Abstract
Despite antiretroviral therapy (ART), a latent reservoir of replication-competent HIV-1 persists in resting memory CD4+ T-cells and precludes cure1-6. Lorenzo-Redondo et al.7 analyzed HIV-1 sequences collected from three individuals during the first six months of ART, discovered specific patterns of sequence evolution, and concluded that viral replication persists during therapy. We believe these evolutionary patterns are artifacts of rapidly decaying viral subpopulations present during the first months of therapy and are not characteristic of the long-lived reservoir. The study therefore provides no evidence that ongoing replication is an additional barrier to cure for treated individuals who consistently maintain low viral loads.
Lorenzo-Redondo et al. collected samples before and three and six months after treatment initiation, when labile viral populations dominate and change rapidly. Prior to treatment, most HIV-1 DNA in resting CD4+ T-cells exists in an unintegrated state decaying with a half-life of days8,9. Another major population of infected resting cells decays with a half-life of weeks10. The latent reservoir of integrated proviruses, observed in blood and lymphoid tissue1, is smaller and decays with half-life of ~4 years5,6. Lifelong persistence of this reservoir is determined by longevity and proliferation of the infected cells11. Initiation of ART blocks new infection from replenishing these populations, revealing their different lifespans. Differential decay causes dramatic shifts in infected cell populations in the first six months of ART, making suspect any conclusions about viral evolution gleaned from this period. Below, we support this claim by simulating differential decay and replicating the analysis of Lorenzo-Redondo et al. on the simulated data. We find that false signals of viral evolution – and ongoing viral replication – often appear.
To estimate size and decay of labile compartments, we examined a cohort of seven early-treated individuals, which we consider comparable to the two early-treated participants in the Lorenzo-Redondo et al. study. Blankson et al.10 used the quantitative viral outgrowth assay (qVOA) on blood samples to detect resting CD4+ T-cells harboring replication-competent HIV-1. At ART initiation, infected cell frequencies greatly exceeded those of individuals on long-term ART. A multi-log, multi-phasic decay over the first year of therapy reduced frequencies to levels typically observed during long-term ART. Fitting to the most extensively sampled individual, we inferred a large, fast-decaying population, a smaller population with slower decay, and a very small persistent reservoir, approximated as constant (Fig. 1). At zero, three, and six months following ART initiation, labile populations comprise 99.99%, 96.2%, and 76% of infected resting cells, respectively, masking the persistent reservoir. The RNA-based assays performed by Lorenzo-Redondo et al. on lymphoid tissue paint a similar picture: the infection decays rapidly over the first three to six months, eventually dwindling to a more stable state >3 orders of magnitude smaller than the pretreatment population (Lorenzo-Redondo et al., Extended Data Figure 1). Regardless of sequencing depth, the limited number of infected cells in a blood draw or tissue biopsy likely prevents the persistent reservoir from being sequenced at early timepoints. The genetic diversity of this reservoir only emerges later. Latency studies are therefore generally restricted to participants who have received suppressive ART for >6 months, a precaution not taken by Lorenzo-Redondo et al.
Brodin et al.12 suggested that decay of labile populations may produce false signals of evolution during treatment, even in the absence of viral replication. We used computer simulations of viral populations during acute infection and treatment to confirm this hypothesis. Simulated virus replicated and seeded subpopulations for four months, and treatment then blocked replication for six months. During treatment, labile subpopulations decayed, while a stable reservoir persisted, as in Fig. 1. Nearly 12,000 simulations were subjected to the tests performed by Lorenzo-Redondo et al: genetic divergence from start to end of therapy, evolutionary rate calculations, and measurement of clock-like signal in maximum-likelihood trees (Supplementary Tables 1 and 2).
We tested a range of parameters defining growth and competition in the pre-treatment viral population and selected 8,000 simulations with realistic viral diversity. Depending on parameter values, up to 57% of simulations produced a false impression of clock-like evolution according to all three tests used by Lorenzo-Redondo et al. (Supplementary Methods). For comparison, of the 17 gene/tissue combinations studied by the authors, 11 (65%) produced a signature of evolution according to the two tests for which statistics were explicitly presented (Lorenzo-Redondo et al., Extended Data Tables 1 & 2). Decay of labile compartments can remove positively selected variants that occurred before treatment, revealing ancestral genotypes – which masquerade as the product of new mutation during treatment. Strong positive selection (anywhere in the genome, not only in the sequenced region) is required to generate a false impression of evolution. We believe that this mechanism is realistic, as rapid selective sweeps, caused by CTL escape mutations with selective coefficients of 20% or more, typify acute infection13.
