A Novel Synthetic TLR-4 Agonist Adjuvant Increases the Protective Response to a Clinical-Stage West Nile Virus Vaccine Antigen in Multiple Formulations.

West Nile virus (WNV) is a mosquito-transmitted member of the Flaviviridae family that has emerged in recent years to become a serious public health threat. Given the sporadic nature of WNV epidemics both temporally and geographically, there is an urgent need for a vaccine that can rapidly provide effective immunity. Protection from WNV infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. Despite many promising E-protein vaccine candidates, there are currently none licensed for use in humans. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) by combining it with a novel synthetic TLR-4 agonist adjuvant. Using the murine model of WNV disease, we find that inclusion of a TLR-4 agonist in either a stable oil-in-water emulsion (SE) or aluminum hydroxide (Alum) formulation provides both dose and dosage sparing functions, whereby protection can be induced after a single immunization containing only 100 ng of WN-80E. Additionally, we find that inclusion of adjuvant with a single immunization reduced viral titers in sera to levels undetectable by viral plaque assay. The enhanced protection provided by adjuvanted immunization correlated with induction of a Th1 T-cell response and the resultant shaping of the IgG response. These findings suggest that inclusion of a next generation adjuvant may greatly enhance the protective capacity of WNV recombinant subunit vaccines, and establish a baseline for future development.

Preclinical and clinical development of a dengue recombinant subunit vaccine.

This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 µg and 50 µg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 µg DEN1-80E and the 50 µg DEN1-80E formulations with Alhydrogel™ were generally well tolerated.

Advances in flavivirus vaccine development.

Flaviviruses comprise a diverse family of viruses that are cumulatively responsible for hundreds of millions of cases of infection annually. The Flavivirus genus includes both insect-vectored viruses, such as yellow fever and dengue, and non-vectored viruses such as HCV; the viruses have a broad range of disease presentation and geographic distribution. No specific antiviral therapies are currently available for the diseases caused by insect-vectored flaviviruses. Thus, efforts have been focused on the prevention of disease, through either vaccination or vector control, rather than on the treatment of infected individuals. While vector control can occasionally be successful in controlling the spread of flavivirus outbreaks, vaccines appear to be a more cost-effective, sustainable, and environmentally friendly approach. A review of vaccines for the medically important flaviviruses presents the full spectrum of vaccine options and complexity levels, and provides examples of successes and major challenges. The insect-borne flavivirus vaccine field is dynamic, with new and improved vaccines being advanced to replace existing vaccines, and novel vaccine approaches being developed for those targets that currently lack an approved vaccine. Advances in scientific knowledge and in the application of new technologies are helping to overcome some of the key challenges that have stymied the field for decades. New, safe and effective vaccines to protect against yellow fever, Japanese encephalitis, tick-borne encephalitis, West Nile and dengue viruses will likely result.

Uncoupling of promitogenic and antiapoptotic functions of IL-2 by Smad-dependent TGF-beta signaling.

TGF-beta opposes proliferative signaling by IL-2 through mechanisms that remain incompletely defined. In a well-characterized CD8(+) T cell model using wild-type and mutated IL-2 receptors, we examined the effects of TGF-beta on distinct IL-2 signaling events in CD8(+) T cells. IL-2 induces c-myc, cyclin D2, and cyclin E in a redundant manner through the Shc and STAT5 pathways. TGF-beta inhibited the ability of either the Shc or STAT5 pathway to induce these genes, as well as T cell proliferation. The inhibitory effects of TGF-beta were reversed by expression of a dominant-negative form of Smad3. TGF-beta did not impair proximal signaling by Shc or STAT5, and induction of some downstream genes, including cytokine-inducible Src homology-2-containing protein (CIS), bcl-x(L), and bcl-2, was spared. Experiments with c-fos, cyclin D2, and CIS reporter genes revealed that promoter-proximal regulatory elements dictate the sensitivity of IL-2 target genes to inhibition by TGF-beta. By leaving the Shc and STAT5 pathways functional while inhibiting their target genes selectively, TGF-beta was found to uncouple the proliferative and antiapoptotic functions of IL-2. Thus, TGF-beta is not a simple antagonist of IL-2, but rather serves to qualitatively modify the IL-2 signal to create a unique pattern of gene expression that neither cytokine can induce independently.