Progress 07/01/21 to 02/28/23

Outputs
Target Audience:The direct audience for this project include, poultry health specialists, flock managers and owners as well as veterinarians. Our current commercialization approach is to collaborate with strategic partners to manufacture and market this novel IBV vaccine. Changes/Problems:The mosaic constructs did not provide adequate protection and so we had to design bicistronic vaccine constructs to increase the antigen payload. What opportunities for training and professional development has the project provided?The project director learnt many new techniques related to the chicken model system such as blood, tears, oral swab collection and subcutaneous and intranasal vaccine administration. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Major goals of the project Goals: The economic success of the poultry industry in the USA hinges on extensive use of vaccines to control infections. Infectious Bronchitis (IB) is an economically important severe respiratory infectious disease of chickens caused by IB coronavirus (IBV). Current IBV Modified Live Vaccines (MLV) elicit a poor immune response and protection is usually short-lived and could require revaccinations. Vaccine development is further compounded by the presence of multiple serotypes or variants of IBV with little or no cross-protection seen among the serotypes. Reversion to virulence is another potential risk associated with widely used MLV for IBV. Preliminary data from our group indicated that a plasmid DNA (pQAC-N) encoding the N protein of IBV adjuvanted with QuilA Chitosan (QAC) nanoparticles, is sufficient to elicit robust humoral immune responses. In this project, our goal is to develop multivalent vaccine constructs that can protect chickens from IB. We have organized our project to address the following aims. Aim I: Characterize safety and immunogenicity of the mosaic DNA vaccine constructs (pQAC-NS). We will examine the safety and immunogenicity of pQAC-NS DNA constructs following intranasal (IN) or in ovo (IO) immunization routes. Aim II: Evaluate the cross-protective efficacy of pQAC-NS. We will examine the cross protective immunity of pQAC-NS constructs against divergent Ark and Cal strains in a standard heterologous vaccine/challenge model system in comparison to relevant MLV. What was accomplished under these goals? Objective I For Objective I of this project mosaic vaccine constructs were synthesized, and the safety and immunogenicity was tested in chickens. The mosaic S and N sequence for M41 and Conn were commercially synthesized with flanking restriction sites and C-terminal 6xHis tag to be cloned into an in-house vaccine expression vector (pCAG, originally from Addgene) to form the new mosaic pQAC-NS construct (mosaic S+ mosaic N). Following the generation of the construct, groups of 1-day old (D1cornish rocks broilers (N=15/group) were immunized with commercially available Mass41 vaccine as a positive control. For the MLV, each chick was immunized using the intranasal (IN) route with a dosage as suggested by the manufacturer. The mosaic pQAC-NS encoding mosaic N and S antigens for Mass41/Conn isolates was given by the SQ route at a dose of 30+30 ug/chick at day 1 and 20 ug + 20 ug at day 14 of age for prime/boost groups. At 21 days of age, all bird groups were challenged with virulent field isolate of Mass41 strain or Conn strain (obtained from APHIS) via eye drop route using a titer of at least 104 EID 50/bird. All chicks were sacrificed at 7 days post challenge. Sera and tears from different time-points were analyzed for humoral response. No untoward effect due to vaccination was observed in any of the vaccinated chickens indicating that pQAC-NS vaccines were safe. The prechallenge (21 DPV) serum IgY titers showed no statistically significant differences between the vaccinated and the sham vaccinated chickens. Objective II For Objective II, the protective efficacy of the vaccine was tested in chickens challenged with M41 and Conn strains. Chickens vaccinated in Objective I were challenged at 21 days of age. All bird groups were challenged with virulent field isolate of Mass41 strain or Conn strain (obtained from APHIS) via eye drop route using a titer of at least 104 EID 50/bird. All chicks were sacrificed at 7 days post challenge. Viral shedding in tears and swabs were quantitated by qRT-PCR standardized and validated in-house. The chickens that were sham vaccinated and challenged with M41 strain had significantly higher viral load compared to chickens vaccinated with mosaic vaccine and challenged with M41. No such differences were observed when the challenge was with the Conn strain. In addition, no differences in clinical severity scores were observed between any of the vaccine groups compared with the sham vaccine group. Based on the modest protective response of the mosaic vaccines we hypothesized that if the antigen expression by the plasmid DNA is improved we may observe better protection. To achieve this, we constructed a bicistronic vector that expressed both the S and N proteins of M41 from the same plasmid backbone using an IRES sequence (dubbed S-I-N). Chickens (N=9) were prime vaccinated at day 1 and boosted at D14 with the bicistronic S-I-N (60 ug prime and 200 ug boost at D14) construct or individual S (30 ug prime and 100 ug boost) and N (30 ug prime and 100 ug boost) constructs (dubbed SN) mixed together. At day 28 post vaccination chickens were challenged with M41 strain as mentioned above. The immune response as measured by IgY, and the clinical severity and viral loads were evaluated post challenge. The IgY humoral immune response data showed that chickens vaccinated with bicistronic S-I-N vaccine had similar IgY titers compared to SN vaccine. However, both the vaccine groups were significantly higher than the sham vaccinated chickens. No differences in clinical severity were observed between any of the groups. The viral titers in chickens vaccinated with the bicistronic S-I-N vaccine showed significantly lower viral loads compared to sham vaccinated as well as SN vaccinated chickens. This data indicated that increasing the antigen dose can significantly improve the vaccine performance of our IBV vaccines.

