Gene expression analysis for the characterization of immune response to vaccines against neglected diseases

Emerging infectious diseases and neglected tropical diseases represent a wide range of diseases caused by a great variety of pathogens, which affect billions of people worldwide. Although pharmaceutical industries have focused for years on eradicating emerging diseases, recently their efforts moved to research and development of drugs to control neglected diseases. In this thesis, we employ transcriptomic analyses to evaluate the efficacy and safety of three vaccine platforms directed against diseases from both categories: the rVSV-ZEBOV-GP vaccine against Ebola virus infection, the ChAd63-KH vaccine for post-kala-azar dermal leishmaniasis, and the novel iNTS-GMMA vaccine targeting invasive non-typhoidal Salmonella infections. The rVSV-ZEBOV- GP vaccine is a live, replication competent VSV vector that expresses the glycoprotein of Zaire ebolavirus, which received a full marketing authorization by EMA in 2021, for adult administration. ChAd63-KH is a replication defective simian adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB,whose safety and immunogenicity have been already assessed in a phase 1 study. iNTS-GMMA is a novel platform containing modified S.Typhimurium and S.Enteriditis outern membrane vesicles, cyrrently under evaluation in human studies. For each vaccine, whole-blood samples were collected at different timepoints and stored at -80°C in PAXGene tubes. RNA was extracted with PAXGene blood RNA extraction QIAcube Connect system and quantified with RNA High Sensitivity Assay kit for Qubit™ 4 Fluorometer. Sequencing libraries were prepared using the Illumina® Ribo-Zero Plus Kit starting from 40 ng of total RNA. The bioinformatic analysis was conducted with the software R. For each vaccine we investigated gene expression across timepoints, performing differential gene expression (DGE) analysis and enrichment analysis, with DEseq2 and tmod packages respectively. Generally, all vaccine platforms elicited innate immune response soon after the administration with upregulated blood transcriptional modules related interferon responsiveness, dendritic cell and monocyte activation, and antigen presentation, while antibody production was detectable 7 days after injection. The transcriptional data supported the known immunogenicity of the ChAd63-KH vaccine.For iNTS-GMMA, we further explored B-cell receptor (BCR) repertoires using the MiXCR pipeline, identifying approximately 4,000 distinct clonotypes. In the rVSV-ZEBOV-GP pediatric cohort, correlations between gene expression and antibody titers were investigated to identify early transcriptional determinants of humoral immunity.