Viral receptor-binding site antibodies with diverse germline origins.

Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by 11 different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B cell targets.

Selective targeting of mutated calreticulin by the monoclonal antibody INCA033989 inhibits oncogenic function of MPN.

Mutations in calreticulin (mutCALR) are the second most common drivers of myeloproliferative neoplasms (MPNs) and yet, the current therapeutic landscape lacks a selective agent for mutCALR-expressing MPNs. Here we show that the monoclonal antibody INCA033989 selectively targets mutCALR-positive cells. INCA033989 antagonized mutCALR-driven signaling and proliferation in engineered cell lines and primary CD34+ cells from patients with MPN. No antibody binding or functional activity was observed in cells lacking mutCALR. In a mouse model of mutCALR-driven MPN, treatment with a INCA033989 mouse surrogate antibody effectively prevented the development of thrombocytosis and accumulation of megakaryocytes in the bone marrow. INCA033989 reduced the pathogenic self-renewal of mutCALR-positive disease-initiating cells in both primary and secondary transplantations, illustrating its disease-modifying potential. In summary, we describe a novel mutCALR-targeted therapy for MPNs, a monoclonal antibody that selectively inhibits the oncogenic function of MPN cells without interfering with normal hematopoiesis.

Metaproteomic analysis using the Galaxy framework.

Metaproteomics characterizes proteins expressed by microorganism communities (microbiome) present in environmental samples or a host organism (e.g. human), revealing insights into the molecular functions conferred by these communities. Compared to conventional proteomics, metaproteomics presents unique data analysis challenges, including the use of large protein databases derived from hundreds or thousands of organisms, as well as numerous processing steps to ensure high data quality. These challenges limit the use of metaproteomics for many researchers. In response, we have developed an accessible and flexible metaproteomics workflow within the Galaxy bioinformatics framework. Via analysis of human oral tissue exudate samples, we have established a modular Galaxy-based workflow that automates a reduction method for searching large sequence databases, enabling comprehensive identification of host proteins (human) as well as "meta-proteins" from the nonhost organisms. Downstream, automated processing steps enable basic local alignment search tool analysis and evaluation/visualization of peptide sequence match quality, maximizing confidence in results. Outputted results are compatible with tools for taxonomic and functional characterization (e.g. Unipept, MEGAN5). Galaxy also allows for the sharing of complete workflows with others, promoting reproducibility and also providing a template for further modification and enhancement. Our results provide a blueprint for establishing Galaxy as a solution for metaproteomic data analysis. All MS data have been deposited in the ProteomeXchange with identifier PXD001655 (http://proteomecentral.proteomexchange.org/dataset/PXD001655).

The influenza C virus CM2 protein can alter intracellular pH, and its transmembrane domain can substitute for that of the influenza A virus M2 protein and support infectious virus production.

The influenza C virus CM2 protein and a chimeric influenza A virus M2 protein (MCM) containing the CM2 transmembrane domain were assessed for their ability to functionally replace the M2 protein. While all three proteins could alter cytosolic pH to various degrees when expressed from cDNA, only M2 and MCM could at least partially restore infectious virus production to M2-deficient influenza A viruses. The data suggest that while the CM2 ion channel activity is similar to that of M2, sequences in the extracellular and/or cytoplasmic domains play important roles in infectious virus production.

Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses.

Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.

Mutations in the membrane-proximal region of the influenza A virus M2 protein cytoplasmic tail have modest effects on virus replication.

Influenza A virus encodes M2, a proton channel that has been shown to be important during virus entry and assembly. In order to systematically investigate the role of the membrane-proximal residues in the M2 cytoplasmic tail in virus replication, we utilized scanning and directed alanine mutagenesis in combination with transcomplementation assays and recombinant viruses. The membrane-proximal residues 46 to 69 tolerated numerous mutations, with little, if any, effect on virus replication, suggesting that protein structure rather than individual amino acid identity in this region may be critical for M2 protein function.

Simultaneous affinity maturation and developability enhancement using natural liability-free CDRs.

ABBREVIATIONS: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; ka: association rate; kd: dissociation rate; KD: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.

