Measuring HIV-1—Specific T Cell Immunity: How Valid Are Current Assays?
- 1Vaccine Clinical Research Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
- 2Partners AIDS Research Center, Massachusetts General Hospital, Charlestown, and Division of AIDS, Harvard Medical School, Boston
- Reprints or correspondence: Dr. Marcus Altfeld, Partners AIDS Research Center, 149 13th St., Charlestown, MA 02129 (maltfeld{at}partners.org).
The development of an effective HIV-1 vaccine to either prevent virus transmission or halt disease progression represents one of the major challenges in biomedical research. To date, >95 clinical trials of different HIV vaccine candidates have been completed, involving >26,000 volunteers. Although the specific functional ability and phenotype required of vaccine-induced cells to protect against HIV infection or limit transmission are unknown [1], efficacy trials afford the opportunity to uncover mechanisms and predictors of protection. To advance HIV vaccine development, it is essential to (1) identify the immune responses that candidate vaccines elicit based on standardized assays, (2) compare and prioritize responses between candidate regimens to guide product advancement decisions, and (3) define the protective mechanisms of viral containment.
Because success in eliciting broadly neutralizing antibodies has been limited to date and because data on the importance of the cytotoxic T lymphocyte (CTL) response in controlling HIV exist, recent vaccine candidates advancing in clinical trials have been focused primarily on inducing cellular immunity. Virusspecific CD8+ T cell responses are thought to play an important role in the control of natural HIV-1 infection, on the basis of the following observations: (1) the first appearance of HIV-1—specific CD8+ T cell responses in primary infection coincides with the decline of peak viremia [2, 3]; (2) depletion of CD8+ T cells in simian immunodeficiency virus (SIV)—infected rhesus macaques results in increased viremia [4]; (3) polymorphisms in HLA class I alleles restricting CD8+ T cell responses are associated with differential HIV-1 disease outcome [5, 6]; and (4) HIV-1 evades virus-specific CD8+ T cell—mediated immune pressure by selecting for variant epitope sequences [7, 8]. However, despite this strong evidence for the important role of CD8+ T cell immunity in the control of HIV-1 infection, current assays to quantify T cell immunity by measuring antigen-specific cytokine production or cytotoxic activity have failed to correlate HIV-1 specific CD8+ T cell responses with protection from infection or control of viral replication [9–10].
After HIV-1 infection of a cell and reverse transcription of the viral RNA, the viral genome is integrated into the host cell genomic DNA. Once active viral replication starts, resulting in translation of viral proteins, parts of the viral proteome are processed and presented on the surface of infected cells by HLA class I molecules, allowing target cell recognition by the T cell receptor of epitope-specific CD8+ T cells. The rapid recognition and subsequent elimination of HIV-1—infected cells before production of new viruses represent critical effector functions of virus-specific CD8+ T cells that are not reflected in the quantification of cytokine secretion or cytotoxic activity after stimulation with high concentrations of exogenous peptide. In 1996, Drs. Otto Yang, Paul Johnson, and Bruce Walker described an in vitro HIV-1 replication inhibition assay that measured the ability of CD8+ T cells to inhibit viral replication in autologous CD4+ T cells infected with laboratory strains of HIV-1 [11]. Studies by these investigators, as well as by additional groups adapting this in vitro assay, have demonstrated that virus-specific CD8+ T cells can inhibit, and under some conditions fully suppress, HIV-1 replication in vitro in a dose-dependent manner through direct cytotoxic activity as well as through the secretion of antiviral cytokines [12–16].
In new data published in this issue of the Journal, Bennett et al. compare the cross-clade antiviral activity of HIV-1—specific CD8+ T cells using the in vitro viral replication inhibition assay with a peptide-based cytolysis assay [17]. The latter assay uses exogenous clade-specific peptides to detect the cross-reactive cytotoxic activity of HIV-1—specific CD8+ T cells, similar to a large number of previous studies measuring cross-clade activity of CD8+ T cells by use of exogenous peptide. In striking contrast to this broad cross-clade detection of HIV-1—specific CTLs, the direct antiviral activity of the same CTL clones, measured using the viral replication inhibition assay, was focused against the autologous B clade sequence and exhibited little or no cross-clade activity. Bennett et al. further demonstrated that the functional avidity of variant epitope sequences for the major histocompatibility complex (MHC) molecule was significantly reduced, compared with wild-type sequences.
Together, these data suggest that the cross-clade antiviral activity of HIV-1—specific CTLs is significantly less in comparison with cross-clade recognition assays employing exogenously added peptides and also question the validity of these types of assays as surrogates for assessing antiviral T cell activity in HIV-1 vaccine trials. There are similar data showing significant impairment of the ability of CD8+ T cells to kill target cells infected with variant viruses in the SIV-infected macaque model, despite broad cross-reactive responses identified in peptide-based assays (David Watkins, personal communication). Therefore, caution must be used when interpreting the cross-reactivity of HIV-1—specific CD8+ T cells to nonphysiological stimulation with peptides. At a minimum, the levels of wild-type and variant peptide required to initiate the cascade of effector functions must be titrated. In nonhuman primate studies, a repeated low-dose SIV challenge of vaccinated animals could be useful to assess the protective potential of such cells [18].
