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

Abstract

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.

Molecular Farming Strategy for the Rapid Production of Protein-Based Reagents for Use in Infectious Disease Diagnostics

“…The sensitivity of the ELISA was validated using inactivated IBDV-vaccinated sera and the sera from experimentally infected and recovered specific pathogen free (SPF) chickens. The VP3-based ELISA allowed for the determination of chickens vaccinated with inactivated IBDV, but no reactivity was observed when using VP2-vaccinated chick sera [55].…”

Diagnosing Infectious Diseases in Poultry Requires a Holistic Approach: A Review

“…Recent developments based upon recombinant antigens and proteins have improved assay specificity and facilitated standardization of interpretation. Recent developments enable the differentiation between birds being vaccinated with chemically inactivated viruses or with viral vector vaccines and birds being infected with field virus, as shown for, e.g., avian influenza virus, NDV, ILTV, IBDV and FAdV [269][270][271][272].…”

Development of SARS-CoV-2 neutralizing antibody detection assay by using recombinant plant-produced proteins

“…The vaccine candidates, monoclonal antibodies and other non-pharmaceutical proteins (industrial enzymes, growth factors, cytokines, anti-microbial peptides) produced in plants were summarized in recent reviews [ 7 , 22 , 23 ]. In addition to vaccines and therapeutics, plant-produced proteins can be used as diagnostic reagents to develop lateral flow strip test or ELISA for infectious disease diagnosis [24] , [25] , [26] , [27] . By utilizing plant transient expression system, the recombinant proteins can be produced in 1-2 weeks after the cloning of desired gene in plant expression vector [12] .…”