Site‐directed mutagenesis of <scp>HLA</scp> molecules reveals the functional epitope of a human <scp>HLA‐A1</scp>/<scp>A36</scp>‐specific monoclonal antibody

Meng, Shi-Yuan; Bezstarosti, Suzanne; Singh, Ujjwala; Yap, Michelle; Scott, Laura J.; Petrosyan, Naiiry; Quiroz, Fred; Eps, Ned Van; Hui, Eric Ka-Wai; Suh, David; Zhu, Qing; Pei, Rui; Claas, Frans H.J.; Lowe, David; Heidt, Sebastiaan

doi:10.1111/tan.14895

Cited by 4 publications

(3 citation statements)

References 12 publications

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“…The technique involves targeting specific amino acids within the antigen sequence to alter them and determine their interaction with a specific ligand [ 20 , 21 , 22 ]. The first step in site-directed mutagenesis is to identify the candidate amino acids that are likely to be part of the epitope.…”

Section: Resultsmentioning

confidence: 99%

Epitopes and Mimotopes Identification Using Phage Display for Vaccine Development against Infectious Pathogens

Palma¹

2023

Vaccines

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Traditional vaccines use inactivated or weakened forms of pathogens which could have side effects and inadequate immune responses. To overcome these challenges, phage display has emerged as a valuable tool for identifying specific epitopes that could be used in vaccines. This review emphasizes the direct connection between epitope identification and vaccine development, filling a crucial gap in the field. This technique allows vaccines to be engineered to effectively stimulate the immune system by presenting carefully selected epitopes. Phage display involves screening libraries of random peptides or gene/genome fragments using serum samples from infected, convalescent, or vaccinated individuals. This method has been used to identify epitopes from various pathogens including SARS-CoV-2, Mycobacterium tuberculosis, hepatitis viruses, H5N1, HIV-1, Human T-lymphotropic virus 1, Plasmodium falciparum, Trypanosoma cruzi, and Dirofilaria repens. Bacteriophages offer advantages such as being immunogenic carriers, low production costs, and customization options, making them a promising alternative to traditional vaccines. The purpose of this study has been to highlight an approach that encompasses the entire process from epitope identification to vaccine production using a single technique, without requiring additional manipulation. Unlike conventional methods, phage display demonstrates exceptional efficiency and speed, which could provide significant advantages in critical scenarios such as pandemics.

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Section: Resultsmentioning

confidence: 99%

Epitopes and Mimotopes Identification Using Phage Display for Vaccine Development against Infectious Pathogens

Palma¹

2023

Vaccines

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“…Occasionally, the reactivity patterns of human HLA mAbs are highly complex with multiple potential SA AA outside a 3.5 Å radius could potentially form the functional epitope. We have recently shown that these complicated reactivity patterns can be resolved by site‐directed mutagenesis of HLA molecules to determine the crucial SA AA for binding of a mAbs to its target HLA 33 . Such strategy will be of interest for a number of HLA class II mAbs for which we have not yet been able to identify the functional epitope.…”

Section: Discussionmentioning

confidence: 99%

“…We have recently shown that these complicated reactivity patterns can be resolved by site-directed mutagenesis of HLA molecules to determine the crucial SA AA for binding of a mAbs to its target HLA. 33 Such strategy will be of interest for a number of HLA class II mAbs for which we have not yet been able to identify the functional epitope. The increasing number of newly generated human HLA-specific mAbs with unique specificities together with mutation studies (when necessary), as well as adsorption elution studies will contribute significantly to the antibody verification endeavour.…”

Section: Discussionmentioning

confidence: 99%

Antibody verification of HLA class I and class II eplets by human monoclonal HLA antibodies

Kramer,

Bezstarosti,

Franke‐van Dijk

et al. 2024

HLA

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In solid organ transplantation, formation of de novo donor‐specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA‐specific monoclonal antibodies (mAbs) were generated from HLA‐reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA‐EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II‐specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA‐DR‐specific eplets, and conversion of five eplets (one HLA‐A, one HLA‐B, two HLA‐DR, and one HLA‐DP), from provisionally verified to truly antibody‐verified. Finally, one HLA‐DQ‐specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA‐specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting.

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Site‐directed mutagenesis of HLA molecules reveals the functional epitope of a human HLA‐A1/A36‐specific monoclonal antibody

Meng

Bezstarosti

Singh

et al. 2022

HLA

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Eplet 44KM is currently listed in the HLA Epitope Registry but does not adhere to the eplet definition of an amino acid configuration within a 3.5 Å radius. Eplet 44KM has been previously redefined to the antibody‐verified reactivity pattern 44K/150V/158V, based on reactivity analysis of monoclonal antibody VDK1D12. Since the three residues are always simultaneously present on common HLA alleles, methods to define which residue is crucial for antibody‐induction and binding are limited. In this proof‐of‐concept study, we performed site‐directed mutagenesis to narrow down the antibody‐verified reactivity pattern 44K/150V/158V to a single amino acid and defined 44K as the eplet or functional epitope of mAb VDK1D12.

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Site‐directed mutagenesis of HLA molecules reveals the functional epitope of a human HLA‐A1/A36‐specific monoclonal antibody

Cited by 4 publications

References 12 publications

Epitopes and Mimotopes Identification Using Phage Display for Vaccine Development against Infectious Pathogens

Epitopes and Mimotopes Identification Using Phage Display for Vaccine Development against Infectious Pathogens

Antibody verification of HLA class I and class II eplets by human monoclonal HLA antibodies

Site‐directed mutagenesis of HLA molecules reveals the functional epitope of a human HLA‐A1/A36‐specific monoclonal antibody

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