Biology subjects

Kaczmarek, J. Z.

Publications and source records attributed to Kaczmarek, J. Z..

3 recordsLinked to original sources

bioRxiv2024

Wild-type and single-O-antigen repeat outer-membrane vesicles induce equivalent protection against homologous and heterologous Salmonella challenge

Lipopolysaccharide O-antigen is an immunodominant target of protective antibodies. Variation in O-antigen structures limits antibody-mediated cross-protection between closely-related pathogens including Salmonella Typhimurium (STm) and S. Enteritidis (SEn). Bacterial outer membrane vesicles (OMV) are vaccine platforms presenting surface antigens in their natural conformations. To assess how O-antigen lengths impact antibody responses and control of homologous or heterologous infection, mice were immunized with STm-OMV containing wild-type O-antigen unit repeats (wt-OMV), [≤]1 O-antigen unit (wzy-OMV), or no O-antigen units (wbaP-OMV) respectively and challenged with either STm or SEn. Unexpectedly, anti-STm LPS IgG and protection to STm were comparable after immunization with either wt-OMV or wzy-OMV. Anti-porin responses were elevated after immunization with wzy-OMV and wbaP-OMV. A single immunization with any OMV induced minimal cross-protection against SEn, except in blood. In contrast, boosting with O-antigen-expressing OMV enhanced control of SEn infections by >10-fold. These results suggest that i) Antibody to single or variable-length O-antigen units are comparably protective against Salmonella; ii) Antigens other than immunodominant O-antigens may be targets of cross-reactive antibodies that moderate bacterial burdens; iii) Boosting can enhance the level of cross-protection against related Salmonella serovars and iv) High tissue burdens of Salmonella can be present in the absence of detectable bacteraemia.

immunology↗

bioRxiv2023

Epitope mapping of SARS-CoV 2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls.

Understanding protein-protein interaction is essential when designing drugs or investigating biological processes. A variety of techniques can be employed in order to map the regions on proteins that are involved in binding eg., CryoEM, X-ray spectroscopy, linear epitope mapping, or mass spectrometry-based methods. The most commonly utilized mass spectrometry-based techniques are cross-linking and hydrogen-deuterium exchange (HDX). An alternative technique for identifying residues on the three-dimensional structure of proteins, that are involved in binding, can be hydroxyl radical protein footprinting (HRPF). However, this method is currently hampered by high initial cost and complex experimental setup. Here we set out to present a generally applicable method using Fenton chemistry for mapping of epitopes in a standard mass spectrometry laboratory. Furthermore, the described method illustrates the importance of controls on several levels when performing mass spectrometry-based epitope mapping. In particular, the inclusion of a negative antibody control has not previously been widely utilized in epitope mapping by HRPF analysis. In order to limit the number of false positives, we further introduced quantification by TMT labelling, thereby allowing for direct comparison between sample conditions and biological triplicates. Lastly, up to six technical replicates were incorporated in the experimental setup in order to achieve increased depth of the final analysis. Both binding and opening of regions on receptor-binding domain (RBD) from SARS-CoV-2 Spike Protein, Alpha, and Delta variants, were observed. The negative control antibody experiment combined with the high overlap between biological triplicates resulted in the exclusion of 40% of the significantly changed regions, including both binding and opening regions. The final identified binding region was mapped to a three-dimensional structure and agrees with the literature for neutralizing antibodies towards SARS-CoV-2 Spike Protein. The presented method is straightforward to implement for the analysis of HRPF in a generic MS-based laboratory. The high reliability of the data was achieved by increasing the number of technical and biological replicates combined with negative antibody controls.

biochemistry↗

bioRxiv2023

Deep physico-chemical characterization of individual serum antibody responses against SARS-CoV-2 RBD using a dual titration microspot assay

Antigen specific humoral immunity can be characterized by the analysis of serum antibodies. While serological assays for the measurement of specific antibody levels are available, these are not quantitative in the biochemical sense. Yet, understanding humoral immune responses quantitatively on the systemic level would need a universal, complete, quantitative, comparable measurement method of antigen specific serum antibodies of selected immunoglobulin classes. Here we describe a fluorescent, dual-titration immunoassay, which provides the physico-chemical parameters that are both necessary and sufficient to quantitatively characterize the humoral immune response. We define the theory of the approach that is based on physical chemistry. For validation of theory, we used recombinant receptor binding domain of SARS-CoV-2 as antigen on microspot arrays and varied the concentration of both the antigen and serum antibodies from infected persons to obtain a measurement matrix of binding data. Both titration curves were simultaneously fitted using an algorithm based on the generalized logistic function and adapted for analyzing thermodynamic variables of binding. We obtained equilibrium affinity constants and chemical potentials for distinct antibody classes. These variables reflect the quality and the effective quantity of serum antibodies, respectively. The proposed fluorescent dual-titration microspot immunoassay can generate truly quantitative serological data that is suitable for immunological, medical and systems biological analysis.

immunology↗