There was no association between the degree of progression (NP or SP) and any of the variables associated with disease progression (e.g., age, sex and high alcohol intake) or with chronic hepatitis B and/ or C.
All 19 LTNP carried HIV-1 subtype B strains. Analysis of nef showed that all but one had a functional nef gene, with no alterations or defects in their functional domains (Table 3). In this patient (number 3) molecular analysis revealed a deletion of 199 bp. This deletion removed the highly conserved acidic domain, (PXX)4 and PKC motifs, and placed downstream sequences out of frame, which resulted in a premature stop codon. This patient has maintained stable CD4 T-cell counts for more than 16 years. No duplications of the nuclear factor (NF)-κB binding elements that could compensate this deletion were found. All of the patients with a complete nef gene showed some amino acid features (T15, N51, H102, L170 and E182) which previously have been associated with non-progressive HIV-1 infection. In fact, their ‘Nef progression score’ was within the range assigned by others  to NP. However, no differences were observed when comparing NP and SP in this study.
Genetic analysis of vpr showed that all 19 LTNP carried a functional gene. The Vpr peptide was 96 amino acids long in all but one individual, who had an insertion of three amino acids at the C-terminal end. The R77Q change was found in seven (36.8%) patients. There were no significant differences when comparing NP and SP: 4/11 (33%) and 3/7 (42.5%), respectively. However, patients with detectable plasma viraemia and/or higher CD38 expression tended to show R77Q more frequently than patients with undetectable viral load and minimal immune activation.
No patient was homozygous for the Δ32 CCR5 genotype, which was found in heterozygosity in only three subjects (nos. 14, 15 and 17). All patients with Δ32 CCR5 were SP (Table 3).
Virus replicative capacity and co-receptor usage
The replicative capacity (RC) could be measured in viruses from only seven individuals (three NP and four SP) from whom samples were available. Overall, RC values were lower in LTNP with respect to wild-type viruses taken as controls. However, there was a wide variability in the RC among isolates from LTNP, ranging from 5% to 93%. The median RC values were 40% in NP and 74.5% in SP.
Viral co-receptor tropism was examined in the seven patients tested for RC. All individuals were infected with R5 viruses. However, longitudinal samples collected from one patient (number 15) belonging to the SP group showed a shift from exclusively R5 viruses at baseline to a mixed X4/R5 virus population in samples collected on follow-up. This switch preceded a CD4 T-cell decline of 80 × 106 cells/l per year. Interestingly, virus tropism characterization of the two most recent blood specimens collected from this patient show only R5 viruses.
Activation of T-cell subsets
Levels of CD8 T-cell activation were low in all LTNP but three, all of whom had detectable viraemia and showed a progressive CD4 T-cell decline. There was a trend towards an association between T-cell activation and disease progression. Patients belonging to the SP group showed slightly higher activation of CD8 T cells in respect to NP, although the difference did not reach statistical significance (P = 0.08), probably due to the small size of the study population (Table 3). Activation of CD4 T cells was also lower in LTNP than in controls, but there were no significant differences between NP and SP.
HIV specific CTL responses
The mean number of peptide pools to which patients responded was similar (4 ± 1) in LTNP and a group of controls (HIV progressors). For each pool of peptides, a positive CTL response was detected in LTNP and controls, respectively: 93% and 86% to Gag; 92% and 68% to Pol; 93% and 81% to Env; 87% and 100% to Nef; 40% and 47% to regulatory peptides.
There were no differences in the level of CTL responses against each pool, nor in the level of total responses between LTNP and controls: Gag, 0.52% versus 0.82%; Pol, 0.37% versus 0.43%; Env, 0.32% versus 0.31%; Nef, 0.46% versus 0.53%; Reg, 0.07% versus 0.14%; total, 1.84% versus 2.23%. In both groups, the peptide pool that contributed most to the total CTL response was Gag (28.7% in LTNP versus 32.8% in controls), followed by Nef (23.9% versus 28.7%), Env (22.2% versus 17%), Pol (21.3% versus 13%) and lastly regulatory peptides (3.7% versus 8.2%), respectively. Finally, there were no significant differences in the CTL response when comparing NP and SP.
