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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

Selectant VLPs elicit high-titer peptide-specific antibody responses but variable AMA1-reactive IgG titers.Groups of mice were immunized twice with 10mer selectant VLPs or wild-type MS2 VLPs. Serum was taken two-weeks following the boost. (A) Vaccine-elicited peptide titers were assessed by ELISA using recombinant PP7 bacteriophage VLPs displaying the NWDPTQFPGK peptide (in a surface-exposed AB loop similar to that of MS2) as the target antigen. (B) Antibody responses against native AMA1. In 3/10 vaccinated mice we observed low-titer cross-reactivity; these are referred to as “responders”. The remaining mice were characterized as “non-responders”. Mean OD405 values are shown for each group. Error bars represent standard error of the mean, SEM.
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pone.0132560.g001: Selectant VLPs elicit high-titer peptide-specific antibody responses but variable AMA1-reactive IgG titers.Groups of mice were immunized twice with 10mer selectant VLPs or wild-type MS2 VLPs. Serum was taken two-weeks following the boost. (A) Vaccine-elicited peptide titers were assessed by ELISA using recombinant PP7 bacteriophage VLPs displaying the NWDPTQFPGK peptide (in a surface-exposed AB loop similar to that of MS2) as the target antigen. (B) Antibody responses against native AMA1. In 3/10 vaccinated mice we observed low-titer cross-reactivity; these are referred to as “responders”. The remaining mice were characterized as “non-responders”. Mean OD405 values are shown for each group. Error bars represent standard error of the mean, SEM.

Mentions: We investigated the immunogenicity of the 10mer selectant (MS2-NWDPTQFPGK) by immunizing a group of ten Balb/C mice with 10 μg of VLPs. Mice were immunized twice, at a two-week interval, and two weeks after the boost peptide-specific and recombinant AMA1-reactive IgG titers were measured by ELISA (Fig 1). To detect peptide-specific responses we constructed a recombinant PP7 bacteriophage VLP that displayed the NWDPTQFPGK peptide and used this VLP as a target antigen in an ELISA. All of the mice immunized with the 10mer selectant VLP had high-titer peptide-specific IgG antibody levels (end point dilution titers of 104−105; Fig 1A). However, only a subset (3 of 10, termed ‘responders’) of the mice immunized with MS2-NWDPTQFPGK VLPs produced antibodies that cross-reacted with recombinant AMA1, although these antibodies were of low titer (Fig 1B). The majority of mice immunized with MS2-NWDPTQFPGK VLPs were ‘non-responders’, in that they failed to elicit AMA1-reactive antibodies. All of the immunized mice, including non-responders, had similarly high peptide titers, so we could not attribute the variable response to gross quantitative differences in the antibody responses in individual mice. We considered the possibility that mice may have a low frequency of B cells that are capable of producing AMA1-reactive antibodies. While this may be the case, the use of higher antigen doses (25 μg) failed to increase the percentage of responders (data not shown).


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)

Selectant VLPs elicit high-titer peptide-specific antibody responses but variable AMA1-reactive IgG titers.Groups of mice were immunized twice with 10mer selectant VLPs or wild-type MS2 VLPs. Serum was taken two-weeks following the boost. (A) Vaccine-elicited peptide titers were assessed by ELISA using recombinant PP7 bacteriophage VLPs displaying the NWDPTQFPGK peptide (in a surface-exposed AB loop similar to that of MS2) as the target antigen. (B) Antibody responses against native AMA1. In 3/10 vaccinated mice we observed low-titer cross-reactivity; these are referred to as “responders”. The remaining mice were characterized as “non-responders”. Mean OD405 values are shown for each group. Error bars represent standard error of the mean, SEM.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132560.g001: Selectant VLPs elicit high-titer peptide-specific antibody responses but variable AMA1-reactive IgG titers.Groups of mice were immunized twice with 10mer selectant VLPs or wild-type MS2 VLPs. Serum was taken two-weeks following the boost. (A) Vaccine-elicited peptide titers were assessed by ELISA using recombinant PP7 bacteriophage VLPs displaying the NWDPTQFPGK peptide (in a surface-exposed AB loop similar to that of MS2) as the target antigen. (B) Antibody responses against native AMA1. In 3/10 vaccinated mice we observed low-titer cross-reactivity; these are referred to as “responders”. The remaining mice were characterized as “non-responders”. Mean OD405 values are shown for each group. Error bars represent standard error of the mean, SEM.
Mentions: We investigated the immunogenicity of the 10mer selectant (MS2-NWDPTQFPGK) by immunizing a group of ten Balb/C mice with 10 μg of VLPs. Mice were immunized twice, at a two-week interval, and two weeks after the boost peptide-specific and recombinant AMA1-reactive IgG titers were measured by ELISA (Fig 1). To detect peptide-specific responses we constructed a recombinant PP7 bacteriophage VLP that displayed the NWDPTQFPGK peptide and used this VLP as a target antigen in an ELISA. All of the mice immunized with the 10mer selectant VLP had high-titer peptide-specific IgG antibody levels (end point dilution titers of 104−105; Fig 1A). However, only a subset (3 of 10, termed ‘responders’) of the mice immunized with MS2-NWDPTQFPGK VLPs produced antibodies that cross-reacted with recombinant AMA1, although these antibodies were of low titer (Fig 1B). The majority of mice immunized with MS2-NWDPTQFPGK VLPs were ‘non-responders’, in that they failed to elicit AMA1-reactive antibodies. All of the immunized mice, including non-responders, had similarly high peptide titers, so we could not attribute the variable response to gross quantitative differences in the antibody responses in individual mice. We considered the possibility that mice may have a low frequency of B cells that are capable of producing AMA1-reactive antibodies. While this may be the case, the use of higher antigen doses (25 μg) failed to increase the percentage of responders (data not shown).

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