Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine.

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Immunogenicity and protective efficacy of an inactivated Newcastle disease virus vaccine encapsulated in poly-(lactic-co-glycolic acid) nanoparticles.

An immunization experiment was conducted in specific pathogen-free chickens with the inactivated Newcastle disease virus (NDV) vaccine encapsulated in the poly-(lactic-co-glycolic) acid (PLGA) nanoparticles (NP) to evaluate its immunogenicity and protective efficacy. The NDV vaccine was prepared by inactivating one virulent Indian strain of NDV belonging to Genotype VII by using beta-propiolactone. PLGA nanoparticles encapsulating inactivated NDV were prepared by the solvent evaporation method. Scanning electron microscopy and zeta sizer analysis revealed that the (PLGA+NDV) NP were spherical, with an average size of 300 nm, having a zeta potential of -6 mV. The encapsulation efficiency and loading efficiency were 72% and 2.4%, respectively. On immunization trial in chicken, the (PLGA+NDV) NP induced significantly (P < 0.0001) higher levels of HI and IgY antibodies with the peak HI titer of 28 and higher expression of IL-4 mRNA. The consistency of higher antibody levels suggests slow and pulsatile release of the antigens from the (PLGA+NDV) NP. The nano-NDV vaccine also induced cell mediated immunity with higher expression of IFN-γ indicating strong Th1 mediated immune responses in contrast to the commercial oil adjuvanted inactivated NDV vaccine. Further, the (PLGA+NDV) NP afforded 100% protection against the virulent NDV challenge. Our results suggested that PLGA NP have adjuvant potential on induction of humoral as well as Th1 biased cell mediated immune responses and also enhanced protective efficacy of the inactivated NDV vaccine. This study provides an insight for development of PLGA NP based inactivated NDV vaccine using the same genotype circulating in the field as well as for other avian diseases at exigencies.

Complete Genome Sequence of a Virulent Duck Enteritis Virus (DEV/India/IVRI-2016) Isolated from Southern India.

Molecular characterization of Indian isolates of duck enteritis virus (DEV) so far has been limited to a few selected genomic regions. Here, we report the complete genome sequence of an isolate, DEV/India/IVRI-2016, from southern India that is 158,091 bp in length.

Evaluation of a Lipopolysaccharide and Resiquimod Combination as an Adjuvant with Inactivated Newcastle Disease Virus Vaccine in Chickens.

Various toll-like receptor (TLR) agonists have shown potential as adjuvants with different vaccines in both human and livestock species, including chickens. Our previous studies on combination of lipopolysaccharide (LPS; TLR4 agonist) and resiquimod (R-848; TLR7 agonist) showed the synergistic up-regulation of pro-inflammatory Th1 and Th2 cytokines in chicken peripheral blood mononuclear cells (PMBCs). Hence, the present study aimed to explore the combined adjuvant effect of LPS and R-848 with inactivated Newcastle disease virus (NDV) vaccine in chickens. Two weeks-old SPF chickens were immunized with inactivated NDV vaccine along with a combination of LPS and R-848 as an adjuvant with suitable control groups. A booster dose was given two weeks later. Antibody responses were assessed by enzyme linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test, while cell-mediated immune responses were analyzed by a lymphocyte transformation test (LTT) and flow cytometry following vaccination. Two weeks post-booster, the birds were challenged with a velogenic strain of NDV, and protection against clinical signs, mortality and virus shedding was analyzed. The results indicated that inactivated NDV vaccine with R-848 induced significantly higher humoral and cellular immune responses with 100% protection against mortality and viral shedding following a virulent NDV challenge. However, the combination of LPS and R-848 along with inactivated NDV vaccine produced poor humoral and cellular immune responses and could not afford protection against challenge infection and virus shedding when compared to the vaccine-alone group, indicating the deleterious effects of the combination on antigen-specific immune responses. In conclusion, the combination of LPS and R-848 showed the inhibitory effects on antigen-specific humoral, cellular and protective immune responses when used as an adjuvant with inactivated NDV vaccines in chickens. This inhibitory effect might have occurred due to systemic cytokine storm. A nanoparticle-based delivery of the combination of LPS and R-848 for slow and sustained release could be tried as an alternative method to explore the synergistic effect of the combination as an adjuvant in chickens.

