Current status of virus-vectored vaccines against pathogens that affect poultry.

The most effective strategies for the control of disease in poultry are vaccination and biosecurity. Vaccines useful against pathogens affecting poultry must be safe, effective with a single dose, inexpensive, applicable by mass vaccination methods, and able to induce a protective immune response in the presence of maternal antibodies. Viral vector meet some of these characteristics and if the attenuated virus used as vector infects birds, the vaccine will have the advantage of being bivalent. Thus, viral vectors are currently a tool of choice for the development of new poultry vaccines. This review describes the main viruses used as vectors for the delivery and in vivo expression of antigens of poultry pathogens. It also presents the methodologies most frequently used to obtain recombinant viral vectors and summarizes the state-of-the-art related to vectored vaccines in poultry (some of them currently licensed), the pathogens targeted and their antigens, and the ability of these vaccines to induce an effective immune response. Finally, the review discusses the results of a few studies comparing recombinant viral vector vaccines and live-attenuated vaccines in vaccine matching challenges, and mentions strategies and future researches that can help to improve the efficacy of vectored vaccines in poultry birds.

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication.

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.

Induction of Both Local Immune Response in Mice and Protection in a Rabbit Model by Intranasal Immunization with Modified Vaccinia Ankara Virus Expressing a Secreted Form of Bovine Herpesvirus 1 Glycoprotein D.

In this study, we evaluated the immunogenicity and efficacy of mucosal delivery of a recombinant modified vaccinia Ankara virus (MVA) expressing the secreted version of bovine herpesvirus type 1 (BoHV-1) glycoprotein D (MVA-gDs) without addition of adjuvant in two animal models. First, we demonstrated the capability of MVA-gDs of inducing both local and systemic anti-gD humoral immune response after intranasal immunization of mice. Then, we confirmed that two doses of MVA-gDs administered intranasally to rabbits induced systemic anti-gD antibodies and conferred protection against BoHV-1 challenge. Our results show the potential of using MVA as a vector for the rational design of veterinary vaccines capable of inducing specific and protective immune responses both at local and systemic level.

Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens.

Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.

Vaccine strategies against Babesia bovis based on prime-boost immunizations in mice with modified vaccinia Ankara vector and recombinant proteins.

In this study, a recombinant modified vaccinia virus Ankara vector expressing a chimeric multi-antigen was obtained and evaluated as a candidate vaccine in homologous and heterologous prime-boost immunizations with a recombinant protein cocktail. The chimeric multi-antigen comprises immunodominant B and T cell regions of three Babesia bovis proteins. Humoral and cellular immune responses were evaluated in mice to compare the immunogenicity induced by different immunization schemes. The best vaccination scheme was achieved with a prime of protein cocktail and a boost with the recombinant virus. This scheme induced high level of specific IgG antibodies and secreted IFN and a high degree of activation of IFNγ(+) CD4(+) and CD8(+) specific T cells. This is the first report in which a novel vaccine candidate was constructed based on a rationally designed multi-antigen and evaluated in a prime-boost regime, optimizing the immune response necessary for protection against bovine babesiosis.

Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens

Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.

Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens.

A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry.

Recombinant MVA expressing secreted glycoprotein D of BoHV-1 induces systemic and mucosal immunity in animal models.

Bovine herpesvirus-1 (BoHV-1) infection is distributed worldwide and the development of new tools to fight against this pathogen has become extremely important. In this work a recombinant modified vaccinia virus Ankara (MVA) vector expressing the secreted version of glycoprotein D, MVA-gDs, was obtained and evaluated as a candidate vaccine. First, the correct expression, antigenicity, and N-glycosylation of glycoprotein D were confirmed by molecular techniques. Then MVA-gDs was used as parenteral immunogen in BALB/C mice in which a specific anti-gD humoral immune response was induced and maintained for 7 mo. Two doses of MVA-gDs supplemented with cholera toxin delivered by intranasal immunization induced IgA anti-gD humoral immune responses in nasal and bronchopulmonary washes, as well as IgG anti-gD antibodies in serum samples. In order to evaluate the protection conferred by MVA-gDs immunization, a rabbit BoHV-1 challenge assay was performed. A shorter viral excretion period and a reduction in the number of animals shedding BoHV-1 was observed in the group immunized with recombinant MVA-gDs. In conclusion our data encourage further studies to evaluate MVA-gDs, alone or combined with other immunogens, as a candidate vaccine for BoHV-1.