Identification of the inducible CD8+ T-cell functions responsible for virus control in HIV-1-infected humans will profoundly impact vaccine design. To this end, it is important to delineate the functional antiviral capacity of HIV-1-specific CD8+ T cells elicited by vaccine strategies and to compare those capacities with CD8+ T-cell inhibitory activities found in HIV-1-infected subjects who control virus production. T cells are remarkably heterogeneous and can be characterized by their capacity to express numerous functions including cytolysis, cytokine secretion, and proliferation. Some of these functions, as well cellular homing properties and differentiation stage, can be inferred from cellular phenotypes. In this study, we demonstrated that vaccination against HIV-1 Env and Gag-Pol by DNA priming followed by rAd5 boosting elicited CD8+ T-cell-mediated antiviral activity. In both vaccinees and in chronically infected HIV-1+ virus controllers, this activity correlated with HIV-1-specific CD107a or MIP-1β expression from HIV-1-specific T cells. Moreover, for vaccinees or virus controllers, purified memory CD8+ T cells from a wide range of differentiation stages were capable of significantly inhibiting virus replication.
The quality of CD8+
T cells can be assessed by examining multiple parameters simultaneously (22
). Various functions may have antiviral activity; for example, CD107a expression is associated with cytolytic activity and may reflect the ability of antigen-specific CD8+
T cells to eliminate infected cells. Similarly, MIP-1β secretion can directly inhibit infection of CCR5-using viruses by blocking the viral coreceptor (7
). Finally, effector cytokines such as IFN-γ or TNF-α may have antiviral activity, although TNF-α can also activate HIV-1 replication through NF-κB activation. Identifying which of these factors is responsible for antiviral activity by individual CD8+
T cells can be ascertained with multiparameter flow cytometry. Here, we report on the role of multiple CD8+
T-cell functions in inhibiting viral replication in vitro
In general, the total expression of IFN-γ, CD107a, or MIP-1β correlated with viral inhibition. However, it is important to note that these cytokines are not expressed independently but are coordinately regulated in many T cells. By dissecting these responses down to individual combinations of these cytokines, we found that the viral inhibition was most closely defined by MIP-1β or CD107a expression. Indeed, the secretion of IFN-γ in the absence of these two functions was not correlated with inhibition. These data suggested that IFN-γ is a surrogate for CD107a and/or MIP-1β because of high coexpression but is not likely to contribute to virus inhibition in this assay. Likewise, TNF-α or IL-2 secretion was not observed to be characteristic of antiviral cells.
We also examined the relationship of the CD8+ T-cell phenotype to antiviral activity. In general, vaccinees have HIV-1-specific cells of an earlier differentiation stage than do controllers, and the chronically infected have the most-differentiated cells. Nonetheless, we did not identify strong correlations between the phenotype of HIV-1-specific CD8+ T cells and the magnitude of antiviral activity in either vaccinees or infected subjects. To further delineate the relationship of differentiation stage to inhibition, we isolated pure populations of CD8+ T cells based on their phenotypically defined differentiation stage and quantified the ability of these cells to effect viral inhibition. Overall, we found that cells of any memory phenotype could inhibit HIV-1, whether of the central memory or effector memory categories.
A goal of vaccination is to elicit a strong and durable memory response—a characteristic of central memory cells. However, due to the rapidity in which HIV-1 establishes a latent infection (18
), it will be important to arm HIV-1-specific T cells to respond rapidly to infection; thus it is presumed that effector T cells will also be needed. Our results suggest that whether a vaccine elicits central memory- or effector memory-biased CD8+
T-cell responses, a strong antiviral activity can be achieved. A vaccine-elicited CD8+
T-cell-mediated antiviral response could help control early viral replication and reduce the risk of established infection and subsequent transmission. The magnitude and breadth of a vaccine elicited virus inhibitory response needed to impact in vivo
virus replication are still unknown. The DNA/rAd5 vaccination induced CD8+
virus inhibitory responses that were 0.7-log less than HIV-1+
virus controllers; it is not clear if the level of CD8+
T-cell responses achieved by this vaccination strategy would be sufficient for controlling virus replication in vivo
. Further optimization of vaccine regimens to induce these responses will be important.
The genetic determinants HLA B*57 (2
) and HLA B*27 (12
) are strongly associated with virus control. An unresolved question is whether T-cell-based vaccines can elicit functional CD8+
T cells in subjects without these MHC alleles. We found that HIV-1-infected individuals bearing these alleles did not have substantially greater inhibitory activity. Importantly, our study also demonstrates that T-cell-based vaccines can effectively target genetically diverse populations and elicit CD8 inhibitory activity even in individuals who are not genetically predisposed to have better control of HIV-1 replication.
T cells that can inhibit a diverse panel of HIV-1 isolates are an important goal for vaccine design; thus assay systems need to be established to measure this heterologous virus inhibition, akin to the way neutralizing antibodies are systematically studied (15
). In this study, we used a panel of HIV-1 enveloped viruses that include several distinct transmitted/founder viruses and viruses with different coreceptor usage. We found that although all viruses were inhibited, there were some differences in sensitivity, with two of the transmitted viruses (CH040 and CH058) having the most sensitivity to CD8 virus inhibition. Importantly, measurement of inhibitory activity against a wide panel of virus isolates may reveal the potential breadth or coverage of the CD8 virus inhibitory response elicited by a given vaccine approach.
In summary, we show that the expression of MIP-1β and CD107a by HIV-1-specific CD8+ T cells, in either the setting of vaccination or natural infection, most closely corresponds to antiviral activity. In contrast, it appears that the phenotype of the HIV-1-specific CD8+ T cells is less important to this activity. We also demonstrate that, with respect to virus inhibition, HIV-1 vaccines can stimulate anti-HIV CD8+ T-cell subsets similar in function to what is found in HIV-1+ virus controllers. Quantifying these functions will be important for the optimization of vaccine strategies attempting to elicit CD8-mediated antiviral activity.