The Antibody Response to Plasmodium falciparum: Cues for Vaccine Design and the Discovery of Receptor-Based Antibodies.

Plasmodium falciparum remains a serious public health problem and a continuous challenge for the immune system due to the complexity and diversity of the pathogen. Recent advances from several laboratories in the characterization of the antibody response to the parasite have led to the identification of critical targets for protection and revealed a new mechanism of diversification based on the insertion of host receptors into immunoglobulin genes, leading to the production of receptor-based antibodies. These advances have opened new possibilities for vaccine design and passive antibody therapies to provide sterilizing immunity and control blood-stage parasites.

Structure, receptor recognition, and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein.

The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.

Broadly neutralizing antiviral antibodies.

A fascinating aspect of viral evolution relates to the ability of viruses to escape the adaptive immune response. The widely held view has been that the great variability of viral glycoproteins would be an absolute obstacle to the development of antibody-based therapies or vaccines that could confer broad and long-lasting protection. In the past five years, new approaches have been developed to interrogate human memory B cells and plasma cells with high efficiency and to isolate several broadly neutralizing antiviral antibodies against highly variable pathogens such as HIV-1 and influenza virus. These antibodies not only provide new tools for prophylaxis and therapy for viral diseases but also identify conserved epitopes that may be used to design new vaccines capable of conferring broader protection.

Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology.

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.

Clonal structure, stability and dynamics of human memory B cells and circulating plasmablasts.

Memory B cells persist for a lifetime and rapidly differentiate into antibody-producing plasmablasts and plasma cells upon antigen re-encounter. The clonal relationship and evolution of memory B cells and circulating plasmablasts is not well understood. Using single-cell sequencing combined with isolation of specific antibodies, we found that in two healthy donors, the memory B cell repertoire was dominated by large IgM, IgA and IgG2 clonal families, whereas IgG1 families, including those specific for recall antigens, were of small size. Analysis of multiyear samples demonstrated stability of memory B cell clonal families and revealed that a large fraction of recently generated plasmablasts was derived from long-term memory B cell families and was found recurrently. Collectively, this study provides a systematic description of the structure, stability and dynamics of the human memory B cell pool and suggests that memory B cells may be active at any time point in the generation of plasmablasts.

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion.

Recent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism.

Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses ∼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.

Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

An Unbiased Screen for Human Cytomegalovirus Identifies Neuropilin-2 as a Central Viral Receptor.

Characterizing cell surface receptors mediating viral infection is critical for understanding viral tropism and developing antiviral therapies. Nevertheless, due to challenges associated with detecting protein interactions on the cell surface, the host receptors of many human pathogens remain unknown. Here, we build a library consisting of most single transmembrane human receptors and implement a workflow for unbiased and high-sensitivity detection of receptor-ligand interactions. We apply this technology to elucidate the long-sought receptor of human cytomegalovirus (HCMV), the leading viral cause of congenital birth defects. We identify neuropilin-2 (Nrp2) as the receptor for HCMV-pentamer infection in epithelial/endothelial cells and uncover additional HCMV interactors. Using a combination of biochemistry, cell-based assays, and electron microscopy, we characterize the pentamer-Nrp2 interaction and determine the architecture of the pentamer-Nrp2 complex. This work represents an important approach to the study of host-pathogen interactions and provides a framework for understanding HCMV infection, neutralization, and the development of novel anti-HCMV therapies.

Dynamics in protein translation sustaining T cell preparedness.

In response to pathogenic threats, naive T cells rapidly transition from a quiescent to an activated state, yet the underlying mechanisms are incompletely understood. Using a pulsed SILAC approach, we investigated the dynamics of mRNA translation kinetics and protein turnover in human naive and activated T cells. Our datasets uncovered that transcription factors maintaining T cell quiescence had constitutively high turnover, which facilitated their depletion following activation. Furthermore, naive T cells maintained a surprisingly large number of idling ribosomes as well as 242 repressed mRNA species and a reservoir of glycolytic enzymes. These components were rapidly engaged following stimulation, promoting an immediate translational and glycolytic switch to ramp up the T cell activation program. Our data elucidate new insights into how T cells maintain a prepared state to mount a rapid immune response, and provide a resource of protein turnover, absolute translation kinetics and protein synthesis rates in T cells ( https://www.immunomics.ch ).

A Human Bi-specific Antibody against Zika Virus with High Therapeutic Potential.

