Human Neutralizing Monoclonal Antibodies Research Articles

Plant production of recombinant antigens containing the receptor binding domain (RBD) of two SARS-CoV-2 variants.

The aim of this work was to rapidly produce in plats two recombinant antigens (RBDw-Fc and RBDo-Fc) containing the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 variants Wuhan and Omicron as fusion proteins to the Fc portion of a murine IgG2a antibody constant region (Fc). The two recombinant antigens were expressed in Nicotiana benthamiana plants, engineered to avoid the addition of N-linked plant-typical sugars, through vacuum agroinfiltration and showed comparable purification yields (about 35mg/kg leaf fresh weight). Their Western blotting and Coomassie staining evidenced the occurrence of major in planta proteolysis in the region between the RBD and Fc, which was particularly evident in RBDw-Fc, the only antigen bearing the HRV 3C cysteine protease recognition site. The two RBD N-linked glycosylation sites showed very homogeneous profiles free from plant-typical sugars, with the most abundant glycoform represented by the complex sugar GlcNAc4Man3. Both antigens were specifically recognised in Western Blot analysis by the anti-SARS-CoV-2 human neutralizing monoclonal antibody J08-MUT and RBDw-Fc was successfully used in competitive ELISA experiments for binding to the angiotensin-converting enzyme 2 receptor to verify the neutralizing capacity of the serum from vaccinated patients. Both SARS-Cov-2 antigens fused to a murine Fc region were rapidly and functionally produced in plants with potential applications in diagnostics.

Antiviral mechanisms of two broad-spectrum monoclonal antibodies for rabies prophylaxis and therapy.

Rabies is an acute and lethal encephalomyelitis caused by lyssaviruses, among which rabies virus (RABV) is the most prevalent and important for public health. Although preventable through the post-exposure administration of rabies vaccine and immunoglobulins (RIGs), the disease is almost invariably fatal since the onset of clinical signs. Two human neutralizing monoclonal antibodies (mAbs), RVC20 and RVC58, have been shown to be effective in treating symptomatic rabies. To better understand how these mAbs work, we conducted structural modeling and in vitro assays to analyze their mechanisms of action, including their ability to mediate Fc-dependent effector functions. Our results indicate that both RVC20 and RVC58 recognize and lock the RABV-G protein in its pre-fusion conformation. RVC58 was shown to neutralize more potently the extra-cellular virus, while RVC20 mainly acts by reducing viral spreading from infected cells. Importantly, RVC20 was more effective in promoting effector functions compared to RVC58 and 17C7-RAB1 mAbs, the latter of which is approved for human rabies post-exposure treatment. These results provide valuable insights into the multiple mechanisms of action of RVC20 and RVC58 mAbs, offering relevant information for the development of these mAbs as treatment for human rabies.

A fully human neutralizing monoclonal antibody targeting a highly conserved epitope of the human cytomegalovirus glycoprotein B.

Human cytomegalovirus causes severe diseases in children (by congenital infection) and immunocompromised patients. Treatment with antiviral agents, such as ganciclovir, is limited by their toxicity. In this study, we investigated the effectiveness of a fully human neutralizing monoclonal antibody to inhibit human cytomegalovirus infection and viral cell-to-cell spread. We isolated a potent neutralizing antibody, EV2038 (IgG1 lambda), targeting human cytomegalovirus glycoprotein B using Epstein-Barr virus transformation. This antibody inhibited human cytomegalovirus infection by all four laboratory strains and 42 Japanese clinical isolates, including ganciclovir-resistant isolates, with a 50% inhibitory concentration (IC50) ranging from 0.013 to 0.105 μg/mL, and 90% inhibitory concentration (IC90) ranging from 0.208 to 1.026 μg/mL, in both human embryonic lung fibroblasts (MRC-5) and human retinal pigment epithelial (ARPE-19) cells. Additionally, EV2038 prevented cell-to-cell spread of eight clinical viral isolates, with IC50 values ranging from 1.0 to 3.1 μg/mL, and IC90 values ranging from 13 to 19 μg/mL, in ARPE-19 cells. EV2038 recognized three discontinuous sequences on antigenic domain 1 of glycoprotein B (amino acids 549-560, 569-576, and 625-632), which were highly conserved among 71 clinical isolates from Japan and the United States. Pharmacokinetics study in cynomolgus monkeys suggested the potential efficacy of EV2038 in vivo, the concentration of which in serum remained higher than the IC90 values of cell-to-cell spread until 28 days after intravenous injection of 10 mg/kg EV2038. Our data strongly support EV2038 as a promising candidate and novel alternative for the treatment of human cytomegalovirus infection.

Potent universal beta-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel β-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human β-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human β-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.

Growth, Antigenicity, and Immunogenicity of SARS-CoV-2 Spike Variants Revealed by a Live rVSV-SARS-CoV-2 Virus.

