analysis and search for APOBEC‐editing to describe genetic diversity in samples issued from an endemic region. We included 15 Congolese samples including 12 saliva and whole blood samples of people living with HIV and 3 KS biopsies samples, to perform KSHV qPCR and NGS Illumina followed by KSHV typing on ORFs K1 and K15. Furthermore, we looked for APOBEC3B‐mutations in KSHV‐whole‐genome sequences. KSHV viral load was detectable in all samples and ranged from 374 to 72799 copies/10 ⁶ cells. NGS allowed us to have 8 full sequences out of the 15 samples analyzed. Phylogenetic analysis on KSHV‐whole‐genome resulted in distinct phylogenetic clustering between the genomic sequences of Congolese KSHV and those derived from Western countries. KSHV ORF‐K1 subtypes A5, B and C were identified. On KSHV ORF‐K15, only the P subtype was observed. We found virtually no APOBEC3B‐induced mutations in KSHV genome. KSHV sequences from Congo samples present phylogenetic particularities but remain close to the whole genome sequences found in Sub‐Saharan Africa. We were able to observe, using bioinformatics tools, only very few mutations due to APOBEC3B on KSHV genome.

Dès le début de la pandémie de COVID-19, des outils de biologie moléculaire permettant la détection et l’analyse du génome du SARS-CoV-2 ont été développés et déployés à une échelle sans précédent. Les techniques de PCR ont été utilisées pour diagnostiquer quotidiennement des millions de personnes, tandis que la démocratisation du séquençage de nouvelle génération (NGS) a permis de générer rapidement des centaines de milliers de génomes viraux. Ces outils ont été une source d’information majeure dans l’étude et la compréhension de la physiopathologie du SARS-CoV-2, mais également dans le suivi en temps réel de la dynamique de la pandémie et de l’évolution de ce virus. Nous avons entrepris cette thèse afin d’évaluer comment ces outils de biologie moléculaire peuvent contribuer à l’étude et la surveillance du SARS-CoV-2, dans le cadre de trois études répondants à différents objectifs. Avec l’étude PhyloCoV, nous avons tout d’abord apporté une description précise de l’évolution de la diversité virale observée au cours de l’année 2020 en région Ile-de-France, grâce au séquençage de 736 génomes du SARS-CoV-2 provenant de patients et de personnels soignant des CHU Pitié-Salpêtrière et Bichat Claude-Bernard. Cette étude nous a également permis de développer une méthode de clustering phylogénétique permettant la détection des transmissions intra-hospitalières de la COVID-19. Ensuite, dans le cadre d’une étude observationnelle au sein du GH Sorbonne Université, nous avons estimé la fréquence et la mortalité associées aux transmissions nosocomiales du SARS-CoV-2. Entre septembre et novembre 2020, 209 cas de COVID-19 nosocomial ont été identifiés, représentant 13,9% des patients hospitalisés infectés par le SARS-CoV-2 et associés à une mortalité de 31%. Le séquençage des génomes viraux a permis de réfuter près d’un tiers des transmissions nosocomiales et de révéler de précieuses informations sur les voies de transmission au sein des services hospitaliers. Finalement, avec l’étude COCOPREV, nous avons étudié l’évolution du génome du SARS-CoV-2 au cours de l’infection chez des patients traités par des anticorps monoclonaux, majoritairement immunodéprimés. Plusieurs mutations de résistance émergent dans la protéine SPIKE sous les trois traitements étudiés. Les patients traités avec du Tixagevimab/Cilgavimab présentent un risque 5 fois plus élevé de développer des mutations au niveau des résidus R346 et K444. A la différence des autres mutations retrouvées, les substitutions R346 et K444 ont plusieurs fois émergé dans différents variants grâce à leur impact sur le fitness du SARS-CoV-2. Elles ont finalement été sélectionnées par les variants BQ.1 et XBB, variants actuellement majoritaires. En conclusion, nous avons démontré l’utilité des outils de biologie moléculaire dans la surveillance de la transmission du SARS-CoV-2 dans les hôpitaux et dans la population en général, ainsi que dans l'identification de mutations de résistance émergentes chez des patients traités par des anticorps monoclonaux. Ces outils continueront très probablement d'être utilisés dans le cadre de la pandémie de COVID-19 mais également dans la surveillance de l’émergence et de la propagation d’autres virus.

