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Open Access
Studies on immunogenicity of recombinant fiber protein of Inclusion Body Hepatitis-Hydropericardium Syndrome (IBH-HPS) virus with special reference to formulation of a candidate subunit vaccine
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-02) Pandey, Garima; Rajesh Kumar
In the present study, recombinant fiber protein of strain FAdV- 2/11 of Fowl adenovirus D isolate PANTNAGAR/HA-14/R-21 was studied for its immunogenicity in terms of effect of dose and adjuvant on the immune response of chicken against IBH-HPS virus. SPF chickens were immunized with different doses of recombinant fiber protein with FCA and 25μg/bird dose provided best protection. In second experiment, broilers were immunized with 25μg/bird along with different adjuvants viz ; montanide, resiquimod, saponin and FCA and different immune parameters were studied. Macrophage function test revealed that resiquimod group has released maximum concentration of nitric oxide. Cell mediated and humoral immune responses were analyzed by cutaneous basophil hypersentivity test, lymphocytes proliferation test and serum neutralization test, respectively. Maximum thickness of foot web was observed in montanide group at 72hrs post inoculation of DNCB. Montanide group showed maximum Tcell response at 21st DPI. Neutralization index of montanide group was highest at 28th DPI. Viral DNA in faeces was detected in all groups at 7th DPC, in montanide group 3 out of 6 faecal samples were positive while on 10th DPC only challenged control group was positive for viral DNA in faeces. 25μg/bird dose with montanide showed best immune response in broilers against challenged with virulent FAdV-2/11. Immunoinformatics analysis of Fiber protein of FAdV-2/11 revealed 21 continuous B cell epitopes with 13 epitopes having surface accessibility and 19 epitopes were antigenic as predicted by BepiPred, Emini surface accessible and Kolaskar and Taogankar antigenicity method, respectively. Out of four models predicted by SWISS MODEL, model 1 was best and verified by different servers. Three discontinuous epitopes were predicted by Ellipro tool. Sixteen epitopes, strongly linked with the MHC alleles were determined by MHC Class-I binding tool and six core peptides have been predicted by MHC Class-II binding tool. Secondary and tertiary structures were predicted by PesiPred V4.0 and Phyre2 tool, respectively. Physico-chemical properties of fiber protein were predicted by ProtParam tool. 0.5002 antigenicity score was predicted by VaxiJen v2 server and thus, fiber protein was proved antigenic and immunogenic. Therefore, fiber protein can be used for development of promising peptide vaccines. Present study concludes that a recombinant subunit vaccine candidate containing recombinant fiber protein (25μg/bird) with montanide as adjuvant may be formulated for further field trials.
Open Access
Egg derived antibodies (IgY) against Outer membrane proteins of multi-drug resistant Salmonella Typhimurium: Production and evaluation of therapeutic potential
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-03) Tiwari, Aakanksha; Rajesh Kumar
Keeping in view, the emerging problem of anti microbial resistance in Salmonella Typhimurium, the present research work was carried out to produce and assess a therapeutic alternative against the bacteria. In the present study, attempts were made to revive glycerol stocks of 150 field isolates and 14 isolates were successfully revived. These isolates were characterized culturally and subjected to ABST. The isolate showing resistance against six groups of antibiotics was selected for further study. This isolate was also characterized molecularly and serotyped as Salmonella Typhimurium with antigen types- 4,5,12:i:1,2. After confirmation of the culture, OMPs were isolated with a concentration of 7.28 mg/ml and SDS-PAGE analysis revealed prominent OMP bands ranging from 11-90 kDa. OMPs were used for hyperimmunizing the RIR layers with a suitable adjuvant subcutaneously. Eggs were collected and IgY was isolated by dextran sulphate method. The purified IgY preparation revealed bands of 63 kDa and 27 kDa in SDS-PAGE, corresponding to the heavy and the light chains. Total protein concentration of IgY preparation by Lowry method was 14.246 mg/ml and specific IgY concentration by RID was 12.023 mg/ml, indicating 84.40% purity. The specificity of IgY against the OMPs was indicated by positive reaction in AGID, CIE, Western blot and Dot Enzyme Immunoassay. Titre of antibody in serum and egg yolk at weekly basis was also determined by ELISA. The maximum antibody titre was observed at 13th week and 11th week in the egg yolk and serum, respectively. It was also observed that IgY was stable at temperatures ranging from -40 ̊ C to 37 ̊ C as indicated by ELISA and SDS-PAGE. In-vitro efficacy testing of different concentrations of IgY against the bacteria showed results in a dose-dependent manner and significant difference was observed between the different combinations. Massive adherence and penetration of bacteria in Vero cells was observed in the negative and culture control unlike in the IgY treated bacterial Vero cells. In vivo experiment in mice, prophylactic group showed no mortality and bacterial count in the faecal swabs and in the organs was significantly less. It indicates that prophylactic activity of IgY is much stronger than the therapeutic activity.
Open Access
Studies on immunogenicity of recombinant fiber protein of Inclusion Body Hepatitis- Hydropericardium Syndrome (IBH-HPS) virus with special reference to formulation of a candidate subunit vaccine
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-02) Pandey, Garima; Rajesh Kumar
In the present study, recombinant fiber protein of strain FAdV- 2/11 of Fowl adenovirus D isolate PANTNAGAR/HA-14/R-21 was studied for its immunogenicity in terms of effect of dose and adjuvant on the immune response of chicken against IBH-HPS virus. SPF chickens were immunized with different doses of recombinant fiber protein with FCA and 25μg/bird dose provided best protection. In second experiment, broilers were immunized with 25μg/bird along with different adjuvants viz ; montanide, resiquimod, saponin and FCA and different immune parameters were studied. Macrophage function test revealed that resiquimod group has released maximum concentration of nitric oxide. Cell mediated and humoral immune responses were analyzed by cutaneous basophil hypersentivity test, lymphocytes proliferation test and serum neutralization test, respectively. Maximum thickness of foot web was observed in montanide group at 72hrs post inoculation of DNCB. Montanide group showed maximum Tcell response at 21st DPI. Neutralization index of montanide group was highest at 28th DPI. Viral DNA in faeces was detected in all groups at 7th DPC, in montanide group 3 out of 6 faecal samples were positive while on 10th DPC only challenged control group was positive for viral DNA in faeces. 25μg/bird dose with montanide showed best immune response in broilers against challenged with virulent FAdV-2/11. Immunoinformatics analysis of Fiber protein of FAdV-2/11 revealed 21 continuous B cell epitopes with 13 epitopes having surface accessibility and 19 epitopes were antigenic as predicted by BepiPred, Emini surface accessible and Kolaskar and Taogankar antigenicity method, respectively. Out of four models predicted by SWISS MODEL, model 1 was best and verified by different servers. Three discontinuous epitopes were predicted by Ellipro tool. Sixteen epitopes, strongly linked with the MHC alleles were determined by MHC Class-I binding tool and six core peptides have been predicted by MHC Class-II binding tool. Secondary and tertiary structures were predicted by PesiPred V4.0 and Phyre2 tool, respectively. Physico-chemical properties of fiber protein were predicted by ProtParam tool. 0.5002 antigenicity score was predicted by VaxiJen v2 server and thus, fiber protein was proved antigenic and immunogenic. Therefore, fiber protein can be used for development of promising peptide vaccines. Present study concludes that a recombinant subunit vaccine candidate containing recombinant fiber protein (25μg/bird) with montanide as adjuvant may be formulated for further field trials.