scholarly journals On a mouse monoclonal antibody that neutralizes all four dengue virus serotypes

2009 ◽  
Vol 90 (4) ◽  
pp. 799-809 ◽  
Author(s):  
Ravikumar Rajamanonmani ◽  
Celine Nkenfou ◽  
Paula Clancy ◽  
Yin Hoe Yau ◽  
Susana Geifman Shochat ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Entry ◽  
Cross Reactivity ◽  
Binding Activity ◽  
Single Chain ◽  
Viral Attachment ◽  
Denv Serotypes ◽  
Humoral Immune ◽  
Single Chain Fv ◽  
Domain Iii

The flavivirus envelope glycoprotein (E) is responsible for viral attachment and entry by membrane fusion. Its ectodomain is the primary target of the humoral immune response. In particular, the C-terminal Ig-like domain III of E, which is exposed at the surface of the viral particle, forms an attractive antigen for raising protective monoclonal antibodies (mAb). 9F12, a mouse mAb raised against a dengue virus (DENV) serotype 2 recombinant domain III, cross-reacts with corresponding domains from the other three DENV serotypes and also with West Nile virus. mAb 9F12 binds with nanomolar affinity to a conserved epitope that maps to the viral surface comprising residues 305, 307, 310 and 330 of the E protein. mAb 9F12 neutralizes all four DENV serotypes in plaque reduction assays. We expressed a single-chain Fv from 9F12 that retains the binding activity of the parent mAb. Adsorption and fusion inhibition assays indicate that mAb 9F12 prevents early steps of viral entry. Its virus inhibition activity and broad cross-reactivity makes mAb 9F12 a suitable candidate for optimization and humanization into a therapeutic antibody to treat severe infections by dengue.

scholarly journals Phage display demonstrates durable differences in serological profile by route of inoculation in primary infections of non-human primates with Dengue Virus 1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jayant V. Rajan ◽  
Michael McCracken ◽  
Caleigh Mandel-Brehm ◽  
Greg Gromowski ◽  
Simon Pollett ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Phage Display ◽  
Denv Infection ◽  
Phage Library ◽  
Humoral Immune ◽  
Linear Peptide ◽  
Humoral Immune Responses ◽  
High Resolution Map ◽  
Domain Iii ◽  
Inoculation Route

AbstractNatural dengue virus (DENV) infections occur by mosquito bite but how the inoculation route affects the humoral immune response is unknown. We serologically profiled 20 non-human primates (NHP) from a prior study of DENV1 infection where animals were inoculated by mosquito (N = 10) or subcutaneous injection (N = 10). Using a comprehensive, densely tiled and highly redundant pan-flavivirus programmable phage library containing 91,562 overlapping 62 amino acid peptides, we produced a high-resolution map of linear peptide sequences enriched during DENV seroconversion. Profiles in mosquito-inoculated and subcutaneously-inoculated animals were similar up to 90 days after primary infection, but diverged at 1 year with differences in sero-reactivity in the Envelope (E; residues 215–406; p < 0.08), and Nonstructural-3 (NS3; residues 549–615; p < 0.05) proteins in mosquito-inoculated versus subcutaneously-inoculated animals. Within the E protein, residues 339–384 in domain III accounted for > 99% of the observed sero-reactivity difference. Antibody breadth did not vary by mode of inoculation. The differential reactivity to E domain III seen by phage display validated orthogonally by ELISA, but did not correlate with late neutralization titers. Serological profiling of humoral immune responses to DENV infection in NHP by programmable phage display demonstrated durable differences in sero-reactivity by route of inoculation.

scholarly journals Generation and Selection of a Panel of Pan-Filovirus Single-Chain Antibodies using Cell-Free Ribosome Display

2018 ◽  
Author(s):  
Adinarayana Kunamneni ◽  
Elizabeth C. Clarke ◽  
Chunyan Ye ◽  
Steven B. Bradfute ◽  
Ravi Durvasula
Keyword(s):  
Hemorrhagic Fever ◽  
Cross Reactivity ◽  
Free Ribosome ◽  
Rapid Diagnostics ◽  
Single Chain ◽  
Ribosome Display ◽  
Single Chain Antibodies ◽  
Single Chain Fv ◽  
New Generation ◽  
Selection Of

