Antigen-specific proliferation responses.

<p>PBLs (<i>n</i> = 5 for each group) were obtained from immunized chickens on days 35, and lymphocytes were stimulated with 400 PFU of inactivated rHVT-<i>pmpD</i>-N. The results were examined as the stimulation index, calculated as the mean counts per minute (cpm) values of stimulated and non-stimulated wells. The values were shown as dot plots and means. ^a–cBars with the different superscripts indicate statistically significant difference among three groups.</p

Confirmation of PmpD-N protein expression in HVT vector by immunoblotting assay and indirect immunofluorescence.

<p>(A) The PmpD-N expression in rHVT-<i>pmpD</i>-N was detected by immunoblot with <i>C</i>. <i>psittaci</i> strain 6BC-specific polyclonal antibodies. Lane M, pre-stained protein ladder; Lane 1, cell lysate post inoculation with rHVT-<i>pmpD</i>-N; Lane 2, cell lysate post inoculation with parental HVT. The black arrow indicates the approximately size of 43kDa. (B) Indirect immunofluorescence analysis of PmpD-N expression in CEF cells. CEF cells on glass coverslips were infected with rHVT-<i>pmpD</i>-N, then incubated with mouse anti-PmpD-N polyclonal antibody of <i>C</i>. <i>psittaci</i> and chicken anti-HVT polyclonal serum, and then reacted with goat anti-mouse IgG conjugated with Alexa Fluor 488 (green fluorescence, shown in the lower left panel) and goat anti-chicken IgY labelled with Alexa Fluor 568 (red fluorescence, shown in the lower right panel), respectively. Finally, cell nuclei were stained with DAPI (blue fluorescence, shown in the top right panel). The merged image is shown in the top left panel. The expression of the targeted protein is indicated by white arrows in top left panel. (C) Parental HVT control. CEF cells on glass coverslips were infected with parental HVT, and then the process of test and the panel meaning are the same as those shown in Fig 1B.</p

T lymphocyte subsets induced by immunization with rHVT-<i>pmpD</i>-N and parental HVT.

<p>T cells were purified from the blood of immunized chickens (<i>n</i> = 5 for each group) on day 35. The values were shown as dot plots and means. (A) The proportion of T cell subsets in PBLs by flow cytometric analysis. ^a–bBars in the same T lymphocyte subsets with the different superscripts indicate significantly different among three groups. (B) The ratio of CD4+/CD8+ among three groups. ^a–bBars with the different superscripts indicate statistically significant difference among three groups.</p

Protective efficacy of rHVT-<i>pmpD</i>-N and parental HVT against challenge with very virulent MDV strain RB-1B.

<p>^a Twenty-five 1-day-old SPF chickens in the rHVT-<i>pmpD</i>-N group were immunized with 8000 PFUs of rHVT-<i>pmpD</i>-N, while 20 SPF chickens in parental HVT group were immunized with 8000 PFUs of parental HVT (recovered from BAC-clone). A total of 20 chickens in the CEF control group were inoculated with uninfected CEF (1.3 × 10⁵ cells per dose) as the negative control. Seven days after immunization, 10 chickens from each group were inoculated intraperitoneally with a virulent strain of MDV RB-1B (1000 PFUs/chicken) and then monitored for 60 days.</p><p>Protective efficacy of rHVT-<i>pmpD</i>-N and parental HVT against challenge with very virulent MDV strain RB-1B.</p

Humoral immune responses.

<p>Sera from CEF control group (n = 10), parental HVT group (n = 10) and rHVT-<i>pmpD</i>-N group (n = 15) were analyzed by ELISA assay for anti-PmpD-N antibody using plates coated with PmpD-N as the antigens as described in material and method section. Results are expressed as absorbance at 450nm/630nm. A serum sample was considered positive when its OD value exceeded 0.045. The values were shown as means ± standard deviation. ** Indicates <i>P</i> < 0.01 when rHVT-<i>pmpD</i>-N & parental HVT group or CEF control group.</p

Pharyngeal shedding of challenge strain and chlamydial loads in lungs after challenge.

