Limits...
Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display.

Guo N, Duan H, Kachko A, Krause BW, Major ME, Krause PR - EBioMedicine (2015)

Bottom Line: Thus, using mRNA display to interrogate mAbs permits high resolution identification of functional peptide antigens that direct targeted immune responses, supporting its use in vaccine reverse engineering for pathogens against which potent neutralizing mAbs are available.After the identified peptides were injected into mice, the mice produced their own antibodies with characteristics similar to the original antibody.This approach can provide previously unavailable information about antibody binding and could also be useful in developing new vaccines.

View Article: PubMed Central - PubMed

Affiliation: Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Ave, Silver Spring, MD 20993, United States.

ABSTRACT

Unlabelled: Vaccine reverse engineering is emerging as an important approach to vaccine antigen identification, recently focusing mainly on structural characterization of interactions between neutralizing monoclonal antibodies (mAbs) and antigens. Using mAbs that bind unknown antigen structures, we sought to probe the intrinsic features of antibody antigen-binding sites with a high complexity peptide library, aiming to identify conformationally optimized mimotope antigens that capture mAb-specific epitopes. Using a high throughput sequencing-enhanced messenger ribonucleic acid (mRNA) display approach, we identified high affinity binding peptides for a hepatitis C virus neutralizing mAb. Immunization with the selected peptides induced neutralizing activity similar to that of the original mAb. Antibodies elicited by the most commonly selected peptides were predominantly against specific epitopes. Thus, using mRNA display to interrogate mAbs permits high resolution identification of functional peptide antigens that direct targeted immune responses, supporting its use in vaccine reverse engineering for pathogens against which potent neutralizing mAbs are available.

Research in context: We used a large number of randomly produced small proteins ("peptides") to identify peptides containing specific protein sequences that bind efficiently to an antibody that can prevent hepatitis C virus infection in cell culture. After the identified peptides were injected into mice, the mice produced their own antibodies with characteristics similar to the original antibody. This approach can provide previously unavailable information about antibody binding and could also be useful in developing new vaccines.

No MeSH data available.


Related in: MedlinePlus

Binding of selected peptides to the selection mAb41. (A) Binding of synthetic peptides to mAb41 at various concentrations. Biotinylated peptides were added to streptavidin-coated microtiter plates. mAb 41 was applied as the primary antibody in a 10-fold dilution series. HRP-conjugated goat anti-mouse antibody was used as the detection antibody. Results shown are the mean of two replicates performed in one of three independent experiments, each of which showed similar results. (B) Competition for binding to mAb41 between p41_1 and wild type pB. 1 μg biotinylated pB was attached to streptavidin-coated plates. 12 ng of mAb41in 100 μl PBS containing 5% milk was added in the presence of increasing amount of p41_1 as indicated. The bound mAb41 was detected using an HRP-conjugated goat anti-mouse antibody. An unrelated peptide from the mRNA display library was used as a negative control. Mean values are graphed and error bars represent SEM of sample replicates. (C) Measurement of binding affinity of selected peptides to mAb41 by Octet RED. Biotinylated peptides were immobilized on Streptavidin biosensors (Fortebio) and Fabs of mAb41 were used as analyte in a 2-fold dilution series ranging from 125 nM to 7.8 nM. Sensorgram data for peptide p41_1 are shown. (D) Binding constants of mAb41-selected peptides to mAb41 Fabs were obtained by fitting sensorgrams (blue) with a 1:1 model (red) using ForteBio Data Analysis Software. p41_2 is not included due to unsatisfactory curve fitting.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4563141&req=5