Background: Reemergence of human herpesvirus 8 (HHV-8)–induced Kaposi sarcoma (KS) in people living with HIV (PLWH) despite antiretroviral therapy (ART) poses a clinical challenge because they already have favorable CD4 T-cell numbers and undetectable viral loads. We observed that clinical presentation in PLWH on ART resembled classic KS found in older HIV-uninfected patients and hypothesized that immunosenescence may thus play a role in occurrence of KS on ART. We compared viral and immune factors implicated in the development of KS in ART-treated PLWH (HIV KS) and HIV-uninfected classic KS patients (cKS), compared to controls without KS (HIV Control, cControls respectively). Methods: Plasma, peripheral blood mononuclear cell, and skin tissues were obtained from 11 HIV KS and 11 cKS patients and 2 groups of age-matched controls. Results: HIV KS participants were younger than cKS (aged 53 vs 75 years). HHV-8 genotypes did not differ between groups. Despite the younger age and a lower CD4/CD8 ratio, activated, exhausted, and senescent T-cell frequencies were similar between HIV KS and cKS. Anti–HHV-8 immunoglobulin G levels were higher and circulating HHV-8 DNA lower in HIV KS compared with cKS. Circulating platelet-derived growth factors AA-BB and granulocyte colony-stimulating factors were higher in HIV KS We observed similar levels of HHV-8 DNA and PD-1 expression in skin lesions from HIV KS and cKS patients. Conclusions: Altogether, early immune senescence could be involved in the development of KS in ART-treated PLWH. Higher anti–HHV-8 immunoglobulin G levels could be linked with lower circulating viral load. Such insights should help developing therapeutical strategies to prevent development and treat KS in PLWH on ART.

Background: High-risk patients, often immunocompromised and not responding to vaccine, continue to experience severe coronavirus disease 2019 (COVID-19) and death. Monoclonal antibodies (mAbs) were shown to be effective to prevent severe COVID-19 for these patients. Nevertheless, concerns about the emergence of resistance mutations were raised. Methods: We conducted a multicentric prospective cohort study, including 264 patients with mild to moderate COVID-19 at high risk for progression to severe COVID-19 and treated early with casirivimab/imdevimab, sotrovimab, or tixagevimab/cilgavimab. We sequenced the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome during follow-up and searched for emerging spike mutations. Results: Immunocompromised patients have a 6-fold increased risk of developing mutations, which are associated with a prolonged duration of viral clearance but no clinical worsening. Emerging P337S/R/L/H, E340D/K/A/Q/V/G, and K356T/R substitutions in patients treated with sotrovimab are associated with higher viral RNA loads for up to 14 days post–treatment initiation. Tixagevimab/cilgavimab is associated with a 5-fold increased risk of developing mutations. R346K/I/T/S and K444R/N/M substitutions associated with tixagevimab/cilgavimab have been identified in multiple SARS-CoV-2 lineages, including BQ.1 and XBB. Conclusions: The probability of emerging mutations arising in response to mAbs is significant, emphasizing the crucial need to investigate these mutations thoroughly and assess their impact on patients and the evolutionary trajectory of SARS-CoV-2.

Reprogrammed metabolism is regarded as a hallmark of cancer and offers a selective advantage to tumor cells during carcinogenesis. The redox equilibrium is necessary for growth, spread and the antioxidant pathways are boosted following Reactive Oxygen Species (ROS) production to prevent cell damage in tumor cells. Human herpesvirus 8 (HHV-8), the etiologic agent of Kaposi sarcoma KS and primary effusion lymphoma (PEL), is an oncogenic virus that disrupts cell survival-related molecular signaling pathways leading to immune host evasion, cells growths, angiogenesis and inflammatory tumor-environment. We recently reported that primaquine diphosphate causes cell death by apoptosis in HHV-8 infected PEL cell lines in vivo and exhibits therapeutic anti-tumor activity in mice models and advanced KS. Our findings also suggest that the primaquine-induced apoptosis in PEL cells is mostly influenced by ROS production and targeting the redox balance could be a new approach to treat HHV-8 related diseases. In this review, we summarized the knowledge about the influence of ROS in cancer development; more specifically, the proof of evidence from our work and from the literature that redox pathways are important for the development of HHV-8 pathologies.

