Rapid exploration of the epitope coverage produced by an Ebola survivor to guide the discovery of therapeutic antibody cocktails

ABSTRACT Background Development of successful neutralizing antibodies is dependent upon broad epitope coverage to increase the likelihood of achieving therapeutic function. Recent advances in synthetic biology have allowed us to conduct an epitope binning study on a large panel of antibodies identified to bind to Ebola virus glycoprotein with only published sequences. Methods and Results A rapid, first-pass epitope binning experiment revealed seven distinct epitope families that overlapped with known structural epitopes from the literature. A focused set of antibodies was selected from representative clones per bin to guide a second-pass binning that revealed previously unassigned epitopes, confirmed epitopes known to be associated with neutralizing antibodies, and demonstrated asymmetric blocking of EBOV GP from allosteric effectors reported from literature. Conclusions Critically, this workflow allows us to probe the epitope landscape of EBOV GP without any prior structural knowledge of the antigen or structural benchmark clones. Incorporating epitope binning on hundreds of antibodies during early stage antibody characterization ensures access to a library’s full epitope coverage, aids in the identification of high quality reagents within the library that recapitulate this diversity for use in other studies, and ultimately enables the rational development of therapeutic cocktails that take advantage of multiple mechanisms of action such as cooperative synergistic effects to enhance neutralization function and minimize the risk of mutagenic escape. The use of high-throughput epitope binning during new outbreaks such as the current COVID-19 pandemic is particularly useful in accelerating timelines due to the large amount of information gained in a single experiment.

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Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines

Nature Communications ◽

10.1038/s41467-021-22867-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Linling He ◽

Anshul Chaudhary ◽

Xiaohe Lin ◽

Cindy Sou ◽

Tanwee Alkutkar ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Ebola Virus ◽

Heptad Repeat ◽

T Cell Epitopes ◽

Vaccine Strategy ◽

Cell Responses ◽

Self Assembling ◽

Protein Nanoparticles ◽

Main Target ◽

Repertoire Sequencing

AbstractEbola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

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Persistence of Viremia and Production of Neutralizing Antibodies Differentially Regulated by Polymorphic APOBEC3 and BAFF-R Loci in Friend Virus-Infected Mice

Journal of Virology ◽

10.1128/jvi.02516-09 ◽

2010 ◽

Vol 84 (12) ◽

pp. 6082-6095 ◽

Cited By ~ 27

Author(s):

Sachiyo Tsuji-Kawahara ◽

Tomomi Chikaishi ◽

Eri Takeda ◽

Maiko Kato ◽

Saori Kinoshita ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Early Stage ◽

Chromosome 15 ◽

Cell Repertoire ◽

Viral Loads ◽

Functional Polymorphisms ◽

B Cell Repertoire ◽

Retroviral Replication ◽

Different Strains ◽

Host Genes

ABSTRACT Several host genes control retroviral replication and pathogenesis through the regulation of immune responses to viral antigens. The Rfv3 gene influences the persistence of viremia and production of virus-neutralizing antibodies in mice infected with Friend mouse retrovirus complex (FV). This locus has been mapped within a narrow segment of mouse chromosome 15 harboring the APOBEC3 and BAFF-R loci, both of which show functional polymorphisms among different strains of mice. The exon 5-lacking product of the APOBEC3 allele expressed in FV-resistant C57BL/6 (B6) mice directly restricts viral replication, and mice lacking the B6-derived APOBEC3 exhibit exaggerated pathology and reduced production of neutralizing antibodies. However, the mechanisms by which the polymorphisms at the APOBEC3 locus affect the production of neutralizing antibodies remain unclear. Here we show that the APOBEC3 genotypes do not directly affect the B-cell repertoire, and mice lacking B6-derived APOBEC3 still produce FV-neutralizing antibodies in the presence of primed T helper cells. Instead, higher viral loads at a very early stage of FV infection caused by either a lack of the B6-derived APOBEC3 or a lack of the wild-type BAFF-R resulted in slower production of neutralizing antibodies. Indeed, B cells were hyperactivated soon after infection in the APOBEC3- or BAFF-R-deficient mice. In contrast to mice deficient in the B6-derived APOBEC3, which cleared viremia by 4 weeks after FV infection, mice lacking the functional BAFF-R allele exhibited sustained viremia, indicating that the polymorphisms at the BAFF-R locus may better explain the Rfv3-defining phenotype of persistent viremia.

