Structural and mechanistic insights into disease-associated endolysosomal exonucleases PLD3 and PLD4.

Endolysosomal exonucleases PLD3 and PLD4 (phospholipases D3 and D4) are associated with autoinflammatory and autoimmune diseases. We report structures of these enzymes, and the molecular basis of their catalysis. The structures reveal an intra-chain dimer topology forming a basic active site at the interface. Like other PLD superfamily members, PLD3 and PLD4 carry HxKxxxxD/E motifs and participate in phosphodiester-bond cleavage. The enzymes digest ssDNA and ssRNA in a 5'-to-3' manner and are blocked by 5'-phosphorylation. We captured structures in apo, intermediate, and product states and revealed a "link-and-release" two-step catalysis. We also unexpectedly demonstrated phosphatase activity via a covalent 3-phosphohistidine intermediate. PLD4 contains an extra hydrophobic clamp that stabilizes substrate and could affect oligonucleotide substrate preference and product release. Biochemical and structural analysis of disease-associated mutants of PLD3/4 demonstrated reduced enzyme activity or thermostability and the possible basis for disease association. Furthermore, these findings provide insight into therapeutic design.

Structural and mechanistic insights into disease-associated endolysosomal exonucleases PLD3 and PLD4.

Endolysosomal exonucleases PLD3 and PLD4 (phospholipases D3 and D4) are associated with autoinflammatory and autoimmune diseases. We report structures of these enzymes, and the molecular basis of their catalysis. The structures reveal an intra-chain dimer topology forming a basic active site at the interface. Like other PLD superfamily members, PLD3 and PLD4 carry HxKxxxxD/E motifs and participate in phosphodiester-bond cleavage. The enzymes digest ssDNA and ssRNA in a 5'-to-3' manner and are blocked by 5'-phosphorylation. We captured structures in apo, intermediate, and product states and revealed a 'link-and-release' two-step catalysis. We also unexpectedly demonstrated phosphatase activity via a covalent 3' phosphistidine intermediate. PLD4 contains an extra hydrophobic clamp that stabilizes substrate and could affect oligonucleotide substrate preference and product release. Biochemical and structural analysis of disease-associated mutants of PLD3/4 demonstrated reduced enzyme activity or thermostability and the possible basis for disease association. Furthermore, these findings provide insight into therapeutic design.

Broadening a SARS-CoV-1-neutralizing antibody for potent SARS-CoV-2 neutralization through directed evolution.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing antibody that neutralizes one virus but not a related virus. Through a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1-neutralizing antibody, to bind to the receptor binding domain of SARS-CoV-2 with >1000-fold increased affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of engineered CR3022 elucidated the molecular mechanisms by which the antibody can accommodate sequence differences in the epitopes between SARS-CoV-1 and SARS-CoV-2. This workflow provides a blueprint for the rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.

Disease in the Pld4thss/thss Model of Murine Lupus Requires TLR9.

Phospholipase D4 (PLD4) is an endolysosomal exonuclease of ssRNA and ssDNA, rather than a phospholipase as its name suggests. Human polymorphisms in the PLD4 gene have been linked by genome-wide association studies to systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. However, B6.129 Pld4-/- mice develop features of a distinct disease, macrophage activation syndrome, which is reversed in mice mutated in TLR9. In this article, we compare a Pld4 null mutant identified on the BALB/c background, Pld4thss/thss, which has distinct phenotypes: short stature, thin hair, and features of systemic lupus erythematosus. All phenotypes analyzed were largely normalized in Pld4thss/thssTlr9-/- mice. Thus, Pld4thss/thss represents a rare model in which mouse lupus etiology is TLR9 dependent. Compared with PLD4-deficient B6 mice, Pld4thss/thss mice had elevated levels of serum IgG, IgG anti-dsDNA autoantibodies, BAFF, and IFN-γ and elevated B cell numbers. Overall, the data suggest that PLD4 deficiency can lead to a diverse array of rheumatological abnormalities depending upon background-modifying genes, and that these diseases of PLD4 deficiency are largely driven by TLR9 recognition of ssDNA.

Fusion of the molecular adjuvant C3d to cleavage-independent native-like HIV-1 Env trimers improves the elicited antibody response.

