Therapeutic Antibodies Research Articles

Challenges and compromises: Predicting unbound antibody structures with deep learning.

Therapeutic antibodies are manufactured, stored and administered in the free state; this makes understanding the unbound form key to designing and improving development pipelines. Prediction of unbound antibodies is challenging, specifically modelling of the CDRH3 loop, where inaccuracies are potentially worse due to a bias in structural data towards antibody-antigen complexes. This class imbalance provides a challenge for deep learning models trained on this data, potentially limiting generalisation to unbound forms. Here we discuss the importance of unbound structures in antibody development pipelines. We explore how the latest generation of structure predictors can provide new insights and assess how conformational heterogeneity may influence binding kinetics. We hypothesise that generative models may address some of these issues. While prediction of antibodies in complex is essential, we should not ignore the need for progress in modelling the unbound form.

Development and characterization of a reverse genetics system for the lineage II Chicava strain of Machupo virus in a guinea pig model.

Machupo virus (MACV) is a New World mammarenavirus (hereafter referred to as "arenavirus") and the etiologic agent of Bolivian hemorrhagic fever (BHF). No vaccine or antiviral therapy exists for BHF, which causes up to 35% mortality in humans. New World arenaviruses evolve separately in different locations. To date, up to eight lineages of MACV have been identified in Bolivia. While the prototype MACV Carvallo strain belongs to lineage I discovered in the Memore Province in the 1960, the MACV lineage II strains have become the dominantly-circulating lineage in the same province since 1993. We report the development of a reverse genetics system for the MACV lineage II Chicava strain, using a pRF42 plasmid encoding the L and S segment genomic RNA under the transcriptional control of a murine DNA-dependent RNA polymerase I promoter sequence. Rescue of the recombinant MACV Chicava strain (rMACV-Chicava) was accomplished by expression of the L protein and nucleoprotein genes of the MACV Carvallo strain in trans in transfected baby hamster kidney (BHK-21) cells. We characterized the multiplication kinetics of rMACV-Chicava in African green monkey kidney epithelial Vero cells, followed by determining the virulence phenotype in outbred Hartley guinea pigs. We demonstrated that the multiplication kinetics in Vero cells, virulence phenotype in guinea pigs, and neutralizing antibody titers are indistinguishable between rMACV-Chicava and the wild-type parental virus. We conclude that rMACV-Chicava provides a useful model system to investigate the emergence of MACV lineage II strains and the guinea pig model has utility for the development of candidate vaccines and therapeutic antibodies for BHF.

Impact of Various Forced Oxidative Stress Factors in Rapid Degradation of mAb: Trastuzumab as a Case Study.

Therapeutic monoclonal antibodies (mAbs) are prone to degradation viaaggregation and fragmentation. In this study, forced degradation of trastuzumab (TmAb) was explored in saline and in-vitro models having H2O2 and exposed to UV light (case study 1), both bleomycin (BML) formulation and ferrous ions (Fe2+) (case study 2), and sodium hypochlorite (NaOCl) (case study 3). Size exclusion chromatography, dynamic light scattering, spectroscopic analysis, and fluorescence microscope image processing was carried out for characterizing TmAb degradation. Saline samples containing TmAb and 0.1% H2O2 incubated at 40ºC for 1h in the presence of UV light showed increased monomer loss by more than 40% compared to TmAb sample without H2O2 exposed to UV light. Saline containing TmAb having both 0.1-unit BML and 0.25mM Fe2+ showed increased monomer loss by more than 50% compared to TmAb in saline having only Fe2+ or BML. A higherTmAb degradation was also observed in saline containing 0.01% NaOCl compared to saline without NaOCl. Samples containing aggregates of mAb showed altered protein structure. Degradation of TmAb in saline increased with time, temperature, and concentrations of H2O2, Fe2+, and NaOCl. At different analysis time points, TmAb monomer loss was higher in saline compared to human serum filtrate, an in-vitro model. Aggregate particles (> 2µm size) of TmAb were also observed in serum containing both Fe2+ and BML. It can be concluded that rapid TmAb degradation significantly enhanced due to various stress factors, and the aggregates couldresult in enhanced immunogenic risk to the patients.

