Development of a Novel Assay Based on Plant-Produced Infectious Bursal Disease Virus VP3 for the Differentiation of Infected From Vaccinated Animals.

Infectious bursal disease virus is the causative agent of Gumboro disease, a severe infection that affects young chickens and is associated with lymphoid depletion in the bursa of Fabricius. Traditional containment strategies are based either on inactivated or live-attenuated vaccines. These approaches have several limitations such as residual virulence or low efficacy in the presence of maternally derived antibodies (MDA) but, most importantly, the impossibility to detect the occurrence of natural infections in vaccinated flocks. Therefore, the development of novel vaccination strategies allowing the differentiation of infected from vaccinated animals (DIVA) is a priority. Recently, commercial vectored and experimental subunit vaccines based on VP2 have been proved effective in protecting from clinical disease and posed the basis for the development of novel DIVA strategies. In this study, an engineered version of the VP3 protein of IBDV (His-VP3) was produced in plants, successfully purified from Nicotiana benthamiana leaves, and used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-VP3 antibodies. The His-VP3 ELISA was validated with a panel of 180 reference sera and demonstrated to have 100% sensitivity (95% CI: 94.7-100.0) and 94.17% specificity (95% CI: 88.4-97.6). To evaluate the application of His-VP3 ELISA as a DIVA test, the novel assay was used to monitor, in combination with a commercial kit, detecting anti-VP2 antibodies, the immune response of chickens previously immunized with an inactivated IBDV vaccine, a recombinant Turkey herpes virus carrying the VP2 of IBDV (HVT-ND-IBD) or with plant-produced VP2 particles. The combined tests correctly identified the immune status of the vaccinated specific pathogen free white-leghorn chickens. Moreover, the His-VP3 ELISA correctly detected MDA against VP3 in commercial broiler chicks and showed that antibody titers fade with time, consistent with the natural decrease of maternally derived immunity. Finally, the novel assay, in combination with a VP2-specific ELISA, demonstrated its potential application as a DIVA test in chickens inoculated with VP2-based vaccines, being able to detect the seroconversion after challenge with a very virulent IBDV strain.

The expression in plants of an engineered VP2 protein of Infectious Bursal Disease Virus induces formation of structurally heterogeneous particles that protect from a very virulent viral strain.

Infectious Bursal Disease Virus (IBDV), the etiological agent of Gumboro disease, causes mortality and immunosuppression in chickens and major losses to poultry industry worldwide. The IBDV major capsid protein VP2 is considered the best candidate for the production of novel subunit vaccines. This structural protein contains the major conformational epitopes responsible for the induction of IBDV neutralizing antibodies in chickens and has been demonstrated able to form supramolecular structures in yeast and insect cells. The aim of this study was to express an engineered version of the VP2 protein (His-pVP2) to verify its ability to self-assemble into virus-like particles in plants. The recombinant VP2 was transiently expressed by agroinfiltration in Nicotiana benthamiana and transmission electron microscopy of sucrose density gradient fractions revealed the presence of a mixed population of differently shaped particles ranging from spherical capsids, with a diameter between ~25 and ~70 nm, to tubular structures, with variable length (from 100 to 400 nm). The recombinant VP2-based particles when used for the intramuscular immunization of specific-pathogen-free chicks resulted able to induce the production of anti-IBDV specific antibodies at titers comparable to those induced by a commercial vaccine. Moreover, all the immunized birds survived to the challenge with a Moroccan very virulent IBDV strain with no major histomorphological alterations of the Bursa of Fabricius, similarly to what obtained with the commercial inactivated vaccine.

Complete genome analysis and time scale evolution of very virulent infectious bursal disease viruses isolated from recent outbreaks in Morocco.

