The first phase could be measured in three patients (C, U, and V). In these patients, viral loads increased rapidly (mean rate of increase r1 = 0.74 ± 0.04/day) until virus peaked at a mean of 7 days after onset of symptoms at an average of 2 × 106 copies/ml plasma. The second phase, which could be measured in all of the non-treated patients, was characterized by rapid clearance (r2 = 0.42 ± 0.20/day), resulting in an average viral density of 0.125 × 106 copies/ml at a median of 21 days after onset of symptoms. During the third phase (quantified for patients B, C, E, F, and G), HIV-1 RNA levels still declined but at a much slower rate (r3 =0.030 ± 0.025/day). The transition between the third phase of slowly declining virus and steady state occurred at a median of 41 days after the onset of symptoms with steady-state viral load approximately 25 000 copies/ml plasma.
Comparison between HIV-RNA decline and viral variability
The samples examined for variations in virus population by direct sequencing were collected at a frequency enabling comparison with HIV-1 RNA levels during PHI in eight patients not receiving treatment . A change of the major sequence was detected during the first month in patients D and F, and during the second month in patient C. In patients B, E, G, and H, no changes of the major sequence were detected during the observation period. The timepoint for transition between phases of rapid and slow decline of RNA levels (t2) was determined in six of these patients (Fig. 3). The transition occurred at a mean of 14 days (range 10–19) in patients C, D, and F, in whom changes of the major sequence were detected during the first 2 months. By contrast, the transition occurred at a mean of 33 days (range 21–54) in patients B, E, and G, in whom similar changes were not recorded.
In patient D, these results were confirmed by cloning of the V3 region derived from viral RNA obtained at day 5 (20 clones), day 8 (20 clones), and day 12 (18 clones). The major sequence detected by direct sequencing at day 28 was not the same as the major sequence detected at days 5, 8, or 12. This sequence, however, was present in 5% of clones from day 5, in 0% of clones from day 8, and in 28% of clones from day 12. As mentioned above, we noted that viral load first increased and then levelled off beginning around day 14 in patient D. The viral load perturbations seen in patient D, therefore, seemed to be related in time to the appearance of a new major variant.
Correlation between recorded peak HIV-RNA level and subsequent HIV levels and T cell subset counts
Peak values of HIV-1 RNA recorded during PHI from each patient were compared with subsequent values at intervals of 50 days, from day 50 to day 600. The correlation coefficients (r) ranged between 0.73 and 1.0 (P < 0.05 for all correlations), thus showing a significant correlation between early viral load, as determined by peak level measured during PHI, and steady-state levels. Peak HIV-1 RNA levels were not correlated, however, with subsequent CD4 and CD8 cell counts (data not shown).
Comparison between treated and non-treated patients
The early peak HIV-1 RNA levels in patients receiving antiretroviral treatment did not differ from those recorded in untreated patients. At 4 weeks after the onset of primary HIV infection, however, treated patients displayed a significantly lower mean HIV-1 RNA level than untreated patients: 2500 copies/ml [95% confidence interval (CI) 450–13 800] compared with 103 000 copies/ml (95% CI 64 500–162 000;P < 0.01).
Antiretroviral treatment was started within 10 days following onset of symptoms (days 0, 3, 4, 6, 7 and 10, respectively) in six patients (Fig. 4) and HIV-1 RNA declined rapidly to < 50 copies/ml; all samples drawn day 69 or later from these patients tested negative. Three patients started treatment later (days 13, 19 and 21, respectively). Because of non-compliance and concomitant intravenous drug abuse, treatment in one patient was discontinued after 6 months, when HIV-1 RNA had declined to undetectable levels in the other two patients.
Patients receiving treatment developed milder CD8 lymphocytosis during the acute phase than patients without treatment (Fig. 5). These differences persisted throughout the study period from week 4 through 12 months of follow-up (P < 0.05). The CD4 cell counts did not differ significantly between the two groups of patients.
This study offers new insights into the time course and kinetics of viremia following onset of symptoms in PHI. The pre-illness pattern of viral dissemination has been clearly established in animal models [23–25] : genitally inoculated virus infects T cells, macrophages, and dendritic cell, followed by a stepwise progression of the infection from regional lymph nodes to distant lymphoid tissues, presumably by migration of infected cells, followed by subsequent spread to the systemic circulation about 1 week after inoculation. It seems reasonable to expect a similar pattern of HIV infection in humans, considering that our results indicate that viremia appears during the week preceding onset of symptoms and that the incubation period is about 2 weeks in most cases.
