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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 selectant VLPs libraries was monitored by capture ELISA.Pooled crude VLP lysates, in duplicate, were applied to wells coated with 4G2 or an isotype control. VLP binding was detected using rabbit anti-MS2 polyclonal antibody. Relative binding was calculated by using the ratio of mean binding to 4G2 with the isotype control. The orange bar represents the final library from a selection using only a 10-mer insert VLP library (i.e. the library from which NWDPTQFPGT was identified). The pink bars represent all the stages of selection, including the original mutagenic library (round 0), using a VLP library with insert sequences randomized from the 10-mer NWDPTQFPGK selectant. The blue bars show the results of the mixed length VLP library selection. This experiment was repeated three times using different dilutions of the VLP libraries; similar ratios were observed in each experiment.
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pone.0132560.g003: Relative binding of selectant VLPs libraries was monitored by capture ELISA.Pooled crude VLP lysates, in duplicate, were applied to wells coated with 4G2 or an isotype control. VLP binding was detected using rabbit anti-MS2 polyclonal antibody. Relative binding was calculated by using the ratio of mean binding to 4G2 with the isotype control. The orange bar represents the final library from a selection using only a 10-mer insert VLP library (i.e. the library from which NWDPTQFPGT was identified). The pink bars represent all the stages of selection, including the original mutagenic library (round 0), using a VLP library with insert sequences randomized from the 10-mer NWDPTQFPGK selectant. The blue bars show the results of the mixed length VLP library selection. This experiment was repeated three times using different dilutions of the VLP libraries; similar ratios were observed in each experiment.

Mentions: Because some proportion of this library already had the ability to bind to 4G2, we performed just two rounds of selections using libraries in which the peptides were displayed at low-valency. Selections were monitored by capture ELISA, by comparing ability of 4G2 versus an isotype control to bind to selected VLPs. As shown in Fig 3, a single round of selection using the mutagenic library resulted in substantial enrichment of the 4G2-binding population of VLPs. After each round of selection, VLPs were deep sequenced and the enrichment of specific peptide selectants was calculated (S2 Table). The original 10mer clone remained the most represented by number of reads total in each round. However, several other peptides were strongly enriched during the selection process.


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 selectant VLPs libraries was monitored by capture ELISA.Pooled crude VLP lysates, in duplicate, were applied to wells coated with 4G2 or an isotype control. VLP binding was detected using rabbit anti-MS2 polyclonal antibody. Relative binding was calculated by using the ratio of mean binding to 4G2 with the isotype control. The orange bar represents the final library from a selection using only a 10-mer insert VLP library (i.e. the library from which NWDPTQFPGT was identified). The pink bars represent all the stages of selection, including the original mutagenic library (round 0), using a VLP library with insert sequences randomized from the 10-mer NWDPTQFPGK selectant. The blue bars show the results of the mixed length VLP library selection. This experiment was repeated three times using different dilutions of the VLP libraries; similar ratios were observed in each experiment.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493041&req=5

pone.0132560.g003: Relative binding of selectant VLPs libraries was monitored by capture ELISA.Pooled crude VLP lysates, in duplicate, were applied to wells coated with 4G2 or an isotype control. VLP binding was detected using rabbit anti-MS2 polyclonal antibody. Relative binding was calculated by using the ratio of mean binding to 4G2 with the isotype control. The orange bar represents the final library from a selection using only a 10-mer insert VLP library (i.e. the library from which NWDPTQFPGT was identified). The pink bars represent all the stages of selection, including the original mutagenic library (round 0), using a VLP library with insert sequences randomized from the 10-mer NWDPTQFPGK selectant. The blue bars show the results of the mixed length VLP library selection. This experiment was repeated three times using different dilutions of the VLP libraries; similar ratios were observed in each experiment.
Mentions: Because some proportion of this library already had the ability to bind to 4G2, we performed just two rounds of selections using libraries in which the peptides were displayed at low-valency. Selections were monitored by capture ELISA, by comparing ability of 4G2 versus an isotype control to bind to selected VLPs. As shown in Fig 3, a single round of selection using the mutagenic library resulted in substantial enrichment of the 4G2-binding population of VLPs. After each round of selection, VLPs were deep sequenced and the enrichment of specific peptide selectants was calculated (S2 Table). The original 10mer clone remained the most represented by number of reads total in each round. However, several other peptides were strongly enriched during the selection process.

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