The results of both the STEP and RV144 clinical trials suggest that CD8+
T cell responses alone might neither prevent HIV-1 infection nor lower virus set-point after infection 
. However, given the relatively weak and narrow T-lymphocyte responses were induced by the vaccine protocol used the in the STEP trial 
, and encouraging non-human primate vaccine studies 
it is still important to continue this approach. Thus, features of HIV-specific CD8+
T cells that determine their in vivo
efficacy need to be defined. It has been widely documented that CD8 T cell responses are elicited early following HIV-1 infection and that they are associated with control of viremia as well as with selecting for escape mutants that may impact virus fitness 
. As their most important impact may take place during acute infection, we have focused our study on characterization of the functionality of HIV-specific CD8 T cell responses at this time, concentrating on epitope-specific responses which did or did not drive selection for escape mutations of the transmitted virus.
We evaluated CD8+
T cell responses using 18-mer peptides corresponding to the autologous transmitted/founder viral sequence of seven individuals recruited within 45 days from the onset of acute HIV-1 infection 
. We first studied the total response to the gag
gene product, because of its importance in vaccine design, and then extended the study to epitopes that we had previously characterized for escape mutants because of their potential impact on virus set point 
. We observed that: 1) more than 80% of the epitope specific T cells in acute infection were oligofunctional; 2) MIP-1β-producing T cells were associated with epitopes that escaped; 3) transmitted/founder virus escape mutants in acute infection occur in epitopes that have high entropy at the population level.
Previously in chronically infected patients, multifunctionality of HIV-specific CD8+
T cell responses in the peripheral blood has been shown to be associated with long-term non-progression 
, and it has been related to the CD4 count and inversely related to the virus load at the rectal mucosal level 
. In our study, we characterized the earliest functional responses and observed that the early CD8 T cell response was predominantly mono- or bi-functional. The contribution of T cells with 3-5 functions was small but increased with time from infection and we cannot exclude that they become a major subset as the infection progresses, particularly in those who control the infection well, as previously described. Although we cannot exclude the possibility that the presence of early poly-functional CD8+
T cell responses may predict control of viremia, the observation that the only controller in our cohort displayed a profile of functional responses similar to those who did not control virus replication would suggest otherwise. Our observations are largely in agreement with those made by Streeck and collaborators, who reported an increase in poly-functionality with the appearance of escape mutants and a subsequent decrease in functionality over time 
. In fact, we likewise observed an increase in functionality of epitope-specific CD8+
T cell responses from the early to the HRF time point, although, we showed that only the total CD8+
T cell responses were significantly associated with the appearance of escape mutations. This could be related to the fact that, in our study, responses were detected in most cases before full escape occurred when virus load had not yet reached the virus set-point. The initially low level of multifunctionality might be related to high levels of virus replication that would in turn favor upregulation of inhibitory receptors such as PD1. Such upregulation would have a negative impact on the functionality of the CD8+
T cells – an impact that is rescued in chronically-infected individuals upon control of VL following initiation of therapy 
. By comparing our data to those obtained from individuals immunized with MVA to protect against smallpox virus infection 
, we also hypothesize that HIV-specific CD8 responses may be substantially delayed in acquiring multifunctionality because of impairments in CD4+
T cell populations, which are otherwise preserved and functional in MVA vaccine recipients. It is, therefore, possible that maturation of HIV-1 specific multifunctional CD8+
T cell responses might be affected by both the impact of high levels of virus replication and the early loss of CD4+
T helper function.
In the present study we also sought to characterize whether the functionality of the CD8+
T cell responses generated during acute/early HIV-1 infection may be related to the immune pressure exerted on the transmitted virus. For the 19 ME epitope-specific CD8+
T cell responses studied, the total magnitude of functional CD8+
T cell response at early time points was significantly associated with the early appearance of escape mutations, when responses were mainly oligofunctional. Multifunctional T cells were a minor component of the response. The observation of the overall impact of the magnitude of functional T cell responses on the appearance of escape mutations is in agreement with previous results obtained by the analysis of IFN-γ+
T cell responses, although, in this cohort of patients the IFN-γ+
responses alone did not show significant association with the appearance of escape mutants 
. Other characteristics of the T cell responses to e-ME may also have influenced the rate at which they were escaped. For example, e-ME may generally elicit clonal CD8+
T cell populations with higher avidity 
and/or a better selection of a T cell receptor (TcR) repertoire, as suggested by both human and NHP studies 
. These questions are being addressed separately.
