Walter Reed Army Institute of Research

4 ARTICLES PUBLISHED IN JoVE

Immunology and Infection

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Siriphan Manocheewa¹, Erinn C. Lanxon-Cookson¹, Yi Liu¹, J. Victor Swain¹, Jan McClure¹, Ushnal Rao¹, Brandon Maust¹, Wenjie Deng¹, Justine E. Sunshine¹, Moon Kim¹, 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.

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 Tokarev¹, Matthew Creegan¹, Michael A. Eller¹, Mario Roederer², Diane L. Bolton¹

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.

Immunology and Infection

A High-throughput Shigella-specific Bactericidal Assay

Hailey P. Weerts¹, Jigui Yu², Robert W. Kaminski¹, Moon H. Nahm²

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.

Bioengineering

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Christopher P. Karch^1,2, Peter Burkhard³, Gary R. Matyas¹, 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.