Prophylactic and therapeutic insights into trained immunity: A renewed concept of innate immune memory.

Trained immunity is a renewed concept of innate immune memory that facilitates the innate immune system to have the capacity to remember and train cells via metabolic and transcriptional events to enable them to provide nonspecific defense against the subsequent encounters with a range of pathogens and acquire a quicker and more robust immune response, but different from the adaptive immune memory. Reversing the epigenetic changes or targeting the immunological pathways may be considered potential therapeutic approaches to counteract the hyper-responsive or hypo-responsive state of trained immunity. The efficient regulation of immune homeostasis and promotion or inhibition of immune responses is required for a balanced response. Trained immunity-based vaccines can serve as potent immune stimuli and help in the clearance of pathogens in the body through multiple or heterologous effects and confer protection against nonspecific and specific pathogens. This review highlights various features of trained immunity and its applications in developing novel therapeutics and vaccines, along with certain detrimental effects, challenges as well as future perspectives.

Expression profiles of toll like receptors, MHC and cytokine genes along with viral load in organs of ducklings infected with an Indian isolate of duck enteritis virus.

A comprehensive study on the pathogenicity and host immune response was conducted in White Pekin ducklings after experimental infection with an Indian isolate of duck enteritis virus (DEV). The virus was found to be highly pathogenic and pantropic, which rapidly multiplied in various organs, mainly in the spleen and liver showing higher viral load with severe pathological lesions and caused 100% mortality. Expression profiles of immune gene transcripts in tissues (liver, spleen, brain) revealed upregulation of proinflammatory cytokines IFN-α, IFN- ß, IL-1ß, IL-6 and also iNOS with stimulation of TLRs (TLR-2, 3, 21). IFN-α was robustly upregulated (p < 0.05) especially in liver, might be playing role in antiviral innate immunity. Further, massive upregulation of MHC class-I (p < 0.01), expression of Th1 cytokines (IFN-γ & IL-2) and certain Th2 cytokines (IL-4 & IL-10) suggests stimulation of cell mediated as well as humoral immunity. To our knowledge, we are reporting first time about the robust upregulation of MHC class-I in spleen, liver and brain along with expression of certain cytokines in the peripheral blood mononuclear cells (PBMCs) during experimental DEV infection.

Association of bovine major histocompatibility complex class I (BoLA-A) alleles with immune response to Brucella abortus strain 19 in calves.

In the present study, we analyzed the immune response of calves to Brucella abortus strain 19 vaccine (S19) and its association with MHC class I (BoLA-A) alleles (exons 2-3 and 4-5). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for typing of BoLA-A exon 2-3 with DdeI and TaqI restriction enzymes; and exon 4-5 with HinfI in 45 crossbred calves. The PCR-RFLP analysis revealed five BoLA-A alleles each for exon 2-3 (A10/A19, A19, A18/19, A18 and A31) and exon 4-5 (A, B, C, D and E). Immune response against B. abortus S19 was assessed at the 4th week post vaccination; antibody response by standard tube agglutination test (STAT) and cell-mediated immunity by lymphocyte proliferation and lymphocyte-mediated cytotoxicity assays. Further, the macrophage function in terms of nitrite production was also analyzed. The association analysis of various BoLA-A alleles with the elicitation of immune response revealed that calves with certain defined genotypes induced significantly higher cell-mediated immune response in terms of lymphocyte proliferation with higher stimulation indices (S.I.) of 1.59 (BoLA-A19), 1.49 (A18/19) and 1.52 (HinfI-D); lymphocyte mediated cytotoxicity (55.52 % in A19) and nitrite production (43.40 µM in A31). It is assumed that allelic variants of BoLA-A (exons 2-3 and 4-5) were associated with the differential immune response of calves to B. abortus S19 vaccination. Therefore, further studies on association analysis of MHC class-I genes in large number of cattle may generate more information and might be useful for adapting the alternative approach of exploring genetic resistance in the cattle herd against bovine brucellosis.