The viral dynamics during ‘typical’ PHI is characterized by the following sequence of events. (i) In the previremic phase, HIV disseminates and replicates within the lymphoid system. (ii) The first viremic phase of increasing viral density starts about 1 week after infection when the HIV virus appears in the blood at rapidly increasing levels, initially without clinical symptoms, which usually appear 2 weeks after infection. (iii) The second viremic phase of rapid decay begins at about 3 weeks after infection; virus declines from the peak viremia to about 5–10% of the peak level over about 2 weeks. Clinically this phase is characterized by lymph node enlargement, the appearance of IgG-antibodies, and CD8 lymphocytosis. (iv) The third viremic phase of slow decay occurs from about 5 weeks after infection; during this stage clearance continues but at only 5–10% of the rate seen during the second viremic stage of rapid decline. (v) The fourth viremic phase begins approximately 2 months after infection; during this period, HIV-1 RNA levels approach an approximate steady state between production and clearance but with great individual variation, both in time until, and in level of, the steady state.
Two patients that did not show this typical pattern in our study, patients C and D, both showed evidence of viral sequence change. In patient D, a brief upspike in viral load between days 10 and 20 was temporally associated with a genetic change in env that became evident by direct sequencing on day 12. In patient C, however, the change in env sequence (on day 56) occurred well before the viral load rebound between days 108 and 236, indicating that these events were not temporally associated. It is conceivable, however, that genetic changes in other loci in the virus in patient C were responsible for this rebound.
The pattern of rapid increase and decrease in HIV-1 RNA during the first 5 weeks of PHI is similar to that which is found in most acute infections. In the ‘typical’ HIV infection, however, virus starts to decline more slowly, beginning around week five, eventually hitting an approximate steady state that persists for months or years. This pattern could be quantified in our study as a result of the large number of frequently collected samples. The biphasic decay we observed in untreated patients is remarkably similar to that which occurs during initiation of potent anti-HIV treatment, though it is not clear that it results from the same mechanism. During treatment, the second phase of slower decay has been interpreted to be the decay of long-lived infected cells , which are now thought to consist mainly of resting T cells . However, if most of the virus was produced by long-lived cells during the phase of slow decay in PHI, then initiation of antiretroviral treatment would not be expected to extend the rapid decay further to approximately a 2.0 log10 copies/ml reduction in HIV-1 RNA, which was observed in our patients who received early treatment.
The lower rate of viral clearance that occurs around week 5 could indicate a rebound in target cell populations, reductions in the intensity immunological responses against HIV as antigen levels decrease, or the outgrowth of escape mutants or minor transmitted variants. The last possibility is supported by our finding, although examined in a limited number of patients, of lower clearance rates in patients in which there is an early appearance of a new major HIV sequence [see also 21,22], and also by reports demonstrating rapid selection of cytotoxic T lymphocyte escape virus during PHI [27,28]. Despite the appearance of these escape mutants, HIV-1 RNA continues to decline in these patients, although at a much slower rate than earlier, suggesting that the immune system continues to respond to the new HIV variants generated over the course of the infection. In most patients, a steady state develops between replication and clearance of virus, though the level varies considerably from patient to patient. Interestingly, we found that steady-state viral loads were related to the peak levels measured during primary infection (r = 0.73–1.00;P < 0.05), thus corroborating what has already been demonstrated in a monkey model . It should be emphasized that the peak levels measured in our patients may not be the true peak values, as these are only possible to determine if samples are drawn daily from each patient.
The natural history of PHI has become more difficult to study in recent years since it is now generally recommended that patients with PHI should be given HAART . The rationale for early treatment is that viral load reduction during the earliest phases may prevent the seeding of a latently infected cell compartment, though it is now evident that treatment must be initiated very early during infection to prevent this . Early treatment may also reduce damage to CD4 T cell lymphoproliferative responses [32,33]. Indeed, very early treatment has been shown to lead to a reduction in viral loads in HIV-2-infected macaques that persists even after therapy is withdrawn . We have found, however, that the development of HIV-specific antibodies is at least partly inhibited by early treatment (data not shown). Therefore, the possible influence of the timepoint of initiation of treatment during PHI on both the specific immune response and the reduction of HIV load needs to be further elucidated.
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Keywords:© 2000 Lippincott Williams & Wilkins, Inc.
primary HIV-1 infection; HIV-RNA; treatment