Identification of Neutralizing and Nonneutralizing Epitopes in the Porcine Reproductive and Respiratory Syndrome Virus GP5 Ectodomain

ABSTRACTThe V3 region of HIV-1 gp120 is important for virus-coreceptor interaction and highly immunogenic. Although most anti-V3 antibodies neutralize only the sensitive tier 1 viruses, anti-V3 antibodies effective against the more resistant viruses exist, and a better understanding of these antibodies and their epitopes would be beneficial for the development of novel vaccine immunogens against HIV. The HIV-1 isolate JRFL with its cryptic V3 is resistant to most V3-specific monoclonal antibodies (MAbs). However, the V3 MAb 2424 achieves 100% neutralization against JRFL. 2424 is encoded by IGHV3-53 and IGLV2-28 genes, a pairing rarely used by the other V3 MAbs. 2424 also has distinct binding and neutralization profiles. Studies of 2424-mediated neutralization of JRFL produced with a mannosidase inhibitor further revealed that its neutralizing activity is unaffected by the glycan composition of the virus envelope. To understand the distinct activity of 2424, we determined the crystal structure of 2424 Fab in complex with a JRFL V3 peptide and showed that the 2424 epitope is located at the tip of the V3 crown (307IHIGPGRAFYT319), dominated by interactions with HisP308, ProP313, and ArgP315. The binding mode of 2424 is similar to that of the well-characterized MAb 447-52D, although 2424 is more side chain dependent. The 2424 epitope is focused on the very apex of V3, away from nearby glycans, facilitating antibody access. This feature distinguishes the 2424 epitope from the other V3 crown epitopes and indicates that the tip of V3 is a potential site to target and incorporate into HIV vaccine immunogens.IMPORTANCEHIV/AIDS vaccines are crucial for controlling the HIV epidemics that continue to afflict millions of people worldwide. However, HIV vaccine development has been hampered by significant scientific challenges, one of which is the inability of HIV vaccine candidates evaluated thus far to elicit production of potent and broadly neutralizing antibodies. The V3 loop is one of the few immunogenic targets on the virus envelope glycoprotein that can induce neutralizing antibodies, but in many viruses, parts of V3 are inaccessible for antibody recognition. This study examined a V3-specific monoclonal antibody that can completely neutralize HIV-1 JRFL, a virus isolate resistant to most V3 antibodies. Our data reveal that this antibody recognizes the most distal tip of V3, which is not as occluded as other parts of V3. Hence, the epitope of 2424 is in one of the vulnerable sites on the virus that may be exploited in designing HIV vaccine immunogens.

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Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms17020214 ◽

2016 ◽

Vol 17 (2) ◽

pp. 214 ◽

Cited By ~ 7

Author(s):

Yan-Da Lai ◽

Yen-Yu Wu ◽

Yi-Jiue Tsai ◽

Yi-San Tsai ◽

Yu-Ying Lin ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Cancer Therapy ◽

Phage Display ◽

Neutralizing Antibodies ◽

Phage Display Library ◽

Vascular Endothelial ◽

Endothelial Growth Factor

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A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post–vaccination monitoring

10.21203/rs.2.14955/v2 ◽

2019 ◽

Author(s):

Lihua Wang ◽

Shijiang Mi ◽

Rachel Madera ◽

Llilianne Ganges ◽

Manuel V. Borca ◽

...

Keyword(s):

Monoclonal Antibody ◽

Confidence Interval ◽

Excellent Agreement ◽

Neutralizing Antibodies ◽

Vaccination Campaign ◽

Classical Swine Fever ◽

Enzyme Linked Immunosorbent Assay ◽

Live Virus ◽

Post Vaccination ◽

Neutralizing Monoclonal Antibody

Abstract Background: Virus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post–vaccination monitoring. Results: One monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n=445) and C-strain VNT positive pig sera (n=70), the 6B211 based cELSIA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be detected in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n=139) in parallel, the cELISA showed excellent agreement (Kappa=0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r 2 =0.903, p<0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs). Conclusions: This novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.

