Blood B Cell Depletion Reflects Immunosuppression Induced by Live-Attenuated Infectious Bursal Disease Vaccines

Abstract

Immunosuppression in poultry production is a recurrent problem worldwide, and one of the major viral immunosuppressive agents is Infectious Bursal Disease Virus (IBDV). IBDV infections are mostly controlled by using live-attenuated vaccines. Live-attenuated Infectious Bursal Disease (IBD) vaccine candidates are classified as "mild," "intermediate," "intermediate-plus" or "hot" based on their residual immunosuppressive properties. The immunosuppression protocol described by the European Pharmacopoeia (Ph. Eur.) uses a lethal Newcastle Disease Virus (NDV) infectious challenge to measure the interference of a given IBDV vaccine candidate on NDV vaccine immune response. A Ph. Eur.-derived protocol was thus implemented to quantify immunosuppression induced by one mild, two intermediate, and four intermediate-plus live-attenuated IBD vaccines as well as a pathogenic viral strain. This protocol confirmed the respective immunosuppressive properties of those vaccines and virus. In the search for a more ethical alternative to Ph. Eur.-based protocols, two strategies were explored. First, ex vivo viral replication of those vaccines and the pathogenic strain in stimulated chicken primary bursal cells was assessed. Replication levels were not strictly correlated to immunosuppression observed in vivo. Second, changes in blood leukocyte counts in chicks were monitored using a Ph. Eur. - type protocol prior to lethal NDV challenge. In case of intermediate-plus vaccines, the drop in B cells counts was more severe. Counting blood B cells may thus represent a highly quantitative, faster, and ethical strategy than NDV challenge to assess the immunosuppression induced in chickens by live-attenuated IBD vaccines.

Keywords: B cells; IBDV; immunosuppression; live-attenuated vaccine; replication; vaccine safety.

Figure 1

Implementation of a European Pharmacopoeia-derived protocol for IBDV immunosuppression testing. (A) Layout of animal experiments 1 and 2. (B) Percentage of clinically protected chicks observed after velogenic Newcastle Disease Virus Challenge. Different letters indicate statistically significant differences (p < 0.05) between groups (within one given experiment) using Fisher's exact test with FDR adjustment method for multiple pairwise comparisons. (C) Serological response to NDV vaccination during experiments 1 and 2. Horizontal bars indicate the median of each group. Different letters indicate statistically significant differences (p < 0.05) between groups (within one given experiment) using Kruskal-Wallis test.

Figure 2

Replication curves of IBDV vaccine and pathogenic strains in ex vivo chicken bursal cells. (A) Layout of the experiment. (B) Viral growth kinetics of IBDV vaccine and pathogenic strains. Intermediate vaccine strains appear in blue grey while intermediate-plus and pathogenic strains are in black. Supernatants collected at the indicated time-points were titrated on freshly isolated bursal cells. Viral titers, expressed as median tissue culture infective doses (TCID₅₀) were calculated using the Read and Muench formula. Icons at each time-point indicate the median value obtained from a total of five biological replicates. Different letters indicate statistically significant differences (p < 0.05) between groups using Kruskall-Wallis test. (C) Example of flow cytometry dot-plot. P1: IBDV-negative dead cells; P2: IBDV-positive dead cells; P3: IBDV-negative live cells; P4: IBDV-positive live cells. (D) Percentages of live infected cells at 16 and 23 h post-infection. Dots indicate values for cells from individual bursae (color-coded). Box-plots indicate extreme values, quartiles and median. Different letters indicate statistically significant differences (p < 0.05) between groups using Kruskal-Wallis test.

Figure 3

Implementation of a modified European Pharmacopoeia-derived protocol for IBDV immunosuppression testing. (A) Layout of animal experiment 3. Extra analyses compared to animal experiments 1 and 2 appear in red text. (B) Percentage of clinically protected chicks observed after velogenic Newcastle Disease Virus challenge. Different letters indicate statistically significant differences (p < 0.05) between groups using Fisher's exact test with FDR adjustment method for multiple pairwise comparisons. (C) Serological response to NDV vaccination during experiment 3. Horizontal bars indicate the median of each group. Different letters indicate statistically significant differences (p < 0.05) between groups using Kruskal-Wallis test. (D) Blood B cell concentrations prior to NDV vaccination (“15 days,” left panel), and prior to NDV challenge (“29 days,” right panel). Different letters indicate statistically significant differences (p < 0.05) between groups using Kruskal-Wallis test.