Tyrosines in the influenza A virus M2 protein cytoplasmic tail are critical for production of infectious virus particles.

The cytoplasmic tail of the influenza A virus M2 protein is required for the production of infectious virions. In this study, critical residues in the M2 cytoplasmic tail were identified by single-alanine scanning mutagenesis. The tyrosine residue at position 76, which is conserved in >99% of influenza virus strains sequenced to date, was identified as being critical for the formation of infectious virus particles using both reverse genetics and a protein trans-complementation assay. Recombinant viruses encoding M2 with the Y76A mutation demonstrated replication defects in MDCK cells as well as in primary differentiated airway epithelial cell cultures, defects in the formation of filamentous virus particles, and reduced packaging of nucleoprotein into virus particles. These defects could all be overcome by a mutation of serine to tyrosine at position 71 of the M2 cytoplasmic tail, which emerged after blind passage of viruses containing the Y76A mutation. These data confirm and extend our understanding of the significance of the M2 protein for infectious virus particle assembly.

A single donor is sufficient to produce a highly functional in vitro antibody library.

Antibody complementarity determining region diversity has been considered to be the most important metric for the production of a functional antibody library. Generally, the greater the antibody library diversity, the greater the probability of selecting a diverse array of high affinity leads. According to this paradigm, the primary means of elevating library diversity has been by increasing the number of donors. In the present study we explored the possibility of creating an in vitro antibody library from a single healthy individual, showing that the number of lymphocytes, rather than the number of donors, is the key criterion in the production of a diverse and functional antibody library. We describe the construction of a high-quality phage display library comprising 5 × 109 human antibodies by applying an efficient B cell extraction protocol from a single donor and a targeted V-gene amplification strategy favoring specific antibody families for their improved developability profiles. Each step of the library generation process was followed and validated by next generation sequencing to monitor the library quality and diversity. The functionality of the library was tested using several therapeutically relevant targets for which a vast number of different antibodies with desired biophysical properties were obtained.

A human CD137×PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade.

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.

Influenza immunization elicits antibodies specific for an egg-adapted vaccine strain.

For broad protection against infection by viruses such as influenza or HIV, vaccines should elicit antibodies that bind conserved viral epitopes, such as the receptor-binding site (RBS). RBS-directed antibodies have been described for both HIV and influenza virus, and the design of immunogens to elicit them is a goal of vaccine research in both fields. Residues in the RBS of influenza virus hemagglutinin (HA) determine a preference for the avian or human receptor, α-2,3-linked sialic acid and α-2,6-linked sialic acid, respectively. Transmission of an avian-origin virus between humans generally requires one or more mutations in the sequences encoding the influenza virus RBS to change the preferred receptor from avian to human, but passage of a human-derived vaccine candidate in chicken eggs can select for reversion to avian receptor preference. For example, the X-181 strain of the 2009 new pandemic H1N1 influenza virus, derived from the A/California/07/2009 isolate and used in essentially all vaccines since 2009, has arginine at position 226, a residue known to confer preference for an α-2,3 linkage in H1 subtype viruses; the wild-type A/California/07/2009 isolate, like most circulating human H1N1 viruses, has glutamine at position 226. We describe, from three different individuals, RBS-directed antibodies that recognize the avian-adapted H1 strain in current influenza vaccines but not the circulating new pandemic 2009 virus; Arg226 in the vaccine-strain RBS accounts for the restriction. The polyclonal sera of the three donors also reflect this preference. Therefore, when vaccines produced from strains that are never passaged in avian cells become widely available, they may prove more capable of eliciting RBS-directed, broadly neutralizing antibodies than those produced from egg-adapted viruses, extending the established benefits of current seasonal influenza immunizations.

Study of Andes virus entry and neutralization using a pseudovirion system.

