Understanding How Immune Responses to AIDS Vaccine Candidates are Measured
What are the limitations of current methods used to analyze immune responses to AIDS vaccine candidates and what new strategies are being explored?
Researchers do not measure the efficacy of a vaccine candidate—its actual ability to protect against HIV infection or control disease progression in individuals who become HIV infected despite vaccination—until the candidate is tested in large trials that involve thousands of volunteers who are potentially at risk of acquiring HIV. Instead, during the early stages of clinical evaluation, researchers primarily evaluate the safety of the candidate as well as its ability to trigger an immune response against HIV. The ability of a candidate vaccine to induce immune responses is referred to as its immunogenicity, and evaluating immunogenicity is one way that researchers can determine which candidates are worth pursuing in larger trials.
Researchers utilize different tests known as assays to determine the immunogenicity of AIDS vaccine candidates and different types of assays are used to measure different types of immune responses. Antibodies—Y-shaped proteins that latch on to the virus and stop it from infecting human cells—are most commonly measured using an ELISA or enzyme-linked immunosorbent assay (for more on how ELISA works, see VAX August 2007 Primer on Understanding Immunogenicity).
But many of the vaccine candidates that are currently undergoing clinical testing induce primarily cellular immune responses—both CD4+ and CD8+ T cells—against HIV, and not antibodies. Researchers measure and categorize the cellular immune responses induced by a vaccine candidate in many different ways.
To study HIV-specific CD4+ and CD8+ T-cell responses, researchers isolate these cells from blood samples taken from volunteers in AIDS vaccine trials who received the candidate vaccine. They then expose these cells to the HIV fragment, or antigen, that was included in the vaccine candidate. This stimulates some of the immune cells and causes them to secrete certain proteins, known as cytokines, which can then be measured.
There are many different cytokines that play an important role in the immune response against a virus or bacteria. Some have direct antiviral activity, while others work more indirectly by activating other types of immune cells.
The ELISPOT assay is used to detect secretion of a single cytokine by both CD4+ and CD8+ T cells that are induced by a vaccine candidate. It is most commonly used to measure the release of a specific cytokine called interferon-gamma (IFN-γ; see VAX August 2007 Primer on Understanding Immunogenicity).
Measuring multiple cytokines
Another assay that can measure the ability of CD4+ and CD8+ T cells to secrete a broad range of cytokines is known as multi-parameter flow cytometry. As its name suggests, multi-parameter flow cytometry has a distinct advantage over ELISPOT assays in that it can measure the secretion of multiple cytokines simultaneously. This helps researchers more thoroughly define the cellular immune responses induced by a vaccine candidate.
In flow cytometry, cells or parts of cells are tagged with fluorescent probes that then flow through a beam of light, usually from a laser. Cells with different characteristics scatter the light in different ways, allowing them to be analyzed and sorted based on their ability to secrete different cytokines.
While ELISPOT and flow cytometry assays provide useful data, they are not perfect tools. There are some indications, based on the results of clinical trials, that the ability of a vaccine candidate to induce cells that secrete cytokines is not necessarily an accurate predictor of whether an AIDS vaccine candidate will be effective. For instance, in the recently conducted STEP trial that tested Merck’s adenovirus serotype 5-based vaccine candidate, the ELISPOT assay analysis showed the candidate induced high levels of T cells secreting the cytokine IFN-γ, but the vaccine was still found not to be effective in preventing or controlling HIV infection.
Another assay now being assessed in clinical trials measures the specific function of immune cells induced in response to an AIDS vaccine candidate, rather than cytokine secretion, which is just a signal of immune activation. One of these so-called functional assays is known as the viral inhibition assay. It measures whether CD8+ “killer” T cells taken from blood samples of volunteers who received an AIDS vaccine candidate in a clinical trial are actually capable of doing their job and killing HIV-infected cells. Researchers isolate CD8+ T cells from blood of a vaccinated trial volunteer and combine them with HIV-infected cells in the lab to see if they are able to inhibit the virus. This approach is just now starting to be utilized in clinical trials of AIDS vaccine candidates.
Since researchers do not know precisely what immune responses against HIV will help control the virus or prevent infection altogether, it is important to study several different assays to infer as much as possible about the immune responses induced by vaccine candidates.