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Identification of an Immunogenic Mimic of a Conserved Epitope on the Plasmodium falciparum Blood Stage Antigen AMA1 Using Virus-Like Particle (VLP) Peptide Display.

Crossey E, Frietze K, Narum DL, Peabody DS, Chackerian B - PLoS ONE (2015)

Bottom Line: Most of the selected VLPs failed to reliably elicit AMA1 specific antibodies.However, one VLP consistently induced antibodies that cross-reacted with AMA1.Taken together, these data demonstrate that VLP-peptide display can identify immunogenic mimics of a complex conformational epitope and illustrate the promise and challenges of this approach.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM 87131, United States of America.

ABSTRACT
We have developed a peptide display platform based on VLPs of the RNA bacteriophage MS2 that combines the high immunogenicity of VLP display with affinity selection capabilities. Random peptides can be displayed on the VLP surface by genetically inserting sequences into a surface-exposed loop of the viral coat protein. VLP-displayed peptides can then be isolated by selection using antibodies, and the VLP selectants can then be used directly as immunogens. Here, we investigated the ability of this platform to identify mimotopes of a highly conserved conformational epitope present on the Plasmodium falciparum blood-stage protein AMA1. Using 4G2, a monoclonal antibody that binds to this epitope and is a potent inhibitor of erythrocyte invasion, we screened three different VLP-peptide libraries and identified specific VLPs that bound strongly to the selecting mAb. We then tested the ability of a handful of selected VLPs to elicit anti-AMA1 antibody responses in mice. Most of the selected VLPs failed to reliably elicit AMA1 specific antibodies. However, one VLP consistently induced antibodies that cross-reacted with AMA1. Surprisingly, this VLP bound to 4G2 more weakly than the other selectants we identified. Taken together, these data demonstrate that VLP-peptide display can identify immunogenic mimics of a complex conformational epitope and illustrate the promise and challenges of this approach.

No MeSH data available.


Related in: MedlinePlus

Relative binding of VLP affinity selectants to 4G2.ELISA plates were coated with 500ng of selected VLPs or rAMA1 and different amounts of 4G2 were applied. End-point dilution titers were determined as the reciprocal of the highest sera dilution with an OD greater than 2-fold higher than the no antibody control. The error bars represent the standard error of the mean of triplicate measurements.
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pone.0132560.g005: Relative binding of VLP affinity selectants to 4G2.ELISA plates were coated with 500ng of selected VLPs or rAMA1 and different amounts of 4G2 were applied. End-point dilution titers were determined as the reciprocal of the highest sera dilution with an OD greater than 2-fold higher than the no antibody control. The error bars represent the standard error of the mean of triplicate measurements.

Mentions: We chose eight 4G2-selected VLPs from the mutagenic and the mixed libraries to test further. These included three VLPs from the mutagenic library and five VLPs from the random library that were representative of major sequence families. Plasmids encoding VLP selectants were constructed, verified by sequence analysis, and then used to generate VLPs. 4G2 binding to each of the VLPs was confirmed by end-point dilution ELISA (Fig 5). The three VLPs selected from the mutagenic library all bound strongly to 4G2, and slightly stronger (~10-fold) than the parental clone (NWDPIQFPGK) from which they were derived. In general, the VLPs from the mixed library selection also showed strong binding to 4G2, but two selectants from one family (VTHDGLEGQM and VTHDAWRPD) bound 100–1000 fold less than other selectant VLPs.


Identification of an Immunogenic Mimic of a Conserved Epitope on the Plasmodium falciparum Blood Stage Antigen AMA1 Using Virus-Like Particle (VLP) Peptide Display.

Crossey E, Frietze K, Narum DL, Peabody DS, Chackerian B - PLoS ONE (2015)

Relative binding of VLP affinity selectants to 4G2.ELISA plates were coated with 500ng of selected VLPs or rAMA1 and different amounts of 4G2 were applied. End-point dilution titers were determined as the reciprocal of the highest sera dilution with an OD greater than 2-fold higher than the no antibody control. The error bars represent the standard error of the mean of triplicate measurements.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493041&req=5

pone.0132560.g005: Relative binding of VLP affinity selectants to 4G2.ELISA plates were coated with 500ng of selected VLPs or rAMA1 and different amounts of 4G2 were applied. End-point dilution titers were determined as the reciprocal of the highest sera dilution with an OD greater than 2-fold higher than the no antibody control. The error bars represent the standard error of the mean of triplicate measurements.
Mentions: We chose eight 4G2-selected VLPs from the mutagenic and the mixed libraries to test further. These included three VLPs from the mutagenic library and five VLPs from the random library that were representative of major sequence families. Plasmids encoding VLP selectants were constructed, verified by sequence analysis, and then used to generate VLPs. 4G2 binding to each of the VLPs was confirmed by end-point dilution ELISA (Fig 5). The three VLPs selected from the mutagenic library all bound strongly to 4G2, and slightly stronger (~10-fold) than the parental clone (NWDPIQFPGK) from which they were derived. In general, the VLPs from the mixed library selection also showed strong binding to 4G2, but two selectants from one family (VTHDGLEGQM and VTHDAWRPD) bound 100–1000 fold less than other selectant VLPs.

Bottom Line: Most of the selected VLPs failed to reliably elicit AMA1 specific antibodies.However, one VLP consistently induced antibodies that cross-reacted with AMA1.Taken together, these data demonstrate that VLP-peptide display can identify immunogenic mimics of a complex conformational epitope and illustrate the promise and challenges of this approach.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM 87131, United States of America.

ABSTRACT
We have developed a peptide display platform based on VLPs of the RNA bacteriophage MS2 that combines the high immunogenicity of VLP display with affinity selection capabilities. Random peptides can be displayed on the VLP surface by genetically inserting sequences into a surface-exposed loop of the viral coat protein. VLP-displayed peptides can then be isolated by selection using antibodies, and the VLP selectants can then be used directly as immunogens. Here, we investigated the ability of this platform to identify mimotopes of a highly conserved conformational epitope present on the Plasmodium falciparum blood-stage protein AMA1. Using 4G2, a monoclonal antibody that binds to this epitope and is a potent inhibitor of erythrocyte invasion, we screened three different VLP-peptide libraries and identified specific VLPs that bound strongly to the selecting mAb. We then tested the ability of a handful of selected VLPs to elicit anti-AMA1 antibody responses in mice. Most of the selected VLPs failed to reliably elicit AMA1 specific antibodies. However, one VLP consistently induced antibodies that cross-reacted with AMA1. Surprisingly, this VLP bound to 4G2 more weakly than the other selectants we identified. Taken together, these data demonstrate that VLP-peptide display can identify immunogenic mimics of a complex conformational epitope and illustrate the promise and challenges of this approach.

No MeSH data available.


Related in: MedlinePlus