GFP Labelled Human Parainfluenza Virus Type 3

Case StudyHuman parainfluenza virus type III (HPIV3) is a common respiratory pathogen that infects children and can be fatal to vulnerable populations, including the immunocompromised. There is currently no effective vaccine or treatment, and it results in tens of thousands of hospitalizations each year. To identify protective antibodies against HPIV3, researchers screened the B cell repertoires in the peripheral blood, tonsils, and spleen of healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies target two non-overlapping epitopes of the HPIV3 F protein, with the majority targeting the apical region. Prophylactic administration of one of these antibodies, PI3-E12, effectively protected cotton rats from HPIV3 infection. In addition, PI3-E12 could be used to therapeutically suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.

To focus on B cells producing neutralizing antibodies, the researchers sorted single B cells onto irradiated 3T3 feeder cells expressing CD40L, IL-2, and IL-21 to allow for higher throughput screening of culture supernatants for neutralization prior to antibody cloning. In general, more than half of the sorted B cells and 87% of the sorted IgD- B cells produced antibody levels detectable by enzyme-linked immunosorbent assay (ELISA) (Figure 1a). It was found that 14% of IgD- HPIV3 preF-bound B cells sorted from tonsils produced HPIV3 neutralizing antibodies, compared with 5% from spleen and 2% from peripheral blood (Figure 1b). From these cultures, four additional HPIV3 neutralizing mAbs were cloned, named PI3-A3, PI3-B5, PI3-A10, and PI3-A12 (Figure 1c). None of these antibodies neutralized the related virus HPIV1. All of the newly described antibodies bound tightly to the HPIV3 pre-F conformation without any detectable binding to the post-F conformation (Figure 1c), as expected because B cells binding to the post-F conformation were excluded during the sorting process. In contrast, the previously described antibody PIA174 bound weakly to the post-F conformation. When the antibodies were administered in vivo in anticipation, the researchers confirmed that none of the antibodies bound to permeabilized HEp-2 cells (Figure 1d), a common assessment of autoreactivity.

Figure 1. High-throughput screening of human PBMCs, tonsils, and spleens for B cells producing anti-HPIV3 neutralizing antibodies. a) Supernatants of total B cells and IgD− B cells of unknown specificity (N = 2 donors) individually sorted and expanded on irradiated IL-21+/IL-2+/CD40L+ 3T3 feeder cells were assayed for IgG by ELISA. (b) Supernatants of HPIV3 preF-specific B cells individually sorted and expanded on feeder cells were subjected to a plaque reduction neutralization screen. (c) Monoclonal antibodies isolated from the neutralization screen were analyzed for apparent affinity (KD) and neutralizing potency. Neutralizing titers of HPIV3-specific monoclonal antibodies were determined by 60% plaque reduction neutralization tests on Vero cells using green fluorescent protein (GFP)-labeled HPIV3 or HPIV1. (d) Autoreactive antibody assay in HEp-2 cells using mAb against HPIV3 preF. (Boonyaratanakornkit J, et al., 2021)