Transient antiretroviral therapy selecting for common HIV-1 mutations substantially accelerates the appearance of rare mutations
© Shiri and Welte; licensee BioMed Central Ltd. 2008
Received: 25 July 2008
Accepted: 14 November 2008
Published: 14 November 2008
Highly selective antiretroviral (ARV) regimens such as single dose nevirapine (NVP) used for prevention of mother to child transmission (PMTCT) in resource-limited settings produce transient increases in otherwise marginal subpopulations of cells infected by mutant genomes. The longer term implications for accumulation of further resistance mutations are not fully understood.
We develop a new strain-differentiated hybrid deterministic-stochastic population dynamic type model of healthy and infected cells. We explore how the transient increase in a population of cells transcribed with a common mutation (modelled deterministically), which occurs in response to a short course of monotherapy, has an impact on the risk of appearance of rarer, higher-order, therapy-defeating mutations (modelled stochastically).
Scenarios with a transient of a magnitude and duration such as is known to occur under NVP monotherapy exhibit significantly accelerated viral evolution compared to no-treatment scenarios. We identify a possibly important new biological timescale; namely, the duration of persistence, after a seminal mutation, of a sub-population of cells bearing the new mutant gene, and we show how increased persistence leads to an increased probability that a rare mutant will be present at the moment at which a new treatment regimen is initiated.
Even transient increases in subpopulations of common mutants are associated with accelerated appearance of further rarer mutations. Experimental data on the persistence of small subpopulations of rare mutants, in unfavourable environments, should be sought, as this affects the risk of subverting later regimens.
The rapidity of human immunodeficiency virus (HIV) replication, combined with its high reverse transcriptase error rate , leads to rapid viral evolution, in particular the emergence of drug resistance. Treatment that is unable to sufficiently inhibit viral replication allows the appearance and/or selection of drug-resistant strains. Further accumulation of resistant variants may limit therapeutic efficacy and jeorpadize subsequent treatment options.
A single dose nevirapine (NVP) regimen for prevention of mother to child transmission (PMTCT) is a well known example of a suboptimal regimen that inevitably, if temporarily, exerts selective pressure in favour of resistant strains. This is still a major concern in developing countries where a prophylactic regimen of single dose NVP is widely used for PMTCT . Given the high frequency of mutation, some minority resistant mutants are always preexisting, albeit in trace quantities, at the moment therapy is initiated. Because of the long half-life of single dose NVP, with blood levels detectable up to 2–3 weeks after exposure [3, 4], the duration of sub-therapeutic NVP concentrations may present a significant hazard of developing resistance for the mother. There is a risk of treatment failure after single dose NVP exposure, if the treatment includes a NNRTI . The question arises whether, and to what extent, a transient treatment-induced boost to an otherwise marginal subpopulation results in increased risk of accumulation of further resistance mutations that could potentially increase the risk of subsequent NNRTI-based treatment failure.
In the search for better PMTCT regimens, improved efficacy has been demonstrated for a number of short course regimens for PMTCT in resource-limited settings. For example, 1) use of single dose NVP with additional short course of zidovudine/lamivudine during 3–7 days postpartum , 2) addition of single dose NVP to zidovudine short course during the antenatal period  and, recently, 3) use of intrapartum single dose of combined tenofovir/emtricitabine taken after antenatal short course of zidovudine plus intrapartum single dose NVP . These regimens improve on single dose NVP either in efficacy for PMTCT or reduction of NVP resistance in the mother, or both. However they appear suboptimal in that they select for NNRTI-resistant strains and therefore increase the mothers' risk of virologic failure for subsequent NNRTI-based therapy. For example, in the MASHI study  a total of 218 women started post partum NVP-based therapy after they had received zidovudine from 34 weeks of gestation through delivery. Of these, 112 had received single dose NVP, whilst the rest had received a placebo during labour. After 6 months of post partum treatment with a NVP-based regimen, women without prior NVP exposure were less likely to have virologic failure compared to women who had received intrapartum NVP. Strikingly, of women who started NVP-based therapy within 6 months, 41.7% from the single dose NVP group, but none from the control group, had virologic failure.
