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Measuring Cellular Immunity to Influenza: Methods of Detection, Applications and Challenges.

Coughlan L, Lambe T - Vaccines (Basel) (2015)

Bottom Line: Vaccination can result in an effective, albeit strain-specific antibody response and there is a need for vaccines that can provide superior, long-lasting immunity to influenza.However, the field lacks consensus on the correlates of protection for cellular immunity in reducing severe influenza infection, transmission or disease outcome.Furthermore, unlike serological methods such as the standardized haemagglutination inhibition assay, there remains a large degree of variation in both the types of assays and method of reporting cellular outputs.

View Article: PubMed Central - PubMed

Affiliation: The Jenner Institute, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX1 7DQ, UK. lynda.coughlan@ndm.ox.ac.uk.

ABSTRACT
Influenza A virus is a respiratory pathogen which causes both seasonal epidemics and occasional pandemics; infection continues to be a significant cause of mortality worldwide. Current influenza vaccines principally stimulate humoral immune responses that are largely directed towards the variant surface antigens of influenza. Vaccination can result in an effective, albeit strain-specific antibody response and there is a need for vaccines that can provide superior, long-lasting immunity to influenza. Vaccination approaches targeting conserved viral antigens have the potential to provide broadly cross-reactive, heterosubtypic immunity to diverse influenza viruses. However, the field lacks consensus on the correlates of protection for cellular immunity in reducing severe influenza infection, transmission or disease outcome. Furthermore, unlike serological methods such as the standardized haemagglutination inhibition assay, there remains a large degree of variation in both the types of assays and method of reporting cellular outputs. T-cell directed immunity has long been known to play a role in ameliorating the severity and/or duration of influenza infection, but the precise phenotype, magnitude and longevity of the requisite protective response is unclear. In order to progress the development of universal influenza vaccines, it is critical to standardize assays across sites to facilitate direct comparisons between clinical trials.

No MeSH data available.


Related in: MedlinePlus

Schematic overview of Whole Blood Assay (A) Day 1: whole blood samples are stimulated with antigen, protein export inhibited and samples incubated overnight; (B) Samples are prepared for cryopreservation. Red cells are lysed and stimulated cells frozen down for future analysis by flow cytometry.
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vaccines-03-00293-f003: Schematic overview of Whole Blood Assay (A) Day 1: whole blood samples are stimulated with antigen, protein export inhibited and samples incubated overnight; (B) Samples are prepared for cryopreservation. Red cells are lysed and stimulated cells frozen down for future analysis by flow cytometry.

Mentions: The specific details of individual protocols such as choice of stimulating antigen, concentration of antigen and controls to measure responses to different pathogens require optimization; however the general procedure is relatively similar. In brief, published protocols involve pre-coating polyprolypene tubes with CD28 and CD49a co-stimulatory antibodies which enhance the specific immune response [111], by increasing the observed frequency of cytokine responding CD4+T-cells in response to antigen [111]. Heparinized whole blood and antigen is added at an assay-dependent concentration and samples incubated for 5–6 h at 37 °C after which Brefeldin A and/or monensin is added to help retain cytokines intracellularly (e.g., IFN-γ). Samples are then incubated at 37 °C for a further 5 h after which EDTA is added to detach T-cells from the polyprolypene wall, erythrocytes are lysed and white cells fixed and cryopreserved in foetal calf serum (FCS) containing 10% DMSO (Figure 3). Following cryopreservation, phenotyping of cells by flow cytometry using tetramer staining or ICS can be performed off site where resources and specialised equipment are more readily available.


Measuring Cellular Immunity to Influenza: Methods of Detection, Applications and Challenges.

Coughlan L, Lambe T - Vaccines (Basel) (2015)

Schematic overview of Whole Blood Assay (A) Day 1: whole blood samples are stimulated with antigen, protein export inhibited and samples incubated overnight; (B) Samples are prepared for cryopreservation. Red cells are lysed and stimulated cells frozen down for future analysis by flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494351&req=5

vaccines-03-00293-f003: Schematic overview of Whole Blood Assay (A) Day 1: whole blood samples are stimulated with antigen, protein export inhibited and samples incubated overnight; (B) Samples are prepared for cryopreservation. Red cells are lysed and stimulated cells frozen down for future analysis by flow cytometry.
Mentions: The specific details of individual protocols such as choice of stimulating antigen, concentration of antigen and controls to measure responses to different pathogens require optimization; however the general procedure is relatively similar. In brief, published protocols involve pre-coating polyprolypene tubes with CD28 and CD49a co-stimulatory antibodies which enhance the specific immune response [111], by increasing the observed frequency of cytokine responding CD4+T-cells in response to antigen [111]. Heparinized whole blood and antigen is added at an assay-dependent concentration and samples incubated for 5–6 h at 37 °C after which Brefeldin A and/or monensin is added to help retain cytokines intracellularly (e.g., IFN-γ). Samples are then incubated at 37 °C for a further 5 h after which EDTA is added to detach T-cells from the polyprolypene wall, erythrocytes are lysed and white cells fixed and cryopreserved in foetal calf serum (FCS) containing 10% DMSO (Figure 3). Following cryopreservation, phenotyping of cells by flow cytometry using tetramer staining or ICS can be performed off site where resources and specialised equipment are more readily available.

Bottom Line: Vaccination can result in an effective, albeit strain-specific antibody response and there is a need for vaccines that can provide superior, long-lasting immunity to influenza.However, the field lacks consensus on the correlates of protection for cellular immunity in reducing severe influenza infection, transmission or disease outcome.Furthermore, unlike serological methods such as the standardized haemagglutination inhibition assay, there remains a large degree of variation in both the types of assays and method of reporting cellular outputs.

View Article: PubMed Central - PubMed

Affiliation: The Jenner Institute, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX1 7DQ, UK. lynda.coughlan@ndm.ox.ac.uk.

ABSTRACT
Influenza A virus is a respiratory pathogen which causes both seasonal epidemics and occasional pandemics; infection continues to be a significant cause of mortality worldwide. Current influenza vaccines principally stimulate humoral immune responses that are largely directed towards the variant surface antigens of influenza. Vaccination can result in an effective, albeit strain-specific antibody response and there is a need for vaccines that can provide superior, long-lasting immunity to influenza. Vaccination approaches targeting conserved viral antigens have the potential to provide broadly cross-reactive, heterosubtypic immunity to diverse influenza viruses. However, the field lacks consensus on the correlates of protection for cellular immunity in reducing severe influenza infection, transmission or disease outcome. Furthermore, unlike serological methods such as the standardized haemagglutination inhibition assay, there remains a large degree of variation in both the types of assays and method of reporting cellular outputs. T-cell directed immunity has long been known to play a role in ameliorating the severity and/or duration of influenza infection, but the precise phenotype, magnitude and longevity of the requisite protective response is unclear. In order to progress the development of universal influenza vaccines, it is critical to standardize assays across sites to facilitate direct comparisons between clinical trials.

No MeSH data available.


Related in: MedlinePlus