Monoclonal Antibodies to Bluetongue Virus Define Two Neutralizing Epitopes and a Hemagglutinating Epitope

ABSTRACTHuman papillomavirus (HPV) is the etiological agent for all cervical cancers, a significant number of other anogenital cancers, and a growing number of head and neck cancers. Two licensed vaccines offer protection against the most prevalent oncogenic types, 16 and 18, responsible for approximately 70% of cervical cancer cases worldwide and one of these also offers protection against types 6 and 11, responsible for 90% of genital warts. The vaccines are comprised of recombinantly expressed major capsid proteins that self-assemble into virus-like particles (VLPs) and prevent infection by eliciting neutralizing antibodies. Adding the other frequently identified oncogenic types 31, 33, 45, 52, and 58 to a vaccine would increase the coverage against HPV-induced cancers to approximately 90%. We describe the generation and characterization of panels of monoclonal antibodies to these five additional oncogenic HPV types, and the selection of antibody pairs that were high affinity and type specific and recognized conformation-dependent neutralizing epitopes. Such characteristics make these antibodies useful tools for monitoring the production and potency of a prototype vaccine as well as monitoring vaccine-induced immune responses in the clinic.

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Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS–CoV–2 Spike

10.1101/2021.02.03.429355 ◽

2021 ◽

Author(s):

Carl Graham ◽

Jeffrey Seow ◽

Isabella Huettner ◽

Hataf Khan ◽

Neophytos Kouphou ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Infectious Virus ◽

Antigenic Drift ◽

Host Cells ◽

Receptor Binding Domain ◽

Neutralization Activity ◽

Neutralizing Activity ◽

Neutralizing Epitopes

The interaction of the SARS–CoV–2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS–CoV–2 variants has revealed mutations arising in the RBD, the N–terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike–reactive monoclonal antibodies from SARS–CoV–2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD–specific. None of the S2–specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD–specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan–dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD–specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

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Location of neutralizing epitopes defined by monoclonal antibodies generated against the outer capsid polypeptide, VP1, of foot-and-mouth disease virus A12

Virus Research ◽

10.1016/0168-1702(84)90006-6 ◽

1984 ◽

Vol 1 (6) ◽

pp. 489-500 ◽

Cited By ~ 28

Author(s):

B.H. Robertson ◽

D.O. Morgan ◽

D.M. Moore

Keyword(s):

Monoclonal Antibodies ◽

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Outer Capsid ◽

Neutralizing Epitopes

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Neutralizing epitopes of the serotypes of bluetongue virus present in the United States

Journal of General Virology ◽

10.1099/0022-1317-73-8-1947 ◽

1992 ◽

Vol 73 (8) ◽

pp. 1947-1952 ◽

Cited By ~ 21

Author(s):

P. V. Rossitto ◽

N. J. MacLachlan

Keyword(s):

United States ◽

Bluetongue Virus ◽

The United States ◽

Neutralizing Epitopes

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Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

Applied and Environmental Microbiology ◽

10.1128/aem.71.6.2894-2901.2005 ◽

2005 ◽

Vol 71 (6) ◽

pp. 2894-2901 ◽

Cited By ~ 1

Author(s):

Gregory D. Wiens ◽

Jennifer Owen

Keyword(s):

Amino Acids ◽

Monoclonal Antibodies ◽

Biological Activities ◽

Infected Fish ◽

Transposon Mutagenesis ◽

Renibacterium Salmoninarum ◽

Amino Terminal ◽

Bacterial Kidney Disease ◽

Transposon Mutants ◽

Neutralizing Epitopes

ABSTRACT Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.

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Monoclonal Antibodies Targeting the HR2 Domain and the Region Immediately Upstream of the HR2 of the S Protein Neutralize In Vitro Infection of Severe Acute Respiratory Syndrome Coronavirus

Journal of Virology ◽

10.1128/jvi.80.2.941-950.2006 ◽

2006 ◽

Vol 80 (2) ◽

pp. 941-950 ◽

Cited By ~ 41

Author(s):

Kuo-Ming Lip ◽

Shuo Shen ◽

Xiaoming Yang ◽

Choong-Tat Keng ◽

Aihua Zhang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Polyclonal Antibodies ◽

Heptad Repeat ◽

Functional Domain ◽

Protein Fragment ◽

S Protein ◽

Linear Epitopes ◽

Respiratory Coronavirus ◽

Neutralizing Epitopes

ABSTRACT We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. T. Keng, A. Zhang, S. Shen, K. M. Lip, B. C. Fielding, T. H. Tan, C. F. Chou, C. B. Loh, S. Wang, J. Fu, X. Yang, S. G. Lim, W. Hong, and Y. J. Tan, J. Virol. 79:3289-3296, 2005). In this study, monoclonal antibodies (MAbs) were raised against this fragment to identify the linear neutralizing epitopes in the functional domain and to investigate the mechanisms involved in neutralization. Eighteen hybridomas secreting the S protein-specific MAbs were obtained. Binding sites of these MAbs were mapped to four linear epitopes. Two of them were located within the HR2 region and two immediately upstream of the HR2 domain. MAbs targeting these epitopes showed in vitro neutralizing activities and were able to inhibit cell-cell membrane fusion. These results provide evidence of novel neutralizing epitopes that are located in the HR2 domain and the spacer region immediately upstream of the HR2 of the S protein.

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Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on the Clostridium botulinum Neurotoxin Type A

Applied and Environmental Microbiology ◽

10.1128/aem.67.7.3201-3207.2001 ◽

2001 ◽

Vol 67 (7) ◽

pp. 3201-3207 ◽

Cited By ~ 27

Author(s):

Han-Chung Wu ◽

Chia-Tsui Yeh ◽

Yue-Ling Huang ◽

Lih-Jeng Tarn ◽

Chien-Cheng Lung

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Clostridium Botulinum ◽

Neuromuscular Junctions ◽

Competitive Inhibition ◽

Lethal Dose ◽

Humoral Response ◽

Type A ◽

Phage Clone ◽

Neutralizing Epitopes

ABSTRACT Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 103 and 104 times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D5) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K5). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.

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