Nanobody screening and machine learning guided identification of cross-variant anti-SARS-CoV-2 neutralizing heavy-chain only antibodies.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to persist, demonstrating the risks posed by emerging infectious diseases to national security, public health, and the economy. Development of new vaccines and antibodies for emerging viral threats requires substantial resources and time, and traditional development platforms for vaccines and antibodies are often too slow to combat continuously evolving immunological escape variants, reducing their efficacy over time. Previously, we designed a next-generation synthetic humanized nanobody (Nb) phage display library and demonstrated that this library could be used to rapidly identify highly specific and potent neutralizing heavy chain-only antibodies (HCAbs) with prophylactic and therapeutic efficacy in vivo against the original SARS-CoV-2. In this study, we used a combination of high throughput screening and machine learning (ML) models to identify HCAbs with potent efficacy against SARS-CoV-2 viral variants of interest (VOIs) and concern (VOCs). To start, we screened our highly diverse Nb phage display library against several pre-Omicron VOI and VOC receptor binding domains (RBDs) to identify panels of cross-reactive HCAbs. Using HCAb affinity for SARS-CoV-2 VOI and VOCs (pre-Omicron variants) and model features from other published data, we were able to develop a ML model that successfully identified HCAbs with efficacy against Omicron variants, independent of our experimental biopanning workflow. This biopanning informed ML approach reduced the experimental screening burden by 78% to 90% for the Omicron BA.5 and Omicron BA.1 variants, respectively. The combined approach can be applied to other emerging viruses with pandemic potential to rapidly identify effective therapeutic antibodies against emerging variants.

A Cell-penetrating bispecific antibody suppresses hepatitis B virus replication and secretion.

Hepatitis B virus (HBV) represents one of the major pathogenic factor that leads to chronic liver diseases and the development of hepatocellular carcinoma (HCC). The currently approved anti-HBV drugs cannot eradicate the virus or block the development of HCC. HBV nucleocapsid consists of the hepatitis B core antigen (HBcAg) and the HBV relaxed-circular partially double-stranded DNA (rcDNA), indispensable in virus replication. The present study reported a cell-penetrating bispecific antibody targeting HBcAg and preS1, fused with the cell-penetrating peptide R9TAT, named Anti-preS1 × Anti-HBcAg-R9TAT. The antibody could recognize preS1 and HBcAg and internalize into living cells efficiently, suppressing the extracellular hepatitis B surface antigen (HBsAg) and hepatitis B envelope antigen, and the intracellular HBsAg and HBcAg in vitro. This cell-penetrating bispecific antibody is a novel approach to suppressing HBV replication and secretion and is a promising anti-HBV therapeutic antibody candidate.

A design space for the filtration of challenging monoclonal antibodies using Planova™ S20N, a new regenerated cellulose virus removal filter.

Virus removal by filtration is a crucial step in ensuring the safety of therapeutic antibodies and other biopharmaceutical products by mitigating the risk of endogenous and adventitious viral contamination. However, there are monoclonal antibodies (mAb) that are difficult to filter effectively using virus removal filters (i.e., challenging mAbs), necessitating the creation of guidelines for designing suitable filtration conditions for these challenging molecules. This study presents a filtration design space for filtration conditions using a new regenerated cellulose membrane filter with a representative challenging mAb. The filter demonstrated that filtration throughput is adaptable across a wide range of conditions for low to medium mAb concentrations, indicating its suitability for introduction into platform processes for related biopharmaceutical products.

Potential risk factors of protein aggregation in syringe handling during antibody drug dilution for intravenous administration.

Protein aggregation, a major concern in biopharmaceutical quality control, can be accelerated by various stresses during clinical handling. This study investigated potential aggregation risk factors during dilution process with syringe handling for intravenous administration. Using γ-globulin and IgG solutions as surrogate models of antibody therapeutics, we examined the effects of high sliding speeds and piston operations of the syringe on protein aggregation during saline dilution. Our results revealed that elevated sliding speeds promoted proteinaceous subvisible and/or visible particle formation, which was further enhanced by piston operations. The proteinaceous particle formation was presumed to be caused by fine air bubbles generated due to rapid pressure changes arising from shear stress during needle passage. While polysorbate 20 effectively suppressed the particle formation induced by the syringe handling at sufficient concentrations, its protective effect became inadequate under high dilution conditions, as exemplified by those encountered in low-body-weight patient protocols. Different proteins exhibited varying susceptibility to the syringe-induced aggregation. These findings demonstrate that the combination of syringe handling and dilution conditions could significantly impact protein stability during clinical handling, particularly for less stable biopharmaceuticals. A deeper understanding of these factors is crucial for developing more robust formulations and establishing safer handling practices for biopharmaceuticals.

