Periodic Reporting for period 3 - HIV B Cell Function (Understanding HIV-specific B cell function and viral immunogenicity)
Reporting period: 2021-06-01 to 2022-11-30
Understanding HIV-specific B cell function and viral immunogenicity
This project aims to address two key interrelated research questions. Firstly, why do broadly neutralizing antibodies only develop in certain HIV-positive individuals? Secondly, do non-neutralizing antibodies limit the development of broadly neutralizing antibodies in HIV infection and immunization?
Previous work in the field of HIV broadly neutralizing antibodies has shown that their development is a rare event both at the population level and within the B cell repertoire of an individual with broadly neutralizing sera. It has been noted that some virological parameters and peripheral T cell phenotypes are associated with increased broadly neutralizing antibody development, but none of these has been found to be predictive. This is most probably due to widespread immune dysregulation in untreated HIV infection where broadly neutralizing antibodies develop, and due to the rarity of broadly neutralizing antibody B cells within the total repertoire. Therefore, we are using a single cell antigen-specific approach to address the question of why broadly neutralizing antibodies only develop in certain HIV-positive individuals. Moreover, while prior work in the HIV broadly neutralizing antibody field has focused on isolation of antibodies with remarkable breadth, little attention has been given to non-neutralizing or strain-specific antibodies from these individuals. Consequently, differential data between broadly neutralizing antibodies and non-neutralizing antibodies is normally generated using a handful of non-neutralizing antibodies from the 1990/2000s. Simultaneously, there is a widely held view that non-neutralizing antibodies can restrict the development of broadly neutralizing antibodies by competing for antigen without substantial data to support this. In this project we are directly comparing antibodies of different functionality raised within the same individual to decipher whether other epitopes act as decoys to distract the immune system from making broadly neutralizing antibodies. In addition, basic immunology research into B cell activation has shown that B cells encoding antibodies of higher affinity are more readily activated and so are the ultimate output of affinity maturation. However, these studies have mainly been performed in mice and using simplistic model antigens, so how this relates to complicated HIV antigens with multiple epitopes is unclear. Hence, in this project we will examine the relationship between antibody affinity, epitope specificity and B cell activation in the context of HIV.
Thus, the key objectives of this ERC starting grant are to (1) investigate the immunophenotypes of B cells associated with broadly neutralizing antibodies; (2) create B cell lines from individual patients to study broadly neutralizing antibodies, strain-specific neutralizing antibodies and non-neutralizing antibodies induced by the same HIV exposure; and (3) compare B-cell receptor (BCR) activation of broadly neutralizing antibodies, strain-specific neutralizing antibodies and non-neutralizing antibodies by HIV envelope following infection and vaccination. To achieve this, we are using a combination of cells from HIV-positive individuals, cell lines, pseudotype neutralization assays, viral mutagenesis, binding competition analysis, recombinant monoclonal antibody and protein purification, single-cell flow cytometry, and RNA sequencing. The concepts we discover in our system will also be relevant to antibody responses against other highly variable viral pathogens and the regulation of B cell responses more widely.
This project aims to address two key interrelated research questions. Firstly, why do broadly neutralizing antibodies only develop in certain HIV-positive individuals? Secondly, do non-neutralizing antibodies limit the development of broadly neutralizing antibodies in HIV infection and immunization?
Previous work in the field of HIV broadly neutralizing antibodies has shown that their development is a rare event both at the population level and within the B cell repertoire of an individual with broadly neutralizing sera. It has been noted that some virological parameters and peripheral T cell phenotypes are associated with increased broadly neutralizing antibody development, but none of these has been found to be predictive. This is most probably due to widespread immune dysregulation in untreated HIV infection where broadly neutralizing antibodies develop, and due to the rarity of broadly neutralizing antibody B cells within the total repertoire. Therefore, we are using a single cell antigen-specific approach to address the question of why broadly neutralizing antibodies only develop in certain HIV-positive individuals. Moreover, while prior work in the HIV broadly neutralizing antibody field has focused on isolation of antibodies with remarkable breadth, little attention has been given to non-neutralizing or strain-specific antibodies from these individuals. Consequently, differential data between broadly neutralizing antibodies and non-neutralizing antibodies is normally generated using a handful of non-neutralizing antibodies from the 1990/2000s. Simultaneously, there is a widely held view that non-neutralizing antibodies can restrict the development of broadly neutralizing antibodies by competing for antigen without substantial data to support this. In this project we are directly comparing antibodies of different functionality raised within the same individual to decipher whether other epitopes act as decoys to distract the immune system from making broadly neutralizing antibodies. In addition, basic immunology research into B cell activation has shown that B cells encoding antibodies of higher affinity are more readily activated and so are the ultimate output of affinity maturation. However, these studies have mainly been performed in mice and using simplistic model antigens, so how this relates to complicated HIV antigens with multiple epitopes is unclear. Hence, in this project we will examine the relationship between antibody affinity, epitope specificity and B cell activation in the context of HIV.