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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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Conformational and binding studies of the peptide isolated from phage display library by anti-mouse CTLA-4 monoclonal antibody

Peptide Science — Present and Future ◽

10.1007/0-306-46864-6_277 ◽

2006 ◽

pp. 810-812

Author(s):

Y. Ito ◽

Y. Oomura ◽

K. Ezaki ◽

S. Kojima ◽

T. Fukumoto ◽

...

Keyword(s):

Monoclonal Antibody ◽

Phage Display ◽

Phage Display Library ◽

Binding Studies

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Defining critical residues m the epitope for a hiv-neutralizing monoclonal antibody using phage display and peptide array technologies

Gene ◽

10.1016/0378-1119(93)90252-x ◽

1993 ◽

Vol 137 (1) ◽

pp. 63-68 ◽

Cited By ~ 31

Author(s):

Cindy L. Jellis ◽

Thomas J. Cradick ◽

Paul Rennert ◽

Paul Salinas ◽

James Boyd ◽

...

Keyword(s):

Monoclonal Antibody ◽

Phage Display ◽

Peptide Array ◽

Neutralizing Monoclonal Antibody ◽

Critical Residues

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Isolation of and Characterization of Neutralizing Antibodies to Covid-19 from a Large Human Naïve scFv Phage Display Library

10.1101/2020.05.19.104281 ◽

2020 ◽

Cited By ~ 6

Author(s):

Andy Q. Yuan ◽

Likun Zhao ◽

Lili Bai ◽

Qingwu Meng ◽

Zhenguo Wen ◽

...

Keyword(s):

Phage Display ◽

Neutralizing Antibodies ◽

Epitope Mapping ◽

Neutralization Assay ◽

Phage Display Library ◽

Antibody Library ◽

Antibody Phage ◽

Antibody Phage Display ◽

Human Receptor

AbstractSARS-CoV-2 (Covid-19) has caused currently ongoing global plague and imposed great challenges to health managing systems all over the world, with millions of infections and hundreds of thousands of deaths. In addition to racing to develop vaccines, neutralizing antibodies (nAbs) to this virus have been extensively sought and are expected to provide another prevention and therapy tool against this frantic pandemic. To offer fast isolation and shortened early development, a large human naïve phage display antibody library, was built and used to screen specific nAbs to the receptor-binding domain, RBD, the key for Covid-19 virus entry through a human receptor, ACE2. The obtained RBD-specific antibodies were characterized by epitope mapping, FACS and neutralization assay. Some of the antibodies demonstrated spike-neutralizing property and ACE2-competitiveness. Our work proved that RBD-specific neutralizing binders from human naïve antibody phage display library are promising candidates to for further Covid-19 therapeutics development.

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A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post–vaccination monitoring

10.21203/rs.2.14955/v3 ◽

2020 ◽

Author(s):

Lihua Wang ◽

Shijiang Mi ◽

Rachel Madera ◽

Llilianne Ganges ◽

Manuel V. Borca ◽

...

Keyword(s):

Monoclonal Antibody ◽

Confidence Interval ◽

Excellent Agreement ◽

Neutralizing Antibodies ◽

Vaccination Campaign ◽

Classical Swine Fever ◽

Enzyme Linked Immunosorbent Assay ◽

Live Virus ◽

Post Vaccination ◽

Neutralizing Monoclonal Antibody

Abstract Background: Virus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post–vaccination monitoring. Results: One monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n=445) and C-strain VNT positive pig sera (n=70), the 6B211 based cELSIA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be detected in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n=139) in parallel, the cELISA showed excellent agreement (Kappa=0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r 2 =0.903, p<0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs). Conclusions: This novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.

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Convergent Evolution of Neutralizing Antibodies to Staphylococcus aureus γ-Hemolysin C That Recognize an Immunodominant Primary Sequence-Dependent B-Cell Epitope

mBio ◽

10.1128/mbio.00460-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 2

Author(s):

David N. Hernandez ◽

Kayan Tam ◽

Bo Shopsin ◽

Emily E. Radke ◽

Karen Law ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Phage Display ◽

B Cell ◽

Neutralizing Antibodies ◽

Ex Vivo ◽

Cell Epitope ◽

Phage Display Library ◽

Structural Basis ◽

Peptide Fragments ◽

Content Type

ABSTRACT Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the γ-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved β-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.

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