A single droplet dispensing system for high-throughput screening and reliable recovery of rare events.

Microfluidic droplet sorting has emerged as a powerful technique for a broad spectrum of biomedical applications ranging from single cell analysis to high-throughput drug screening, biomarker detection and tissue engineering. However, the controlled and reliable retrieval of selected droplets for further off-chip analysis and processing is a significant challenge in droplet sorting, particularly in high-throughput applications with low expected hit rates. In this study, we present a microfluidic platform capable of sorting and dispensing individual droplets with minimal loss rates. We demonstrate our direct transfer mechanism by placing selected droplets containing hybridoma cells into microwells, eliminating the need for manual and often lossy handling steps. Sorted droplets are dispensed via a novel 3D-printed dispensing nozzle, enabling precise and controlled placement of selected single droplets into individual wells without affecting the microfluidic sorting flow. The sorting and transfer process is monitored in real time, which provides feedback and quality control of the entire workflow. Our integrated microfluidic system holds great potential for applications requiring high-throughput droplet sorting with minimal sample loss and precise dispensing into microwells, such as screening for therapeutical antibodies or monoclonal cells.

Cathepsins: Novel opportunities for antibody therapeutics in cancer.

Cathepsins, the most abundant lysosomal proteases, have key functions in cell maintenance and homeostasis. They are overexpressed and hypersecreted in cancer and associated with poor prognosis. Secreted cathepsins display pro-tumour activities in the tumour microenvironment and thus represent interesting molecular targets in oncology. Recently, several antibody-based cancer therapies have targeted the pro-tumour activity of the extracellular cathepsin pool, altering several cancer hallmarks, but not the intracellular cathepsin levels that are often crucial for cell homeostasis. In this mini-review, we describe advances in antibodies against extracellular cathepsins in cancer, and their effect on the proteolytic cascade, matrix remodelling, proliferation, and modulation of the anti-cancer immune response. We also discuss the add-on value of combination strategies (anti-cathepsin antibodies with chemotherapy and/or biologics) that make anti-cathepsin antibodies a new opportunity for disease management.

Monitoring M-Protein, Therapeutic Antibodies, and Polyclonal Antibodies in a Multiparametric Mass Spectrometry Assay Provides Insight into Therapy Response Kinetics in Patients with Multiple Myeloma.

Background/Objectives: Multiple Myeloma (MM) is a hematologic malignancy caused by clonally expanded plasma cells that produce a monoclonal immunoglobulin (M-protein), a personalized biomarker. Recently, we developed an ultra-sensitive mass spectrometry method to quantify minimal residual disease (MS-MRD) by targeting unique M-protein peptides. Therapeutic antibodies (t-Abs), key in MM treatment, often lead to deep and long-lasting responses. However, t-Abs can significantly decrease the total polyclonal immunoglobulin (Ig) levels which require supplemental IgG infusion. Here, we demonstrate the simultaneous monitoring of M-proteins, t-Abs, and polyclonal Ig-titers using an untargeted mass spectrometry assay, offering a comprehensive view of therapy response. Methods: Sera collected between 2013 and 2024 from four patients and cerebrospinal fluid (CSF) from one patient who received various t-Abs were analyzed with MS-MRD. M-protein sequences were obtained with a multi-enzyme de novo protein sequencing approach. Unique peptides for M-proteins and t-Abs were selected based on linearity, sensitivity, and slope coefficient in serial dilutions. Ig constant regions were monitored using isotype-specific peptides. Results: The MS-MRD multiplex analysis provided detailed information on drug concentrations and therapy response kinetics. For example, in two patients with refractory disease over five lines of therapy, the MS-MRD analysis showed that the deepest responses were achieved with bispecific t-Ab (teclistamab) treatment. M-protein and t-Ab were also detectable in the CSF of one patient with MS-MRD. Conclusions: This proof-of-concept study shows that the multiplex monitoring of the M-protein, any t-Ab combination, and all Ig-isotypes within one mass spectrometry run is feasible and provides unique insight into therapy response kinetics.

