Summary: Type I interferons (IFNs) inhibit viral replication and cell growth and enhance the immune response, and therefore have many clinical applications. IFN‐α2b ranks third in world market use for a biopharmaceutical, behind only insulin and erythropoietin. The average annual cost of IFN‐α2b for the treatment of hepatitis C infection is $26 000, and is therefore unavailable to the majority of patients in developing countries. Therefore, we expressed IFN‐α2b in tobacco chloroplasts, and transgenic lines were grown in the field after obtaining United States Department of Agriculture Animal and Plant Health Inspection Service (USDA‐APHIS) approval. Stable, site‐specific integration of transgenes into chloroplast genomes and homoplasmy through several generations were confirmed. IFN‐α2b levels reached up to 20% of total soluble protein, or 3 mg per gram of leaf (fresh weight). Transgenic IFN‐α2b had similar in vitro biological activity to commercially produced PEG‐Intron™ when tested for its ability to protect cells against cytopathic viral replication in the vesicular stomatitis virus cytopathic effect (VSV CPE) assay and to inhibit early‐stage human immunodeficiency virus (HIV) infection. The antitumour and immunomodulating properties of IFN‐α2b were also seen in vivo. Chloroplast‐derived IFN‐α2b increased the expression of major histocompatibility complex class I (MHC I) on splenocytes and the total number of natural killer (NK) cells. Finally, IFN‐α2b purified from chloroplast transgenic lines (cpIFN‐α2b) protected mice from a highly metastatic tumour line. This demonstration of high levels of expression of IFN‐α2b, transgene containment and biological activity akin to that of commercial preparations of IFN‐α2b facilitated the first field production of a plant‐derived human blood protein, a critical step towards human clinical trials and commercialization.

The chloroplast bioreactor is an alternative to fermentation-based systems for production of vaccine antigens and biopharmaceuticals. We report here expression of the plague F1-V fusion antigen in chloroplasts. Site-specific transgene integration and homoplasmy were confirmed by PCR and Southern blotting. Mature leaves showed the highest level of transgene expression on the third day of continuous illumination, with a maximum level of 14.8% of the total soluble protein. Swiss Webster mice were primed with adjuvant-containing subcutaneous (s.c.) doses of F1-V and then boosted with either adjuvanted s.c. doses (s.c. F1-V mice) or unadjuvanted oral doses (oral F1-V mice). Oral F1-V mice had higher prechallenge serum immunoglobulin G1 (IgG1) titers than s.c. F1-V mice. The corresponding serum levels of antigen-specific IgG2a and IgA were 2 and 3 orders of magnitude lower, respectively. After vaccination, mice were exposed to an inhaled dose of 1.02 × 10⁶CFU of aerosolizedYersinia pestisCO92 (50% lethal dose, 6.8 × 10⁴CFU). All control animals died within 3 days. F1-V given s.c. (with adjuvant) protected 33% of the immunized mice, while 88% of the oral F1-V mice survived aerosolizedY. pestischallenge. A comparison of splenicY. pestisCFU counts showed that there was a 7- to 10-log reduction in the mean bacterial burden in survivors. Taken together, these data indicate that oral booster doses effectively elicit protective immune responses in vivo. In addition, this is the first report of a plant-derived oral vaccine that protected animals from liveY. pestischallenge, bringing the likelihood of lower-cost vaccines closer to reality.

Eradication of HIV infection depends on the elimination of a small, but stable population of latently infected T cells. After the discontinuation of therapy, activation of latent virus can rekindle infection. To purge this reservoir, it is necessary to define cellular signaling pathways that lead to activation of latent HIV. We used the SCID-hu (Thy/Liv) mouse model of HIV latency to analyze a broad array of T cell-signaling pathways and show in primary, quiescent cells that viral induction depends on the activation of two primary intracellular signaling pathways, protein kinase C or nuclear factor of activated T cells (NF-AT). In contrast, inhibition or activation of other important T cell stimulatory pathways (such as mitogen-activated protein kinase, calcium flux, or histone deacetylation) do not significantly induce virus expression. We found that the activation of NF-κB is critical to viral reactivation; however, all pathways that stimulate NF-κBdonot reactivate latent virus. Our studies further show that inhibition of NF-κB does not prevent activation of HIV by NF-AT, indicating that these pathways can function independently to activate the HIV LTR. Thus, we define several molecular pathways that trigger HIV reactivation from latency and provide evidence that latent HIV infection is maintained by the functional lack of particular transcription factors in quiescent cells.

The host cell activation state impacts the nature of human immunodeficiency virus infection. Activated cells facilitate productive infections; quiescent cells enable the virus to enter a latent state, the major obstacle to viral clearance. We wanted to understand how these differences affected viral gene expression. In quiescent cells activated prior to infection, viral RNA was seen 12 h postinfection; when cells were stimulated postinfection, viral RNA was not seen until 36 h postinfection. Up-regulation of viral RNA in latently infected cells occurred within 2 h poststimulation. This hierarchy also held true for viral protein production. These results may explain the rapid reemergence of viremia following termination of therapy.