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Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.
Copyright © 2020 Elsevier Inc. All rights reserved.
Stem cells in stratified epithelia are generally believed to adhere to a non-hierarchical single-progenitor model. Using lineage tracing and genetic label-retention assays, we show that the hard palatal epithelium of the oral cavity is unique in displaying marked proliferative heterogeneity. We identify a previously uncharacterized, infrequently-dividing stem cell population that resides within a candidate niche, the junctional zone (JZ). JZ stem cells tend to self-renew by planar symmetric divisions, respond to masticatory stresses, and promote wound healing, whereas frequently-dividing cells reside outside the JZ, preferentially renew through perpendicular asymmetric divisions, and are less responsive to injury. LRIG1 is enriched in the infrequently-dividing population in homeostasis, dynamically changes expression in response to tissue stresses, and promotes quiescence, whereas Igfbp5 preferentially labels a rapidly-growing, differentiation-prone population. These studies establish the oral mucosa as an important model system to study epithelial stem cell populations and how they respond to tissue stresses.
Copyright © 2019 Elsevier Inc. All rights reserved.
-induced gastritis is the strongest risk factor for gastric adenocarcinoma, a malignancy preceded by a series of well-defined histological stages, including metaplasia. One microbial constituent that augments cancer risk is the type 4 secretion system (T4SS), which translocates the oncoprotein CagA into host cells. Aberrant stem cell activation is linked to carcinogenesis, and Lrig1 (leucine-rich repeats and Ig-like domains 1) marks a distinct population of progenitor cells. We investigated whether microbial effectors with carcinogenic potential influence Lrig1 progenitor cells ex vivo and via lineage expansion within -infected gastric mucosa. Lineage tracing was induced in (Lrig1/YFP) mice that were uninfected or subsequently infected with or an isogenic mutant (nonfunctional T4SS). In contrast to infection with wild-type (WT) for 2 wk, infection for 8 wk resulted in significantly increased inflammation and proliferation in the corpus and antrum compared with uninfected or mice infected with the mutant. WT -infected mice harbored significantly higher numbers of Lrig1/YFP epithelial cells that coexpressed UEA1 (surface cell marker). The number of cells coexpressing intrinsic factor (chief cell marker), YFP (lineage marker), and GSII lectin (spasmolytic polypeptide-expressing metaplasia marker) were increased only by WT In human samples, Lrig1 expression was significantly increased in lesions with premalignant potential compared with normal mucosa or nonatrophic gastritis. In conclusion, chronic infection stimulates Lrig1-expressing progenitor cells in a -dependent manner, and these reprogrammed cells give rise to a full spectrum of differentiated cells.
Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The "breathing" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.
Understanding antigenic variation in influenza virus strains and how the human immune system recognizes strains are central challenges for vaccinologists. Antibodies directed to the 2 major viral surface membrane proteins, hemagglutinin (HA) and neuraminidase (NA), mediate protection against reinfection following natural infection or vaccination, but HA and NA protein sequences in field strains are highly variable. The central questions are how to achieve protective antibody responses in a higher proportion of individuals and how to induce responses with more breadth and durability. Studies using isolation of human monoclonal antibodies followed by structural and functional characterization revealed conserved antigenic sites recognized by broadly cross-reactive antibodies. The antigenic landscape on HA and NA proteins is coming into focus to inform studies of the correlates and mechanisms of immunity. Understanding the antibody determinants of influenza immunity points the way toward development and testing of next-generation vaccines with potential to confer broadly protective immunity.
© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: email@example.com.
Influenza is a yearly threat to global public health. Rapid changes in influenza surface proteins resulting from antigenic drift and shift events make it difficult to readily identify antibodies with broadly neutralizing activity against different influenza subtypes with high frequency, specifically antibodies targeting the receptor binding domain (RBD) on influenza HA protein. We developed an optimized computational design method that is able to optimize an antibody for recognition of large panels of antigens. To demonstrate the utility of this multistate design method, we used it to redesign an antiinfluenza antibody against a large panel of more than 500 seasonal HA antigens of the H1 subtype. As a proof of concept, we tested this method on a variety of known antiinfluenza antibodies and identified those that could be improved computationally. We generated redesigned variants of antibody C05 to the HA RBD and experimentally characterized variants that exhibited improved breadth and affinity against our panel. C05 mutants exhibited improved affinity for three of the subtypes used in design by stabilizing the CDRH3 loop and creating favorable electrostatic interactions with the antigen. These mutants possess increased breadth and affinity of binding while maintaining high-affinity binding to existing targets, surpassing a major limitation up to this point.
