Measurement of immune responses directed specifically against HIV is critical for understanding the interplay between the virus and the host immune system. By characterizing the immunological correlates of protection against HIV infection, such measurements will aid in the development of efficacious prophylactic vaccines. Immune responses to HIV can be measured by a variety of methods. Humoral immunity is detected by neutralizing antibody assays, while cellular immunity is investigated using a variety of techniques, including cytotoxic T-lymphocyte (CTL) assays for detection of CD8+ T cells with cytolytic ability; ELISpot and intracellular cytokine staining assays for detection of cytokine-secreting T cells; HLA tetramer binding assays for the enumeration of antigen-specific T cells; and lymphoproliferative assays (LPAs) for detection of HIV-specific CD4+ T cells.
| Humoral Immune Responses|
| Neutralizing Antibody Assays|
Sterilizing immunity against HIV infection is expected to require the presence of HIV-specific neutralizing antibodies. Such antibodies prevent infection by binding to regions of the HIV envelope that are required for viral attachment and entry into host cells. Antibody-mediated neutralization of different T-cell line adapted HIV strains is measured by MT-2 cell killing assays.(1) MT-2 is a CD4+ human lymphoblastoid cell line that is highly permissive to cytopathic infection with CXCR4-utilizing strains of HIV.(2) In these assays, cell-free virus is incubated with multiple dilutions of serum samples at 37°C for 1 hour before MT-2 cells are added. Any neutralizing antibodies present in the serum will bind to the virus and limit its ability to infect the MT-2 cells. Neutralization is measured by staining viable cells with Finter's neutral red when cytopathic effects in control wells (cells plus virus but no serum sample) are >70% but <100%. Neutralizing titers are expressed as the reciprocal of the plasma dilution required to protect 50% of cells from virus-induced killing. This 50% cutoff corresponds to a 90% reduction in viral antigen synthesis.(3) For standardization purposes, positive control serum samples with known average titers are tested in parallel. Deviation of more than 2-fold in experimental titer of the positive control serum from its known average titer requires that the assay be repeated. Serum samples are heat treated prior to use in these assays in order to inactivate the complement system, because components of activated complement can deposit on virions and target them to complement receptors on the MT-2 cells, enhancing infection and interfering with assessment of antibody neutralization. Activation of complement is not an issue for PBMC assays (see below) because normal CD4+ T cells do not express complement receptors.
Although MT-2 killing assays are useful for determining neutralization against T-cell line adapted (CXCR4-tropic) strains of virus, they are not useful for determining neutralization against most primary isolates, which are CCR5-tropic. Neutralization of primary isolates of HIV is assayed in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) by monitoring for a reduction in p24 Gag core antigen synthesis.(4) As with the MT-2 assays, known concentrations of virus are incubated with various dilutions of serum samples for 1 hour at 37°C before addition of PHA-stimulated cells. After an overnight incubation the cells are washed and then cultured in fresh interleukin-2 (IL-2) containing growth medium. Culture supernatants are collected on a daily basis and assayed for p24 using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Concentrations of p24 in virus control wells (virus plus cells but with no serum) are calculated for each harvest day and are used to construct a viral replication curve. Neutralization is measured at a time before peak p24 production occurs as determined from this curve. Neutralization titers are the reciprocal of the dilution at which p24 synthesis is reduced by 80% relative to a negative control serum.
| Cellular Immune Responses|
The immunological correlates of protection against HIV are not known. While neutralizing antibodies may be necessary to achieve sterilizing immunity, they have proven difficult to induce by vaccination.(5) It is likely that cellular immunity, particularly CD8+ cytotoxic T lymphocyte, will be critical in limiting viral replication if initial HIV infection cannot be prevented.(6,7) Indeed, depletion of CD8+ lymphocytes in nonhuman primate models results in an increase in plasma viral load.(8-10) In addition, increasing evidence suggests that induction of HIV-specific CD4+ T cells may also be critical for sustained control of viral replication.(11-14) For these reasons, current trials of potential HIV vaccines are designed to induce HIV-specific T-cell responses in addition to neutralizing antibodies.