Use of Quantum Dots as Nanotheranostic Agents: Emerging Applications in Rare Genetic Diseases.

Rare genetic diseases (RGDs) affect a small percentage of the global population but collectively have a substantial impact due to their diverse manifestations. Although the precise reasons behind these diseases remain unclear, roughly 80% of cases are genetically linked. Recent efforts focus on understanding pathology and developing new diagnostic and therapeutic approaches for RGDs. However, there persists a gap between fundamental research and clinical therapeutic approaches, where advancements innanotechnology offer promising improvements. In this context, nanosized light-emitting quantum dots (QDs), ranging from 2-10nm, are promising materials for diverse applications. Their size-tunable light emission, high quantum yield, and photostability allow for precise tracking of cargo. Additionally, QDs can be functionalized with therapeutic agents, antibodies, or peptides to target specific cellular pathways, enhancing treatment efficacy while minimizing side effects. By combining diagnostic and therapeutic capabilities in a single platform, QDs thus offer a versatile and powerful approach to tackle rare genetic disorders. Despite several reviews on various therapeutic applications of QDs, their utilization in the specific domain of RGDs is not well documented. This review highlight QDs' potential in diagnosing and treating certain RGDs and addresses the challenges limiting their application.

Improved Structure-Based Histidine pKa Prediction for pH-Responsive Protein Design.

The near neutral pKa of histidine is commonly exploited to engineer pH-sensitive biomolecules. For example, histidine mutations introduced in the complementarity-determining region (CDR) of therapeutic antibodies can enhance selectivity for antigens in the acidic microenvironment of solid tumors or increase dissociation rates in the acidic early endosomes of cells. While solvent-exposed histidines typically have a pKa near 6.5, interacting histidines can experience pKa shifts of up to 4 pH units in either direction, making histidine one of the most variable titratable residues. To assist in selecting potential histidine mutation sites, pKa prediction software should achieve an accuracy significantly better than the current standard of around 1.0 pH unit. However, the limited availability of experimental histidine pKa measurements hinders the use of AI-based methods. This study evaluates histidine pKa predictions using Amber force field electrostatics combined with a continuum solvent model, previously calibrated in the solvated interaction energy (SIE) function for binding affinity predictions. By incorporating limited rotameric sampling, proton optimization, and an empirical correction for buried side-chains, the method achieves a mean unsigned error of 0.4 pH units across a diverse set of 91 histidines from 38 distinct protein structures obtained from the PKAD database. This approach should improve the in-silico design of pH-responsive mutations. The method is implemented in the software program JustHISpKa (https://mm.nrc-cnrc.gc.ca/software/JustHISpKa).

A type of cryptic epitope-binding antibody on SARS-CoV-2 RBD retains the neutralization against SARS-CoV-2 variants and sarbecoviruses.

The continuing emergence of SARS-CoV-2 variants has posed a great challenge to vaccination strategies. Therefore, the development of broad-spectrum protective antibodies and universal vaccines remains urgently needed. In this study, we isolated two broadly neutralizing mAbs, nCoV-R48 and nCoV-R70, from a vaccinated person. These two mAbs neutralize the SARS-CoV-2 prototype and variants, including Alpha, Beta, Gamma, Delta, BQ1.1, XBB, BF.7, and the recently emerged KP.2. Moreover, nCoV-R48 and nCoV-R70 also neutralize representative sarbecoviruses GD/1/2019 and SARS-CoV. We determined the epitopes of these two mAbs using X-ray crystallography and Cryo-EM. The structural analysis shows that nCoV-R48 and nCoV-R70 recognize the same cryptic epitope on the RBD. These antibodies belong to the RBD-8 class, which exhibits broad neutralizing potency by inducing S1 shedding. This cryptic epitope provides guidance for developing universal therapeutic antibodies against COVID-19.

Current Status of Gout Arthritis: Current Approaches to Gout Arthritis Treatment: Nanoparticles Delivery Systems Approach.

