Optimized polypeptide for a subunit vaccine against avian reovirus.

Avian reovirus (ARV) is a disease-causing agent. The disease is prevented by vaccination with a genotype-specific vaccine while many variants of ARV exist in the field worldwide. Production of new attenuated vaccines is a long-term process and in the case of fast-mutating viruses, an impractical one. In the era of molecular biology, vaccines may be produced by using only the relevant protein for induction of neutralizing antibodies, enabling fast adjustment to the emergence of new genetic strains. Sigma C (SC) protein of ARV is a homotrimer that facilitates host-cell attachment and induce the production and secretion of neutralizing antibodies. The aim of this study was to identify the region of SC that will elicit a protective immune response. Full-length (residues 1-326) and two partial fragments of SC (residues 122-326 and 192-326) were produced in Escherichia coli. The SC fragment of residues 122-326 include the globular head, shaft and hinge domains, while eliminating intra-capsular region. This fragment induces significantly higher levels of anti-ARV antibodies than the shorter fragment or full length SC, which neutralized embryos infection by the virulent strain to a higher extent compared with the antibodies produced in response to the whole virus vaccine. Residues 122-326 fragment is assumed to be folded correctly, exposing linear as well as conformational epitopes that are identical to those of the native protein, while possibly excluding suppressor sequences. The results of this study may serve for the development of a recombinant subunit vaccine for ARV.

Interferon-containing supernatants increase Marek's disease herpesvirus genomes and gene transcription levels, but not virion replication in vitro.

Viruses encounter the innate immune system immediately after infection of the host; specifically, soluble molecules that are both directly lethal and that initiate acquired immunity. Using the oncogenic Marek's disease alpha-herpesvirus (MDV) model, we quantified the effect of a interferon-containing supernatants (ICS), on MDV replication, gene transcription and antigen expression kinetics. We used an established cell culture system and a well-defined virulent MDV (RB-1B). RB-1B was cultured without ICS, or pretreated and then continuously treated with ICS. We compared (i) RB-1B infectivity; (ii) RB-1B growth by microscopy; (iii) numbers of cells expressing RB-1B antigens by flow cytometry; (iv) RB-1B-DNA load per cell by duplex real-time PCR, and (v) gene transcription kinetics for key MDV-life stages by duplex real-time reverse-transcriptase PCR (RT-PCR). ICS inhibited RB-1B infection, completion of productive life cycle and cell-to-cell infection. The numbers of cells expressing glycoprotein B (a kinetically late antigen) greatly decreased, but the numbers of cells expressing pp38 (a kinetically early antigen) decreased only slightly. The two greatest effects were increases in both RB-1B-DNA per infected cell and pp38 mRNA. We propose MDV has evolved to increase specific gene transcription and genome copies per cell to compensate for ICS. We speculate that the bi-directional shared pp38/origin of replication promoter, is central to this mechanism.

The impact of PEGylation on protein immunogenicity.

Covalent attachment of PEG (PEGylation) is widely used to improve the pharmaceutical properties of therapeutic proteins. The applicability and safety of this method have been proven by the use of various PEGylated pharmaceutical proteins approved by the Food and Drug Administration (FDA). One of the properties attributed to PEGylation is immunogenicity reduction of the PEGylated protein. In this study, the impact of PEGylation on immunogenicity was tested and compared for two proteins (chicken IgY and horse IgG) in two strains of mice (Balb/c and C57BL/6) for two routes of administration (i.v. and i.m.) and two sizes of PEG (5 kD and 20 kD). The influence of PEG was shown to be inconsistent between the mouse strains and routes of administration, even with the same tested protein. Consequently, immunogenicity reduction by PEGylation cannot be predicted or assumed; it must be tested on an individual case basis.

Wide-range protection against avian reovirus conferred by vaccination with representatives of four defined genotypes.

Many isolates of the contagious avian reovirus have been characterized, mainly based on the sequence of their sigma C protein. These isolates have been classified into four genotypes. Currently available vaccines are of limited effectiveness, likely due to the existence of many variants. The aim of this study was to test the efficacy of a vaccine consisting of a mixture of prototypes (representatives) of the four defined genotypic groups of avian reovirus. The prototypes were selected based on their distance from the isolates within each genotype. All prototypes were found to be virulent. Antibodies produced against each of the prototypes neutralized all members of its genotype. Birds were then vaccinated with a mixture of the four prototypes. Results suggest that the 4-valent vaccine can prevent disease and confer broad protection against field isolates of avian reovirus.