AbstractFiloviruses, which include ebolaviruses and marburgvirus, can cause outbreaks of highly lethal hemorrhagic fever. This disease causes significant morbidity and mortality in humans and non-human primates, with human fatality rates reaching 90% during some outbreaks. Currently, there are a lack of licensed vaccines or antivirals for these viruses. Since early symptoms of filovirus infection mimic more common diseases, there is a strong unmet public health and biodefense need for broad-spectrum filovirus rapid diagnostics. We have generated a panel of mouse single-chain Fv-antibodies (scFvs) to filovirus glycoproteins (GPs) using cell-free ribosome display and determined their cross-reactivity profiles to all known filovirus species. Two scFvs (4-2 and 22-1) were able to detect all known Ebolavirus and Marburgvirus species. This is the first report on ribosome display scFvs that can detect a broad set of filovirus GPs, which demonstrates their potential use in the development of a new generation of rapid diagnostic immunoassays.

scholarly journals Humoral Immune Response in a Mouse Model Induced With Dengue Virus-like Particles Serotypes 1 and 4 Produced in Silkworm Larvae

2021 ◽  
Author(s):  
Doddy Irawan ◽  
Sabar Pambudi ◽  
Enoch Y. Park
Keyword(s):  
Immune Response ◽  
Dengue Virus ◽  
Protein Domain ◽  
Titration Calorimetry ◽  
Heparin Binding ◽  
Global Public Health ◽  
Silkworm Larvae ◽  
Virus Like Particles ◽  
Humoral Immune ◽  
Domain Iii

Abstract Dengue is an arboviral disease, which threatens almost half the global population, and has emerged as the most significant of current global public health challenges. In this study, we prepared dengue virus-like particles (DENV-LPs) consisting of Capsid-Premembrane-Envelope (CprM/E) and Premembrane-Envelope (prM/E) polypeptides from serotype 1 and 4, which were expressed in the silkworms using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid. 1CprME, 1prME, 4CprME, and 4prME expressed proteins in hemolymph and molecular weight of the purified proteins were 55 kDa, respectively. The purified polypeptides formed spherical Dengue virus-like particles (DENV-LPs) with approximately 30–55 nm in diameter. The immunoelectron microscopy (IEM) images revealed antigens to the surface of a lipid bilayer of DENV-LPs. The heparin-binding assay shows a positive relationship between absorbance and the quantity of E protein domain III (EDIII), which was supported by the isothermal titration calorimetry assay, showing a moderate binding affinity between heparin and DENV-LP. The high correlation between patient sera and DENV-LP reactivities revealed that these DENV-LPs shared similar epitopes with the natural dengue virus. IgG elicitation studies in mice have demonstrated that DENV-LP/CPrMEs elicits a stronger immune response than DENV-LP/prMEs, which lends credence to this claim.

scholarly journals Receptor-activated human α2-macroglobulin interacts with the envelope protein of dengue virus and protects virions from temperature-induced inactivation through multivalent binding

2014 ◽  
Vol 95 (12) ◽  
pp. 2668-2676 ◽  
Author(s):  
Vivian Huerta ◽  
Patricia Toledo ◽  
Noralvis Fleitas ◽  
Alejandro Martín ◽  
Dianne Pupo ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Specific Binding ◽  
Interaction Network ◽  
Denv Infection ◽  
Virus Binding ◽  
Denv Serotypes ◽  
Α2 Macroglobulin ◽  
Multivalent Binding ◽  
Domain Iii ◽  
Amyloid P

Based on the hypothesis that interactions between virions and serum components may influence the outcome of dengue virus (DENV) infections, we decided to use affinity chromatography with domain III from the envelope (E) protein of DENV2 (DIIIE2) as a ligand to isolate virus-binding proteins from human plasma. This approach yielded serum amyloid P (SAP) and α2-macroglobulin (α2M) as novel viral interactors. After confirming the specific binding of both SAP and α2M to DIIIE2 by ELISA, the latter interaction was examined in greater detail. We obtain evidence suggesting that the binding species was actually the receptor-activated form of α2M (α2M*), that α2M* could bind monovalently to recombinant domain III from all four DENV serotypes with affinities in the micromolar range ranking as DENV4>DENV1~DENV2>DENV3 and that this interaction exhibited a strong avidity effect when multivalent binding was favoured (K D 8×10−8 M for DIIIE2). We also showed that α2M* bound to DENV virions of the four serotypes, protecting the virus from temperature-induced inactivation in the absence of serum and enhancing infectivity. The latter effect exhibited an ED50 of 2.9×10−8 M, also suggesting an avidity effect due to multivalent binding. These results will further contribute to the characterization of the virus–host factor interaction network during human DENV infection.

scholarly journals Type- and Subcomplex-Specific Neutralizing Antibodies against Domain III of Dengue Virus Type 2 Envelope Protein Recognize Adjacent Epitopes

