B cells do not play a role in vaccine-mediated immunity against Marek's disease

Abstract

Background: Marek's disease virus (MDV), a highly oncogenic α-herpesvirus, is the etiological agent of Marek's disease (MD) in chickens. The antiviral activity of vaccine-induced immunity against MD reduces the level of early cytolytic infection, production of cell-free virions in the feather follicle epithelial cells (FFE), and lymphoma formation. Despite the success of several vaccines that have greatly reduced the economic losses from MD, the mechanism of vaccine-induced immunity is poorly understood.

Methods: To provide insight into possible role of B cells in vaccine-mediated protection, we bursectomized birds on day of hatch and vaccinated them eight days later. The birds were challenged 10 days post vaccination with or without receiving adoptive lymphocytes from age-matched control birds prior to inoculation. The study also included vaccinated/challenged and non-vaccinated challenged intact birds. Flowcytometric analysis of PBMN cells were conducted twice post bursectomy to confirm B cell depletion and assess the effect of surgery on T cell population. Immunohistochemical analysis and viral genome copy number assessment in the skin samples at termination was performed to measure the replication rate of MDV in the FFE of the skin tissues of the challenged birds.

Results: The non-vaccinated/challenged birds developed typical clinical signs of MD while the vaccinated/challenged and bursectomized, vaccinated/challenged groups with or without adoptive lymphocyte transfer, were fully protected with no sign of transient paralysis, weight loss, or T cell lymphomas. Immunohistochemical analysis and viral genome copy number evaluation in the skin samples revealed that unlike the vaccinated/challenged birds a significant number of virus particles were produced in the FFE of the non-vaccinated/challenged birds at termination. In the bursectomized, vaccinated/challenged groups, only a few replicating virions were detected in the skin of birds that received adoptive lymphocytes prior to challenge.

Conclusions: The study shows that B cells do not play a critical role in MD vaccine-mediated immunity.

Keywords: B cells; Bursectomy; Marek’s disease; Marek’s disease virus; Vaccination.

Fig. 1

The flow cytogram depicts the percentage of B cells in the tested blood samples from the control (Panel A) and the bursectomized birds (Panel B) at 7 days post-surgery. Blood samples from three birds per group were pooled, lymphocytes isolated and 100 μl of total cells at 1 × 10⁶ cells/ml was used for cell surface antigen analysis. B cells were stained with RPE labeled anti B cells monoclonal antibody (Bu1-RPE).

Fig. 2

Bar graph showing the percentages of B cells, CD4⁺ T cells, and CD8⁺ T cells in the tested blood samples at 7 days post bursectomy. Comparative analysis is made between the untreated control and the bursectomized birds. Same total blood samples were used for staining of B cells and double staining of CD4⁺, and CD8⁺ T cells. B cells, CD4⁺ T cells, and CD8⁺ T cells were stained with monoclonal antibodies Bu1-RPE, CD4-PE, and CD8α-FITC, respectively (See M and M).

Fig. 3

The flow cytogram depicts the percentage of B cells in the tested blood samples from the control (Panel A) and bursectomized birds (Panel B) at 41 days post bursectomy (24 days post challenge). Blood samples from three birds per group were pooled, lymphocytes isolated and 100 μl of total cells at 1 × 10⁶ cells/ml was used for cell analysis. B cells were stained with RPE labeled anti B cells monoclonal antibody (Bu1-RPE).

Fig. 4

Bar graph showing the percentages of B cells, CD4⁺ T cells, and CD8⁺ T cells in the tested blood samples at 41 days post bursectomy (24 days post challenge). Comparative analysis was made among birds from all five groups including the untreated control, bursectomized birds with adoptive lymphocyte transfer that were vaccinated/challenged, bursectomized, vaccinated/challenged, un-bursectomized, vaccinated/challenged, and un-bursectomized, un-vaccinated/challenged. Same total blood samples were used for staining of B cells and double staining of CD4⁺, and CD8⁺ T cells. B cells, CD4⁺ T cells, and CD8⁺ T cells were stained with monoclonal antibodies Bu1-RPE, CD4-PE, and CD8α-FITC, respectively.

Fig. 5

Immunohistochemical analysis of MDV antigen in the skin samples of all 5 groups. Anti-gB monoclonal antibody was used for detection of virus particles in the skin tissues of challenged groups. Panel A depicts skin sample from a control bird with no MDV antigen detected. Panel B is showing significant viral replication in the FFE of a bird from un-bursectomized, un-vaccinated/challenged group (Arrow). Panel C represents the skin sample from B cell-depleted, vaccinated, adoptive lymphocyte received, and challenged bird with minor MDV antigen detection in the FFE (Arrows). Panel D is depicting skin sample from a bursectomized, vaccinated/challenged bird that exhibits no sign of viral antigen. Panel E is the skin sample from a vaccinated/challenged bird with intact bursa of Fabricius with minor replication of MDV in the pulp region of one feather shaft (Arrow). Immunohistochemistry was performed on the skin tissues of three individual birds per group.

Fig. 6

The genome copy number of MDV was based on the DNA samples isolated from skin tissues of treated birds at termination (57 dpi). Despite detection of minor replication of the virus in the skin of couple of treated groups, the genome of MDV was only detected in the skin samples of non-vaccinated/challenged birds. DNA samples were isolated from three individual birds of each group and genome copy number analysis was performed in duplicates for each sample.