scholarly journals Two Distinct Lysosomal Targeting Strategies Afford Trojan Horse Antibodies With Pan-Filovirus Activity

2021 ◽  
Vol 12 ◽  
Author(s):  
Ariel S. Wirchnianski ◽  
Anna Z. Wec ◽  
Elisabeth K. Nyakatura ◽  
Andrew S. Herbert ◽  
Megan M. Slough ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Sequence Divergence ◽  
Proteolytic Cleavage ◽  
Trojan Horse ◽  
Viral Glycoprotein ◽  
High Sequence Divergence ◽  
Niemann Pick ◽  
Sites Of Action ◽  
A Site ◽  
Blocking Antibodies

Multiple agents in the family Filoviridae (filoviruses) are associated with sporadic human outbreaks of highly lethal disease, while others, including several recently identified agents, possess strong zoonotic potential. Although viral glycoprotein (GP)-specific monoclonal antibodies have demonstrated therapeutic utility against filovirus disease, currently FDA-approved molecules lack antiviral breadth. The development of broadly neutralizing antibodies has been challenged by the high sequence divergence among filovirus GPs and the complex GP proteolytic cleavage cascade that accompanies filovirus entry. Despite this variability in the antigenic surface of GP, all filoviruses share a site of vulnerability—the binding site for the universal filovirus entry receptor, Niemann-Pick C1 (NPC1). Unfortunately, this site is shielded in extracellular GP and only uncovered by proteolytic cleavage by host proteases in late endosomes and lysosomes, which are generally inaccessible to antibodies. To overcome this obstacle, we previously developed a ‘Trojan horse’ therapeutic approach in which engineered bispecific antibodies (bsAbs) coopt viral particles to deliver GP:NPC1 interaction-blocking antibodies to their endo/lysosomal sites of action. This approach afforded broad protection against members of the genus Ebolavirus but could not neutralize more divergent filoviruses. Here, we describe next-generation Trojan horse bsAbs that target the endo/lysosomal GP:NPC1 interface with pan-filovirus breadth by exploiting the conserved and widely expressed host cation-independent mannose-6-phosphate receptor for intracellular delivery. Our work highlights a new avenue for the development of single therapeutics protecting against all known and newly emerging filoviruses.

scholarly journals Trypsin Treatment Unlocks Barrier for Zoonotic Bat Coronavirus Infection

2019 ◽  
Vol 94 (5) ◽  
Author(s):  
Vineet D. Menachery ◽  
Kenneth H. Dinnon ◽  
Boyd L. Yount ◽  
Eileen T. McAnarney ◽  
Lisa E. Gralinski ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Proteolytic Cleavage ◽  
New Host ◽  
Proteolytic Activation ◽  
Host Restriction ◽  
Human Lung Cells ◽  
Protease Treatment ◽  
A Site ◽  
Blocking Antibodies ◽  
Bat Coronavirus

ABSTRACT Traditionally, the emergence of coronaviruses (CoVs) has been attributed to a gain in receptor binding in a new host. Our previous work with severe acute respiratory syndrome (SARS)-like viruses argued that bats already harbor CoVs with the ability to infect humans without adaptation. These results suggested that additional barriers limit the emergence of zoonotic CoV. In this work, we describe overcoming host restriction of two Middle East respiratory syndrome (MERS)-like bat CoVs using exogenous protease treatment. We found that the spike protein of PDF2180-CoV, a MERS-like virus found in a Ugandan bat, could mediate infection of Vero and human cells in the presence of exogenous trypsin. We subsequently show that the bat virus spike can mediate the infection of human gut cells but is unable to infect human lung cells. Using receptor-blocking antibodies, we show that infection with the PDF2180 spike does not require MERS-CoV receptor DPP4 and antibodies developed against the MERS spike receptor-binding domain and S2 portion are ineffective in neutralizing the PDF2180 chimera. Finally, we found that the addition of exogenous trypsin also rescues HKU5-CoV, a second bat group 2c CoV. Together, these results indicate that proteolytic cleavage of the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs. Coupled with receptor binding, proteolytic activation offers a new parameter to evaluate the emergence potential of bat CoVs and offers a means to recover previously unrecoverable zoonotic CoV strains. IMPORTANCE Overall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding and proteolytic cleavage of the spike are critical factors that must be considered for evaluating the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue that other tissues, including the digestive tract, could be a site for future coronavirus emergence events in humans.