Copyright © 2019 the Author(s). Published by PNAS.
Ebolaviruses Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) cause human disease with high case fatality rates. Experimental monovalent vaccines, which all utilize the sole envelope glycoprotein (GP), do not protect against heterologous ebolaviruses. Human parainfluenza virus type 3-vectored vaccines offer benefits, including needle-free administration and induction of mucosal responses in the respiratory tract. Multiple approaches were taken to induce broad protection against the three ebolaviruses. While GP consensus-based antigens failed to elicit neutralizing antibodies, polyvalent vaccine immunization induced neutralizing responses to all three ebolaviruses and protected animals from death and disease caused by EBOV, SUDV, and BDBV. As immunization with a cocktail of antigenically related antigens can skew the responses and change the epitope hierarchy, we performed comparative analysis of antibody repertoire and Fc-mediated protective mechanisms in animals immunized with monovalent versus polyvalent vaccines. Compared to sera from guinea pigs receiving the monovalent vaccines, sera from guinea pigs receiving the trivalent vaccine bound and neutralized EBOV and SUDV at equivalent levels and BDBV at only a slightly reduced level. Peptide microarrays revealed a preponderance of binding to amino acids 389 to 403, 397 to 415, and 477 to 493, representing three linear epitopes in the mucin-like domain known to induce a protective antibody response. Competition binding assays with monoclonal antibodies isolated from human ebolavirus infection survivors demonstrated that the immune sera block the binding of antibodies specific for the GP glycan cap, the GP1-GP2 interface, the mucin-like domain, and the membrane-proximal external region. Thus, administration of a cocktail of three ebolavirus vaccines induces a desirable broad antibody response, without skewing of the response toward preferential recognition of a single virus. The symptoms of the disease caused by the ebolaviruses Ebola, Bundibugyo, and Sudan are similar, and their areas of endemicity overlap. However, because of the limited antigenic relatedness of the ebolavirus glycoprotein (GP) used in all candidate vaccines against these viruses, they protect only against homologous and not against heterologous ebolaviruses. Therefore, a broadly specific pan-ebolavirus vaccine is required, and this might be achieved by administration of a cocktail of vaccines. The effects of cocktail administration of ebolavirus vaccines on the antibody repertoire remain unknown. Here, an in-depth analysis of the antibody responses to administration of a cocktail of human parainfluenza virus type 3-vectored vaccines against individual ebolaviruses was performed, which included analysis of binding to GP, neutralization of individual ebolaviruses, epitope specificity, Fc-mediated functions, and protection against the three ebolaviruses. The results demonstrated potent and balanced responses against individual ebolaviruses and no significant reduction of the responses compared to that induced by individual vaccines.
Copyright © 2019 American Society for Microbiology.
Synthetically engineered DNA-encoded monoclonal antibodies (DMAbs) are an in vivo platform for evaluation and delivery of human mAb to control against infectious disease. Here, we engineer DMAbs encoding potent anti-Zaire ebolavirus (EBOV) glycoprotein (GP) mAbs isolated from Ebola virus disease survivors. We demonstrate the development of a human IgG1 DMAb platform for in vivo EBOV-GP mAb delivery and evaluation in a mouse model. Using this approach, we show that DMAb-11 and DMAb-34 exhibit functional and molecular profiles comparable to recombinant mAb, have a wide window of expression, and provide rapid protection against lethal mouse-adapted EBOV challenge. The DMAb platform represents a simple, rapid, and reproducible approach for evaluating the activity of mAb during clinical development. DMAbs have the potential to be a mAb delivery system, which may be advantageous for protection against highly pathogenic infectious diseases, like EBOV, in resource-limited and other challenging settings.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
The epithelial-mesenchymal transition (EMT) endows carcinoma cells with traits needed to complete many of the steps leading to metastasis formation, but its contributions specifically to the late step of extravasation remain understudied. We find that breast cancer cells that have undergone an EMT extravasate more efficiently from blood vessels both in vitro and in vivo. Analysis of gene expression changes associated with the EMT program led to the identification of an EMT-induced cell-surface protein, podocalyxin (PODXL), as a key mediator of extravasation in mesenchymal breast and pancreatic carcinoma cells. PODXL promotes extravasation through direct interaction of its intracellular domain with the cytoskeletal linker protein ezrin. Ezrin proceeds to establish dorsal cortical polarity, enabling the transition of cancer cells from a non-polarized, rounded cell morphology to an invasive extravasation-competent shape. Hence, the EMT program can directly enhance the efficiency of extravasation and subsequent metastasis formation through a PODXL-ezrin signaling axis.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.