A bead-based multiplex assay covering all coronaviruses pathogenic for humans for sensitive and specific surveillance of SARS-CoV-2 humoral immunity.

Serological assays measuring antibodies against SARS-CoV-2 are key to describe the epidemiology, pathobiology or induction of immunity after infection or vaccination. Of those, multiplex assays targeting multiple antigens are especially helpful as closely related coronaviruses or other antigens can be analysed simultaneously from small sample volumes, hereby shedding light on patterns in the immune response that would otherwise remain undetected. We established a bead-based 17-plex assay detecting antibodies targeting antigens from all coronaviruses pathogenic for humans: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV strains 229E, OC43, HKU1, and NL63. The assay was validated against five commercial serological immunoassays, a commercial surrogate virus neutralisation test, and a virus neutralisation assay, all targeting SARS-CoV-2. It was found to be highly versatile as shown by antibody detection from both serum and dried blot spots and as shown in three case studies. First, we followed seroconversion for all four endemic HCoV strains and SARS-CoV-2 in an outbreak study in day-care centres for children. Second, we were able to link a more severe clinical course to a stronger IgG response with this 17-plex-assay, which was IgG1 and IgG3 dominated. Finally, our assay was able to discriminate recent from previous SARS-CoV-2 infections by calculating the IgG/IgM ratio on the N antigen targeting antibodies. In conclusion, due to the comprehensive method comparison, thorough validation, and the proven versatility, our multiplex assay is a valuable tool for studies on coronavirus serology.

Toxin exposure and HLA alleles determine serum antibody binding to toxic shock syndrome toxin 1 (TSST-1) of Staphylococcus aureus.

Life-threatening toxic shock syndrome is often caused by the superantigen toxic shock syndrome toxin-1 (TSST-1) produced by Staphylococcus aureus. A well-known risk factor is the lack of neutralizing antibodies. To identify determinants of the anti-TSST-1 antibody response, we examined 976 participants of the German population-based epidemiological Study of Health in Pomerania (SHIP-TREND-0). We measured anti-TSST-1 antibody levels, analyzed the colonization with TSST-1-encoding S. aureus strains, and performed a genome-wide association analysis of genetic risk factors. TSST-1-specific serum IgG levels varied over a range of 4.2 logs and were elevated by a factor of 12.3 upon nasal colonization with TSST-1-encoding S. aureus. Moreover, the anti-TSST-1 antibody levels were strongly associated with HLA class II gene loci. HLA-DRB1*03:01 and HLA-DQB1*02:01 were positively, and HLA-DRB1*01:01 as well as HLA-DQB1*05:01 negatively associated with the anti-TSST-1 antibody levels. Thus, both toxin exposure and HLA alleles affect the human antibody response to TSST-1.

A Comprehensive View on the Human Antibody Repertoire Against Staphylococcus aureus Antigens in the General Population.

Our goal was to provide a comprehensive overview of the antibody response to Staphylococcus aureus antigens in the general population as a basis for defining disease-specific profiles and diagnostic signatures. We tested the specific IgG and IgA responses to 79 staphylococcal antigens in 996 individuals from the population-based Study of Health in Pomerania. Using a dilution-based multiplex suspension array, we extended the dynamic range of specific antibody detection to seven orders of magnitude, allowing the precise quantification of high and low abundant antibody specificities in the same sample. The observed IgG and IgA antibody responses were highly heterogeneous with differences between individuals as well as between bacterial antigens that spanned several orders of magnitude. Some antigens elicited significantly more IgG than IgA and vice versa. We confirmed a strong influence of colonization on the antibody response and quantified the influence of sex, smoking, age, body mass index, and serum glucose on anti-staphylococcal IgG and IgA. However, all host parameters tested explain only a small part of the extensive variability in individual response to the different antigens of S. aureus.

Technical report: xMAPr - High-dynamic-range (HDR) quantification of antigen-specific antibody binding.

Here, we demonstrate a high-dynamic-range quantification of antibody binding to single antigens in a multiplexed suspension bead array format. Using a dilution-based approach and the newly developed data analysis tool the quantitative dynamic range is increased by three orders of magnitude in the selected samples. The strong increase in dynamic range results in a more robust data basis for downstream analyses.

In vivo proteomics identifies the competence regulon and AliB oligopeptide transporter as pathogenic factors in pneumococcal meningitis.

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens.

A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 105 S. aureus cells, 578 proteins were identified. Increased abundance of proteins required for oxidative stress response, amino acid biosynthesis, and fermentation together with decreased abundance of ribosomal proteins and nucleotide reductase NrdEF was observed in post-infection samples compared to the pre-infection state.