Crude Inactivated Influenza A Virus Adjuvated with a Bispecific Antibody Complex Targeting Chicken CD40 and AIV M2e Confers Protection Against Lethal HPAI Challenge in Chickens.

In vivo targeting an immunogen to the CD40 receptor expressed on professional antigen-presenting cells (APCs) dramatically enhances speed, magnitude, and quality of the immune response. Our previous evaluation of this strategy in poultry was limited to immunogenicity studies using CD40-targeted synthetic peptides, which demonstrated significant antigen-specific serum IgG and tracheal IgA levels <1 week after primary administration. In this study, this antibody-guided immunization strategy was modified to permit incorporation of inactivated highly pathogenic avian influenza virions (in lieu of short synthetic peptides) as the immunogen by simply mixing a bispecific antibody complex (anti-CD40/M2e) with crude inactivated virus before injection. Adjuvated avian influenza virus (AIV) induced significant hemagglutination inhibition titers up to 6 weeks postimmunization. In efficacy studies, administration of a single vaccine dose yielded 56%-64% survival against challenge with highly pathogenic H5N1, and 100% protection was achieved upon boosting. These results represent a feasible strategy to effectively target whole inactivated influenza A virus to chicken APCs, regardless of AIV clade and without phenotyping or purifying the virus from crude allantoic fluid. The data represent proof of principle for the unique prophylactic efficacy and versatility of a CD40-targeting adjuvation strategy that can in principle also be harnessed in other poultry vaccines.

Evaluation of recombinant Salmonella vaccines to provide cross-serovar and cross-serogroup protection.

Historically, Salmonella vaccines have been either live attenuated or killed bacterin vaccines that fail to offer cross-serogroup protection, which limits risk mitigation and protection of consumers. Subunit recombinant vaccines which possess highly conserved antigens offer potential to provide cross-serogroup protection, and the ability to express immune-enhancing molecules that promote recognition by the immune system. Six Salmonella subunit vaccine candidates were developed in either attenuated S. Enteritidis (SE) or S. Typhimurium (ST) that cell-surface express antigenic epitopes of high mobility group box 1 immune-enhancing sequence (H), peptidoglycan associated lipoprotein (P), and Omp18 protein Cj0113 (C) in different pattern arrangements for evaluation against S. Heidelberg (SH) challenge in broilers. In exp. 1, chicks were orally vaccinated with SE-CPH, SE-HCP, SE-CHP, ST-CPH, ST-HCP, or ST-CHP at 1 × 107 cfu/chick, or saline on d 1 and d 14. On d 17 all birds were challenged with 6 × 106 cfu/chick SH, and ceca collected on d 23 and d 28. On d 23 only SE-CPH reduced (P < 0.05) SH recovery at 0.34 ± 0.23 log10 cfu when compared to control at 1.19 ± 0.26 log10 cfu. On d 28, SE-CPH and ST-HCP reduced SH recovery at 0.40 ± 0.40 and 0.51 ± 0.26 log10 cfu, respectively in comparison to control at 1.36 ± 0.23 log10 cfu. For exp. 2, chicks were orally vaccinated with 1 × 108 cfu/chick SE-CPH, SE-HCP, SE-CHP or saline on d 1. At d 7 all chicks were orally challenged with 7 × 106 cfu/chick SH and ceca collected on d 28 and d 35. SE-CPH reduced (P < 0.05) SH recovery on d 28 when compared to control (6.16 ± 0.13 vs. 4.71 ± 0.55 log10 cfu). In exp 3, chicks were vaccinated by spray in a commercial vaccination cabinet with SE-CPH vaccination, 1.6 × 107 cfu/chick, or saline. Birds were challenged on d 14 with 3 × 107 cfu/chick SH and ceca collected on d 18 and d 25. SE-CPH reduced SH recovery (P < 0.05) on d 18, 2.75 ± 0.05 log10 cfu, and d 25, 1.89 ± 0.43 log10 cfu, as compared to control chickens at 5.6 ± 0.37 (d 18) and 3.98 ± 0.5 log10 cfu (d 25). The results of these experiments suggest that cross-serogroup protection is possible using these SE and ST-vectored subunit vaccines.

Orexin system is expressed in avian muscle cells and regulates mitochondrial dynamics.

Orexin A and B, orexigenic peptides produced primarily by the lateral hypothalamus that signal through two G protein-coupled receptors, orexin receptors 1/2, have been implicated in the regulation of several physiological processes in mammals. In avian (nonmammalian vertebrates) species; however, the physiological roles of orexin are not well defined. Here, we provide novel evidence that not only is orexin and its related receptors 1/2 (ORXR1/2) expressed in chicken muscle tissue and quail muscle (QM7) cell line, orexin appears to be a secretory protein in QM7 cells. In vitro administration of recombinant orexin A and B (rORX-A and B) differentially regulated prepro-orexin expression in a dose-dependent manner with up-regulation for rORX-A (P < 0.05) and downregulation for rORX-B (P < 0.05) in QM7 cells. While both peptides upregulated ORXR1 expression, only a high dose of rORX-B decreased the expression of ORXR2 (P < 0.05). The presence of orexin and its related receptors and the regulation of its own system in avian muscle cells indicate that orexin may have autocrine, paracrine, and/or endocrine roles. rORXs differentially regulated mitochondrial dynamics network. While rORX-A significantly induced the expression of mitochondrial fission-related genes (DNM1, MTFP1, MTFR1), rORX-B increased the expression of mitofusin 2, OPA1, and OMA1 genes that are involved in mitochondrial fusion. Concomitant with these changes, rORXs differentially regulated the expression of several mitochondrial metabolic genes (av-UCP, av-ANT, Ski, and NRF-1) and their related transcriptional regulators (PPARγ, PPARα, PGC-1α, PGC-1ß, and FoxO-1) without affecting ATP synthesis. Taken together, our data represent the first evidence of the presence and secretion of orexin system in the muscle of nonmammalian species and its role in mitochondrial fusion and fission, probably through mitochondrial-related genes and their related transcription factors.