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Self-Replicating RNA Viruses for RNA Therapeutics

Molecules ◽

10.3390/molecules23123310 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3310 ◽

Cited By ~ 13

Author(s):

Kenneth Lundstrom

Keyword(s):

Clinical Trials ◽

Phase I ◽

Tumor Cells ◽

Neutralizing Antibodies ◽

Ebola Virus ◽

Vaccine Development ◽

Rna Viruses ◽

Antibody Responses ◽

Phase Iii ◽

Phase I Clinical Trials

Self-replicating single-stranded RNA viruses such as alphaviruses, flaviviruses, measles viruses, and rhabdoviruses provide efficient delivery and high-level expression of therapeutic genes due to their high capacity of RNA replication. This has contributed to novel approaches for therapeutic applications including vaccine development and gene therapy-based immunotherapy. Numerous studies in animal tumor models have demonstrated that self-replicating RNA viral vectors can generate antibody responses against infectious agents and tumor cells. Moreover, protection against challenges with pathogenic Ebola virus was obtained in primates immunized with alphaviruses and flaviviruses. Similarly, vaccinated animals have been demonstrated to withstand challenges with lethal doses of tumor cells. Furthermore, clinical trials have been conducted for several indications with self-amplifying RNA viruses. In this context, alphaviruses have been subjected to phase I clinical trials for a cytomegalovirus vaccine generating neutralizing antibodies in healthy volunteers, and for antigen delivery to dendritic cells providing clinically relevant antibody responses in cancer patients, respectively. Likewise, rhabdovirus particles have been subjected to phase I/II clinical trials showing good safety and immunogenicity against Ebola virus. Rhabdoviruses have generated promising results in phase III trials against Ebola virus. The purpose of this review is to summarize the achievements of using self-replicating RNA viruses for RNA therapy based on preclinical animal studies and clinical trials in humans.

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Trehalose 6,6′-Dimycolate (Cord Factor) of Mycobacterium tuberculosis Induces Corneal Angiogenesis in Rats

Infection and Immunity ◽

10.1128/iai.68.10.5991-5997.2000 ◽

2000 ◽

Vol 68 (10) ◽

pp. 5991-5997 ◽

Cited By ~ 35

Author(s):

Norio Saita ◽

Nagatoshi Fujiwara ◽

Ikuya Yano ◽

Kazuhiko Soejima ◽

Kazuo Kobayashi

Keyword(s):

Mycobacterium Tuberculosis ◽

Neutralizing Antibodies ◽

Early Stage ◽

Vascular Endothelial ◽

Cytokine Network ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Cord Factor ◽

Factor Alpha ◽

Endothelial Growth Factor

ABSTRACT Neovascularization or angiogenesis is required for the progression of chronic inflammation. The mechanism of inflammatory neovascularization in tuberculosis remains unknown. Trehalose 6,6′-dimycolate (TDM) purified from Mycobacterium tuberculosis was injected into rat corneas. TDM challenge provoked a local granulomatous response in association with neovascularization. Neovascularization was seen within a few days after the challenge, with the extent of neovascularization being dose dependent, although granulomatous lesions developed 14 days after the challenge. Cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8), IL-1β, and vascular endothelial growth factor (VEGF), were found in lesions at the early stage (within a few days after the challenge) and were detectable until day 21. Neovascularization was inhibited substantially by neutralizing antibodies to VEGF and IL-8 but not IL-1β. Treatment with anti-TNF-α antibodies resulted in partial inhibition. TDM possesses pleiotropic activities, and the cytokine network plays an important role in the process of neovascularization.

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Molecular Identification and Genetic Characterization of Early-Stage Multiple Primary Lung Cancer by Large-Panel Next-Generation Sequencing Analysis

Frontiers in Oncology ◽

10.3389/fonc.2021.653988 ◽

2021 ◽

Vol 11 ◽

Author(s):

Guotian Pei ◽

Mingwei Li ◽

Xianjun Min ◽

Qiang Liu ◽

Dasheng Li ◽

...

Keyword(s):

Lung Cancer ◽

Next Generation Sequencing ◽

Early Stage ◽

Primary Lung Cancer ◽

Advanced Stage ◽

Next Generation ◽

Molecular Method ◽

Multiple Primary ◽

Large Panel ◽

Generation Sequencing

ObjectiveThe incidence of early stage multiple primary lung cancer (MPLC) has been increasing in recent years, while the ideal strategy for its diagnosis and treatment remains controversial. The present study conducted genomic analysis to identify a new molecular classification method for accurately predicting the diagnosis and therapy for patients with early stage MPLC.MethodsA total of 240 tissue samples from 203 patients with multiple-non-small-cell lung cancers (NSCLCs) (n = 30), early stage single-NSCLC (Group A, n = 94), and advanced-stage NSCLC (Group B, n = 79) were subjected to targeted multigene panel sequencing.ResultsThirty patients for whom next-generation sequencing was performed on >1 tumor were identified, yielding 45 tumor pairs. The frequencies of EGFR, TP53, RBM10, ERBB2, and CDKN2A mutations exhibited significant differences between early and advanced-stage NSCLCs. The prevalence of the EGFR L858R mutation in early stage NSCLC was remarkably higher than that in advanced-stage NSCLC (P = 0.047). The molecular method classified tumor pairs into 26 definite MPLC tumors and four intrapulmonary metastasis (IM) tumors. A high rate of discordance in driver genetic alterations was found in the different tumor lesions of MPLC patients. The prospective Martini histologic prediction of MPLC was discordant with the molecular method for three patients (16.7%), particularly in the prediction of IM (91.7% discordant).ConclusionsComprehensive molecular evaluation allows the unambiguous delineation of clonal relationships among tumors. In comparison, the Martini and Melamed criteria have notable limitations in the recognition of IM. Our results support the adoption of a large panel to supplement histology for strongly discriminating NSCLC clonal relationships in clinical practice.

