Engineered immunogens to elicit antibodies against conserved coronavirus epitopes.

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employ computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant receptor binding domain. These engineered proteins bind with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interact with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicit sera with broad betacoronavirus reactivity and protect as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.

Nanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive antibody response against diverse coronavirus subgenera.

Multivalent antigen display is a fast-growing area of interest toward broadly protective vaccines. Current nanoparticle-based vaccine candidates demonstrate the ability to confer antibody-mediated immunity against divergent strains of notably mutable viruses. In coronaviruses, this work is predominantly aimed at targeting conserved epitopes of the receptor binding domain. However, targeting conserved non-RBD epitopes could limit the potential for antigenic escape. To explore new potential targets, we engineered protein nanoparticles displaying coronavirus prefusion-stabilized spike (CoV_S-2P) trimers derived from MERS-CoV, SARS-CoV-1, SARS-CoV-2, hCoV-HKU1, and hCoV-OC43 and assessed their immunogenicity in female mice. Monotypic SARS-1 nanoparticles elicit cross-neutralizing antibodies against MERS-CoV and protect against MERS-CoV challenge. MERS and SARS nanoparticles elicit S1-focused antibodies, revealing a conserved site on the S N-terminal domain. Moreover, mosaic nanoparticles co-displaying distinct CoV_S-2P trimers elicit antibody responses to distant cross-group antigens and protect male and female mice against MERS-CoV challenge. Our findings will inform further efforts toward the development of pan-coronavirus vaccines.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein elicits antibodies targeting a membrane-proximal epitope.

IMPORTANCE: Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants, infecting all children by age 5. RSV also causes substantial morbidity and mortality in older adults, and a vaccine for older adults based on a prefusion-stabilized form of the viral F glycoprotein was recently approved by the FDA. Here, we investigate a set of antibodies that belong to the same public clonotype and were isolated from individuals vaccinated with a prefusion-stabilized RSV F protein. Our results reveal that these antibodies are highly potent and recognize a previously uncharacterized antigenic site on the prefusion F protein. Vaccination with prefusion RSV F proteins appears to boost the elicitation of these neutralizing antibodies, which are not commonly elicited by natural infection.

Engineered Immunogens to Elicit Antibodies Against Conserved Coronavirus Epitopes.

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employed computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant RBD. These engineered proteins bound with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interacted with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicited sera with broad betacoronavirus reactivity and protected as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.

Structure-Based Stabilization of SOSIP Env Enhances Recombinant Ectodomain Durability and Yield.

The envelope glycoprotein (Env) is the main focus of human immunodeficiency virus type 1 (HIV-1) vaccine development due to its critical role in viral entry. Despite advances in protein engineering, many Env proteins remain recalcitrant to recombinant expression due to their inherent metastability, making biochemical and immunological experiments impractical or impossible. Here, we report a novel proline stabilization strategy to facilitate the production of prefusion Env trimers. This approach, termed "2P," works synergistically with previously described SOSIP mutations and dramatically increases the yield of recombinantly expressed Env ectodomains without altering the antigenic or conformational properties of near-native Env. We determined that the 2P mutations function by enhancing the durability of the prefusion conformation and that this stabilization strategy is broadly applicable to evolutionarily and antigenically diverse Env constructs. These findings provide a new Env stabilization platform to facilitate biochemical research and expand the number of Env variants that can be developed as future HIV-1 vaccine candidates. IMPORTANCE Recent estimates have placed the number of new human immunodeficiency virus type 1 (HIV-1) infections at approximately 1.5 million per year, emphasizing the ongoing and urgent need for an effective vaccine. The envelope (Env) glycoprotein is the main focus of HIV-1 vaccine development, but, due to its inherent metastability, many Env variants are difficult to recombinantly express in the relatively large quantities that are required for biochemical studies and animal trials. Here, we describe a novel structure-based stabilization strategy that works synergistically with previously described SOSIP mutations to increase the yield of prefusion HIV-1 Env.

A novel humanized Chi3l1 blocking antibody attenuates acetaminophen-induced liver injury in mice.