Utilization of rye as energy source affects bacterial translocation, intestinal viscosity, microbiota composition, and bone mineralization in broiler chickens.

Two independent trials were conducted to evaluate the utilization of rye as energy source on bacterial translocation (BT), intestinal viscosity, gut integrity, gut microbiota composition, and bone mineralization, when compared with a traditional cereal (corn) in broiler chickens. In each experiment, day-of-hatch, broiler chickens were randomly assigned to either a corn or a rye diet (n = 20 chickens/group). At 10 d of age, in both experiments, 12 chickens/group were randomly selected, and given an oral gavage dose of fluorescein isothiocyanate dextran (FITC-d). After 2.5 h of oral gavage, blood samples were collected to determine the passage of FITC-d. The liver was collected from each bird to evaluate BT. Duodenum, ileum, and cecum gut sections were collected to evaluate intestinal viscosity and to enumerate gut microbiota. Tibias were collected for observation of bone parameters. Broilers fed with rye showed increased (p < 0.05) intestinal viscosity, BT, and serum FITC-d. Bacterial enumeration revealed that chickens fed with rye had increased the number of total lactic acid bacteria in all three sections of the gastrointestinal tract evaluated when compared to chickens fed with corn. Chickens fed with rye also had significantly higher coliforms in duodenum and ileum, whereas the total number of anaerobes increased only in duodenum. A significant reduction in bone strength and bone mineralization was observed in chickens fed with rye when compared with corn fed chickens. In conclusion, rye evoked mucosal damage in chickens that alter the intestinal viscosity, increased leakage through the intestinal tract, and altered the microbiota composition as well as bone mineralization. Studies to evaluate dietary inclusion of selected DFM candidates that produce exogenous enzymes in rye fed chickens are currently being evaluated.

Passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination.

Birds transfer maternal antibodies (MAb) to their offspring through the egg yolk where the antibody is absorbed and enters the circulatory system. Maternal antibodies provide early protection from disease, but may interfere with the vaccination efficacy in the chick. MAb are thought to interfere with vaccine antigen processing that reduces the subsequent immune response. Once MAb titers are depleted, the chick will respond to vaccination, but they are also susceptible to viral infection. This study examines the effect of MAb on seroconversion to different viral-vectored avian influenza virus (AIV) vaccines. Chicks were given passively transferred antibodies (PTA) using AIV hyperimmunized serum, and subsequently vaccinated with a fowlpox-AIV recombinant vaccine (FPr) or a Newcastle disease virus-AIV recombinant vaccine (NDVr). Our results indicate that passively transferred antibodies led to significant reduction of seroconversion and clinical protection from virulent challenge in recombinant virus vaccinated chicks thus demonstrating maternal antibody interference to vaccination. The passive antibody transfer model system provides an important tool to evaluate maternal antibody interference to vaccination.

Development of DIVA (differentiation of infected from vaccinated animals) vaccines utilizing heterologous NA and NS1 protein strategies for the control of triple reassortant H3N2 influenza in turkeys.

Since 2003, triple reassortant (TR) swine H3N2 influenza viruses containing gene segments from human, avian, and swine origins have been detected in the U.S. turkey populations. The initial outbreak that occurred involved birds that were vaccinated with the currently available H3 swine- and avian-origin influenza vaccines. Antigenically, all turkey swine-lineage TR H3N2 isolates are closely related to each other but show little or no antigenic cross-reactivity with the avian origin or swine origin influenza vaccine strains that are currently being used in turkey operations. These results call for re-evaluation of currently available influenza vaccines being used in turkey flocks and development of more effective DIVA (differentiation of infected from vaccinated animals) vaccines. In this study, we selected one TR H3N2 strain, A/turkey/OH/313053/04 (H3N2) that showed broad cross reactivity with other recent TR turkey H3N2 isolates, and created NA- and NS-based DIVA vaccines using traditional reassortment as well as reverse genetics methods. Protective efficacy of those vaccines was determined in 2-week-old and 80-week-old breeder turkeys. The reassortant DIVA vaccines significantly reduced the presence of challenge virus in the oviduct of breeder turkeys as well as trachea and cloaca shedding of both young and old breeder turkeys, suggesting that proper vaccination could effectively prevent egg production drop and potential viral contamination of eggs in infected turkeys. Our results demonstrate that the heterologous NA and NS1 DIVA vaccines together with their corresponding serological tests could be useful for the control of TR H3N2 influenza in turkeys.