Periodic Reporting for period 1 - mABSPN (Antibody-based therapy against Streptococcus pneumoniae) Reporting period: 2018-06-15 to 2020-06-14 Summary of the context and overall objectives of the project The goal of this project was to unravel whether complement-enhancing monoclonal antibodies can be se as a new therapeutic approach against the gram-positive bacteria Streptococcus pneumoniae (pneumococcus). One third of the annual deaths occurring in the word are estimated to be due infectious diseases and, particularly, respiratory infections are responsible of the death of 4 million people every year. According to World Health Organization (WHO), pneumonia kills more children worldwide than any other disease, being S. pneumoniae the most common cause of sever pneumonia among children under 5 years old in developing countries, as well as in children and adults over 60 years in Europe and the Unites States. Pneumococcus, is indeed the main cause of community acquired pneumoniae, non-epidemic bacterial meningitis and acute otitis media, as one of the major causes of bacterial sepsis. The search of effective treatments to fight against infectious diseases has been, since many years, among the main challenge of medicine. To reduce the great impact on morbidity and mortality of S. pneumoniae world-wide, health care efforts over the past 50 years have concentrated on the development of serotype-specific vaccines. Current available vaccines target the polysaccharide capsule (CPS), which is considered the most important virulence factor of pneumococcus due its role preventing phagocytosis killing and its variability. There are more than 90 different capsule serotypes described so far that induce a type-specific immune response. Although widespread vaccination has been proven highly effective for lowering invasive pneumococcal disease (IPD), a major contributing factor for the still large burden of disease is the limited serotype coverage provided by current vaccines and the associated emergence of IPD caused by non-vaccine serotypes, however other factors are also at play. These includes, poor immunogenicity of some serotypes and the uncertainly efficacy of current immunization practice in high-risk groups. Finally, the emergence of strains with high level of antibiotic resistance is jeopardizing the use of antibiotic therapies and highlights the strong need to develop new therapeutic strategies against pneumococcal infections. In this project we study whether monoclonal antibodies against S. pneumoniae that improve the activity of our immune system can be used as therapeutic antibodies. Because complement activation is crucial for antibody-dependent killing of S. pneumoniae, development of complement-activating antibodies represents an attractive strategy for antibacterial therapy. Therefore, the main objectives of this projects include the identification of antibodies against S. pneumoniae driving potent complement activation and unravel whether monoclonal antibodies eliciting complement are effective in pneumococcal killing. At its conclusion, this project shows that the use of complement-enhancing antibodies is a promising new therapeutic approach against S. pneumoniae 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 Prevention of pneumococcal disease relies on an efficient recognition and clearance of the invading pathogen by the complement system and professional phagocytes. Antibody-mediated complement activation is crucial to eliminate S. pneumoniae infections. In addition, it is known that complement activation is more efficient when bacterium-bound IgGs form ordered hexamer structures. The non-covalent interaction of six IgG molecules through Fc-Fc domains provide an optimal docking platform for the avid binding of C1, the first complement component. In this project, we generated several capsule-specific monoclonal antibodies and studied their potential to induce complement activation on encapsulated pneumococci. Our data show that wild-type anti-capsular antibodies have a poor capacity to induce complement activation (likely because they do not form IgG hexamers). Furthermore, we show that this limitation can be overcome by introduction of a single amino acid mutations that enhances Fc-Fc domain contacts. In this way, we achieved a strikingly enhanced complement activation on several S. pneumoniae clinical isolates. Using freshly isolated human neutrophils, we confirmed that engineered antibodies potently increased phagocytosis of pneumococci by neutrophils, even achieving complete bacterial killing of some serotypes. Moreover, the most promising engineered antibody was used to evaluate its greater potential in comparison with its wild-type counterpart antibody in an in vivo infection model of pneumococcal bacteremic pneumonia. Overall, this study revealed that Fc-engineered mutants of anti-capsule polysaccharide antibodies potently increase complement-mediated killing of pneumococci, both in vitro and in vivo. A manuscript describing this work is in preparation and will be submitted soon. Finally, considering the limitations of targeting specific capsule types and, in order to identify which bacterial antigens should be targeted to elicit not only a potent bacterial killing, but also a serotype independent pneumococcal protection, the fellow has been trained to develop methods to isolate human B cells recognizing bacteria and sequence human antibodies against S. pneumoniae. 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) This project has advanced our understanding of how antibodies target encapsulated S. pneumoniae strains and provided critical insights to understand why some anti-capsular IgGs fail to induce a potent complement activation and phagocytosis of pneumococcus. Furthermore, we have demonstrated that antibodies targeting CPS can be modified to enhance their capacity effectively eliminate S. pneumoniae. By the introduction of single point mutations in the antibody’s Fc domain that enhance antibody hexamerization, we achieved a potent enhanced phagocytic killing of highly virulent and important S. pneumoniae serotypes. We have also demonstrated in an in vivo model the enhanced functionality of engineered antibodies to protect mice from bacteremic pneumonia. The development of high-throughput sequencing method of B cell receptors will enable further molecular and functional analysis of individuals antibody repertoire against S. pneumoniae. Using this information, we can strongly enhance the immune response against pneumococci. Complement-enhancing antibodies could be a promising strategy to target either vaccine escape serotypes or combat antibiotic-resistant pneumococcal infections. Overall, the results derived from this project provide a proof of concept for the use of complement-enhancing monoclonal antibodies as a new therapeutic approach against S. pneumoniae infections.