Acetaminophen (APAP) overdose is a leading cause of acute liver injury in the USA. The chitinase 3-like-1 (Chi3l1) protein contributes to APAP-induced liver injury (AILI) by promoting hepatic platelet recruitment. Here, we report the development of a Chi3l1-targeting antibody as a potential therapy for AILI. By immunizing a rabbit successively with the human and mouse Chi3l1 proteins, we isolated cross-reactive monoclonal antibodies (mAbs) from single memory B cells. One of the human and mouse Chi3l1 cross-reactive mAbs was humanized and characterized in both in vitro and in vivo biophysical and biological assays. X-ray crystallographic analysis of the lead antibody C59 in complex with the human Chi3l1 protein revealed that the kappa light contributes to majority of the antibody-antigen interaction; and that C59 binds to the 4α-5ß loop and 4α-helix of Chi3l1, which is a functional epitope and hotspot for the development of Chi3l1 blocking antibodies. We humanized the C59 antibody by complementarity-determining region grafting and kappa chain framework region reverse mutations. The humanized C59 antibody exhibited similar efficacy as the parental rabbit antibody C59 in attenuating AILI in vivo. Our findings validate Chi3l1 as a potential drug target for AILI and provide proof of concept of developing Chi3l1 blocking antibody as a therapy for the treatment of AILI.

A single, improbable B cell receptor mutation confers potent neutralization against cytomegalovirus.

Cytomegalovirus (CMV) is a leading cause of infant hearing loss and neurodevelopmental delay, but there are no clinically licensed vaccines to prevent infection, in part due to challenges eliciting neutralizing antibodies. One of the most well-studied targets for CMV vaccines is the viral fusogen glycoprotein B (gB), which is required for viral entry into host cells. Within gB, antigenic domain 2 site 1 (AD-2S1) is a target of potently neutralizing antibodies, but gB-based candidate vaccines have yet to elicit robust responses against this region. We mapped the genealogy of B cells encoding potently neutralizing anti-gB AD-2S1 antibodies from their inferred unmutated common ancestor (UCA) and characterized the binding and function of early lineage ancestors. Surprisingly, we found that a single amino acid heavy chain mutation A33N, which was an improbable mutation rarely generated by somatic hypermutation machinery, conferred broad CMV neutralization to the non-neutralizing UCA antibody. Structural studies revealed that this mutation mediated key contacts with the gB AD-2S1 epitope. Collectively, these results provide insight into potently neutralizing gB-directed antibody evolution in a single donor and lay a foundation for using this B cell-lineage directed approach for the design of next-generation CMV vaccines.

A Germline-Targeting Chimpanzee SIV Envelope Glycoprotein Elicits a New Class of V2-Apex Directed Cross-Neutralizing Antibodies.

HIV-1 and its SIV precursors share a broadly neutralizing antibody (bNAb) epitope in variable loop 2 (V2) at the envelope glycoprotein (Env) trimer apex. Here, we tested the immunogenicity of germ line-targeting versions of a chimpanzee SIV (SIVcpz) Env in human V2-apex bNAb heavy-chain precursor-expressing knock-in mice and as chimeric simian-chimpanzee immunodeficiency viruses (SCIVs) in rhesus macaques (RMs). Trimer immunization of knock-in mice induced V2-directed NAbs, indicating activation of V2-apex bNAb precursor-expressing mouse B cells. SCIV infection of RMs elicited high-titer viremia, potent autologous tier 2 neutralizing antibodies, and rapid sequence escape in the canonical V2-apex epitope. Six of seven animals also developed low-titer heterologous plasma breadth that mapped to the V2-apex. Antibody cloning from two of these animals identified multiple expanded lineages with long heavy chain third complementarity determining regions that cross-neutralized as many as 7 of 19 primary HIV-1 strains, but with low potency. Negative stain electron microscopy (NSEM) of members of the two most cross-reactive lineages confirmed V2 targeting but identified an angle of approach distinct from prototypical V2-apex bNAbs, with antibody binding either requiring or inducing an occluded-open trimer. Probing with conformation-sensitive, nonneutralizing antibodies revealed that SCIV-expressed, but not wild-type SIVcpz Envs, as well as a subset of primary HIV-1 Envs, preferentially adopted a more open trimeric state. These results reveal the existence of a cryptic V2 epitope that is exposed in occluded-open SIVcpz and HIV-1 Env trimers and elicits cross-neutralizing responses of limited breadth and potency. IMPORTANCE An effective HIV-1 vaccination strategy will need to stimulate rare precursor B cells of multiple bNAb lineages and affinity mature them along desired pathways. Here, we searched for V2-apex germ line-targeting Envs among a large set of diverse primate lentiviruses and identified minimally modified versions of one chimpanzee SIV Env that bound several human V2-apex bNAb precursors and stimulated one of these in a V2-apex bNAb precursor-expressing knock-in mouse. We also generated chimeric simian-chimpanzee immunodeficiency viruses and showed that they elicit low-titer V2-directed heterologous plasma breadth in six of seven infected rhesus macaques. Characterization of this antibody response identified a new class of weakly cross-reactive neutralizing antibodies that target the V2-apex, but only in occluded-open Env trimers. The existence of this cryptic epitope, which in some Env backgrounds is immunodominant, needs to be considered in immunogen design.

Nanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive protection across divergent subgroups.

Multivalent antigen display is a fast-growing area of interest towards broadly protective vaccines. Current nanoparticle-based vaccine candidates demonstrate the ability to confer antibody-mediated immunity against divergent strains of notably mutable viruses. In coronaviruses, this work is predominantly aimed at targeting conserved epitopes of the receptor-binding domain. However, targeting other conserved non-RBD epitopes could further limit the potential for antigenic escape. To further explore new potential targets, we engineered protein nanoparticles displaying CoV_S-2P trimers derived from MERS-CoV, SARS-CoV-1, SARS-CoV-2, hCoV-HKU1, and hCoV-OC43 and assessed their immunogenicity in mice. Monotypic SARS-1_S-2P nanoparticles elicited cross-neutralizing antibodies against MERS_S and protected against MERS-CoV challenge. MERS and SARS-I53_dn5 nanoparticles elicited S1-focused antibodies, revealing a conserved site on the NTD. Moreover, mosaic nanoparticles co-displaying distinct CoV_S-2P trimers elicited antibody responses to distant cross-group antigens while protecting against MERS challenge despite diminished valency of MERS_S-2P. Our findings will inform further efforts towards the development of pan-coronavirus vaccines.

Structural rearrangements allow nucleic acid discrimination by type I-D Cascade.

CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3' binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.

Efficient discovery of SARS-CoV-2-neutralizing antibodies via B cell receptor sequencing and ligand blocking.

Although several monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for coronavirus disease 2019 (COVID-19) therapy, development was generally inefficient, with lead generation often requiring the production and testing of numerous antibody candidates. Here, we report that the integration of target-ligand blocking with a previously described B cell receptor-sequencing approach (linking B cell receptor to antigen specificity through sequencing (LIBRA-seq)) enables the rapid and efficient identification of multiple neutralizing mAbs that prevent the binding of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The combination of target-ligand blocking and high-throughput antibody sequencing promises to increase the throughput of programs aimed at discovering new neutralizing antibodies.

Structural basis for HCMV Pentamer recognition by neuropilin 2 and neutralizing antibodies.

Human cytomegalovirus (HCMV) encodes multiple surface glycoprotein complexes to infect a variety of cell types. The HCMV Pentamer, composed of gH, gL, UL128, UL130, and UL131A, enhances entry into epithelial, endothelial, and myeloid cells by interacting with the cell surface receptor neuropilin 2 (NRP2). Despite the critical nature of this interaction, the molecular determinants that govern NRP2 recognition remain unclear. Here, we describe the cryo-EM structure of NRP2 bound to Pentamer. The high-affinity interaction between these proteins is calcium dependent and differs from the canonical carboxyl-terminal arginine (CendR) binding that NRP2 typically uses. We also determine the structures of four neutralizing human antibodies bound to the HCMV Pentamer to define susceptible epitopes. Two of these antibodies compete with NRP2 binding, but the two most potent antibodies recognize a previously unidentified epitope that does not overlap the NRP2-binding site. Collectively, these findings provide a structural basis for HCMV tropism and antibody-mediated neutralization.

