Towards unveiling virulence dynamics and effective prevention of neonatal Group B Streptococcus infections: A comprehensive investigation

Abstract

Background: Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a bacterium commonly found as an opportunistic commensal in the gastrointestinal and vaginal tracts of a significant number of healthy women. However, it can transition into an invasive pathogen capable of causing severe diseases, particularly in neonates born to GBS colonised mothers. GBS infection in neonates presents in two distinct clinical manifestations: early-onset GBS (EOGBS) disease, occurring within the first week of life, and late-onset GBS (LOGBS) disease, manifesting between 7 and 89 days after birth. The majority of invasive GBS neonatal diseases are attributed to serotype III, specifically belonging to the hypervirulent lineage ST17. The objective of this thesis was to investigate the mechanisms underlying differences in virulence and disease causation between GBS III ST17 vs. ST19 and EOGBS vs. LOGBS strains using in vitro and in vivo models. Additionally, the study aimed to assess the efficacy of a novel GBS protein-based vaccine in protection against infection with the GBS III ST17 strain. It was hypothesized that GBS strains of different sequence types undergo dynamic changes in their genome and phenotypic alterations in response to environmental tissue conditions, potentially impacting their capacity to cause invasive infections. These adaptive changes in various GBS sequence types may provide valuable insights for developing novel preventive and therapeutic approaches. Another hypothesis proposed that vaccination with the novel GBS protein-based vaccine could stimulate the production of high levels of protective antibodies, offering defense against invasive GBS disease. The development of an effective GBS protein-based vaccine targeting pregnant women holds the potential to significantly reduce the burden of GBS disease and save thousands of neonates from mortality annually.

Methods: GBS serotype III clinical isolates obtained from early-onset and late-onset cases, categorised into either ST17 or ST19, were included in the study. In vitro phenotypic investigations were conducted, comprising adhesion and invasion assays using both the vaginal epithelial cell line (VK2) and the human brain endothelial cell line (HBECs), along with capsule thickness assays. A mouse model of both vaginal colonisation and sepsis was used to examine the in vivo colonisation and virulence of these strains. Furthermore, comparative genomic analyses were performed on whole-genome sequenced (WGS) EOGBS and LOGBS strains. In the vaccine candidate immunisation experiments, antibodies in mouse sera were measured using IgG ELISA, and the functionality of the antibodies was assessed via opsonophagocytic killing assay (OPKA).

Results: The data indicate distinct differences between ST17 and ST19 isolates. In addition, a detailed analysis of EOGBS and LOGBS strains within ST17 and ST19 exhibits increased adherence and invasive capacities of EOGBS compared to LOGBS when interacting with HBECs. Furthermore, EOGBS strains also demonstrate reduced capsule thickness in contrast to LOGBS. In the mouse model of sepsis, a lower survival rate and higher bacterial load in the brains of mice infected with EOGBS strains were observed compared to those infected with LOGBS. Comparative genomic analysis showed differences in virulence-associated genes between EOGBS and LOGBS strains. Moreover, the results of the vaccination studies revealed that immunising mice with the novel protein vaccine candidate induced functional protective antibodies that protected mice against invasive GBS III ST17 infection.

Conclusion: The in vitro and in vivo characterization of GBS, along with the comparative genomic analysis conducted in this thesis, offer valuable insights into the virulence mechanisms of EOGBS and LOGBS strains. In addition, the promising results obtained from the preclinical development of a novel protein-based vaccine candidate prompt further investigation of this vaccine in murine models of vaginal colonisation and vertical transmission as well as human studies.