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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

Neutralization of HCVcc GT1a/2a chimeric virus by polyclonal sera from mice immunized with selected peptides. (A) ELISA endpoint titers of polyclonal sera against immunizing peptides. Mice were immunized with peptides as indicated (n = 4 or 5 for each peptide). Polyclonal sera after the second boost were measured for ELISA endpoint titers.(B) Neutralizing ID50 titers of cell culture HCV GT1a/2a chimeric virus by antisera against selected peptides (n = 4 or 5 mice for each peptide). For mice with ID50 < 1:20, 1:10 was used in the mean ID50 calculation. Results shown are the mean of two replicates performed in one of three independent experiments, each of which showed similar results. (C) Anti-p41_1 sera were either mock depleted (mock-D) or depleted using peptide p41_1 (p41_1-D). Depleted sera (1:1000 dilution) were tested for binding to p41_1 by ELISA. (D) HCVcc 1a/2a chimeric virus neutralization after p41_1 depletion of anti-41_1 sera (dilution 1:40). (C) and (D): Similar results observed in n = 3 samples; results from the mouse serum with the strongest neutralizing activity are shown. (Error bars represent SEM of sample duplicates. *p < 0.05, **p < 0.01, Student t test, unpaired).
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f0020: Neutralization of HCVcc GT1a/2a chimeric virus by polyclonal sera from mice immunized with selected peptides. (A) ELISA endpoint titers of polyclonal sera against immunizing peptides. Mice were immunized with peptides as indicated (n = 4 or 5 for each peptide). Polyclonal sera after the second boost were measured for ELISA endpoint titers.(B) Neutralizing ID50 titers of cell culture HCV GT1a/2a chimeric virus by antisera against selected peptides (n = 4 or 5 mice for each peptide). For mice with ID50 < 1:20, 1:10 was used in the mean ID50 calculation. Results shown are the mean of two replicates performed in one of three independent experiments, each of which showed similar results. (C) Anti-p41_1 sera were either mock depleted (mock-D) or depleted using peptide p41_1 (p41_1-D). Depleted sera (1:1000 dilution) were tested for binding to p41_1 by ELISA. (D) HCVcc 1a/2a chimeric virus neutralization after p41_1 depletion of anti-41_1 sera (dilution 1:40). (C) and (D): Similar results observed in n = 3 samples; results from the mouse serum with the strongest neutralizing activity are shown. (Error bars represent SEM of sample duplicates. *p < 0.05, **p < 0.01, Student t test, unpaired).

Mentions: Since the selected peptides bound mAb41 with high affinity, we hypothesized that these peptides might mimic native antigen in presentation of critical epitopes to the immune system and thus might be useful in inducing immune responses that resemble mAb41. We immunized BALB/c mice (n = 4 or 5 for each immunogen) with the top five peptides selected by mAb41. Polyclonal sera were named after the immunizing peptides, i.e. anti-p41_1 is the serum obtained from mice immunized with p41_1. Because mAb41 was cloned from mice previously immunized with pA peptide (Fig. 2B), we immunized mice with the original wild type pA as a control. Polyclonal sera from immunized mice showed binding reactivity to the immunizing peptides with titers in the 1:104 to 105 range for most animals (Fig. 4A), with the exception of p41_2, which showed lower titers (below 2 × 103) and were therefore excluded from further analysis. Since mAb41 specifically neutralizes HCV GT1a, we tested the capacity of these polyclonal sera to neutralize HCV GT1a in vitro using 1a/2a chimeric HCVcc.


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)

Neutralization of HCVcc GT1a/2a chimeric virus by polyclonal sera from mice immunized with selected peptides. (A) ELISA endpoint titers of polyclonal sera against immunizing peptides. Mice were immunized with peptides as indicated (n = 4 or 5 for each peptide). Polyclonal sera after the second boost were measured for ELISA endpoint titers.(B) Neutralizing ID50 titers of cell culture HCV GT1a/2a chimeric virus by antisera against selected peptides (n = 4 or 5 mice for each peptide). For mice with ID50 < 1:20, 1:10 was used in the mean ID50 calculation. Results shown are the mean of two replicates performed in one of three independent experiments, each of which showed similar results. (C) Anti-p41_1 sera were either mock depleted (mock-D) or depleted using peptide p41_1 (p41_1-D). Depleted sera (1:1000 dilution) were tested for binding to p41_1 by ELISA. (D) HCVcc 1a/2a chimeric virus neutralization after p41_1 depletion of anti-41_1 sera (dilution 1:40). (C) and (D): Similar results observed in n = 3 samples; results from the mouse serum with the strongest neutralizing activity are shown. (Error bars represent SEM of sample duplicates. *p < 0.05, **p < 0.01, Student t test, unpaired).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4563141&req=5

f0020: Neutralization of HCVcc GT1a/2a chimeric virus by polyclonal sera from mice immunized with selected peptides. (A) ELISA endpoint titers of polyclonal sera against immunizing peptides. Mice were immunized with peptides as indicated (n = 4 or 5 for each peptide). Polyclonal sera after the second boost were measured for ELISA endpoint titers.(B) Neutralizing ID50 titers of cell culture HCV GT1a/2a chimeric virus by antisera against selected peptides (n = 4 or 5 mice for each peptide). For mice with ID50 < 1:20, 1:10 was used in the mean ID50 calculation. Results shown are the mean of two replicates performed in one of three independent experiments, each of which showed similar results. (C) Anti-p41_1 sera were either mock depleted (mock-D) or depleted using peptide p41_1 (p41_1-D). Depleted sera (1:1000 dilution) were tested for binding to p41_1 by ELISA. (D) HCVcc 1a/2a chimeric virus neutralization after p41_1 depletion of anti-41_1 sera (dilution 1:40). (C) and (D): Similar results observed in n = 3 samples; results from the mouse serum with the strongest neutralizing activity are shown. (Error bars represent SEM of sample duplicates. *p < 0.05, **p < 0.01, Student t test, unpaired).
Mentions: Since the selected peptides bound mAb41 with high affinity, we hypothesized that these peptides might mimic native antigen in presentation of critical epitopes to the immune system and thus might be useful in inducing immune responses that resemble mAb41. We immunized BALB/c mice (n = 4 or 5 for each immunogen) with the top five peptides selected by mAb41. Polyclonal sera were named after the immunizing peptides, i.e. anti-p41_1 is the serum obtained from mice immunized with p41_1. Because mAb41 was cloned from mice previously immunized with pA peptide (Fig. 2B), we immunized mice with the original wild type pA as a control. Polyclonal sera from immunized mice showed binding reactivity to the immunizing peptides with titers in the 1:104 to 105 range for most animals (Fig. 4A), with the exception of p41_2, which showed lower titers (below 2 × 103) and were therefore excluded from further analysis. Since mAb41 specifically neutralizes HCV GT1a, we tested the capacity of these polyclonal sera to neutralize HCV GT1a in vitro using 1a/2a chimeric HCVcc.

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