Modeling Infectious Bursal Disease Virus (IBDV) Antigenic Drift In Vitro

Abstract

Infectious bursal disease virus (IBDV) vaccines do not induce sterilizing immunity, and vaccinated birds can become infected with field strains. Vaccine-induced immune selection pressure drives the evolution of antigenic drift variants that accumulate amino acid changes in the hypervariable region (HVR) of the VP2 capsid, which may lead to vaccine failures. However, there is a lack of information regarding how quickly mutations arise, and the relative contribution different residues make to immune escape. To model IBDV antigenic drift in vitro, we serially passaged a classical field strain belonging to genogroup A1 (F52/70) ten times, in triplicate, in the immortalized chicken B cell line, DT40, in the presence of sub-neutralizing concentrations of sera from birds inoculated with IBDV vaccine strain 2512, to generate escape mutants. This assay simulated a situation where classical strains may infect birds that have suboptimal vaccine-induced antibody responses. We then sequenced the HVR of the VP2 capsid at passage (P) 5 and 10 and compared the sequences to the parental virus (P0), and to the virus passaged in the presence of negative control chicken serum that lacked IBDV antibodies. Two escape mutants at P10 had the same mutations, D279Y and G281R, and a third had mutations S251I and D279N. Furthermore, at P5, the D279Y mutation was detectable, but the G281R mutation was not, indicating the mutations arose with different kinetics.

Keywords: HVR; antigenic drift; escape mutant; hypervariable region; immune escape; infectious bursal disease virus (IBDV).

Figure 1

Generation of IBDV escape mutants. 100 TCID₅₀ of IBDV strain F52/70 was mixed with 1:5000 of either (a) hyperimmune serum obtained from inoculating chickens with IBDV vaccine strain 2512, or with (b) negative control serum that did not contain any anti-IBDV antibodies. The mixture was incubated for 1 h at 37 °C, and added to DT40 cells, which were then incubated for 3 days at 37 °C, lysed, and the quantity of virus determined by a TCID₅₀ assay. Then, virus at each passage was mixed with increasing concentrations of serum and passaged onto fresh DT40 cells. This protocol was repeated for ten passages. The sequences of the HVR of the viruses at passages 5 and 10 were determined by Sanger sequencing.

Figure 2

Passage of F52/70 in sub-neutralizing concentrations of anti-2512 serum led to immune escape in DT40 cells. The F52/70 virus that was passaged 10 times in serum lacking IBDV antibodies (wt P10), and the F52/70 virus that was passaged 10 times in anti-2512 serum to generate an escape mutant (escape P10), were titrated in triplicate in the presence of either negative control serum (neg serum) or anti-2512 serum (pos serum) at a dilution of 1:100. The virus titer was quantified, expressed as log₁₀ TCID₅₀/mL, and plotted on the y axis. A one-way ANOVA with Tukey post hoc comparisons was conducted, and results were considered significantly different when p < 0.05 (*), ns = “not significant”.

Figure 3

Alignment of the HVR amino acid sequence from IBDV strain F5/70 passaged in positive or negative serum. (a) The amino acid sequence of the VP2 HVR of the IBDV strain F52/70 that is in GenBank (Accession number AY321953) was aligned to the following sequences: the stock F52/70 virus at passage (P)0, the F52/70 virus that had been passaged 5 times in negative serum lacking anti-IBDV antibodies (Negative sera) (blue shading), and the F52/70 that had been passaged 5 times in increasing concentrations of anti-2512 serum (Positive sera) (pink shading). (b) The F52/70 GenBank sequence was aligned to the stock F52/70 virus at P0, the F52/70 virus at P10 in Negative sera (blue shading), and the three independently generated escape mutants comprised of the F52/70 virus at P10 in Positive serum (pink shading). Amino acids that were identical to the F52/70 GenBank sequence were labelled with a dot, and amino acids that were different from F52/70 were assigned their lettered code. The four hydrophilic loops within the HVR (loops P-BC, P-DE, P-FG, and P-HI) were boxed.

Figure 4

Structural modelling of the HVRs. The predicted structure of the HVR of VP2 of IBDV strain F52/70 was modelled using AlphaFold. Images were generated with PyMol, and the side view (upper panels) and end-on/axial tip view (lower panels) of each molecule were displayed. The HVR was depicted as solid grey, and the predicted structure of the parental F52/70 virus before passage (P0) was compared to the F52/70 virus that was passaged 10 times in serum that lacked IBDV antibodies (P10 (-) Serum), and the three escape mutants that were passaged 10 times in anti-2512 serum (P10 (+) Serum). Amino acid differences between the passaged viruses and the P0 HVR were highlighted in green. The P10 (-) Serum virus contained the S251I, A270E, and S317R mutations, the first and second P10 (+) Serum viruses contained the D279Y and G281R mutations, and the last P10 (+) Serum virus contained the S251I and D279N mutations.