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Walter Reed Army Institute of Research

4 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses
Siriphan Manocheewa 1, Erinn C. Lanxon-Cookson 1, Yi Liu 1, J. Victor Swain 1, Jan McClure 1, Ushnal Rao 1, Brandon Maust 1, Wenjie Deng 1, Justine E. Sunshine 1, Moon Kim 1, Morgane Rolland 3,4, James I. Mullins 1,2
1Department of Microbiology, University of Washington, 2Departments of Medicine and Laboratory Medicine, University of Washington, 3U.S Military HIV Research Program, Walter Reed Army Institute of Research, 4Henry M. Jackson Foundation

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

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Immunology and Infection

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques
Andrey Tokarev 1, Matthew Creegan 1, Michael A. Eller 1, Mario Roederer 2, Diane L. Bolton 1
1US Military HIV Research Program, Henry M. Jackson Foundation, Walter Reed Army Institute of Research, 2Vaccine Research Center, NIAID, NIH

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

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Immunology and Infection

A High-throughput Shigella-specific Bactericidal Assay
Hailey P. Weerts 1, Jigui Yu 2, Robert W. Kaminski 1, Moon H. Nahm 2
1Department of Enteric Infections, Bacterial Diseases Branch, Walter Reed Army Institute of Research, 2University of Alabama at Birmingham

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

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Bioengineering

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation
Christopher P. Karch 1,2, Peter Burkhard 3, Gary R. Matyas 1, Zoltan Beck 1,2
1U.S. Military HIV Research Program, Walter Reed Army Institute of Research, 2Henry M. Jackson Foundation for the Advancement of Military Medicine, 3Alpha-O Peptides AG

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

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