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Introduction: Immunogenicity, the unwanted immune response triggered by therapeutic antibodies, poses significant challenges in biotherapeutic development. This response can lead to the production of anti-drug antibodies, potentially compromising the efficacy and safety of treatments. The internalization of therapeutic antibodies into dendritic cells (DCs) is a critical factor influencing immunogenicity. Using monoclonal antibodies, with differences in non-specific cellular uptake, as tools to explore the impact on the overall risk of immunogenicity, this study explores how internalization influences peptide presentation and subsequently T cell activation. Materials and methods: To investigate the impact of antibody internalization on immunogenicity, untargeted toolantibodies with engineered positive or negative charge patches were utilized. Immature monocyte-derived DCs (moDCs), known for their physiologically relevant high endocytic activity, were employed for internalization assays, while mature moDCs were used for MHC-II associated peptide proteomics (MAPPs) assays. In addition to the lysosomal accumulation and peptide presentation, subsequent CD4+ T cell activation has been assessed. Consequently, a known CD4+ T cell epitope from ovalbumin was inserted into the tool antibodies to evaluate T cell activation on a single, shared epitope. Results: Antibodies with positive charge patches exhibited higher rates of lysosomal accumulation and epitope presentation compared to those with negative charge patches or neutral surface charge. Furthermore, a direct correlation between internalization rate and presentation on MHC-II molecules could be established. To explore the link between internalization, peptide presentation and CD4+ T cell activation, tool antibodies containing the same OVA epitope were used. Previous observations were not altered by the insertion of the OVA epitope and ultimately, an enhanced CD4+ T cell response correlated with increased internalization in DCs and peptide presentation. Discussion: These findings demonstrate that the biophysical properties of therapeutic antibodies, particularly surface charge, play a crucial role in their internalization into DCs. Antibodies internalized faster and processed by DCs, are also more prone to be presented on their surface leading to a higher risk of triggering an immune response. These insights underscore the importance of considering antibody surface charge and other properties that enhance cellular accumulation during the preclinical development of biotherapeutics to mitigate immunogenicity risks.

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Introduction: Immunogenicity refers to the ability of a substance, such as a therapeutic drug, to elicit an immune response. While beneficial in vaccine development, undesirable immunogenicity can compromise the safety and efficacy of therapeutic proteins by inducing anti-drug antibodies (ADAs). These ADAs can reduce drug bioavailability and alter pharmacokinetics, necessitating comprehensive immunogenicity risk assessments starting at early stages of drug development. Given the complexity of immunogenicity, an integrated approach is essential, as no single assay can universally recapitulate the immune response leading to the formation of anti-drug antibodies. Methods: To better understand the Dendritic Cell (DC) contribution to immunogenicity, we developed two flow cytometry-based assays: the DC internalization assay and the DC activation assay. Monocyte-derived dendritic cells (moDCs) were generated from peripheral blood mononuclear cells (PBMCs) and differentiated over a five-day period. The internalization assay measured the accumulation rate of therapeutic antibodies within moDCs, while the activation assay assessed the expression of DC activation markers such as CD40, CD80, CD86, CD83, and DC-SIGN (CD209). To characterize these two assays further, we used a set of marketed therapeutic antibodies. Results: The study highlights that moDCs differentiated for 5 days from freshly isolated monocytes were more prone to respond to external stimuli. The internalization assay has been shown to be highly sensitive to the molecule tested, allowing the use of only 4 donors to detect small but significant differences. We also demonstrated that therapeutic antibodies were efficiently taken up by moDCs, with a strong correlation with their peptide presentation on MHC-II. On the other hand, by monitoring DC activation through a limited set of activation markers including CD40, CD83, and DC-SIGN, the DC activation assay has the potential to compare a series of compounds. These two assays provide a more comprehensive understanding of DC function in the context of immunogenicity, highlighting the importance of both internalization and activation processes in ADA development. Discussion: The DC internalization and activation assays described here address key gaps in existing immunogenicity assessment methods by providing specific and reliable measures of DC function. The assays enhance our ability to pre-clinically evaluate the immunogenic potential of biotherapeutics, thereby improving their safety and efficacy. Future work should focus on further validating these assays and integrating them into a holistic immunogenicity risk assessment framework.

