Expression, purification and characterisation of recombinant Escherichia coli derived chicken interleukin-12.

Zoonotic viruses, such as H5N1 Avian Influenza, pose major threats to both animals and humans, and with this in mind there is a need for the development of new anti-viral strategies. The cytokine interleukin-12 (IL-12) is known to play a pivotal regulatory role in the anti-viral response due to its role in the induction of the key anti-viral cytokine IFN-gamma. Therefore, strategies which provide a means for the production of therapeutic quantities of IL-12 may be of major benefit. Here we describe the development of biologically active Escherichia coli (E. coli) derived chicken IL-12 (ChIL-12). The single chain ChIL-12 gene was cloned into the pET32b expression vector, transformed into the BL-21 E. coli strain and expression induced with IPTG. Over expressed protein was solubilised with zwittergent detergent and isolated utilising Nickel ion affinity chromatography. Biological activity was determined as ChIL-12 stimulated proliferation of pre-treated T-cells in vitro. This study is the first example of a biologically active E. coli derived IL-12 from a non-mammalian vertebrate subsequently providing a means for testing the anti-viral therapeutic potential of ChIL-12 in an in vivo model.

Discovery and Synthesis of C-Nucleosides as Potential New Anti-HCV Agents.

Nucleoside analogues have long been recognized as prospects for the discovery of direct acting antivirals (DAAs) to treat hepatitis C virus because they have generally exhibited cross-genotype activity and a high barrier to resistance. C-Nucleosides have the potential for improved metabolism and pharmacokinetic properties over their N-nucleoside counterparts due to the presence of a strong carbon-carbon glycosidic bond and a non-natural heterocyclic base. Three 2'CMe-C-adenosine analogues and two 2'CMe-guanosine analogues were synthesized and evaluated for their anti-HCV efficacy. The nucleotide triphosphates of four of these analogues were found to inhibit the NS5B polymerase, and adenosine analogue 1 was discovered to have excellent pharmacokinetic properties demonstrating the potential of this drug class.

The chicken TH1 response: potential therapeutic applications of ChIFN-γ.

The outcomes of viral infections are costly in terms of human and animal health and welfare worldwide. The observed increase in the virulence of some viruses and failure of many vaccines to stop these infections has lead to the apparent need to develop new anti-viral strategies. One approach to dealing with viral infection may be to employ the therapeutic administration of recombinant cytokines to act as 'immune boosters' to assist in augmenting the host response to virus. With this in mind, a greater understanding of the immune response, particularly cell mediated T-helper-1 (TH1) type responses, is imperative to the development of new anti-viral and vaccination strategies. Following the release of the chicken genome, a number of TH1-type cytokines have been identified, including chicken interleukin-12 (ChIL-12), ChIL-18 and interferon-γ ChIFN-γ), highlighting the nature of the TH1-type response in this non-mammalian vertebrate. To date a detailed analysis of the in vivo biological function of these cytokines has been somewhat hampered by access to large scale production techniques. This review describes the role of TH-1 cytokines in immune responses to viruses and explores their potential use in enhancing anti-viral treatment strategies in chickens. Furthermore, this review focuses on the example of ChIFN-γ treatment of Chicken Anemia Virus (CAV) infection. CAV causes amongst other things thymocyte depletion and thymus atrophy, as well as immunosuppression in chickens. However, due to vaccination, clinical disease appears less often, nevertheless, the subclinical form of the disease is often associated with secondary complicating infections due to an immunocompromised state. Since CAV-induced immunosuppression can cause a marked decrease in the immune response against other pathogens, understanding this aspect of the disease is critically important, as well as providing insights into developing new control approaches. With increasing emphasis on developing alternative control programs for poultry diseases, novel therapeutic strategies provide one approach. We show here that the in ovo administration of ChIFN-γ impacts the depletion of T-cell precursors during CAV infection. Therefore, it appears that ChIFN-γ may have the potential to be used as a novel therapeutic reagent to impact virus infection and alter immunosuppression caused by CAV and potentially other pathogens.