SARS-CoV-2 is an emerging coronavirus threatening human health and the economy worldwide. As an RNA virus, variants emerge during the pandemic and potentially influence the efficacy of the anti-viral drugs and vaccines. Eight spike variants harboring highly recurrent mutations were selected and introduced into a replication-competent recombinant VSV in place of the original G protein (rVSV-SARS-CoV-2). The resulting mutant viruses displayed similar growth curves in vitro as the wild-type virus and could be neutralized by sera from convalescent COVID-19 patients. Several variants, especially Beta strain, showed resistance to human neutralizing monoclonal antibodies targeting the receptor-binding domain (RBD). A single dose of rVSV-SARS-CoV-2 Beta variant could elicit enhanced and broad-spectrum neutralizing antibody responses in human ACE2 knock-in mice and golden Syrian hamsters, while other mutants generated antibody levels comparable to the wild-type. Therefore, our results will be of value to the development of next-generation vaccines and therapeutic antibodies.

Broad-Spectrum Anti-coronavirus Vaccines and Therapeutics to Combat the Current COVID-19 Pandemic and Future Coronavirus Disease Outbreaks.

While the COVID-19 pandemic caused by SARS-CoV-2 is continuing, it may become worse in the coming winter months with a high potential for the emergence and spread of escape variants of SARS-CoV-2. SARS-related CoVs (SARSr-CoVs) from bats may also cause outbreaks of emerging coronavirus diseases in the future. These predictions call for the development of broad-spectrum anti-coronavirus vaccines and therapeutics to combat the current COVID-19 pandemic and future emerging coronavirus disease epidemics. In this review, we describe advances and challenges in the development of broad-spectrum vaccines and neutralizing antibodies against lineage B betacoronaviruses (β-CoV-Bs), including SARS-CoV-2, SARS-CoV, and SARSr-CoVs, as well as peptide-based pan-CoV fusion inhibitors and their potential in the prevention and treatment of COVID-19 and other human coronavirus infections.

In Silico Antibody Mutagenesis for Optimizing Its Binding to Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2.

Coronavirus disease 2019 (COVID-19) is an ongoing global pandemic, and there are currently no FDA-approved medicines for treatment or prevention. Inspired by promising outcomes for convalescent plasma treatment, the development of antibody drugs (biologics) to block SARS-CoV-2 infection has been the focus of drug discovery, along with tremendous efforts in repurposing small-molecule drugs. In the past several months, experimentally, many human neutralizing monoclonal antibodies (mAbs) were successfully extracted from plasma of recovered COVID-19 patients. Currently, several mAbs targeting the SARS-CoV-2’s spike glycoprotein (S-protein) are in clinical trials. With known atomic structures of the mAb and S-protein complex, it becomes possible to investigate in silico the molecular mechanism of mAb’s binding with S-protein and to design more potent mAbs through protein mutagenesis studies, complementary to existing experimental efforts. Leveraging today’s superb computing power, we propose a fully automated in silico protocol for quickly identifying possible mutations in a mAb (e.g., CB6) to enhance its binding affinity for S-protein for the design of more efficacious therapeutic mAbs.

A panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD. A subset of the antibodies exert their inhibitory activity by abrogating binding of the RBD to the human ACE2 receptor. The human monoclonal antibodies described here represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.

Adult and Cord Blood-Derived High-Affinity gB-CAR-T Cells Effectively React Against Human Cytomegalovirus Infections.

Human cytomegalovirus (HCMV) reactivations are associated with lower overall survival after transplantations. Adoptive transfer of HCMV-reactive expanded or selected T cells can be applied as a compassionate use, but requires that the human leukocyte antigen-matched donor provides memory cells against HCMV. To overcome this, we developed engineered T cells expressing chimeric antigen receptors (CARs) targeted against the HCMV glycoprotein B (gB) expressed upon viral reactivation. Single-chain variable fragments (scFvs) derived from a human high-affinity gB-specific neutralizing monoclonal antibody (SM5-1) were fused to CARs with 4-1BB (BBL) or CD28 (28S) costimulatory domains and subcloned into retroviral vectors. CD4+ and CD8+ T cells obtained from HCMV-seronegative adult blood or cord blood (CB) transduced with the vectors efficiently expressed the gB-CARs. The specificity and potency of gB-CAR-T cells were demonstrated and compared in vitro using the following: 293T cells expressing gB, and with mesenchymal stem cells infected with a HCMV TB40 strain expressing Gaussia luciferase (HCMV/GLuc). BBL-gB-CAR-T cells generated with adult or CB demonstrated significantly higher in vitro activation and cytotoxicity performance than 28-gB-CAR-T cells. Nod.Rag.Gamma (NRG) mice transplanted with human CB CD34+ cells with long-term human immune reconstitution were used to model HCMV/GLuc infection in vivo by optical imaging analyses. One week after administration, response to BBL-gB-CAR-T cell therapy was observed for 5/8 mice, defined by significant reduction of the bioluminescent signal in relation to untreated controls. Response to therapy was sporadically associated with CAR detection in spleen. Thus, exploring scFv derived from the high-affinity gB-antibody SM5-1 and the 4-1BB signaling domain for CAR design enabled an in vitro high on-target effect and cytotoxicity and encouraging results in vivo. Therefore, gB-CAR-T cells can be a future clinical option for treatment of HCMV reactivations, particularly when memory T cells from the donors are not available.

