Since the first demonstration of in vivo gene expression from an injected RNA molecule almost two decades ago, the field of RNA-based therapeutics is now taking significant strides, with many cancer and infectious disease targets entering clinical trials. Critical to this success has been advances in the knowledge and application of delivery formulations. Currently, various lipid nanoparticle (LNP) platforms are at the forefront, but the encapsulation approach underpinning LNP formulations offsets the synthetic and rapid-response nature of RNA vaccines. Second, limited stability of LNP formulated RNA precludes stockpiling for pandemic readiness. Here, we show the development of a two-vialed approach wherein the delivery formulation, a highly stable nanostructured lipid carrier (NLC), can be manufactured and stockpiled separate from the target RNA, which is admixed prior to administration. Furthermore, specific physicochemical modifications to the NLC modulate immune responses, either enhancing or diminishing neutralizing antibody responses. We have combined this approach with a replicating viral RNA (rvRNA) encoding Zika virus (ZIKV) antigens and demonstrated a single dose as low as 10 ng can completely protect mice against a lethal ZIKV challenge, representing what might be the most potent approach to date of any Zika vaccine.
The emergence and re-emergence of highly virulent viral pathogens with the potential to cause a pandemic creates an urgent need for the accelerated discovery of antiviral therapeutics. Antiviral human monoclonal antibodies (mAbs) are promising candidates for the prevention and treatment of severe viral diseases, but their long development timeframes limit their rapid deployment and use. Here, we report the development of an integrated sequence of technologies, including single-cell mRNA-sequence analysis, bioinformatics, synthetic biology and high-throughput functional analysis, that enables the rapid discovery of highly potent antiviral human mAbs, the activity of which we validated in vivo. In a 78-d study modelling the deployment of a rapid response to an outbreak, we isolated more than 100 human mAbs that are specific to Zika virus, assessed their function, identified that 29 of these mAbs have broadly neutralizing activity, and verified the therapeutic potency of the lead candidates in mice and non-human primate models of infection through the delivery of an antibody-encoding mRNA formulation and of the respective IgG antibody. The pipeline provides a roadmap for rapid antibody-discovery programmes against viral pathogens of global concern.
The X region of human T-cell leukemia virus type I (HTLV-I) encodes two proteins that regulate viral gene expression. The tax protein is the product of the transactivator gene and has been shown to up-regulate the expression of some cellular genes controlling T-cell replication, including that of the interleukin-2 (IL-2) T-cell growth hormone and the a chain of its receptor (IL-2R). Several studies have shown that tax transactivation of the IL-2R a-chain promoter is mediated by binding sites for the transcriptional activator NF-KB, and this mechanism has also been implicated in the tax activation of IL-2 promoter activity. The rex gene product of HTLV-I regulates viral protein production by influencing mRNA expression and has been implicated in the stabilization of IL-2R a-chain mRNA. In the present studies, the ability of the tax and rex proteins to transactivate IL-2 gene expression has been reinvestigated. The ability of the tax protein to transactivate IL-2 promoter activity appears, at least in part, to be mediated by the recognition sequence for a DNA-binding complex known as CD28RC. Consistent with this hypothesis is the observation that tax-mediated activation of IL-2 gene expression is resistant to the immunosuppressive affects of cyclosporin A, a property postulated for the CD28RC binding complex. Unexpectedly, this tax-mediated up-regulation of IL-2 expression is synergized by the presence of the rex protein. These findings demonstrate that transactivation of IL-2 gene expression by tax is augmented by mechanisms distinct from NF-cB and raise the possibility that rex, as well as tax, contributes to the oncogenic capability of HTLV-I by altering the expression of the IL-2 gene in T cells infected with this retrovirus. * Corresponding author. pression of the IL-2 gene is tightly controlled, and activation of this gene product requires multiple signals. At the transcriptional level, the expression of the human IL-2 gene is activated by several transcription factors (for a review, see reference 46). One of these is the inducible NFAT-1 protein which is necessary but not sufficient for IL-2 gene activity through the T-cell receptor. The abrogation of IL-2 promoter activity by the immunosuppressant cyclosporin A has been shown to be due to a block in the activity of this transcription factor (12). Other proteins, such as NF-KB, AP-1 (from c-fos and c-jun), and octamer binding proteins, are also reported to be involved but not sufficient for IL-2 gene activation. The studies presented here show that transactivation of the IL-2 promoter by tax is mediated, at least in part, by the binding site for the CD28RC complex which is activated by CD28 stimulation of T cells (13). In addition, endogenous IL-2 production and IL-2 promoter activity are shown to be resistant to the immunosuppressive effects of cyclosporin A in the presence of tax. This response appears to be synergized by the presence of rex. These data support the hypothesis that IL-2 gene activation in the presence of HTLV-I proteins may be via a pathway...
Synthetic biology has allowed for the industrial production of supply-limited sesquiterpenoids such as the antimalarial drug artemisinin and β-farnesene. One of the only unmodified animal products used in medicine is squalene, a triterpenoid derived from shark liver oil, which when formulated into an emulsion is used as a vaccine adjuvant to enhance immune responses in licensed vaccines. However, overfishing is depleting deep-sea shark populations, leading to potential supply problems for squalene. We chemically generated over 20 squalene analogues from fermentation-derived β-farnesene and evaluated adjuvant activity of the emulsified compounds compared to shark squalene emulsion. By employing a desirability function approach that incorporated multiple immune readouts, we identified analogues with enhanced, equivalent, or decreased adjuvant activity compared to shark squalene emulsion. Availability of a library of structurally related analogues allowed elucidation of structure-function relationships. Thus, combining industrial synthetic biology with chemistry and immunology enabled generation of sustainable terpenoid-based vaccine adjuvants comparable to current shark squalene-based adjuvants while illuminating structural properties important for adjuvant activity.
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