Bovino Interferon gamma Kit Elisa (A270342)

The aim of this work was to evaluate the immunogenicity and efficacy of DNA and MVA vaccines encoding the RVFV glycoproteins Gn and Gc in an ovine model of RVFV infection. Adult sheep of both sexes were challenged 12 weeks after the last immunization and clinical, virological, biochemical and immunological consequences, were analyzed. Strategies based on immunization with homologous DNA or heterologous DNA/MVA prime-boost were able to induce a rapid in vitro neutralizing antibody response as well as IFN? production after in vitro virus specific re-stimulation. In these animals we observed reduced viremia levels and less clinical signs when compared with mock-immunized controls. In contrast, sheep inoculated with a homologous MVA prime-boost showed increased viremia correlating with the absence of detectable neutralizing antibody responses, despite of inducing cellular responses after the last immunization. However, faster induction of neutralizing antibodies and IFN? production after challenge were found in this group when compared to the mock vaccinated group, indicative of a primed immune response. In conclusion, these results suggest that vaccination strategies based on DNA priming were able to mount and maintain specific anti-RVFV glycoprotein immune responses upon homologous or heterologous booster doses, warranting further optimization in large animal models of infection.

In most viral infections, recall T cell responses are critical for protection. The magnitude of these secondary responses can also affect the CD8 and CD4 epitope repertoire diversity. Bluetongue virus (BTV) infection in sheep elicits a T cell response that contributes to viremia control and could be relevant for cross-protection between BTV serotypes. Here, we characterized CD4⁺ and CD8⁺ T cell responses during primary and recall responses. During primary immune responses, both CD4⁺ and CD8⁺ T cell populations expanded by 14 days post-infection (dpi). CD4⁺ T cell populations showed a lower peak of expansion and prolonged contraction phase compared to CD8⁺ T cell populations. Recall responses to BTV challenge led to BTV-specific expansion and activation of CD8⁺ but not of CD4⁺ T cells. The evolution of the BTV-specific TCR repertoire was also characterized in response to VP7 peptide stimulation. Striking differences in repertoire development were noted over the time-course of infection. During primary responses, a broader repertoire was induced for MHC-I and MHC-II epitopes. However, during memory responses, a narrowed repertoire was activated towards a dominant motif in VP7 comprising amino acids 139-291. Monocytes were also examined, and expanded during acute infection resolution. In addition, pro-inflammatory cytokine levels increased after BTV inoculation and persisted throughout the experiment, indicative of a prolonged inflammatory state during BTV infections. These findings could have implications for vaccine design as the narrowing memory T cell repertoire induced after BTV re-infection could lead to the development of protective immunodominant TCR repertoires that differs between individual sheep.

Fasciolosis continues to be a major cause of economic losses in the livestock industry and a growing threat to humans. The limited spectrum of effective anthelmintics and the appearance of resistances urge the need for developing an effective vaccine. Most studies have been focused on the use of TH₁-polarizing adjuvants and the use of recombinant Fasciola critical molecules and, despite the efforts, no reproducible protections have been achieved. The F. hepatica MF6p/FhHDM-1 protein is a heme-binding protein also reported to have immunomodulatory properties, constituting a promising target for vaccination and/or as target for the development of new flukicides. Thus, in this study, we investigated the effects of the TH₁-polarizing adjuvant Quil A® on sheep immune response to MF6p/FhHDM-1, and the vaccine potential of both native and synthetic forms of this protein against ovine fasciolosis. Subcutaneous injection of Quil A® alone, i.e., without co-injecting any antigen, expands the antibody repertoire to MF6p/FhHDM-1 triggered by a subsequent primoinfection with metacercariae. This effect was not observed with aluminum hydroxide, the most frequently adjuvant used in commercial vaccines. On the other hand, vaccination with synthetic MF6p/FhHDM-1 in Quil A® prompted a 2-4-week delay in the antibody response induced in sheep by a challenge experimental infection. Moreover, fluke populations stablished showed stunted growth and low antigen release probably due to reduced metabolic activity. These observations suggest that primary circulating antibodies induced by the immunization had harmful effects on fluke development. Such effects could not be demonstrated to be associated to TH₁ immune response linked events (production of IgG₂ isotype antibodies and IFN-?).

Control of ovine brucellosis with subcellular vaccines can solve some drawbacks associated with the use of Brucella melitensis Rev.1. Previous studies have demonstrated that the polymeric antigen BLSOmp31 administered by parenteral route was immunogenic and conferred significant protection against B. ovis in rams. Immunization with BLSOmp31 by conjunctival route could be efficient for the induction of mucosal and systemic immune responses. In this work, we evaluated the conjunctival immunization using a thermoresponsive and mucoadhesive in situ gel composed of Poloxamer 407 (P407) and chitosan (Ch) as vaccine delivery system for BLSOmp31 in rams. Serum samples, saliva, lacrimal, preputial and nasal secretions were analyzed to measure specific IgG and IgA antibodies. Cellular immune response was evaluated in vivo and in vitro. Immunization with BLSOmp31-P407-Ch induced high IgG antibody levels in serum and preputial secretions which remained at similar levels until the end of the experiment. Levels of IgG in saliva, lacrimal and nasal secretions were also higher compared to unvaccinated control group but decreased more rapidly. IgA antibodies were only detected in nasal and preputial secretions. BLSOmp31-P407-Ch stimulated a significant cellular immune response in vivo and in vitro. The induction of systemic and local immune responses indicates a promising potential of P407-Ch for the delivery of BLSOmp31 by conjunctival route.

The measurement of interferon gamma-induced protein 10 (IP-10) in antigen-stimulated whole blood is a sensitive biomarker of Mycobacterium bovis infection in African buffaloes (Syncerus caffer). However, this species often occurs in remote locations and diagnostic samples must be transported to centralised laboratories for processing. In humans, plasma IP-10 is highly stable and this feature contributes to its diagnostic utility; for this reason we aimed to characterize the stability of this molecule in buffaloes. Blood from M. bovis-infected and -uninfected animals was incubated with pathogen-specific peptides, saline and phytohaemagglutinin, respectively. Plasma fractions were harvested and aliquots of selected samples were: (i) stored at different temperatures for various times; (ii) heat treated before storage at RT, and (iii) stored on Protein Saver Cards (PSCs) at RT for either 2 or 8 weeks before measurement of IP-10. Incubation of plasma at 65°C for 20 min caused no loss of IP-10 and this protein could be quantified in plasma stored on PSCs for 2 and 8 weeks. Moreover, for all storage conditions, IP-10 retained its excellent diagnostic characteristics. These features of IP-10 might allow for the heat inactivation of potentially infectious plasma which would facilitate the safe and simple transport of samples.