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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

Characterization of the binding reactivity of anti-p41_1 and anti-p41_3 antisera. Biotinylated peptides were immobilized onto streptavidin-coated plates. Binding of (A) mAb41 (1.2 μg/ml), (B) anti-p41_1, (C) anti-pA, and (D) anti-p41_3 (1:1000 dilution, n = 3 for (B–D)) to pB and mAb 41-selected peptides (p41_1 to p41_5) were measured by ELISA. Mean values are graphed and error bars represent SEM of technical replicates for (A) mAb41, and biological replicates for antisera (B–D), with each biological replicate having technical duplicates.
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f0025: Characterization of the binding reactivity of anti-p41_1 and anti-p41_3 antisera. Biotinylated peptides were immobilized onto streptavidin-coated plates. Binding of (A) mAb41 (1.2 μg/ml), (B) anti-p41_1, (C) anti-pA, and (D) anti-p41_3 (1:1000 dilution, n = 3 for (B–D)) to pB and mAb 41-selected peptides (p41_1 to p41_5) were measured by ELISA. Mean values are graphed and error bars represent SEM of technical replicates for (A) mAb41, and biological replicates for antisera (B–D), with each biological replicate having technical duplicates.

Mentions: To compare binding characteristics of peptide-elicited antibodies with those of mAb41, we evaluated anti-p41_1 antisera, anti-pA antisera, and mAb41 for their binding reactivity to pB and to the top peptides selected by mAb41. Since peptides p41_1 to p41_5 were enriched by mAb41 through multiple rounds of selection, as a group they likely reflect the structure of the mAb41 antigen binding site and thus were used as signature peptides for detecting mAb41-like antibodies. As expected, mAb41 bound to all the peptides in the ELISA (Fig. 5A). Anti-p41_1 antisera showed a similar binding profile to that of mAb41 when tested against the other mimotope peptides with the exception of p41_2 (Fig. 5B). In contrast, anti-pA sera from the immunized mice showed no detectable binding to any of the top 5 peptides selected by mAb41 and only bound to pB (Fig. 5C). These results suggest that anti-p41_1 sera contains mAb41-like antibodies, while in anti-pA sera, this population is not dominant. Anti-p41_3 antisera showed a binding profile similar to that of anti-p41_1 (Fig. 5D). The reason for the different behavior of p41_2 from that of other mAb41-selected peptides in these assays remains unknown, but may be related to its relatively low binding affinity to mAb41 as assessed by ELISA (Fig. 3A).


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)

Characterization of the binding reactivity of anti-p41_1 and anti-p41_3 antisera. Biotinylated peptides were immobilized onto streptavidin-coated plates. Binding of (A) mAb41 (1.2 μg/ml), (B) anti-p41_1, (C) anti-pA, and (D) anti-p41_3 (1:1000 dilution, n = 3 for (B–D)) to pB and mAb 41-selected peptides (p41_1 to p41_5) were measured by ELISA. Mean values are graphed and error bars represent SEM of technical replicates for (A) mAb41, and biological replicates for antisera (B–D), with each biological replicate having technical duplicates.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0025: Characterization of the binding reactivity of anti-p41_1 and anti-p41_3 antisera. Biotinylated peptides were immobilized onto streptavidin-coated plates. Binding of (A) mAb41 (1.2 μg/ml), (B) anti-p41_1, (C) anti-pA, and (D) anti-p41_3 (1:1000 dilution, n = 3 for (B–D)) to pB and mAb 41-selected peptides (p41_1 to p41_5) were measured by ELISA. Mean values are graphed and error bars represent SEM of technical replicates for (A) mAb41, and biological replicates for antisera (B–D), with each biological replicate having technical duplicates.
Mentions: To compare binding characteristics of peptide-elicited antibodies with those of mAb41, we evaluated anti-p41_1 antisera, anti-pA antisera, and mAb41 for their binding reactivity to pB and to the top peptides selected by mAb41. Since peptides p41_1 to p41_5 were enriched by mAb41 through multiple rounds of selection, as a group they likely reflect the structure of the mAb41 antigen binding site and thus were used as signature peptides for detecting mAb41-like antibodies. As expected, mAb41 bound to all the peptides in the ELISA (Fig. 5A). Anti-p41_1 antisera showed a similar binding profile to that of mAb41 when tested against the other mimotope peptides with the exception of p41_2 (Fig. 5B). In contrast, anti-pA sera from the immunized mice showed no detectable binding to any of the top 5 peptides selected by mAb41 and only bound to pB (Fig. 5C). These results suggest that anti-p41_1 sera contains mAb41-like antibodies, while in anti-pA sera, this population is not dominant. Anti-p41_3 antisera showed a binding profile similar to that of anti-p41_1 (Fig. 5D). The reason for the different behavior of p41_2 from that of other mAb41-selected peptides in these assays remains unknown, but may be related to its relatively low binding affinity to mAb41 as assessed by ELISA (Fig. 3A).

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