Inactivated Wild-Type Mumps Virus

Over the past decade, mumps virus (MuV) has caused outbreaks in highly vaccinated populations. Suboptimal T-cell immunity may play a role in the susceptibility of vaccinated individuals to mumps. Here, researchers developed an IFNγ ELISPOT assay to assess MuV-specific T-cell responses in peripheral blood mononuclear cells (PBMCs) from healthy (vaccinated) donors and mumps patients. Various methods for in vitro MuV-specific stimulation of PBMCs were compared, using live or inactivated MuV or MuV-infected autologous antigen-presenting cells, i.e., Epstein Barr virus-transformed B lymphoblastoid cell lines (EBV-BLCLs) or (mitogen-preactivated) PBMCs, to measure their ability to recall IFNγ-producing responding cells by ELISPOT. Flow cytometric confirmation data demonstrated that primarily T cells and NK cells produced IFNγ upon stimulation with live MuV. MuV-specific T cell responses were enumerated by ELISPOT by depleting CD56⁺ NK cells from PBMCs prior to stimulation with live MuV. This assay allows the assessment of memory MuV-specific T cell responses in MuV-vaccinated or infected individuals. In addition, this study provides evidence that live MuV induces IFNγ production not only by T cells but also by NK cells.

Among the MuV stimulations studied, the live 24-hour MuV stimulation method was preferred for reasons of assay sensitivity and practicality. In addition, the inactive 24-hour MuV stimulation method was selected for further analysis to allow comparison of the two methods that may have different antigen presentation pathways. Consistent with the ELISPOT results, intracellular IFNγ staining by flow cytometry in the live cell population detected more cells producing IFNγ after live MuV stimulation than after inactivated MuV stimulation (Figure 1A). MuV-specific IFNγ production was found in the CD3⁺ T cell compartment after both live and inactivated MuV stimulation (Figure 1A and B). In addition, a significant proportion of CD56-expressing NK cells produced IFNγ after live MuV stimulation but not after inactivated MuV stimulation (Figure 1A and B). In line with this finding, when only isolated CD56⁺ cells (representing mainly NK cells) were incubated with live or inactivated MuV, indeed only live MuV induced IFNγ SFC in ELISPOT (Figure 1C). IFNγ production by NK cells stimulated by live MuV varied greatly between donors. Although the frequency of NK cells in PBMCs (5-10%) is much lower than that of CD4⁺ and CD8⁺ T cells (60-70% and 15-20%, respectively), the data suggest that live MuV stimulation may induce a large number of IFNγ-producing NK cells in PBMCs.

Figure 1. MuV-specific IFNγ production by T cells and NK cells. (Han W G H, et al., 2016)