Subodh Kishore

Immunological evaluation of mannosylated chitosan nanoparticles based foot and mouth disease virus DNA vaccine, pVAC FMDV VP1-OmpA in guinea pigs.

A DNA vaccine for foot and mouth disease (FMD) based on mannosylated chitosan nanoparticles was evaluated in guinea pigs. The DNA construct was comprised of FMD virus full length-VP1 gene and outer membrane protein A (Omp A) gene of Salmonella typhimurium as a Toll-like receptor (TLR)-ligand in pVAC vector. Groups of guinea pigs immunized either intramuscularly or intra-nasally were evaluated for induction of virus neutralizing antibodies, Th1(IgG2) and Th2 (IgG1) responses, lymphocyte proliferation, reactive nitrogen intermediate production, secretory IgA for naso-mucosal immune response and protection upon homotypic type O virulent FMD virus challenge. The results indicate the synergistic effect of OmpA on the immunogenic potential of FMD DNA vaccine construct delivered using mannosylated chitosan nano-particles by different routes of administration. These observations suggest the substantial improvement in all the immunological parameters with enhanced protection in guinea pigs.

Co-administration of flagellin augments immune responses to inactivated foot-and-mouth disease virus (FMDV) antigen.

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal virus known that affects livestock health and production. This study aimed to investigate the effect of flagellin, a toll-like receptor 5 agonist, on the immune responses to inactivated FMDV antigen in guinea pig model. Our results showed that the co-administration of flagellin with FMDV antigen through intradermal route induces earlier and higher anti-FMDV neutralizing antibody responses as compared to FMDV antigen alone. Both IgG1 and IgG2 antibody-isotype responses were enhanced, but the IgG1/IgG2 ratios were relatively low, indicative of TH1 type of immune activation. On live viral challenge, flagellin+FMDV immunized guinea pigs showed 70% (7 out of 10) protection rate as compared to 40% (4 out of 10) in FMDV alone immunized guinea pigs. The results demonstrate that the co-administration of flagellin augments immune responses (preferably TH1 type) and protective efficacy against FMDV in guinea pigs.

Immune response and viral persistence in Indian buffaloes (Bubalus bubalis) infected with foot-and-mouth disease virus serotype Asia 1.

ABSTRACT Despite their potential role in the spread of foot-and-mouth disease (FMD), the immune response and viral persistence in FMD virus (FMDV)-infected Indian buffaloes (Bubalus bubalis) have been unexplored. We found similar kinetics of neutralizing antibody responses in the sera and secretory fluids of buffaloes following experimental FMDV Asia 1 infection, but the lymphocyte-proliferative response in infected buffaloes was of low magnitude. Despite inducing a significant systemic and secretory immune response, viral persistence seems to be a common outcome in buffaloes following FMDV Asia 1 infection, which is associated with a weak cellular immune response.

Enhanced mucosal immune response in cattle persistently infected with foot-and-mouth disease virus

The mucosal immune response to foot-and-mouth disease virus (FMDV) type Asia 1 was examined in experimentally infected cattle by assaying antibodies by the virus-neutralizing test (VNT) and IgA ELISA in two secretory fluids, oesophageal pharyngeal fluid (OPF) and oro-nasal fluid (ONF). Out of 17 animals infected by the intradermo-lingual route, 12 became persistently infected (carriers), as defined by positive antigen capture RT-PCR reactions for FMDV RNA in OPF samples collected at 28 days or later after exposure. This proportion of carriers (71%) with FMDV Asia 1 is comparable to other serotypes of the virus. When the two groups were examined, the carriers and non-carriers showed no difference in the serum antibody titre until the end of the experiment at 182 days post-infection (DPI). However, despite an initial similarity significantly higher neutralizing antibody titres and FMDV-specific IgA response were detected among the carriers than the non-carriers in both of the secretory fluids. The response was higher and more stable in ONF compared to OPF. Thus, mucosal antibody assays have the potential to be used as a means of differentiating carrier from non-carrier cattle. Furthermore, the findings are consistent with the higher mucosal antibody response in carriers being an effect of persistent infection rather than the cause.

Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells

Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria. They not only represent a potential platform for development of novel vaccines but also provide a tool for efficient adjuvant and antigen delivery system. In the present study, we investigated the interaction between BGs of Escherichia coli (E. coli) and bovine monocyte-derived dendritic cells (MoDCs). MoDCs are highly potent antigen-presenting cells and have the potential to act as a powerful tool for manipulating the immune system. We generated bovine MoDCs in vitro from blood monocytes using E. coli expressed bovine GM-CSF and IL-4 cytokines. These MoDCs displayed typical morphology and functions similar to DCs. We further investigated the E. coli BGs to induce maturation of bovine MoDCs in comparison to E. coli lipopolysaccharide (LPS). We observed the maturation marker molecules such as MHC-II, CD80 and CD86 were induced early and at higher levels in BG stimulated MoDCs as compared to the LPS stimulated MoDCs. BG mediated stimulation induced significantly higher levels of cytokine expression in bovine MoDCs than LPS. Both pro-inflammatory (IL-12 and TNF-α) and anti-inflammatory (IL-10) cytokines were induced in MoDCs after BGs stimulation. We further analysed the effects of BGs on the bovine MoDCs in an allogenic mixed lymphocyte reaction (MLR). We found the BG-treated bovine MoDCs had significantly (p<0.05) higher capacity to stimulate allogenic T cell proliferation in MLR as compared to the LPS. Taken together, these findings demonstrate the E. coli BGs induce a strong activation and maturation of bovine MoDCs.

