Periodic Reporting for period 2 - AutoEngineering (Engineering antibodies in B cells using endogenous AID activity)
Reporting period: 2023-01-01 to 2024-06-30
Despite decades of research, effective vaccines against some viruses that evolve to escape from antibody responses remain elusive. Although antiviral therapeutics are available to control or even cure some of the respective diseases, elimination programs for viruses can fail, and poor access to care can lead to a rise in new cases of infection. Novel preventive measures and vaccination strategies are thus highly demanded.
The AutoEngineering project aims to develop a vaccination strategy through an innovative approach to engineering B cells. Natural DNA breaks acquired during B cell maturation are exploited to equip antibodies with extra-domains that confer superior reactivity to pathogens. The concept builds on “design-by-nature”, namely, the discovery of LAIR1- and LILRB1-containing antibodies. Exons encoding for pathogen-receptors can be integrated in the antibody gene during B cell development and maturation, translating into insert-containing antibodies. The latter confer broad reactivity to Plasmodium in more than 5 % of individuals living in malaria-endemic regions. However, the natural repertoire of insert antibodies is limited, because their generation depends on the transcription of the respective receptors in B cells. Integration of major pathogen receptors into antibodies would be desirable, as a virus is unlikely to escape its own receptor.
This project breaks new ground for two developments: i) B cell engineering independent of nucleases; and ii) novel insert-antibodies preventing immune escape. Therefore, the project aims to gain insights into the natural mechanism of insert acquisition and exploit it for B cell engineering. Insert-antibodies containing viral receptors shall be designed to equip B cells with antibodies of exceptional potency. Besides, our tool, specifically developed to study insertions in the antibody gene, will be explored to predict engineering outcomes and define signatures of DNA repair.
The AutoEngineering project aims to develop a vaccination strategy through an innovative approach to engineering B cells. Natural DNA breaks acquired during B cell maturation are exploited to equip antibodies with extra-domains that confer superior reactivity to pathogens. The concept builds on “design-by-nature”, namely, the discovery of LAIR1- and LILRB1-containing antibodies. Exons encoding for pathogen-receptors can be integrated in the antibody gene during B cell development and maturation, translating into insert-containing antibodies. The latter confer broad reactivity to Plasmodium in more than 5 % of individuals living in malaria-endemic regions. However, the natural repertoire of insert antibodies is limited, because their generation depends on the transcription of the respective receptors in B cells. Integration of major pathogen receptors into antibodies would be desirable, as a virus is unlikely to escape its own receptor.
This project breaks new ground for two developments: i) B cell engineering independent of nucleases; and ii) novel insert-antibodies preventing immune escape. Therefore, the project aims to gain insights into the natural mechanism of insert acquisition and exploit it for B cell engineering. Insert-antibodies containing viral receptors shall be designed to equip B cells with antibodies of exceptional potency. Besides, our tool, specifically developed to study insertions in the antibody gene, will be explored to predict engineering outcomes and define signatures of DNA repair.
In the first part of the project, we improved our methodology to determine the quantity of genomic inserts in antibody genes. Our pipeline was expanded to assess the signatures of DNA repair in antibody recombination scars. We screened healthy donor samples and paved the way to unravel the mechanism of insert acquisition. Furthermore, our methodology has been adapted to mice, and we are now in the process of studying the impact of knock-outs of potentially relevant DNA repair proteins in vitro and in vivo. Working towards a B cell vaccine, we established the basic protocol for nuclease-free B cell engineering as well as CRISPR/Cas-based engineering for comparison. We identified designs to successfully produce recombinant antibodies containing a virus receptor. We prepared in vivo experiments to be performed in the second half of the project, aiming to add insert-antibodies to the mouse repertoire through nuclease-free B cell engineering.
The aims of this project go beyond current attempts to study immune diversity, genetically modify B cells, and engineer antibodies. Expected results at the end of the project are: i) a methodology that exploits antibody recombination DNA-scars for assessing the quality of DNA-break repair; ii) the exploitation of natural DNA breaks for the engineering of B cells via exon-integration, paving the way for in vivo engineering; and iii) novel antibodies containing pathogen receptors that surpass conventional reactivity.