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Monitoring the presence of RNA from emerging pathogenic viruses, such as SARS-CoV-2, in wastewater (WW) samples requires suitable methods to ensure an effective response. Genome sequencing of WW is one of the crucial methods, but it requires high-quality RNA in sufficient quantities, especially for monitoring emerging variants. Consequently, methods for viral concentration and RNA extraction from WW samples have to be optimized before sequencing. The purpose of this study was to achieve high coverage (≥ 90 %) and sequencing depth (at least ≥200×) even for low initial RNA concentrations (< 105 genome copies (GC)/L) in WW. A further objective was to determine the range of SARS-CoV-2 RNA concentrations that allow high-quality sequencing, and the optimal sample volume for analysis. Ultrafiltration (UF) methods were used to concentrate viral particles from large influent samples (up to 500 mL). An RNA extraction protocol using silica beads, neutral phenol-chloroform treatment, and a PCR inhibitor removal kit was chosen for its effectiveness in extracting RNA and eliminating PCR inhibitors, as well as its adaptability for use with large influent samples. Recovery rates ranged from 24 % to 63 % (N = 17) for SARS-CoV-2 naturally present in WW samples. 200 mL WW samples can be enough for UF concentration, as they showed high quality sequencing analyses with between 5 × 104 GC/L and 6 × 103 GC/L. Below 6 × 103 GC/L, high-quality sequencing was also achieved for ∼40 % of the samples using 500 mL of WW. Sequencing analysis for variant detection was performed on 200 mL WW samples with coverage of >95 % and sequencing depth of >1000×. Analyses revealed the predominance of variant EG.5, known as Eris (66 %-100 %). The use of UF methods in combination with a suitable RNA extraction protocol appear promising for sequencing enveloped viruses in WW in a context of viral emergence.

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Over two decades, most cancer vaccines failed clinical development. Key factors may be the lack of efficient priming with tumor-specific antigens and strong immunostimulatory signals. MVX-ONCO-1, a personalized cell-based cancer immunotherapy, addresses these critical steps utilizing clinical-grade material to replicate a successful combination seen in experimental models: inactivated patient's own tumor cells, providing the widest cancer-specific antigen repertoire and a standardized, sustained, local delivery over days of a potent adjuvant achieved by encapsulated cell technology. We conducted an open-label, single-arm, first-in-human phase I study with MVX-ONCO-1 in patients with advanced refractory solid cancer. MVX-ONCO-1 comprises irradiated autologous tumor cells coimplanted with two macrocapsules containing genetically engineered cells producing granulocyte-macrophage colony-stimulating factor. Patients received six immunizations over 9 weeks without maintenance therapy. Primary objectives were safety, tolerability, and feasibility, whereas secondary objectives focused on efficacy and immune monitoring. Data from 34 patients demonstrated safety and feasibility with minor issues. Adverse events included one serious adverse event possibly related to investigational medicinal product and two moderate-related adverse events. More than 50% of the patients with advanced and mainly nonimmunogenic tumors showed clinical benefits, including partial responses, stable diseases, and prolonged survival. In recurrent/metastatic head and neck squamous cell carcinoma, one patient achieved a partial response, whereas another survived for more than 7 years without anticancer therapy for over 5 years. MVX-ONCO-1 is safe, well tolerated, and beneficial across several tumor types. Ongoing phase IIa trials target patients with advanced recurrent/metastatic head and neck squamous cell carcinoma after initial systemic therapy. SIGNIFICANCE: This first-in-human phase I study introduces a groundbreaking approach to personalized cancer immunotherapy, addressing limitations of traditional strategies. By combining autologous irradiated tumor cells as a source of patient-specific antigens and utilizing encapsulated cell technology for localized, sustained delivery of granulocyte-macrophage colony-stimulating factor as an adjuvant, the study shows a very good safety and feasibility profile. This innovative approach holds the promise of addressing tumor heterogeneity by taking advantage of each patient's antigenic repertoire.

