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Final Report Summary - DISCO (A multidisciplinary Doctoral Industrial School on novel preventive strategies against E. Coli infections)

DISCo (A multidisciplinary Doctoral Industrial School on novel preventive strategies against E. Coli infections) was a doctoral school that originates from the common interest of GlaxoSmithKline Vaccines Srl (GSKVACSRL) and the French National Institute for Agronomical Research (INRA) in preventing E. coli infections. Both institutes have a strong background in microbial pathogenesis, supported by a number of ongoing research programs aimed at combating human infectious diseases. In particular, GSKVACSRL, by using a high-throughput in silico genomic approach, named “Reverse Vaccinology”, has recently delivered on the market Bexsero® that have been introduced in vaccination schedule in different countries for the prevention of meningococcal diseases. Starting from this successful achievement, this project was aimed to apply both bioinformatic and experimental approaches (including proteomic, functional and immunological studies) to identify bacterial surface associated determinants able to induce cross-protective antibodies against different E.coli pathotypes. GSKVACSRL had already successfully exploited subtractive Reverse Vaccinology approach to identify novel immunogenic antigens capable of inducing protection against extraintestinal pathogenic E. coli species in a mouse model of sepsis. This doctoral program aimed to extend such analysis to intestinal E.coli pathotypes for which a broadly protective vaccine is still not available, but it would aid in the control of morbidity and mortality associated to diarrheal disease (especially in developing countries). Moreover, GSKVACSRL has provided expertise in “in vitro” and “in vivo” evaluation of adjuvants and on immunization regimens, while INRA has provided the knowledge in studying the protein composition of the cell surface and of different cellular compartments by applying Mass Spectrometry, as well as the access to an EHEC animal model of colonization and the knowledge on the interplay between pathogens and commensal flora.
The Reverse Vaccinology approach was successful in identifying new promising vaccine candidates. By scanning a prototype Enterohemorrhagic (EHEC O157:H7 EDL933 strain) complete annotated genome for the search of potentially surface exposed determinants, 24 novel protein candidates were identified as novel putative vaccine antigens. The presence and conservation of the selected antigens was evaluated by a BLAST analysis on 47 completed E. coli genomes belonging to different E. coli pathotypes. The 24 proteins resulted to be present in Intestinal Pathogenic E. coli, with 5 out of the 24 proteins also present in Extraintestinal E. coli; and 18 of them also present in Enteropathogenic E.coli pathotype genome. The results suggest that there is not an exclusive antigen for all the pathotypes; and that multiple antigens may be needed to cover a wide range of pathogenic strains. The genes encoding for the selected antigens were amplified, expressed and purified as recombinant proteins in E.coli, testing their production levels and solubility. Twenty-four purified antigens were used to immunize mice, using alum hydroxide as adjuvant. Three doses were administrated to mice at day 1, 21 and 35. The final bleed to obtain the mice sera was performed at day 49. Sera were analyzed in many different immunoassays. Three antigens resulted to be the most promising in terms of expression level and immunogenicity. The three antigens were expressed in an E.coli strain engineered to overproduce outer membrane vesicles, also known as generalized modules for membrane antigens (GMMA). GMMA containing each of these three antigens were isolated and tested for protection in an EHEC animal model. One of the three antigens, when expressed in GMMA, induced antibodies able to reduce bacterial colonization in colon and caecum tissues, revealing its potential as potential vaccine antigens against EHEC infection.
The search of new antigens was also complemented by an experimental approach based on the proteosurfaceome analysis of EHEC O157:H7strain grown in different conditions. This approach was based on protein labeling and shotgun proteomics coupled to high-resolution mass spectrometry. The study begun with the development of a labeling protocol based on biotin reagents with different molecular size. This method allowed for a sophisticated and accurate way to differentially explore the cell envelope of EHEC O157:H7 grown in vitro in different metabolic environments as well as of bacteria recovered from the mouse ileal environment. Selected proteins were further characterized for their role in adhesion by the generation of EHEC knock-out strains and the study of their ability to adhere to purified ECM molecules or to a HT29 epithelial cell model. OmpC, OmpA and OmpX were identified as the adhesins responsible for binding to collagen and to HT29 cells, suggesting that they may play an important role in colonization and highlighting their potential as vaccine antigens, since antibodies raised against these adhesins could reduce adhesion and supposedly colonization.
While the search of new antigens by genomic and proteomic approaches was ongoing, the immunological evaluation in mice of different immunization regimens and routes was pursued, using an ExPEC antigen (SslE: Secreted and surface-associated lipoprotein from Escherichia coli) as model. Intranasal, sublingual and intramuscolar routes of immunization were compared to evaluate the immune response induced and to identify the most optimal for the induction of a robust immune response. Priming and boosting experiments were conducted using the same route or different routes of immunization. This analysis has allowed to identify the best protocol of immunization based on intranasal route as primary and intramuscular immunizations as boost. This mixed regimen was the most efficient to generate a high immune response, both at systemic and mucosal level. Furthermore, immunization with the model antigen although inducing a strong immune response, did not cause alteration on the intestinal microbiota. This represents a very important finding, demonstrating that the induction of an immune response at the level of the gut mucosa do not alter the balance between the different commensal species.
Finally, to define the best adjuvant to be used in future vaccine formulations, immunological properties of two different adjuvants (SMIPs) acting as Toll-like Receptor 7 agonists were analysed in vitro and in vivo with the aim to dissect their mechanisms of action. The effect of the two adjuvants on macrophage polarization was tested by measuring the level of mRNA of different mediators in Raw cells, in vitro. Moreover, FACS analysis of purified murine peritoneal macrophages treated in vitro with GSK adjuvants coherently correlated with the gene-expression analysis performed both in the cell line and in the peritoneal macrophages. After all the in vitro evidences accumulated, SMIPs were injected in mice, either in their soluble forms or formulated with a constant dose of Alum, and preliminary data of their effect on the immunogenicity of SslE, selected as model antigen, have been obtained.
Within the DISCo project, results with the potential to be translated into patent applications have been achieved. The new selected antigens could represent important components for the design of novel vaccines able to prevent E.coli infections. This could potentially have a strong impact on Public Health and also reduce costs compared to current medical treatments.
Aiming to improve awareness of general public of the importance of preventive strategies against E. coli infections as an important way to improve quality of life and reduce the risk of life-threatening outbreaks from novel and uncontrolled pathogenic strains, the students have been involved in several outreach activities as well as in the creation of the DISCo website (
Training in both scientific and transferable skills received by the young scientists recruited in DISCo, will allow them to improve their career perspectives. The exposition of the fellows to the most innovative ideas and techniques in the growing field of vaccinology and in particular the reverse vaccinology approach, will educate the next generation of academic or industrial research leaders.

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