Immunogenicity of DNA, mRNA and Subunit Vaccines Against Beak and Feather Disease Virus

Abstract

Background/objectives: Beak and feather disease virus (BFDV) is the causative agent of psittacine beak and feather disease (PBFD), affecting psittacine birds. There is currently no commercial vaccine or treatment for this disease. This study developed a novel BFDV coat protein mRNA vaccine encapsidated by TMV coat protein to form pseudovirions (PsVs) and tested its immunogenicity alongside BFDV coat protein (CP) subunit and DNA vaccine candidates.

Methods: mRNA and BFDV CP subunit vaccine candidates were produced in Nicotiana benthamiana and subsequently purified using PEG precipitation and gradient ultracentrifugation, respectively. The DNA vaccine candidate was produced in E. coli cells harbouring a plasmid with a BFDV1.1mer pseudogenome. Immunogenicity of the vaccine candidates was evaluated in African grey parrot chicks.

Results: Successful purification of TMV PsVs harbouring the mRNA vaccine, and of the BFDV-CP subunit vaccine, was confirmed by SDS-PAGE and western blot analysis. TEM analyses confirmed formation of TMV PsVs, while RT-PCR and RT-qPCR cDNA amplification confirmed encapsidation of the mRNA vaccine candidate within TMV particles. Restriction digests verified presence of the BFDV1.1mer genome in the plasmid. Four groups of 5 ten-week-old African grey parrot (Psittacus erithacus) chicks were vaccinated and received two boost vaccinations 2 weeks apart. Blood samples were collected from all four groups on day 14, 28 and 42, and sera were analysed using indirect ELISA, which showed that all vaccine candidates successfully elicited specific anti-BFDV-CP immune responses. The subunit vaccine candidate showed the strongest immune response, indicated by higher binding titres (>6400), followed by the mRNA and DNA vaccine candidates.

Conclusions: The candidate vaccines present an important milestone in the search for a protective vaccine against PBFD, and their inexpensive manufacture could considerably aid commercial vaccine development.

Keywords: BFDV; DNA vaccine; mRNA vaccine; subunit vaccine.

Figure A1

Amplification plot (A), melt peak (B) and standard curve (C) of pRIC4-BFDV-CP-OAS plasmid DNA.

Figure 1

Schematic diagram showing Budgerigar BFDV coat protein and TMV origin of assembly fusion (BFDV-CP-OAS). The flanking restriction sites were used for cloning into pRIC4, a replicating plant expression vector (diagram not drawn to scale).

Figure 2

Illustration of vaccine administration and blood collection from the African grey parrot chicks throughout the trial of the study.

Figure 3

Expression confirmation of TMV rods encapsidating BFDV-CP-OAS mRNA. Images showing leaves that were harvested 7 dpi when they were very necrotic (A). TEM image showing TMV rods of various sizes, ranging from 50 to 350 nm (indicated by orange arrows). The expected TMV/BFDV-CP-OAS is approximately 89 nm in length. The scale bar is 100 nm. (B). Scale bar indicates 100 nm (B). Coomassie stained gel image of TMV-CP expression in N. benthamiana leaves harvested on 7 dpi (C). Crude: crude samples, PEG: 4% PEG-precipitated samples. PageRuler™ Prestained Protein Ladder Plus in kDa (Thermo Fisher Scientific). Black arrow indicates the size of TMV-CP, approximately 17.5 kDa.

Figure 4

TEM analysis of TMV/BFDV-CP-OAS extractions prepared from leaf material harvested 7 dpi and purified using PEG precipitation. These are representative images indicating that neither filter-sterilisation or heat-treatment had any negative effects on the abundance and integrity of TMV rods. (A) filtered sample, (B) heated sample, (C) control sample. Arrows indicate sample TMV rods. Scale bar indicates 100 nm. (D) Quantification of TMV/BFDV-CP-OAS samples using BSA standards ranging from 15 to 0.47 µg. Samples were either filtered using 0.45 µM syringe filter (filtered), heated at 70 °C for 20 min (heated), or not treated at all (control). A band of approximately 15 kDA was observed in both the treated and control samples, as indicated by the orange arrow. M: PageRuler^TM Prestained Protein Ladder Plus (Thermo Fischer Scientific).

Figure 5

PCR using RNA (in red rectangle) and cDNA (in blue rectangle) samples as template. GeneRuler^TM 1 kb DNA Ladder in bp (M), No template control (−VE), Plasmid DNA as control (+VE). RNA samples were treated with two different concentrations of DNase to evaluate the most effective concentration. First strand of cDNA was synthesised using both RNA treatments.

