SHLD1 is part of the Shieldin (SHLD) complex, which acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end resection and promote DNA repair via non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and repair of RAG-induced DSBs in XLF-deficient cells, the function of SHLD during these processes remains elusive. Here we report that SHLD1 is dispensable for lymphocyte development and RAG-mediated V(D)J recombination, even in the absence of XLF. By contrast, SHLD1 is essential for restricting resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR by NHEJ and alternative end-joining. Finally, we show that this SHLD1 function is required for orientation-specific joining of AID-initiated DSBs. Our data thus suggest that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms: SHLD-independent synapsis of V(D)J segments and switch regions within chromatin, and SHLD-dependent protection of AID-DSB ends against resection.

Background/Objectives: An assay for protein content is essential but insufficient for quality control of acellular pertussis vaccines, which might consist of up to five components, each needing individual quantification. Generally, purified pertussis antigens such as pertussis toxin (PTx), filamentous haemagglutinin (FHA), and pertactin (PRN) should be detoxified or stabilized chemically before being formulated into vaccine bulk. The use of chemical agents like formaldehyde and glutaraldehyde can alter the immunological reactivity of these antigens, rendering direct assays by methods such as ELISA ineffective. Methods: In this study, a simple method based on single radial diffusion (SRD) using low concentrations of polyclonal antisera against PT toxoid (PTd), FHA, and PRN was developed. By adding a detergent, diffusible subunits are produced regardless of the original physical state of the antigens, making it suitable for quantifying these antigens after chemical treatment. Results: The assay has shown good specificity, accuracy, and precision. Furthermore, it can differentiate between preparations with the same protein concentration but different antigenic contents. A significant positive correlation between the antigen content and the in vivo immunogenicity has also been demonstrated. Conclusions: An assay for quality control and consistency monitoring of combined vaccines containing acellular pertussis antigen components has been established.

Background/Objectives: Chemically or genetically detoxified pertussis toxin (PTx) is a crucial antigen component of the acellular pertussis vaccine. Chemical detoxification using glutaraldehyde generally causes significant structural changes to the toxin. However, how these structural changes in PTx affect its antigenic properties remains unclear. Additionally, there is limited knowledge regarding how many alterations in antigenic properties impact immunogenicity. Methods: To investigate the impact of structural changes on antigenic properties, we developed a sandwich ELISA to quantify the neutralizing epitopes on PTx. Subsequently, we analyzed different PTx toxoid (PTd) preparations with the assay. Additionally, we assessed the immunogenicity of various acellular pertussis vaccine candidates containing these PTd preparations. Finally, the assay was applied to evaluate the consistency of commercial batches of PTx and PTd intermediates. Results: The assay demonstrated reasonable specificity, accuracy, and precision, and it was sensitive enough to quantify variations in neutralizing epitopes among different PTd samples that shared the same protein concentration. Importantly, we found a positive correlation between the number of neutralizing epitopes in detoxified PTx and its immunogenicity, indicating that the amount of neutralizing epitopes present determines the immunogenicity of glutaraldehyde-inactivated PTx. Moreover, commercial batches of PTx and PTd intermediates exhibited minor variations in neutralizing epitopes. Conclusions: These findings have significant implications for developing acellular pertussis vaccines as they highlight the importance of preserving the neutralizing epitopes of PTx during detoxification to ensure the vaccine’s effectiveness. This assay is also valuable for the quality control of PTd as it more accurately represents the actual antigenic changes of PTx.

