Important note: Information in this article was accurate in December 2000. The state of the art may have changed since the publication date. IAVI Report - December 2000 / January 2001
Patricia Kahn
Over the past few years, the mounting evidence that protection against HIV will require cellular immune responses (as well as antibodies) has fueled the development of vaccine candidates aimed at stimulating this arm of the immune system.
As these candidates now move into the early rounds of clinical trials, vaccine researchers face the task of figuring out how well they induce T-cell responses and what kind(s) of responses they induce. They will also need to compare responses among different candidates, to address issues such as which HIV antigens or vaccine platforms are the most immunogenic.
As straightforward as these questions seem, however, they represent new scientific terrain: no preventive vaccine trials have ever used careful quantitation of T-cell responses as an endpoint. The methods to do so simply didn't exist — the available assays were difficult to carry out and only semi-quantitative.
But that has changed, thanks to several new, far more precise T-cell assays taking root in immunology labs. Although all of them need further development, including clarification of just what functions the T-cells they measure have, these new techniques represent a huge boon for clinical studies of HIV vaccines — assuming they work as well in detecting vaccine-induced responses as they do in virus-infected people, who usually show much stronger immune responses than vaccinees.
Over the past year, much effort has gone into vetting these assays for use in HIV vaccine trials. That has involved analyzing and optimizing a myriad of parameters, from specificity, sensitivity and reproducibility, to ease of use in the developing countries where trials will take place. Here we present a rundown of the assays and an update of which ones are on the list for upcoming trials. This issue featured prominently in the recent meeting of NIH's AIDS Vaccine Research Committee (22-23 January 2001) and was the topic of two consultative IAVI meetings (18 April 2000 and 17 January 2001).
CTL killing assay: The workhorse of the older methods, this assay measures the ability of cells removed from whole blood (peripheral blood monocytic cells, or PBMC) to kill target cells expressing a specific antigen. The assay is carried out after culturing PBMCs for roughly two weeks, together with cells expressing the specific antigen. Killing is then measured as the amount of radioactivity released when the cultured PBMCs are mixed with radioactively-labelled target cells.
The assay's big advantages are that it measures a clear, important T-cell function, and it has been used in all HIV vaccine trials so far that looked at T-cell responses. For both these reasons, it remains on the list for NIH's HIV Vaccine Trial Network (HVTN). But besides being cumbersome to carry out, CTL assays are not very quantitative and are relatively insensitive. Also, they work poorly on previously frozen cells — a huge drawback for vaccine trials. Yet another disadvantage is in determining whether the responding cells belong to the CD4+ or CD8+ subset, which can only be done by the laborious procedure of depleting one or the other cell population with antibodies.
Limiting Dilution Analysis (LDA): This method quantitates the bulk lytic assay described above by culturing dilutions of PBMCs. For example, if a 1:100 dilution of PBMCs still shows lytic activity when tested after the two-week culture but a 1:1000 dilution does not, it means that the numbers of cells able to kill targets is between 0.1% and 1% of the fresh PBMC. While this method does yield quantitative data, it is even more cumbersome than the CTL assay and therefore ill—suited for scale-up.
ELISpot: The most widely used of the new generation of assays, ELISpot measures the number of T-cells secreting a specific cytokine, such as interferon-gamma or tumor necrosis factor-alpha, that serves as a marker of T-cell effectors. The starting PBMCs are first stimulated with antigen (whole protein or peptide) for 6-24 hours and then mixed with enzymatically-tagged cytokine antibodies. The mixture is then chemically treated so that bound antibody—cell complexes (i.e., cytokine-secreting cells) are stained blue.
Its advantages are many. ELISpot is highly sensitive, quantitative and easy to perform, even in low-tech settings, and it can be scaled up for large numbers of samples. A big breakthrough was the finding that, although the assay initially performed poorly on thawed cells, the problem was solved by using peptides rather than whole proteins as the stimulating antigen. Scoring the assay (counting stained cells) is increasingly being done with automated image analyzers, reducing some of the subjectivity that arose with manual counts and therefore enhancing the assay's reproducibility — a key to using it in multi-site trials. The remaining disadvantages include a high background in many peoples' hands, which makes it harder to distinguish low responders from background with confidence, and its inability to distinguish CD4+ from CD8+ responders without the cumbersome cell separation procedure mentioned above.
Yet overall the ELISpot assay is a major step forward for the field. It will be a key T-cell assay in HVTN trials, and Kent Weinhold of Duke University in Durham, principal investigator (PI) of the HVTN's Central Immunology Laboratory, says that he and Julie McElrath (PI of the University of Washington, Seattle laboratory site) are well on their way towards validating it. Merck is also using ELISpot (and has validated it extensively) in its HIV vaccine program, including the ongoing DNA vaccine Phase I study, and IAVI will use it as well.
