Methods for Improving the Safety and Efficacy of Vaccines against Emerging Virus Pathogens

Principal Investigator: L. Markoff, MD
Office / Division / Lab: OVRR / DVP / LVVD

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Overview

Public Health Issue: In the past 20 years, diseases caused by novel viruses have emerged or increased dramatically in incidence. This phenomenon has stimulated a world-wide effort to develop new vaccines or to improve on the efficacy of existing ones. Live virus vaccines are desirable, because such vaccines have the greatest potential to elicit long-lasting protective immunity and are usually the most cost-effective. Many of the emerging viral pathogens are members of the genus flavivirus, including dengue, West Nile, Yellow Fever, and Japanese encephalitis (JE) viruses. Dengue and West Nile viruses are potential and real current threats to the US public health, respectively. The incidence of encephalitis caused by JE and of Yellow Fever is on the rise in Asia and South America, respectively. Our object is also to provide a link between FDA and US public health authorities, on one side, and organizations conducting global efforts to control these diseases, such as the WHO and the Pediatric Dengue Vaccine Initiative (PDVI), on the other side.

Regulatory Contribution: The staff of LVVD views our mission as the conduct of state of the art review of INDs and BLAs through our parallel pursuit of research goals that remain relevant to the regulatory mission of CBER and which enable us to provide insight into scientific issues that relate to the review process. Since the products we review are live or subunit or DNA-vectored virus vaccines derived by molecular techniques, we have elected to emphasize the study of mechanisms of flavivirus replication in our laboratory, especially as these phenomena affect virulence. Thus our research not only includes the role of virus genetic elements in replication in specific cell types and in animals, but also the role of virus genes in eliciting cell death and innate immune responses. By this approach, we hope to elucidate ways in which the safety of live vaccines can be enhanced. We believe that maintenance of a state-of-the-art research effort in this area enhances our capabilities as reviewers of the relevant products.

Research Approach: Most of the live flavivirus vaccines under development are based on an "infectious DNA". This term refers to a cloned DNA that can be transcribed into RNA in a test tube, such that the RNA product is infectious when put into cells. Using this approach, mutations can be introduced into virus genes by site-directed mutagenesis of DNA. This approach has huge advantages in comparison to the techniques that were available when existing live vaccines, like the Sabin polio vaccine and yellow fever vaccine were invented, because it also avoids the necessity of growing the vaccine virus in cells that have not been well characterized for the presence of occult, novel viruses that could potentially contaminate the vaccine virus preparation. Therefore, most of the research in our laboratory is also based on the construction of infectious DNAs and the insertion of site-specific mutations into cloned virus genomes. We have generated infectious DNA copies of the genomes of dengue, West Nile, and Japanese encephalitis viruses. These "flaviviruses" are all spread by mosquito bites, and dengue and West Nile viruses are potential and real current threats to the US public health, respectively. We introduce mutations into the infectious DNAs in the laboratory and then observe the effects of those mutations on the growth of viruses that result. Some of the mutant viruses are tested in mice and/or monkeys in order to evaluate both safety and efficacy. Results are analyzed so as to correlate the effects of mutations on virus virulence (as measured by host range specificity, replication efficiency, ability to cause cell death or to stimulate the innate immune system).

Mission Relevance and Outcomes: This work is directly relevant to the products we regulate, which include several that consist of novel live vaccine candidates targeted at these same pathogens. In addition, we have been successful in creating a candidate Dengue virus vaccine, and we are working on a novel West Nile virus vaccine (both are patented).

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Publications

RNA 2008 Dec;14(12):2645-56
Genome 3'-end repair in dengue virus type 2.
Teramoto T, Kohno Y, Mattoo P, Markoff L, Falgout B, Padmanabhan R

Vaccine 2008 Nov 5;26(47):5981-8
Attenuated West Nile viruses bearing 3'SL and envelope gene substitution mutations.
Yu L, Robert Putnak J, Pletnev AG, Markoff L

Virology 2008 Apr 25;374(1):170-85
Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.
Yu L, Nomaguchi M, Padmanabhan R, Markoff L

Vaccine 2007 Feb 26;25(10):1727-34
Neurologic disease associated with 17D-204 yellow fever vaccination: A report of 15 cases.
McMahon AW, Eidex RB, Marfin AA, Russell M, Sejvar JJ, Markoff L, Hayes EB, Chen RT, Ball R, Braun MM, Cetron M; the Yellow Fever Working Group

Antimicrob Agents Chemother 2006 Apr;50(4):1320-9
Triaryl pyrazoline compound inhibits flavivirus RNA replication.
Puig-Basagoiti F, Tilgner M, Forshey BM, Philpott SM, Espina NG, Wentworth DE, Goebel SJ, Masters PS, Falgout B, Ren P, Ferguson DM, Shi PY

J Virol 2005 Feb;79(4):2309-24
The topology of bulges in the long stem of the flavivirus 3' stem-loop is a major determinant of RNA replication competence.
Yu L, Markoff L

J Biol Chem 2004 Mar 26;279(13):12141-51
Requirements for West Nile virus (-)- and (+)-strand subgenomic RNA synthesis in vitro by the viral RNA-dependent RNA polymerase expressed in Escherichia coli.
Nomaguchi M, Teramoto T, Yu L, Markoff L, Padmanabhan R