Thus, the key objectives of this ERC starting grant are to (1) investigate the immunophenotypes of B cells associated with broadly neutralizing antibodies; (2) create B cell lines from individual patients to study broadly neutralizing antibodies, strain-specific neutralizing antibodies and non-neutralizing antibodies induced by the same HIV exposure; and (3) compare B-cell receptor (BCR) activation of broadly neutralizing antibodies, strain-specific neutralizing antibodies and non-neutralizing antibodies by HIV envelope following infection and vaccination. To achieve this, we are using a combination of cells from HIV-positive individuals, cell lines, pseudotype neutralization assays, viral mutagenesis, binding competition analysis, recombinant monoclonal antibody and protein purification, single-cell flow cytometry, and RNA sequencing. The concepts we discover in our system will also be relevant to antibody responses against other highly variable viral pathogens and the regulation of B cell responses more widely.
We have access to samples from HIV-positive individuals who were not on treatment and so are likely to have broadly neutralizing antibodies. Our goal was to identify the samples with the highest activity and to map the epitopes underlying their neutralization breadth. Our first step was to screen samples for breadth by assessing serum activity in a luciferase-based pseudotype neutralization assay against viral strains selected to represent global HIV diversity. The data from these screens revealed which individuals had neutralization breadth. This data also allowed us to streamline the methods used to epitope map the sera (viral mutagenesis and addition of decoy proteins to neutralization assay). This has enabled us to generate recombinant HIV antigen-baits for samples with neutralization breadth. These will now be used in single cell flow cytometry and downstream RNA sequencing, and for antibody characterization. Furthermore, we have successfully characterised broadly neutralizing antibodies, strain-specific and non-neutralizing antibodies from the same HIV exposure. To date, our work suggests that previously identified non-neutralizing and strain-specific antibody epitopes are also found alongside broadly neutralizing antibodies but that these functionally inferior antibodies do not compete for the same epitopes, nor have higher affinity for antigen despite being more frequent than broadly neutralizing antibodies. We are expanding these findings by analyzing more HIV infected individuals and have established a study of HIV patients receiving vaccinations to compare to B cell responses to those during infection.
This project has advanced beyond the state of the art in two main ways. Firstly, because we have established a collaboration with Dr James E Voss (The Scripps Research Institute) to use Crispr-cas9 to engineer the IgG locus of B cells so that they encode the sets of antibodies identified in Objective 2. This will enable us to undertake ground-breaking work on the relationship between antibody affinity and epitope specificity on B cell activation. This technology allows these studies to be performed in B cell lines with a uniform background such that heterogeneity within B cells does not obfuscate the results. This additional data will further enhance our ERC work and our understanding of why some epitopes are more readily recognized upon infection and immunization. Secondly, we have adapted and optimized a single-cell RNA sequencing method for use with rare populations of quiescent cells. This will allow us to draw the most impactful conclusions from our single-cell studies of HIV-specific B cells rather than being limited to cell surface marker expression and pre-defined transcriptomic array data. We anticipate being able to define the phenotype of broadly neutralizing antibody producing B cells in unparalleled detail. That this technique is applicable to other antigen-specific B cells in both immunization and vaccine responses will enhance the importance, impact and dissemination of our work. Overall, we plan to consolidate and extend our current impactful findings and prepare our work for publication and dissemination.