A novel human anti-BAFF neutralizing monoclonal antibody derived from in vitro immunization

B-cell activating factor (BAFF) plays a key role in the normal regulation of B cell development and immune response. Its abnormal expression level is accompanied by the occurrence of various autoimmune diseases. Therefore, BAFF is an effective target for the treatment of such diseases. Here, we report a new anti-BAFF monoclonal antibody. Based on improved in vitro immunization method, we used a recombinant BAFF containing unnatural amino acid p-nitro-phenylalanine (pNO2Phe) as an antigen to trigger immune response in vitro. The plasma cells were sorted by flow cytometry (FACS), and the antibody library was constructed based on the sorted plasma cells. The high affinity antigen-binding fragments were panned by phage display technology, and finally the anti-BAFF human IgG was obtained. The antibody demonstrated its ability to neutralize BAFF effectively both in vitro and in vivo. We propose that this novel full-length human anti-BAFF monoclonal antibody is a promising therapeutic candidate for the treatment of autoimmune diseases.

A protective human monoclonal antibody targeting the West Nile virus E protein preferentially recognizes mature virions

West Nile virus (WNV), a member of the Flavivirus genus, is a leading cause of viral encephalitis in the United States1. The development of neutralizing antibodies against the flavivirus envelope (E) protein is critical for immunity and vaccine protection2. Previously identified candidate therapeutic mouse and human neutralizing monoclonal antibodies (mAbs) target epitopes within the E domain III lateral ridge and the domain I-II hinge region, respectively3. To explore the neutralizing antibody repertoire elicited by WNV infection for potential therapeutic application, we isolated 10 mAbs from WNV-infected individuals. MAb WNV-86 neutralized WNV with a 50% inhibitory concentration (IC50) of 2 ng/mL, one of the most potently neutralizing flavivirus-specific antibodies ever isolated. WNV-86 targets an epitope in E domain II, and preferentially recognizes mature virions lacking an uncleaved form of the chaperone protein prM, unlike most flavivirus-specific antibodies4. In vitro selection experiments revealed a neutralization escape mechanism involving a glycan addition to E domain II. Finally, a single dose of WNV-86 administered two days post-infection protected mice from lethal WNV challenge. This study identifies a highly potent human neutralizing mAb with therapeutic potential that targets an epitope preferentially displayed on mature virions.

Prophylactic and therapeutic efficacy of mAb treatment against MERS-CoV in common marmosets

The high case-fatality rate of confirmed MERS-CoV infections underlines the urgent need for an effective treatment to reduce the disease severity and mortality. REGN3051 and REGN3048 are two fully human neutralizing monoclonal antibodies (mAb) against MERS-CoV that reduced virus replication in mice expressing human DPP4 upon prophylactic and therapeutic treatment. Here, we evaluated the prophylactic and therapeutic efficacy of REGN3048 and REGN3051 in the common marmoset model of MERS-CoV infection. Intravenous administration of mAb resulted in high levels of MERS-CoV-neutralizing activity in circulating blood. When animals were treated with mAbs one day before challenge, respiratory disease was less severe and, in animals treated with both REGN3048 and REGN3051, viral loads in the lungs were reduced. However, therapeutic treatment on day one after challenge was less efficacious as it did not prevent the development of severe respiratory disease and all treated animals developed bronchointerstitial pneumonia of similar severity as the control animals. Thus, mAb administration may be more effective in a prophylactic treatment regimen rather than treatment of MERS.

Mouse and Human Monoclonal Antibodies Protect against Infection by Multiple Genotypes of Japanese Encephalitis Virus.

ABSTRACTJapanese encephalitis virus (JEV) remains a leading cause of viral encephalitis worldwide. Although JEV-specific antibodies have been described, an assessment of their ability to neutralize multiple genotypes of JEV has been limited. Here, we describe the development of a panel of mouse and human neutralizing monoclonal antibodies (MAbs) that inhibit infection in cell culture of four different JEV genotypes tested. Mechanism-of-action studies showed that many of these MAbs inhibited infection at a postattachment step, including blockade of virus fusion. Mapping studies using site-directed mutagenesis and hydrogen-deuterium exchange with mass spectrometry revealed that the lateral ridge on domain III of the envelope protein was a primary recognition epitope for our panel of strongly neutralizing MAbs. Therapeutic studies in mice demonstrated protection against lethality caused by genotype I and III strains when MAbs were administered as a single dose even 5 days after infection. This information may inform the development of vaccines and therapeutic antibodies as emerging strains and genotypic shifts become more prevalent.

