The association between the strength of the virus-specific CT8-TL response and the resolution of acute-stage viremia has been illustrated for both natural HIV-1 and experimental SIV infection (10
). CT8-TL-mediated immune pressure also results in the emergence of viral escape mutants, and viral escape is considered a major obstacle to the development of T-cell-based prophylactic vaccines (27
The current study accurately monitored viral sequence variations within three well-defined CD8-TL epitopes by monitoring the experimental infection of cynomolgus and rhesus macaques with SIVmac239. We employed ultradeep pyrosequencing to quantify viral escapes in these CD8-TL epitopes in the SIV-infected macaques. Unlike the single-genome amplification technique, which provides sequence information from only a limited number of viral clones (typically less than 50 clones per single sample), ultradeep pyrosequencing readily allows for 1,000 or more sequence reads of an epitope of interest. This in-depth coverage of the three epitopes Nef103-111RM9, Tat28-35SL8, and Gag181-189CM9 permitted us to carefully dissect sequential CT8-TL-driven viral strain changes restricted by Mafa-A1*063 (RM9) and Mamu-A1*001 (SL8 and CM9).
We systematically computed the rates of escape, revealing substantial differences among escape-prone and escape-resistant epitopes by employing ultradeep pyrosequencing data, which measured the proportion of each wild and mutant clone more precisely than any conventional Sanger sequencing. Mathematical models have framed the escape phenomenon as the interplay between the susceptibility to CD8-TL killing and the viral fitness cost for the wild type versus escape variants; the rate of escape is given by the difference between the rate of CT8-TL killing and the fitness cost of the variant. The relative contribution of CT8-TL killing and viral fitness cost to the escape kinetics differs from epitope to epitope. This is exemplified by the rapid escape of the SL8 epitope but the much slower escape of the CM9 epitope in the Indian rhesus macaques. Despite the presence of strong and early CD8-TL responses to CM9, the high fitness cost of CM9 epitope mutants and the requirement of compensatory substitutions within extraepitopic regions to restore viral fitness result in the delayed emergence of CD8-TL escape mutations within this epitope (22
Another element that may contribute to slower escape is the diminished quantity and quality of CD8+
T-cell responses at the chronic stage. The frequency of CD8+
T cells specific for a single epitope has been shown to peak at around 2 weeks postinfection and to decrease around 20-fold as the set point is being approached (36
). The quality and effectiveness of the CD8+
T lymphocyte response also may diminish over time due to chronic antigenic stimulation, the impairment of CD4+
T cells, and damage to lymphoid tissue (56
Our estimates for the rate of the first viral CD8-TL escape within the escape-prone epitopes RM9 and SL8 were greater than some previous estimates (7
) but comparable with others (16
). Our estimates also are consistent with those based on quantitative real-time PCR (qRT-PCR) assays (36
). The basis of qRT-PCR is different from that of cloning and sequencing; it tracks viral loads of both wild-type and predefined escape mutants. The limitation of this approach is that it permits the measurement of only those mutant viruses for which the assay was designed. Table summarizes these previous estimates along with our estimates. The high-resolution ultradeep pyrosequencing data in the current study provided more accurate estimates for the rate of SIV escape than these earlier studies, which generated escape rates ranging from 0.19 to 1.07 day−1
Estimates of timing of escape and rate of escapea
Our finding using the SIV/macaque model closely parallels the recent findings from the analysis of viral sequences obtained from three subjects with primary HIV-1 infection (24
). Specifically, our data show that more diverse sequence variants, reflected by higher Shannon entropy scores, appear in the viral decline phase after the peak of viremia. Following this period of diversification, viral sequences became homogenized with the positive selection of a single dominant epitope variant at the viral set point. This feature was observed in various HIV-1 epitopes, including Rev9-26
, and Env822-839
Our estimate of the rate of viral escape during the acute phase of SIVmac239 infection of macaques was found to be comparable to the rate for viral escape during the acute HIV-1 infection of humans. From a recent study of HIV-1 escape (24
), when we limited the analysis to the cases where the timing of escape was within 20 days of documented HIV-1 infection after the first screening, the average rate of CD8-TL escape was 0.33 day−1
. We note that all three acute subjects analyzed in reference 24
were in Fiebig stage II at the first screening according to reference 50
. Here, the Fiebig staging system classifies the status of HIV-1-infected subjects based on an orderly appearance of viral RNA, antigen, and antibodies in plasma during early infection (18
). On average, Fiebig stage II (viral RNA+
, before seroconversion) corresponds to around 22 days postinfection (29
). Hence, 20 days of documented HIV-1 infection after the first screening corresponds to around 42 days postinfection, which is very similar to the timeframe analyzed in our macaque study. While the average timing of HIV-1 escape is greater than that of SIV escape, 34.8 ± 5.2 days (HIV-1) versus 22.1 ± 3.78 days (SIV), the rates of escape were comparable, 0.33 ± 0.09 day−1
(HIV-1) versus 0.36 ± 0.36 day−1
(SIV). We compared the rates of HIV-1 and SIV CD8-TL escape in Table .