The SARS-CoV-2 neutralizing antibodies response is the best indicator of effective protection after infection and/or vaccination, but its evaluation requires tedious cell-based experiments using an infectious virus. We analyzed, in 105 patients with various histories of SARS-CoV-2 infection and/or vaccination, the neutralizing response using a virus neutralization test (VNT) against B.1, Alpha, Beta and Omicron variants, and compared the results with two surrogate assays based on antibody-mediated blockage of the ACE2-RBD interaction (Lateral Flow Boditech and ELISA Genscript). The strongest response was observed for recovered COVID-19 patients receiving one vaccine dose. Naïve patients receiving 2 doses of mRNA vaccine also demonstrate high neutralization titers against B.1, Alpha and Beta variants, but only 34.3% displayed a neutralization activity against the Omicron variant. On the other hand, non-infected patients with half vaccination schedules displayed a weak and inconstant activity against all isolates. Non-vaccinated COVID-19 patients kept a neutralizing activity against B.1 and Alpha up to 12 months after recovery but a decreased activity against Beta and Omicron. Both surrogate assays displayed a good correlation with the VNT. However, an adaptation of the cut-off positivity was necessary, especially for the most resistant Beta and Omicron variants. We validated two simple and reliable surrogate neutralization assays, which may favorably replace cell-based methods, allowing functional analysis on a larger scale.

Previous reports have shown a variability in the diagnostic performance of RDTs. In the era of SARS-CoV-2 variants and the use of RDT, mutation associated with these variants could affect the test performance.

Human Herpesvirus 8 (HHV-8) is associated with three main severe orphan malignancies, Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD), and primary effusion lymphoma (PEL), which present few therapeutic options. We identified the antimalarial primaquine diphosphate (PQ) as a promising therapeutic candidate for HHV-8-associated PEL and KS. Indeed, PQ strongly reduced cell viability through caspase-dependent apoptosis, specifically in HHV-8-infected PEL cells. Reactive oxygen species (ROS)- and endoplasmic reticulum (ER) stress-mediated apoptosis signaling pathways were found to be part of the in vitro cytotoxic effect of PQ. Moreover, PQ treatment had a clinically positive effect in a nonobese diabetic (NOD)/SCID xenograft PEL mouse model, showing a reduction in tumor growth and an improvement in survival. Finally, an exploratory proof-of-concept clinical trial in four patients harboring severe KS was conducted, with the main objectives to assess the efficacy, the safety, and the tolerability of PQ, and which demonstrated a positive efficacy on Kaposi’s sarcoma-related lesions and lymphedema.

France went through three deadly epidemic waves due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing major public health and socioeconomic issues. We proposed to study the course of the pandemic along 2020 from the outlook of two major Parisian hospitals earliest involved in the fight against COVID-19. Genome sequencing and phylogenetic analysis were performed on samples from patients and health care workers (HCWs) from Bichat (BCB) and Pitié-Salpêtrière (PSL) hospitals. A tree-based phylogenetic clustering method and epidemiological data were used to investigate suspected nosocomial transmission clusters. Clades 20A, 20B and 20C were prevalent during the spring wave and, following summer, clades 20A.EU2 and 20E.EU1 emerged and took over. Phylogenetic clustering identified 57 potential transmission clusters. Epidemiological connections between participants were found for 17 of these, with a higher proportion of HCWs. The joint presence of HCWs and patients suggest viral contaminations between these two groups. We provide an enhanced overview of SARS-CoV-2 phylogenetic changes over 2020 in the Paris area, one of the regions with highest incidence in France. Despite the low genetic diversity displayed by the SARS-CoV-2, we showed that phylogenetic analysis, along with comprehensive epidemiological data, helps to identify and investigate healthcare associated clusters.