Development of a serologic assay and novel vaccine approach to mitigate turkey arthritis reovirus
Requested fund from MTRPC: $70,000
From 9/1/2021to 8/30/2023
Tamer Sharafeldin, DVM, MVSc, PhD
Assistant Professor (Veterinary Pathologist), South Dakota State University Animal Disease Research and Diagnostic Laboratory
N campus Dr, Brookings, SD, 57007Tamer.Sharafeldin@sdstate.edu Oﬃce: 605/688-6544
The main objectives are to improve the diagnostics capability and the vaccination strategy to limit the economic losses induced by turkey arthritis reovirus. To fulﬁll these objectives, we outlined 3 speciﬁc aims to be completed through clear and measurable laboratory and experimental procedures as follows:
1. Develop a sensitive and speciﬁc Indirect Fluorescent Antibody (IFA) assay to detect turkey reovirus antibodies.
2. Develop and evaluate turkey reovirus-ISCOMs vaccines.
3. In vitro and in vivo evaluation of BacMam vector carrying turkey reovirus genes.
Develop a sensitive and speciﬁc Indirect Fluorescent Antibody (IFA) assay to detect turkey reovirus antibodies: After thorough discussion among the research team considering the logistics of diagnostics, we decided to develop ELISA instead of IFA.
TARV-SigmaC (S1 gene) sequence was codon optimized and cloned into pET-28a plasmid then transformed into competent DE3 E. coli. The expression of target gene was veriﬁed by western blot and once veriﬁed, the expressed protein was puriﬁed and concentrated to be used to coat plates for ELISA. Standardization and optimization of Turkey reovirus ELISA assay was performed to determine of the appropriate antigen concentration, blocking buﬀer, blocking Ime, sample diluent and the conjugated anti-turkey HRP. During the standardization, sera from known positive and known negative are used. We are currently validating the assay by testing ﬁeld serum samples, and we establish relative sensitivity and speciﬁcity. The result of our developed assay is compared to the results of the commercially available chicken reovirus ELISA kit. So far, we received nearly500 serum samples with the help of turkey veterinarians in Minnesota, South Dakota, North Carolina, and other states. We expect to have nearly
1000 serum samples for validation. So far, the assay is showing very promising results, and we expect to deploy it in fall 2023.
Develop and evaluate turkey reovirus-ISCOMs vaccines: The ISCOMATRIX was developed in our laboratory by using Quil A:Cholesterol: Phospholipid in ratio of 5:1:1.This suspension was dialyzed, and ﬁltrated. The suspension was visualized the negative staining electron microscopy to verify the assembly of cage like structure particles(Fig.1). The puriﬁed, and concentrated TARV-SC antigen, which was produced in E. coli expression system, was mixed with the ISCOMATRIX suspension. The ISCOM-TARV-SC integration in the mix was veriﬁed using immunogold electron microscopy (Fig2). The protein was quantiﬁed, and the ﬁnal protein concentration is0.5 mg/ ml. The vaccine is ready to be administrated to SPF turkeys to test the eﬃcacy and safety (Trial start on September 16/date of receiving the SPF turkeys).
Fig1: ISCOMATRIX particles of nearly 35nm of diameter.
Fig2: ISCOMATRIX particles (Icosahedral) conjugated with the SigmaC protein (spiral shape)
In vitro and in vivo evaluation of BacMam vector carrying turkey reovirus genes: Sequences of TARV- sigma C & B were codon optimize and cloned to baculovirus transfer vector. Both the cloned vector and the linearized baculovirus DNA were transfected to sf-9 cells. The success of gene transfer from the shuttle vector to the backbone of baculovirusis conﬁrmed through the visualization under fluorescent microscopy. Once the virus assembly is conﬁrmed by the positive green fluorescent signal. The virus was propagated on sf-9 cells and the expression of the cloned genes were conﬁrmed by western blot. Once the western blot conﬁrmed the expression of both TARV-sigmaC and SigmaB, both recombinant viruses were propagated and titrated. The r-baculoviruses were mixed 1:1 before vaccine administration. The ﬁrst trial showed that birds vaccinated with 2 doses of the vaccine via oral/intranasal routes at 2 and 4 weeks had a signiﬁcantly lower tendon lesion scores after challenge at 5 weeks of age compared with the non-vaccinated control(Fig3). A second trial to conﬁrm safety and eﬃcacy is to start on September 16 upon the arrival of the SPF turkeys.
Fig3: The histologic lesions score of turkeys which received BAcMam-SigmaC-SigmaB vaccine vs controls. The higher lesions scores reﬂect more disease and less protection. Turkeys received vaccines had signiﬁcantly lower tendon histologic lesion scores after challenge compared with challenge control group which did not receive the vaccine. The group that received 2 doses of the BacMam-SigmaC-SigmaB vaccine had no signiﬁcant diﬀerence after challenge in comparison with the negative control group which was not challenged.
Final comment: The ELISA assay is developed, and validation process is currently performed. The assay will be oﬀered for diagnostics this fall. Both vaccines were developed and there combinant baculovirus showed a promising result. SPF turkeys delayed submission created some delay. We expect submitting the ﬁnal report by the end of fall 2023.