T H E   N I H   C A T A L Y S T     J U L Y  –  A U G U S T   2005

Postbac Poster Day, May 5, 2005: A Small Sampler


by Aarthi Ashok
and Annie Nyugen

From Virus to Vaccine: Creating a Live Attenuated Vaccine for Dengue, Type 2

Caroline Agrawal, Boston University.
Preceptors: Steve Whitehead and Joseph Blaney, NIAID, Laboratory of Infectious Diseases

Dengue is a mosquito-borne virus with serotypes 1, 2, 3, and 4, which are prevalent in most of the tropical areas of the world. Dengue causes a flu-like illness that can result in severe or fatal complications when a previously infected individual encounters a secondary infection with a different serotype of the virus. No effective vaccines are currently available.

Agrawal was interested in designing a live attenuated vaccine strain of dengue virus in the laboratory primarily because of its immediate clinical applications. She adopted a reverse genetics approach, using PCR mutagenesis to modify the genome of a dengue serotype 2 strain in order to attenuate the virus.

She has been able to design two candidate vaccine strains: a dengue type 2 strain with a 30-nucleotide deletion in the 3'-UTR sequence (D-30) and a 3-nucleotide deletion in the NS3 viral protein that has both helicase and protease activities; and a dengue type 2/4 chimera that has the structural protein of a type 2 strain in a type 4 background in addition to two point substitutions in the NS5 polymerase protein.

The ability of these candidate vaccine strains to grow in Vero cells, the intended vaccine production cell line, was then assessed. Although the type 2/4 chimera candidate grew efficiently in these cells, Agrawal and colleagues were surprised to find that the type 2 D-30 strain with the single amino acid deletion in NS3 was unable to grow in these cells. Agrawal is currently exploring the possible reasons for this surprising finding.

In future studies, the candidate strains will be injected directly into human liver tumor cells in SCID mice to assess their level of attenuation. If these candidate strains show promise in the rodent model, they will be used to vaccinate rhesus monkeys prior to challenge with wild-type virus. The titer of neutralizing antibody in these monkeys and the development of significant viremia or side effects will be closely monitored. If these strains are attenuated in monkeys, they will be included in a tetravalent dengue vaccine formulation to be used in human clinical trials.

Agrawal plans to continue to study viruses during her graduate education at Duke University in Chapel Hill, N.C.

Aarthi Ashok

Caroline Agrawal


RNase H1 and Cell Viability: In vitro RNAi Knockdown of Rnase H1

Robert C. Wirka, University of Wisconsin, Madison.
Preceptor: Robert Crouch, NICHD, Laboratory of Molecular Genetics

Ribonucleases H (RNases H) are important enzymes required for the removal of RNA in the RNA-DNA hybrids formed during DNA replication in eukaryotic cells and during the conversion of the viral RNA genome to DNA during HIV replication. In the latter case, the virally encoded reverse transcriptase (RT) contains the RNase H activity.

Wirka was interested in studying the function of RNase H1 during normal cell growth and proliferation. Previous experiments in their lab had shown that RNase H1 gene knockout deprives mice of the ability to replicate mitochondrial DNA and is thus lethal.

Because cells from these mice could not be grown up to examine any cellular defects, Wirka decided to knock out RNase H1 function using siRNA. To this end, he constructed a RNase H1-GFP fusion construct that was transfected into human osteosarcoma cells by electroporation. He also co-transfected this construct with various anti-RNase H1 siRNAs and quantified the knockdown of expression by FACS analysis.

Wirka was able to obtain specific knockdown of RNase H1 expression using this system, with siRNA electroporation efficiencies estimated at 99 percent, RNase H1-GFP knockdown at about 70 percent, and construct transfection efficiencies at about 70 percent.

He plans to continue using and optimizing this siRNA strategy to knock down expression of endogenous RNase H1 in untransfected human osteosarcoma cells. Using RT-PCR and Western blot analysis, he hopes to determine the efficiency of knockdown of RNase H1 expression in these cells.

RNase H is critical for the production of infectious HIV and is therefore an excellent target for therapeutic drugs, Wirka notes. This research, he says, may inform the development of HIV RNase H drugs that are extremely specific for the viral enzyme.

"This has been an excellent experience for me," says Wirka, who hopes to remain involved in research during his clinical training at The Cleveland Clinic Lerner College of Medicine.

Aarthi Ashok

Robert Wirka


Glycodendrimers: Novel Inhibitors of HIV-1 Infection

Benitra Johnson, South Carolina State University, Orangeburg.
Preceptors: Robert Blumenthal and Anu Puri, NCI, Laboratory of Experimental and Computational Biology

Glycodendrimers are synthetic multivalent carbohydrate conjugates derived from analogous moieties found on cell membrane surfaces; they play a role in the binding interactions of certain pathogens with mammalian cells.

Previous studies have shown that glycodendrimers inhibit viral entry into host cells.

Johnson carried out experiments using a reporter-gene–based assay to measure the effectiveness of a novel glycodendrimer in blocking HIV-1 infectivity.

Successful viral entry and integration into the host cell’s genome leads to the expression of the luciferase reporter gene; quantification of luciferase luminescence serves as an indirect measure of HIV infection. Preliminary data, Johnson reported, indicate that glycodendrimers effectively inhibit HIV-1 entry.

Although the mechanism of inhibition remains unknown, Johnson offers two possible hypotheses: There may be some form of modulation of the virus-cell interaction and/or there may be some interaction between the glycodendrimers and the host cell, leading to altered cell physiology and abortion of infection.

Continuing glycodendrimer research, she says, may lead to additional avenues to counter HIV, which persists despite the expanding marketplace of antiviral agents.

Johnson is contemplating applying to medical school.

Annie Nguyen

Benitra Johnson


Magnetic Resonance Imaging in Experimental Cerebral Ischemia

Naomi Lewin, University of Maryland, College Park.
Preceptor: Lawrence Latour, NINDS, Stroke Branch

The aim of Lewin’s research is to develop noninvasive MRI techniques to track cerebral changes in animal models of stroke throughout the course of stroke induction and recovery.

Stroke is induced in the rat by inserting a silicon-coated suture into the middle cerebral artery; the suture is removed after a given amount of time.

The acute phase of stroke is characterized by cell death resulting from ischemia, or decreased blood flow, either by direct or indirect causes such as apoptosis and inflammation.

Two MRI techniques were used to observe neurological damage in rats. Perfusion-weighted MRI using gadolinium contrast agent reveals light regions on the mean-transit-time map that signify decreased blood flow—which is interpreted to reflect areas at risk for damage.

In addition, diffusion-weighted MRI, in which light regions indicate areas of cell death and cell swelling, delineate actual lesions.

Lewin noted that the use of both MRI techniques is better than either alone in tracking and interpreting the course of ischemia and cell death in the lab and in the clinical setting.

Lewin’s lab aims to investigate potential markers of cerebral damage in rats and then translate the findings to human diagnosis. Ultrasmall superpara-magnetic iron oxide particles are among markers of inflammation under investigation.

Lewin plans to continue research in neuroscience as part of an MD/PhD program at the Weill Medical College of Cornell University in New York City, which she will enter this fall.

Annie Nguyen

Naomi Lewin



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