Zika virus (ZIKV), a mosquito-borne flavivirus, causes devastating congenital birth defects. We isolated a human monoclonal antibody (mAb), ZKA190, that potently cross-neutralizes multi-lineage ZIKV strains. ZKA190 is highly effective in vivo in preventing morbidity and mortality of ZIKV-infected mice. NMR and cryo-electron microscopy show its binding to an exposed epitope on DIII of the E protein. ZKA190 Fab binds all 180 E protein copies, altering the virus quaternary arrangement and surface curvature. However, ZIKV escape mutants emerged in vitro and in vivo in the presence of ZKA190, as well as of other neutralizing mAbs. To counter this problem, we developed a bispecific antibody (FIT-1) comprising ZKA190 and a second mAb specific for DII of E protein. In addition to retaining high in vitro and in vivo potencies, FIT-1 robustly prevented viral escape, warranting its development as a ZIKV immunotherapy.

Publisher Correction: An immunoregulatory and tissue-residency program modulated by c-MAF in human TH17 cells.

In the version of this article initially published, in the legend to Fig. 1b, the description of the frequency of TH17-IL-10+ clones was incomplete for the first group; this should read as follows: "...13 experiments with clones isolated from CCR6+CCR4+CXCR3- T cells...". Also, the label along the vertical axis of the bottom right plot in Figure 5b was incomplete; the correct label is 'IFN-γ+ cells (%)'. Finally, in the first sentence of the final paragraph of the final Results subsection, the description of the regions analyzed was incorrect; that sentence should begin: "DNA motif-enrichment analysis of the subset-specific H3K27ac-positive regions...". The errors have been corrected in the HTML and PDF versions of the article.

L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity.

Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses.

Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes.

Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.

An immunoregulatory and tissue-residency program modulated by c-MAF in human TH17 cells.

Different types of effector and memory T lymphocytes are induced and maintained in protective or pathological immune responses. Here we characterized two human CD4+ TH17 helper cell subsets that, in the recently activated state, could be distinguished on the basis of their expression of the anti-inflammatory cytokine IL-10. IL-10+ TH17 cells upregulated a variety of genes encoding immunoregulatory molecules, as well as genes whose expression is characteristic of tissue-resident T cells. In contrast, IL-10- TH17 cells maintained a pro-inflammatory gene-expression profile and upregulated the expression of homing receptors that guide recirculation from tissues to blood. Expression of the transcription factor c-MAF was selectively upregulated in IL-10+ TH17 cells, and it was bound to a large set of enhancer-like regions and modulated the immunoregulatory and tissue-residency program. Our results identify c-MAF as a relevant factor that drives two highly divergent post-activation fates of human TH17 cells and provide a framework with which to investigate the role of these cells in physiology and immunopathology.

Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors.

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.

Social network architecture of human immune cells unveiled by quantitative proteomics.

The immune system is unique in its dynamic interplay between numerous cell types. However, a system-wide view of how immune cells communicate to protect against disease has not yet been established. We applied high-resolution mass-spectrometry-based proteomics to characterize 28 primary human hematopoietic cell populations in steady and activated states at a depth of >10,000 proteins in total. Protein copy numbers revealed a specialization of immune cells for ligand and receptor expression, thereby connecting distinct immune functions. By integrating total and secreted proteomes, we discovered fundamental intercellular communication structures and previously unknown connections between cell types. Our publicly accessible (http://www.immprot.org/) proteomic resource provides a framework for the orchestration of cellular interplay and a reference for altered communication associated with pathology.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Structural basis of malaria RIFIN binding by LILRB1-containing antibodies.

Some Plasmodium falciparum repetitive interspersed families of polypeptides (RIFINs)-variant surface antigens that are expressed on infected erythrocytes1-bind to the inhibitory receptor LAIR1, and insertion of DNA that encodes LAIR1 into immunoglobulin genes generates RIFIN-specific antibodies2,3. Here we address the general relevance of this finding by searching for antibodies that incorporate LILRB1, another inhibitory receptor that binds to ß2 microglobulin and RIFINs through their apical domains4,5. By screening plasma from a cohort of donors from Mali, we identified individuals with LILRB1-containing antibodies. B cell clones isolated from three donors showed large DNA insertions in the switch region that encodes non-apical LILRB1 extracellular domain 3 and 4 (D3D4) or D3 alone in the variable-constant (VH-CH1) elbow. Through mass spectrometry and binding assays, we identified a large set of RIFINs that bind to LILRB1 D3. Crystal and cryo-electron microscopy structures of a RIFIN in complex with either LILRB1 D3D4 or a D3D4-containing antibody Fab revealed a mode of RIFIN-LILRB1 D3 interaction that is similar to that of RIFIN-LAIR1. The Fab showed an unconventional triangular architecture with the inserted LILRB1 domains opening up the VH-CH1 elbow without affecting VH-VL or CH1-CL pairing. Collectively, these findings show that RIFINs bind to LILRB1 through D3 and illustrate, with a naturally selected example, the general principle of creating novel antibodies by inserting receptor domains into the VH-CH1 elbow.