2007 ◽  
Vol 81 (23) ◽  
pp. 12816-12826 ◽  
Author(s):  
Soila Sukupolvi-Petty ◽  
S. Kyle Austin ◽  
Whitney E. Purtha ◽  
Theodore Oliphant ◽  
Grant E. Nybakken ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Surface Display ◽  
Viral Envelope ◽  
Yeast Surface Display ◽  
Neutralizing Activity ◽  
Denv Serotypes ◽  
E Protein ◽  
Lateral Ridge ◽  
Domain Iii

ABSTRACT Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.

scholarly journals Generation and Characterization of Human Monoclonal scFv Antibodies against Helicobacter pylori Antigens

2002 ◽  
Vol 70 (8) ◽  
pp. 4158-4164 ◽  
Author(s):  
Nicole Reiche ◽  
Andreas Jung ◽  
Thomas Brabletz ◽  
Tanja Vater ◽  
Thomas Kirchner ◽  
...  
Keyword(s):  
Immune Response ◽  
Helicobacter Pylori ◽  
Phage Display ◽  
Humoral Immune Response ◽  
Vaccine Development ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Humoral Immune ◽  
Single Chain Fv ◽  
H Pylori

ABSTRACT Infection with Helicobacter pylori is chronic despite a vigorous cellular and humoral immune response and causes severe pathology in some patients. In this study, phage display was used as a new approach in order to investigate the role of the host's humoral immune response in the pathogenesis of H. pylori gastritis. Human monoclonal single-chain Fv (scFv) antibody fragments against H. pylori cell lysate and the H. pylori urease were isolated from an immune phage display library, constructed from peripheral blood lymphocytes of an H. pylori-infected patient. After affinity selection, 23% of the clones tested showed binding activity against a lysate of the H. pylori Sydney strain in enzyme-linked immunosorbent assay (ELISA) and 9% bound the H. pylori urease. Further characterization by PCR-fingerprint analysis and sequencing revealed that two closely related H. pylori binders and one antiurease scFv could be isolated. The selected scFvs were highly specific as analyzed by ELISA and immunoblots using various bacterial lysates and recombinant proteins. Analysis of the humoral immune response following H. pylori infection using human monoclonal antibodies might contribute to a better understanding of the pathogenesis of the disease. Moreover, using immune phage display libraries, it might be possible for relevant epitopes of H. pylori antigens to be determined, which might be of use for vaccine development.

scholarly journals The Phosphatidylserine and Phosphatidylethanolamine Receptor CD300a Binds Dengue Virus and Enhances Infection

2015 ◽  
Vol 90 (1) ◽  
pp. 92-102 ◽  
Author(s):  
Xavier Carnec ◽  
Laurent Meertens ◽  
Ophélie Dejarnac ◽  
Manuel Perera-Lecoin ◽  
Mohamed Lamine Hafirassou ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Entry ◽  
Polyclonal Antibodies ◽  
Denv Infection ◽  
Health Concern ◽  
Target Cells ◽  
Apoptotic Cells ◽  
Dengue Disease ◽  
Denv Serotypes ◽  
Viral Particles

ABSTRACT Dengue virus (DENV) is the etiological agent of the major human arboviral disease. We previously demonstrated that the TIM and TAM families of phosphatidylserine (PtdSer) receptors involved in the phagocytosis of apoptotic cells mediate DENV entry into target cells. We show here that human CD300a, a recently identified phospholipid receptor, also binds directly DENV particles and enhances viral entry. CD300a facilitates infection of the four DENV serotypes, as well as of other mosquito-borne viruses such as West Nile virus and Chikungunya virus. CD300a acts as an attachment factor that enhances DENV internalization through clathrin-mediated endocytosis. CD300a recognizes predominantly phosphatidylethanolamine (PtdEth) and to a lesser extent PtdSer associated with viral particles. Mutation of residues in the IgV domain critical for phospholipid binding abrogate CD300a-mediated enhancement of DENV infection. Finally, we show that CD300a is expressed at the surface of primary macrophages and anti-CD300a polyclonal antibodies partially inhibited DENV infection of these cells. Overall, these data indicate that CD300a is a novel DENV binding receptor that recognizes PtdEth and PtdSer present on virions and enhance infection. IMPORTANCE Dengue disease, caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease of humans and is a major global health concern. The molecular bases of DENV-host cell interactions during virus entry are poorly understood, hampering the discovery of new targets for antiviral intervention. We recently discovered that the TIM and TAM proteins, two receptor families involved in the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, interact with DENV particles-associated PtdSer through a mechanism that mimics the recognition of apoptotic cells and mediate DENV infection. In this study, we show that CD300a, a novel identified phospholipid receptor, mediates DENV infection. CD300a-dependent DENV infection relies on the direct recognition of phosphatidylethanolamine and to a lesser extent PtdSer associated with viral particles. This study provides novel insights into the mechanisms that mediate DENV entry and reinforce the concept that DENV uses an apoptotic mimicry strategy for viral entry.

