Decreased breadth of the antibody response to the spike protein of SARS-CoV-2 after repeated vaccination.

Introduction: The rapid development of vaccines to prevent COVID-19 has raised the need to compare the capacity of different vaccines in terms of developing a protective humoral response. Previous studies have shown inconsistent results in this area, highlighting the importance of further research to evaluate the efficacy of different vaccines. Methods: This study utilized a highly sensitive and reliable flow cytometry method to measure the titers of IgG1 isotype antibodies in the blood of healthy volunteers after receiving one or two doses of various vaccines administered in Spain. The method was also used to simultaneously measure the reactivity of antibodies to the S protein of the original Wuhan strain and variants B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.617.1 (Kappa). Results: Significant differences were observed in the titer of anti-S antibodies produced after a first dose of the vaccines ChAdOx1 nCov-19/AstraZeneca, mRNA-1273/Moderna, BNT162b2/Pfizer-BioNTech, and Ad26.COV.S/Janssen. Furthermore, a relative reduction in the reactivity of the sera with the Alpha, Delta, and Kappa variants, compared to the Wuhan strain, was observed after the second boosting immunization. Discussion: The findings of this study provide a comparison of different vaccines in terms of anti-S antibody generation and cast doubts on the convenience of repeated immunization with the same S protein sequence. The multiplexed capacity of the flow cytometry method utilized in this study allowed for a comprehensive evaluation of the efficacy of various vaccines in generating a protective humoral response. Future research could focus on the implications of these findings for the development of effective COVID-19 vaccination strategies.

Validating Flow Cytometry as a Method for Quantifying Bdellovibrio Predatory Bacteria and Its Prey for Microbial Ecology.

Bdellovibrio bacteriovorus is a predatory, Gram-negative bacteria that feeds on many pathogenic bacteria and has been investigated as a possible solution for mitigating biofilms in different fields. The application depends on more fundamental ecological studies into the dynamics between Bdellovibrio and their prey. To do so requires an accurate, reliable, and, preferably rapid, way of enumerating the cells. Flow cytometry (FCM) is potentially a rapid, accurate, and inexpensive tool for this, but it has yet to be validated in the enumeration of Bdellovibrio. In this study, we developed a protocol to measure the number of Bdellovibrio in samples of various densities using FCM and compared the results with those of other methods: optical density (OD), PFU assay (PFU), and quantitative PCR (qPCR). We observed a strong correlation between values obtained using FCM and PFU (ρ = 0.923) and FCM and qPCR (ρ = 0.987). Compared to optical density there was a much weaker correlation (ρ = 0.784), which was to be expected given the well-documented uncertainty in converting optical density (OD) to cell numbers. The FCM protocol was further validated by demonstrating its ability to distinguish and count mixed populations of Bdellovibrio and the prey Pseudomonas. Thus, the accuracy of FCM as well as its speed and reproducibility make it a suitable alternative for measuring Bdellovibrio cell numbers, especially where many samples are required to capture the dynamics of predator-prey interactions. IMPORTANCE The rise of antibiotic resistance and the unwanted growth of bacteria is a universally growing problem. Predatory bacteria can be used as a biological alternative to antibiotics because they grow by feeding on other bacteria. To apply this effectively requires further study and a deeper understanding of the forces that drive a prey population to elimination. Initially, such studies require more reliable methods to count these cells. Flow cytometry (FCM) is potentially a rapid, accurate, and inexpensive tool for this, but it has yet to be validated for predatory bacteria. This study develops a protocol to count the predatory bacteria Bdellovibrio bacteriovorus and its Pseudomonas prey using FCM and compare the results with those of other methods, demonstrating its ability for studies into B. bacteriovorus predation dynamics. This could lead to the use of B. bacteriovorus for killing bacterial biofilms in fields, such as drinking water and agriculture.

Quantification of BCL-2 Family Members by Flow Cytometry.

Flow cytometry is a powerful technique for the detection and quantification of cell surface and intracellular proteins. It enables the ability to measure the expression levels of specific proteins in a cell population of interest without the need to physically separate out the cells from within a heterogeneous population by using the appropriate cell-specific markers. It also requires fewer cells than other traditional techniques such as Western blotting. Here we describe a robust and reproducible method to measure the expression levels of the BCL-2 family members, BCL-2, BCL-XL, and MCL-1 by quantitative flow cytometry (QFCM) using validated antibodies.

Implementing a routine flow cytometry assay for nucleated red blood cell counts in cord blood units.

INTRODUCTION: As required by standards organizations, Héma-Québec Cord Blood Bank performs enumeration of nucleated red blood cells (NRBCs) in cord blood units (CBUs). This study presents the validation and implementation approaches developed to transfer the routine NRBC enumeration from the manual blood film method to a flow cytometric assay. METHODS: The flow cytometry method was adapted from Tsuji (Cytometry, 37, 1999, 291). This assay was validated to assess the specificity, detection limit, repeatability, and reproducibility of the method, including interoperator and interlaboratory testing. Finally, postimplementation follow-up and adjustments were performed for CBU over a 7-month period. RESULTS: Blood film and flow cytometry NRBC enumerations showed a strong correlation (n = 40; Pearson's r correlation = 0.90). Validation was successful as exemplified by the correlation in interlaboratory testing (n = 30; r = 0.98). During implementation, our routine laboratory analyses revealed that CBU with low NRBC content (≤2%), representing 26% of all CBU tested, resulted in 15% of repeated reading and/or staining and was the principal source of nonconformity. Small adjustments in the standard operating procedures (SOPs), including a fixed 200-event setting in the NRBC gate for the second reading of the replicates, have completely solved this issue. CONCLUSION: Flow cytometric NRBC enumerations, now implemented in Héma-Québec Public Cord Blood Bank, is an improvement in the efficiency of our operations by integrating the count for NRBC into our flow cytometry platform.

Robust G2 pausing of adult stem cells in Hydra.

Hydra is a freshwater hydrozoan polyp that constantly renews its two tissue layers thanks to three distinct stem cell populations that cannot replace each other, epithelial ectodermal, epithelial endodermal, and multipotent interstitial. These adult stem cells, located in the central body column, exhibit different cycling paces, slow for the epithelial, fast for the interstitial. To monitor the changes in cell cycling in Hydra, we established a fast and efficient flow cytometry procedure, which we validated by confirming previous findings, as the Nocodazole-induced reversible arrest of cell cycling in G2/M, and the mitogenic signal provided by feeding. Then to dissect the cycling and differentiation behaviors of the interstitial stem cells, we used the AEP_cnnos1 and AEP_Icy1 transgenic lines that constitutively express GFP in this lineage. For the epithelial lineages we used the sf-1 strain that rapidly eliminates the fast cycling cells upon heat-shock and progressively becomes epithelial. This study evidences similar cycling patterns for the interstitial and epithelial stem cells, which all alternate between the G2 and S-phases traversing a minimal G1-phase. We also found interstitial progenitors with a shorter G2 that pause in G1/G0. At the animal extremities, most cells no longer cycle, the epithelial cells terminally differentiate in G2 and the interstitial progenitors in G1/G0. At the apical pole ~80% cells are post-mitotic differentiated cells, reflecting the higher density of neurons and nematocytes in this region. We discuss how the robust G2 pausing of stem cells, maintained over weeks of starvation, may contribute to regeneration.