Neisseria gonorrhoeae (Ng) is a human pathogen that is responsible for gonorrhoea, a sexually-transmitted infectious disease that is difficult to treat due to widespread antibiotic resistance and lack of a suitable vaccine. The 4CMenB vaccine, currently licensed for the closely related bacteria Neisseria meningitidis, has been found to induce a cross-reactive immune response with observational studies indicating approximately 30-40% of cross protection against Ng. The adaptive immune response is governed by Immunoglobulin G glycoproteins (IgGs), for which their glycoforms play an important, yet not fully explored role in their function. Hence, a detailed characterisation of these IgG glycoforms is crucial to understanding the impact of this vaccination and thus provide a road map to determining vaccine responders from non-responders.
This study is the first of its kind to elucidate both the total IgG glycomic profile and subclass-specific IgG (IgG1, IgG2, IgG3, and IgG4) glycopeptides derived from 4CMenB-vaccinated patients’ sera (collected at 0m, 3m, 6m and 12m) using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) and Orbitrap Fusion mass spectrometer coupled to the nano-liquid chromatography system, respectively. The total IgG glycomic profiling by xCGE-LIF allowed for the sensitive, robust, and selective glycoprofiling of glycans, including their structural isomers and sialylation linkage (alpha2-3 or alpha 2-6), which can be separated based on their size, charge and shape with high peak capacity. This technique enabled the identification of 30 to 35 glycan structures corresponding to the total IgG glycome.
The Orbitrap Fusion LC-MS system, coupled with the stepped-HCD fragmentation strategy combining three different collision energies into a single MS2 scan, was subsequently employed for the simultaneous determination of the subclass-specific IgGs and their corresponding glycoforms. As a result, the stepped HCD spectra with high resolution ion detection contained a richer set of fragment ions that were useful for structure-specific intact IgG glycopeptide analysis and facilitated the confident assignment (BYONIC scoring >100) of each intact glycopeptide, in addition to differentiating between the IgG3/IgG4 isotypes which have the same peptide mass. In total, 14 intact IgG glycopeptides comprising fucosylated, galactosylated, bisecting and sialylated bi-antennary glycoforms were detected along with their afucosylated and agalactosylated counterparts across the various IgG subclasses.
These findings provide a valuable insight into the patient-specific adaptive immune responses to 4CMenB vaccination to facilitate translation into rapid diagnostics for clinical evaluation and vaccine response monitoring.