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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

Antibody responses in VLP-immunized mice boosted with recombinant AMA1.(A) Anti-peptide responses in responder mice are enhanced by boosting with 25μg recombinant rAMA1 (with incomplete Freund’s adjuvant). Mice were immunized with selectant VLPs and then boosted with recombinant AMA1. Mice were segregated into responders (red circles) and non-responders (blue circles). Also shown are the anti-peptide responses in mice immunized with selectant VLPs prior to boosting with rAMA1 (grey circles). As controls, groups of mice were immunized with wild-type MS2 VLPs (open circles) or recombinant AMA1 alone (black circles). Immunization with recombinant AMA1 boosts anti-peptide titers, but only in animals that had AMA1-cross-reactive titers present after VLP-selectant immunization. Mean and SEM are shown for each group. (B) Competition of immune sera with 4G2 for AMA1 binding. Sera from immunized mice were incubated with 250 ng rAMA1 on an ELISA plate in the presence of 100ng mAb 4G2. Serum was pooled from mice immunized with selectant VLPs boosted with rAMA1 (responders and non-responders), rAMA1 alone, or, as a control, wild-type MS2 VLPs. Data represent triplicate wells, normalized to the value of the no serum control. Mean OD405 and SEM are shown at each dilution.
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pone.0132560.g002: Antibody responses in VLP-immunized mice boosted with recombinant AMA1.(A) Anti-peptide responses in responder mice are enhanced by boosting with 25μg recombinant rAMA1 (with incomplete Freund’s adjuvant). Mice were immunized with selectant VLPs and then boosted with recombinant AMA1. Mice were segregated into responders (red circles) and non-responders (blue circles). Also shown are the anti-peptide responses in mice immunized with selectant VLPs prior to boosting with rAMA1 (grey circles). As controls, groups of mice were immunized with wild-type MS2 VLPs (open circles) or recombinant AMA1 alone (black circles). Immunization with recombinant AMA1 boosts anti-peptide titers, but only in animals that had AMA1-cross-reactive titers present after VLP-selectant immunization. Mean and SEM are shown for each group. (B) Competition of immune sera with 4G2 for AMA1 binding. Sera from immunized mice were incubated with 250 ng rAMA1 on an ELISA plate in the presence of 100ng mAb 4G2. Serum was pooled from mice immunized with selectant VLPs boosted with rAMA1 (responders and non-responders), rAMA1 alone, or, as a control, wild-type MS2 VLPs. Data represent triplicate wells, normalized to the value of the no serum control. Mean OD405 and SEM are shown at each dilution.

Mentions: To better characterize the differences between the responders and non-responders, we boosted MS2-NWDPTQFPGK VLP-immunized mice with 25 μg of recombinant AMA1 (rAMA1) protein and compared antibody responses to mice that were only immunized with rAMA1. Fig 2A shows the subsequent peptide-specific responses in these groups of animals. Immunization with rAMA1 alone elicited antibodies against AMA1 (not shown), but failed to induce antibodies that recognized the 10mer 4G2 mimotope. Boosting non-responder mice with rAMA1 failed to increase anti-peptide IgG titers. However, boosting the responder group with recombinant AMA1 increased the NWDPTQFPGK peptide-specific IgG titers. This provides indirect evidence that immunization with MS2-NWDPTQFPGK VLPs elicited low-titer 4G2-like antibody responses in a subset of animals.


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)

Antibody responses in VLP-immunized mice boosted with recombinant AMA1.(A) Anti-peptide responses in responder mice are enhanced by boosting with 25μg recombinant rAMA1 (with incomplete Freund’s adjuvant). Mice were immunized with selectant VLPs and then boosted with recombinant AMA1. Mice were segregated into responders (red circles) and non-responders (blue circles). Also shown are the anti-peptide responses in mice immunized with selectant VLPs prior to boosting with rAMA1 (grey circles). As controls, groups of mice were immunized with wild-type MS2 VLPs (open circles) or recombinant AMA1 alone (black circles). Immunization with recombinant AMA1 boosts anti-peptide titers, but only in animals that had AMA1-cross-reactive titers present after VLP-selectant immunization. Mean and SEM are shown for each group. (B) Competition of immune sera with 4G2 for AMA1 binding. Sera from immunized mice were incubated with 250 ng rAMA1 on an ELISA plate in the presence of 100ng mAb 4G2. Serum was pooled from mice immunized with selectant VLPs boosted with rAMA1 (responders and non-responders), rAMA1 alone, or, as a control, wild-type MS2 VLPs. Data represent triplicate wells, normalized to the value of the no serum control. Mean OD405 and SEM are shown at each dilution.
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pone.0132560.g002: Antibody responses in VLP-immunized mice boosted with recombinant AMA1.(A) Anti-peptide responses in responder mice are enhanced by boosting with 25μg recombinant rAMA1 (with incomplete Freund’s adjuvant). Mice were immunized with selectant VLPs and then boosted with recombinant AMA1. Mice were segregated into responders (red circles) and non-responders (blue circles). Also shown are the anti-peptide responses in mice immunized with selectant VLPs prior to boosting with rAMA1 (grey circles). As controls, groups of mice were immunized with wild-type MS2 VLPs (open circles) or recombinant AMA1 alone (black circles). Immunization with recombinant AMA1 boosts anti-peptide titers, but only in animals that had AMA1-cross-reactive titers present after VLP-selectant immunization. Mean and SEM are shown for each group. (B) Competition of immune sera with 4G2 for AMA1 binding. Sera from immunized mice were incubated with 250 ng rAMA1 on an ELISA plate in the presence of 100ng mAb 4G2. Serum was pooled from mice immunized with selectant VLPs boosted with rAMA1 (responders and non-responders), rAMA1 alone, or, as a control, wild-type MS2 VLPs. Data represent triplicate wells, normalized to the value of the no serum control. Mean OD405 and SEM are shown at each dilution.
Mentions: To better characterize the differences between the responders and non-responders, we boosted MS2-NWDPTQFPGK VLP-immunized mice with 25 μg of recombinant AMA1 (rAMA1) protein and compared antibody responses to mice that were only immunized with rAMA1. Fig 2A shows the subsequent peptide-specific responses in these groups of animals. Immunization with rAMA1 alone elicited antibodies against AMA1 (not shown), but failed to induce antibodies that recognized the 10mer 4G2 mimotope. Boosting non-responder mice with rAMA1 failed to increase anti-peptide IgG titers. However, boosting the responder group with recombinant AMA1 increased the NWDPTQFPGK peptide-specific IgG titers. This provides indirect evidence that immunization with MS2-NWDPTQFPGK VLPs elicited low-titer 4G2-like antibody responses in a subset of animals.

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