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

Abstract

Chlamydia psittaci (C. psittaci) 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 C. psittaci and Marek’s disease was developed and examined. The 5'-terminus of pmpD gene (pmpD-N) encoding the N-terminal fragment of polymorphic membrane protein D of C. psittaci 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-pmpD-N) was recovered from primary chicken embryo fibroblast (CEF) cells by transfection of modified HVT BAC DNA containing the pmpD-N gene. The rHVT-pmpD-N construct was confirmed to express PmpD-N by immunoblot and immunofluorescence. The rHVT-pmpD-N was stable during 20 passages in vitro. The growth kinetics of rHVT-pmpD-N was comparable to that of parental HVT in vitro and in vivo. One-day-old SPF chickens inoculated subcutaneously with rHVT-pmpD-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-pmpD-N-immunized birds as compared to the parental HVT. All chickens vaccinated with rHVT-pmpD-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 C. psittaci CB7 strain, a significant decrease in respiratory distress, lesions and Chlamydia load was found in the rHVT-pmpD-N-vaccinated group compared to the parental HVT. In conclusion, our study suggests that the rHVT-pmpD-N live vaccine may be viable as a candidate dual vaccine that provides protection against both very virulent MDV and C. psittaci.

Chlamydial polymorphic membrane proteins: regulation, function and potential vaccine candidates

“…97 A recent study demonstrated that chickens inoculated with a recombinant herpesvirus of turkeys (HTV) expressing C. psittaci PmpD displayed increased PmpD-specific antibodies and significantly elevated CD4+ T levels. 98 Collectively, these studies suggest that Pmps may be good candidates for a chlamydial vaccine because they are outer membrane proteins that are recognized by antigen presenting cells, degraded, processed and presented to CD4+ T cells. Additionally, these chlamydial proteins have been shown to activate T cells and induce cytokine and antibody production that are important in the resolution of a chlamydial infection.…”

A Promising Recombinant Herpesvirus of Turkeys Vaccine Expressing PmpD-N of Chlamydia psittaci Based on Elongation Factor-1 Alpha Promoter

“…The HD11 chicken macrophage cell line was maintained in RPMI-1640 medium with 10% FBS (v/v). The DNA of C. psittaci strain CB7 was extracted and stored in our lad as reported previously ( 10 ).…”

“…The recombinant HVT was generated as described previously ( 10 ). The recombinant HVT bacterial artificial chromosome (BAC) was based on EF-1α promoter, which was from the vector of pEF6/V5-His (Invitrogen, Carlsbad, CA, USA).…”

“…The major outer membrane protein (MOMP) and the autotransported polymorphic membrane protein D (PmpD) of C. psittaci are proved to be good vaccine candidates ( 8 – 10 ). PmpD has more merit than MOMP because PmpD is conserved and can elicit early immune-mediated neutralization of an ongoing chlamydial infection ( 11 , 12 ).…”

“…In our previous study, a recombinant HVT expressing PmpD-N based on cytomegalovirus (CMV) promoter (rHVT-CMV- pmpD ) was constructed to successfully elicit an exceptional cellular and humoral immunity, and finally proved to confer a partial protection in chickens against C. psittaci challenge infection ( 10 ). In this study, we developed another recombinant HVT vaccine expressing PmpD-N based on elongation factor-1 alpha (EF-1α) promoter (rHVT-EF- pmpD ), and its morphological and immunological characterization was analyzed in comparison with rHVT-CMV- pmpD in vitro .…”