Evaluation of immune responses induced by vectored recombinant HVT vaccines using Gumboro disease as a model

  • Fiona Ingrao

Student thesis: Doc typesDoctor of Veterinary Sciences


Herpesvirus of turkey (HVT) is a nonpathogenic alphaherpesvirus commonly used in chicken as a vaccine against Marek’s disease (MD) because of its antigenic relationship with MD virus (MDV). Technological
progress in the molecular genetic manipulation of viral genomic material allowed the generation of recombinant vaccines using HVT as a vector. Recombinant HVT (rHVT) can serve as efficient live replicative vaccines into which foreign genes coding for protective antigens are inserted and subsequently expressed. These vaccines are therefore bivalent and present the advantage of generating immune responses targeting both the vector strain and the foreign antigen. Moreover, rHVT vaccines establish a persistent viremia in chickens, promoting long-lasting protective immunity, and are widely recognized as safe and efficacious against several avian pathologies.
In this thesis, the studies have been principally focused on infectious bursal disease (IBD), also
called Gumboro disease, which is an immunosuppressive avian pathology representing a major threat to poultry producers in many countries worldwide. Considering the economic impact of IBD and the limitations of classical vaccines, the use of new generation vaccines was required. In the last decade, two
rHVT vaccines against IBD (rHVT-IBD) have been licensed and used successfully for in ovo and day-old
immunization of commercial chickens. Thus far, there is scarce knowledge available regarding the interactions between the rHVT-IBD and host immune system, and the immune mechanisms involved in the stimulation of protective immunity.
The first chapter of this thesis focused on the characterization of the insert expressed by rHVT-IBD. The two commercially available rHVT-IBD vaccines contain a viral gene coding for the immunogenic capsid protein VP2 from two different strains of IBDV. Therefore, the expression of
VP2 produced by both vaccines was characterized in vitro. A real-time PCR specific to VP2 DNA was also optimized to detect the VP2 protein expressed by both rHVT-IBD in situ following day-old vaccination and allowed the differentiation of vaccinated birds from unvaccinated. In addition, it
highlighted a rapid widespread distribution of rHVT-IBD throughout host tissues and an increased
rHVT-IBD genome load in the feather follicles of vaccinated chickens. The higher viral DNA load
detected in this tissue is an interesting characteristic since feathers are known to be associated with rHVT replication.
Afterwards, the immune responses triggered by rHVT-IBD vaccines in the host were investigated. Although humoral immunity is considered to play an essential role in the protection against IBDV, low insert-specific antibodies titers following rHVT-IBD vaccination were reported in recent literature, highlighting the probable role played by cell-mediated immunity (CMI). However, despite the increasing availability of tools to measure cellular responses in chicken over the last few years, the contribution of vaccines-associated CMI is still poorly documented. Therefore, a challenge of the 12 present work was to implement ex vivo antigen recall stimulation assay to monitor VP2 cell-mediated responses induced by rHVT-IBD vaccination. The findings indicated that IBDV-specific CMI could be measured from 3 weeks postvaccination through the detection of chicken interferon-gamma (ChIFNγ) produced by splenocytes in response to an ex vivo antigenic stimulation, by using enzyme-linked immunospot (ELISPOT) assay. Interestingly, this approach has proved to be more sensitive in detecting ChIFNγ than ELISA test, making ELISPOT assay a useful tool for the characterization of vaccination-induced cellular immune responses.
Although HVT vectored vaccines are known to generate slowly-rising humoral responses, their
capability of inducing early protection, in spite of the absence of detectable insert-specific antibodies,
remains unclear. Moreover, the underlying immunological mechanisms involving CMI early post-vaccination have not yet been understood. Therefore, the second chapter of this thesis further focused
on investigating the immune responses triggered early after rHVT-IBD vaccination. In a first
experiment, the phenotypic analysis of lymphocytes subsets revealed an increased percentage of splenic and circulating CD8+ cells at 7 days postvaccination (dpv). In parallel, uantification of
the expression of genes associated with innate and adaptive immune responses demonstrated an upregulation of cytotoxicity-associated genes in spleenand lung of rHVT-HVT-vaccinated chickens from 7 dpv, while the expression of target genes associated with innate immunity did not appear to be influenced.
To determine the contribution of cytotoxic responses in the mounting of immunity against IBDV without using tedious classical CTL assays, an experimental in vivo model of stimulation of IBDV-specific immune responses was developed.
Two-week-oldchickens were vaccinated with rHVT-IBD and were inoculated with an attenuated IBDV two weeks after immunization in order to stimulate VP2-specific immune responses. Expression profiling of genes associated with adaptive immune responses demonstrated a significant upregulation of CD4, CD8, perforin, and IFNγ in the bursa of vaccinated birds at 7 days postinoculation. In the lung, CD8, granzymes,and perforin transcripts levels were also significantly upregulated in rHVT-IBD-vaccinated chickens at 7 days post-IBDV inoculation, suggesting the activation of specific cellular immune responses. These results support the assumption that rHVT-IBD-specific cytotoxic responses are stimulated within the first two weeks following the immunization.
The third and last part of the present work aimed to investigate the specific tropism exhibited by rHVT-IBD strain for particular lymphocyte subsets through the detection of the VP2 insert in unsorted and magnetically-sorted peripheral blood and splenic lymphocytes isolated from vaccinated SPF chickens. This experiment revealed that the vaccine was detectable in bulk splenic lymphocyte
populations isolated from rHVT-IBD-vaccinated SPF chickens, but also in B and T cells-enriched
Date of Award11 Jan 2018
Original languageEnglish
Awarding Institution
  • University of Namur
SponsorsCeva Santé Animale
SupervisorBenoit Muylkens (Supervisor), Benedicte Lambrecht (Co-Supervisor), Xavier De Bolle (President), Gwenaëlle DAUPHIN (Jury), Laurent Gillet (Jury), Thierry VAN DEN BERG (Jury) & Nicolas Eterradossi (Jury)

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