Neutralization Mechanism of a Monoclonal Antibody Targeting a Porcine Circovirus Type 2 Cap Protein Conformational Epitope

ABSTRACT Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections. IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.

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Purification of Porcine Circovirus Type 2 Using an Affinity Chromatography Based on a Neutralizing Monoclonal Antibody against Viral Capsid Protein

Pathogens ◽

10.3390/pathogens10121564 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1564

Author(s):

Haiqiao Bian ◽

Chong Yu ◽

Yanwu Wei ◽

Li Feng ◽

Changming Liu ◽

...

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Affinity Chromatography ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Enzyme Linked Immunosorbent Assay ◽

Chromatography Method ◽

Viral Adsorption

Porcine circovirus type 2 (PCV2) is a DNA virus without an envelope. The viral particle is icosahedral and has a diameter of approximately 17 nm. In order to obtain the purified virus, a broad-spectrum monoclonal antibody 3A5 against PCV2 was coupled to CNBr-activated SepharoseTM 4B, and an affinity chromatography was established for PCV2 purification. A total of 6.5 mg of purified PCV2a/LG with 97% purity was obtained from 120 mL of the viral culture medium, and only PCV2 was detected by electron microscopy. No significant changes in the antigenic characteristics of the purified virus were detected by a capture enzyme-linked immunosorbent assay (ELISA). Furthermore, the titer of the purified PCV2 was 100 times higher than that of the unpurified virus. This affinity chromatography method was also used to purify PCV2b/LN590516 and PCV2d/SD446F16, and the purified viruses were detected by electron microscopy, capture ELISA, and virus titration, respectively. The results showed that these two strains can be successfully purified, but the yield is lower than that of the PCV2a strain. In addition, the purified virus could be used to study the viral adsorption and invasion of PK15 cells using indirect immunofluorescence assays. A large number of PCV2 signals were detected to transfer from the cellular surface to the periphery of the nucleus of the PK15 cells after 30 min of adsorption of the PCV2 to the PK15 cells. The affinity chromatography is a simple and convenient tool to obtain PCV2 with high purity. It could be applied for virus structure analysis, antibody preparation, and viral adsorption and invasion research.

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Effect of Porcine Circovirus Type 2 (PCV2) Vaccination of the Dam on PCV2 Replication In Utero

Clinical and Vaccine Immunology ◽

10.1128/cvi.00455-08 ◽

2009 ◽

Vol 16 (6) ◽

pp. 830-834 ◽

Cited By ~ 27

Author(s):

D. M. Madson ◽

A. R. Patterson ◽

S. Ramamoorthy ◽

N. Pal ◽

X. J. Meng ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Specific Antigen ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Reproductive Failure ◽

Porcine Circovirus ◽

Serum Samples ◽

Pcv2 Vaccination ◽

Group 3

ABSTRACT The aims of this study were to determine if porcine circovirus type 2 (PCV2) vaccination of the dam is effective in preventing fetal PCV2 infection and reproductive failure. Twelve pregnant, PCV2-naïve sows were randomly divided into four groups, with three sows in each group. Group 1 sows served as noninoculated, nonvaccinated negative controls, group 2 sows were vaccinated with a commercially available PCV2 vaccine at 28 days of gestation and were not inoculated, group 3 sows were vaccinated at 28 days of gestation and inoculated with PCV2b at 56 days of gestation, and group 4 sows were inoculated with PCV2b but were not vaccinated. Serum samples from all sows were collected weekly throughout the gestation period, and sows were allowed to farrow naturally. At parturition, sow colostrum samples, presuckle serum samples, and tissues from the piglets were collected. Reproductive failure was not observed under the study conditions. PCV2 vaccination induced PCV2-specific immunoglobulin G and serum neutralizing antibodies in sows from groups 2 and 3 and prevented detectable PCV2 viremia in the dams after challenge. In group 3, PCV2 DNA was detected in colostrum samples, fetuses, and live-born pigs; however, microscopic lesions and PCV2-specific antigen were not present in any of the fetuses in this group. The results from this study indicate that vertical transmission of PCV2 can occur in PCV2-vaccinated dams.

