Our results suggest that in the individuals studied, the time between primary HIV-1 infection and AIDS could be subdivided into three phases based on patterns of viral evolution over the C2-V5 region of env. The phases were defined by changes in viral divergence from the founder strain and intra-time point diversity; these definitions in turn were reinforced by observed changes in the prevalence of X4 viruses (probably including dualtropic X4/R5 viruses). In the first phase, both viral population divergence and diversity increased linearly, with diversification essentially keeping pace with divergence at a rate of about 1% per year. The beginning of the second phase was signaled by a leveling off, or decrease, in diversity. In this phase, the viral population continued to diverge from the founder strain at the same rate, while diversity continued to plateau or constrict. Finally, the increase in divergence also slowed or stabilized, marking the beginning of the third phase, the latter being characterized by a decline in diversity.
The peaking of diversity was strongly supported by statistical analysis. Although similar support for the point of divergence stabilization was not found, the validity of this concept, as well as the general framework put forward here, is strongly supported by the consistency of the patterns described among participants and by the close linkages observed between the three defined phases and the patterns of X4 virus prevalence and T-cell subset landmarks. Thus, the second phase was strongly associated with X4 viruses, which in most participants first appeared very close to the beginning of this phase and peaked in prevalence very close to the end of it. The third phase in turn was closely associated in time with the decline of CD4+
T cells to <200/μl and with the failure of T-cell homeostasis and a loss of phenotypically naive (CD45RA+
T cells (48a). The proposed framework is also generally consistent with previous sporadic reports of constriction of HIV diversity (15
) and similar rates of divergence from the founder strain (39
). Here, we have extended these previous reports by systematically documenting the consistency of these patterns and rates.
Other arguments support the idea that the proposed framework, though derived from a small number of participants, may have broad applicability. (i) Participants were studied in substantial depth from seroconversion to advanced disease in those developing disease progression. (ii) The findings were quite consistent. (iii) Participants were representative of most HIV-1-infected people in terms of the rate of disease progression (median survival time = 9.1 years after seroconversion compared to 10.2 years among the MACS participants as a whole [56
]) and occurrence of T-cell homeostasis failure before AIDS (seen in ~75% of MACS participants who develop AIDS [24
]). (iv) The patterns were seen even in two participants who were initially selected for lack of progression of HIV disease but exhibited evidence of progression as follow-up advanced.
Taken together, a summary sequence of events can be inferred (Fig. ) that describes the virological and T-cell changes that occur in the asymptomatic period of HIV infection. It should be stressed that these phases constitute general trends found in the six individuals we studied who developed AIDS during the period of analysis, although all nine experienced the initial events we described. The sequence begins with the detection of X4 viruses and the attainment of peak viral diversity at a variable time after seroconversion (4 to 9 years in these participants). If the time of X4 virus appearance is taken as time zero, then peak diversity occurs an average of 0.3 years later, followed by X4 peak representation at 1.5 years, divergence stabilization at 2.2 years, T-cell homeostasis failure at 2.5 years, and CD4+
cell transition through 200 cells/μl at 2.9 years. Based on a study of 212 seroconverters and 1,129 seroprevalant infections within the MACS cohort, the occurrence of clinical AIDS-defining conditions follows T-cell homeostasis failure by 1.7 years (24
), or approximately 4.2 years from the emergence of X4 viruses.
FIG. 6 Schematic illustration of proposed consistent patterns in development of HIV disease in moderate progressors. (a) Clinical phases of HIV infection as well as typical patterns of CD4+ and CD3+ T cells and plasma viral RNA loads. The initial (more ...)
Several caveats should be noted. It is not clear whether the patterns and sequence of events observed can be generalized to people with other rates (i.e., very rapid or very slow) of disease progression or over shorter observation periods (23
). Seven of the participants we studied were typical progressors, whereas two (participants 9 and 11), were initially chosen as examples of nonprogressors but subsequently did progress. Second, the length of the intervals between the noted events is variable within the individuals we studied; the durations estimated here are not meant to set limits and are mentioned only as a frame of reference for the evaluation of additional HIV-infected individuals. Third, we drew our inferences about viral evolutionary patterns on only 650 bp of env
; other genes and gene regions evolve more slowly and may not exhibit the same patterns.