Emerging of very virulent infectious bursal disease virus (vvIBDV) genotype in poultry flocks in Morocco were characterized. VP2 sequence analysis showed that the strains of Moroccan vvIBDV genotypes clustered separately from classic and vaccine strains reference of IBDV. The full-length genome of four Moroccan vvIBDV strains was determined, in order to get a more exhaustive molecular characterization allowing to conduct the evolution time scale and speculations on their origin. In a phylogenetic tree, nucleotide sequences of segment A and B formed a common branch with those vvIBDV references strains published in GenBank, but they clearly grouped into a distinct subcluster. An alignment of deduced amino acid sequences segment B, confirmed the presence of the conserved TDN tripeptide found in all of the vvIBDV genotype and revealed the presence of 2 substitutions I472L and E688D specific for the vvIBDV Moroccan isolates. The deduced amino acid sequences of segment A genes showed the presence of the "signature" typical of the vvIBDV genotype and revealed the presence of 7 aa substitutions specific for the vvIBDV Moroccan strains. The evolution rate for IBDV VP2 gene was estimated at 5.875 × 10-4 substitutions/site/year. The estimation of the time to most common recent ancestor of Moroccan vvIBDV based on the VP2 sequences available was 31 years, corresponding to 3 years earlier than the first vvIBDV case detection in layers in the country.

Functional characterization of a plant-produced infectious bursal disease virus antigen fused to the constant region of avian IgY immunoglobulins.

Infectious bursal disease virus (IBDV) is the cause of an economically important highly contagious disease of poultry, and vaccines are regarded as the most beneficial interventions for its prevention. In this study, plants were used to produce a recombinant chimeric IBDV antigen for the formulation of an innovative subunit vaccine. The fusion protein (PD-FcY) was designed to combine the immunodominant projection domain (PD) of the viral structural protein VP2 with the constant region of avian IgY (FcY), which was selected to enhance antigen uptake by avian immune cells. The gene construct encoding the fusion protein was transiently expressed in Nicotiana benthamiana plants and an extraction/purification protocol was set up, allowing to reduce the contamination by undesired plant compounds/proteins. Mass spectrometry analysis of the purified protein revealed that the glycosylation pattern of the FcY portion was similar to that observed in native IgY, while in vitro assays demonstrated the ability of PD-FcY to bind to the avian immunoglobulin receptor CHIR-AB1. Preliminary immunization studies proved that PD-FcY was able to induce the production of protective anti-IBDV-VP2 antibodies in chickens. In conclusion, the proposed fusion strategy holds promises for the development of innovative low-cost subunit vaccines for the prevention of avian viral diseases.

Molecular characterization and phylogenetic analysis of very virulent infectious bursal disease virus circulating in Morocco during 2016-2017.

Very virulent infectious bursal disease virus (vvIBDV), the cause of significant economic losses in many poultry-producing areas, has been present in Morocco since 1991. In spite of the introduction of vaccination, disease outbreaks are frequently observed. To ascertain if vaccines failure may be due to the emergence of new strains, the aim of this study was to perform for the first time the molecular characterization of vvIBDV strains circulating in Morocco by focusing on the hypervariable region (HVR) of the VP2 protein, which is frequently used for molecular epidemiology and phylogenetic studies. Field samples of haemorrhagic bursae of Fabricius were collected for molecular characterization in different parts of the country during 2016-2017 from 48 chicken flocks showing symptoms of disease. In a phylogenetic tree, nucleotide sequences containing the VP2 HVR of 13 samples that were positive for vvIBDV formed a common branch with those of vvIBDV references strains published in GenBank, but they clearly grouped into a distinct subcluster. An alignment of the deduced amino acid sequences, in addition to confirming the presence of the "signature" typical of the vvIBDV HVR, also revealed the presence of substitutions in hydrophilic loops that are known to be involved in the elicitation of neutralizing antibodies. One of these substitutions is unique to the Moroccan isolates. These results represent the first molecular characterization of vvIBDV isolates in Morocco and may indicate that one of the causes of vaccine ineffectiveness is antigenic drift.