Douglas Horejsh

Anti–Severe Acute Respiratory Syndrome Coronavirus Immune Responses: The Role Played by Vγ9Vδ2 T Cells

Abstract Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV) strain. Analyses of T cell repertoires in health care workers who survived SARS-CoV infection during the 2003 outbreak revealed that their effector memory Vγ9Vδ2 T cell populations were selectively expanded ∼3 months after the onset of disease. No such expansion of their αβ T cell pools was detected. The expansion of the Vγ9Vδ2 T cell population was associated with higher anti–SARS-CoV immunoglobulin G titers. In addition, in vitro experiments demonstrated that stimulated Vγ9Vδ2 T cells display an interferon-γ–dependent anti–SARS-CoV activity and are able to directly kill SARS-CoV–infected target cells. These findings are compatible with the possibility that Vγ9Vδ2 T cells play a protective role during SARS

T-cell response profiling to biological threat agents including the SARS coronavirus

The emergence of pathogens such as SARS and the increased threat of bioterrorism has stimulated the development of novel diagnostic assays for differential diagnosis. Rather than focusing on the detection of an individual pathogen component, we have developed a T cell profiling system to monitor responses to the pathogens in an array format. Using a matrix of antigens specific for different pathogens, a specific T cell profile was generated for each individual by monitoring the intracellular production of interferon-gamma by flow cytometry. This assay allows for the testing of multiple proteins or peptides at a single time and provides a quantitative and phenotypic assessment of CD4(+) and CD8(+) responding cells. We present profiling examples for several positive individuals, including those vaccinated with the smallpox and anthrax vaccines. We also show antigen optimization for the SARS-hCoV, as studies revealed that these proteins contain peptides which cross-react with more common coronaviruses, a cause of the common cold. The T cell array is an early and sensitive multiplex measure of active infection, exposure to a pathogen, or effective, recent vaccination.

Flow Cytometry and T-Cell Response Monitoring after Smallpox Vaccination

Orthopoxvirus zoonosis or smallpox as result of bioterrorism or biological warfare represents a risk for epidemic spread. By monitoring T-cell responses by flow cytometry, we observed a recall response after recent vaccination against smallpox. When the high similarity between the orthopoxviruses is considered, this rapid assay that uses vaccinia antigens could identify recently exposures.

Simultaneous control of DNA and RNA processing efficiency using a nucleic acid calibration set

PCR-based detection techniques enables reliable and sensitive nucleic acid target detection. However, quantitative determination methods often fail to control for the efficiency of nucleic acid extraction, reverse transcription, and PCR amplification. This problem is even more prominent when working with clinical samples due to target sequence loss during nucleic acid processing or the co-purification of PCR inhibitors (1,2). Handling processes are often assumed to approach 100% efficiency in the laboratory, even if practical experience shows that this efficiency can be much lower. This inability to ensure accuracy can lead to significant error in uncalibrated DNA sample quantitation. The additional need for reverse transcription of RNA may further increase the quantitative error rate, as yet another enzymatic process is involved. Nucleic acid controls have been developed based upon known sequences to calibrate either DNA or RNA handling; DNA calibrators have been used to control for the amplification of target sequences using realtime PCR methods (3–8), while RNA calibrators have been developed to test reverse transcription and amplification efficiencies (9–11). A nonpathogenic viral particle carrying a sequence for use as an external positive control of extraction and amplification has also been described (12). Unfortunately, most of the established processing controls are only suitable for limited applications (i.e., either DNA or RNA detection). Cross-contamination of biological samples or minute detection from natural sources reveals the need for completely synthetic sequences, with no homology to sequences in the nucleic acid databases. It is, therefore, beneficial to design an internal, synthetic calibration system that can control for both DNA and RNA processing steps in a single tube. This set includes both RNA and DNA targets with identical primer binding sites and, thus, primer binding efficiency, but easily distinguishable sequence characteristics, allowing for simultaneous detection, quantitation, and calibration of nucleic acid processing efficiency. A 150-bp randomly generated nucleic acid sequence was developed for use as a short control (SC). A GCrich 75-bp sequence was inserted in the middle of the 150-bp sequence to generate a 225-bp sequence, long control (LC). Besides size, the two sequences were designed to have easily distinguishable probe binding sites with a predicted product melting temperature difference of 4°C. Calibrator sequences have been published as GenBank® accession nos. EF143258 (DNA control, LC) and EF143257 (RNA control, SC). Simultaneous control of DNA and RNA processing efficiency using a nucleic acid calibration set

BeadCons: detection of nucleic acid sequences by flow cytometry.

Molecular beacons are single‐stranded nucleic acid structures with a terminal fluorophore and a distal, terminal quencher. These molecules are typically used in real‐time PCR assays, but have also been conjugated with solid matrices. This unit describes protocols related to molecular beacon–conjugated beads (BeadCons), whose specific hybridization with complementary target sequences can be resolved by cytometry. Assay sensitivity is achieved through the concentration of fluorescence signal on discrete particles. By using molecular beacons with different fluorophores and microspheres of different sizes, it is possible to construct a fluid array system with each bead corresponding to a specific target nucleic acid. Methods are presented for the design, construction, and use of BeadCons for the specific, multiplexed detection of unlabeled nucleic acids in solution. The use of bead‐based detection methods will likely lead to the design of new multiplex molecular diagnostic tools.