How relevant is the observation that the T cell responses detected using exogenous peptides does not correlate with the antiviral activity of these same T cells to evaluating the immune responses induced by candidate vaccines? In the HIV Vaccine Trials Network, a tiered algorithm is used to evaluate T cell responses to candidate vaccines (figure 1). Tier 1 assays include the interferon—γ enzymelinked immunospot (ELISpot) assay [19] and the 8-color intracellular cytokine flow cytometric assay [20]. Both assays are robust and validated and can be performed with cryopreserved peripheral blood mononuclear cells, permitting batch testing as well as retrospective immunogenicity studies. An important drawback of these assays, in particular in the study by Bennett et al., is that, although CD8+ T cells may secrete cytokines in response to cells loaded with synthetic peptides, providing information about their frequency, such results do not imply that the detected CTLs can recognize the epitope in the context of an HIV-infected cell. Tier 2 assays include determinations of the epitope specificities and breadth of T cell responses. The optimal epitope is confirmed by examining the functional avidity and the definition of the HLA-restricting molecule. This information can show whether certain epitopes are more readily targeted by candidate vaccines, whether they reside within conserved or variable regions of HIV proteins, whether they are commonly recognized during acute and/or chronic infection, and whether they are susceptible to escape mutation. Tier 3 assays determine functional, phenotypic, and molecular profiles of T cell responses elicited by candidate vaccines and after infection in those vaccinees who become infected.
Figure 1.
Tiered approach to measuring HIV-1—specific T cell responses. ELISpot, enzyme-linked immunospot; ICS, intracellular cytokine staining; IFN, interferon, MHC, major histocompatibility complex; TCR, T cell receptor. Adapted with permission from Julie McElrath.
Taken together, the assays currently employed to evaluate T cell responses primarily use exogenous antigen and quantify the immunogenicity of vaccines, but they might fail to evaluate the actual effectiveness of the vaccine-induced T cells. The addition of assays in this algorithm to assess the antiviral activity of HIV-1—specific T cells directly may be an important consideration for future product-advancement decisions. Importantly, a vaccine efficacy trial inducing HIV-1—specific T cell responses was halted in September 2007 because the product was unable to protect the volunteers from HIV-1 infection or to lower viral load in infected individuals, despite the induction of robust HIV-1—specific CD4+ and CD8+ T cell responses secreting cytokines in response to exogenous peptides in earlier trials [21]. This trial was testing a recombinant nonreplicating adenovirus vector encoding Gag, Pol, and Nef proteins from HIV-1 clade B. Although disappointing, the data from this trial will provide important information to guide the future design and evaluation of candidate vaccines. Most importantly, it challenges the algorithms currently used to assess and advance vaccine products.
It has been difficult to determine which functional capabilities of antigen-specific T cells are necessary to protect against HIV-1 infection or disease progression. Unless there is substantial antiviral activity against variant viruses, both within the same clade as well as potentially between different clades, it is unlikely that a single immunogen will elicit responses effective against the many possible viruses to which vaccinees might be exposed. As peptide-based assays do not approximate the ability of CD8+ T lymphocytes to suppress the replication of the virus in autologous CD4+ T lymphocytes, the need for novel assays is urgent. The viral inhibition assay used in the study by Bennett et al. might serve as a suitable alternative, though this assay has significant limitations. Bennett et al. used a small number of epitope-specific CTL clones that were maintained in long-term in vitro cultures. It is possible that epitope-specific CTL clones, which use a single T cell receptor, might exhibit less cross-reactivity than primary epitope-specific CD8+ T cell populations that are normally polyclonal. In addition, the viral replication inhibition assay is labor intensive and requires separation of CD4+ and CD8+ T cells and measurement of HIV p24 over time; the cell-killing ability can vary with manipulation and culture of lymphocyte populations; in vitro HIV replication can vary greatly and is dependent on input virus strain and cellular activation; and assay standardization and validation will be complex despite the availability of clonal CTLs and target cell lines. Despite these challenges, the in vitro viral replication inhibition assay might represent an attractive alternative for obtaining a physiological correlate of the antiviral activity of HIV-1—specific CD8+ T cells, in particular if freshly isolated virus-specific effector cells are used, as recently described by Saez-Cirion et al. [22].
The accurate assessment of the antiviral activity of HIV-1—specific CD8+ T cells is a crucial step toward identifying the immune correlates of vaccine efficacy. There is a tendency to consider CD8+ lymphocyte responses as equivalent, perhaps in the same way that the Env-specific antibodies were once thought to be equally effective in mediating antiviral activity. It is possible that there is a hierarchy of efficacious CD8+ lymphocyte responses in which the most effective CD8+ lymphocytes may be an analogue of a potent and broad neutralizing antibody. For vaccine development, it will be important to define the most effective antiviral activities of CD8+ lymphocyte responses against viral epitopes that are presented early on the surface of infected cells, preferably before Nef-mediated downregulation of MHC class I [13, 16, 17]. A benchmark toward this goal is to identify CD8+ T lymphocytes directed against these viral epitopes that exhibit strong antiviral activity.
- Received October 4, 2007.
- Accepted October 5, 2007.
- © 2008 by the Infectious Diseases Society of America
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