The deposition of monosodium urate (MSU) crystals within joint spaces produces a painful inflammatory condition known as gout, a specific form of arthritis. The condition calls for a combined curative and preventive management model. A new development in the approach to gout is that of NLRP3-targeted biologic agents, such as monoclonal therapies, to provide more accurate treatment by blocking specific pro-inflammatory cytokines. Nanoparticle drug delivery enhances biological availability and delivery to targets, which may increase therapeutic efficacy and decrease general toxicity. The preventive approach again cannot be ignored, mainly keeping up certain modifications in diet and weight, along with pharmacological therapies to reduce uric acid (UA) levels and to decrease the frequency of acute attacks. The advancement of genetic profiling of patients and biomarker discoveries drives the trend towards building individualized medicine and care, quickly gaining ground as the most effective method of delivering treatments to individual patients, moving away from one-size-fits-all treatments. The following paper aims to provide an updated account of the management of gout with a focus on recent developments, in order to enhance these approaches, the quality of life for patients with gout, and the standard of gout treatment.

A comparison between different chemical fractionation methods for immunoglobulin preparation

ABSTRACT Background Application of antibodies in therapeutics and diagnostics are growing Continually. Herein, we aimed to find the most qualified immunoglobulin (Ig) chemical preparation method. Methods A rabbit was immunized against recombinant SARS-CoV-2 nucleocapsid (NP) and reactive polyclonal antibodies were prepared using the ammonium sulfate (AS), caprylic acid (CA), polyethylene glycol (PEG), and caprylic acid/ammonium sulfate (CA/AS) methods. Different antibody solutions were analyzed by SDS-PAGE and subsequently quantified by ImageJ software for further analysis in terms of Ig purity, Ig recovery, and albumin impurity. Ultimately, the prepared antibodies were assessed via Western blotting and ELISA to evaluate their ability to bind NP. Results Prepared Ig solutions via the CA/AS method had the highest Ig purity (followed by CA, PEG, and AS) and lowest albumin impurity (followed by CA, AS, and PEG). The PEG method had the highest recovery followed by AS, CA, and CA/AS methods. Moreover, antibodies prepared via different methods demonstrated comparable binding capacities to NP in ELISA and Western blotting. Conclusions CA/AS, closely followed by CA, proved to be the most qualified method for the preparation of Ig yielding the highest Ig purity while the PEG method resulted in the highest Ig recovery rate.

Emerging Trends in Neuroblastoma Diagnosis, Therapeutics, and Research.

This review explores the current understanding and recent advancements in neuroblastoma, one of the most common extracranial solid pediatric cancers, accounting for ~ 15% of childhood cancer-related mortality. The hallmarks of NBL, including angiogenesis, metastasis, apoptosis resistance, cell cycle dysregulation, drug resistance, and responses to hypoxia and ROS, underscore its complex biology. The tumor microenvironment's significance in disease progression is acknowledged in this study, along with the pivotal role of cancer stem cells in sustaining tumor growth and heterogeneity. A number of molecular signatures are being studied in order to better understand the disease, with many of them serving as targets for the development of new therapeutics. This includes inhibitor therapies for NBL patients, which notably concentrate on ALK signaling, MDM2, PI3K/Akt/mTOR, Wnt, and RAS-MAPK pathways, along with regulators of epigenetic mechanisms. Additionally, this study offers an extensive understanding of the molecular therapies used, such as monoclonal antibodies and CAR-T therapy, focused on both preclinical and clinical studies. Radiation therapy's evolving role and the promise of stem cell transplantation-mediated interventions underscore the dynamic landscape of NBL treatment. This study has also emphasized the recent progress in the field of diagnosis, encompassing the adoption of artificial intelligence and liquid biopsy as a non-intrusive approach for early detection and ongoing monitoring of NBL. Furthermore, the integration of innovative treatment approaches such as CRISPR-Cas9, and cancer stem cell therapy has also been emphasized in this review.

Decoding the molecular interplay of CD20 and therapeutic antibodies with fast volumetric nanoscopy.

Elucidating the interaction between membrane proteins and antibodies requires whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) achieves molecular resolution but is restricted to two-dimensional imaging owing to long acquisition times. We have developed two-dye imager (TDI) probes that enable ~15-fold faster imaging. Combining TDI-DNA-PAINT and LLS microscopy on immunological B cells revealed the oligomeric states and interaction of endogenous CD20 with the therapeutic monoclonal antibodies (mAbs) rituximab, ofatumumab, and obinutuzumab. Our results demonstrate that CD20 is abundantly expressed on microvilli that bind mAbs, which leads to an antibody concentration-dependent B cell polarization and stabilization of microvilli protrusions. These findings could aid rational design of improved immunotherapies targeting tumor-associated antigens.