CD8+ T cells exert their antiviral effects by direct lysis of infected cells and by secretion of antiviral cytokines (eg, interferon gamma [IFN-gamma]) or chemokines (eg, MIP1-alpha, RANTES). Cellular immunoassays have been developed to measure both of these types of effector function.
| Cytotoxic T-Lymphocyte (CTL) Assays|
Chromium release assays determine the ability of CD8+ T cells to lyse target cells expressing specific HIV antigens. Briefly, freshly isolated PBMCs from an infected or vaccinated individual are stimulated in vitro with HIV antigens for 2 weeks in order to expand HIV-specific T cells. Such expansion is required because HIV-specific CD8+ T cells are normally present at frequencies that are too low to detect by this method if cells are used directly ex vivo. These expanded T-cell lines are then incubated with autologous target cells (normally B-lymphoblastoid cell lines derived by transformation of autologous B cells with Epstein-Barr virus) that have been labeled with the radioisotope 51Cr and infected with vaccinia expressing the HIV antigens of interest. HIV-specific T cells will recognize and lyse the target cells, resulting in liberation of the radioactive label into the culture supernatant. Spontaneous lysis is measured in replicate wells containing radiolabeled targets in the absence of effector cells, and maximum lysis is measured in replicate wells containing detergent. Radioactivity of the supernatants is measured using scintillation counting. Specimens with HIV-1-specific lysis exceeding 15% [calculated as: (lysis minus spontaneous lysis) /(maximum lysis minus spontaneous lysis) X 100] after correcting for lysis against mock-infected target cells are considered positive.
CTL assays are extremely laborious due to the in vitro stimulation period and preparation of autologous target cells required for these assays. In addition, they can only be reliably performed on freshly isolated, not cryopreserved, PBMCs. For these reasons, a number of assays designed to measure other effector functions of CD8+ T cells, notably cytokine secretion, have been developed.
| Interferon gamma (IFN-gamma) ELISpot Assays|
These assays measure the ability of T cells to secrete IFN-gamma in response to a specific antigen. They are based on ELISA methodology. PBMCs from the individual being tested are added along with specific HIV peptides to a 96-well nitrocellulose plate that has been coated with antibodies to IFN-gamma. Control wells containing PBMCs and medium alone, without the HIV peptides, are assayed in parallel. During an overnight incubation at 37°C HIV-specific T cells present among the PBMCs will be stimulated by their cognate HIV peptide to secrete IFN-gamma. The plate-bound anti-IFN-gamma antibody captures this IFN-gamma. The following day, the PBMCs are removed and the wells washed before addition of a second anti-IFN-gamma antibody, which is labeled with biotin. This second antibody recognizes a different epitope on IFN-gamma than the first coating antibody. A "sandwich" of IFN-gamma between two anti-IFN-gamma antibodies is thus produced. Excess unbound antibody is removed by washing before addition of a streptavidin-enzyme conjugate. Streptavidin binds with extraordinarily high avidity to the biotin linked to the second anti-IFN-gamma antibody, creating a very stable biotin/streptavidin complex bound to the enzyme (typically horseradish peroxidase [HRP]). The enzyme substrate is added, which results in the formation of a colored product, or spot, where the streptavidin/biotin complex resides. Each spot corresponds to an antigen-specific cell that has secreted IFN-g in response to its cognate HIV peptide. The number of spots corresponds to the number of antigen-specific T cells present among the PBMCs. HIV-specific CD8 T-cell responses can be distinguished from HIV-specific CD4 T-cell responses in these assays by depletion of CD8+ T cells prior to the assay using magnetic beads labeled with anti-CD8 antibodies. These CD8-depleted cells are tested in parallel with the undepleted PBMC. If responses are seen in the PBMC wells but not the corresponding CD8-depleted wells, the responses are due to HIV-specific CD8+ T cells. If responses persist in the CD8-depleted wells, these are due to HIV-specific CD4+ T cells.