scholarly journals Inoculation by mosquito induces durable differences in serological profile in non-human primates infected with DENV1

2020 ◽  
Author(s):  
Jayant V. Rajan ◽  
Michael McCracken ◽  
Caleigh Mandel-Brehm ◽  
Greg Gromowski ◽  
Simon Pollett ◽  
...  
Keyword(s):  
Immune Response ◽  
Dengue Virus ◽  
Phage Display ◽  
Post Infection ◽  
Antibody Binding ◽  
Phage Library ◽  
Mosquito Bite ◽  
Humoral Immune ◽  
One Year ◽  
Domain Iii

ABSTRACTNatural dengue virus (DENV) infections are delivered by mosquito bite but how the route of inoculation route could shape the humoral immune response is not well understood. Here, we serologically profiled 20 non-human primates (NHP) from a prior study of DENV1 infection in which the animals were inoculated by mosquito (N=10) or subcutaneous injection (N=10). Using a comprehensive, densely tiled and highly redundant pan-flavivirus programmable phage library containing 91,562 overlapping 62 amino acid peptides, we produced a high-resolution map of linear peptide sequences enriched during DENV seroconversion. We found that serological profiles in mosquito-inoculated and subcutaneously-inoculated animals were similar up to 90 days after primary infection, but diverged at 1 year. We found differences in sero-reactivity, as indicated by the median area under the curve (AUC) in the Envelope (E; residues 215-406; p < 0.08), and Nonstructural-3 (NS3; residues 549-615; p < 0.05) proteins in mosquito-inoculated versus subcutaneously-inoculated animals. Within the E protein, residues 339-384 in domain III accounted for >99% of the total AUC difference across residues 215-406. Antibody breadth did not vary by mode of inoculation. The differential reactivity to E domain III (EDIII) seen by phage display validated orthogonally by ELISA, but did not correlate with late neutralization titers. Serological profiling of humoral immune responses to DENV infection in NHP by programmable phage display demonstrated durable differences in sero-reactivity by route of inoculation. These findings could have implications for DENV diagnostics and evaluation of vaccines.IMPORTANCEDengue virus (DENV) infections are transmitted by mosquito bite, but how being infected by mosquito bite affects the immune response is not known. In this study, we analyzed antibodies produced by rhesus macaques infected with DENV using programmable phage display, a high-throughput method for characterizing what viral protein derived peptides serum antibodies bind to. We found that while there was no difference in antibody binding profiles at early timepoints post-infection, at one year post-infection, there were substantial differences in the antibody binding profiles of macaques who were infected by mosquito bite versus those that were infected by injection. In general, antibodies in the macaques inoculated by mosquito maintained higher levels of sero-reactivity, with a strong signal still present one year post-infection. The findings we report could have implications for DENV diagnostics and evaluation of DENV vaccines.

scholarly journals A New Approach in DENV Diagnostic based on Linear Peptides Obtained by Phage Display

2021 ◽  
Author(s):  
Mirna Burciaga-Flores ◽  
Tanya A. Camacho-Villegas ◽  
Pavel H. Lugo-Fabres ◽  
Abel Gutiérrez-Ortega ◽  
José Esteban Muñoz-Medina ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Phage Display ◽  
Early Stage ◽  
In Silico Analysis ◽  
Cross Reactivity ◽  
Diagnostic Tools ◽  
Correct Identification ◽  
Binding Modes ◽  
Ns1 Protein ◽  
Denv Serotypes

Abstract Dengue is a viral disease caused by any of the four distinct dengue virus (DENV) serotypes that circulate in many parts of the world. DENV now co-circulates with Zika and Chikungunya viruses (ZIKV and CHIKV) in many regions of the Americas. Having this in mind, plus the fact that DENV clinical diagnosis persists as a difficult task, due to the similarity in symptoms, as well as false-positive results by the cross-reactivity of the IgG and IgM against these three viruses, correct identification of DENV at an early stage of the disease is essential to minimise transmission and prevent potentially devastating sequelae. Here, by phage display, we isolate specific peptides for dengue virus NS1 protein. The specificity of the linear peptides as diagnostic tools for DENV NS1 protein in sera samples was investigated, and the selected peptides showed the ability to recognise DENV, and no cross-reactivity was shown. Moreover, in silico analysis was performed to assess the possible binding modes of these peptides to DENV-NS1, using a molecular docking approach. These peptides are suitable for use in an ELISA assay for dengue virus detection in human serum and the possibility to adapt these peptides to PoC platforms.

Sign in / Sign up

Export Citation Format

Share Document