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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

Sera from mice immunized with VLPs displaying the VTHDAWRPD peptide binds to the 4G2 epitope.(A) Reactivity of IgG in pooled sera from mice immunized with VLPs displaying the VTHDAWRPD peptide (closed circles) against recombinant AMA1 from 3D7, FVO, and L32 strains. Pooled preimmune serum (open circles) was used as a negative control. (B) Anti-AMA1 reactivity of pooled sera from mice immunized with VLPs displaying the VTHDAWRPD peptide (diluted 1:40) or AMA1 (diluted 1:1,000) in the presence (grey bars) or absence (black bars) of mAb 4G2. (C) Immunofluorescence. Immunosera binds to native AMA1 on merozoites. Slides were stained with pooled preimmune sera (1:25 dilution; left panel), pooled sera from mice immunized with VLP-VTHDAWRPD (1:25; middle panel), or, as a positive control, the anti-AMA1 mAb 1E9 (right panel).
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pone.0132560.g007: Sera from mice immunized with VLPs displaying the VTHDAWRPD peptide binds to the 4G2 epitope.(A) Reactivity of IgG in pooled sera from mice immunized with VLPs displaying the VTHDAWRPD peptide (closed circles) against recombinant AMA1 from 3D7, FVO, and L32 strains. Pooled preimmune serum (open circles) was used as a negative control. (B) Anti-AMA1 reactivity of pooled sera from mice immunized with VLPs displaying the VTHDAWRPD peptide (diluted 1:40) or AMA1 (diluted 1:1,000) in the presence (grey bars) or absence (black bars) of mAb 4G2. (C) Immunofluorescence. Immunosera binds to native AMA1 on merozoites. Slides were stained with pooled preimmune sera (1:25 dilution; left panel), pooled sera from mice immunized with VLP-VTHDAWRPD (1:25; middle panel), or, as a positive control, the anti-AMA1 mAb 1E9 (right panel).

Mentions: ELISA binding data suggested that MS2-VTHDAWRPD VLPs elicited antibodies that bound to the 4G2 epitope on AMA1. To confirm this, we assessed the ability of these sera to bind to AMA1 from other P. falciparum strains, compete with 4G2 for binding to AMA1, and to bind to native parasite. Although AMA1 is polymorphic and has strain-specific variable regions, the 4G2 epitope is highly conserved between strains. We tested serum reactivity to AMA1 from three strains of P. falciparum, 3D7, FVO, and L32. The sequences of the FVO and L32 AMA1 antigens differ from 3D7 by 19 and 24 amino acids, respectively. However, none of these amino acid differences are located in the putative 4G2 binding region. As shown in Fig 7A, sera from vaccinated mice bound to AMA1-3D7, AMA1-FVO and, to a lesser extent, AMA1-L32. To discount the possibility that immunosera from VLP-immunized mice bound to a domain of AMA1 outside of the 4G2 epitope, we assessed that ability of 4G2 to block serum binding to AMA1. Serum binding to rAMA1 was measured by ELISA in the absence or presence of 4G2. As shown in Fig 7B, pretreatment of AMA1 with 4G2 substantially decreased the binding of IgG from MS2-VTHDAWRPD VLP immunized mice to AMA1, consistent with the hypothesis that this sera binds to the 4G2 epitope. As expected, pretreatment with 4G2 had only a modest effect on the binding of polyclonal sera from mice immunized with AMA1-3D7 to recombinant AMA1, since this sera presumably binds to multiple epitopes on the protein. Sera from MS2-VTHDAWRPD VLP-vaccinated mice failed to inhibit 4G2 binding to AMA1 (data not shown); presumably this is because the anti-AMA1 titers induced upon vaccination with VLPs were low. Next, to test whether these sera bound to native PfAMA1, we assessed binding to 3D7 merozoites by immunofluorescence. As shown in Fig 7C, serum from mice immunized with the VLP selectant showed a similar staining pattern on merozoites as a control anti-AMA1 mAb (1E9). Thus, taken together, these data support the contention that the VTHDAWRPD peptide, when displayed on an MS2 VLP, is an immunogenic mimic of the 4G2 epitope.