To support our argument with actual sequence data and without assuming a selection coefficient, we simulated reservoir seeding and post-treatment decay using HIV-1 gag sequences obtained from three individuals during untreated acute infection14. Appearance of post-treatment “evolution” again depended on pre-treatment dynamics. The individual with most extensive sequence changes, suggesting strong pre-treatment selection, passed a phylogenetic test of forward evolution in 92 of 100 replicate simulations (Supplementary Methods, Supplementary Table 3, Extended Data Table 1).
Fig. 2 illustrates how decay of labile compartments produces the appearance of evolution in an example simulation (Replicate 91 of Parameter Set 48, Supplementary Table 2). Before treatment, the population diverges through mutation and selection. When treatment starts, the most diverged sequences die out, resulting in a decline in divergence. If divergence is measured not from the origin of infection, but rather (as by Lorenzo-Redondo et al.) from the most common genotype at treatment initiation, this retreat towards the origin is misinterpreted as increasing divergence (Fig. 2A). After ~2 months of treatment, this trend slows, as labile compartments (the source of more recently produced virus) decay. If a time-structured tree is constructed only from sequences collected during treatment (Fig. 2B), then a pattern of forward evolution appears. Maximum-likelihood phylogenetic analysis suggests the same pattern, and there are even three internal branches (three separate “novel mutations”) leading exclusively to sequences sampled at the later two timepoints (Fig. 2C). Including sequences sampled before the start of treatment and rooting at the actual infection origin reveals the truth (Fig. 2D): sequences diverge for four months and then the pattern of divergence reverses (i.e., sequences sampled at treatment initiation tend towards rightmost leaves of the tree).
We have not aimed to show that 100% of viral replication ceases during suppressive ART; this “absolute negative claim” is neither believable nor strictly necessary. The question relevant to HIV cure research is not whether any replication occurs, but whether sufficient replication occurs to fuel viral persistence during ART. Insufficient, or “subcritical” replication may contribute to residual viremia, but does not cause long-term persistence and re-seeding of the latent reservoir. Most importantly, subcritical replication is not a barrier to HIV cure15.
What we do claim is that, even in the complete absence of viral replication, misleading evolutionary signatures of high-level replication are expected to appear in the first six months of ART. Adding multiple anatomical compartments or other elaborations to our model could increase realism but would not disturb this basic conclusion. Decay of labile populations confounds evolutionary analysis, and so the observations of Lorenzo-Redondo et al. are insufficient evidence for ongoing replication. We thus remain unconvinced that ongoing replication contributes to reservoir stability during ART, and we encourage repetition of these studies using replication-competent viruses from HIV-1-infected individuals after >1 year of suppressive ART.
Competing Financial Interests
The authors declare no competing financial interests
Author Contributions
Wrote the manuscript: DISR, ALH, SBL, RFS. Simulations and phylogenetic analyses: DISR, ALH, RFS. Estimation of labile compartments: SBL, RFS.
Methods
Simulations used a stochastic model of birth, death, and mutation of infected cells. Upon birth, a productively infected cell gives rise to another infected cell, which may experience mutation. A productively infected cell may transition to any state identified in Fig. 1. Maximum-likelihood trees were constructed using PhyML (HKY85 model; single rate category; estimation of base frequencies, transition/transversion ratio, and proportion invariant sites; best of NNI/SPR). The inferred root was chosen from the earliest timepoint to maximize R2 of the root-to-tip regression. The time-structured tree was constructed using BEAST v2.4.4. Details are provided in Supplementary Methods.
Acknowledgements
DISR acknowledges support from amfAR Fellowship # 109511-61-RKRL and National Institutes of Health grant R01GM117591. ALH acknowledges support from National Institutes of Health grant DP50D019851 and Bill & Melinda Gates Foundation award OPP1148627.