Limits...
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 PBMC isolation for IFN-γ ELISpot Assay. (A) Heparinised blood is added to lymphoprep-containing leucosep tubes and samples centrifuged for 13 min at 1000 ×g without brake. A plasma sample is taken before mononuclear/PBMCs are collected. Cells are washed and counted before being used for an ELISPOT assay; (B) Schematic diagram outlining the methodology behind an IFN-γ ELISpot assay; (C) Example of spot forming units following development of an ELISpot assay, showing antigen-specific IAV responses, negative control medium alone (R10) and positive control (PHA/SEB).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494351&req=5

vaccines-03-00293-f001: Schematic overview of PBMC isolation for IFN-γ ELISpot Assay. (A) Heparinised blood is added to lymphoprep-containing leucosep tubes and samples centrifuged for 13 min at 1000 ×g without brake. A plasma sample is taken before mononuclear/PBMCs are collected. Cells are washed and counted before being used for an ELISPOT assay; (B) Schematic diagram outlining the methodology behind an IFN-γ ELISpot assay; (C) Example of spot forming units following development of an ELISpot assay, showing antigen-specific IAV responses, negative control medium alone (R10) and positive control (PHA/SEB).

Mentions: Alternatively, the ELISPOT assay (Figure 1A–C) is an extremely sensitive and accurate method for detection of antigen-specific T-cells (or B-cells), allowing for identification of a single cell secreting a particular cytokine (e.g., IFN-γ). The technique was first developed by Czerkinsky and colleagues in the 1980s [72] and has subsequently been accepted as one of the most validated assays for human clinical trials [73,74]. The method involves the isolation of PBMCs and addition of a set number of cells to a capture antibody-coated plate (e.g., anti-IFN-γ). Cells are then stimulated with a pre-determined concentration of specific antigen (e.g., peptide, virus or whole protein antigen). In the presence of the stimulus, antigen-specific T-cells will secrete cytokine (e.g., IFN-γ) which can be captured by the antibody used to coat the plate. Following an assay-dependent period of stimulation (usually 18–20 h), the cells are removed by washing and the bound cytokine typically detected using a secondary detection reagent conjugated to an enzymatic label (e.g., alkaline phosphatase—ALP). This enzyme catalyzes the colorimetric spot formation when in the presence of a chromogenic substrate (e.g., 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt—BCIP). Other enzymes which can be used for development of ELISPOTs include horseradish peroxidase, followed by addition of the chromogen 3,3',5,5'-tetramethylbenzidine (TMB). The choice of controls is also of critical importance in the ELISPOT assay. Most immunologists choose to stimulate with the polyclonal mitogen phytohaemagglutinin (PHA) as a positive control for the assay. However, Cytomegalovirus/Epstein Barr virus (CMV/EBV) peptide pools which consist of epitopes presented by broad range of HLA alleles are also included for quality control and standardisation purposes [75]. The use of whole antigen or peptide pools spanning large antigens ensures that the ELISPOT assay is not limited by HLA restriction to the same extent as ICS-based tetramer assays (see below). Furthermore, the use of the ELISPOT assay is attractive because it allows enumeration of the number of antigen-specific cells through calculation of the number of spot forming units (SFU). Additionally, the assay can be used on fresh or frozen PBMCs [76].


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

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

Schematic overview of PBMC isolation for IFN-γ ELISpot Assay. (A) Heparinised blood is added to lymphoprep-containing leucosep tubes and samples centrifuged for 13 min at 1000 ×g without brake. A plasma sample is taken before mononuclear/PBMCs are collected. Cells are washed and counted before being used for an ELISPOT assay; (B) Schematic diagram outlining the methodology behind an IFN-γ ELISpot assay; (C) Example of spot forming units following development of an ELISpot assay, showing antigen-specific IAV responses, negative control medium alone (R10) and positive control (PHA/SEB).
© Copyright Policy
Related In: Results  -  Collection

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

vaccines-03-00293-f001: Schematic overview of PBMC isolation for IFN-γ ELISpot Assay. (A) Heparinised blood is added to lymphoprep-containing leucosep tubes and samples centrifuged for 13 min at 1000 ×g without brake. A plasma sample is taken before mononuclear/PBMCs are collected. Cells are washed and counted before being used for an ELISPOT assay; (B) Schematic diagram outlining the methodology behind an IFN-γ ELISpot assay; (C) Example of spot forming units following development of an ELISpot assay, showing antigen-specific IAV responses, negative control medium alone (R10) and positive control (PHA/SEB).
Mentions: Alternatively, the ELISPOT assay (Figure 1A–C) is an extremely sensitive and accurate method for detection of antigen-specific T-cells (or B-cells), allowing for identification of a single cell secreting a particular cytokine (e.g., IFN-γ). The technique was first developed by Czerkinsky and colleagues in the 1980s [72] and has subsequently been accepted as one of the most validated assays for human clinical trials [73,74]. The method involves the isolation of PBMCs and addition of a set number of cells to a capture antibody-coated plate (e.g., anti-IFN-γ). Cells are then stimulated with a pre-determined concentration of specific antigen (e.g., peptide, virus or whole protein antigen). In the presence of the stimulus, antigen-specific T-cells will secrete cytokine (e.g., IFN-γ) which can be captured by the antibody used to coat the plate. Following an assay-dependent period of stimulation (usually 18–20 h), the cells are removed by washing and the bound cytokine typically detected using a secondary detection reagent conjugated to an enzymatic label (e.g., alkaline phosphatase—ALP). This enzyme catalyzes the colorimetric spot formation when in the presence of a chromogenic substrate (e.g., 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt—BCIP). Other enzymes which can be used for development of ELISPOTs include horseradish peroxidase, followed by addition of the chromogen 3,3',5,5'-tetramethylbenzidine (TMB). The choice of controls is also of critical importance in the ELISPOT assay. Most immunologists choose to stimulate with the polyclonal mitogen phytohaemagglutinin (PHA) as a positive control for the assay. However, Cytomegalovirus/Epstein Barr virus (CMV/EBV) peptide pools which consist of epitopes presented by broad range of HLA alleles are also included for quality control and standardisation purposes [75]. The use of whole antigen or peptide pools spanning large antigens ensures that the ELISPOT assay is not limited by HLA restriction to the same extent as ICS-based tetramer assays (see below). Furthermore, the use of the ELISPOT assay is attractive because it allows enumeration of the number of antigen-specific cells through calculation of the number of spot forming units (SFU). Additionally, the assay can be used on fresh or frozen PBMCs [76].

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