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Potent Therapy and Transcriptional Profile of Combined Erythropoietin Derived Peptide CHBP and Caspase-3 siRNA against Kidney Ischemia/Reperfusion Injury in mice

10.21203/rs.3.rs-50459/v1 ◽

2020 ◽

Author(s):

Yuanyuan Wu ◽

Weiwei Chen ◽

Yufang Zhang ◽

Aifen Liu ◽

Cheng Yang ◽

...

Keyword(s):

Caspase 3 ◽

Ischemia Reperfusion Injury ◽

Early Stage ◽

Synergistic Effects ◽

Ischemia Reperfusion ◽

Combined Treatment ◽

Specific Treatment ◽

Transcriptional Profile ◽

Apoptosis And Inflammation ◽

Active Caspase

Abstract Cause-specific treatment and timely diagnosis are still not available for acute kidney injury (AKI) apart from supportive therapy and serum creatinine measurement. A novel erythropoietin-derived cyclic helix B surface peptide (CHBP) protects kidneys against AKI with different causes, but the underlying mechanism is not fully defined. Herein, we investigated the transcriptional profile of renoprotection induced by CHBP and its potential synergistic effects with siRNA targeting caspase-3, an executing enzyme of apoptosis and inflammation, (CASP3siRNA) on ischemia/reperfusion (IR)-induced AKI. Utilizing a mouse model with 30-min renal bilateral ischemia and 48-h reperfusion, the renoprotection of CHBP or CASP3siRNA was demonstrated in renal function and structure, active caspase-3 and HMGB1 expression. Combined treatment of CHBP and CASP3siRNA further preserved kidney structure, and reduced active caspase-3 and HMGB1. Furthermore, differentially expressed genes (DEGs) were identified with fold change > 1.414 and P < 0.05. In IR kidneys, 281 DEGs induced by CHBP were mainly involved in promoting cell division and improving cellular function and metabolism (up-regulated STAT5B and SLC22A7). The additional administration of CASP3siRNA caused 504 and 418 DEGs in IR + CHBP kidneys with or without NCsiRNA, with 37 genes in common. These DEGs were associated with modulated apoptosis and inflammation (up-regulated BCL6, SLPI and SERPINA3M), and immunity, injury and microvascular homeostasis (up-regulated CFH and GREM1, and down-regulated ANGPTL2). This proof-of-effect study indicated the potent renoprotection of CASP3siRNA upon CHBP at the early stage of IR-induced AKI. Underlying genes, BCL6, SLPI, SERPINA3M, GREM1 and ANGPTL2, might be potential new biomarkers for clinical applications.

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Synergistic actions of stem cell factor and other burst-promoting activities on proliferation of CD34+ highly purified blood progenitors expressing HLA-DR or different levels of c-kit protein

Blood ◽

10.1182/blood.v84.12.4099.bloodjournal84124099 ◽

1994 ◽

Vol 84 (12) ◽

pp. 4099-4106 ◽

Cited By ~ 25

Author(s):

Y Sonoda ◽

H Sakabe ◽

Y Ohmisono ◽

S Tanimukai ◽

S Yokota ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Factor ◽

Early Stage ◽

Human Peripheral Blood ◽

Synergistic Effects ◽

Target Population ◽

Additive Effect ◽

Gm Csf ◽

Stage Of Development ◽

Hla Dr

We studied the synergistic effects of stem cell factor (SCF) and other burst-promoting activities (BPAs) such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-9 on proliferation of human peripheral blood-derived highly purified progenitors. SCF, IL-3, GM-CSF, and IL-9 showed significant BPA when CD34+HLA-DR+ cells were used as the target population. IL-3 exerted the most potent BPA, and GM-CSF supported approximately 40% to 70% of the erythroid burst-forming units that are responsive to IL-3. SCF and IL-9 showed much weaker BPA than that of IL-3 or GM-CSF. Combinations of IL- 3 with other BPAs did not show synergistic actions supporting erythroid- burst formation. However, GM-CSF showed a significant additive effect with IL-9 or SCF. When CD34+c-kithigh cells were used as the target, SCF showed a much stronger BPA. Also, a distinct additive effect between SCF and IL-3 or GM-CSF on erythrocyte-containing mixed colony formation was observed. On the other hand, when CD34+c-kitlow cells were used as the target, SCF, IL-3, and GM-CSF could express BPA. In contrast, IL-9 alone failed to support erythroid-burst formation. Because CD34+c-kithigh cells weakly expressed CD34 antigen, these cells appeared to be more mature progenitors than CD34+c-kitlow cells. These results suggest that IL-9 acts on more mature progenitors than those of SCF, IL-3, or GM-CSF and that the primary target of SCF is multipotential progenitors at the very early stage of development.