scholarly journals Trypsin treatment unlocks barrier for zoonotic coronaviruses infection

2019 ◽  
Author(s):  
Vineet D. Menachery ◽  
Kenneth H. Dinnon ◽  
Boyd L. Yount ◽  
Eileen T. McAnarney ◽  
Lisa E. Gralinski ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Proteolytic Cleavage ◽  
Human Gut ◽  
New Host ◽  
Proteolytic Activation ◽  
Host Restriction ◽  
Human Lung Cells ◽  
Protease Treatment ◽  
A Site ◽  
Blocking Antibodies

AbstractTraditionally, the emergence of coronaviruses (CoVs) has been attributed to a gain in receptor binding in a new host. Our previous work with SARS-like viruses argued that bats already harbor CoVs with the ability to infect humans without adaptation. These results suggested that additional barriers limit the emergence of zoonotic CoV. In this work, we describe overcoming host restriction of two MERS-like bat CoVs using exogenous protease treatment. We found that the spike protein of PDF2180-CoV, a MERS-like virus found in a Ugandan bat, could mediate infection of Vero and human cells in the presence of exogenous trypsin. We subsequently show that the bat virus spike can mediate infection of human gut cells, but is unable to infect human lung cells. Using receptor-blocking antibodies, we show that infection with the PDF2180 spike does not require MERS-CoV receptor DPP4 and antibodies developed against the MERS spike receptor-binding domain and S2 portion are ineffective in neutralizing the PDF2180 chimera. Finally, we found that addition of exogenous trypsin also rescues replication of HKU5-CoV, a second MERS-like group 2c CoV. Together, these results indicate that proteolytic cleavage of the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs. Coupled with receptor binding, proteolytic activation offers a new parameter to evaluate emergence potential of CoVs and offer a means to recover previously unrecoverable zoonotic CoV strains.ImportanceOverall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding and proteolytic cleavage of the spike are critical factors that must be considered for evaluating the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue that other tissues, including the digestive tract, could be a site for future coronavirus emergence events in humans.

scholarly journals Structural characterization and duplication modes of pseudogenes in plants

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Flavia Mascagni ◽  
Gabriele Usai ◽  
Andrea Cavallini ◽  
Andrea Porceddu
Keyword(s):  
Evolutionary Rate ◽  
Populus Trichocarpa ◽  
Sequence Divergence ◽  
Strand Break ◽  
Whole Genome ◽  
Flanking Sequence ◽  
Plant Genomes ◽  
High Sequence Divergence ◽  
Flanking Regions

AbstractWe identified and characterized the pseudogene complements of five plant species: four dicots (Arabidopsis thaliana, Vitis vinifera, Populus trichocarpa and Phaseolus vulgaris) and one monocot (Oryza sativa). Retroposition was considered of modest importance for pseudogene formation in all investigated species except V. vinifera, which showed an unusually high number of retro-pseudogenes in non coding genic regions. By using a pipeline for the classification of sequence duplicates in plant genomes, we compared the relative importance of whole genome, tandem, proximal, transposed and dispersed duplication modes in the pseudo and functional gene complements. Pseudogenes showed higher tendencies than functional genes to genomic dispersion. Dispersed pseudogenes were prevalently fragmented and showed high sequence divergence at flanking regions. On the contrary, those deriving from whole genome duplication were proportionally less than expected based on observations on functional loci and showed higher levels of flanking sequence conservation than dispersed pseudogenes. Pseudogenes deriving from tandem and proximal duplications were in excess compared to functional loci, probably reflecting the high evolutionary rate associated with these duplication modes in plant genomes. These data are compatible with high rates of sequence turnover at neutral sites and double strand break repairs mediated duplication mechanisms.