f0015: Binding of selected peptides to the selection mAb41. (A) Binding of synthetic peptides to mAb41 at various concentrations. Biotinylated peptides were added to streptavidin-coated microtiter plates. mAb 41 was applied as the primary antibody in a 10-fold dilution series. HRP-conjugated goat anti-mouse antibody was used as the detection antibody. Results shown are the mean of two replicates performed in one of three independent experiments, each of which showed similar results. (B) Competition for binding to mAb41 between p41_1 and wild type pB. 1 μg biotinylated pB was attached to streptavidin-coated plates. 12 ng of mAb41in 100 μl PBS containing 5% milk was added in the presence of increasing amount of p41_1 as indicated. The bound mAb41 was detected using an HRP-conjugated goat anti-mouse antibody. An unrelated peptide from the mRNA display library was used as a negative control. Mean values are graphed and error bars represent SEM of sample replicates. (C) Measurement of binding affinity of selected peptides to mAb41 by Octet RED. Biotinylated peptides were immobilized on Streptavidin biosensors (Fortebio) and Fabs of mAb41 were used as analyte in a 2-fold dilution series ranging from 125 nM to 7.8 nM. Sensorgram data for peptide p41_1 are shown. (D) Binding constants of mAb41-selected peptides to mAb41 Fabs were obtained by fitting sensorgrams (blue) with a 1:1 model (red) using ForteBio Data Analysis Software. p41_2 is not included due to unsatisfactory curve fitting.

Mentions: The five most abundant selected peptides (p41-1 to p41-5, Fig. 2B) were chemically synthesized for further analysis. By ELISA, mAb41 showed concentration-dependent binding to the mRNA display-selected peptides (Fig. 3A). Peptide p41_1, the most abundant peptide selected by mAb41, was used as a competitor of wild-type peptide B (pB, a shorter version of pA, Fig. 2B) for binding to mAb41 to confirm that the selected peptides interact with the antigen-binding sites of the selection antibody. Increasing amounts of p41_1 inhibited binding of mAb41 to immobilized pB (Fig. 3B). This binding was not appreciably affected by a negative control peptide from a mRNA display library that is not enriched by mAb41 (Fig. 3B), implying that p41_1 binds to the same antigen binding site on mAb41 as does wild type pB. Similar results were obtained using p41_3 as competitor (Figure S4A). Using the Octet binding assay, dissociation constants (Kd) of mAb41 Fabs with mAb41-selected peptides (with the exception of p41_2 which could not be evaluated due to unsatisfactory curve fitting) were within the 11–70 nanomolar range, similar to the affinity of mAb41 Fab fragments (Kd = 13 nM) for wild type pB (Fig. 3C–D and Figure S4B–E).


Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display.

Guo N, Duan H, Kachko A, Krause BW, Major ME, Krause PR - EBioMedicine (2015)

Binding of selected peptides to the selection mAb41. (A) Binding of synthetic peptides to mAb41 at various concentrations. Biotinylated peptides were added to streptavidin-coated microtiter plates. mAb 41 was applied as the primary antibody in a 10-fold dilution series. HRP-conjugated goat anti-mouse antibody was used as the detection antibody. Results shown are the mean of two replicates performed in one of three independent experiments, each of which showed similar results. (B) Competition for binding to mAb41 between p41_1 and wild type pB. 1 μg biotinylated pB was attached to streptavidin-coated plates. 12 ng of mAb41in 100 μl PBS containing 5% milk was added in the presence of increasing amount of p41_1 as indicated. The bound mAb41 was detected using an HRP-conjugated goat anti-mouse antibody. An unrelated peptide from the mRNA display library was used as a negative control. Mean values are graphed and error bars represent SEM of sample replicates. (C) Measurement of binding affinity of selected peptides to mAb41 by Octet RED. Biotinylated peptides were immobilized on Streptavidin biosensors (Fortebio) and Fabs of mAb41 were used as analyte in a 2-fold dilution series ranging from 125 nM to 7.8 nM. Sensorgram data for peptide p41_1 are shown. (D) Binding constants of mAb41-selected peptides to mAb41 Fabs were obtained by fitting sensorgrams (blue) with a 1:1 model (red) using ForteBio Data Analysis Software. p41_2 is not included due to unsatisfactory curve fitting.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4563141&req=5

