Protective mAb Research Articles

Antibody-Fab and -Fc features promote Mycobacterium tuberculosis restriction.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a leading cause of death by an infectious disease globally, with no efficacious vaccine. Antibodies are implicated in Mtb control, but the mechanisms of antibody action remain poorly understood. We assembled a library of TB monoclonal antibodies (mAb) and screened for the ability to restrict Mtb in mice, identifying protective antibodies targeting known and novel antigens. To dissect the mechanism of mAb-mediated Mtb restriction, we optimized a protective lipoarabinomannan-specific mAb through Fc-swapping. In vivo analysis of these Fc-variants revealed a critical role for Fc-effector function in Mtb restriction. Restrictive Fc-variants altered distribution of Mtb across innate immune cells. Single-cell transcriptomics highlighted distinctly activated molecular circuitry within innate immune cell subpopulations, highlighting early activation of neutrophils as a key signature of mAb-mediated Mtb restriction. Therefore, improved antibody-mediated restriction of Mtb is associated with reorganization of the tissue-level immune response to infection and depends on the collaboration of antibody Fab and Fc.

Bispecific antibodies targeting two glycoproteins on SFTSV exhibit synergistic neutralization and protection in a mouse model

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high fatality rate of up to 30% caused by SFTS virus (SFTSV). However, no specific vaccine or antiviral therapy has been approved for clinical use. To develop an effective treatment, we isolated a panel of human monoclonal antibodies (mAbs). SF5 and SF83 are two neutralizing mAbs that recognize two viral glycoproteins (Gn and Gc), respectively. We found that their epitopes are closely located, and we then engineered them as several bispecific antibodies (bsAbs). Neutralization and animal experiments indicated that bsAbs display more potent protective effects than the parental mAbs, and the cryoelectron microscopy structure of a bsAb3 Fab-Gn-Gc complex elucidated the mechanism of protection. In vivo virus passage in the presence of antibodies indicated that two bsAbs resulted in less selective pressure and could efficiently bind to all single parental mAb-escape mutants. Furthermore, epitope analysis of the protective mAbs against SFTSV and RVFV indicated that they are all located on the Gn subdomain I, where may be the hot spots in the phleboviruses. Collectively, these data provide potential therapeutic agents and molecular basis for the rational design of vaccines against SFTSV infection.

Functional and structural investigation of a broadly neutralizing SARS-CoV-2 antibody.

Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance. Here, we isolated mAbs from donors with breakthrough infection with Omicron subvariants using a single-B cell screening platform. We identified a mAb, O5C2, which possesses broad-spectrum neutralization and antibody-dependent cell-mediated cytotoxic activities against SARS-CoV-2 variants, including EG.5.1. Single-particle analysis by cryo-electron microscopy revealed that O5C2 targeted an unusually large epitope within the receptor-binding domain of spike protein that overlapped with the angiotensin-converting enzyme 2 binding interface. Furthermore, O5C2 effectively protected against BA.5 Omicron infection in vivo by mediating changes in transcriptomes enriched in genes involved in apoptosis and interferon responses. Our findings provide insights into the development of pan-protective mAbs against SARS-CoV-2.

Design and characterization of protective pan-ebolavirus and pan-filovirus bispecific antibodies.

Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.

In vivo delivery of engineered synthetic DNA-encoded SARS-CoV-2 monoclonal antibodies for pre-exposure prophylaxis in non-human primates

ABSTRACT COVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.

Salmonella typhimurium targeting with monoclonal antibodies prevents infection in mice.

Salmonella is a prevalent foodborne and waterborne pathogens threating global public health and food safety. Given the diversity of Salmonella serotypes and the emergence of antibiotic-resistant strains, there is an urgent need for the development of broadly protective therapies. This study aims to prepare monoclonal antibodies (Mabs) with broad reactivity against multi-serotype Salmonella strains, potentially offering cross-protection. We prepared two Mabs F1D4 and B7D4 against protein FliK and BcsZ, two potential vaccine candidates against multi-serotype Salmonella. The two Mabs belonging to IgG1 isotype exhibited high titers of 1:256,000 and 1:512,000 respectively, as well as broad cross-reactivity against 28 different serotypes of Salmonella strains with percentages of 89.29% and 92.86%, correspondingly. Neutralizing effects of the two Mabs on Salmonella growth, adhesion, invasion and motility was evaluated in vitro using bacteriostatic and bactericidal activity with and without complement and bacterial invasion inhibition assay. Additionally, cytotoxicity assays, animal toxicity analyses, and pharmacokinetic evaluations demonstrated the safety and sustained effectiveness of both Mabs. Furthermore, F1D4 or B7D4-therapy in mice challenged with S. Typhimurium LT2 exhibited milder organs damage and lower Salmonella colonization, as well as the higher relative survival of 86.67% and 93.33% respectively. This study produced two broadly reactive and potential cross protective Mabs F1D4 and B7D4, which offered new possibilities for immunotherapy of salmonellosis.

