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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

mRNA display selection combined with HTS. The DNA library used contains a T7 promoter (T7), a CMV Translation enhancer (TE), a 15 or 27-mer random region ((Trimer)15or27) and a constant region (3′ constant) encoding the peptide QLRNSCA. “Trimer” represents the mixture of 20 trimer (codon) phosphoramidites (Glen Research), each encoding one amino acid. In vitro transcription, ligation to a puromycin linker (green), in vitro translation and reverse transcription (RT) were performed as described in the Materials and methods section. mRNA/DNA–peptide fusions were applied to protein G magnetic beads (ProG beads) complexed with monoclonal antibodies (mAb) for selection. The regenerated DNA library was converted to an Illumina sequencing library by PCR amplification using forward and reverse primers containing Illumina adapters and subjected to MiSeq sequencing.
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f0005: mRNA display selection combined with HTS. The DNA library used contains a T7 promoter (T7), a CMV Translation enhancer (TE), a 15 or 27-mer random region ((Trimer)15or27) and a constant region (3′ constant) encoding the peptide QLRNSCA. “Trimer” represents the mixture of 20 trimer (codon) phosphoramidites (Glen Research), each encoding one amino acid. In vitro transcription, ligation to a puromycin linker (green), in vitro translation and reverse transcription (RT) were performed as described in the Materials and methods section. mRNA/DNA–peptide fusions were applied to protein G magnetic beads (ProG beads) complexed with monoclonal antibodies (mAb) for selection. The regenerated DNA library was converted to an Illumina sequencing library by PCR amplification using forward and reverse primers containing Illumina adapters and subjected to MiSeq sequencing.

Mentions: To validate our experimental system, we first performed mRNA display selection using FLAG M2 mAb as “selection antibody” using a 15-mer library. After each round of selection, nucleic acid sequences linked to the peptides that remained bound to the M2 mAb were PCR-amplified and subjected to Illumina MiSeq HTS sequencing (Fig. 1). We deduced peptide sequences from the HTS results, and ranked the peptides by their frequency within the HTS run as a reflection of their relative affinity for the selection antibody. After 2 rounds of selection with FLAG M2 mAb, the consensus motif DYKXXD homologous to the FLAG epitope (DYKDDDDK) was readily identified among the most frequent peptide sequences obtained (Figure S1), indicating a valid experimental system. Consistent with a recent report (Olson et al., 2012), these results showed that HTS provided a large number of sequences for pattern identification, and reduced the number of mRNA display selection rounds needed to identify peptide binders compared to conventional mRNA display using a low-throughput sequencing method.


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)

mRNA display selection combined with HTS. The DNA library used contains a T7 promoter (T7), a CMV Translation enhancer (TE), a 15 or 27-mer random region ((Trimer)15or27) and a constant region (3′ constant) encoding the peptide QLRNSCA. “Trimer” represents the mixture of 20 trimer (codon) phosphoramidites (Glen Research), each encoding one amino acid. In vitro transcription, ligation to a puromycin linker (green), in vitro translation and reverse transcription (RT) were performed as described in the Materials and methods section. mRNA/DNA–peptide fusions were applied to protein G magnetic beads (ProG beads) complexed with monoclonal antibodies (mAb) for selection. The regenerated DNA library was converted to an Illumina sequencing library by PCR amplification using forward and reverse primers containing Illumina adapters and subjected to MiSeq sequencing.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0005: mRNA display selection combined with HTS. The DNA library used contains a T7 promoter (T7), a CMV Translation enhancer (TE), a 15 or 27-mer random region ((Trimer)15or27) and a constant region (3′ constant) encoding the peptide QLRNSCA. “Trimer” represents the mixture of 20 trimer (codon) phosphoramidites (Glen Research), each encoding one amino acid. In vitro transcription, ligation to a puromycin linker (green), in vitro translation and reverse transcription (RT) were performed as described in the Materials and methods section. mRNA/DNA–peptide fusions were applied to protein G magnetic beads (ProG beads) complexed with monoclonal antibodies (mAb) for selection. The regenerated DNA library was converted to an Illumina sequencing library by PCR amplification using forward and reverse primers containing Illumina adapters and subjected to MiSeq sequencing.
Mentions: To validate our experimental system, we first performed mRNA display selection using FLAG M2 mAb as “selection antibody” using a 15-mer library. After each round of selection, nucleic acid sequences linked to the peptides that remained bound to the M2 mAb were PCR-amplified and subjected to Illumina MiSeq HTS sequencing (Fig. 1). We deduced peptide sequences from the HTS results, and ranked the peptides by their frequency within the HTS run as a reflection of their relative affinity for the selection antibody. After 2 rounds of selection with FLAG M2 mAb, the consensus motif DYKXXD homologous to the FLAG epitope (DYKDDDDK) was readily identified among the most frequent peptide sequences obtained (Figure S1), indicating a valid experimental system. Consistent with a recent report (Olson et al., 2012), these results showed that HTS provided a large number of sequences for pattern identification, and reduced the number of mRNA display selection rounds needed to identify peptide binders compared to conventional mRNA display using a low-throughput sequencing method.

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