Publications

Progress 07/01/22 to 02/28/23

Outputs
Target Audience: Nothing Reported Changes/Problems:The mosaic constructs did not provide adequate protection and so we had to design bicistronic vaccine constructs to increase the antigen payload. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective II For Objective II, the protective efficacy of the vaccine was tested in chickens challenged with M41 and Conn strains. Chickens vaccinated in Objective I were challenged at 21 days of age. All bird groups were challenged with virulent field isolate of Mass41 strain or Conn strain (obtained from APHIS) via eye drop route using a titer of at least 104 EID 50/bird. All chicks were sacrificed at 7 days post challenge. Viral shedding in tears and swabs were quantitated by qRT-PCR standardized and validated in-house. The chickens that were sham vaccinated and challenged with M41 strain had significantly higher viral load compared to chickens vaccinated with mosaic vaccine and challenged with M41. No such differences were observed when the challenge was with the Conn strain. In addition, no differences in clinical severity scores were observed between any of the vaccine groups compared with the sham vaccine group. Based on the modest protective response of the mosaic vaccines we hypothesized that if the antigen expression by the plasmid DNA is improved we may observe better protection. To achieve this, we constructed a bicistronic vector that expressed both the S and N proteins of M41 from the same plasmid backbone using an IRES sequence (dubbed S-I-N). Chickens (N=9) were prime vaccinated at day 1 and boosted at D14 with the bicistronic S-I-N (60 ug prime and 200 ug boost at D14) construct or individual S (30 ug prime and 100 ug boost) and N (30 ug prime and 100 ug boost) constructs (dubbed SN) mixed together. At day 28 post vaccination chickens were challenged with M41 strain as mentioned above. The immune response as measured by IgY, and the clinical severity and viral loads were evaluated post challenge. The IgY humoral immune response data showed that chickens vaccinated with bicistronic S-I-N vaccine had similar IgY titers compared to SN vaccine. However, both the vaccine groups were significantly higher than the sham vaccinated chickens. No differences in clinical severity were observed between any of the groups. The viral titers in chickens vaccinated with the bicistronic S-I-N vaccine showed significantly lower viral loads compared to sham vaccinated as well as SN vaccinated chickens. This data indicated that increasing the antigen dose can significantly improve the vaccine performance of our IBV vaccines.

Publications

Progress 07/01/21 to 06/30/22

Outputs
Target Audience:The direct audience for this project include, poultry health specialists, flock managers and owners as well as veterinarians. Our current commercialization approach is to collaborate with strategic partners to manufacture and market this novel IBV vaccine. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project director learnt many new techniques related to the chicken model system such as blood, tears, oral swab collection and subcutaneous and intranasal vaccine administration. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The mosaic S and N sequence for M41 and Conn were commercially synthesized with flanking restriction sites and C-terminal 6xHis tag to be cloned into an in-house vaccine expression vector (pCAG, originally from Addgene) to form the new mosaic pQAC-NS construct (mosaic S+ mosaic N). Following the generation of the construct, groups of 1-day old (D1cornish rocks broilers (N=15/group) were immunized with commercially available Mass41 vaccine as a positive control. For the MLV, each chick was immunized using the intranasal (IN) route with a dosage as suggested by the manufacturer. The mosaic pQAC-NS encoding mosaic N and S antigens for Mass41/Conn isolates was given by the SQ route at a dose of 30+30 ug/chick at day 1 and 20 ug + 20 ug at day 14 of age for prime/boost groups. At 21 days of age, all bird groups were challenged with virulent field isolate of Mass41 strain or Conn strain (obtained from APHIS) via eye drop route using a titer of at least 104 EID 50/bird. All chicks were sacrificed at 7 days post challenge. Sera and tears from different time-points were analyzed for humoral response. No untoward effect due to vaccination was observed in any of the vaccinated chickens indicating that pQAC-NS vaccines were safe. The prechallenge (21 DPV) serum IgY titers showed no statistically significant differences between the vaccinated and the sham vaccinated chickens.

Publications