New Neutralizing Epitope Exposed on the Domain II of Tick-Borne Encephalitis Virus Envelope Glycoprotein E.

Orthoflavivirus encephalitidis, formerly tick-borne encephalitis virus (TBEV), belongs to the Orthoflavivirus genus. TBEV is transmitted by tick bites and infection with TBEV can lead to serious disorders of the central nervous system. In this study, a new protective monoclonal mouse antibody (mAb) FVN-32, with high binding activity to glycoprotein E of TBEV, was selected and examined in post exposure prophylaxis in a mouse model of TBEV infection. BALB/c mice were injected mAb FVN-32 at doses of 200 μg, 50 μg, and 12.5 μg per mouse one day after a TBEV challenge. mAb FVN-32 showed 37.5% protective efficacy when administered at doses of 200 μg and 50 μg per mouse. The epitope for protective mAb FVN-32 was localized in TBEV glycoprotein E domain I+II, using a set of truncated fragments of glycoprotein E. Additionally, the target site recognized by mAb FVN-32 was defined using combinatorial libraries of peptides. Three-dimensional modeling revealed that the site is dspatially close to the fusion loop, but does not come into contact with it, and is localized in a region between 247 and 254 amino acid residues on the envelope protein. This region is conserved among TBEV-like orthoflaviviruses.

Expression and purification of conserved, protective streptococcal antigens for monoclonal antibody isolation

Abstract Streptococcus pyogenesis a significant cause of infectious morbidity and mortality globally. Antibiotics remain the essential treatment for S. pyogenesinfection however, as resistance against long-used antibiotics grows amongst bacterial pathogens and antibiotic failure becomes increasingly common in S. pyogenestreatment, the necessity of alternative treatment methods escalates. Here, we have selected conserved streptococcal antigens to express in Escherichia coliin pursuit of isolating protective human monoclonal antibodies (mAbs) for streptococcal infection. We have successfully expressed the serine proteinase ScpC (180 kDa) which impedes the recruitment of neutrophils to the infection site. In addition, we have produced SpyAD (48.9 kDa), a protein that is conserved across streptococcal emm types and is shown to play an essential role in bacterial adhesion and division. Both proteins are important virulence factors and are highly conserved across emmtypes, making them promising targets for future vaccination and therapeutic treatments. Following the expression and purification of the antigens, SpyAD and ScpC are used to screen human peripheral blood mononuclear cells (PBMCs) for antigen specific B-cells via flow cytometry. This work confirms the use of E. colias an expression system for streptococcal antigens and the employment of such antigens in protective mAb isolation. Supported by the NIH Post-Baccalaureate Research Education Program

A non-neutralizing glycoprotein B monoclonal antibody protects against herpes simplex virus disease in mice.

There is an unmet need for monoclonal antibodies (mAbs) for prevention or as adjunctive treatment of herpes simplex virus (HSV) disease. Most vaccine and mAb efforts focus on neutralizing antibodies, but for HSV this strategy has proven ineffective. Preclinical studies with a candidate HSV vaccine strain, ΔgD-2, demonstrated that non-neutralizing antibodies that activate Fcγ receptors (FcγRs) to mediate antibody-dependent cellular cytotoxicity (ADCC) provide active and passive protection against HSV-1 and HSV-2. We hypothesized that this vaccine provides a tool to identify and characterize protective mAbs. We isolated HSV-specific mAbs from germinal center and memory B cells and bone marrow plasmacytes of ΔgD-2-vaccinated mice and evaluated these mAbs for binding, neutralizing, and FcγR-activating activity and for protective efficacy in mice. The most potent protective mAb, BMPC-23, was not neutralizing but activated murine FcγRIV, a biomarker of ADCC. The cryo-electron microscopic structure of the Fab-glycoprotein B (gB) assembly identified domain IV of gB as the epitope. A single dose of BMPC-23 administered 24 hours before or after viral challenge provided significant protection when configured as mouse IgG2c and protected mice expressing human FcγRIII when engineered as a human IgG1. These results highlight the importance of FcR-activating antibodies in protecting against HSV.

