Periodic Reporting for period 1 - EarlyMicroAbs (Deciphering the antibody-microbiota axis in early life)
Berichtszeitraum: 2023-06-01 bis 2025-11-30
Overview
The term “gut microbiota” refers to the trillions of microbes, which affect human health in numerous ways. Antibodies play a central role in immune regulation of the gut microbiota. To understand how healthy individuals maintain homeostasis, it is crucial to identify which microbial species and which of their surface structures are recognized by the immune system and at what point in life this recognition begins. We aim to link BCR sequences with functional antigens (work package 1, WP1), integrate these with antibody-binding data from phage display immunoprecipitation sequencing (PhIP-Seq) libraries in healthy adults (WP2), and explore associations with early-life immunity and allergies (WP3).
WP 1:
Antibodies can target nearly any possible antigen with their sequences determining antibody structure and specificity. Despite this diversity, most individuals produce antibodies against similar bacterial and viral antigens, indicating convergence in immune recognition. Understanding whether this arises from shared BCR gene usage, rearrangements, or somatic mutations is crucial for revealing the mechanisms of adaptive immunity.
Recently, novel single-cell RNA sequencing (scRNA-seq) methods combined with tetramer staining have enabled the retrieval of paired heavy and light chain antibody sequences from individual B cells, linked to antigen specificity against up to dozen of antigens. In contrast, phage display techniques allow to study antibody epitope repertoires against thousands of antigens by combining phage-displayed antigen libraries with serum profiling.
We aim to improve these methods, allowing us to link antibody sequence to antigen specificity in a high-throughput manner at the single-cell level, supporting vaccine development and therapeutic antibody research.
WP 2:
The commensal gut microbiota plays a critical role in the development of the human adaptive immune system and are involved in both health and disease. Conversely, bacteria and viruses within the microbiome are targeted by antibodies, and the immune system orchestrates the maintenance of homeostasis.
In the gut and other mucosal sites such as the mouth or lungs, IgA antibodies dominate the immune response. In contrast, serum contains approximately 75% IgG, along with smaller amounts of IgA, IgM, IgE, and IgD. Both IgG and IgA antibodies against gut microbiota have been reported in serum. Here, we apply the PhIP-Seq technology to compare systemic (blood) antibody repertoires with those found at mucosal sites (mouth and gut) and to investigate the binding of different immunoglobulin classes. This project will provide novel data on antigen repertoires of immunoglobulin classes that have not been systematically studied before. By combining antibodies with phage-displayed synthetic libraries, PhIP-Seq technology will allow us to profile immunoglobulins against hundreds of thousands of bacterial, viral, and allergenic epitopes.
WP 3:
Already the earliest experiences and exposures in an infant’s life can profoundly influence their health well into adulthood. Factors such as the mode of delivery, breastfeeding versus formula feeding and the introduction of solid foods act as critical modulators in this process. These elements shape the infant’s exposure to a diverse array of both beneficial and pathogenic microorganisms. Such early encounters play a significant role in how the immune system develops and becomes ‘programmed’ for life. Antibodies are key players in this developmental phase. During the initial weeks, infants benefit from protective maternal antibodies, however this passive protection wanes after the first few months, requiring the infant to start producing its own immune defences as soon as possible. Throughout the first and formative years, every encounter can trigger an immune response with largely unknown consequences. A major gap in current understanding is identifying the specific microbial structures targeted by these early antibodies. Even more critically, we lack a comprehensive picture of how failures in this initial immune programming might ‘misdirect’ the immune system, potentially leading to immune-mediated conditions such as allergy and asthma.
The term “gut microbiota” refers to the trillions of microbes, which affect human health in numerous ways. Antibodies play a central role in immune regulation of the gut microbiota. To understand how healthy individuals maintain homeostasis, it is crucial to identify which microbial species and which of their surface structures are recognized by the immune system and at what point in life this recognition begins. We aim to link BCR sequences with functional antigens (work package 1, WP1), integrate these with antibody-binding data from phage display immunoprecipitation sequencing (PhIP-Seq) libraries in healthy adults (WP2), and explore associations with early-life immunity and allergies (WP3).
WP 1:
Antibodies can target nearly any possible antigen with their sequences determining antibody structure and specificity. Despite this diversity, most individuals produce antibodies against similar bacterial and viral antigens, indicating convergence in immune recognition. Understanding whether this arises from shared BCR gene usage, rearrangements, or somatic mutations is crucial for revealing the mechanisms of adaptive immunity.
Recently, novel single-cell RNA sequencing (scRNA-seq) methods combined with tetramer staining have enabled the retrieval of paired heavy and light chain antibody sequences from individual B cells, linked to antigen specificity against up to dozen of antigens. In contrast, phage display techniques allow to study antibody epitope repertoires against thousands of antigens by combining phage-displayed antigen libraries with serum profiling.
We aim to improve these methods, allowing us to link antibody sequence to antigen specificity in a high-throughput manner at the single-cell level, supporting vaccine development and therapeutic antibody research.
WP 2:
The commensal gut microbiota plays a critical role in the development of the human adaptive immune system and are involved in both health and disease. Conversely, bacteria and viruses within the microbiome are targeted by antibodies, and the immune system orchestrates the maintenance of homeostasis.