Anti-HIV Passive Immunization: New Weapons in the Arsenal

Anti-HIV passive immunization with human neutralizing monoclonal antibodies (nmAbs) has made exciting gains: (i) identification of the HIV envelope V2 apex as a new in vivo protective epitope, (ii) a novel clade C SHIV for challenge studies, and (iii) a highly protective, trispecific nmAb. Potent, broad-spectrum protection by nmAbs holds promise.

Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset

Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings.

A supporting role of Chinese National Immortalized Cell Bank in life science research.

A biorepository of human samples is essential to support the research of life science. Lymphoblastoid B cell line (LCL), which is easy to be prepared and can reproduce indefinitely, is a convenient form of sample preservation. LCLs are established from human B cells transformed by Epstein-Barr virus (EBV). Chinese National Immortalized Cell Bank has preserved human LCLs from different ethnic groups in China. As there are many studies on the nature of LCLs and public available resources with genome-wide data for LCLs, they have been widely applied in genetics, immunology, pharmacogenetics/genomics, regenerative medicine, cancer pathogenesis and immunotherapy, screening and generation of fully human neutralizing monoclonal antibodies and study on EBV pathogenesis. Here, we review the characteristics of LCLs and their contributions to scientific research, and introduce preserved samples in Chinese National Immortalized Cell Bank to the scientific community. We hope this bank can support more areas in the scientific research.

Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in infant macaques.

Prevention of mother to child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested anti-HIV-1 human neutralizing monoclonal antibodies (NmAb) as post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with SHIVSF162P3. On days 1, 4, 7, and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h following administration. Replicating virus was found in multiple tissues by day 1 in animals without treatment. All NmAb-treated macaques were free of virus in blood and tissues at 6 months post-exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged following CD8+ T cell depletion. These results suggest early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs.

Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1.

The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays.

Towards the prophylactic and therapeutic use of human neutralizing monoclonal antibodies for Middle East respiratory syndrome coronavirus (MERS-CoV).

In 2012 September, Saudi Arabian businessman died from acute respiratory failure, which was caused by Middle East respiratory syndrome coronavirus (MERS-CoV) (1). The MERS-CoV is the first lineage C of the genus Betacoronavirus that is known to infect humans (2). As of December 2nd, 2014, a total of 927 cases of human MERS-CoV infection with 338 deaths have been reported by the World Health Organization (WHO) (3). Due to high fatality rate of 35% and clusters of human-to-human transmission, MERS-CoV is of global concern for a potential of pandemic. Contrary to spreading of human MERS-CoV infection worldwide, its specific treatment or vaccine is currently unavailable although interferon-α2b and ribavirin has been reported to improve outcome in MERS-CoV-infected rhesus macaques (4). Thus, agent development for MERS-CoV infection is an urgent issue for the world researcher.

Are anti-HIV IgAs good guys or bad guys?

An estimated 90% of all HIV transmissions occur mucosally. Immunoglobulin A (IgA) molecules are important components of mucosal fluids. In a vaccine efficacy study, in which virosomes displaying HIV gp41 antigens protected most rhesus monkeys (RMs) against simian-human immunodeficiency virus (SHIV), protection correlated with vaginal IgA capable of blocking HIV transcytosis in vitro. Furthermore, vaginal IgG exhibiting virus neutralization and/or antibody-dependent cellular cytotoxicity (ADCC) correlated with prevention of systemic infection. In contrast, plasma IgG had neither neutralizing nor ADCC activity. More recently, a passive mucosal immunization study provided the first direct proof that dimeric IgAs (dIgAs) can prevent SHIV acquisition in RMs challenged mucosally. This study compared dimeric IgA1 (dIgA1), dIgA2, or IgG1 versions of a human neutralizing monoclonal antibody (nmAb) targeting a conserved HIV Env epitope. While the nmAb neutralization profiles were identical in vitro, dIgA1 was significantly more protective in vivo than dIgA2. Protection was linked to a new mechanism: virion capture. Protection also correlated with inhibition of transcytosis of cell-free virus in vitro. While both of these primate model studies demonstrated protective effects of mucosal IgAs, the RV144 clinical trial identified plasma IgA responses to HIV Env as risk factors for increased HIV acquisition. In a secondary analysis of RV144, plasma IgA decreased the in vitro ADCC activity of vaccine-induced, Env-specific IgG with the same epitope specificity. Here we review the current literature regarding the potential of IgA – systemic as well as mucosal – in modulating virus acquisition and address the question whether anti-HIV IgA responses could help or harm the host.