Antimicrobial Resistance (AMR) is perhaps the most emerging alarm in health. It already causes 700,000 deaths per year and the forecast for 2050 is 10 million deaths, more than cancer today. WHO, UN General Assembly, World Bank, G20, EU, UK and USA governments call for new antibiotics, but the pipeline for new antibiotics is not very promising. Here we propose to use new technologies to develop human monoclonal antibodies and vaccines against three AMR bacteria such as gonococcus, pneumococcus and E.coli. The technology defined as reverse vaccinology 2.0, already successful for viral infections, will be used for bacterial vaccines. Taking advantage of the recent possibility of high throughput cloning of human B cells from convalescent or vaccinated people we aim to find targets difficult or impossible to be discovered using conventional technologies. B cells will be cloned from people convalescent from target infections and from people vaccinated with Men B vaccine which confers some protection against gonococcus. The antibodies produced by the clones will be screened for their ability to bind, intoxicate or kill bacteria using a novel high-throughput microscopy platform that rapidly captures digital images and also with conventional, lower throughput technologies such as bactericidal, opsono-phagocytosis and FACS assays. The selected antibodies, will be expressed as full length and used for passive immunization in animal models and tested for protection in vivo. Finally, those antibodies that will provide the best protection in the above assays, will be used to identify the recognized antigens. Selected antigens will be expressed and tested in vaccine formulations. Fab fragments can be used to make co-crystals with the antigen and determine the crystal structure of the new antigens, for the development of structure-based antigen design. In conclusion we expect to enable development of human monoclonal antibodies and vaccines against AMR.

The COVID-19 pandemic caught the world unprepared. Vaccines and monoclonal antibodies (mAbs), developed in record time, mitigated the health and economic damages, however our reaction has always been one step behind the virus evolution, and emerging variants repeatedly escaped our interventions. The omicron variant escaped humoral immunity generated by most vaccines and mAbs by mutating immunodominant epitopes. The extremely potent mAb developed by our laboratory also lost potency against omicron. Here we propose to develop vaccines and monoclonals neutralizing existing and future variants of SARS-CoV-2 and other coronaviruses, by targeting immunologically subdominant regions which are less susceptible to antigenic variation. We will isolate mAbs from individuals who had infection and multiple vaccinations, whose repertoire is enriched in B cells encoding broadly neutralizing antibodies, to build a map of the broadly shared epitopes. Structural prediction and clustering of the immune repertoire through deep neural networks will be used to improve the breadth of coverage of the mAbs. The Monte Carlo-based sequence design of Rosetta and free energy perturbation calculations will be used to in-silico “design protein-binding proteins” and identify newly designed immunogens which can be loaded on nanoparticles and used as vaccines. This approach will provide broadly protective mAbs and vaccines proactively designed to neutralize all variants of SARS-CoV-2 and new coronaviruses that are very likely to jump from animals to humans in the future. If successful, the approach to map subdominant epitopes and use of genomic and structural information to design mAbs and vaccines targeting subdominant, broadly conserved epitopes, will pave the way to approach other pathogens with high antigenic variability such as influenza and HIV viruses, Plasmodium spp. and antibiotic resistant bacteria. This will strongly increase European competitiveness in fighting infections.

This proposal originates from recent results obtained in the course of the Advanced ERC Project “OMVac”, the scope of which is to exploit the unique adjuvanticity properties of bacterial Outer Membrane Vesicles (OMVs) for developing innovative vaccines against infectious diseases and cancer. In particular, Synthetic Biology was applied to engineer OMVs with FAT1, a tumour associated antigen expressed in most primary and metastatic colorectal cancers (CRC). Using cancer models in immunocompetent mice, immunization with FAT1-decorated OMVs inhibited subcutaneous growth of FAT1-positive CT26 cancer cells and protection correlated with an increase in tumour infiltration of CD4+/CD8+ T cells and concomitant decrease of Treg and MDSCs. These promising results prompted the submission of the present proposal which has as main objectives: 1) the demonstration that FAT1-OMV immunization can synergise with the protective activity of checkpoint inhibitors, and 2) the development of a scalable FAT1-OMV production and purification process which could allow testing FAT1-OMV/checkpoint inhibitor combination in the clinical setting.

This proposal intends to shed light on the interplay between cancer immunity and gut microbiome as a way to optimize personalized cancer vaccines and immunotherapy. The project originates from two milestone discoveries. First, to be effective cancer immunotherapies have to target CD4+/CD8+ T cell neo-epitopes, which originate from tumor mutations. Second, the gut microbiome influences the effectiveness of anti-PD-1/PD-L1 antibody immunotherapy both in animal models and in humans. We also recently showed in a mouse model that oral gavages with Bifidobacterial cocktails improved the therapeutic power of neo-epitope-based cancer vaccines. How microbiome affects anti-cancer immunity has not been fully elucidated yet and a deep understanding of the underlying mechanisms has the potential to substantially improve cancer immunotherapy. Since microbiome antigens are processed and presented by antigen-presenting cells and microbiome-induced T cells represent large fraction of the peripheral T cell repertoire, our hypothesis is that this large repertoire includes T cells which cross-react with cancer neo-epitopes (“molecular mimicry (MM)”). Depending upon the composition of gut microbiome, cross-reacting T cells can positively or negatively modulate anti-tumor immunity. To demonstrate the role of MM in cancer immunity this project intends (i) to select the cross-reactive T cell epitopes as predicted by meta-omics analysis of gut microbiome and exome/transcriptome analysis of cancer cell lines, (ii) to formulate vaccines containing different combination of cross-reactive epitopes, and (iii) to test vaccine anti-tumor activities in normal mice, gnotobiotic mice and mice with engineered microbiome. The ultimate goals are: 1) to provide new criteria for neo-epitope selection in personalized cancer vaccines, 2) to develop prognostic tools based on microbiome analysis, and 3) to define microbial species to be used as immune-potentiators in patients undergoing cancer therapy.

ISV is a promising immunotherapy strategy which envisages the direct injection of immunomostimulatory reagents into the tumor mass. ISV offers the advantage to reduce off-target toxicity and to induce potent inflammation where cancer antigens have the maximal concentration. Different substances are being exploited in ISV and they include adjuvants, lytic viruses, cytokines/chemokines, antibodies. The rationale is to recruit antigen-presenting cells, T cells, NK cells and macrophages, thus promoting a strong anti-tumor immunity. Two ISV products are available and others are expected to come in the near future. Similarly to other cancer therapies, the current ISV strategies need to be optimized to improve their efficacy. In this proposal we wish to demonstrate the strength of a novel approach of ISV optimization based on two main innovative solutions. First, we plan to couple ISV to the oral administration of Bifidobacterium, a probiotic that, in the context of the Advanced ERC Grant “Vaccibiome”, we have shown to promote the remodeling of the gut microbiome and the tumor infiltration of T cells. Second, we propose to treat tumors with engineered bacterial Outer Membrane Vesicles (OMVs). OMVs are naturally decorated with a number of immunostimulatory molecules and we have already demonstrated the effectiveness of E. coli OMVs in different tumor mouse models. We plan to exploit our proprietary OMV engineering strategies to further potentiate OMV adjuvanticity by decorating them with Flt3L, a chemokine known to recruit immune cells. The combination of Bifidobacterium and Flt3L-OMVs will create a “perfect immunological storm” at the tumor site capable of eliminating both primary and metastatic tumors. Considering its simplicity and low production costs, our Microbiome-ISV therapy has the potential to become a broadly applicable neoadjuvant therapy to be performed before surgery in a large panel of solid tumors.