Laboratory And Preclinical Studies Of Flaviviruses

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

The mosquito-borne members of the Flaviviridae family, contain a single-stranded positive-sense RNA genome and are the cause of yellow fever, dengue fever, Japanese encephalitis, Zika, and West Nile fever syndromes. In recent years, much of our laboratory effort was focused on the development and preclinical testing of dengue virus vaccine candidates suitable for inclusion in a live attenuated tetravalent vaccine. Clinical lots of each of these vaccine candidates were manufactured in prior years and have been evaluated individually and in combination in numerous Phase I and II clinical trials, Optimal tetravalent admixtures have been selected and have now completed a first Phase III evaluation. Although the dengue virus vaccine program is predominantly in a clinical mode at this time, considerable effort is currently devoted to support a number of important functions, including, 1) manufacture, replacement, maintenance, stability/sterility analysis, and distribution of clinical lots of vaccines suitable for study in human subjects, 2) basic research on virus stabilization,, 3) submission and laboratory support of IND applications for the clinical evaluation of tetravalent dengue vaccine formulations, 4) support of the seven companies/institutions that have licensed our vaccine technology or virus products, which includes consultative visits and clinical trial planning, development of manufacturing processes, preparation and shipping of vaccine seed or clinical lot viruses, assistance with sequence analysis, and sharing of IND/clinical trial data, 5) support of collaborations with investigators interested in basic virology or immunology studies, 6) use of immune cells collected from our clinical studies to investigate the innate immune response to vaccination, 7) characterization of epitopes recognized following dengue or Zika virus infection, and 8) refinement/qualification of laboratory assays such as the antibody neutralization assay, and 9) isolation and recombinant construction of suitable DENV-4 and DENV-1 strains for use as a potential clinical challenge strain. With the emergence of Zika virus in the Western hemisphere ur attention remains focused on the development of live attenuated vaccine candidates that would be compatible for co-formulation with our tetravalent dengue vaccine. Initially, recombinant chimeric viruses expressing the prM and E proteins of Zika virus on either the DENV-2 or DENV-4 background were generated and evaluated successfully in rhesus monkeys and human subjects. Although the chimeric candidate appeared to be safe for use in humans, it was observed to be over-attenuated with low infectivity and subsequently limited immunogenicity, even at an increased inoculation potency. Additional candidates were evaluated in preclinical studies in collaboration with LID and include full-length ZIKV candidates containing 3-UTR deletions and microRNA targets that restrict virus replication in tissues such as brain, placenta, epididymis, and certain macrophage types. Three of these candidates have been manufactured under cGMP by Charles River Laboratories and are IND applications are pending. In addition, collaborative studies continue to look at the pathology and immune responses of ZIKV in monkey models of infection and the use of the virus as a cytolytic viral agent against several cancer types. Mosquito-borne Japanese encephalitis virus (JEV) causes the most important viral encephalitis in the Asia Pacific region, accounting for more than 20,000 reported cases and 6,000 deaths annually. Efforts to develop a JEV vaccine continue in our laboratory and it is envisioned that a suitable live attenuated JEV vaccine could be combined with our live attenuated DEN virus vaccine to create a second-generation pentavalent vaccine for the control of these viruses in Southeast Asia. The laboratory has recovered numerous engineered viruses and has evaluated their pathogenicity in mice. The recombinant rJEV virus remains fully virulent in mice and provides a background for the evaluation of attenuating mutations. Sets of mutations derived from the attenuated SA14-14-2 vaccine virus produced in China have been introduced into the rJEV virus clone in order to evaluate the attenuating potential of mutations found in both the structural and non-structural genes. Following earlier evaluation in mice, a number of these recombinant viruses look suitable for evaluation in non-human primates and consideration as vaccine candidates. We are awaiting the re-commissioning of our BSL3 suite to enable us to move the JEV vaccine candidates forward. We plan to investigate the maturation state of DENV produced by live mosquitoes with a central hypothesis that viral the maturation state contributes to overall viral pathogenesis. To accomplish this we will optimize several techniques and variables: identify a specific combination of DENV and mosquito strains capable of producing sufficient virus titers for analysis, develop an efficient mosquito saliva/virus harvesting technique, and optimize molecular and biochemical methods for quantifying levels of viral maturation.