f0015: Binding of selected peptides to the selection mAb41. (A) Binding of synthetic peptides to mAb41 at various concentrations. Biotinylated peptides were added to streptavidin-coated microtiter plates. mAb 41 was applied as the primary antibody in a 10-fold dilution series. HRP-conjugated goat anti-mouse antibody was used as the detection antibody. Results shown are the mean of two replicates performed in one of three independent experiments, each of which showed similar results. (B) Competition for binding to mAb41 between p41_1 and wild type pB. 1 μg biotinylated pB was attached to streptavidin-coated plates. 12 ng of mAb41in 100 μl PBS containing 5% milk was added in the presence of increasing amount of p41_1 as indicated. The bound mAb41 was detected using an HRP-conjugated goat anti-mouse antibody. An unrelated peptide from the mRNA display library was used as a negative control. Mean values are graphed and error bars represent SEM of sample replicates. (C) Measurement of binding affinity of selected peptides to mAb41 by Octet RED. Biotinylated peptides were immobilized on Streptavidin biosensors (Fortebio) and Fabs of mAb41 were used as analyte in a 2-fold dilution series ranging from 125 nM to 7.8 nM. Sensorgram data for peptide p41_1 are shown. (D) Binding constants of mAb41-selected peptides to mAb41 Fabs were obtained by fitting sensorgrams (blue) with a 1:1 model (red) using ForteBio Data Analysis Software. p41_2 is not included due to unsatisfactory curve fitting.
Mentions: The five most abundant selected peptides (p41-1 to p41-5, Fig. 2B) were chemically synthesized for further analysis. By ELISA, mAb41 showed concentration-dependent binding to the mRNA display-selected peptides (Fig. 3A). Peptide p41_1, the most abundant peptide selected by mAb41, was used as a competitor of wild-type peptide B (pB, a shorter version of pA, Fig. 2B) for binding to mAb41 to confirm that the selected peptides interact with the antigen-binding sites of the selection antibody. Increasing amounts of p41_1 inhibited binding of mAb41 to immobilized pB (Fig. 3B). This binding was not appreciably affected by a negative control peptide from a mRNA display library that is not enriched by mAb41 (Fig. 3B), implying that p41_1 binds to the same antigen binding site on mAb41 as does wild type pB. Similar results were obtained using p41_3 as competitor (Figure S4A). Using the Octet binding assay, dissociation constants (Kd) of mAb41 Fabs with mAb41-selected peptides (with the exception of p41_2 which could not be evaluated due to unsatisfactory curve fitting) were within the 11–70 nanomolar range, similar to the affinity of mAb41 Fab fragments (Kd = 13 nM) for wild type pB (Fig. 3C–D and Figure S4B–E).

Bottom Line: Thus, using mRNA display to interrogate mAbs permits high resolution identification of functional peptide antigens that direct targeted immune responses, supporting its use in vaccine reverse engineering for pathogens against which potent neutralizing mAbs are available.After the identified peptides were injected into mice, the mice produced their own antibodies with characteristics similar to the original antibody.This approach can provide previously unavailable information about antibody binding and could also be useful in developing new vaccines.

View Article: PubMed Central - PubMed

Affiliation: Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Ave, Silver Spring, MD 20993, United States.

ABSTRACT

Unlabelled: Vaccine reverse engineering is emerging as an important approach to vaccine antigen identification, recently focusing mainly on structural characterization of interactions between neutralizing monoclonal antibodies (mAbs) and antigens. Using mAbs that bind unknown antigen structures, we sought to probe the intrinsic features of antibody antigen-binding sites with a high complexity peptide library, aiming to identify conformationally optimized mimotope antigens that capture mAb-specific epitopes. Using a high throughput sequencing-enhanced messenger ribonucleic acid (mRNA) display approach, we identified high affinity binding peptides for a hepatitis C virus neutralizing mAb. Immunization with the selected peptides induced neutralizing activity similar to that of the original mAb. Antibodies elicited by the most commonly selected peptides were predominantly against specific epitopes. Thus, using mRNA display to interrogate mAbs permits high resolution identification of functional peptide antigens that direct targeted immune responses, supporting its use in vaccine reverse engineering for pathogens against which potent neutralizing mAbs are available.

Research in context: We used a large number of randomly produced small proteins ("peptides") to identify peptides containing specific protein sequences that bind efficiently to an antibody that can prevent hepatitis C virus infection in cell culture. After the identified peptides were injected into mice, the mice produced their own antibodies with characteristics similar to the original antibody. This approach can provide previously unavailable information about antibody binding and could also be useful in developing new vaccines.

No MeSH data available.


Related in: MedlinePlus