Figure 6

Fold change in BFDV-CP gene copy numbers between samples A, B and C. Average Cq values of two individual experiments in triplicates using RNA samples from a single extraction. The ΔΔCT method was used to quantify the differential expression of BFDV CP in samples B and C, using sample A as the reference control. Error bars represent standard error of means. The BFDV-CP mRNA copies obtained in sample B and C were statistically significantly higher (p = 0.0005 and p < 0.0001) compared to the control sample A (denoted by *** and ****). The mRNA copies in sample C were also significantly higher (p < 0.0001) compared to sample B (denoted by ****).

Figure 7

(A–E). Image of a Thinwall 38 mL Ulta-Clear^TM ultracentrifuge tube showing different fractions (top, middle, bottom bands) after iodixanol (20–50%) density gradient ultracentrifugation of concentrated extract at 175,000× g for 4 h at 4 °C (A). SDS-PAGE and western blot analyses of plant-produced BFDV CP (B–E). pRIC: negative control, empty plant expression vector; BFDV-CP: pRIC with BFDV CP; Lane M: protein ladder; (B) Fractions from density gradient; Middle (Fraction collected at the middle band; Bottom (fraction collected at the bottom band; Top (fraction collected at the top band). Black arrow = BFDV-CP of 28 kDa and blue arrow = BFDV-CP dimer of 56 kDa. Red arrow = plant RuBisCO of 55 kDa. (C) pooled samples from gradient. Blot (D) was probed with 1: 5000 commercial rabbit anti-RuBisCO primary antibody and 1:10,000 anti-rabbit IgG AP-conjugated for the secondary antibody. Blot (E) was probed with 1:2000 anti BFDV CP sera (#6841 final) for primary antibody and 1:10,000 anti-rabbit IgG AP-conjugated for secondary antibody.

Figure 8

(A) Indirect ELISA assay analysing the immune response induced by sera samples from candidate vaccines and PBS control. Antigen: plant-purified BFDV-CP; primary antibody: pooled sera (DAY 1, 14, 28 and 42) from respective vaccine groups (1:100); secondary antibody: anti-bird (1:5000). Data represent averages from four individual experiments (plates) with four replicates in each experiment. Error bars represent standard error of means. Statistical significance was calculated using the two-way RM ANOVA and p-values were calculated based on Tukey’s multiple comparisons test with 95% Cl. Asterisk (*) denotes statistical significance. D14: All three vaccine candidate groups (DNA, BFDV-CP and mRNA) elicited statistically higher (p = 0.0074, p = 0.0007 and p = 0.0016) immune response compared to negative control (denoted by **, *** and ** respectively). BFDV-CP and mRNA vaccines induced statistically higher immune response (p = 0.0008 and p = 0.0024) compared to DNA vaccine (denoted by *** and ** respectively). There was no statistically significant difference (p = 0.6743, denoted by ns) between the immune response induced by BFDV-CP (A 405 nm = 0.354) and mRNA vaccine (A 405 nm = 0.325). D28: the three vaccines, DNA, BFDV-CP and mRNA maintained significantly higher (p = 0.0016, p < 0.0001 and p = 0.0008) immune responses compared to the PBS control (denoted by **, ****, and *** respectively). BFDV-CP and mRNA vaccines elicited 3.4- and 1.3-fold higher immune response (p < 0.0001 and p = 0.0458) compared to the DNA vaccine (denoted by **** and *), and BFDV-CP was 2.6-fold higher (p < 0.0001) compared to mRNA vaccine (denoted by ****). D42: DNA, BFDV-CP and mRNA vaccines still had a much higher immune response (p = 0.0001, p < 0.0001 and p = 0.0073) compared to PBS control (denoted by ***, **** and **). BFDV-CP and mRNA vaccine immune response had increased to 4.2- and 1.8-fold higher (p < 0.0001 and p = 0.0421) compared to DNA vaccine (denoted by **** and *), and BFDV-CP was 2.3-fold higher (p = 0.0016) than mRNA vaccine (denoted by **). (B) Titrations of sera from four vaccine groups: PBS (negative control), DNA, BFDV-CP and mRNA group. Antigen: plant-purified BFDV-CP; primary antibody: pooled sera (DAY 1, 14, 28 and 42) from respective vaccine groups (1:100–1:6400); secondary antibody: anti-bird (1:5000). Data represent averages from two individual experiments (plates) with three replicates in each experiment. Error bars represent standard error of means.