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  1. Biologics Research Projects

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

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

General Overview

In the past 20 years, previously unknown viruses and viruses that were previously geographically restricted have emerged as major threats to public health. This has stimulated a global effort to develop new vaccines or to improve the efficacy of existing ones.

Among the emerging mosquito-borne viral pathogens are dengue, West Nile, Yellow Fever, and Japanese encephalitis (JE) viruses. While dengue is a potential threat to US public health, West Nile viruses are already a real threat. The incidence of encephalitis caused by JE in Asia and of Yellow Fever in South America is on the rise. Most recently, disease caused by the mosquito-borne chikungunya virus has emerged in Africa and South Asia and is a potential threat to the US.

It is possible for attenuated (weakened) versions of these viruses to be used as vaccines against the diseases they cause. Live virus attenuated vaccines hold the greatest potential for stimulating long-lasting protective immunity, and they are usually the most cost-effective strategy for doing so. Our laboratory conducts research on dengue, West Nile, and chikungunya viruses in order to understand how the genetic material of these viruses replicate inside infected cells and how the virus builds copies of itself during infections. This knowledge may inform how to develop safe and effective attenuated strains of these viruses. Indeed, we have patents on attenuated dengue and West Nile viruses, and we collaborated with the National Institute of Allergy and Infectious Diseases of NIH to develop a live, attenuated dengue vaccine that is now in clinical trials.

Our strategy is to alter the ability of viruses to grow in cells by making changes in their genetic material. In some cases, the changes we create reduce the ability of the virus to cause disease. This work helps us to understand the complexities of novel candidate vaccines that may be submitted to FDA for review.

We also provide a link between FDA and US public health authorities and organizations such as the World Health Organization and the Pediatric Dengue Vaccine Initiative (PDVI), which conduct global programs to control these diseases.

Scientific Overview

Flaviviruses contain positive- or "messenger"-sense RNA genomes. It is very difficult to introduce mutations into RNA in a site-specific manner, so most of the live flavivirus vaccines under development are based on use of an "infectious DNA." This term refers to a cloned DNA that can be transcribed into genome RNA that can produce virus after transfection of cells. Thus, mutations can be introduced into RNA virus genes by site-directed mutagenesis of DNA. Infectious DNA has significant advantages compared to techniques used historically to make live vaccines, such as the Sabin polio vaccine and yellow fever vaccine. For example, it avoids the passage of virus in cells that have not been well characterized for the presence of adventitious agents that could contaminate the vaccine virus.

The goal of our research is to study how these RNA viruses replicate and cause disease. To this end, we have previously generated infectious DNA copies of the genomes of dengue, West Nile, and Japanese encephalitis viruses--flaviviruses spread by mosquito bites.

Currently, we are generating an infectious DNA for the RNA genome of the alfavirus, chikungunya virus (CHIKV). CHIKV is the cause of a major epidemic of CHIK fever endemic in Africa and South Asia since 2005. This virus is a potential threat to US health because it is spread by Aedes albopictus mosquitoes, which are abundant in the Eastern US. Furthermore, no one in the US has protective immunity to CHIK.

Our plan for work on CHIKV will follow the pattern we have previously established. We will initially observe the effects of mutations on the replication of mutant chikungunya viruses derived in vitro in a variety of mammalian and insect cell types. We will determine how a given mutation in the CHIKV genome alters virus replication at the level of RNA synthesis, packaging and assembly, and/or virion morphogenesis. Mutant CHIK viruses that fill necessary criteria based on the results are then tested in mice and/or monkey models of disease. These tests assess the effects of mutations on virulence as measured by tissue tropism, replication efficiency as evidenced by titers of virus in blood and organs, and ability to stimulate an immune response comparable to that stimulated by wild-type parent virus.

In similar studies, we previously generated mutants of dengue-2 virus that failed to replicate well in cultured mosquito cells due to a specific mutation in the 3' non-coding region of the genome, a conserved stem-loop structure known as the 3'-SL. Subsequently, this virus was shown to be attenuated in a monkey model. Further we showed that the identical mutation had a similar phenotype in the context of the dengue-1 genome and was a feasible method for generating a tetravalent dengue vaccine. Subsequently, we used a similar strategy to attenuate West Nile virus, during which effort we gained important information regarding the role of the 3'SL in virus replication.


Virology 2017 Jun;506:130-40
Characterization of virus-specific vesicles assembled by West Nile virus non-structural proteins.
Yu L, Takeda K, Gao Y

Vaccine 2014 Apr 17;32(19):2225-30
Stability of neuraminidase in inactivated influenza vaccines.
Sultana I, Yang K, Getie-Kebtie M, Couzens L, Markoff L, Alterman M, Eichelberger MC

Virology 2013 Nov;446(1-2):365-77
Protein-protein interactions among West Nile non-structural proteins and transmembrane complex formation in mammalian cells.
Yu L, Takeda K, Markoff L

N Engl J Med 2013 Feb 21;368(8):689-91
Yellow fever outbreak in Sudan.
Markoff L

J Biol Chem 2011 Jun 24;286(25):22521-34
Identification of cis-acting elements in the 3'-untranslated region of the dengue virus type 2 RNA that modulate translation and replication.
Manzano M, Reichert ED, Polo S, Falgout B, Kasprzak W, Shapiro BA, Padmanabhan R

Vaccine 2011 Mar 21;29(14):2601-6
Influenza neuraminidase-inhibiting antibodies are induced in the presence of zanamivir.
Sultana I, Gao J, Markoff L, Eichelberger MC

Vaccine 2010 Apr 9;28(17):3030-7
Identification of mutations in a candidate dengue 4 vaccine strain 341750 PDK20 and construction of a full-length cDNA clone of the PDK20 vaccine candidate.
Kelly EP, Puri B, Sun W, Falgout B

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


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