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Chapter 35 : Cellular Immune Assays for Evaluation of Vaccine Efficacy

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Abstract:

A greater understanding of the immune response, particularly of cell-mediated immune responses in malaria, tuberculosis (TB), human immunodeficiency virus (HIV), and cancer patients, will lead to improved vaccine design. Currently, in vitro analyses of cellular immune responses are being used for assessments of the efficacy of therapeutic and prophylactic vaccines, for clinical diagnosis, and for studies of immune regulation. This chapter was compiled by authors who have been involved in HIV vaccine testing for 15 years and have been collaborating with or have worked in laboratories in both developed and developing countries. When conducting cellular immunology assays, the integrity of the peripheral blood mononuclear cells (PBMC), especially the cellular membranes, is critical for success. In a separate experiment, the authors assessed the effect of the time from collection to processing on the proportions of lymphocyte subsets (total T cells, CD4 and CD8 T cells, B cells, and NK cells). The standard LPA measures antigen-induced cell division. Cells (usually PBMC) are incubated in the presence of various concentrations of an antigen (specific) or mitogen (nonspecific) stimulus of interest. The percentages of responders to mitogens, recall antigens, and HIV antigens were consistent for comparisons of fresh and cryopreserved PBMC. CD8 T lymphocytes are able to mediate a variety of effector mechanisms and may provide the basis for protective immunity against a diverse array of infectious pathogens and tumors. The influences of host genes, health and nutrition status, and disease burden of patients are taken into consideration by conducting studies in-country.

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
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Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
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FIGURE 1

Effect of time to processing of whole blood on white blood cell subsets. Whole blood was collected from 20 subjects into CPTs (two per subject) and stored at 22 to 25°C for 2 and 30 h prior to processing. Hatched bars, 2 h; white bars, 30 h.

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
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FIGURE 2

Effect of time to processing of whole blood on lymphocyte subsets. Whole blood was collected from 19 subjects into CPTs (two per subject) and stored at 22 to 25°C prior to PBMC separation and measurements of lymphocyte subsets by flow cytometry. Hatched bars, 2 h; white bars, 30 h.

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
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FIGURE 3

Comparison of viability (V), percent lymphocytes (%Ly), and percent neutrophils (%Ne) of PBMC following separation of paired samples from 20 subjects by using CPTs and Leucosep (L) tubes. Hatched bars, CPTs; white bars, Leucosep tubes.

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
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FIGURE 4

Time to processing affects production of IFN-γ. The processing of seven blood samples and ELISPOT assay setup were either performed immediately (solid bars) or after overnight storage of blood at room temperature in a safety cabinet (stippled bars). PBMC were stimulated with a pool of CEF peptides. The average number of background SFC attributable to PBMC only was subtracted.

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
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FIGURE 5

The effect of three different anticoagulants on T-cell function was measured by an ELISPOT assay. White box, heparin; shaded box, EDTA; stippled box, acid citrate dextrose. Ten samples were processed within 6 h of collection, and the secretion of IFN-γ in response to the CEF epitope pool was examined. The data are shown with a box plot of IFN-γ-secret-ing SFC/million PBMC. The box plot shows the distribution of values; from the top to bottom, lines are drawn parallel to the axis, indicating the 95th, 75th, 50th (median), 25th, and 5th percentiles. A Kruskall-Wallis test revealed that there were no significant differences between the CEF-induced responses in PBMC collected in heparin, EDTA, and acid citrate dextrose.

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
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FIGURE 6

Percent viability of stored PBMC over time, indicated by center. PBMC were thawed, and their viability was assessed by a trypan blue exclusion technique. (Reproduced with permission from reference .)

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
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FIGURE 7

Viable cell recovery over time, indicated by center. Reference lines at 10 cells mark the original numbers of PBMC stored per vial. (Reproduced with permission from reference .)

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
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Tables

Generic image for table
TABLE 1

Organizations involved in validating immunogenicity assays for human vaccine trials

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
Generic image for table
TABLE 2

Stages and variables in the separation and cryopreservation of PBMC from whole blood

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35
Generic image for table
TABLE 3

Lymphoproliferative responses in fresh and cryopreserved PBMC from HIV-1-infected individuals

Citation: Cox J, deSouza M, Ratto-Kim S, Ferrari G, Weinhold K, Birx D. 2006. Cellular Immune Assays for Evaluation of Vaccine Efficacy, p 301-314. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch35

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