An effective HIV vaccine likely requires the elicitation of neutralizing antibodies (NAbs) against multiple HIV-1 clades. The recently developed cleavage-independent native flexibly linked (NFL) envelope (Env) trimers exhibit well-ordered conformation and elicit autologous tier 2 NAbs in multiple animal models. Here, we investigated whether the fusion of molecular adjuvant C3d to the Env trimers can improve B- cell germinal center (GC) formation and antibody responses. To generate Env-C3d trimers, we performed a glycine-serine- based (G4S) flexible peptide linker screening and identified a linker range that allowed native folding. A 30-60- amino- acid- long linker facilitates Env-to-C3d association and achieves the secretion of well-ordered trimers and the structural integrity and functional integrity of Env and C3d. The fusion of C3d did not dramatically affect the antigenicity of the Env trimers and enhanced the ability of the Env trimers to engage and activate B cells in vitro. In mice, the fusion of C3d enhanced germinal center formation, the magnitude of Env-specific binding antibodies, and the avidity of the antibodies in the presence of an adjuvant. The Sigma Adjuvant System (SAS) did not affect the trimer integrity in vitro but contributed to altered immunogenicity in vivo, resulting in increased tier 1 neutralization, likely by increased exposure of variable region 3 (V3). Taken together, the results indicate that the fusion of the molecular adjuvant, C3d, to the Env trimers improves antibody responses and could be useful for Env-based vaccines against HIV.

Broadly neutralizing antibodies targeting a conserved silent face of spike RBD resist extreme SARS-CoV-2 antigenic drift.

Developing broad coronavirus vaccines requires identifying and understanding the molecular basis of broadly neutralizing antibody (bnAb) spike sites. In our previous work, we identified sarbecovirus spike RBD group 1 and 2 bnAbs. We have now shown that many of these bnAbs can still neutralize highly mutated SARS-CoV-2 variants, including the XBB.1.5. Structural studies revealed that group 1 bnAbs use recurrent germline-encoded CDRH3 features to interact with a conserved RBD region that overlaps with class 4 bnAb site. Group 2 bnAbs recognize a less well-characterized "site V" on the RBD and destabilize spike trimer. The site V has remained largely unchanged in SARS-CoV-2 variants and is highly conserved across diverse sarbecoviruses, making it a promising target for broad coronavirus vaccine development. Our findings suggest that targeted vaccine strategies may be needed to induce effective B cell responses to escape resistant subdominant spike RBD bnAb sites.

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease.

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against novel pandemic coronaviruses and to more effectively respond to SARS-CoV-2 variants. The emergence of Omicron and subvariants of SARS-CoV-2 illustrates the limitations of solely targeting the receptor-binding domain (RBD) of the spike (S) protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors, which targets a conserved S2 region in the betacoronavirus spike fusion machinery. Select bnAbs showed broad in vivo protection against all three deadly betacoronaviruses, SARS-CoV-1, SARS-CoV-2, and MERS-CoV, which have spilled over into humans in the past two decades. Structural studies of these bnAbs delineated the molecular basis for their broad reactivity and revealed common antibody features targetable by broad vaccination strategies. These bnAbs provide new insights and opportunities for antibody-based interventions and for developing pan-betacoronavirus vaccines.

Rare, convergent antibodies targeting the stem helix broadly neutralize diverse betacoronaviruses.

Humanity has faced three recent outbreaks of novel betacoronaviruses, emphasizing the need to develop approaches that broadly target coronaviruses. Here, we identify 55 monoclonal antibodies from COVID-19 convalescent donors that bind diverse betacoronavirus spike proteins. Most antibodies targeted an S2 epitope that included the K814 residue and were non-neutralizing. However, 11 antibodies targeting the stem helix neutralized betacoronaviruses from different lineages. Eight antibodies in this group, including the six broadest and most potent neutralizers, were encoded by IGHV1-46 and IGKV3-20. Crystal structures of three antibodies of this class at 1.5-1.75-Å resolution revealed a conserved mode of binding. COV89-22 neutralized SARS-CoV-2 variants of concern including Omicron BA.4/5 and limited disease in Syrian hamsters. Collectively, these findings identify a class of IGHV1-46/IGKV3-20 antibodies that broadly neutralize betacoronaviruses by targeting the stem helix but indicate these antibodies constitute a small fraction of the broadly reactive antibody response to betacoronaviruses after SARS-CoV-2 infection.

Asymmetrical forward and reverse developmental trajectories determine molecular programs of B cell antigen receptor editing.

During B lymphopoiesis, B cell progenitors progress through alternating and mutually exclusive stages of clonal expansion and immunoglobulin (Ig) gene rearrangements. Great diversity is generated through the stochastic recombination of Ig gene segments encoding heavy and light chain variable domains. However, this commonly generates autoreactivity. Receptor editing is the predominant tolerance mechanism for self-reactive B cells in the bone marrow (BM). B cell receptor editing rescues autoreactive B cells from negative selection through renewed light chain recombination first at Igκ then Igλ loci. Receptor editing depends on BM microenvironment cues and key transcription factors such as NF-κB, FOXO, and E2A. The specific BM factor required for receptor editing is unknown. Furthermore, how transcription factors coordinate these developmental programs to promote usage of the λ chain remains poorly defined. Therefore, we used two mouse models that recapitulate pathways by which Igλ light chain-positive B cells develop. The first has deleted J kappa (Jκ) genes and hence models Igλ expression resulting from failed Igκ recombination (Igκdel). The second models autoreactivity by ubiquitous expression of a single-chain chimeric anti-Igκ antibody (κ-mac). Here, we demonstrated that autoreactive B cells transit asymmetric forward and reverse developmental trajectories. This imparted a unique epigenetic landscape on small pre-B cells, which opened chromatin to transcription factors essential for Igλ recombination. The consequences of this asymmetric developmental path were both amplified and complemented by CXCR4 signaling. These findings reveal how intrinsic molecular programs integrate with extrinsic signals to drive receptor editing.

Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques.

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting broadly neutralizing antibody responses to CoVs. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein with a two-shot protocol generated potent serum receptor binding domain cross-neutralizing antibody responses to both SARS-CoV-2 and SARS-CoV-1. Furthermore, responses were equally effective against most SARS-CoV-2 variants of concern (VOCs) and some were highly effective against Omicron. This result contrasts with human infection or many two-shot vaccination protocols where responses were typically more SARS-CoV-2 specific and where VOCs were less well neutralized. Structural studies showed that cloned macaque neutralizing antibodies, particularly using a given heavy chain germline gene, recognized a relatively conserved region proximal to the angiotensin converting enzyme 2 receptor binding site (RBS), whereas many frequently elicited human neutralizing antibodies targeted more variable epitopes overlapping the RBS. B cell repertoire differences between humans and macaques appeared to influence the vaccine response. The macaque neutralizing antibodies identified a pan-SARS-related virus epitope region less well targeted by human antibodies that could be exploited in rational vaccine design.

Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape pathways.

The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we used a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduces the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity-matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for antiviral indications would benefit from affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.

Broadly neutralizing antibodies target the coronavirus fusion peptide.

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We used an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. COV44-62 and COV44-79 broadly neutralize alpha- and betacoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than receptor binding domain-specific antibodies. In crystal structures of COV44-62 and COV44-79 antigen-binding fragments with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine residue at the S2' cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.

In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice.

Transplantation of B cells engineered ex vivo to secrete broadly neutralizing antibodies (bNAbs) has shown efficacy in disease models. However, clinical translation of this approach would require specialized medical centers, technically demanding protocols and major histocompatibility complex compatibility of donor cells and recipients. Here we report in vivo B cell engineering using two adeno-associated viral vectors, with one coding for Staphylococcus aureus Cas9 (saCas9) and the other for 3BNC117, an anti-HIV bNAb. After intravenously injecting the vectors into mice, we observe successful editing of B cells leading to memory retention and bNAb secretion at neutralizing titers of up to 6.8 µg ml-1. We observed minimal clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 off-target cleavage as detected by unbiased CHANGE-sequencing analysis, whereas on-target cleavage in undesired tissues is reduced by expressing saCas9 from a B cell-specific promoter. In vivo B cell engineering to express therapeutic antibodies is a safe, potent and scalable method, which may be applicable not only to infectious diseases but also in the treatment of noncommunicable conditions, such as cancer and autoimmune disease.

A broad and potent neutralization epitope in SARS-related coronaviruses.

Many neutralizing antibodies (nAbs) elicited to ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through natural infection and vaccination have reduced effectiveness to SARS-CoV-2 variants. Here, we show that therapeutic antibody ADG20 is able to neutralize SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize a broad range of VOCs, albeit with reduced potency against Omicron. Thus, this conserved and vulnerable site can be exploited for the design of universal vaccines and therapeutic antibodies.

Broadly neutralizing antibodies target the coronavirus fusion peptide.

The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2' cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development. One-Sentence Summary: Rare monoclonal antibodies from COVID-19 convalescent individuals broadly neutralize coronaviruses by targeting the fusion peptide.

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease.

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.

A broad and potent neutralization epitope in SARS-related coronaviruses.

Many neutralizing antibodies (nAbs) elicited to ancestral SARS-CoV-2 through natural infection and vaccination generally have reduced effectiveness to SARS-CoV-2 variants. Here we show therapeutic antibody ADG20 is able to neutralize all SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize all VOCs, albeit with reduced potency against Omicron. Thus, this highly conserved and vulnerable site can be exploited for design of universal vaccines and therapeutic antibodies.

A human antibody reveals a conserved site on beta-coronavirus spike proteins and confers protection against SARS-CoV-2 infection.

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a CoV disease 2019 (COVID-19) convalescent donor that exhibits broad reactivity with human ß-CoVs. Here, we showed that CC40.8 targets the conserved S2 stem helix region of the CoV spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem peptide at 1.6-Å resolution and found that the peptide adopted a mainly helical structure. Conserved residues in ß-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted that CC40.8-like bnAbs are relatively rare in human COVID-19 infection, and therefore, their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on ß-CoV spike proteins for protective antibodies that may facilitate the development of pan-ß-CoV vaccines.