Abstract
Herpesvirus of turkeys (HVT), used originally as a vaccine against Marek’s disease (MD), has recently been shown to be a highly effective viral vector for generation of recombinant vaccines that deliver protective antigens of other avian pathogens. Until the recent launch of commercial HVT-vectored dual insert vaccines, most of the HVT-vectored vaccines in the market carry a single foreign gene and are usually developed with slow and less efficient conventional recombination methods. There is immense value in developing multivalent HVT-vectored vaccines capable of inducing simultaneous protection against multiple avian pathogens, particularly to overcome the interference between individual recombinant HVT vaccines. Here we demonstrate the use of a previously developed CRISPR/Cas9 gene editing protocol for the insertion of ILTV gD-gI and the H9N2 AIV hemagglutinin expression cassettes into the distinct locations of the recombinant HVT-IBDV VP2 viral genome, to generate the triple insert HVT-VP2-gDgI-HA recombinant vaccine. The insertion, protein expression, and stability of each insert were then evaluated by PCR, immunostaining and Western blot analyses. The successful generation of the first triple insert recombinant HVT vaccine with the potential for the simultaneous protection against three major avian viral diseases in addition to MD is a major innovation in vaccination-based control of major poultry diseases.
Highlights
Poultry is one of the fastest growing sectors of livestock farming contributing hugely to the GlobalFood Security and alleviation of poverty
The three live replicating viral vectors commonly used in poultry vaccination are Herpesvirus of turkeys (HVT), FPV, and NDV (Newcastle disease virus), and the vectored vaccines are used to protect against diseases such as ND (Newcastle disease), IBD, ILT, AI, and Mycoplasma gallisepticum infections
Insertion of the glycoprotein D-glycoprotein I (gDgI) and HA expression cassettes into the HVT-VP2 genome at HVT65/66 and US2 loci respectively were carried out sequentially using the strategy described previously [23]
Summary
Poultry is one of the fastest growing sectors of livestock farming contributing hugely to the GlobalFood Security and alleviation of poverty. Poultry production is expected to grow by 24% in the decade and it is estimated that poultry will account for the largest proportion of livestock output. This growth will only be feasible if losses from infectious diseases are successfully controlled, mainly through the use of effective vaccines. Vaccines 2020, 8, 97 clear that recombinant vaccines could become an important new tool in protecting poultry against infectious diseases with increased efficiency [1]. The three live replicating viral vectors commonly used in poultry vaccination are HVT (herpesvirus of turkeys), FPV (fowlpox virus), and NDV (Newcastle disease virus), and the vectored vaccines are used to protect against diseases such as ND (Newcastle disease), IBD (infectious bursal disease), ILT (infectious laryngotracheitis), AI (avian influenza), and Mycoplasma gallisepticum infections.
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