PROposing Action to ConTrol and Impede betacoronaVirus Emergencies

Project Information

PROACTIVE

Grant agreement ID: 101098201

DOI

10.3030/101098201

EC signature date 31 October 2023

Start date 1 November 2023

End date 31 October 2028

Funded under

• European Research Council (ERC)

Total cost

€ 2 498 750,00

EU contribution

€ 2 498 750,00

Coordinated by

FONDAZIONE TOSCANA LIFE SCIENCES
Italy

Periodic Reporting for period 1 - PROACTIVE (PROposing Action to ConTrol and Impede betacoronaVirus Emergencies)

Reporting period: 2023-11-01 to 2026-04-30

Summary of the context and overall objectives of the project

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 SARSCoV-
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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The PROACTIVE project focused on the execution of antibody discovery and early functional characterization activities as defined in the Description of Action. Peripheral blood mononuclear cells (PBMCs) were obtained from selected donor cohorts, including SARS-CoV-1 survivors vaccinated and/or infected with SARS-CoV-2, vaccinated individuals, and individuals with hybrid and super-hybrid immunity. These cohorts were selected to analyze for B cell repertoires encoding broadly neutralizing antibodies.
PBMCs were processed using single-cell sorting to isolate antigen-specific memory B cells, which were cultured to produce monoclonal antibodies (mAbs). Extensive functional screening was performed to assess neutralization activity against multiple SARS-CoV-2 variants and other coronaviruses. This effort resulted in the isolation of several hundred neutralizing mAbs.
Through iterative selection and characterization, a subset of antibodies was identified that met the key project criteria: high neutralization potency, broad activity across all tested SARS-CoV-2 variants and SARS-CoV-1, and recognition of subdominant conserved epitopes. These findings demonstrate the successful completion of Work Packages 1 and 2.
In parallel, initial activities foreseen under Work Package 3 were initiated by prioritizing selected antibodies for downstream analyses aimed at assessing viral escape under antibody-mediated selective pressure. The technical and scientific achievements of reporting period 2 establish a robust foundation for subsequent structural, computational, and in vivo validation phases of the project.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

PROACTIVE generated results that go beyond the current state of the art in coronavirus immunology and antibody discovery. While most existing therapeutic monoclonal antibodies (mAbs) and vaccine approaches target immunodominant and highly variable regions of the SARS-CoV-2 spike protein, PROACTIVE successfully identified a class of human monoclonal antibodies recognizing conserved, subdominant epitopes shared across sarbecoviruses. These epitopes are under lower selective pressure and therefore represent more stable and durable targets for both therapy and prevention.
By exploiting rare immune repertoires derived from SARS-CoV-1 survivors subsequently exposed to SARS-CoV-2 antigens, as well as from individuals with hybrid and super-hybrid immunity, the project demonstrated that broad neutralization breadth can be naturally achieved in humans and systematically isolated. The identified antibodies exhibit very high potency and neutralize all tested SARS-CoV-2 variants as well as SARS-CoV-1, a level of breadth that is not achieved by currently approved mAb therapies.
Beyond antibody discovery, the generation of large, high-quality datasets linking antibody sequence, germline usage, functional breadth, and immune history provides an unprecedented resource to understand how repeated heterologous antigenic exposures shape the human immune repertoire. These results open the way to rational, data-driven design of antibody cocktails and vaccine antigens targeting conserved coronavirus vulnerabilities, thereby advancing pandemic preparedness strategies beyond variant-reactive approaches toward truly proactive and mutation-resistant interventions.