Pervaiz A. Dar

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.

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.

DNA prime-protein boost strategy with replicase-based DNA vaccine against foot-and-mouth disease in bovine calves.

The limited efficacy of DNA vaccines against foot-and-mouth disease (FMD) in cattle and other natural hosts has prompted a search for a more effective vaccination regimen. In this study we tested a DNA prime-protein boost vaccination strategy against FMD in bovine calves. We used purified recombinant FMDV specific multi-epitope protein (rMEG990) and an optimized sindbis virus replicase-based DNA vaccine expressing this protein (pSinCMV-Vac-MEG990). We demonstrate that vaccination with a low dose of pSinCMV-Vac-MEG990 (10 μg/animal) and subsequently boosting with rMEG990 resulted in induction of neutralizing antibodies, IFN-γ production and protection against homologous virus challenge. However, vaccination with a high dose of pSinCMV-Vac-MEG990 (100 μg/animal) and boosting with rMEG990 resulted in significantly lower immune responses and more severity to the challenge test. Additionally, we show that the post-vaccinal IFN-γ levels in animals correlated positively to their protection against FMDV challenge. These findings suggest that a replicase-based DNA vaccine in proper prime-boost combination may offer an efficient vaccine strategy against FMDV and that IFN-γ could be used as an additional immune parameter to predict protection against FMDV infection.

Infection with foot-and-mouth disease virus (FMDV) induces a natural killer (NK) cell response in cattle that is lacking following vaccination

Natural killer (NK) cells play a role in innate antiviral immunity by directly lysing virus-infected cells and producing antiviral cytokines such as interferon gamma (IFN-γ). We developed a system for characterizing the bovine NK response to foot-and-mouth disease virus (FMDV), which causes a disease of cloven-hoofed animals and remains a threat to livestock industries throughout the world. IL-2 stimulation of PBMC resulted in poor killing of human K562 cells, which are often used as NK target cells, while lysis of the bovine BL3.1 cell line was readily detected. Depletion of NKp46-expressing cells revealed that 80% of the killing induced by IL-2 could be attributed to NKp46(+) cells. In order to characterize the response of NK cells against FMDV in vivo, we infected groups of cattle with three different strains of the virus (A24 Cruzeiro, O1 Manisa, O Hong Kong) and evaluated the cytolytic ability of NK cells through the course of infection. We consistently observed a transient increase in cytolysis, although there was variation in magnitude and kinetics. This increase in cytolysis remained when CD3(+) cells were removed from the preparation of lymphocytes, indicating that cytolysis was independent of MHC-T cell receptor interaction or γδ T cell activation. In contrast, animals monitored following vaccination against FMDV did not exhibit any increase in NK killing. These data suggest that NK cells play a role in the host immune response of cattle against FMDV, and contrast with the suppression of NK activity previously observed in swine infected with FMDV.

Sindbis virus replicase-based DNA vaccine construct encoding FMDV-specific multivalent epitope gene: studies on its immune responses in guinea pigs.

Foot‐and‐mouth disease (FMD) is still a perennial global menace affecting livestock health and production. It is imperative to figure out new ways to curb this disease. In this study, a sindbis virus replicase‐based DNA vaccine, pSinCMV‐Vac‐MEG990, encoding a multivalent epitope gene (representing tandemly linked VP1 C‐terminal halves of three foot‐and‐mouth disease virus (FMDV) serotypes) was constructed. In vitro transfection studies in BHK‐21 cells revealed that the construct was able to express FMDV‐specific antigen but does not overproduce the antigen. Immunization of guinea pigs with the construct at dose rate of 10, 5, 2 and 1 μg per animal through intramuscular route showed significant neutralizing antibody induction at all doses against all serotype tested as compared to non‐immunized controls. On viral challenge of guinea pigs 4 week post‐immunization with 1000 GPID50 of FMDV serotype A, it was observed that the immunization not only delayed the appearance and reduced the severity of FMD lesions significantly (P < 0.05) but also provided complete protection in several guinea pigs. In fact, two of six and one of six guinea pigs were completely protected in 10 and 5 μg immunized groups, respectively. These results suggest that the development of the replicase‐based DNA vaccine may provide a promising approach as an alternative vaccine strategy for controlling FMD.

Bacterial flagellin—a potent immunomodulatory agent

Flagellin is a subunit protein of the flagellum, a whip-like appendage that enables bacterial motility. Traditionally, flagellin was viewed as a virulence factor that contributes to the adhesion and invasion of host cells, but now it has emerged as a potent immune activator, shaping both the innate and adaptive arms of immunity during microbial infections. In this review, we summarize our understanding of bacterial flagellin and host immune system interactions and the role flagellin as an adjuvant, anti-tumor and radioprotective agent, and we address important areas of future research interests.