Further analysis of the proportion of epitope-specific T cells producing individual cytokines revealed for the first time that MIP-1β+
T cells constituted greater than 80% of the cells participating in the early responses and, therefore, were associated with the appearance of escape mutants. The contribution of MIP-1β+
T cells to the overall response declined with time for the l-ME and nME responses, suggesting that persistent antigenic stimulation might also be an important factor in selecting for clones whose functional profile is dominated more by IFN-γ+
production as part of the maturation process 
. Among the CD8+
responding cells, we observed that CD8+
T cells were the two multifunctional subsets most highly contributing to the early responses. We have recently demonstrated that CD8+
T cell mediated antiviral function in virus controllers and vaccinees is associated with Clade B Env and Gag-specific MIP-1β and CD107a expression as monofunctional and dual-functional populations, suggesting the importance of both cytolytic and non-cytolytic mechanisms in control of HIV replication 
T cells can suppress HIV-1 replication noncytolytically through the secretion of a number of soluble factors, including the B-chemokines, MIP1α, MIP1β and RANTES, which can potently inhibit CCR5 tropic virus infection 
. We have found that the secretion of MIP-1α, MIP-1β, IP-10, MIG, IL-1, and interferon gamma correlated most strongly with soluble noncytolytic suppression 
. Using the ability of histone hyperacetylation to alter the expression of immune-related genes, we identified that MIP-1α and IP-10 were also among the genes that were down-regulated by histone hyperacetylation; again supporting the idea that they contributed to soluble CD8+
T cell mediated suppression 
. Taken together, these observations suggest that T cells involved in selecting for escape mutants during acute HIV-1 infection may possess a combination of activities that include both killing and inhibitory activity. This possibility has also been suggested in the SIV model where loss of HIV-infected CD4+
T cells was not prevented by administration of antiretroviral therapy in CD8-depleted animals, suggesting that CD8+
T cell functions other than cytotoxic activity might contribute to early control of virus replication and recovery of CD4 counts 
Neither the magnitude, the phenotype, nor the functionality of CD8+ T cell responses at time of HRF showed any association with the appearance of escape mutants. These lack of associations could be related to several factors: 1) our inability to detect the true peak of responses; 2) the fact that the maturation of responding cells may be impaired by high levels of virus replication; and 3) rapid appearance of escape mutants.
Our data indicate that high entropy at the population level is significantly associated with the appearance of escape within the first six months post-infection, extending observations made in previous studies of escape in acute infection 
. Epitopes that exhibited delayed escape despite an early response were associated with low entropy at the population level, hence higher fitness costs may be associated with change in these epitopes. Our finding is in line with results from earlier studies that have demonstrated the role of fitness costs of sequence changes in/around CD8+ T cell epitopes in constraining escape from epitope-specific responses 
. Delayed escape was also associated with less vigorous CD8+
T cell responses as revealed by lower total response magnitudes assessed by ICS.
Overall, our data show that fully multifunctional (5+) Gag-specific CD8+ T cell responses are not detectable during the initial weeks following HIV-1 infection in the response to the transmitted virus. Moreover, the relative contribution of 3- and 4-multifunctional subsets to the total response is small, although it increases with time from initial infection. Importantly, the magnitude of epitope-specific CD8+ T cell responses including the frequency of epitope-specific MIP-1β -producing cells is associated with the order of appearance of escape mutants. The order in which different epitopes escaped CD8 responses is also associated with the population diversity (entropy) of the epitopes. These findings underscore the important role that the magnitude of the CD8+ T cell responses plays in exerting pressure on replication of transmitted/founder HIV-1 in the early stages of infection, and illustrate the importance of considering both epitope entropy and functional responses beyond the cytotoxic properties of epitope-specific CD8+ T cells for vaccine design.