Synergistic Protection against Secondary Pneumococcal Infection by Human Monoclonal Antibodies Targeting Distinct Epitopes.

Streptococcus pneumoniae persists as a leading cause of bacterial pneumonia despite the widespread use of polysaccharide-based vaccines. The limited serotype coverage of current vaccines has led to increased incidence of nonvaccine serotypes, as well as an increase in antibiotic resistance among these serotypes. Pneumococcal infection often follows a primary viral infection such as influenza virus, which hinders host defense and results in bacterial spread to the lungs. We previously isolated human monoclonal Abs (mAbs) against the conserved surface Ag pneumococcal histidine triad protein D (PhtD), and we demonstrated that mAbs to this Ag are protective against lethal pneumococcal challenge prophylactically and therapeutically. In this study, we elucidated the mechanism of protection of a protective anti-pneumococcal human mAb, PhtD3, which is mediated by the presence of complement and macrophages in a mouse model of pneumococcal infection. Treatment with mAb PhtD3 reduced blood and lung bacterial burden in mice, and mAb PhtD3 is able to bind to bacteria in the presence of the capsular polysaccharide, indicating exposure of surface PhtD on encapsulated bacteria. In a mouse model of secondary pneumococcal infection, protection mediated by mAb PhtD3 and another mAb targeting a different epitope, PhtD7, was reduced; however, robust protection was restored by combining mAb PhtD3 with mAb PhtD7, indicating a synergistic effect. Overall, these studies provide new insights into anti-pneumococcal mAb protection and demonstrate, to our knowledge, for the first time, that mAbs to pneumococcal surface proteins can protect against secondary pneumococcal infection in the mouse model.

High-density binding to Plasmodium falciparum circumsporozoite protein repeats by inhibitory antibody elicited in mouse with human immunoglobulin repertoire.

Antibodies targeting the human malaria parasite Plasmodium falciparum circumsporozoite protein (PfCSP) can prevent infection and disease. PfCSP contains multiple central repeating NANP motifs; some of the most potent anti-infective antibodies against malaria bind to these repeats. Multiple antibodies can bind the repeating epitopes concurrently by engaging into homotypic Fab-Fab interactions, which results in the ordering of the otherwise largely disordered central repeat into a spiral. Here, we characterize IGHV3-33/IGKV1-5-encoded monoclonal antibody (mAb) 850 elicited by immunization of transgenic mice with human immunoglobulin loci. mAb 850 binds repeating NANP motifs with picomolar affinity, potently inhibits Plasmodium falciparum (Pf) in vitro and, when passively administered in a mouse challenge model, reduces liver burden to a similar extent as some of the most potent anti-PfCSP mAbs yet described. Like other IGHV3-33/IGKV1-5-encoded anti-NANP antibodies, mAb 850 primarily utilizes its HCDR3 and germline-encoded aromatic residues to recognize its core NANP motif. Biophysical and cryo-electron microscopy analyses reveal that up to 19 copies of Fab 850 can bind the PfCSP repeat simultaneously, and extensive homotypic interactions are observed between densely-packed PfCSP-bound Fabs to indirectly improve affinity to the antigen. Together, our study expands on the molecular understanding of repeat-induced homotypic interactions in the B cell response against PfCSP for potently protective mAbs against Pf infection.

Human Monoclonal Antibodies against NS1 Protein Protect against Lethal West Nile Virus Infection.

ABSTRACTEnvelope protein-targeted vaccines for flaviviruses are limited by concerns of antibody-dependent enhancement (ADE) of infections. Nonstructural protein 1 (NS1) provides an alternative vaccine target that avoids this risk since this protein is absent from the virion. Beyond its intracellular role in virus replication, extracellular forms of NS1 function in immune modulation and are recognized by host-derived antibodies. The rational design of NS1-based vaccines requires an extensive understanding of the antigenic sites on NS1, especially those targeted by protective antibodies. Here, we isolated human monoclonal antibodies (MAbs) from individuals previously naturally infected with WNV, mapped their epitopes using structure-guided mutagenesis, and evaluated their efficacy in vivo against lethal WNV challenge. The most protective epitopes clustered at three antigenic sites that are exposed on cell surface forms of NS1: (i) the wing flexible loop, (ii) the outer, electrostatic surface of the wing, and (iii) the spaghetti loop face of the β-ladder. One additional MAb mapped to the distal tip of the β-ladder and conferred a lower level of protection against WNV despite not binding to NS1 on the surface of infected cells. Our study defines the epitopes and modes of binding of protective anti-NS1 MAb antibodies following WNV infection, which may inform the development of NS1-based countermeasures against flaviviruses.