The ELISpot assay is also working well in labs in Entebbe, Durban and Nairobi, among other developing country settings, so transferring the technology should not prove to be an obstacle for vaccine trials. At IAVI's recent consultative meeting, Philip Goulder (Oxford University) described its use in a collaborative project based in Durban (with Hoosen Coovadia of the University of Natal and Bruce Walker of Massachusetts General Hospital) to map HIV epitopes recognized by people infected with HIV subtype C. By optimizing the procedure step by step, Goulder said they have reduced background by 10-100 fold, allowing them to score low responses far more reliably.
He also described their use of a peptide "matrix" — a defined set of pools containing 6-7 peptides each, where each peptide is present in several different pools. The method is an effective internal control for avoiding false positives, Goulder said, since each peptide recognized by a given individual should give a positive reaction in every pool containing it. The matrix method is also a quick way to identify the specific epitope(s) being recognized — a use that Goulder says is the real strength of ELISpot.
Intracellular cytokine staining (ICC): Like ELISpot, this assay also measures cytokine-producing cells following antigen stimulation, in this case detecting the intracellular (rather than secreted) form. Detection is based on fluorescent labelling of cells, which are then counted using a fluorescence-activated cell sorter (FACS). Sensitivity is similar to ELISpot, although this still varies from lab to lab, and it works well with thawed cells.
ICC offers several advantages over ELISpot. One is the simplicity of sample preparation: it works with whole blood (i.e., without separating PBMCs first); alternatively, samples can be processed through a few simple steps, then fixed, and frozen for later workup. Another plus is that cells can be stained for several markers simultaneously, so that a single assay can detect not only cytokine-positive cells but other characteristics of the responding cells, for example, whether they are CD4+ or CD8+. Its biggest disadvantage is that it requires an expensive FACS machine and personnel trained to use it, making it far from trivial to introduce in developing country settings. Yet that has also proven solvable: the method is well-established at several African sites, for example the Ugandan Virus Research Institute (UVRI) in Entebbe.
Another perceived weakness of the assay — wide variability among labs — was addressed by Holden Maecker of Becton Dickenson (BD; San Jose), the company developing this methodology, at IAVI's January meeting. He described a study in which the same samples were assayed at four different sites and found to show about 25% variation. But variation was reduced by half when BD collected the computer data sets from the four sites and re-set the fluorescence parameters, suggesting that the actual biological variation is smaller than it first appears. He also reported that BD is working on automating much of the assay. IAVI plans to use this method alongside ELISpot in its upcoming trials, while the HVTN is looking at it in the just-launched HVTN 203 trial (see below).
Tetramer staining: This assay uses flow cytometry to measure CD8+ T-cells that recognize (and bind) a specific HIV epitope. It works by mixing the cell sample with four molecules of a single epitope (as peptide) joined to a class I HLA molecule (a molecule on the surface of CD8+ cells that helps display the epitope to the immune system). Key advantages are that it directly measures cells with receptors for a specific epitope without prior antigen stimulation, it is highly quantitative and sensitive and it can be used on thawed cells. But tetramer assays require knowing the HLA type of each person being sampled and having a specific epitope known to be recognized by that HLA type — effectively eliminating it for broad screening of diverse populations. Yet it is very useful for in-depth analysis of responses in specific populations where this information is available.
Topping the list for the near future is the need to better understand the cells detected by each of these assays — what their functions are and how they overlap. One effort was described by the HVTN's Kent Weinhold at the recent AVRC meeting. Using stored samples from previous HIV vaccine trials, Weinhold reported that the CTL assay often correlates with ELISpot but with exceptions in both directions, suggesting that these assays detect overlapping, non-identical cell populations. The HVTN is now embarking on a large, systematic analysis — also including the ICC and proliferation assays, and comparing fresh and frozen cells — in the newly launched HVTN 203 Phase II trial (canarypox vaccine with or without gp120 boost) with 330 volunteers.
Another issue, raised by Francis Gotch (Imperial College, London) at IAVI's latest meeting, is the potential differences in immune responses and assay performance due to immune status of the study volunteers in different countries. Gotch spends time at the UVRI in Entebbe, where volunteers generally have a high infection load, particularly with helminths (intestinal worms). They also tend to have higher background levels in some of the immune assays, increases that are not seen in Africans living in London — suggesting that they stem from a more highly activated immune system. Gotch recommends that volunteers for HIV vaccine trials get treated beforehand for worms — a simple, inexpensive procedure that also provides immediate benefit to participants.
Observations like these bring the focus back to the bottom line: understanding how to best use these assays in the settings where they are needed most.
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©2000. The IAVI Report.
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