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The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.

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[This corrects the article DOI: 10.1016/j.omtm.2022.07.017.].

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Encapsulated cell therapy holds a great potential to deliver sustained levels of highly potent therapeutic proteins to patients and improve chronic disease management. A versatile encapsulation device that is biocompatible, scalable, and easy to administer, retrieve, or replace has yet to be validated for clinical applications. Here, we report on a cargo-agnostic, macroencapsulation device with optimized features for protein delivery. It is compatible with adherent and suspension cells, and can be administered and retrieved without burdensome surgical procedures. We characterized its biocompatibility and showed that different cell lines producing different therapeutic proteins can be combined in the device. We demonstrated the ability of cytokine-secreting cells encapsulated in our device and implanted in human skin to mobilize and activate antigen-presenting cells, which could potentially serve as an effective adjuvant strategy in cancer immunization therapies. We believe that our device may contribute to cell therapies for cancer, metabolic disorders, and protein-deficient diseases.

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Nowadays, combination antiretroviral therapy (cART) is the standard treatment for all people with human immunodeficiency virus (HIV-1). Although cART is effective in treating productive infection, it does not eliminate latent reservoirs of the virus. This leads to lifelong treatment associated with the occurrence of side effects and the development of drug-resistant HIV-1. Suppression of viral latency is therefore the major hurdle to HIV-1 eradication. Multiple mechanisms exist to regulate viral gene expression and drive the transcriptional and post-transcriptional establishment of latency. Epigenetic processes are amongst the most studied mechanisms influencing both productive and latent infection states. The central nervous system (CNS) represents a key anatomical sanctuary for HIV and is the focal point of considerable research efforts. However, limited and difficult access to CNS compartments makes understanding the HIV-1 infection state in latent brain cells such as microglial cells, astrocytes, and perivascular macrophages challenging. This review examines the latest advances on epigenetic transformations involved in CNS viral latency and targeting of brain reservoirs. Evidence from clinical studies as well as in vivo and in vitro models of HIV-1 persistence in the CNS will be discussed, with a special focus on recent 3D in vitro models such as human brain organoids. Finally, the review will address therapeutic considerations for targeting latent CNS reservoirs.

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[This corrects the article DOI: 10.3389/fcimb.2023.1190867.].

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Immunogenicity, defined as the ability to provoke an immune response, can be either wanted (i.e., vaccines) or unwanted. The latter refers to an immune response to protein or peptide therapeutics, characterized by the production of anti-drug antibodies, which may affect the efficacy and/or the safety profiles of these drugs. Consequently, evaluation of the risk of immunogenicity early in the development of biotherapeutics is of critical importance for defining their efficacy and safety profiles. Here, we describe and validate a fit-for-purpose FluoroSpot-based in vitro assay for the evaluation of drug-specific T cell responses. A panel of 24 biotherapeutics with a wide range of clinical anti-drug antibody response rates were tested in this assay. We demonstrated that using suitable cutoffs and donor cohort sizes, this assay could identify most of the compounds with high clinical immunogenicity rates (71% and 78% for sensitivity and specificity, respectively) while we characterized the main sources of assay variability. Overall, these data indicate that the dendritic cell and CD4+ T cell restimulation assay published herein could be a valuable tool to assess the risk of drug-specific T cell responses and contribute to the selection of clinical candidates in early development.

