Neutralizing antibodies (nAbs) are a feature of adaptive immunity that are produced in response to a pathogen beginning anywhere from a few days to weeks following infection. In some cases, such as with elderly or immunocompromised patients, there is a clinical benefit to administering exogenously produced nAbs, as this can reduce the likelihood of severe disease progression in these individuals. However, the development and production of nAbs is laborious, necessitating the implementation of new techniques and technologies to speed discovery efforts. In this study, researchers from the National University of Singapore demonstrate how a droplet microfluidics platform can be used to rapidly screen for and sort single cells producing nAbs. In contrast to screening using fluorescence-activated cell sorting (FACS) and display systems, screening using droplet-based platforms can demonstrate true virus neutralization rather than binding affinity. In addition, the high-throughput capacity of droplet-based platforms offers massive time savings in comparison to hybridoma or single B-cell-based functional screening workflows. Here, the authors present a high-throughput workflow for enrichment of nAbs based on their functional activity against the Chikungunya virus.
The key feature of the droplet-based workflow established by Lin & Tay et al is the ability to identify virus neutralization function at the single-cell level, then select the cells secreting these functional nABs for enrichment and analysis. To accomplish this, they first examined the bulk infection behavior of the Chikungunya virus (CHIKV-ZSGreen) in host HEK 293T cells. The CHIKV-ZSGreen strain encodes a fluorescent protein under control of a subgenomic promoter, so cells that become infected with CHIKV will express green fluorescence. In the bulk assay, over 73% of HEK 293T cells expressed green fluorescence 22 hours after CHIKV exposure, indicating infection. However, when the anti-CHIKV monoclonal nAb (8B10) was allowed to bind to CHIKV before susceptible cells were added, only 9.75% of cells expressed green fluorescence. This confirmed the effectiveness of the nAb 8B10 against CHIKV and informed the design of the in-droplet functional assay.
The droplet-based, single-cell virus neutralization assay is comprised of four steps. First, single antibody-secreting cells (ASCs) are encapsulated in droplets using a flow-focusing microfluidic chip. Because of the small droplet volume, nABs accumulate rapidly over a short period of time, and clonal expansion is not necessary. So, after 24 hours, CHIKV is added to the droplets by picoinjection, and the droplets are allowed to incubate for 3 hours. This is followed by the addition of host cells by picoinjection; for the droplet nuetralization assay, an initial concentration of 100 million cells per mL was used to deliver an average of 8 cells to each droplet. After 18-22 hours, the droplets that contain infected cells can be visualized by their green fluorescence. Selecting for and collecting droplets that express a lower level of green fluorescence would thus be expected to enrich for nAbs. To accomplish this sorting at high throughput, the droplets were injected into a dielectrophoretic sorter using a sorting threshold of 0.48 V.
This workflow was validated on a mixed population of ASCs consisting of cells that secrete 8B10 nAbs and cells that do not secrete nAbs at a 1:2 ratio. To assess the efficacy of the workflow, the nAb-secreteing cells were stained red and the non-secreting cells were stained blue prior to being added to droplets; it is important to note that this staining was not used for cell sorting, but simply to assess the percentage of nAB-secreting cells in the droplets selected by the dielectrophoretic sorter during downstream analysis. Using FACS, it was determined that the droplet-based functional screening assay resulted in an ASC enrichment ratio of 1.90-2.75. While this represents a modest enrichment, the authors suggest that there are opportunities for host cell and reporter virus engineering that would improve their permissiveness and infectivity, resulting in a more specific functional screening assay. Importantly, the droplet-based workflow showed similar enrichment of nAbs (enrichment ratio of 2.42) in a more complex scenario that better resembled physiological samples, where the proportion of target nAb-secreting cells was low (~2%) and there was an excess of unrelated antibodies (anti-SARS-COV-2) present in the sample. Thus, this workflow represents a promising first step for enriching functional antibodies from primary samples. Combined with RNA sequencing of the antibody-coding genes, this workflow should greatly improve the characterization and development of functional nAbs.