Advanced Polymeric Nanoparticles for Cancer Immunotherapy: Materials Engineering, Immunotherapeutic Mechanism and Clinical Translation.

Cancer immunotherapy, which leverages immune system components to treat malignancies, has emerged as a cornerstone of contemporary therapeutic strategies. Yet, critical concerns about the efficacy and safety of cancer immunotherapies remain formidable. Nanotechnology, especially polymeric nanoparticles (PNPs), offers unparalleled flexibility in manipulation-from the chemical composition and physical properties to the precision control of nanoassemblies. PNPs provide an optimal platform to amplify the potency and minimize systematic toxicity in a broad spectrum of immunotherapeutic modalities. In this comprehensive review, the basics of polymer chemistry, and state-of-the-art designs of PNPs from a physicochemical standpoint for cancer immunotherapy, encompassing therapeutic cancer vaccines, in situ vaccination, adoptive T-cell therapies, tumor-infiltrating immune cell-targeted therapies, therapeutic antibodies, and cytokine therapies are delineated. Each immunotherapy necessitates distinctively tailored design strategies in polymeric nanoplatforms. The extensive applications of PNPs, and investigation of their mechanisms of action for enhanced efficacy are particularly focused on. The safety profiles of PNPs and clinical research progress are discussed. Additionally, forthcoming developments and emergent trends of polymeric nano-immunotherapeutics poised to transform cancer treatment paradigms into clinics are explored.

Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D.

The naturally occurring mutation E484D in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can render viral entry ACE2 independent and imdevimab resistant. Here, we investigated whether the cellular proteins ASGR1, DC-SIGN, and TMEM106B, which interact with the viral S protein, can contribute to these processes. Employing S protein-pseudotyped particles, we found that expression of ASGR1 or DC-SIGN jointly with TMEM106B allowed for robust entry of mutant E484D into otherwise non-susceptible cells, while this effect was not observed upon separate expression of the single proteins and upon infection with SARS-CoV-2 wild type (WT). Furthermore, expression of ASGR1 or DC-SIGN conferred ACE2 independence and imdevimab resistance to entry of mutant E484D but not WT, and entry under those conditions was dependent on endogenous TMEM106B. These results suggest that engagement of certain cellular lectins can direct SARS-CoV-2 mutant E484D to an ACE2-independent, TMEM106B-dependent entry pathway that is not inhibited by imdevimab.IMPORTANCEThe interaction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with the ACE2 receptor determines the viral cell tropism and is the key target of the neutralizing antibody response. Here, we show that SARS-CoV-2 with a single, naturally occurring mutation in the spike protein, E484D, can use the cellular lectins ASGR1 and DC-SIGN in conjunction with TMEM106B for ACE2-independent entry and evasion of therapeutic antibodies. These results suggest that engagement of cellular lectins might modulate target cell choice of SARS-CoV-2 and might allow evasion of certain neutralizing antibodies.

Simultaneous Blockade of CD209 and CD209L by Monoclonal Antibody Does Not Provide Sufficient Protection Against Multiple Viral Infections InVivo.

Many virus species, including Ebola virus, Marburg virus, SARS-CoV-2, dengue virus (DENV) and Zika virus (ZIKV), exploit CD209 and CD209L as alternative or attachment receptors for viral cis- or trans-infection. Thus, CD209 and CD209L may be critical targets for the development of therapeutic monoclonal blocking antibody drugs to disrupt the infection process caused by multiple viruses. Here, we produced a human chimeric monoclonal blocking antibody that simultaneously blocks CD209 and CD209L, namely 7-H7-B1. We show that 7-H7-B1 effectively blocks multiple pseudotyped or live viral infections invitro, including SARS-CoV, SARS-CoV-2, Ebola virus, Marburg virus, ZIKV and DENV infections. However, the 7-H7-B1 mAb does not provide favourable protection against Zaire Ebola virus or ZIKV infection in hCD209 knock-in mice invivo. Thus, our findings indicate that although CD209 and CD209L are critical for multiple viral infections invitro, they may play only a partial role in viral infections invivo.