Role of the multiple telomeric repeat arrays in integration, persistence, and efficacy of the commercial CVI988 vaccine

Abstract

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes fatal T cell lymphomas in chickens. Oncogenic MDV strains can integrate their genome into the host telomeres of latently infected and tumor cells. This integration process is facilitated by telomeric repeat arrays (TMR) present at the ends of the MDV genome, which consist of the hexanucleotide (TTAGGG)_n that is identical to host telomere sequences. In addition, integration of the virus genome is crucial for the development of lymphomas. Live-attenuated vaccines play a vital role in protecting chickens against this deadly disease, yet our understanding of their biology remains limited. Intriguingly, the commercial gold standard MDV vaccine, the live-attenuated MDV strain CVI988, also possesses TMR at the ends of its genome. In this study, we investigated the role of the multiple TMR arrays (mTMR) in vaccine virus integration, latency, reactivation, and protection against very virulent MDV. Our data revealed that the mTMR present in CVI988 are important for virus genome integration and maintenance in latently infected cells in vitro. In addition, virus latency, reactivation, and vaccine efficacy were reduced in an mTMR deleted mutant compared to the wild-type vaccine. These results provide valuable insights into the biology of this important vaccine virus and shed light on the roles of the mTMR in vaccine integration, latency, and protection against very virulent MDV.IMPORTANCEMarek's disease virus (MDV) is an oncogenic herpesvirus and causes lethal lymphomas in chickens. The gold standard vaccine is the live-attenuated MDV strain CVI988 (a.k.a. Rispens). CVI988 is extensively used in chickens worldwide due to its high efficacy in preventing disease and lymphomas. The CVI988 vaccine harbors telomere arrays (TMR) at the ends of its genome. TMR facilitate genome integration of oncogenic MDV strains into the host telomeres. This study provides critical insights into the biology of the widely used MDV vaccine strain CVI988, demonstrating the crucial role of mTMR in viral genome integration, latency, and protection against very virulent MDV. Furthermore, our findings enhance the understanding of MDV vaccine biology and may guide future strategies to improve Marek's disease control.

Keywords: CVI988; MDV vaccine; Rispens; genome integration; herpesvirus; telomeres; vaccine effectiveness.

Fig 1

Generation and in vitro characterization of the CVI988 ΔmTMR mutant. (A) Schematic representation of the CVI988 genome with deletion of the internal repeat regions (ΔIR_LR-HR) and deletion of mTMR (ΔmTMR). (B) Plaque-size assays comparing cell-to-cell spread properties of CVI988 and the ΔmTMR mutant. The box plots depict the mean plaque diameters from three independent experiments, including minimum and maximum values, a line at the median, and a “+” at the mean (P > 0.05, unpaired t test, n > 50 per experiment). (C) Multi-step growth kinetics assays comparing replication properties of CVI988 and the ΔmTMR mutant. Viral genome copy numbers were quantified using qPCR. The means of three independent experiments, along with standard deviations, are presented as copy numbers per million cells (P > 0.05, Mann-Whitney test). (D) Representative metaphase chromosomes (4′,6-diamidino-2-phenylindole [DAPI] stain, blue) showing integrated virus (Cy3 streptavidin, red) in 855-19T cells infected with CVI988 (left) and ΔmTMR (right). Scale bars represent 10 µm. (E) Comparison of virus maintenance after 855-19 T cell infections with CVI988 and ΔmTMR. Maintenance of viral genomes in the T cell line was assessed by qPCR analysis at 1 dpi and 14 dpi (*P < 0.05, Mann-Whitney test, n = 3).

Fig 2

Assessment of the vaccine efficacy of CVI988 and the ΔmTMR mutant in vivo. (A) Marek’s disease incidence (clinical disease signs) in mock-vaccinated and CVI-vaccinated or ΔmTMR-vaccinated and 686-challenged chickens. A Kaplan-Meier analysis was performed to determine statistical significance (****P < 0.0001, Mantel-Cox test). (B) Tumor incidence of infected chickens at termination of the experiment. Results are presented as the percentage of affected chickens per group (****P < 0.0001, χ² test). Mock group, n = 10; CVI988 and ΔmTMR groups, n = 25. (C) Mean number of tumor-affected organs per chicken with lymphomas in mock-vaccinated (n = 9) and ΔmTMR-vaccinated (n = 2) and 686-challenged chickens (with standard deviations [error bars]).