A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D.

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.

Proteo-Genomic Analysis Identifies Two Major Sites of Vulnerability on Ebolavirus Glycoprotein for Neutralizing Antibodies in Convalescent Human Plasma.

Three clinically relevant ebolaviruses – Ebola (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) viruses, are responsible for severe disease and occasional deadly outbreaks in Africa. The largest Ebola virus disease (EVD) epidemic to date in 2013-2016 in West Africa highlighted the urgent need for countermeasures, leading to the development and FDA approval of the Ebola virus vaccine rVSV-ZEBOV (Ervebo®) in 2020 and two monoclonal antibody (mAb)-based therapeutics (Inmazeb® [atoltivimab, maftivimab, and odesivimab-ebgn] and Ebanga® (ansuvimab-zykl) in 2020. The humoral response plays an indispensable role in ebolavirus immunity, based on studies of mAbs isolated from the antibody genes in peripheral blood circulating ebolavirus-specific human memory B cells. However, antibodies in the body are not secreted by circulating memory B cells in the blood but rather principally by plasma cells in the bone marrow. Little is known about the protective polyclonal antibody responses in convalescent plasma. Here we exploited both single-cell antibody gene sequencing and proteomic sequencing approaches to assess the composition of the ebolavirus glycoprotein (GP)-reactive antibody repertoire in the plasma of an EVD survivor. We first identified 1,512 GP-specific mAb variable gene sequences from single cells in the memory B cell compartment. Using mass spectrometric analysis of the corresponding GP-specific plasma IgG, we found that only a portion of the large B cell antibody repertoire was represented in the plasma. Molecular and functional analysis of proteomics-identified mAbs revealed recognition of epitopes in three major antigenic sites - the GP head domain, the glycan cap, and the base region, with a high prevalence of neutralizing and protective mAb specificities that targeted the base and glycan cap regions on the GP. Polyclonal plasma antibodies from the survivor reacted broadly to EBOV, BDBV, and SUDV GP, while reactivity of the potently neutralizing mAbs we identified was limited mostly to the homologous EBOV GP. Together these results reveal a restricted diversity of neutralizing humoral response in which mAbs targeting two antigenic sites on GP – glycan cap and base – play a principal role in plasma-antibody-mediated protective immunity against EVD.

Antibodies targeting epitopes on the cell-surface form of NS1 protect against Zika virus infection during pregnancy

There are no licensed therapeutics or vaccines available against Zika virus (ZIKV) to counteract its potential for congenital disease. Antibody-based countermeasures targeting the ZIKV envelope protein have been hampered by concerns for cross-reactive responses that induce antibody-dependent enhancement (ADE) of heterologous flavivirus infection. Nonstructural protein 1 (NS1) is a membrane-associated and secreted glycoprotein that functions in flavivirus replication and immune evasion but is absent from the virion. Although some studies suggest that antibodies against ZIKV NS1 are protective, their activity during congenital infection is unknown. Here we develop mouse and human anti-NS1 monoclonal antibodies that protect against ZIKV in both non-pregnant and pregnant mice. Avidity of antibody binding to cell-surface NS1 along with Fc effector functions engagement correlate with protection in vivo. Protective mAbs map to exposed epitopes in the wing domain and loop face of the β-platform. Anti-NS1 antibodies provide an alternative strategy for protection against congenital ZIKV infection without causing ADE.

A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver

Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an invitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.

Binding Sites of Anti-Lcr V Monoclonal Antibodies Are More Critical than the Avidities and Affinities for Passive Protection against Yersinia pestis Infection in a Bubonic Plague Model.

Plague is a zoonotic disease that is caused by Yersinia pestis. Monoclonal antibodies (mAbs) that bind to the V-antigen, a virulence factor that is produced by Y. pestis, can passively protect mice from plague. An analysis of protective mAbs that bind to V-antigen was made to assess binding sites, avidities, and affinities. Anti-V mAbs were screened for their efficacy in a murine model of plague. Antigen-binding sites of protective V mAbs were determined with a linear peptide library, V-antigen fragment, competitive binding, and surface plasmon resonance. The avidities to the V-antigen was determined by ELISA, and affinities of the mAbs to the V-antigen were determined by surface plasmon resonance. The most protective mAb 7.3 bound to a unique conformational site on the V-antigen, while a less protective mAb bound to a different conformational site located on the same V-antigen fragment as mAb 7.3. The avidity of mAb 7.3 for the V-antigen was neither the strongest overall nor did it have the highest affinity for the V-antigen. The binding site of the most protective mAb was critical in its ability to protect against a lethal plague challenge.