We have described the main features and subsequent outcome of a cohort of 19 LTNP established in 1997. Most of these individuals were first recognized as HIV-1 seropositive in 1985 and 1987, although we have evidence of infection in one of them since 1979. Thus, at the time of cohort establishment all of these subjects had been infected with HIV-1 for more than 10 years. An important finding in our study was the recognition of a different outcome in the study population during the subsequent 6 years of follow-up. Twelve subjects (NP) have remained asymptomatic with stable CD4 T-cell counts while the other seven (SP) have experienced a significant decline of these cells. A similar delayed diminution of CD4 T cells has been reported in other LTNP [12,13]. We could not find any association of this phenomenon with some of the potential cofactors such as age, sex, alcohol intake or other infectious agents, such as hepatitis viruses.
The majority of individuals assigned to the group of NP had undetectable viral load at all time points. On the contrary, all subjects showing slow progressive disease had detectable levels of viraemia. Thus, the ability of the virus to replicate with more or less efficiency was clearly one critical determinant of the distinct outcome seen in this cohort of LTNP, as has been suggested by others [17,18]. Although the virus replicative capacity was reduced in almost all of the patients, slightly lower values were observed in NP than in SP. These findings are in agreement with a causal relationship between viral replication and CD4 T-cell depletion. Whether it results from direct cell killing by the virus and/or from indirect mechanisms such as immune activation is open to debate . Most subjects in this cohort of LTNP showed low T-cell activation, which tended to be lower in NP than in SP. Thus, it might be argued that low immune activation could have contributed to preserve these patients from CD4 T-cell depletion.
Specific HIV CTL responses are important in restricting viral replication and probably also in delaying disease progression. However, in our study we did not find differences in the level of CTL responses when comparing LTNP with a control group of drug-naive HIV progressors. Thus, our findings do not support a critical role of CTL responses in halting HIV progression. If relevant, qualitative parameters within the CTL response could be the main determinants of HIV control, as suggested by others .
Non-syncytium inducing strains seem to be less deleterious to the host immune system  and along with a low replicative capacity, may determine slower progression of HIV disease . In our cohort, six out of seven LTNP maintained R5 viruses during the entire follow-up. The single patient who showed a shift from R5 to mixed R5/X4 viruses at some point, suffered a coincident rapid loss of CD4 T lymphocytes. The emergence of viral variants capable of using an expanded range of coreceptors most probably explained this adverse outcome in this individual.
The Δ32-CCR5 allele seems to provide a continuous protective effect during the course of HIV-1 infection, reducing the risk of disease progression by 31% when present in heterozygosity . None of our patients presented this allele in homozygosis and only three carried the Δ32 deletion in heterozygosis. Since all of them belonged to the SP group, our findings do not support a critical role of CCR5 as cause of LTNP. The protection by the CCR5 delta phenotype is lost when the infecting strain is able to induce syncytia . Interestingly, one of our patients with the Δ32 deletion was the subject who showed a virus with mixed CCR5/CXCR4 tropism. At the time of its appearance, the patient showed a significant CD4 T-cell depletion, perhaps indirectly reflecting a protective effect of Δ32-CCR5.
We could not find any other virological feature in these LTNP which could distinguish those showing slow progression and those without any progression at all. Large deletions in nef are rarely present in HIV isolates from infected persons, although they seem to be more common in LTNP [25,26]. Only one of our patients presented a major deletion that rendered a non-functional nef gene. Moreover, specific amino acid changes in Nef, which have been associated with LTNP , did not differ among NP and SP. Likewise, we did not find differences between vpr genes from NP and SP. The change R77Q was present in 33% and 42% of NP and SP, respectively. These percentages are clearly below those reported originally among LTNP, which were in the range of 80% . Interestingly, most LTNP with detectable viraemia in our cohort presented the R77Q change, and it could be hypothesized that the presence of this mutation might have ameliorated T-cell destruction in those individuals despite them harbouring detectable viral load.
In summary, LTNP seem to be a heterogeneous small subset of HIV-1 infected individuals. No unique viral or host factors explain the absence of disease progression. Even very low levels of HIV replication seem to result in progressive CD4 T-cell depletion, although in these patients it may take decades to become manifest. Whether this applies to all LTNP or whether a subset of them will maintain their CD4 T-cell numbers indefinitely is unclear.
The authors thank J. Gonzalez-Lahoz for his continuous support.
Sponsorship: Supported in part by grants from Comunidad Autónoma de Madrid, Asociación Investigación y Educación en SIDA, FIPSE, and Red de Investigación en SIDA (RIS G03/173).
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Keywords:© 2004 Lippincott Williams & Wilkins, Inc.
long-term non-progressors; disease progression; natural history; viral fitness