Central tolerance shapes the neutralizing B cell repertoire against a persisting virus in its natural host.

Viral mimicry of host cell structures has been postulated to curtail the B cell receptor (BCR) repertoire against persisting viruses through tolerance mechanisms. This concept awaits, however, experimental testing in a setting of natural virus-host relationship. We engineered mouse models expressing a monoclonal BCR specific for the envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV), a naturally persisting mouse pathogen. When the heavy chain of the LCMV-neutralizing antibody KL25 was paired with its unmutated ancestor light chain, most B cells underwent receptor editing, a behavior reminiscent of autoreactive clones. In contrast, monoclonal B cells expressing the same heavy chain in conjunction with the hypermutated KL25 light chain did not undergo receptor editing but exhibited low levels of surface IgM, suggesting that light chain hypermutation had lessened KL25 autoreactivity. Upon viral challenge, these IgMlow cells were not anergic but up-regulated IgM, participated in germinal center reactions, produced antiviral antibodies, and underwent immunoglobulin class switch as well as further affinity maturation. These studies on a persisting virus in its natural host species suggest that central tolerance mechanisms prune the protective antiviral B cell repertoire.

Multifunctional human monoclonal antibody combination mediates protection against Rift Valley fever virus at low doses.

The zoonotic Rift Valley fever virus (RVFV) can cause severe disease in humans and has pandemic potential, yet no approved vaccine or therapy exists. Here we describe a dual-mechanism human monoclonal antibody (mAb) combination against RVFV that is effective at minimal doses in a lethal mouse model of infection. We structurally analyze and characterize the binding mode of a prototypical potent Gn domain-A-binding antibody that blocks attachment and of an antibody that inhibits infection by abrogating the fusion process as previously determined. Surprisingly, the Gn domain-A antibody does not directly block RVFV Gn interaction with the host receptor low density lipoprotein receptor-related protein 1 (LRP1) as determined by a competitive assay. This study identifies a rationally designed combination of human mAbs deserving of future investigation for use in humans against RVFV infection. Using a two-pronged mechanistic approach, we demonstrate the potent efficacy of a rationally designed combination mAb therapeutic.

VelcroVax: a "Bolt-On" Vaccine Platform for Glycoprotein Display.

Having varied approaches to the design and manufacture of vaccines is critical in being able to respond to worldwide needs and newly emerging pathogens. Virus-like particles (VLPs) form the basis of two of the most successful licensed vaccines (against hepatitis B virus [HBV] and human papillomavirus). They are produced by recombinant expression of viral structural proteins, which assemble into immunogenic nanoparticles. VLPs can be modified to present unrelated antigens, and here we describe a universal "bolt-on" platform (termed VelcroVax) where the capturing VLP and the target antigen are produced separately. We utilize a modified HBV core (HBcAg) VLP with surface expression of a high-affinity binding sequence (Affimer) directed against a SUMO tag and use this to capture SUMO-tagged gp1 glycoprotein from the arenavirus Junín virus (JUNV). Using this model system, we have solved the first high-resolution structures of VelcroVax VLPs and shown that the VelcroVax-JUNV gp1 complex induces superior humoral immune responses compared to the noncomplexed viral protein. We propose that this system could be modified to present a range of antigens and therefore form the foundation of future rapid-response vaccination strategies. IMPORTANCE The hepatitis B core protein (HBc) forms noninfectious virus-like particles, which can be modified to present a capturing molecule, allowing suitably tagged antigens to be bound on their surface. This system can be adapted and provides the foundation for a universal "bolt-on" vaccine platform (termed VelcroVax) that can be easily and rapidly modified to generate nanoparticle vaccine candidates.

Contrasting Modes of New World Arenavirus Neutralization by Immunization-Elicited Monoclonal Antibodies.

Transmission of the New World hemorrhagic fever arenaviruses Junín virus (JUNV) and Machupo virus (MACV) to humans is facilitated, in part, by the interaction between the arenavirus GP1 glycoprotein and the human transferrin receptor 1 (hTfR1). We utilize a mouse model of live-attenuated immunization with envelope exchange viruses to isolate neutralizing monoclonal antibodies (NAbs) specific to JUNV GP1 and MACV GP1. Structures of two NAbs, termed JUN1 and MAC1, demonstrate that they neutralize through disruption of hTfR1 recognition. JUN1 utilizes a binding mode common to all characterized infection- and vaccine-elicited JUNV-specific NAbs, which involves mimicking hTfR1 binding through the insertion of a tyrosine into the receptor-binding site. In contrast, MAC1 undergoes a tyrosine-mediated mode of antigen recognition distinct from that used by the reported anti-JUNV NAbs and the only other characterized anti-MACV NAb. These data reveal the varied modes of GP1-specific recognition among New World arenaviruses by the antibody-mediated immune response. IMPORTANCE The GP1 subcomponent of the New World arenavirus GP is a primary target of the neutralizing antibody response, which has been shown to be effective in the prevention and treatment of infection. Here, we characterize the structural basis of the antibody-mediated immune response that arises from immunization of mice against Junín virus and Machupo virus, two rodent-borne zoonotic New World arenaviruses. We isolate a panel of GP1-specific monoclonal antibodies that recognize overlapping epitopes and exhibit neutralizing behavior, in vitro. Structural characterization of two of these antibodies indicates that antibody recognition likely interferes with GP1-mediated recognition of the transferrin receptor 1. These data provide molecular-level detail for a key region of vulnerability on the New World arenavirus surface and a blueprint for therapeutic antibody development.

Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera.

SARS-CoV-2 has caused over 2 million deaths in little over a year. Vaccines are being deployed at scale, aiming to generate responses against the virus spike. The scale of the pandemic and error-prone virus replication is leading to the appearance of mutant viruses and potentially escape from antibody responses. Variant B.1.1.7, now dominant in the UK, with increased transmission, harbors 9 amino acid changes in the spike, including N501Y in the ACE2 interacting surface. We examine the ability of B.1.1.7 to evade antibody responses elicited by natural SARS-CoV-2 infection or vaccination. We map the impact of N501Y by structure/function analysis of a large panel of well-characterized monoclonal antibodies. B.1.1.7 is harder to neutralize than parental virus, compromising neutralization by some members of a major class of public antibodies through light-chain contacts with residue 501. However, widespread escape from monoclonal antibodies or antibody responses generated by natural infection or vaccination was not observed.

Recent Advances in Bunyavirus Glycoprotein Research: Precursor Processing, Receptor Binding and Structure.

The Bunyavirales order accommodates related viruses (bunyaviruses) with segmented, linear, single-stranded, negative- or ambi-sense RNA genomes. Their glycoproteins form capsomeric projections or spikes on the virion surface and play a crucial role in virus entry, assembly, morphogenesis. Bunyavirus glycoproteins are encoded by a single RNA segment as a polyprotein precursor that is co- and post-translationally cleaved by host cell enzymes to yield two mature glycoproteins, Gn and Gc (or GP1 and GP2 in arenaviruses). These glycoproteins undergo extensive N-linked glycosylation and despite their cleavage, remain associated to the virion to form an integral transmembrane glycoprotein complex. This review summarizes recent advances in our understanding of the molecular biology of bunyavirus glycoproteins, including their processing, structure, and known interactions with host factors that facilitate cell entry.

Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera.

The race to produce vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began when the first sequence was published, and this forms the basis for vaccines currently deployed globally. Independent lineages of SARS-CoV-2 have recently been reported: UK, B.1.1.7; South Africa, B.1.351; and Brazil, P.1. These variants have multiple changes in the immunodominant spike protein that facilitates viral cell entry via the angiotensin-converting enzyme-2 (ACE2) receptor. Mutations in the receptor recognition site on the spike are of great concern for their potential for immune escape. Here, we describe a structure-function analysis of B.1.351 using a large cohort of convalescent and vaccinee serum samples. The receptor-binding domain mutations provide tighter ACE2 binding and widespread escape from monoclonal antibody neutralization largely driven by E484K, although K417N and N501Y act together against some important antibody classes. In a number of cases, it would appear that convalescent and some vaccine serum offers limited protection against this variant.

The antigenic anatomy of SARS-CoV-2 receptor binding domain.

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.

Molecular rationale for antibody-mediated targeting of the hantavirus fusion glycoprotein.

The intricate lattice of Gn and Gc glycoprotein spike complexes on the hantavirus envelope facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Through study of a neutralizing monoclonal antibody termed mAb P-4G2, which neutralizes the zoonotic pathogen Puumala virus (PUUV), we provide a molecular-level basis for antibody-mediated targeting of the hantaviral glycoprotein lattice. Crystallographic analysis demonstrates that P-4G2 binds to a multi-domain site on PUUV Gc and may preclude fusogenic rearrangements of the glycoprotein that are required for host-cell entry. Furthermore, cryo-electron microscopy of PUUV-like particles in the presence of P-4G2 reveals a lattice-independent configuration of the Gc, demonstrating that P-4G2 perturbs the (Gn-Gc)4 lattice. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.

High-resolution crystal structure of arthropod Eiger TNF suggests a mode of receptor engagement and altered surface charge within endosomes.