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Antiviral Effect of Epigallocatechin Gallate via Impairing Porcine Circovirus Type 2 Attachment to Host Cell Receptor

Viruses ◽

10.3390/v12020176 ◽

2020 ◽

Vol 12 (2) ◽

pp. 176 ◽

Cited By ~ 8

Author(s):

Jiarong Li ◽

Dongfeng Song ◽

Shengnan Wang ◽

Yadong Dai ◽

Jiyong Zhou ◽

...

Keyword(s):

Amino Acids ◽

Heparan Sulfate ◽

Epigallocatechin Gallate ◽

Antiviral Effect ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Cell Surface Receptor ◽

Porcine Circovirus ◽

Arginine Residues

The green tea catechin epigallocatechin gallate (EGCG) exhibits antiviral activity against various viruses. Whether EGCG also inhibits the infectivity of circovirus remains unclear. In this study, we demonstrated the antiviral effect of EGCG on porcine circovirus type 2 (PCV2). EGCG targets PCV2 virions directly and blocks the attachment of virions to host cells. The microscale thermophoresis assay showed EGCG could interact with PCV2 capsid protein in vitro with considerable affinity (Kd = 98.03 ± 4.76 μM), thereby interfering with the binding of the capsid to the cell surface receptor heparan sulfate. The molecular docking analysis of capsid–EGCG interaction identified the key amino acids which formed the binding pocket accommodating EGCG. Amino acids ARG51, ASP70, ARG73 and ASP78 of capsid were found to be critical for maintaining the binding, and the arginine residues were also essential for the electrostatic interaction with heparan sulfate. The rescued mutant viruses also confirm the importance of the key amino acids of the capsid to the antiviral effect of EGCG. Our findings suggest that catechins could act as anti-infective agents against circovirus invasion, as well as provide the basic information for the development and synthesis of structure-based anti-circovirus drugs.

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A broad spectrum monoclonal antibody against porcine circovirus type 2 for antigen and antibody detection

Applied Microbiology and Biotechnology ◽

10.1007/s00253-019-09715-0 ◽

2019 ◽

Vol 103 (8) ◽

pp. 3453-3464 ◽

Cited By ~ 4

Author(s):

Liping Huang ◽

Yanwu Wei ◽

Deli Xia ◽

Dan Liu ◽

Hongzhen Zhu ◽

...

Keyword(s):

Monoclonal Antibody ◽

Broad Spectrum ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Antibody Detection ◽

Porcine Circovirus

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Studies on Porcine Circovirus Type 2 Vaccination of 5-Day-Old Piglets

Clinical and Vaccine Immunology ◽

10.1128/cvi.05318-11 ◽

2011 ◽

Vol 18 (11) ◽

pp. 1865-1871 ◽

Cited By ~ 18

Author(s):

K. C. O'Neill ◽

H. G. Shen ◽

K. Lin ◽

M. Hemann ◽

N. M. Beach ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Porcine Circovirus Type ◽

Positive Control ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Respiratory Syndrome Virus ◽

Control Group ◽

Associated Lesions

ABSTRACTPorcine circovirus type 2 (PCV2) vaccines have become widely used since they became available in 2006. It is not uncommon for producers to use PCV2 vaccines in pigs younger than what is approved by manufacturers. The objective of this study was to determine the efficacy of a chimeric and a subunit PCV2 vaccine administered at 5 or 21 days of age. Forty-eight PCV2-naïve piglets were randomly divided into six groups of eight pigs each. Vaccination was done at day 5 or day 21, followed by triple challenge with PCV2, porcine parvovirus (PPV), and porcine reproductive and respiratory syndrome virus (PRRSV) at day 49. Vaccinated pigs seroconverted to PCV2 approximately 14 days postvaccination and had a detectable neutralizing antibody response by 21 days postvaccination regardless of age at vaccination. At day 49, the pigs vaccinated with the chimeric vaccine had significantly higher levels of neutralizing antibodies than the pigs vaccinated with the subunit vaccine. After challenge, vaccinated pigs had significantly decreased levels of PCV2 viremia and a decreased prevalence and severity of microscopic lesions compared to the positive-control group, which had severe lymphoid lesions associated with abundant PCV2 antigen, compatible with PCV-associated disease. The results of this study indicate that, under the conditions of this study, vaccination of PCV2-naïve pigs at day 5 or day 21 resulted in development of a detectable humoral immune response and provided reduction or complete protection against PCV2 viremia and PCV2-associated lesions after triple challenge with PCV2, PPV, and PRRSV.

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