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. 2021 Feb 16;9(2):159.
doi: 10.3390/vaccines9020159.

A Novel Effective and Safe Vaccine for Prevention of Marek's Disease Caused by Infection with a Very Virulent Plus (vv+) Marek's Disease Virus

Yifei Liao  1 , Sanjay M Reddy  1 , Owais A Khan  1   2 , Aijun Sun  1   3 , Blanca Lupiani  1
Affiliations

A Novel Effective and Safe Vaccine for Prevention of Marek's Disease Caused by Infection with a Very Virulent Plus (vv+) Marek's Disease Virus

Yifei Liao et al. Vaccines (Basel). .

Abstract

Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that causes rapid onset lymphoma in chickens. Marek's disease (MD) is effectively controlled using vaccination; however, MDV continues to break through vaccinal immunity, due to the emergence of highly virulent field strains. Earlier studies revealed that deletion of the meq gene from MDV resulted in an attenuated virus that protects against MD in chickens challenged with highly virulent field strains. However, the meq deleted virus retains the ability to induce significant lymphoid organ atrophy. In a different study, we found that the deletion of the vIL8 gene resulted in the loss of lymphoid organ atrophy in inoculated chickens. Here, we describe the generation of a recombinant MDV from which both meq and vIL8 genes were deleted. In vitro studies revealed that the meq and vIL8 double deletion virus replicated at levels similar to the parental very virulent plus (vv+) virus. In addition, in vivo studies showed that the double deletion mutant virus (686BAC-ΔMeqΔvIL8) conferred protection comparable to CVI988, a commercial vaccine strain, when challenged with a vv+ MDV virus, and significantly reduced lymphoid organ atrophy, when compared to meq null virus, in chickens. In conclusion, our study describes the development of a safe and effective vaccine candidate for prevention of MD in chickens.

Keywords: Marek’s disease virus; Meq; lymphoid organ atrophy; vIL8; vaccine.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
In vitro characterization of meq and vIL8 single and double deletion mutant viruses. (A) Genomic structure of serotype 1 Marek’s disease virus (MDV) and location of meq and vIL8 genes. UL: unique long; US: unique short; TRL and IRL: terminal and internal repeat long; TRS and IRS: terminal and internal repeat short. (B) Genomic analysis of meq and/or vIL8 deletion viruses. DNA of 686BAC (lanes 1 and 5), 686BAC-ΔMeq (lanes 2 and 6), 686BAC-ΔvIL8 (lanes 3 and 7) and 686BAC-ΔMeqΔvIL8 (lanes 4 and 8) were digested with BamHI or EcoRI, followed by agarose gel electrophoresis. Asterisks indicate different bands due to deletion of the meq and/or vIL8 genes. M: 1 kb plus ladder. (C) PCR analysis of viral genome isolated from infected chicken embryonic fibroblasts (CEF), using primers specific for MDV ribonucleotide reductase (RR), and meq and vIL8 flanking primers. Lane 1: 686 virus; lane 2: 686BAC virus; lane 3: 686BAC-ΔMeq virus; lane 4: 686BAC-ΔvIL8 virus; lane 5: 686BAC-ΔMeqΔvIL8 virus. (D) Immunofluorescence assay (IFA). 686BAC, 686BAC-ΔMeq, 686BAC-ΔvIL8 or 686BAC-∆Meq∆vIL8 BAC transfected CEF were subjected to IFA using MDV pp38 specific monoclonal antibody and FITC conjugated secondary antibody. Scale bar = 100 µm. (E) In vitro growth kinetics. CEF were infected with 100 plaque-forming units (PFU) of 686BAC, 686BAC-ΔMeq, 686BAC-ΔvIL8 and 686BAC-ΔMeqΔvIL8 viruses. Infected cells were trypsinized, diluted, and co-seeded with fresh CEF at the indicated time points, and plaques were counted 6 days post infection. Each point represents two independent experiments and data present average plaque numbers ± standard error of the mean (SEM).
Figure 2
Figure 2
In vivo replication and reactivation of meq and vIL8 single and double deletion mutant viruses. (A) Virus genome copy number in spleen of inoculated chickens. Genomic DNA were extracted from splenocytes of inoculated chickens at 5, 14, and 56 days post-inoculation (dpi) and virus genome copy number was measured by qPCR. 686BAC and 686BAC-ΔvIL8 viruses inoculated chickens were not tested (NT) at day 56, as all the chickens in those groups had died. Results are presented as average MDV genome copies per GAPDH copy of three chickens, and error bars represent standard error of the mean (SEM). (B) Virus reactivation assay. CEF were co-seeded with 106 peripheral blood lymphocytes (PBL) isolated from 686BAC, 686BAC-ΔMeq, 686BAC-ΔvIL8, or 686BAC-ΔMeqΔvIL8 virus inoculated chickens at 14 dpi. Plaques were counted 7 days after infection. Data present average plaque numbers of three chickens ± SEM.
Figure 3
Figure 3
Replication of meq and vIL8 double deletion virus in lymphoid organs and feather follicle epithelium (FFE) of inoculated chickens. At 5 or 14 days post-inoculation (dpi), lymphoid organs (spleen, thymus, and bursa) and feather follicle epithelium (FFE) were collected, respectively, from chickens inoculated with 686BAC or 686BAC-∆Meq∆vIL8 viruses, or negative control chickens. Tissue samples were frozen immediately using the O.C.T. (optimal cutting temperature) compound and 6 to 8 µm-thick cryostat sections were stained with MDV pp38 specific monoclonal antibody. Scale bar = 50 µm.
Figure 4
Figure 4
Pathogenesis studies of meq and vIL8 single and double deletion mutant viruses. One-day-old SPF MDV Ab– chickens were inoculated with 2000 plaque-forming units (PFU) of 686BAC, 686BAC-ΔMeq, 686BAC-ΔIL8 or 686BAC-ΔMeqΔIL8 viruses, or kept uninoculated and served as negative control. Chickens were maintained in isolation for 65 days and daily mortality was recorded. (A) Lymphoid organ atrophy. At 14 days post-inoculation (dpi), five chickens from each group were euthanized, the lymphoid organs collected, and the relative bursa and thymus to body weight was determined. Results represent mean value with error bars representing standard error of the mean. **: p < 0.01. (B) Survival curves of chickens inoculated with the indicated viruses or uninoculated control group.
Figure 5
Figure 5
Protection studies of meq and vIL8 double deletion virus in MDV maternal antibodies negative (Ab–) and positive (Ab+) chickens. Fifteen one-day-old SPF MDV Ab– (A) and commercial MDV Ab+ (B) chickens were unvaccinated or vaccinated with 2000 plaque-forming units (PFU) of 686BAC-∆Meq, 686BAC-∆Meq∆vIL8, or CVI988/Rispens subcutaneously. Five days later, vaccinated and unvaccinated control chickens were challenged subcutaneously with 500 PFU of MDV 686 strain virus. Survival curves of each group are presented. The trends of chicken survival over time were examined with LogRank and Wilcoxon tests. ns: no significant difference to CVI988/Rispens vaccinated group.
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