Caractérisation de nouveaux facteurs de la réparation des cassures double-brin de l'ADN dans les lymphocytes B: La diversité génétique des récepteurs d'antigènes des lymphocytes B repose sur deux processus de recombinaison site-spécifique de l'ADN : la recombinaison V(D)J qui est initiée dans les cellules B immatures par la nucléase RAG (Recombination activating genes 1 et 2) et la commutation isotypique (class switch recombination, CSR) qui est induite dans les cellules B matures par la cytidine désaminase AID (activation induced cytidine deaminase). Des erreurs lors de ces processus de recombinaison peuvent avoir un impact sur le développement des lymphocytes et peuvent aussi provoquer une instabilité du génome conduisant à des syndromes d'immunodéficience plus ou moins sévère et au développement de cancers lymphoïdes. Les cassures doubles brins de l'ADN (CDB) générées par ces enzymes induisent une réponse cellulaire dépendante de la protéine kinase Ataxia telangectasia mutated (ATM) lors de laquelle la protéine 53BP1 s'accumule aux sites endommagés pour favoriser la réparation de type jonction d'extrémités non-homologues (non-homologous end-joining, NHEJ). Alors que la recombinaison V(D)J et la CSR toutes deux monopolisent la voie 53BP1-NHEJ pour la réparation des CDB induites, ces deux réactions semblent avoir des modes de régulation différents. En effet, alors que la recombinaison V(D)J peut avoir lieu sans la protéine 53BP1, le CSR est fortement impacté par une déficience en 53BP1. Récemment, notre laboratoire et d'autres groupes avons identifié deux nouvelles protéines de la réparation des CDB, C20orf196 (SHLD1) et FAM35A (SHLD2), qui forment avec REV7 (également appelée MAD2L2) et CTC-534A2.2 (SHLD3) le complexe shieldin qui agit en aval de 53BP1 pour promouvoir la réparation par NHEJ. Au cours de mon travail de thèse, j'ai investigué le rôle de SHLD1, SHLD2 et MAD2L2 dans la recombinaison V(D)J et la CSR. Pour ce faire, j'ai inactivé génétiquement Shld1, Shld2 et Mad2l2 dans deux systèmes cellulaires permettant la modélisation de la recombinaison V(D)J et le CSR, respectivement les cellules de type immature pro-B immortalisées par la kinase Abelson (v-abl) et les cellules B CH12F3. J'ai montré que les protéines SHLD1, SHLD2 et MAD2L2 sont essentielles pour la CSR mais dispensables pour la recombinaison de la V(D)J. j'ai aussi montré qu'une diminution de l'expression de TRIP13, un régulateur nouvellement identifié de MAD2L2-Shieldin, n'a pas d'impact sur la recombinaison V(D)J. Enfin, en utilisant un modèle murin de déficience de SHLD1, j'ai confirmé que les cellules immortalisée v-Abl pro-B Shld1-/- ont une recombinaison V(D)J normale. Dans une deuxième partie de mon travail, j'ai testé si le complexe shieldin pouvait avoir des fonctions redondantes avec le facteur du NHEJ XLF lors de la réparation des CDB induites par RAG. J'ai pour ce fait généré une série de clones de cellules v-Abl pro-B déficients pour 53BP1-shieldin et/ou XLF et j'ai testé la recombinaison V(D)J dans ces différents clones. J'ai ainsi montré que les cellules inactivées pour XLF et SHL2, REV7 et 53BP1 ont une recombinaison V(D)J défectueuse par rapport aux clones cellulaires B inactivés pour XLF et SHLD1. Dans l'ensemble, mes études apportent de nouvelles connaissances sur les mécanismes de diversification des gènes codants pour les récepteurs d'antigènes des lymphocytes et, en particulier, démontrent que l'axe 53BP1-Shieldin joue des fonctions uniques lors de la réparation des CDB générées par les enzymes RAG et AID.

Chemically or genetically detoxified pertussis toxin (PTx) is a crucial antigen component of the acellular pertussis vaccine. Chemical detoxification using glutaraldehyde generally causes significant structural changes to the toxin. However, how these structural changes in PTx affect its antigenic properties remains unclear. Additionally, there is limited knowledge regarding how many alterations in antigenic properties impact immunogenicity. To investigate the impact of structural changes on antigenic properties, we developed a sandwich ELISA to quantify the neutralizing epitopes on PTx. Subsequently, we analyzed different PTx toxoid (PTd) preparations with the assay. Additionally, we assessed the immunogenicity of various acellular pertussis vaccine candidates containing these PTd preparations. Finally, the assay was applied to evaluate the consistency of commercial batches of PTx and PTd intermediates. The assay demonstrated reasonable specificity, accuracy, and precision, and it was sensitive enough to quantify variations in neutralizing epitopes among different PTd samples that shared the same protein concentration. Importantly, we found a positive correlation between the number of neutralizing epitopes in detoxified PTx and its immunogenicity, indicating that the amount of neutralizing epitopes present determines the immunogenicity of glutaraldehyde-inactivated PTx. Moreover, commercial batches of PTx and PTd intermediates exhibited minor variations in neutralizing epitopes. These findings have significant implications for developing acellular pertussis vaccines, as they highlight the importance of preserving the neutralizing epitopes of PTx during detoxification to ensure the vaccine's effectiveness. This assay is also valuable for the quality control of PTd, as it more accurately represents the actual antigenic changes of PTx.