The tumour necrosis factor alpha (TNFα) superfamily of proteins are critical in numerous biological processes, such as in development and immunity. Eiger is the sole TNFα member described in arthropods such as in the important model organism Drosophila. To date there are no structural data on any Eiger protein. Here we present the structure of the TNF domain of Eiger from the fall armyworm Spodoptera frugiperda (SfEiger) to 1.7 Å from a serendipitously obtained crystal without prior knowledge of the protein sequence. Our structure confirms that canonical trimerization is conserved from ancestral TNFs and points towards a mode of receptor engagement. Furthermore, we observe numerous surface histidines on SfEiger, potentially acting as pH switches following internalization into endosomes. Our data contributes to the genome annotation of S. frugiperda, a voracious agricultural pest, and can serve as a basis for future structure-function investigations of the TNF system in related arthropods such as Drosophila.

The methyltransferase domain of the Sudan ebolavirus L protein specifically targets internal adenosines of RNA substrates, in addition to the cap structure.

Mononegaviruses, such as Ebola virus, encode an L (large) protein that bears all the catalytic activities for replication/transcription and RNA capping. The C-terminal conserved region VI (CRVI) of L protein contains a K-D-K-E catalytic tetrad typical for 2'O methyltransferases (MTase). In mononegaviruses, cap-MTase activities have been involved in the 2'O methylation and N7 methylation of the RNA cap structure. These activities play a critical role in the viral life cycle as N7 methylation ensures efficient viral mRNA translation and 2'O methylation hampers the detection of viral RNA by the host innate immunity. The functional characterization of the MTase+CTD domain of Sudan ebolavirus (SUDV) revealed cap-independent methyltransferase activities targeting internal adenosine residues. Besides this, the MTase+CTD also methylates, the N7 position of the cap guanosine and the 2'O position of the n1 guanosine provided that the RNA is sufficiently long. Altogether, these results suggest that the filovirus MTases evolved towards a dual activity with distinct substrate specificities. Whereas it has been well established that cap-dependent methylations promote protein translation and help to mimic host RNA, the characterization of an original cap-independent methylation opens new research opportunities to elucidate the role of RNA internal methylations in the viral replication.

Structural basis of cholesterol binding by a novel clade of dendritic cell modulators from ticks.

Two crystal structures of Japanin, an 18 kDa immune-modulatory lipocalin from the Brown Ear Tick (Rhipicephalus appendiculatus), have been determined at 2.2 and 2.4 Å resolution. In both crystal forms the protein is in complex with cholesterol, which sits in a closed pocket at the centre of the lipocalin barrel. Both crystal forms are dimers, which are also observed in solution. Molecular modelling suggests that previously-described members of a tick protein family bearing high sequence homology to Japanin are also likely to bind cholesterol or cholesterol derivatives.

Structural dissection of human metapneumovirus phosphoprotein using small angle x-ray scattering.

The phosphoprotein (P) is the main and essential cofactor of the RNA polymerase (L) of non-segmented, negative-strand RNA viruses. P positions the viral polymerase onto its nucleoprotein-RNA template and acts as a chaperone of the nucleoprotein (N), thereby preventing nonspecific encapsidation of cellular RNAs. The phosphoprotein of human metapneumovirus (HMPV) forms homotetramers composed of a stable oligomerization domain (Pcore) flanked by large intrinsically disordered regions (IDRs). Here we combined x-ray crystallography of Pcore with small angle x-ray scattering (SAXS)-based ensemble modeling of the full-length P protein and several of its fragments to provide a structural description of P that captures its dynamic character, and highlights the presence of varyingly stable structural elements within the IDRs. We discuss the implications of the structural properties of HMPV P for the assembly and functioning of the viral transcription/replication machinery.

Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein.

Non-segmented, (-)RNA viruses cause serious human diseases. Human metapneumovirus (HMPV), an emerging pathogen of this order of viruses (Mononegavirales) is one of the main causes of respiratory tract illness in children. To help elucidate the assembly mechanism of the nucleocapsid (the viral RNA genome packaged by the nucleoprotein N) we present crystallographic structures of HMPV N in its assembled RNA-bound state and in a monomeric state, bound to the polymerase cofactor P. Our structures reveal molecular details of how P inhibits the self-assembly of N and how N transitions between the RNA-free and RNA-bound conformational state. Notably, we observe a role for the C-terminal extension of N in directly preventing premature uptake of RNA by folding into the RNA-binding cleft. Our structures suggest a common mechanism of how the growth of the nucleocapsid is orchestrated, and highlight an interaction site representing an important target for antivirals.

X-ray structure and activities of an essential Mononegavirales L-protein domain.

