Characterizing infection of B cells with wild-type and vaccine strains of measles virus

Abstract

Acute infection with measles virus (MeV) causes transient immunosuppression often leading to secondary infections. MeV infection of B lymphocytes results in changes in the antibody repertoire and memory B cell populations for which the mechanism is unknown. In this study, we characterize the infection of primary B cells with wild-type and vaccine strains of MeV. Vaccine-infected B cells were characterized by a higher percentage of cells positive for viral protein, a higher level of viral transcription and reduced cell death compared to wild-type infected cells, regardless of B cell subtype. Vaccine-infected cells showed more production of TNF-α and IL-10 but less production of IL-8 compared to wild-type infected cells. IL-4 and IL-6 levels detected were increased during both vaccine and wild-type infection. Despite evidence of replication, measles-infected B cells did not produce detectable viral progeny. This study furthers our understanding of the outcomes of MeV infection of human B cells.

Keywords: Immunology; Virology.

Graphical abstract

Figure 1

MeV protein expression and transcription in infected BJAB cells (A–C) Measles hemagglutinin (H) expression in FL-15 and GFP expression in EZ-GFP infected BJAB cells were analyzed by flow cytometry (A) (n = 5). Transcription of MeV N gene normalized to RNaseP was measured by rRT-PCR in cells infected with EZ-GFP (B) and FL-15 (C) (n = 3). Statistics were determined using multiple t-tests. Error bars represent standard deviation (∗∗ = p < 0.01, ∗∗∗ = p < 0.005, ∗∗∗∗ = p < 0.001).

Figure 2

MeV protein expression in infected B lymphocytes (A–D) B cells isolated from whole blood of two healthy human donors were infected with EZ-GFP or FL-15. Example gating strategy used for analysis, cells were gated on singlets, lymphocytes, viability, and CD19 (A). The number of infected cells was determined relative to cells stained at 0 h-post infection (B). For MeV vaccine infected B cells, GFP expression was measured at 0 h and 24 h (C) and 0 h and 48 h (D) in two separate infections, each performed in triplicate. Surface expression of MeV H protein in wild-type MeV infected cells was measured with a mixture of CV1/CV4 antibodies directed against the H protein. Expression was measured at 0 h and 24 h (C) and 0 h and 48 h (D) in two separate infections, each performed in triplicate. Results from both donors are combined in each figure C and D. Statistical analyses were also performed to assess differences between EZ-GFP and FL-15 at 24 (C) and 48 h (D). Statistics were determined using multiple t-tests. Error bar represents standard deviation (n = 4, ∗∗∗ = p < 0.005, ∗∗ = p < 0.01).

Figure 3

MeV N gene transcription following in vitro infection of human B lymphocytes (A–D) B cells isolated from fresh blood were infected and assessed for N gene expression. N gene expression was measured via rRT-PCR using RNA from B cells infected with EZ-GFP or FL-15. N gene transcription at 0 h and 24 h (A, B) and at 0 h and 48 h (C, D) was normalized to RNaseP in two separate experiments. Fold change in N gene transcription is shown above columns. Infections were performed in triplicate in cells isolated from two healthy donors in two separate infections. Error bars represent standard deviation (n = 4, significance was determined by Student’s t test ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.005).

Figure 4

MeV protein expression in B cell subtypes following infection (A–F) Freshly thawed B cells from human donors, provided by STEMCell, were infected in vitro. Cells were gated on live CD19⁺CD20⁺ B cells and subdivided into memory and naive subtypes by CD27 and IgD expression (A) IgD-CD27⁺ (Switched Memory), IgD+CD27⁺ (Non-switched Memory), IgD+CD27⁻ (Naive), IgD-CD27⁻ (Double-Negative). MeV H protein or recombinant GFP expression corresponding to infected cells was evaluated in each B cell subtype with the gate set based infected cells stained at 0 h. Expression of GFP in EZ-GFP infected B cell subtypes (C) and hemagglutinin (H) in FL-15 infected B cell subtypes (D) was evaluated by the example gating strategy of B cells 0 and 48 h post-infection (B). Statistical analyses were performed to assess differences in infections between subtypes during infection with EZ-GFP (E) or FL-15 (F). Infections were performed in two separate donors in triplicate. Error bars represent standard deviation and significance was determined by multiple t-tests (n = 4. n.s. = non-significant, ∗∗∗∗∗ = p < 0.0001, ∗∗∗ = p < 0.005, ∗∗ = p < 0.01, ∗ = p < 0.05, ⇔ = conditions compared in the statistical analysis).

Figure 5

Measurement of progeny MeV in B lymphocytes at 48 h infection (A–D) B cells were isolated from fresh blood and infected. Viral titers in supernatant or lysate from B cells (panels A and B) or BJAB cells (panels C and D) infected with EZ-GFP (panels A and C) or FL-15 (panels B and D) were measured by endpoint dilution in using Vero/hSLAM cells. Virus inoculum was not removed from B cells (A and B) but viral inoculum was removed after 2 h of incubation from BJAB cells (C and D). Viral titer was plotted as the log PFU/mL. Error bars represent standard deviation. Experiments were performed in two separate donors in duplicate (n = 4).

Figure 6

Cell viability following MeV infection B cells isolated from whole blood of two healthy human donors were infected with EZ-GFP and FL-15 with infections performed in triplicate. Infected cells were assessed for viability using a fluorescent amine-reactant dye. The gating strategy from Figure 4A was used to determine boundaries for loss of viability. Loss of viability included dying and dead cells measured at 0, 24, and 48 h in infected and uninfected cells. Error bars represent standard deviation. Statistics were determined using multiple t-tests (n = 4, ∗∗∗ = p < 0.001, ∗∗ = p < 0.01, ∗ = p < 0.05).

Figure 7

Cytokine protein production in B cells following MeV infection (A–E) Previously frozen B cells (STEMCell) from two healthy donors were infected with EZ-GFP or FL-15 at an MOI of 1. Supernatants were collected and stored at −80°C. Supernatants from 24 h post-infection and 48 h post-infection were assessed for TNF-α (A), IL-4 (B), IL-6 (C), IL-8 (D), and IL-10 (E) using a multiplex bead based Luminex assay. Statistical differences between each condition were assessed at individual timepoints using multiple t-tests between each condition and from 24 to 48 h post-infection. Stimulation and infection were performed in two separate donors in duplicate (n = 4, ∗∗∗ = p < 0.001, ∗∗ = p < 0.01, ∗ = p < 0.05).