Longitudinal data demonstrating the role of cytotoxic T lymphocytes (CTL) in the course of HIV -infection are available mainly from clade B-infected individuals and have revealed the persistence of strong and broad CTL responses in long-term non-progressors [1–3]. The breadth of CTL directed against HIV proteins was stable over time and was shown to be negatively correlated with viral load and disease progression . Despite low CD4 T-cell numbers and a persistently activated immune system [5–7], HIV disease progression in clade C-infected Ethiopians is comparable to that in developed nations . To compare individuals with fast and slow disease progression, HIV-specific T-cell responses were analysed in the course of HIV infection in 10 antiretroviral-naive HIV-1 subtype C-infected Ethiopian factory workers who participated in a long-term cohort study on HIV-1 incidence and progression [9,10].
Almost all subjects in the slow CD4 T-cell decline group had stable CD4 T-cell levels, varying from 100 to 966 cells/μl at the first HIV-positive visit and from 56 to 1061 cells/μl after 5 years of follow-up, with a maximum CD4 T-cell loss of 13 cells/μl per year (Fig. 1a), which is smaller than the median yearly CD4 T-cell loss in HIV-infected Ethiopians (32 cells/μl blood) . The viral load ranged from 1.9 to 4.3 log and declined in two subjects (subjects A and D) and increased in three subjects (subjects B, C, and E). CD8 T-cell numbers remained stable between 500 and 600 cells/μl in two subjects (A and E) and increased in the other three subjects (75–95 cells/μl per year).
CD4 T-cell counts in individuals with a relatively fast CD4 T-cell decline ranged from 310 to 826 cells/μl at the first visit, and declined to 74–456 cells/μl at the last visit. The yearly CD4 T-cell loss in the fast CD4 T-cell decline group ranged from 48 to 176 cells/μl, which is significantly higher than that in the slow CD4 T-cell decline group (P = 0.009, Mann–Whitney test; Fig. 1a). CD8 T-cell counts declined in all subjects, which coincided with a severe depletion of the CD4 T-cell counts at the last timepoints analysed. The viral load increased in all cases except one.
The CD8 T-cell count in the fast CD4 T-cell decline group at intake was significantly higher (772–2429 cells/μl) than in the slow CD4 T-cell decline group (514–1000 cells/μl; P = 0.047). CD4 T-cell counts (P = 0.92) and viral load (P = 0.14) at intake were no different between the two groups (Mann–Whitney test).
Over a timeframe of 3–5 years, HIV-1 subtype C Gag-specific CTL responses were evaluated by IFN-γ enzyme-linked immunospot assay after stimulation with a total pool of 49 clade-C (isolate 96 ZM 651.8) specific synthetic Gag peptides (20 amino acids in length overlapping by 10 amino acids), and 14 subpools each containing seven peptides (final concentration 2 μg/ml for each peptide) to identify the specific peptide(s) responsible for the observed T-cell responses .
Six out of the 10 study subjects, three with a fast and three with a slow CD4 T-cell decline, showed CTL directed to one or more HIV-1 Gag peptides [responders, > 100 spots/106 peripheral blood mononuclear cells (PBMC); Fig. 1b], whereas the remaining four lacked detectable Gag-specific T-cell responses despite a good response to mitogens (phytohaemagglutinin; non-responders) confirming our previous cross-sectional study in which 40% of individuals also lacked detectable Gag-specific T-cell responses (A. Tsegaye et al., 2006, in preparation). The lack of HIV-1 Gag-specific responses persisted over time during a follow-up of 4–5 years and was not explained by differences in stages of disease progression, as non-responsiveness was observed irrespective of the rate of CD4 T-cell decline.
The magnitude of the total anti-Gag CTL response was below 800 spot-forming cells (SFC)/106 PBMC in the fast CD4 T-cell decline group. In two of the individuals with fast CD4 T-cell decline, CTL responses declined to less than 200 SFC/106 PBMC. In contrast, in the slow CD4 T-cell decline group, the total response ranged from 600 to 6000 SFC/106 PBMC at the first timepoint, and persisted over time (Fig. 1b). Similarly, Betts et al.  showed a role for HIV-specific CTL in long-term survivors with a slow rate of CD4 T-cell loss (18 cells/μl per year).
Most peptides targeted by the slow CD4 T-cell decline group were from the P24 region of Gag, whereas responses against p17 (amino acids 31–70) dominated in the fast CD4 T-cell decline group. As a whole, more conserved epitopes that are less prone to CTL escape are found in the p24 region of Gag , and may thus give a persistent CTL response leading to CD4 T-cell preservation. Remarkably, all of the individuals with a slow CD4 T-cell decline responded to the p24 peptide 261IYKRWIILGLNKIVRMYSPV280, whereas none of the individuals in the fast CD4 T-cell decline group did, suggesting that a persistent response to this p24 peptide may be beneficial. The three individuals responding to this peptide shared human leukocyte antigen alleles belonging to the B27 supertype (Fig. 1b), making it likely that the response against this peptide is B27 restricted and suggesting a protective role for this supertype.
CTL responses from two of the three responders in the slow CD4 T-cell decline group were directed against several Gag peptides (six and nine peptides) including the peptide 170MFTALSEGATPQDLNTMLNT189, which contains the most dominantly targeted epitope in South African individuals, TL9 [14,15]. In contrast, the breadth of the Gag-specific CTL response in the group with a relatively fast CD4 T-cell decline was narrow; the three responders in this group responded to one, two, and three peptides each.
Taken together, poor and declining CD8 T-cell responses mainly directed to p17 Gag coincided with fast CD4 T-cell decline, whereas high, persistent and broad CD8 T-cell responses mainly directed to p24 Gag correlated with slow CD4 T-cell loss. Our finding that T cells directed against dominant peptides persisted over time is promising for the design and development of effective vaccines appropriate for the local population. Furthermore, the identified peptides are part of the recently identified immunodominant epitope-rich long amino acid stretches in South African individuals , which contain multiple epitopes that are frequently targeted. Therefore, our data suggest that vaccines focusing on p24 Gag might be appropriate for subtype C-prevailing African regions.
This study is part of the Ethiopian–Netherlands AIDS Research Project (ENARP), a collaborative effort of the Ethiopian Health and Nutrition Research Institute, the Amsterdam Municipal Health Service, the Department of Clinical Viro-Immunology, Sanquin Research and the Academic Medical Center of the University of Amsterdam. The kind collaboration of ENARP cohort participants is gratefully acknowledged. The HIV-1 subtype C Gag peptides complete set was obtained through the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.
Sponsorship: ENARP is a bilateral project financially supported by the Netherlands Ministry of Foreign Affairs and the Ethiopian Ministry of Health.
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