Our report of comparable rates of very early escapes within humans and macaques is contradictory to a previous meta-analysis study of SIV and HIV-1 CTL escape events (7
). Either higher levels of CD8+
T cells targeting each peptide or a more efficient killing mechanism by CTL in macaques was suggested to explain the difference (7
). Our calculation quantitatively indicates that HIV-1 and SIV escape with comparable rates if we focus on very early events within around 1 or 2 months postinfection. Our quantification is consistent with reference 36
, which reported the association of the rate of escape with the timing of escape during primary SIV infections.
We observed an association between the timing of the first escape and the peak timing of the peak CD8-TL response to the escape-prone epitopes. The correlation coefficient was 0.76, and the correlation was statistically significant (P
= 0.027). Because viral CD8-TL escape is a surrogate indicator for functional CD8-TL activity, these findings suggest that an early peak in the acute CD8-TL response is more important for the effective CD8-TL-mediated containment of acute virus replication than the peak magnitude of the acute CD8-TL response. However, note that none of the animals in our study were able to control SIV replication during the chronic phase, including the three vaccinated animals that exhibited a very robust anamnestic CD8-TL response. Thus, it remains unclear what characteristic(s) of the early CD8-TL response best predict the sustained immune-mediated suppression of virus replication at the steady state. Numerous studies have addressed how different facets of CD8-TL responses, the magnitude, the breadth, and the functional capacity, are related to the level of the containment of HIV-1 and SIV (2
Viral CTL escape kinetics are characterized by the rapid loss of the transmitted epitope concurrently with a decline from the acute-phase peak of viremia. In the phase of falling viral load, the level of transmitted epitopes decays exponentially (Fig. ), which is consistent with the trend predicted by the acute-sequence evolution model (9
). However, the rate of loss during this period is 438 times faster than that predicted by the acute-sequence evolution model, which is premised on the progressive but nonselective replacement of the transmitted virus population through random base substitutions that result from error-prone reverse transcription (9
). The rapid loss of the transmitted epitope strongly supports the existence of a strong selective pressure mediated by CT8-TL responses.
Despite the robust selective pressure, the transmitted (wild-type) RM9 and SL8 epitopes were found to persist as a minor population in the host, even at the viral set point, in all animals (8
). The percentage of the transmitted (wild-type) epitope remaining at day 140 ranged from 0.04 to 12.2% (8
). Thus, despite strong CT8-TL responses against the transmitted virus and a low fitness cost associated with mutations in the SL8 and RM9 epitopes, the transmitted virus population could not be eradicated in most of the animals. Conversely, more than half of CD8-TL escape mutant epitopes were utterly cleared within the same time frame. Some of these mutant epitopes existed in a greater proportion than founder epitopes during the viral decline stage. Our observation may imply that the initial formation of a reservoir of SIV infection is preferentially driven by transmitted, founder epitopes rather than transient acute escape epitopes despite the fact that the population size of some of the acute escape epitope clones was larger than that of the transmitted clone during the phase of acute viremia decline.
Previous results obtained from human subjects with primary HIV-1 infection have shown the complete loss of the transmitted (wild-type) epitope obtained within a similar time period, 159 days after the first preseroconversion screening (24
). We believe that this discrepancy can be explained most plausibly by the difference in methodology used in our study versus that of Goonetilleke et al., who performed single-genome amplification (SGA) as a prelude to sequence analysis (24
). In this earlier study, only a limited number of clones were analyzed, around 50- to 100-fold fewer than that in the present study. It is likely that the limited number of clones analyzed accounts for the failure to detect the transmitted epitope at late time points postinfection. According to our previous power analysis (29
), if we sample 50 clones, we can be 95% confident that the undetected transmitted epitope comprises less than 5.8% of the total virus population. However, if we increase the sample size to 1,414 (as in the present study), we can be 95% confident that the undetected transmitted (wild-type) epitope comprises less than 0.21% of the total virus population. This calculation suggests that the greater depth of coverage possible using ultradeep pyrosequencing compared to that of single-genome amplification permits a much greater sensitivity for the detection of rare virus sequence variants, including residual virus that contains wild-type epitopes. Quantitative real-time PCR (qRT-PCR) assays observed the persistence of the wild-type epitope in SIV infections (36
). We do not know the source of the persistence of the transmitted epitope. The transmitted epitope at the viral set point may be derived from productively infected or latently infected CD4+
T cells, cellular sources other than CD4+
T cells, and/or follicular dendritic cell-associated virions (11
In conclusion, our study first estimated the rate of CD8-TL-mediated viral escape and the timing of escape by employing high-throughput pyrosequencing data in experimental SIV infections. The rates of escape during very early periods of HIV-1 and SIV infections were unexpectedly comparable, implying the prominent role of CD8-TL in shaping viral evolution during early SIV and HIV-1 infections.