Hatchability and mortality following infection of chick embryos with subgroup a rous sarcoma virus

An investigation, using Rhode Island Red, New Hampshire, Columbian and Australorp, was conducted to study the correlation between chorioallantoic membrane (CAM)-infection phenotype to challenge with Bryan strain of Rous sarcoma virus (BS-RSV) of subgroup A and subsequent mortality, following hatching. A total of 806 CAMs and 438 chicks over nine hatch-replicates in three experiments provided evidence that infection via an inoculation of the CAM induced rapid and high mortality, about 72%, averaged over the four breeds, from a fibrosarcomatous liver tumour (LT) in CAM-susceptible chicks. In contrast, only 14% of chicks inoculated via the yolk sac died from liver tumours. Infection via the CAM reduced hatchability by about 28% (P<0.01) as compared to the hatchability of embryos infected via the yolk sac. About 21% of chicks which developed no pocks on the CAM after RSV inoculation developed liver tumours and died.

Kinetics of cytokine expression in bovine PBMCs and whole blood after in vitro stimulation with foot-and-mouth disease virus (FMDV) antigen

The interest in analysing antigen-specific cytokine responses has substantially increased in recent years, in part due to their use in assessing vaccine efficacy. In the present study, the kinetics of IL-2, IL-4 and IFN-γ expression was determined in bovine PBMCs by real-time PCR and in whole blood by cytokine-release assay after in vitro stimulation with recall foot-and-mouth disease virus (FMDV) antigen. The results showed that the cytokine mRNA of IL-2 and IFN-γ in PBMCs were induced early (peak induction at 6 h), whereas the IL-4 mRNA showed delayed induction (peaked at 24 h). In contrast, the kinetics of cytokine proteins in whole blood was different and required the accumulation of the proteins before being optimally detected. The peak accumulation of cytokine protein in whole blood was recorded at 72 h for IL-2 and IL-4, and 96 h for IFN-γ. The findings of this study are of importance when selecting an optimal time points for measuring antigen-specific cytokine expression in cattle.

An effective mannosylated chitosan nanoparticle DNA vaccine for FMD virus.

We are reporting you an effective DNA vaccine for FMD virus, and it was prepared using mannosylated chitosan (MC) nanoparticles to form the FMDV-pVAC-VP1-OmpA complex particles. These particles were characterized for their physical properties like morphology, size and charge prior evaluating the actual vaccine delivery effect of MC-nanoparticles. The immunological evaluation indicated that the 20 μg of DNA vaccine complexed with MC nano-particles was found optimum in inducing the immune response in G pigs as measured by FMDV specific neutralising antibodies and Th1/Th2 responses using micro SNT and ELISA, respectively.

The relationship between cellular immune response to foot-and-mouth disease virus Asia 1 and viral persistence in Indian cattle (Bos indicus)

Foot-and-mouth disease (FMD), the most contagious animal disease, is associated with persistent viral infection in ruminants, despite the induction of systemic immune response. The present study was performed to decipher the relation between the persistent FMD virus (FMDV) infection and cellular immune response in Indian cattle (Bosindicus) following experimental inoculation of FMDV Asia 1. Persistent viral infection (carriers) was detected by antigen capture RT-PCR on the oesophageal-pharyngeal fluid. Viral excretion was found to be intermittent and strongly variable among the persistently infected Indian cattle. Lymphocyte proliferative (LP) response, assessed as reactivity of peripheral blood mononuclear cells to FMDV Asia 1 antigen (Ag) was of low magnitude indicating a weak primary cellular immune response following infection. LP response to FMDV Ag was higher among the non-carriers than carriers of FMDV Asia 1. An enhanced LP response was associated with the lack of virus shedding in the OPF. The findings of this study are suggestive of relationship between cellular immune response and virus excretion during persistence of FMDV Asia 1 in infected cattle.

Kinetics of immune response to foot-and-mouth disease virus (type Asia 1) in experimental cattle

Humoral and mucosal (secretory antibody)immune response to FMDV type Asia 1 in cattle was analyzed after vaccination and infection using virus neutralizing test (VNT). Vaccination (1/16th the usual dose) failed to protect cattle from generalized clinical disease following experimental FMDV Asia 1 infection. Our results showed that infection induced higher and prolonged serum antibody titres indicating antigen mass is important for optimal immune response. Experimental FMDV infection induced significant secretory antibody (mucosal) response in cattle. Though, there was no difference in the serum antibody response between the cattle that developed generalized infection (unprotected) and those with only localized infection (protected), secretory antibody response differed, wherein the unprotected cattle had higher secretory response than protected cattle. Thus, FMDV Asia 1 infection stimulates a similar serum antibody response and a unique secretory antibody response among the infected cattle.