Broadly inhibitory antibodies against severe malaria virulence proteins.

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

Highly protective antimalarial antibodies via precision library generation and yeast display screening.

The monoclonal antibody CIS43 targets the Plasmodium falciparum circumsporozoite protein (PfCSP) and prevents malaria infection in humans for up to 9 mo following a single intravenous administration. To enhance the potency and clinical utility of CIS43, we used iterative site-saturation mutagenesis and DNA shuffling to screen precise gene-variant yeast display libraries for improved PfCSP antigen recognition. We identified several mutations that improved recognition, predominately in framework regions, and combined these to produce a panel of antibody variants. The most improved antibody, CIS43_Var10, had three mutations and showed approximately sixfold enhanced protective potency in vivo compared to CIS43. Co-crystal and cryo-electron microscopy structures of CIS43_Var10 with the peptide epitope or with PfCSP, respectively, revealed functional roles for each of these mutations. The unbiased site-directed mutagenesis and screening pipeline described here represent a powerful approach to enhance protective potency and to enable broader clinical use of antimalarial antibodies.

Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit.

Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection. We previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 spike, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and binds to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is maintained against the Alpha, Delta, Gamma and Omicron variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region. Negative stain electron microscopy and a 1.74 Å crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs.

The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria.

L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAb's ontogeny and mechanisms of binding PfCSP will facilitate vaccine development. Here, we isolate mAbs clonally related to L9 and show that this B cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9κ) with clonally related heavy chains results in chimeric mAbs that cross-link two NVDPs, cross-react with NANP, and more potently neutralize sporozoites in vivo compared with their original light chain. Structural analyses reveal that the chimeric mAbs bound minor repeats in a type-1 ß-turn seen in other repeat-specific antibodies. These data highlight the importance of L9κ in binding NVDP on PfCSP to neutralize sporozoites and suggest that PfCSP-based immunogens might be improved by presenting ≥2 NVDPs.

Isolation and characterization of cross-neutralizing coronavirus antibodies from COVID-19+ subjects.

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects.

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterized 198 antibodies isolated from four COVID19+ subjects and identified 14 SARS-CoV-2 neutralizing antibodies. One targeted the NTD, one recognized an epitope in S2 and twelve bound the RBD. Three anti-RBD neutralizing antibodies cross-neutralized SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency rather than the antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. The anti-S2 antibody also neutralized SARS-CoV-1 and all four cross-neutralizing antibodies neutralized the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

Design of Alphavirus Virus-Like Particles Presenting Circumsporozoite Junctional Epitopes That Elicit Protection against Malaria.

The most advanced malaria vaccine, RTS,S, includes the central repeat and C-terminal domains of the Plasmodium falciparum circumsporozoite protein (PfCSP). We have recently isolated human antibodies that target the junctional region between the N-terminal and repeat domains that are not included in RTS,S. Due to the fact that these antibodies protect against malaria challenge in mice, their epitopes could be effective vaccine targets. Here, we developed immunogens displaying PfCSP junctional epitopes by genetic fusion to either the N-terminus or B domain loop of the E2 protein from chikungunya (CHIK) alphavirus and produced CHIK virus-like particles (CHIK-VLPs). The structural integrity of these junctional-epitope-CHIK-VLP immunogens was confirmed by negative-stain electron microscopy. Immunization of these CHIK-VLP immunogens reduced parasite liver load by up to 95% in a mouse model of malaria infection and elicited better protection than when displayed on keyhole limpet hemocyanin, a commonly used immunogenic carrier. Protection correlated with PfCSP serum titer. Of note, different junctional sequences elicited qualitatively different reactivities to overlapping PfCSP peptides. Overall, these results show that the junctional epitopes of PfCSP can induce protective responses when displayed on CHIK-VLP immunogens and provide a basis for the development of a next generation malaria vaccine to expand the breadth of anti-PfCSP immunity.

Structural characterization of a nonhydrolyzing UDP-GlcNAc 2-epimerase from Neisseria meningitidis serogroup A.

Bacterial nonhydrolyzing UDP-N-acetylglucosamine 2-epimerases catalyze the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). UDP-ManNAc is an important intermediate in the biosynthesis of certain cell-surface polysaccharides, including those in some pathogenic bacteria, such as Neisseria meningitidis and Streptococcus pneumoniae. Many of these epimerases are allosterically regulated by UDP-GlcNAc, which binds adjacent to the active site and is required to initiate UDP-ManNAc epimerization. Here, two crystal structures of UDP-N-acetylglucosamine 2-epimerase from Neisseria meningitidis serogroup A (NmSacA) are presented. One crystal structure is of the substrate-free enzyme, while the other structure contains UDP-GlcNAc substrate bound to the active site. Both structures form dimers as seen in similar epimerases, and substrate binding to the active site induces a large conformational change in which two Rossmann-like domains clamp down on the substrate. Unlike other epimerases, NmSacA does not require UDP-GlcNAc to instigate the epimerization of UDP-ManNAc, although UDP-GlcNAc was found to enhance the rate of epimerization. In spite of the conservation of residues involved in binding the allosteric UDP-GlcNAc observed in similar UDP-GlcNAc 2-epimerases, the structures presented here do not contain UDP-GlcNAc bound in the allosteric site. These structural results provide additional insight into the mechanism and regulation of this critical enzyme and improve the structural understanding of the ability of NmSacA to epimerize modified substrates.

Structural basis for potent neutralization of SARS-CoV-2 and role of antibody affinity maturation.

SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determine the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain. The structure reveals that CV30 binds to an epitope that overlaps with the human ACE2 receptor binding motif providing a structural basis for its neutralization. CV30 also induces shedding of the S1 subunit, indicating an additional mechanism of neutralization. A germline reversion of CV30 results in a substantial reduction in both binding affinity and neutralization potential indicating the minimal somatic mutation is needed for potently neutralizing antibodies against SARS-CoV-2.

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 in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.

Characterization of neutralizing antibodies from a SARS-CoV-2 infected individual.

B cells specific for the SARS-CoV-2 S envelope glycoprotein spike were isolated from a COVID-19-infected subject using a stabilized spike-derived ectodomain (S2P) twenty-one days post-infection. Forty-four S2P-specific monoclonal antibodies were generated, three of which bound to the receptor binding domain (RBD). The antibodies were minimally mutated from germline and were derived from different B cell lineages. Only two antibodies displayed neutralizing activity against SARS-CoV-2 pseudo-virus. The most potent antibody bound the RBD in a manner that prevented binding to the ACE2 receptor, while the other bound outside the RBD. Our study indicates that the majority of antibodies against the viral envelope spike that were generated during the first weeks of COVID-19 infection are non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 spike-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive/therapeutic potential and can serve as templates for vaccine-design.

Structural basis for potent neutralization of SARS-CoV-2 and role of antibody affinity maturation.

SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determined the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain (RBD). The structure reveals CV30's epitope overlaps with the human ACE2 receptor binding site thus providing the structural basis for its neutralization by preventing ACE2 binding.

Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation.

Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes, and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject 21 days after the onset of clinical disease. 45 S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation with limited clonal expansion, and three bound the receptor-binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine design.

Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6 reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein.

Effectors are microbial-derived secreted proteins with an essential function in modulating host immunity during infections. CfAvr4, an effector protein from the tomato pathogen Cladosporium fulvum and the founding member of a fungal effector family, promotes parasitism through binding fungal chitin and protecting it from chitinases. Binding of Avr4 to chitin is mediated by a carbohydrate-binding module of family 14 (CBM14), an abundant CBM across all domains of life. To date, the structural basis of chitin-binding by Avr4 effector proteins and of recognition by the cognate Cf-4 plant immune receptor are still poorly understood. Using X-ray crystallography, we solved the crystal structure of CfAvr4 in complex with chitohexaose [(GlcNAc)6] at 1.95Å resolution. This is the first co-crystal structure of a CBM14 protein together with its ligand that further reveals the molecular mechanism of (GlcNAc)6 binding by Avr4 effector proteins and CBM14 family members in general. The structure showed that two molecules of CfAvr4 interact through the ligand and form a three-dimensional molecular sandwich that encapsulates two (GlcNAc)6 molecules within the dimeric assembly. Contrary to previous assumptions made with other CBM14 members, the chitohexaose-binding domain (ChBD) extends to the entire length of CfAvr4 with the reducing end of (GlcNAc)6 positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc)6 enabled the elucidation of the precise topography and amino acid composition of Avr4's ChBD and further showed that these residues do not individually mediate the recognition of CfAvr4 by the Cf-4 immune receptor. Instead, the studies highlighted the dependency of Cf-4-mediated recognition on CfAvr4's stability and resistance against proteolysis in the leaf apoplast, and provided the evidence for structurally separating intrinsic function from immune receptor recognition in this effector family.

The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the Nucleus and Regulates Defense Hormone Expression during Plant Innate Immunity.

Plants employ cell-surface pattern recognition receptors (PRRs) to detect pathogens. Although phytohormones produced during PRR signaling play an essential role in innate immunity, a direct link between PRR activation and hormone regulation is unknown. EFR is a PRR that recognizes bacterial EF-Tu and activates immune signaling. Here we report that EFR regulates the phytohormone jasmonic acid (JA) through direct phosphorylation of a receptor-like cytoplasmic kinase, BIK1. The BIK1 structure revealed that the EFR-phosphorylated sites reside on a uniquely extended loop away from the BIK1 kinase core domain. Phosphomimetic mutations of these sites resulted in increased phytohormones and enhanced resistance to bacterial infections. In addition to its documented plasma membrane localization, BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors involved in the JA and salicylic acid (SA) regulation. These findings demonstrate the mechanistic basis of signal transduction from PRR to phytohormones, mediated through a PRR-BIK1-WRKY axis.

Structural Analysis of an Avr4 Effector Ortholog Offers Insight into Chitin Binding and Recognition by the Cf-4 Receptor.

Chitin is a key component of fungal cell walls and a potent inducer of innate immune responses. Consequently, fungi may secrete chitin-binding lectins, such as the Cf-Avr4 effector protein from the tomato pathogen Cladosporium fulvum, to shield chitin from host-derived chitinases during infection. Homologs of Cf-Avr4 are found throughout Dothideomycetes, and despite their modest primary sequence identity, many are perceived by the cognate tomato immune receptor Cf-4. Here, we determined the x-ray crystal structure of Pf-Avr4 from the tomato pathogen Pseudocercospora fuligena, thus providing a three-dimensional model of an Avr4 effector protein. In addition, we explored structural, biochemical, and functional aspects of Pf-Avr4 and Cf-Avr4 to further define the biology of core effector proteins and outline a conceptual framework for their pleiotropic recognition by single immune receptors. We show that Cf-Avr4 and Pf-Avr4 share functional specificity in binding (GlcNAc)6 and in providing protection against plant- and microbial-derived chitinases, suggesting a broader role beyond deregulation of host immunity. Furthermore, structure-guided site-directed mutagenesis indicated that residues in Pf-Avr4 important for binding chitin do not directly influence recognition by Cf-4 and further suggested that the property of recognition is structurally separated or does not fully overlap with the virulence function of the effector.