Bioinspired Design and Oriented Synthesis of Immunogenic Site-Specifically Penicilloylated Peptides

Abstract

β-Lactam antibiotics allergy is recognized as a public health concern. By covalently binding to serum proteins, penicillins are known to form immunogenic complexes. The latter are recognized and digested by antigen-presenting cells into drug-hapten peptides leading to the immunization of treated persons and IgE-mediated hypersensitivity reactions encompassing anaphylaxis. If type I allergic reactions to drugs are often unpredictable, they are known to be dependent on CD4 T-cells. This fundamental study revisits the chemical basis of the benzylpenicillin (BP) allergy with the aim of identifying immunologically relevant biomimetic benzylpenicilloylated peptides through the analysis of BP-conjugated human serum albumin (BP-HSA) profile and the evaluation of the naı̈ve CD4 T-cell responses to candidate BP-HSA-derived peptides. The chemical structures of BP-HSA bioconjugates synthesized in vitro at both physiological and basic pH were investigated by mass spectrometry. From the ten most representative lysine residues grafted by BP-hapten, HSA-bioinspired 15-mer peptide sequences were designed and the potential T-cell epitope profile of each peptide was predicted using two complementary in silico approaches, i.e., HLA class II binding prediction tools from the Immune Epitope Database and Analysis Resource (IEDB) and computational alanine scanning mutagenesis. Twelve structurally diversified benzylpenicilloylated peptides (BP-Ps) were selected and synthesized with the aid of a flexible synthesis pathway using an original benzylpenicilloylated lysine monomer as common precursor. In order to corroborate their predicted "epitope" profile, the naı̈ve CD4 T-cell response specific to BP was evaluated through a coculture approach. To our knowledge, this study showed for the first time the ability of bioinspired peptides structurally stemming from BP-HSA to be recognized by naı̈ve CD4 T-cells thus identifying a pre-existing T-cell repertoire for penicillin molecules bound to proteins. It also established a promising model approach expandable to other most frequently used penicillin classes of antibiotics to reveal biomimetic drug-modified antigenic peptides relevant for qualitative and quantitative drug allergy studies.

Identification of T‐cell epitopes from benzylpenicillin conjugated to human serum albumin and implication in penicillin allergy

“…BP‐HSA was produced as described previously . The presence of BP on HSA was confirmed using Western blots and mass spectrometry MALDI‐TOF analysis and is published in a companion work . BP‐binding sites on HSA were identified through tryptic digestion of BP‐HSA bioconjugates followed by MALDI‐MS and nano‐LC‐MS/MS analysis.…”

“…Twelve 15 mer long BP‐haptenated peptides identified as potential T‐cell epitopes were selected using the MHC II binding prediction tool available at the IEDB (http://www.iedb.org). A percentile rank was generated allowing a uniform comparison of peptide's binding affinity to HLA alleles across different predictors. A peptide was considered a good binder for a specific allele if its IEDB predicted consensus percentile rank was ≤20 .…”

“…A peptide was considered a good binder for a specific allele if its IEDB predicted consensus percentile rank was ≤20 . BP‐haptenated peptides were synthesized using a BP‐lysine monomer and standard Fmoc chemistry. They were purified by RP‐HPLC to approximately 90% purity as determined by analytical HPLC.…”

“…Penicillin‐binding sites on HSA have been found both in vivo using sera obtained from patients and in vitro by synthesizing bioconjugates all of which mapped at lysine residues . We have recently used a combination of immunological and high‐resolution MS (mass spectrometry) approaches to identify benzylpenicillin (BP)‐binding sites on HSA …”

“…We selected candidate peptides using an in silico approach addressing peptide binding to HLA molecules. BP‐haptenated peptides were synthesized using an original BP‐lysine monomer . With the selected haptenated peptides, we were able to define BP‐haptenated peptides recognized by naïve T cells.…”