In a study published in the March 27 issue of Science, Ashley Haase and colleagues offer an unprecedented insight into the battle between host and virus. The study employs two different models: SIV infection in rhesus macaques and LCMV infection in mice. Many years ago, Haase pioneered a technique called in situ hybridization (ISH) that allows the visualization and quantification of virus-infected cells in tissue samples by staining the viral RNA with silver grains. He has now married this approach to a complementary method that identifies virus-specific CD8 T cells in tissues, using a modified version of the widely used tetramer assay (tetramers can identify CD8 T cells based on the specific epitope they are targeting). Tissue images captured using the two techniques are overlayed to create what Haase describes as a “battlefield map” which shows the spatial organization of both virus-infected cells and virus-specific CD8 T cells.
In the Science paper Haase describes results obtained in a study of rhesus macaques challenged intravaginally with SIVmac239. Staining of both viral RNA and SIV Gag-specific CD8 T cells showed that the number of detectable conjugates correlated with the magnitude of the viral load reduction from peak (around days 10-14 post-challenge) to day 21. This finding held true in both cervix and lymph nodes. Haase also used the images to calculate an effector to target cell ratio (E:T ratio) and found that there was a statistically significant correlation between the E:T ratio and the extent of post-peak viral load control.
To explore the value of the approach in another model, Haase collaborated with Rafi Ahmed’s group at Emory University to study the impact of E:T ratios in murine LCMV infection. Two strains of LCMV were compared: the Armstrong strain, which causes a time-limited acute infection that is rapidly cleared, and clone 13, which establishes a chronic persistent infection. The results demonstrate that the different outcomes are associated with clone 13’s rapid infection of far greater numbers of macrophages and fibroreticular cells compared to the Armstrong strain. This propensity of clone 13 causes the number of infected target cells to far outnumber effector cells. In contrast, the Armstrong strain infected a limited number of cells which were rapidly outnumbered by effector cells, leading to rapid clearance. Specifically, the E:T ratio for the Armstrong strain was 4 at day 3 post-infection, which was higher by a factor of 20 than the ratio for clone 13 at the same timepoint.
Haase and colleagues conclude that “location, timing, and numbers of effectors and targets all count in determining outcome.” The methodology, which Haase dubs ISTH, will now be used to ascertain if there are vaccination strategies that can create enough effector cells to overwhelm SIV at the portal of entry, before systemic infection can take hold. Ultimately, the hope is that this research can guide the development of a protective HIV vaccine.
This image from the paper shows the individual images of
Gag-specific CD8 T cells (top right), SIV-infected cells (middle right) and the
two images overlayed (bottom right). CREDIT: Science Magazine, 27 March 2009, Li
et al., pp. 1726 – 1729. Reproduced for non-profit educational presentation use
only.
Science 27 March 2009: Vol. 323. no. 5922, pp. 1726 - 1729
DOI: 10.1126/science.1168676
REPORTS
Visualizing Antigen-Specific and Infected Cells in Situ Predicts Outcomes in Early Viral Infection
Qingsheng Li,1 Pamela J. Skinner,2 Sang-Jun Ha,3 Lijie Duan,1 Teresa L. Mattila,2 Aaron Hage,2 Cara White,2 Daniel L. Barber,4 Leigh O'Mara,3 Peter J. Southern,1 Cavan S. Reilly,5 John V. Carlis,6 Christopher J. Miller,7 Rafi Ahmed,3 Ashley T. Haase1
1 Department of Microbiology, Medical School, University of Minnesota, Minneapolis, MN 55455, USA.
2 Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, MN55108, USA.
3 Emory Vaccine Center, Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA.
4 Immunobiology Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
5 Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, MN 55455, USA.
6 Department of Computer Science and Engineering, Institute of Technology, University of Minnesota, Minneapolis, MN 55455, USA.
7 Center for Comparative Medicine, California National Primate Research Center, University of California, Davis, CA 95616, USA.
In the early stages of viral infection, outcomes depend on a race between expansion of infection and the immune response generated to contain it. We combined in situ tetramer staining with in situ hybridization to visualize, map, and quantify relationships between immune effector cells and their targets in tissues. In simian immunodeficiency virus infections in macaques and lymphocytic choriomeningitis virus infections in mice, the magnitude and timing of the establishment of an excess of effector cells versus targets were found to correlate with the extent of control and the infection outcome (i.e., control and clearance versus partial or poor control and persistent infection). This method highlights the importance of the location, timing, and magnitude of the immune response needed for a vaccine to be effective against agents of persistent infection, such as HIV-1.
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