Characterization of lactic acid bacteria mobilome through whole-genome sequencing and use of Streptococcus gordonii as live bacterial vector for COVID-19 vaccine development

In the first part of the thesis, whole-genome sequencing analysis contributed to enlarge current knowledge of bacterial mobilome. Sequence analysis of the Streptococcus pneumoniae clinical strain BM6001 allowed for mobilome characterization, which accounts for 15.54% of the whole genome and includes (i) the ICE Tn5253 composite; (ii) the novel IME Tn7089; (iii) the novel transposon Tn7090; (iv) 3 prophages and 2 satellite prophages; (v) 5 genomic islands; (vi) 72 insertion sequences; (vii) 69 RUPs; (viii) 153 BOX elements; and (ix) 31 SPRITEs. BM6001 clusters in a small lineage with five S. pneumoniae historical strains, but it is distantly related to the lineage due to its unique mobilome. Mobilome analysis of the probiotic Lactobacillus crispatus strain M247 allowed identification of the novel IME Tn7088 carrying an adaptation module homologous to the listeriolysin S locus of Listeria monocytogenes, and the novel siphovirus prophage phiM247. In the second part of the thesis, a S. pneumoniae nisin inducible host-vector expression system based on ICE Tn5253 was constructed and validated. The system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecalis. In all bacterial species the amount of heterologous protein produced correlated with the nisin concentration used for induction. Finally, a mucosal vaccine against SARS-CoV-2, the etiologic agent of COVID-19, was developed. The host-vector system designed for integrating heterologous genes into the Streptococcus gordonii chromosome was employed to express the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein on the bacterial surface. This system, using the plasmid-based integration strategy pSMB55, anchors the heterologous protein to the surface as a fusion with the signal sequence, N’-terminal and C’- terminal anchor domain derived from the Streptococcus pyogenes M6 surface protein. In a murine model, mucosal priming with the recombinant live vaccine vector via intranasal administration followed by subcutaneous boosting with un-adjuvanted spike protein demonstrated the efficacy of the heterologous prime-boost approach. This vaccination strategy induced a robust humoral and cellular immune response, generating spike-specific memory B cells, crucial for a quicker and more potent immune response upon re-exposure to the pathogen, as well as long-term immunity.