scholarly journals A Fully Protective Congenital CMV Vaccine Requires Neutralizing Antibodies to Viral Pentamer and gB Glycoprotein Complexes but a pp65 T-Cell Response Is Not Necessary

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1467
Author(s):  
K. Yeon Choi ◽  
Alistair McGregor
Keyword(s):  
T Cell ◽  
Cell Response ◽  
Neutralizing Antibodies ◽  
T Cell Response ◽  
Control Group ◽  
Type I ◽  
Cmv Infection ◽  
Viral Glycoprotein ◽  
Congenital Cmv Infection ◽  
Congenital Cmv

A vaccine against congenital cytomegalovirus infection is a high priority. Guinea pig cytomegalovirus (GPCMV) is the only congenital CMV small animal model. GPCMV encodes essential glycoprotein complexes for virus entry (gB, gH/gL/gO, gM/gN) including a pentamer complex (gH/gL/GP129/GP131/GP133 or PC) for endocytic cell entry. The cohorts for protection against congenital CMV are poorly defined. Neutralizing antibodies to the viral glycoprotein complexes are potentially more important than an immunodominant T-cell response to the pp65 protein. In GPCMV, GP83 (pp65 homolog) is an evasion factor, and the GP83 mutant GPCMV has increased sensitivity to type I interferon. Although GP83 induces a cell-mediated response, a GP83-only-based vaccine strategy has limited efficacy. GPCMV attenuation via GP83 null deletion mutant in glycoprotein PC positive or negative virus was evaluated as live-attenuated vaccine strains (GP83dPC+/PC-). Vaccinated animals induced antibodies to viral glycoprotein complexes, and PC+ vaccinated animals had sterilizing immunity against wtGPCMV challenge. In a pre-conception vaccine (GP83dPC+) study, dams challenged mid-2nd trimester with wtGPCMV had complete protection against congenital CMV infection without detectable virus in pups. An unvaccinated control group had 80% pup transmission rate. Overall, gB and PC antibodies are key for protection against congenital CMV infection, but a response to pp65 is not strictly necessary.

scholarly journals Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus

2016 ◽  
Vol 113 (42) ◽  
pp. 11931-11936 ◽  
Author(s):  
Wenqian He ◽  
Gene S. Tan ◽  
Caitlin E. Mullarkey ◽  
Amanda J. Lee ◽  
Mannie Man Wai Lam ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Neutralizing Antibodies ◽  
Influenza A ◽  
Critical Role ◽  
Host Cells ◽  
Viral Glycoprotein ◽  
Effector Functions ◽  
Dependent Cell

The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising “universal” influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

Strong genetic subdivision in Leptobrachium hendricksoni (Anura: Megophryidae) in Southeast Asia

2018 ◽  
Vol 39 (1) ◽  
pp. 99-111
Author(s):  
Gordon Draškić ◽  
Sansareeya Wangkulangkul ◽  
Iñigo Martínez-Solano ◽  
Judit Vörös
Keyword(s):  
Southeast Asia ◽  
Sequence Divergence ◽  
Population Divergence ◽  
Geological History ◽  
Base Pairs ◽  
Sea Levels ◽  
Phylogenetic Studies ◽  
High Sequence Divergence ◽  
Southern Thailand ◽  
Rising Sea Levels

Many biodiversity hotspots are located in areas with a complex geological history, like Southeast Asia, where species diversity may still be far underestimated, especially in morphologically conservative groups like amphibians. Recent phylogenetic studies on the frog genusLeptobrachiumfrom Southeast Asia revealed the presence of deeply divergent mitochondrial clades inLeptobrachium hendricksonifrom Malaysia and Sumatra but populations from Thailand have not been studied so far. In this study, we re-evaluate patterns of intraspecific genetic diversity inL. hendricksonibased on the analysis of combined sequences of mitochondrial 12S and 16S genes (1310 base pairs) including for the first time samples from southern Thailand. Thai populations ofL. hendricksoniformed a distinct clade with respect to populations from central and southern Malaysia and Sumatra. High sequence divergence between lineages from Thailand, Malaysia and Sumatra suggests the possible presence of cryptic species inL. hendricksoni. Divergence withinL. hendricksonidates back to the late Miocene, around 6 Mya, when lineages from Thailand, north Malaysia and Sumatra split from a lineage in south Malaysia, at about the same time as rising sea levels isolated the Thai-Malay peninsula. Subsequent splits took place later in the Pliocene, around 4.5 and 2.6 Mya. Our results highlight the role of geological history in promoting population divergence and speciation.

