Synergy of subgroup J avian leukosis virus and Eimeria tenella to increase pathogenesis in specific-pathogen-free chickens.

To investigate the effects of co-infections of subgroup J avian leukosis virus (ALV-J) and Eimeria tenella on the pathogenesis in specific-pathogen-free (SPF) white leghorn chickens, groups of chickens were infected with ALV-J strain NX0101 at one day of age or with E. tenella at 14 days of age or both. The control group was left uninfected and was mock-inoculated with phosphate buffer saline (PBS). Mortality rates, body weights, cecal lesions, and viremia of infected chickens in each group were evaluated. Immune status was evaluated by measuring several parameters: immune organ weight/body weight index, specific humoral responses to inactivated NDV vaccine and to inoculated E. tenella, proportions of blood CD3+CD4+ and CD3+CD8α+ lymphocytes and transcriptional levels of cytokines in blood and cecal tonsils. The results show that co-infections of ALV-J and E. tenella induced a higher mortality rate and a lower body weight in SPF chickens compared to single-pathogen infection. In co-infected chickens, ALV-J accelerated the disease symptoms induced by E. tenella, and the E. tenella extended the ALV-J viremia. Thymus atrophy, decrease in the humoral response levels to pathogens and the NDV vaccine, modifications in the blood lymphocyte sub-populations and transcriptional cytokine disorders were found in co-infected chickens compared to chickens infected with one pathogen alone and to controls. We underline a synergy between ALV-J and E. tenella that results in increasing pathogenesis in SPF chickens.

Evaluation of a chimeric multi-epitope-based DNA vaccine against subgroup J avian leukosis virus in chickens.

The prokaryotic expressed recombinant chimeric multi-epitope protein X (rCMEPX) had been evaluated with good immunogenicity and protective efficacy against subgroup J avian leukosis virus (ALV-J) in our previous study. In the present research, we cloned the chimeric multi-epitope gene X into the eukaryotic expression vector pVAX1 to evaluate its potency as a DNA vaccine. The purified recombinant gp85 protein and rCMEPX were used as positive controls and a DNA prime-protein boost strategy was also studied. Six experimental groups of 7-day-old chickens (20 per group) were immunized intramuscularly three times at 2weeks interval with PBS, gp85, rCMEPX, pVAX1, pVAX-X and pVAX-X+rCMEPX respectively. The antibody titers and cellular immune responses were assayed after immunization. The efficacy of immunoprotection against the challenge of ALV-J NX0101 strain was also examined. The results showed that the DNA vaccine could elicit both neutralizing antibodies and cellular responses. Immune-challenge experiments showed good protection efficacy against ALV-J infection. Particularly, the regimen involving one priming pVAX-X and twice recombinant rCMEPX boosting, induced the highest antibody titers in all immunized groups. Our results suggest that the constructed chimeric multi-epitope DNA has potential for a candidate vaccine against ALV-J when used in proper prime-boost combinations. The data presented here may provide an alternative strategy for vaccine design in chicken ALV-J prevention.

Evaluation of a multi-epitope subunit vaccine against avian leukosis virus subgroup J in chickens.

The intricate sequence and antigenic variability of avian leukosis virus subgroup J (ALV-J) have led to unprecedented difficulties in the development of vaccines. Much experimental evidence demonstrates that ALV-J mutants have caused immune evasion and pose a challenge for traditional efforts to develop effective vaccines. To investigate the potential of a multi-epitope vaccination strategy to prevent chickens against ALV-J infections, a recombinant chimeric multi-epitope protein X (rCMEPX) containing both immunodominant B and T epitope concentrated domains selected from the major structural protein of ALV-J using bioinformatics approach was expressed in Escherichia coli Rosetta (DE3). Its immunogenicity and protective efficacy was studied in chickens. The results showed that rCMEPX could elicit neutralizing antibodies and cellular responses, and antibodies induced by rCMEPX could specifically recognize host cell naturally expressed ALV-J proteins, which indicated that the rCMEPX is a good immunogen. Challenge experiments showed 80% chickens that received rCMEPX were well protected against ALV-J challenge. This is the first report of a chimeric multi-epitope protein as a potential immunogen against ALV-J.

Design and construction of a chimeric multi-epitope gene as an epitope-vaccine strategy against ALV-J.

In the present study, we designed and constructed a chimeric multi-epitope gene of ALV-J to develop a potential multi-epitope vaccine using a reverse vaccinology approach. The chimeric gene includes 4 multi-epitope concentrated fragments (Gag (278-376aa), Pol (784-855aa), Env (Gp85:145-156aa and Gp37:412-538aa) screened from major structural proteins of ALV-J using epitope prediction software. The recombinant chimeric multi-epitope protein (rCMEPX) encoded by the cloned chimeric gene was successfully expressed using an Escherichia coli expression system. The rCMEPX was induced optimally at 37°C for 4.0 h with 0.5mM IPTG. The identity and purity of the expressed rCMEPX was analyzed on a SDS-PAGE. The specific recognition of the purified rCMEPX by the chicken anti-ALV-J serum on a western analysis demonstrated a good immunoreactivity of the expressed rCMEPX, which indicates that the construction and expression of the multi-epitope based chimeric gene for ALV-J vaccine development is successful. The antigenicity and reactionogenicity of the rCMEPX were evaluated by western blot and indirect ELISA. Our results showed good reactionogenicity, specificity, and sensitivity for the expressed rCMEPX, suggesting that it may be a promising vaccine candidate against ALV-J infections.