Despite these concerns, recognition of the aforementioned patterns of HIV-1 evolution suggests a way to reconcile conflicting reports about the relationship between the rate of HIV-1 disease progression and the degree and rate of increase in viral diversity (15
). Because only extensive sampling can detect the complex pattern of viral evolution we describe, viral diversification could appear spuriously slow if viral populations are assessed infrequently, e.g., only early and late in infection (thereby missing the point of peak diversity) or in people with highly atypical disease pathogenesis (e.g., the slowest and the most rapid progressors). A previous study of env
sequences by Shankarappa et al. (71
) suggested the existence of a diversity peak with continuing divergence from the founder strain, although sampling was less frequent and potentially compromised by resampling (44
In this study, X4 viruses were detected in the blood of all nine participants and grew to predominance in six. This represents a significantly higher frequency of detection of X4 viruses than in approximately 50% of the individuals observed in previous studies of people progressing to AIDS (36
). Previous studies have used phenotypic assays to detect X4 viruses, as opposed to the genotypic analysis done here. Both methodologies can miss detection of X4 viruses on occasion. However, we also noted that the representation of X4 viruses peaked and then diminished over time, which could lead to falsely low estimates of the proportion of people who have circulating X4 viruses if they were sampled subsequent to such a decline. Our higher detection rate by sequence analysis could also reflect the fact that PBMC DNA harbors defective and latent proviruses for extended periods (12
) and thus may provide a record of past transient replication of X4 viruses. Evidence for the transience of X4 viruses has also been seen in previous studies (30
). We have also detected transient representation of X4 viruses as well as a similar pattern of viral evolutionary dynamics in the two other patients we have studied throughout infection: a gay man with rapidly progressing disease (43
) and a perinatally infected child (40a
). Overall, our results support the view that X4 viruses are likely to occur in significantly more than 50% of people who develop AIDS. It should be noted that X4 viruses have often been linked to AIDS in HIV-1 subtype A, B, D, and E infections (18
), have been found with significant frequency in early HIV-1 subtype C infections (79
), but are seen at low frequency in cases of late-stage subtype C infections (59
). Thus, it has not been established that our findings apply to infections with all HIV-1 subtypes.
Interestingly, the timing of evolution and rapidity of outgrowth of X4 viruses relative to the point of peak viral diversity appeared to differ by mutation. X4 viruses with a mutation at position 306 (participants 5 and 8) increased rapidly, reaching a peak level shortly after initial detection. In contrast, viruses with a mutation at position 320 (participants 1, 2, and 3) reached peak levels more gradually, and the appearance of the position 319 mutation was most variable and tended to be found at low levels (participants 3, 6, 7, and 8 but not 9) and in individuals with other X4-specific mutations. Thus, although these data are preliminary, the specific X4 mutation may be important to viral growth properties and in disease progression.
These observations suggest an important role for X4 viruses in T-cell destruction late in the progression of HIV-1 infection in the participants studied. This is plausible in view of the preferential expression of CXCR4 on naive T cells (9
) and on thymocytes (50
), which are needed to replenish memory T cells (6
). Among other recent findings suggesting a differential impact of R5 and X4 viruses on T-cell populations (5
) is the mediation of apoptosis of CD8+
cells by X4 Env (29
), a mechanism that may apply to CXCR4-expressing naive CD4+
cells as well.
Our data also suggest that the degree and rapidity of immune reconstitution (1
) that occurs under HAART may differ according to the phase at which HAART is initiated, with greater reconstitution possible prior to some of the milestones we described. For example, it may be that people treated with HAART before X4 viruses are detected will be able to restore lost immunological functioning more effectively than those whose treatment is begun later. In addition, given the serious long-term side effects of HAART (10
), the ability to predict AIDS onset ~4 years in advance based on detection of X4 viruses (57
) may help refine therapeutic strategies.