Because antigen-specific effector T cells secrete a number of cytokines and chemokines in addition to IFN-gamma that may play an antiviral role, measurement of IFN-gamma alone is not ideal, since it is likely that discrete populations of cells secreting other cytokines and chemokines will be missed. ELISpot assays have been developed to enumerate cells secreting a number of other cytokines, such as IL-2 or tumor necrosis factor alpha (TNF-alpha).
| Intracellular Cytokine Staining (ICS)|
Intracellular cytokine staining (ICS) is a flow cytometry-based method for enumeration of antigen-specific, cytokine-secreting T cells. PBMCs from the subject being tested (typically an infected or vaccinated individual) are stimulated with HIV peptides in the presence of costimulatory antibodies against CD28 and CD49d. Negative control stimulations with PBMCs and costimulatory antibodies but no HIV peptide, and positive control stimulations with superantigens such as staphylococcal enterotoxin B (SEB) are set up in parallel. Stimulations are allowed to proceed for 6 hours at 37°C in the presence of brefeldin A or monesin, which inhibit protein transport through the golgi. Thus any cytokines induced by the HIV peptides in HIV-specific T cells will be prevented from being secreted and will accumulate within the cells. Responding cells can be visualized using fluorescently labeled antibodies that are specific for the cytokines of interest. Responding cells can be further characterized by additional, lineage-specific antibody markers (eg, CD3 and CD8 antibodies for CD8+ T cells). Once fluorescently labeled, the cells are analyzed using a flow cytometer, which possesses lasers and filters to excite specific fluorescent antibodies and measure the released light, permitting enumeration of the responding T cells.
ICS and ELISpot both measure the same effector function but have distinct advantages. In general, ELISpot requires fewer cells and is less technically demanding than ICS. ICS, however, allows precise immunophenotyping using lineage-specific markers to identify the responding cell population or populations. A major advantage of both ICS and ELISpot assays is that, unlike chromium release assays, they can be performed on cryopreserved cells.
| MHC Tetramer Binding Assays|
Whereas the cellular assays mentioned thus far measure effector function, tetramers measure the absolute number of cells that recognize a particular epitope, without providing any information regarding the functionality of these cells.(15,16) HIV-infected cells digest HIV proteins into short peptidic fragments (epitopes), which are bound to MHC class I molecules and displayed at the cell surface. T cells recognize their targets through the interaction of their T-cell receptors (TCR) with these MHC/HIV epitope complexes. MHC tetramers are reagents consisting of four MHC class I molecules bound to the HIV peptide of interest, linked together by a fluorescently labeled streptavidin molecule. These reagents will bind specifically to the TCR of all T cells that recognize that particular MHC/peptide complex. This is a very powerful tool for precise, easy, and rapid enumeration of HIV-specific T cells.
Tetramer technology is limited by several factors. The exact HIV peptide and its restricting MHC class I molecule must be known in order to make the tetramers. Since there are hundreds of different HIV epitopes whose restricting MHC molecules are not defined, this constitutes a major limitation. Furthermore, although MHC class II tetramers are now available for the detection of specific CD4+ T cells, these are even more limited than the class I tetramers by the lack of defined epitopes and known restricting MHC class II molecules. In addition, tetramer staining gives no information regarding the functionality of the stained T cells.
| Lymphoproliferative Assays|
LPAs measure the ability of HIV-specific CD4+ T cells to proliferate in response to their cognate antigen. LPAs are generally performed by incubation of PBMCs with HIV proteins for 6 days at 37°C. The proteins are endocytosed, processed, and presented to HIV-specific CD4+ T cells by macrophages present among the PBMCs. These HIV-specific T cells will start to proliferate in response to activation. After 5 days, thymidine radiolabeled with 3H is added to the culture wells. The proliferating cells take up the radioactive thymidine and incorporate it into newly synthesized DNA. The following day the cells are harvested onto fiber filter mats and the amount of radioactivity is measured. The amount of radioactivity is directly proportional to the amount of proliferation, and therefore, to the number of HIV-specific CD4+ T cells present. LPAs generally do not show proliferation of CD8+ T cells because protein antigens, after endocytosis by macrophages, are processed through the class II antigen-processing pathway. The resulting peptides are presented on MHC class II molecules for recognition by CD4+ T cells. Measurements are normally recorded as stimulation indices (SI). The stimulation index is the counts per minute (cpm) of radioactivity measured in the wells containing HIV protein divided by the cpm measured in control wells containing PBMC but no HIV protein. An SI of 5.0 (ie, a 5-fold increase over background) is considered positive.
We would like to thank Martin Horton (Partnership Design, Chester, UK) for generously designing and providing the ELISpot movie.
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