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The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition.

Goh LY, Hobson-Peters J, Prow NA, Baker K, Piyasena TB, Taylor CT, Rana A, Hastie ML, Gorman JJ, Hall RA - Viruses (2015)

Bottom Line: Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain.Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP.This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

View Article: PubMed Central - PubMed

Affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4072, Australia. l.goh1@uq.edu.au.

ABSTRACT
Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1-35 and 140-210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

No MeSH data available.


Related in: MedlinePlus

Competitive binding profiles of CHIKV CP-specific mAbs in ELISA. Antigens in lysates of CHIKVMAU-infected Vero cells were adsorbed to 96-well plates at a 1/500 dilution prior to incubation with a saturating dilution of purified, unlabelled anti-CP mAbs. Without washing, non-saturating dilutions of biotinylated mAbs were then added as ‘competitor’ antibodies to respective wells. The mean absorbance reading (OD405nm) of four replicates were plotted with bars showing standard error of mean (SEM). 4G2 is a control mAb specific to the E protein of flaviviruses. Assay was optimized to obtain complete inhibition of each biotinylated mAb by its homologous unlabelled competitor.
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viruses-07-02754-f008: Competitive binding profiles of CHIKV CP-specific mAbs in ELISA. Antigens in lysates of CHIKVMAU-infected Vero cells were adsorbed to 96-well plates at a 1/500 dilution prior to incubation with a saturating dilution of purified, unlabelled anti-CP mAbs. Without washing, non-saturating dilutions of biotinylated mAbs were then added as ‘competitor’ antibodies to respective wells. The mean absorbance reading (OD405nm) of four replicates were plotted with bars showing standard error of mean (SEM). 4G2 is a control mAb specific to the E protein of flaviviruses. Assay was optimized to obtain complete inhibition of each biotinylated mAb by its homologous unlabelled competitor.

Mentions: To further define the binding sites of the mAbs from group 1, and determine the topology of their epitopes in the C-terminal half of CP, representative group 1 antibodies were tested in a competitive binding assay against one another using ELISA. Three of the five mAbs in this group (1.7B2, 4.1H11 and 5.2H7) were successfully biotinylated and subsequently assessed for competition against saturating concentrations of each of the five unlabelled mAbs to establish the degree of inhibition exhibited for each pairing. MAbs 1.7B2 and 4.1H11 showed complete two-way inhibition with each other indicating they bind to the same or highly adjacent epitopes (Figure 8). Partial two-way inhibition of both these mAbs with mAb 5.2H7 was also observed, suggesting that the epitope recognized by 5.2H7 is slightly different to the former pair but in the same spatial domain. The partial one-way inhibition of 5.2H7 by the unlabelled mAbs 5.5D11 and 5.5G9, but not by 1.7B2 and 4.1H11, suggests a continuum of overlapping epitopes (Figure 9). The competitive binding result also confirms that these antibodies bind to the same region of the CP.


The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition.

Goh LY, Hobson-Peters J, Prow NA, Baker K, Piyasena TB, Taylor CT, Rana A, Hastie ML, Gorman JJ, Hall RA - Viruses (2015)

Competitive binding profiles of CHIKV CP-specific mAbs in ELISA. Antigens in lysates of CHIKVMAU-infected Vero cells were adsorbed to 96-well plates at a 1/500 dilution prior to incubation with a saturating dilution of purified, unlabelled anti-CP mAbs. Without washing, non-saturating dilutions of biotinylated mAbs were then added as ‘competitor’ antibodies to respective wells. The mean absorbance reading (OD405nm) of four replicates were plotted with bars showing standard error of mean (SEM). 4G2 is a control mAb specific to the E protein of flaviviruses. Assay was optimized to obtain complete inhibition of each biotinylated mAb by its homologous unlabelled competitor.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488721&req=5

viruses-07-02754-f008: Competitive binding profiles of CHIKV CP-specific mAbs in ELISA. Antigens in lysates of CHIKVMAU-infected Vero cells were adsorbed to 96-well plates at a 1/500 dilution prior to incubation with a saturating dilution of purified, unlabelled anti-CP mAbs. Without washing, non-saturating dilutions of biotinylated mAbs were then added as ‘competitor’ antibodies to respective wells. The mean absorbance reading (OD405nm) of four replicates were plotted with bars showing standard error of mean (SEM). 4G2 is a control mAb specific to the E protein of flaviviruses. Assay was optimized to obtain complete inhibition of each biotinylated mAb by its homologous unlabelled competitor.
Mentions: To further define the binding sites of the mAbs from group 1, and determine the topology of their epitopes in the C-terminal half of CP, representative group 1 antibodies were tested in a competitive binding assay against one another using ELISA. Three of the five mAbs in this group (1.7B2, 4.1H11 and 5.2H7) were successfully biotinylated and subsequently assessed for competition against saturating concentrations of each of the five unlabelled mAbs to establish the degree of inhibition exhibited for each pairing. MAbs 1.7B2 and 4.1H11 showed complete two-way inhibition with each other indicating they bind to the same or highly adjacent epitopes (Figure 8). Partial two-way inhibition of both these mAbs with mAb 5.2H7 was also observed, suggesting that the epitope recognized by 5.2H7 is slightly different to the former pair but in the same spatial domain. The partial one-way inhibition of 5.2H7 by the unlabelled mAbs 5.5D11 and 5.5G9, but not by 1.7B2 and 4.1H11, suggests a continuum of overlapping epitopes (Figure 9). The competitive binding result also confirms that these antibodies bind to the same region of the CP.

Bottom Line: Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain.Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP.This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

View Article: PubMed Central - PubMed

Affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4072, Australia. l.goh1@uq.edu.au.

ABSTRACT
Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1-35 and 140-210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

No MeSH data available.


Related in: MedlinePlus