<p>The number of chlamydial inclusions in each group (<i>n</i> = 10 for CEF control group and parental HVT group, <i>n</i> = 15 for rHVT-<i>pmpD</i>-N group) was counted in five randomly selected microscopic fields as described in M&M section. The values were shown as dot plots and medians. (A) Median score of pharyngeal shedding of <i>C</i>. <i>psittaci</i> CB7 strain. (B) Chlamydial loads in lungs after challenge. ^a–bBars in the lungs or pharyngeal swabs with the different superscripts are significantly different.</p

Lesion scores in chickens after challenge.

<p>Gross lesions (<i>n</i> = 10 for CEF control group and parental HVT group, <i>n</i> = 15 for rHVT-<i>pmpD</i>-N group) in target organs were assessed, and higher scores represent more severe lesions. The values were shown as dot plots and medians. ^a–bBars in the same tissue with the different superscripts indicate significantly different among three groups.</p

One-step growth kinetics of rHVT-<i>pmpD</i>-N and parental HVT <i>in vitro</i> and <i>in vivo</i>.

<p>(A) The virus growth was calculated as the fold increase at different time points compared with the 12^th h post infection. The asterisk indicates significant differences of the growth kinetics between rHVT-<i>pmpD</i>-N and parental HVT (<i>P</i><0.05). (B) The replication kinetics of parental HVT (<i>n</i> = 10) and rHVT-<i>pmpD</i>-N (<i>n</i> = 15) in vaccinated chickens was evaluated by real time PCR in PBLs. The HVT genome copies were quantified in 75 ng extracted DNA at different time point. The values were shown as means ± standard deviation.</p

Construction of Recombinant HVT Expressing PmpD, and Immunological Evaluation against <i>Chlamydia psittaci</i> and Marek’s Disease Virus

<div><p><i>Chlamydia psittaci</i> (<i>C</i>. <i>psittaci</i>) is an obligate intracellular zoonotic pathogen that can be transmitted to humans from birds. No efficacious commercial vaccine is available for clearing chlamydial infection due to lack of potential vaccine candidates and effective delivery vehicles. Herpesvirus of turkeys (HVT) is an efficacious commercially available vaccine against Marek’s Disease virus (MDV). In this study, a recombinant HVT-delivered vaccine against <i>C</i>. <i>psittaci</i> and Marek’s disease was developed and examined. The 5'-terminus of <i>pmpD</i> gene (<i>pmpD</i>-N) encoding the N-terminal fragment of polymorphic membrane protein D of <i>C</i>. <i>psittaci</i> was inserted into a nonessential region of HVT genome using reverse genetics based on an infectious bacterial artificial chromosome (BAC) clone of HVT. The recombinant virus (rHVT-<i>pmpD</i>-N) was recovered from primary chicken embryo fibroblast (CEF) cells by transfection of modified HVT BAC DNA containing the <i>pmpD</i>-N gene. The rHVT-<i>pmpD</i>-N construct was confirmed to express PmpD-N by immunoblot and immunofluorescence. The rHVT-<i>pmpD</i>-N was stable during 20 passages <i>in vitro</i>. The growth kinetics of rHVT-<i>pmpD</i>-N was comparable to that of parental HVT <i>in vitro</i> and <i>in vivo</i>. One-day-old SPF chickens inoculated subcutaneously with rHVT-<i>pmpD</i>-N displayed increased PmpD-specific antibody levels and a vigorous PmpD-specific lymphocyte proliferation response using HVT vector or CEF cells as control. Furthermore, the percentage of CD4+ cells was significantly elevated in rHVT-<i>pmpD</i>-N-immunized birds as compared to the parental HVT. All chickens vaccinated with rHVT-<i>pmpD</i>-N or parental HVT were protected completely against challenge with a very virulent strain of Marek’s Disease virus (MDV) RB-1B. Post challenge with <i>C</i>. <i>psittaci</i> CB7 strain, a significant decrease in respiratory distress, lesions and <i>Chlamydia</i> load was found in the rHVT-<i>pmpD</i>-N-vaccinated group compared to the parental HVT. In conclusion, our study suggests that the rHVT-<i>pmpD</i>-N live vaccine may be viable as a candidate dual vaccine that provides protection against both very virulent MDV and <i>C</i>. <i>psittaci</i>.</p></div