Neutralizing monoclonal antibodies against Venezuelan equine encephalitis virus envelope glycoprotein E2 protect from lethal encephalitis

Abstract Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne New World alphavirus responsible for severe morbidity and mortality in humans and equines. The development of therapeutics against VEEV is critical due to its potential for outbreak and aerosolization as a bioterrorist agent. However, effective and widely accessible antiviral agents for VEEV do not exist. Humoral immunity is an important defense against many alphavirus infections, and we thus generated a panel of 17 highly neutralizing murine VEEV-specific monoclonal antibodies (mAbs) using the VEEV vaccine TC-83 and attenuated chimeric SINV-VEEV Trinidad donkey (TRD) subtype IAB. Nine of these mAbs demonstrate 50% effective inhibitory concentrations (EC50) values below 100 ng/ml when tested against enzootic subtype ID and epizootic VEEV subtypes IAB and IC virion envelope glycoproteins. To define specific viral epitopes important for mAb binding, mapping studies including competition assays, HDX, alanine-scanning mutagenesis, and viral escape mutants are ongoing. Preliminary data suggests the epitopes for these mAbs are found in both the A and B domains of the surface virion envelope 2 glycoprotein. Additionally, several of these mAbs confer prophylactic and therapeutic protection in mice aerosol challenged with virulent TRD VEEV. Mechanism of action studies are underway to define the steps in the virus life cycle (viral attachment, entry, fusion, and egress) at which our most potent protective antibodies act. Identification of specific epitopes targeted by these protective mAbs will better inform the development of targeted immunotherapeutics and vaccines against VEEV and related encephalitic alphaviruses.

Structure-Guided Design of a Group B Streptococcus Type III Synthetic Glycan-Conjugate Vaccine.

Identification of glycan functional epitopes is of paramount importance for rational design of glycoconjugate vaccines. We recently mapped the structural epitope of the capsular polysaccharide from type III Group B Streptococcus (GBSIII), a major cause of invasive disease in newborns, by using a dimer fragment (composed of two pentasaccharide repeating units) obtained by depolymerization complexed with a protective mAb. Although reported data had suggested a highly complex epitope contained in a helical structure composed of more than four repeating units, we showed that such dimer conjugated to a carrier protein with a proper glycosylation degree elicited functional antibodies comparably to the full‐length conjugated polysaccharide. Here, starting from the X‐ray crystallographic structure of the polysaccharide fragment–mAb complex, we synthesized a hexasaccharide comprising exclusively the relevant positions involved in binding. Combining competitive surface plasmon resonance and saturation transfer difference NMR spectroscopy as well as in‐silico modeling, we demonstrated that this synthetic glycan was recognized by the mAb similarly to the dimer. The hexasaccharide conjugated to CRM197, a mutant of diphtheria toxin, elicited a robust functional immune response that was not inferior to the polysaccharide conjugate, indicating that it may suffice as a vaccine antigen. This is the first evidence of an X‐ray crystallography‐guided design of a synthetic carbohydrate‐based conjugate vaccine.

Protective activity of anti-lipoteichoic acid monoclonal antibody in single or combination therapies in methicillin-resistant Staphylococcus aureus-induced murine sepsis models

Previously, we generated and screened a panel of monoclonal antibodies (mAbs) against methicillin-resistant Staphylococcus aureus (MRSA) to identify protective mAbs in mouse infection models. One of these mAbs, ZBIA3H, bound to lipoteichoic acid (LTA) and exerted protective effects in a mouse sepsis model. To reinforce the ability of the mAb to protect against infection, combination therapies with the mAb and antibiotics need to be examined. Therefore, herein, we studied the efficacy of ZBIA3H (in combination or alone) in a mouse sepsis model. ZBIA3H improved the survival rate in the mouse models of sepsis induced by highly virulent or refractory S. aureus (community-acquired MRSA strain MW2, vancomycin-intermediate S. aureus strain Mu3, or vancomycin-resistant S. aureus strain VRS1). Furthermore, ZBIA3H remarkably improved the survival rate in combination with antimicrobial agents (vancomycin, daptomycin, or linezolid) in mouse sepsis models. From these results we conclude that anti-LTA mAb ZBIA3H or its humanized form is a promising mAb individually, or in combination with antibiotics, against clinical refractory infection of S. aureus.