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)

Sera from mice immunized with VLPs displaying the VTHDAWRPD peptide binds to the 4G2 epitope.(A) Reactivity of IgG in pooled sera from mice immunized with VLPs displaying the VTHDAWRPD peptide (closed circles) against recombinant AMA1 from 3D7, FVO, and L32 strains. Pooled preimmune serum (open circles) was used as a negative control. (B) Anti-AMA1 reactivity of pooled sera from mice immunized with VLPs displaying the VTHDAWRPD peptide (diluted 1:40) or AMA1 (diluted 1:1,000) in the presence (grey bars) or absence (black bars) of mAb 4G2. (C) Immunofluorescence. Immunosera binds to native AMA1 on merozoites. Slides were stained with pooled preimmune sera (1:25 dilution; left panel), pooled sera from mice immunized with VLP-VTHDAWRPD (1:25; middle panel), or, as a positive control, the anti-AMA1 mAb 1E9 (right panel).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132560.g007: Sera from mice immunized with VLPs displaying the VTHDAWRPD peptide binds to the 4G2 epitope.(A) Reactivity of IgG in pooled sera from mice immunized with VLPs displaying the VTHDAWRPD peptide (closed circles) against recombinant AMA1 from 3D7, FVO, and L32 strains. Pooled preimmune serum (open circles) was used as a negative control. (B) Anti-AMA1 reactivity of pooled sera from mice immunized with VLPs displaying the VTHDAWRPD peptide (diluted 1:40) or AMA1 (diluted 1:1,000) in the presence (grey bars) or absence (black bars) of mAb 4G2. (C) Immunofluorescence. Immunosera binds to native AMA1 on merozoites. Slides were stained with pooled preimmune sera (1:25 dilution; left panel), pooled sera from mice immunized with VLP-VTHDAWRPD (1:25; middle panel), or, as a positive control, the anti-AMA1 mAb 1E9 (right panel).
Mentions: ELISA binding data suggested that MS2-VTHDAWRPD VLPs elicited antibodies that bound to the 4G2 epitope on AMA1. To confirm this, we assessed the ability of these sera to bind to AMA1 from other P. falciparum strains, compete with 4G2 for binding to AMA1, and to bind to native parasite. Although AMA1 is polymorphic and has strain-specific variable regions, the 4G2 epitope is highly conserved between strains. We tested serum reactivity to AMA1 from three strains of P. falciparum, 3D7, FVO, and L32. The sequences of the FVO and L32 AMA1 antigens differ from 3D7 by 19 and 24 amino acids, respectively. However, none of these amino acid differences are located in the putative 4G2 binding region. As shown in Fig 7A, sera from vaccinated mice bound to AMA1-3D7, AMA1-FVO and, to a lesser extent, AMA1-L32. To discount the possibility that immunosera from VLP-immunized mice bound to a domain of AMA1 outside of the 4G2 epitope, we assessed that ability of 4G2 to block serum binding to AMA1. Serum binding to rAMA1 was measured by ELISA in the absence or presence of 4G2. As shown in Fig 7B, pretreatment of AMA1 with 4G2 substantially decreased the binding of IgG from MS2-VTHDAWRPD VLP immunized mice to AMA1, consistent with the hypothesis that this sera binds to the 4G2 epitope. As expected, pretreatment with 4G2 had only a modest effect on the binding of polyclonal sera from mice immunized with AMA1-3D7 to recombinant AMA1, since this sera presumably binds to multiple epitopes on the protein. Sera from MS2-VTHDAWRPD VLP-vaccinated mice failed to inhibit 4G2 binding to AMA1 (data not shown); presumably this is because the anti-AMA1 titers induced upon vaccination with VLPs were low. Next, to test whether these sera bound to native PfAMA1, we assessed binding to 3D7 merozoites by immunofluorescence. As shown in Fig 7C, serum from mice immunized with the VLP selectant showed a similar staining pattern on merozoites as a control anti-AMA1 mAb (1E9). Thus, taken together, these data support the contention that the VTHDAWRPD peptide, when displayed on an MS2 VLP, is an immunogenic mimic of the 4G2 epitope.

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