BRCA1 and BRCA2 genes and associated factors are often mutated in breast and ovarian cancers. BRCA proteins function together with the RAD51 recombinase in DNA double-strand break repair but have separate roles in the metabolism of challenged replication forks. The EU-funded BRCA INSIGHTS project will use a biochemical approach to study the precise functions of these proteins. The objective is to define how BRCA proteins are involved in the initial steps of DNA double-strand break repair, including nucleolytic processing of DNA breaks, RAD51 loading on ssDNA and invasion of homologous DNA template. The study aims to determine the specific separate functions of RAD51, RAD52 and BRCA1/2 complexes in fork remodelling, protection and restart, which prevent pathological degradation of nascent DNA under replication stress.

The overall goal of this project is to understand the molecular mechanisms that lead to the generation of potent and broadly neutralizing antibodies against medically relevant pathogens, and to identify the factors that limit their production in response to infection or vaccination with current vaccines. We will use high-throughput cellular screens to isolate from immune donors clonally related antibodies to different sites of influenza hemagglutinin, which will be fully characterized and sequenced in order to reconstruct their developmental pathways. Using this approach, we will ask fundamental questions with regards to the role of somatic mutations in affinity maturation and intraclonal diversification, which in some cases may lead to the generation of autoantibodies. We will combine crystallography and long time-scale molecular dynamics simulation to understand how mutations can increase affinity and broaden antibody specificity. By mapping the B and T cell response to all sites and conformations of influenza hemagglutinin, we will uncover the factors, such as insufficient T cell help or the instability of the pre-fusion hemagglutinin, that may limit the generation of broadly neutralizing antibodies. We will also perform a broad analysis of the antibody response to erythrocytes infected by P. falciparum to identify conserved epitopes on the parasite and to unravel the role of an enigmatic V gene that appears to be involved in response to bloodstage parasites. The hypotheses tested are strongly supported by preliminary observations from our own laboratory. While these studies will contribute to our understanding of B cell biology, the results obtained will also have translational implications for the development of potent and broad-spectrum antibodies, for the definition of correlates of protection, and for improving vaccine design.

The overall goal of this project is to test, in the human system, several hypotheses related to the role of T helper (Th) subsets in immunity and immunopathology. Using an experimental approach that takes advantage of high throughput culture methods and combines the ex vivo analysis of memory T cells with the in vitro priming of naïve T cells, we will dissect the Th cell response to pathogens, allergens, and self-antigens, in terms of antigen-specificity, tissue tropism, and cytokine production.

We will identify signals and pathways triggered by microbes and allergens that prime polarized Th1, Th2, Th17 and Th22 cells as well as T cells with hybrid phenotypes producing, for instance, IFN-γ and IL-17 or IL-4 and IL-22. We will also address fundamental questions related to tolerance and autoimmunity by measuring frequency and distribution of self-reactive T cells in healthy donors and patients. The analysis of the response to microbes and allergens will address the possibility that different antigens, depending on abundance or location, may drive divergent Th cell responses, thus shedding light on the mechanisms of polarization and immunodominance in vivo. In pilot studies the project will also translate basic findings to the clinical setting, linking polarized Th responses to disease state and severity. Finally, using lentiviral-based approaches for gene silencing and overexpression, we will perform mechanistic studies to understand how environmental factors modulate in Th cells the production of pro- and anti-inflammatory cytokines. The hypotheses tested are strongly supported by preliminary observations from our own laboratory as well as from the biomedical literature. We expect that these studies will significantly expand our basic understanding of T cell biology and will have translational implications for the definition of correlates of protection or disease activity and for the design of improved vaccination and therapeutic strategies.

Immunological memory confers long term protection against pathogens and is the basis of successful vaccination. Following antigenic stimulation long lived plasma cells and memory B cells are maintained for a lifetime, conferring immediate protection and enhanced responsiveness to the eliciting antigen. However, in the case of variable pathogens such as influenza virus, B cell memory is only partially effective, depending on the extent of similarity between the preceding and the new viruses. The B cell response is dominated by serotype-specific antibodies and hetero-subtypic antibodies capable of neutralizing several serotypes appear to be extremely rare. Understanding the basis of broadly neutralizing antibody responses is a critical aspect for the development of more effective vaccines. In this project, we will explore the specificity and dynamics of human antibody responses to influenza virus by using newly developed technological platforms to culture human B cells and plasma cells and to analyze the repertoire of human naïve and memory T cells. High throughput functional screenings, structural analysis and testing in animal models will provide a thorough characterization of the human immune response. The B cell and T cell analysis aims at understanding fundamental aspects of the immune response such as: the selection and diversification of memory B cells; the individual variability of the antibody response, the mechanisms of T-B cooperation and the consequences of the original antigenic sin and of aging on the immune response. This analysis will be complemented by a translational approach whereby broadly neutralizing human monoclonal antibodies will be developed and used: i) for passive vaccination against highly variable viruses; ii) for vaccine design through the identification and production of recombinant antigens to be used as effective vaccines; and iii) for active vaccination in order to facilitate T cell priming and jump start the immune responses.