Andes virus (ANDV), a member of the Hantavirus genus in the family Bunyaviridae, causes an acute disease characteristic of New-World hantaviruses called hantavirus pulmonary syndrome (HPS). HPS is a highly pathogenic disease with a case-fatality rate of 40%. ANDV is the only hantavirus reported to spread directly from human-to-human. The aim of the present study was to develop a quantitative and high-throughput pseudovirion assay to study ANDV infection and neutralization in biosafety level 2 facilities (BSL-2). This pseudovirion assay is based on incorporation of ANDV glycoproteins onto replication-defective vesicular stomatitis virus (VSV) cores in which the gene for the surface G protein has been replaced by that encoding Renilla luciferase. Infection by the pseudovirions can be quantified by luciferase activity of infected cell lysates. ANDV pseudovirions were neutralized by ANDV-specific antisera, and there was good concordance between specificity and neutralization titers of ANDV hamster sera as determined by our pseudovirion assay and a commonly used plaque reduction neutralization titer (PRNT) assay. In addition, the pseudovirions were used to evaluate the requirements for ANDV entry, like pH dependency and the role of beta3 integrin, the reported receptor for other pathogenic hantaviruses, on entry.

The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence.

Influenza A virus particles assemble and bud from plasma membrane domains enriched with the viral glycoproteins but only a small fraction of the total M2 protein is incorporated into virus particles when compared to the other viral glycoproteins. A membrane proximal cholesterol recognition/interaction amino acid consensus (CRAC) motif was previously identified in M2 and suggested to play a role in protein function. We investigated the importance of the CRAC motif on virus replication by generating recombinant proteins and viruses containing amino acid substitutions in this motif. Alteration or completion of the M2 CRAC motif in two different virus strains caused no changes in virus replication in vitro. Viruses lacking an M2 CRAC motif had decreased morbidity and mortality in the mouse model of infection, suggesting that this motif is a virulence determinant which may facilitate virus replication in vivo but is not required for basic virus replication in tissue culture.

Presence of broadly reactive and group-specific neutralizing epitopes on newly described isolates of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease in humans with high rates of mortality. The virus has a tripartite genome composed of a small (S), a medium (M) and a large (L) RNA segment; the M segment encodes the two viral glycoproteins, G(N) and G(C). Whilst relatively few full-length M segment sequences are available, it is apparent that both G(N) and G(C) may exhibit significant sequence diversity. It is unknown whether considerable antigenic differences exist between divergent CCHFV strains, or whether there are conserved neutralizing epitopes. The M segments derived from viral isolates of a human case of CCHF in South Africa (SPU 41/84), an infected tick (Hyalomma marginatum) in South Africa (SPU 128/81), a human case in Congo (UG 3010), an infected individual in Uzbekistan (U2-2-002) and an infected tick (Hyalomma asiaticum) in China (Hy13) were sequenced fully, and the glycoproteins were expressed. These novel sequences showed high variability in the N-terminal region of G(N) and more modest differences in the remainder of G(N) and in G(C). Phylogenetic analyses placed these newly identified strains in three of the four previously described M segment groups. Studies with a panel of mAbs specific to G(N) and G(C) indicated that there were significant antigenic differences between the M segment groups, although several neutralizing epitopes in both G(N) and G(C) were conserved among all strains examined. Thus, the genetic diversity exhibited by CCHFV strains results in significant antigenic differences that will need to be taken into consideration for vaccine development.

Dynamic docking of myosin and actin observed with resonance energy transfer.

Atomic models of myosin subfragment-1 (S1) and the actin filament are docked together using resonance energy-transfer data from both pre- and postpowerstroke conditions. The quality of the resulting best fits discriminated between neck-region orientations of the S1 for a given set of experimental conditions. For measurements of the postpowerstroke states in the presence of ADP, resonance energy-transfer data alone are sufficient to dock the atomic models and provide evidence that S1 exists with at least two neck-region orientations under these conditions. To dock the prepowerstroke state, resonance energy-transfer data were used in combination with previous chemical cross-linking data to determine that a neck-region orientation similar to that of a proposed prepowerstroke state best fit the data. The resulting models determined independently from electron microscopy compare favorably with micrographs from the recent literature. The docking models by resonance energy transfer suggest that the larger movements in the light-chain binding domain are accompanied by twisting and rotating movements of the catalytic domain, causing a tilt of approximately 30 degrees during the weak-to-strong transition. This transition provides the displacement necessary to support motility and force generation.