Research paperDefining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials
Introduction
Historically, protection from disease has been the major determinant of vaccine efficacy, and this is usually attributed to a strong humoral response that neutralizes the invading pathogen or harmful pathogen product (Hilleman, 2000). Due to difficulties raising broadly neutralizing antibodies against HIV, most candidate HIV vaccines in current clinical trials are designed to elicit HIV-specific T cell immunity. As such, characterization of the range of effector functions in vaccine-induced T cells has assumed increasing significance. T cell immunity involved in viral control is presumed to occur either through direct lysis of infected cells or secretion of anti-viral cytokines and chemokines (Yasutomi et al., 1993, Koup et al., 1994, Matano et al., 1998, Jin et al., 1999, Schmitz et al., 1999). Thus, major efforts have been undertaken to standardize assays, primarily IFN-γ ELISpot and ICS by flow cytometry, which can accurately quantify vaccine-induced T cell responses. This information enables characterization of immunogenicity, prioritization of promising vaccine candidates for advancement to larger scale trials, and assessment of correlates of immune protection.
As HIV-1 vaccine evaluation moves forward into multicenter, large-scale phase IIB and III trials throughout the world, it becomes nearly impossible to perform anti-viral T cell assays on freshly isolated PBMC. In response to regulatory requirements that mandate use of validated assays and the growth of the complexities of immunological assays, centralized laboratory facilities have largely replaced small site laboratories. Further, assessments of immune correlates of HIV protection by vaccination entail case-control study designs, necessitating the use of cryopreserved PBMC for retrospective analyses. Thus, it is essential that cryopreserved PBMC retain functional capacity in order to successfully evaluate candidate vaccine immunogenicity and efficacy.
A small number of published studies have examined the impact of sample collection and processing on performance in cellular immune functional assays. Most of the early efforts compared phenotype preservation by flow cytometry using different anticoagulants (Nicholson et al., 1984, Nicholson and Green, 1993, Shalekoff et al., 1998). Findings indicated decreased preservation of T and B cell subsets when whole blood collected in ethylene diamine tetra acetic acid (EDTA) was stored for 24 h (Nicholson et al., 1984, Nicholson and Green, 1993), and decreased expression of activation markers after storage in EDTA or sodium heparin (Shalekoff et al., 1998). A smaller number of publications examined the effect of anticoagulants on the performance of T cells in immunological assays, primarily using the lymphoproliferative assay (LPA) to measure functional competency. These studies showed a gradual and complete loss of proliferation in blood stored in EDTA from 0 to 24 h (Weinberg et al., 1998, Kumar and Satchidanandam, 2000). Measurement of antigen-specific T cell activity by LPA and cytolytic chromium release assays (Weinberg et al., 1998) has typically required freshly isolated PBMC for optimal detection of responses. Shipment and storage of peripheral blood, as well as the type of anticoagulant used in these studies, profoundly affected performance in these assays (Betensky et al., 2000, Sun et al., 2003).
Intracellular cytokine staining (ICS) by flow cytometry and the IFN-γ ELISpot assay (McCutcheon et al., 1997, Scheibenbogen et al., 2000, Kumar et al., 2001, Self et al., 2001, Hudgens et al., 2004, Trigona et al., 2003) can quantify ex vivo virus-specific memory T cells from cryopreserved PBMC (Russell et al., 2003, Maecker et al., 2005). Considerable progress has been made in improving and validating these assays for use in clinical vaccine trials (Russell et al., 2003). No data exist for addressing parameters from venipuncture to isolation/cryopreservation for PBMC recovery/viability and performance in ICS or IFN-γ ELISpot. The requirements for sample preparation and cryopreservation, particularly the optimal time for isolation, have a profound impact on how clinical sites implement future trials aimed at ascertaining induction of memory T cell responses. For this purpose, we studied the effects of different anticoagulants, processing times, processing methods, shipping methods and cryopreservation media on recovery, viability and T cell function in immunological assays in PBMC from HIV-uninfected healthy individuals. We demonstrate that the single most important parameter affecting PBMC performance in the immunoassays is the length of time from venipuncture to cryopreservation.
Section snippets
Study 1: determination of optimal cryopreservation medium and time from venipuncture to blood processing
(See Fig. 1)In the first set of studies, blood from 18 individuals was collected into three different anticoagulants: acid citrate dextrose (ACD), EDTA, and sodium heparin. Half of the blood was processed within 8 h of venipuncture, and half was sent via overnight (O/N) courier (Federal Express) to the Fred Hutchinson Cancer Research Center (FHCRC) laboratory for PBMC isolation and cryopreservation (designated 24 h, which was the approximate time from venipuncture to cryopreservation). All PBMC
Freezing media, anticoagulant and PBMC isolation method do not have an impact on cell recovery/viability
The composition of cryopreservation media can vary among different laboratories. We first examined in Study #1 (Fig. 1) the influence of two commonly used cryopreservants: 10% DMSO in FBS and 10% DMSO/12.5% human serum albumin in RPMI, on PBMC recovery and viability after cryopreservation. Whole blood was drawn from ten donors into ACD, EDTA, or heparin; and PBMC were isolated within either eight or 24 h of venipuncture. The percent viability and recovery were assessed after PBMC thawing and
Discussion
Our findings consistently demonstrate that optimal recovery, viability and function of cryopreserved PBMC for determining antigen-specific immunity using state-of-the-art assays require isolation and cryopreservation within 8 h of venipuncture. Of note, we arbitrarily chose 8 h as our earliest time point for blood processing and PBMC cryopreservation since this is a time interval that can be feasibly implemented in the clinical field setting. However, we recognize that superior PBMC recovery,
Acknowledgements
We thank Jon Hallstrom, Cassandra Beckham, Judy Gordon, Justin Penn and Ruth Baydo for technical support, Mark Deers for data management, Phyllis Stegall for technical editing, Jon Newman for assistance in shipping for all studies, and the site investigators (Drs. Geoffery Gorse, Michael Keefer and Paul Goepfert) for implementation of Study 3.
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These authors contributed equally to these studies.