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Despite many promising results obtained in previous preclinical studies, the clinical development of encapsulated cell technology (ECT) for the delivery of therapeutic proteins from macrocapsules is still limited, mainly due to the lack of an allogeneic cell line compatible with therapeutic application in humans. In our work, we generated an immortalized human myoblast cell line specifically tailored for macroencapsulation. In the present report, we characterized the immortalized myoblasts and described the engineering process required for the delivery of functional therapeutic proteins including a cytokine, monoclonal antibodies and a viral antigen. We observed that, when encapsulated, the novel myoblast cell line can be efficiently frozen, stored, and thawed, which limits the challenge imposed by the manufacture and supply of encapsulated cell-based therapeutic products. Our results suggest that this versatile allogeneic cell line represents the next step toward a broader development and therapeutic use of ECT.

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BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC). METHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4+ T-cell models and ex vivo cultures of PBMCs from HIV+ individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24Gag protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. FINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations. INTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV+ patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies. FUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin ¼, the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation ¼), « Les Amis des Instituts Pasteur à Bruxelles, asbl ¼, the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.

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Suicide Gene Therapy (SGT) aims to introduce a gene encoding either a toxin or an enzyme making the targeted cell more sensitive to chemotherapy. SGT represents an alternative approach to combat pathologies where conventional treatments fail such as pancreatic cancer or the high-grade glioblastoma which are still desperately lethal. We review the possibility to use SGT to treat these cancers which have shown promising results in vitro and in preclinical trials. However, SGT has so far failed in phase III clinical trials thus further improvements are awaited. We can now take advantages of the many advances made in SGT for treating cancer to combat other pathologies such as HIV-1 infection. In the review we also discuss the feasibility to add SGT to the therapeutic arsenal used to cure HIV-1-infected patients. Indeed, preliminary results suggest that both productive and latently infected cells are targeted by the SGT. In the last section, we address the limitations of this approach and how we might improve it.

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Reducing the pool of HIV-1 reservoirs in patients is a must to achieve functional cure. The most prominent HIV-1 cell reservoirs are resting CD4 + T cells and brain derived microglial cells. Infected microglial cells are believed to be the source of peripheral tissues reseedings and the emergence of drug resistance. Clearing infected cells from the brain is therefore crucial. However, many characteristics of microglial cells and the central nervous system make extremely difficult their eradication from brain reservoirs. Current methods, such as the "shock and kill", the "block and lock" and gene editing strategies cannot override these difficulties. Therefore, new strategies have to be designed when considering the elimination of brain reservoirs. We set up an original gene suicide strategy using latently infected microglial cells as model cells. In this paper we provide proof of concept of this strategy.

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A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China at the end of 2019 causing a large global outbreak. As treatments are of the utmost importance, drug repurposing embodies a rich and rapid drug discovery landscape, where candidate drug compounds could be identified and optimized. To this end, we tested seven compounds for their ability to reduce replication of human coronavirus (HCoV)-229E, another member of the coronavirus family. Among these seven drugs tested, four of them, namely rapamycin, disulfiram, loperamide and valproic acid, were highly cytotoxic and did not warrant further testing. In contrast, we observed a reduction of the viral titer by 80% with resveratrol (50% effective concentration (EC50) = 4.6 µM) and lopinavir/ritonavir (EC50 = 8.8 µM) and by 60% with chloroquine (EC50 = 5 µM) with very limited cytotoxicity. Among these three drugs, resveratrol was less cytotoxic (cytotoxic concentration 50 (CC50) = 210 µM) than lopinavir/ritonavir (CC50 = 102 µM) and chloroquine (CC50 = 67 µM). Thus, among the seven drugs tested against HCoV-229E, resveratrol demonstrated the optimal antiviral response with low cytotoxicity with a selectivity index (SI) of 45.65. Similarly, among the three drugs with an anti-HCoV-229E activity, namely lopinavir/ritonavir, chloroquine and resveratrol, only the latter showed a reduction of the viral titer on SARS-CoV-2 with reduced cytotoxicity. This opens the door to further evaluation to fight Covid-19.

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