Differentiating infected from vaccinated animals, and among virulent prototypes of reovirus.

Birds are most susceptible to infection by avian reovirus, genus Orthoreovirus family Reoviridae, at a young age. Although chicks are protected by antibodies transferred from vaccinated maternal flocks, due to the many variants in the field, the efficiency of the vaccines is limited. The level of antibodies against viruses is generally determined by enzyme-linked immunosorbent assay (ELISA), using the whole virus as the antigen. This has some disadvantages: first, the test measures antibodies against all capsid proteins, most of which are irrelevant for neutralizing the virus, and as such does not reflect the real protection status; second, it is impossible to distinguish between vaccine- and infection-derived antibodies. In the case of a virus that changes frequently, a third disadvantage is the inability to distinguish among serotypes. The aim of this study was to develop a test that would address these concerns. Four prototypes of the avian reovirus protein sigma C were used as antigens on the ELISA plate. Sigma C is the main protein inducing neutralizing antibodies and the most variable among strains and isolates, and it is used for reovirus classification. This differentiating ELISA enabled distinguishing between vaccine and field strains of the virus, identifying the infection source, and in the case of vaccination, exclusively determining the level of protective antibodies. Whereas the whole virus detected antibodies against all strains, differentiating ELISA enabled differentiating between infected and vaccinated animals (DIVA) and in most cases, identifying the sigma C genotype. In a field study, a correlation was found between disease symptoms and antibodies identified against virulent strains in the flock. Thus virulent strains can be identified in the field, enabling adjustment of the relevant vaccines.

Differences in genetic background influence the induction of innate and acquired immune responses in chickens depending on the virulence of the infecting infectious bursal disease virus (IBDV) strain.

Previous studies and field observations have suggested that genetic background influences infectious bursal disease virus (IBDV) pathogenesis. However, the influence of the virulence of the infecting IBDV strain and the mechanisms underlying the differences in susceptibility are not known. In the present study IBDV pathogenesis was compared between specific-pathogen-free layer-type (LT) chickens, which are the most susceptible chicken for IBDV and have been used as the model for pathogenesis studies, and broiler-type (BT) chickens, which are known to be less susceptible to clinical infectious bursal disease (IBD). The innate and acquired immune responses were investigated after inoculation of an intermediate (i), virulent (v) or very virulent (vv) strain of IBDV. IBDV pathogenesis was comparable among genetic backgrounds after infection with iIBDV. After infection with vIBDV and vvIBDV, LT birds showed severe clinical disease and mortality, higher bursal lesion scores and IBDV-antigen load relative to BT birds. Circulating cytokine induction varied significantly in both timing and quantity between LT and BT birds and among virus strains (P<0.05). Evaluation of different immune cell populations by flow-cytometric analysis in the bursa of Fabricius provided circumstantial evidence of a stronger local T cell response in BT birds vs. LT birds after infection with the virulent strain. On the other hand, LT birds showed a more significant increase in circulating macrophage-derived immune mediators such as total interferon (IFN) and serum nitrite than BT birds on days 2 and 3 post-vIBDV infection (P<0.05). Stronger stimulation of innate immune reactions especially after vIBDV infection in the early phase may lead to faster and more severe lesion development accompanied by clinical disease and death in LT chickens relative to BT chickens. Interestingly, no significant differences were seen between genetic backgrounds in induction of the IBDV-specific humoral response: timing of IBDV-antibody induction and antibody levels were comparable between BT and LT birds. This study clearly demonstrates a significant influence of chickens' genetic background on disease outcome. The difference between backgrounds in IBDV susceptibility is further influenced by the virulence of the infecting virus strain.

Coated cross-species antibodies by mannosamine-biotin adduct confer protection against snake venom without eliciting humoral immune response.

Passive immunization with cross-species antibodies triggers the patient's immune response, thereby preventing repeated treatment. Mannosamine-biotin adduct (MBA) has been described as a masking agent for immunogenic reduction and here, the immunogenicity and biological activity of MBA-coated horse anti-viper venom (hsIgG) were compared to those of uncoated or PEGylated hsIgG. In in vitro tests, hsIgG binding was not affected by MBA conjugation. The immune response to hsIgG-MBA was about 8-fold and 32-fold lower than to PEG-coated and uncoated hsIgG, respectively. In vivo, hsIgG-MBA showed efficient venom-neutralization activity. We thus demonstrate the feasibility of using MBA as a masking agent for passive immunization with cross-species antibodies.