The L protein of mononegaviruses harbours all catalytic activities for genome replication and transcription. It contains six conserved domains (CR-I to -VI; Fig. 1a). CR-III has been linked to polymerase and polyadenylation activity, CR-V to mRNA capping and CR-VI to cap methylation. However, how these activities are choreographed is poorly understood. Here we present the 2.2-Å X-ray structure and activities of CR-VI+, a portion of human Metapneumovirus L consisting of CR-VI and the poorly conserved region at its C terminus, the +domain. The CR-VI domain has a methyltransferase fold, which besides the typical S-adenosylmethionine-binding site ((SAM)P) also contains a novel pocket ((NS)P) that can accommodate a nucleoside. CR-VI lacks an obvious cap-binding site, and the (SAM)P-adjoining site holding the nucleotides undergoing methylation ((SUB)P) is unusually narrow because of the overhanging +domain. CR-VI+ sequentially methylates caps at their 2'O and N7 positions, and also displays nucleotide triphosphatase activity.

Structural insights into the human metapneumovirus glycoprotein ectodomain.

Human metapneumovirus is a major cause of respiratory tract infections worldwide. Previous reports have shown that the viral attachment glycoprotein (G) modulates innate and adaptive immune responses, leading to incomplete immunity and promoting reinfection. Using bioinformatics analyses, static light scattering, and small-angle X-ray scattering, we show that the extracellular region of G behaves as a heavily glycosylated, intrinsically disordered polymer. We discuss potential implications of these findings for the modulation of immune responses by G.

Novel immunomodulators from hard ticks selectively reprogramme human dendritic cell responses.

Hard ticks subvert the immune responses of their vertebrate hosts in order to feed for much longer periods than other blood-feeding ectoparasites; this may be one reason why they transmit perhaps the greatest diversity of pathogens of any arthropod vector. Tick-induced immunomodulation is mediated by salivary components, some of which neutralise elements of innate immunity or inhibit the development of adaptive immunity. As dendritic cells (DC) trigger and help to regulate adaptive immunity, they are an ideal target for immunomodulation. However, previously described immunoactive components of tick saliva are either highly promiscuous in their cellular and molecular targets or have limited effects on DC. Here we address the question of whether the largest and globally most important group of ticks (the ixodid metastriates) produce salivary molecules that specifically modulate DC activity. We used chromatography to isolate a salivary gland protein (Japanin) from Rhipicephalus appendiculatus ticks. Japanin was cloned, and recombinant protein was produced in a baculoviral expression system. We found that Japanin specifically reprogrammes DC responses to a wide variety of stimuli in vitro, radically altering their expression of co-stimulatory and co-inhibitory transmembrane molecules (measured by flow cytometry) and their secretion of pro-inflammatory, anti-inflammatory and T cell polarising cytokines (assessed by Luminex multiplex assays); it also inhibits the differentiation of DC from monocytes. Sequence alignments and enzymatic deglycosylation revealed Japanin to be a 17.7 kDa, N-glycosylated lipocalin. Using molecular cloning and database searches, we have identified a group of homologous proteins in R. appendiculatus and related species, three of which we have expressed and shown to possess DC-modulatory activity. All data were obtained using DC generated from at least four human blood donors, with rigorous statistical analysis. Our results suggest a previously unknown mechanism for parasite-induced subversion of adaptive immunity, one which may also facilitate pathogen transmission.

Bifunctional lipocalin ameliorates murine immune complex-induced acute lung injury.

Molecules that simultaneously inhibit independent or co-dependent proinflammatory pathways may have advantages over conventional monotherapeutics. OmCI is a bifunctional protein derived from blood-feeding ticks that specifically prevents complement (C)-mediated C5 activation and also sequesters leukotriene B4 (LTB4) within an internal binding pocket. Here, we examined the effect of LTB4 binding on OmCI structure and function and investigated the relative importance of C-mediated C5 activation and LTB4 in a mouse model of immune complex-induced acute lung injury (IC-ALI). We describe two crystal structures of bacterially expressed OmCI: one binding a C16 fatty acid and the other binding LTB4 (C20). We show that the C5 and LTB4 binding activities of the molecule are independent of each other and that OmCI is a potent inhibitor of experimental IC-ALI, equally dependent on both C5 inhibition and LTB4 binding for full activity. The data highlight the importance of LTB4 in IC-ALI and activation of C5 by the complement pathway C5 convertase rather than by non-C proteases. The findings suggest that dual inhibition of C5 and LTB4 may be useful for treatment of human immune complex-dependent diseases.

Crystal structure and functional characterization of an immunomodulatory salivary cystatin from the soft tick Ornithodoros moubata.

The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help to overcome the host's defence during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken in the present study. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and was found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 A (1 A=0.1 nm) and was used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the pro-inflammatory cytokines tumour necrosis factor alpha and interleukin-12, and proliferation of antigen-specific CD4+ T-cells. This suggests that OmC2 may suppress the host's adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.