Expression and characterization of SARS-CoV-2 spike proteins.

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.

A Combination of Receptor-Binding Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS-CoV-2 Spike Neutralization-Escape Mutants.

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.

Potent neutralization of SARS-CoV-2 variants of concern by an antibody with an uncommon genetic signature and structural mode of spike recognition.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.

Efficient discovery of potently neutralizing SARS-CoV-2 antibodies using LIBRA-seq with ligand blocking.

SARS-CoV-2 therapeutic antibody discovery efforts have met with notable success but have been associated with a generally inefficient process, requiring the production and characterization of exceptionally large numbers of candidates for the identification of a small set of leads. Here, we show that incorporating antibody-ligand blocking as part of LIBRA-seq, the high-throughput sequencing platform for antibody discovery, results in efficient identification of ultra-potent neutralizing antibodies against SARS-CoV-2. LIBRA-seq with ligand blocking is a general platform for functional antibody discovery targeting the disruption of antigen-ligand interactions.

Cross-reactive coronavirus antibodies with diverse epitope specificities and Fc effector functions.

The continual emergence of novel coronaviruses (CoV), such as severe acute respiratory syndrome-(SARS)-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. From a recovered SARS-CoV donor sample, we identify and characterize a panel of six monoclonal antibodies that cross-react with CoV spike (S) proteins from the highly pathogenic SARS-CoV and SARS-CoV-2, and demonstrate a spectrum of reactivity against other CoVs. Epitope mapping reveals that these antibodies recognize multiple epitopes on SARS-CoV-2 S, including the receptor-binding domain, the N-terminal domain, and the S2 subunit. Functional characterization demonstrates that the antibodies mediate phagocytosis-and in some cases trogocytosis-but not neutralization in vitro. When tested in vivo in murine models, two of the antibodies demonstrate a reduction in hemorrhagic pathology in the lungs. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses.

An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate. We evaluated RSV F-specific B cell responses before and after vaccination in six participants using complementary B cell sequencing methodologies and identified 555 clonal lineages. DS-Cav1-induced lineages recognized the prefusion conformation of F (pre-F) and were genetically diverse. Expressed antibodies recognized all six antigenic sites on the pre-F trimer. We identified 34 public clonotypes, and structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Thus, vaccination with DS-Cav1 generates a diverse polyclonal response targeting the antigenic sites on pre-F, supporting the development and advanced testing of pre-F-based vaccines against RSV.

Prolonged evolution of the human B cell response to SARS-CoV-2 infection.

A comprehensive understanding of the kinetics and evolution of the human B cell response to SARS-CoV-2 infection will facilitate the development of next-generation vaccines and therapies. Here, we longitudinally profiled this response in mild and severe COVID-19 patients over a period of five months. Serum neutralizing antibody (nAb) responses waned rapidly but spike (S)-specific IgG+ memory B cells (MBCs) remained stable or increased over time. Analysis of 1,213 monoclonal antibodies (mAbs) isolated from S-specific MBCs revealed a primarily de novo response that displayed increased somatic hypermutation, binding affinity, and neutralization potency over time, providing evidence for prolonged antibody affinity maturation. B cell immunodominance hierarchies were similar across donor repertoires and remained relatively stable as the immune response progressed. Cross-reactive B cell populations, likely re-called from prior endemic beta-coronavirus exposures, comprised a small but stable fraction of the repertoires and did not contribute to the neutralizing response. The neutralizing antibody response was dominated by public clonotypes that displayed significantly reduced activity against SARS-CoV-2 variants emerging in Brazil and South Africa that harbor mutations at positions 501, 484 and 417 in the S protein. Overall, the results provide insight into the dynamics, durability, and functional properties of the human B cell response to SARS-CoV-2 infection and have implications for the design of immunogens that preferentially stimulate protective B cell responses.