Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos

Authors: Yu, Qingzhong; LI, YUFENG - Shandong Poultry Research Institute, China; DIMITROV, KIRIL - Consultant; Afonso, Claudio; Spatz, Stephen; Zsak, Laszlo

Submitted to: Vaccine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/25/2019
Publication Date: 1/22/2020
Citation: Yu, Q., Li, Y., Dimitrov, K., Afonso, C.L., Spatz, S.J., Zsak, L. 2020. Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos. Vaccine. 38(4):925-932. https://doi.org/10.1016/j.vaccine.2019.10.074.
DOI: https://doi.org/10.1016/j.vaccine.2019.10.074

Interpretive Summary: The genetic stability of a live virus vaccine is paramount for the vaccine safety and efficacy. In this study, we examined the genetic stability of an experimental vaccine that has been demonstrated to confer protection against Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV) challenges in chickens. The live recombinant vaccine seed virus, rLS/ILTV-gD, was serially passaged in embryonated chicken eggs (ECE) for eight times to mimic NDV vaccine production. The genetic stability and biological properties of egg-passaged vaccine viruses were evaluated by performing next generation sequencing (NGS) analysis, virus titration and virulence assays. The NGS analysis revealed that the egg-passaged vaccine viruses maintained their genome integrity with 13 nucleotide differences between the vaccine seed stock and the egg-passed virus stocks. The biological assessments provided additional evidence that the egg-passaged vaccine viruses retained their growth ability, low virulence and robust level of the antigen expression as the vaccine seed virus, indicating the nucleotide differences were non-detrimental sporadic mutations. These results demonstrated that the rLS/ILTV gD virus is a safe, genetically stable, and potent vaccine candidate after at least eight serial passages in ECE.

Technical Abstract: The Newcastle disease virus (NDV) recombinant expressing the infectious laryngotracheitis virus (ILTV) glycoprotein D (gD) has been demonstrated to confer protection against both virulent NDV and ILTV challenges in chickens. However, there is a concern on the genetic stability of the recombinant vaccine, rLS/ILTV-gD, due to the placement of the foreign gene and its size. In this study, we evaluated the genetic stability and biological properties of the recombinant vaccine after eight serial passages in embryonated chicken eggs (ECE). The vaccine master seed virus at the original egg-passage level 3 (EP3) was diluted and passaged in three separate repetitions (A, B and C) in ECE for eight times (EP4 to EP11). Reverse transcription-polymerase chain reaction analysis of the EP3, EP4, EP5, EP8, and EP11 virus stocks showed that there was no detectable insertion/deletion in the ILTV gD insert region. MiSEQ sequencing analysis of the EP3 and EP11 virus stocks confirmed their genome integrity and revealed just 13 single-nucleotide polymorphisms (SNPs). Biological and immunofluorescent assays provided additional evidence that the EP11 virus stocks was stable, retaining growth kinetics, low pathogenicity, and robust level of gD expression comparable to that of the vaccine master seed virus. This indicated that the SNPs were non-detrimental sporadic mutations. These results demonstrated that the insertion of ILTV gD gene into the NDV LaSota backbone did not affect the genetic stability of the recombinant virus, and that the rLS/ILTV-gD virus is a safe, genetically stable, and potent vaccine candidate after at least eight serial passages in ECE.