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Human interleukin-4 inhibits proliferation of megakaryocyte progenitor cells in culture

Blood ◽

10.1182/blood.v81.3.624.624 ◽

1993 ◽

Vol 81 (3) ◽

pp. 624-630 ◽

Cited By ~ 23

Author(s):

Y Sonoda ◽

Y Kuzuyama ◽

S Tanaka ◽

S Yokota ◽

T Maekawa ◽

...

Keyword(s):

Transforming Growth Factor Beta ◽

Colony Formation ◽

Neutralizing Antibodies ◽

Transforming Growth Factor ◽

Early Stage ◽

Interleukin 4 ◽

Dependent Manner ◽

Inhibitory Effects ◽

Necrosis Factor Alpha

Abstract We studied the effects of recombinant human interleukin-4 (rhIL-4) on megakaryocyte colony formation from enriched hematopoietic progenitors. IL-4 strongly inhibited pure and mixed megakaryocyte colony formation in a dose-dependent manner. Formation of erythroid bursts, eosinophil colonies, and erythrocyte-containing mixed colonies was not affected by the addition of IL-4 as reported previously (Sonoda Y, et al; Blood 75:1615, 1990). Delayed addition experiments suggested that IL-4 acts on an early stage of proliferation of megakaryocyte progenitors. Neutralizing antibodies (antisera) prepared against transforming growth factor beta, tumor necrosis factor alpha, interferon alpha (IFN alpha), and IFN gamma did not affect the inhibitory effects of IL-4 on pure and mixed megakaryocyte colony formation. In addition, the inhibitory effects of IL-4 was also seen in serum-free cultures and in cultures containing highly enriched CD34+, HLA-DR+ cells as a target population. These results indicate that IL-4 may function as one of the negative regulators in human megakaryocytopoiesis in vitro.

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Safety and Immunogenicity of Heterologous and Homologous 2-Dose Regimens of Adenovirus Serotype 26– and Modified Vaccinia Ankara–Vectored Ebola Vaccines: A Randomized, Controlled Phase 1 Study

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa586 ◽

2020 ◽

Cited By ~ 1

Author(s):

Neil Goldstein ◽

Viki Bockstal ◽

Stephan Bart ◽

Kerstin Luhn ◽

Cynthia Robinson ◽

...

Keyword(s):

Immune Responses ◽

Neutralizing Antibodies ◽

Ebola Virus ◽

Phase 1 ◽

Booster Vaccination ◽

Controlled Study ◽

Adenovirus Serotype ◽

Cellular Immune Responses ◽

High Doses ◽

Cellular Immune

Abstract Background This phase 1 placebo-controlled study assessed the safety and immunogenicity of 2-dose regimens of Ad26.ZEBOV (adenovirus serotype 26 [Ad26]) and MVA-BN-Filo (modified vaccinia Ankara [MVA]) vaccines with booster vaccination at day 360. Methods Healthy US adults (N = 164) randomized into 10 groups received saline placebo or standard or high doses of Ad26 or MVA in 2-dose regimens at 7-, 14-, 28-, or 56-day intervals; 8 groups received booster Ad26 or MVA vaccinations on day 360. Participants reported solicited and unsolicited reactogenicity; we measured immunoglobulin G binding, neutralizing antibodies and cellular immune responses to Ebola virus glycoprotein. Results All regimens were well tolerated with no serious vaccine-related adverse events. Heterologous (Ad26,MVA [dose 1, dose 2] or MVA,Ad26) and homologous (Ad26,Ad26) regimens induced humoral and cellular immune responses 21 days after dose 2; responses were higher after heterologous regimens. Booster vaccination elicited anamnestic responses in all participants. Conclusions Both heterologous and homologous Ad26,MVA Ebola vaccine regimens are well tolerated in healthy adults, regardless of interval or dose level. Heterologous 2-dose Ad26,MVA regimens containing an Ebola virus insert induce strong, durable humoral and cellular immune responses. Immunological memory was rapidly recalled by booster vaccination, suggesting that Ad26 booster doses could be considered for individuals at risk of Ebola infection, who previously received the 2-dose regimen.

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