Duncan A Meiklejohn | PubMed Health

PubMed2004

ELISPOT cell rescue.

The enzyme-linked immunospot (ELISPOT) assay is a highly sensitive and reproducible method for quantifying T cell-mediated immune responses, and has been used to measure antigen-specific responses post-vaccination. While there are several advantages of the ELISPOT assay for use in field settings for large-scale vaccination trials, blood draw volumes are often limited, and the number of antigen-specific responses that can be measured is constrained by the limited cell number. We reasoned that it should be possible to salvage and rescue viable cells from a completed ELISPOT assay post-incubation, to use for further experimentation. Here, we show that cells rescued from an ELISPOT plate after assay are viable, and may be used in a second cytokine-producing assay, in a proliferation assay, or to provide a source of DNA for genetic studies such as human leukocyte antigen (HLA) typing. Rescue of cells after an ELISPOT assay will be particularly useful for increasing sample utility and maximizing data collection from T cell assays in vaccine trials.

DNA↗

PubMed2004

R5 human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in primary CD4+ T-cell cultures than X4 HIV-1.

In this report, we present evidence that R5 human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in primary CD4+ T cells than X4 HIV-1. By comparing CD3/CD28-costimulated CD4+ T-cell cultures infected by several X4 and R5 HIV-1 strains, we determined that R5-infected CD4+ T cells produce more virus over time than X4-infected CD4+ T cells. In the first comparison, we found that more cells were infected by the X4-tropic strain LAI than by the R5-tropic strain JR-CSF and yet that higher levels of viral production were detected in the R5-infected cultures. The differential viral production was partially due to the severe cytopathic effects of the X4 virus. We also compared cultures infected with the isogenic HIV-1 strains NL4-3 (X4) and 49.5 (R5). We found that fewer cells were infected by the R5 strain, and yet similar levels of viral production were detected in both infected cultures. Cell death played less of a role in the differential viral production of these strains, as the cell viability remained comparable in both X4- and R5-infected cultures over time. The final comparison involved the primary R5-tropic isolate KP1 and the primary dual-tropic isolate KP2. Although both strains infected similar numbers of cells and induced comparable levels of cytopathicity, viral production was considerably higher in the R5-infected culture. In summary, these data demonstrate that R5 HIV-1 has an increased capacity to replicate in costimulated CD4+ T cells compared to X4 HIV-1.

CD28 Antigens↗