Evaluation of viral cytokine vectors for gene therapy

Abstract

Viral-vectors encoding adjuvant cytokines have applications in vaccine development, gene therapy and immunology research. Virally encoded cytokines have been shown to limit viral-vector replication yet enhance immune responses in animal models. Nevertheless, concerns over the safety of this technology remain, which has prevented licensing for human use. Cytokines are immune modulators that can display context specific properties; therefore the virally encoded cytokines have the potential to elicit different effects depending on the microenvironments in which they are expressed. Hence the evaluation of viral-cytokine-vectors should be performed in a range of immune contexts. In this study, for the first time, the effects of viral-cytokine-vectors were evaluated in mice with Toll-like receptor (TLR) deletions. TLRs have been identified as primary immune sensors that recognize conserved microbial features and induce cytokine programs based on the class of pathogen encountered. Notably, TLRs induce type-l interferons (IFNs) and interleukin (IL)-12 on viral encounter; hence TLR gene knock-out (GKO) mice are susceptible to a range of viral pathogens. In the first part of this study, the role of TLRs in resistance to ectromelia virus (ECTV) was determined. Results indicated that TLR-9, but not TLRs 2/4/7, is critical for resistance to ECTV. Mice with deletion of the TLR adaptor protein MyD88 were also highly susceptible to the infection. MyD88{u207B}/{u207B} mice displayed reduced levels of the inflammatory cytokine IFN-{u03B3} in serum compared to control mice. The requirement of MyD88 for NK cell activation and CD8 T cell expansion following vaccinia virus (VV) infection was also demonstrated. Collectively, the data indicate that MyD88{u207B}/{u207B} mice display profound immune deficiency following poxvirus infections thus providing a novel platform for the evaluation of viral-cytokine-vectors. Two recombinant VVs (rVVs), encoding IFN-{u03B2} or IL-2, with known antiviral effects in wild-type mice, were characterized in MyD88{u207B}/{u207B} mice. Results indicated that IFN-{u03B2} and IL-2 co-expression restored CD8 T cell and NK cell responses against VV in MyD88{u207B}/{u207B} mice, respectively. The adjuvant effect of IFN-{u03B2} was not observed in wild{u00AC}type control mice indicating that the immunobiology of specific co-expressed cytokines can be more perceptible in the absence of host immune responses. Another major finding of the study was that rVV-IL-2, despite restoring NK cell responses in MyD88{u207B}/{u207B} mice, did not display attenuated virus growth as was found in wild-type and athymic nude mice. Administering a higher dose ofthe rVV-IL-2 induced lethality in MyD88{u207B}/{u207B} mice, while the rVV-control strain did not indicating that rVV-IL-2 is highly pathogenic in MyD88{u207B}/{u207B} mice. Hence, rVV-IL-2, previously thought to be a safer vector for immuno-deficient recipients, can be deleterious in the absence of host TH1-type immunity. The converse experiment involved testing rVV-IL-2 in a strongly TH1 polarized context by co-infection with rVV-IL-12. Remarkably the combined rVV-IL-2 / rVV-IL-12 regime induced a MyD88-dependent cytokine storm. In view of the deleterious phenotype, the effect of rVV-IL-2 was further tested in the context of influenza virus type A, a pathogen with strong immune subversion (virulence) characteristics. Results indicated higher susceptibility of wild-type mice to influenza-A infection following rVV-IL-2 infection. In contrast to rVV-IL-2, intranasal (i.n.) delivery of rVV-IFN-{u03B2} protected mice against distinct lethal viral challenges, specifically, Japanese encephalitis virus (JEV), influenza-A virus and ECTV. The antiviral protection was mediated at the lung and brain indicating that i.n. delivery of this vector induces immune-modulation in these therapeutically significant anatomical compartments. The antiviral effect elicited by rVV-IFN-{u03B2} was also shown in MyD88{u207B}/{u207B} mice, indicating that the antiviral effects are not reliant on increased expression of MyD88-dependent TLRs. In conclusion, the studies demonstrate the feasibility of a new research approach that involves studying viral-cytokine-vectors in pattern-recognition receptor GKO mice.