scholarly journals Ebola Viral Glycoprotein Bound to Its Endosomal Receptor Niemann-Pick C1

Cell ◽  
2016 ◽  
Vol 164 (1-2) ◽  
pp. 258-268 ◽  
Author(s):  
Han Wang ◽  
Yi Shi ◽  
Jian Song ◽  
Jianxun Qi ◽  
Guangwen Lu ◽  
...  
Keyword(s):  
Viral Glycoprotein ◽  
Niemann Pick

scholarly journals Protection from cytomegalovirus viremia following glycoprotein B vaccination is not dependent on neutralizing antibodies

2018 ◽  
Vol 115 (24) ◽  
pp. 6273-6278 ◽  
Author(s):  
Ilona Baraniak ◽  
Barbara Kropff ◽  
Lyn Ambrose ◽  
Megan McIntosh ◽  
Gary R. McLean ◽  
...  
Keyword(s):  
Antibody Response ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Natural Infection ◽  
Neutralizing Antibody ◽  
Glycoprotein B ◽  
Solid Organ ◽  
Viral Glycoprotein ◽  
Viral Spread

Human cytomegalovirus (HCMV) is an important pathogen in transplant patients and in congenital infection. Previously, we demonstrated that vaccination with a recombinant viral glycoprotein B (gB)/MF59 adjuvant formulation before solid organ transplant reduced viral load parameters post transplant. Reduced posttransplant viremia was directly correlated with antibody titers against gB consistent with a humoral response against gB being important. Here we show that sera from the vaccinated seronegative patients displayed little evidence of a neutralizing antibody response against cell-free HCMV in vitro. Additionally, sera from seronegative vaccine recipients had minimal effect on the replication of a strain of HCMV engineered to be cell-associated in a viral spread assay. Furthermore, although natural infection can induce antibody-dependent cellular cytotoxicity (ADCC) responses, serological analysis of seronegative vaccinees again presented no evidence of a substantial ADCC-promoting antibody response being generated de novo. Finally, analyses for responses against major antigenic domains of gB following vaccination were variable, and their pattern was distinct compared with natural infection. Taken together, these data argue that the protective effect elicited by the gB vaccine is via a mechanism of action in seronegative vaccinees that cannot be explained by neutralization or the induction of ADCC. More generally, these data, which are derived from a human challenge model that demonstrated that the gB vaccine is protective, highlight the need for more sophisticated analyses of new HCMV vaccines over and above the quantification of an ability to induce potent neutralizing antibody responses in vitro.

scholarly journals Isolation of a human monoclonal antibody specific for the receptor binding domain of SARS-CoV-2 using a competitive phage biopanning strategy

2020 ◽  
Vol 3 (2) ◽  
pp. 95-100 ◽  
Author(s):  
Xin Zeng ◽  
Lingfang Li ◽  
Jing Lin ◽  
Xinlei Li ◽  
Bin Liu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Magnetic Beads ◽  
Human Monoclonal Antibody ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Antibody Library ◽  
Synthetic Antibody ◽  
Blocking Antibodies

Abstract The infection of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 200 000 deaths, but no vaccine or therapeutic monoclonal antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor-binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a “sandwich” complex. Only antibodies competed with ACE2 can bind to the free RBD-His in the supernatant and be subsequently separated by the nickel-nitrilotriacetic acid magnetic beads. rRBD-15 from the competitive biopanning of our synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 and potently inhibit SARS-CoV-2 pseudovirus infection with IC50 values of 12 nM. Nevertheless, rRBD-16 from the standard biopanning can only bind to RBD in vitro, but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.

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