In the gut and other mucosal sites such as the mouth or lungs, IgA antibodies dominate the immune response. In contrast, serum contains approximately 75% IgG, along with smaller amounts of IgA, IgM, IgE, and IgD. Both IgG and IgA antibodies against gut microbiota have been reported in serum. Here, we apply the PhIP-Seq technology to compare systemic (blood) antibody repertoires with those found at mucosal sites (mouth and gut) and to investigate the binding of different immunoglobulin classes. This project will provide novel data on antigen repertoires of immunoglobulin classes that have not been systematically studied before. By combining antibodies with phage-displayed synthetic libraries, PhIP-Seq technology will allow us to profile immunoglobulins against hundreds of thousands of bacterial, viral, and allergenic epitopes.
WP 3:
Already the earliest experiences and exposures in an infant’s life can profoundly influence their health well into adulthood. Factors such as the mode of delivery, breastfeeding versus formula feeding and the introduction of solid foods act as critical modulators in this process. These elements shape the infant’s exposure to a diverse array of both beneficial and pathogenic microorganisms. Such early encounters play a significant role in how the immune system develops and becomes ‘programmed’ for life. Antibodies are key players in this developmental phase. During the initial weeks, infants benefit from protective maternal antibodies, however this passive protection wanes after the first few months, requiring the infant to start producing its own immune defences as soon as possible. Throughout the first and formative years, every encounter can trigger an immune response with largely unknown consequences. A major gap in current understanding is identifying the specific microbial structures targeted by these early antibodies. Even more critically, we lack a comprehensive picture of how failures in this initial immune programming might ‘misdirect’ the immune system, potentially leading to immune-mediated conditions such as allergy and asthma.
For WP1, studying the antibody response to microbiota antigens requires the production of thousands of different antigens in parallel. In the initial experiments, we tested the usability of phage-displayed antigens for antibody-antigen selection.
In WP2, we have adapted the PhIP-seq assay to study IgA, which plays a key role at mucosal sites. As an initial step, we conducted a control experiment to determine the optimal phage-to-antibody ratio that yields the highest number of bound peptides. Moreover, we are currently refining the workflow to mitigate technical issues and improve reproducibility, while also enhancing data analysis and statistical validation.
In WP3, we aim to identify which early immune exposures shape a child’s antibody repertoire and ultimately influence health outcomes. Currently, the precise targets of these antibodies remain still unknown, as do the long-term health implications of early "misdirection" in immune system education. By systematically identifying the targets of these antibodies in blood and breast milk using PhIP-seq, we are gaining important new insights into the complex relationship between the human immune system development and microorganisms.
In WP2, we have adapted the PhIP-seq assay to study IgA, which plays a key role at mucosal sites. As an initial step, we conducted a control experiment to determine the optimal phage-to-antibody ratio that yields the highest number of bound peptides. Moreover, we are currently refining the workflow to mitigate technical issues and improve reproducibility, while also enhancing data analysis and statistical validation.
In WP3, we aim to identify which early immune exposures shape a child’s antibody repertoire and ultimately influence health outcomes. Currently, the precise targets of these antibodies remain still unknown, as do the long-term health implications of early "misdirection" in immune system education. By systematically identifying the targets of these antibodies in blood and breast milk using PhIP-seq, we are gaining important new insights into the complex relationship between the human immune system development and microorganisms.
Regarding WP1, standard methods for antibody research are typically limited to studying only up to ten proteins in parallel. Several computational methods have recently been developed to predict antibody structures, however, structural data for training machine learning models remains limited. Developing a method to link antibody sequences to antigen specificity would enable the collection of data on antibodies targeting multiple antigens in parallel, supporting vaccine development and therapeutic antibody research.
In WP2, the obtained results will shed light on the human adaptive immune response, as each antibody isotype plays a distinct role and is involved in different processes including infection, autoimmunity and allergy. Most studies nowadays focus on IgG, while other classes remain largely unexplored. This project will fill this gap by profiling antigen repertoires across antibody types.
Finally in WP3, by profiling immune system development throughout this key window, we aim to shed light on the maturation of this essential branch of the adaptive immune system in early life. Furthermore, by incorporating breast milk in our analysis, we will be able to demonstrate which specific maternal antibodies continue to protect the child against pathogens even after birth in a depth never seen before. Additionally, by including a cohort of allergic children, and comparing immune profiles with healthy ones, we will also be able to elucidate associations between dysregulation of the microbiota-immune axis impacting human health.
In WP2, the obtained results will shed light on the human adaptive immune response, as each antibody isotype plays a distinct role and is involved in different processes including infection, autoimmunity and allergy. Most studies nowadays focus on IgG, while other classes remain largely unexplored. This project will fill this gap by profiling antigen repertoires across antibody types.
Finally in WP3, by profiling immune system development throughout this key window, we aim to shed light on the maturation of this essential branch of the adaptive immune system in early life. Furthermore, by incorporating breast milk in our analysis, we will be able to demonstrate which specific maternal antibodies continue to protect the child against pathogens even after birth in a depth never seen before. Additionally, by including a cohort of allergic children, and comparing immune profiles with healthy ones, we will also be able to elucidate associations between dysregulation of the microbiota-immune axis impacting human health.