In-vivo mathematical models have been useful in exploring the evolution of drug resistance, suggesting that significant evolution can occur during treatment or before initiation of treatment [9–15]. Based on the models, the authors argued that chances of resistance evolving during treatment are small compared to chances of resistance evolving before suppressive therapy. However these studies did not explore, in any dynamically consistent framework, the emergence/accumulation of multiple mutations in a possibly non constant environment. In this study, we extend these standard models to explicitly investigate the consequences of population dynamical effects amongst common resistant mutants. We show how the deterministic dynamics of the common mutants affects the time taken to produce the rarer mutants.
We start from an ordinary differential equation (ODE)-type model of in-vivo viral replication in the deterministic regime, applicable to cell populations that are large enough for statistical fluctuations to be relatively small (wild-type and common mutant strains). We explicitly add expressions for Poisson rates for the occurrence of rare mutations. Using standard survival analysis, we compute, as a function of time, the probability of avoiding a mutation event. Furthermore, we introduce an additional timescale to the 'survival function' to capture the time over which cells infected by an unfit genome persist before being ecologically overwhelmed. This 'survival function' is a continuous state variable that is incorporated into the system of ODEs without much complexity.
We apply our modelling framework to clinically inspired scenarios. Firstly, we explore the quasi steady state that corresponds to chronic treatment in the presence of two viral populations. We characterize treatment regimes in which rates of appearance of rare mutants are either increased or decreased. Secondly, inspired by regimens used for PMTCT in resource limited countries, we investigate the transient behaviour of the model under a short perturbation of the fitness parameters, such as occurs during a short course of suboptimal therapy. Transient therapy significantly increase the hazards of rare mutations. Thirdly, we explore the interaction between a monotherapy short course and subsequent ongoing antiretroviral therapy (ART). The appendix deals with details of mutation combinatorics.
We develop a hybrid deterministic-stochastic model of healthy and infected T cell populations. Our analysis starts with a standard multi-strain model of in-vivo viral replication that distinguishes cells infected with one of Ns viral strains. These kinds of models have been used to try to understand viral evolution in the context of immune response and antiretroviral therapy [9, 10, 12, 16, 17]. For our purposes, we add a new self-consistent stochastic element to the standard deterministic model of viral evolution.
where Nd is the number of strains which are modelled by a deterministic process, i.e. those strains which are assumed to be present with sufficiently large populations for deterministic models to be sensible. We address the incorporation of rare strains shortly.
Physiologically, HIV transmission occurs either by cell-free viral particles released by infected cells, or by direct cell-to-cell contact. It has been demonstrated that cellular contacts drastically enhance productive viral transfer compared to what is observed with free virus infection . Our model, like previously published models of in vivo HIV dynamics, does not have free virions. Even if free virions are physiologically important, including them for the present purposes would not change any of our conclusions as the dynamical effects appear only at very short time scales. In our basic model, k i is a composite fitness parameter that captures the effective cell-to-cell transmission efficiency via all mechanisms. Antiretroviral therapy with currently known drugs does not affect virion or infected-cell survival, but interferes with some stage of the viral replication cycle, i.e. reduces the values of the fitness parameter k i . This basic model also does not explicitly incorporate the dynamics of immune system response such as clonal expansion of effector cells or feedback linking viral and infected-cell clearance rates to the healthy cell population.
natural healthy cell death rate
[40–50 days] 
productively infected cell death rate
[1–2 days] 
supply rate of target cells
2 × 108 cells day-1
viral strain infectivity
single-point mutation rate
2.5 × 10-5
probability of error free transcription
Implied by ε21
A fraction F of infected cells become virus producing. The others become latent and, on average, take time 1/a to reactivate to become virus-producing cells. For simplicity, we assume that latently infected cells have a much longer lifetime than their activation time. Note that for F = 1, we obtain system (1). By adjusting F (it cannot be very realistically estimated directly from data) we can vary the importance assigned to the presence of latently infected cells, without changing the equilibrium values of healthy and virus-producing infected cells. We do not attempt to capture fine physiological details of latently infected cell dynamics, but rather the concept that these cells can be the source of new productively infected cells and hence give rise slower dynamics than a model with just virus producing infected cells.
The phylogenetic relationships amongst all strains, and the initial conditions, determine the number of continuously and stochastically modelled strains. We adopt the following computational procedure:
1. Initially run the deterministic model with populations for Nd strains, and survival functions for avoiding the Ns - Nd rare mutants.
2. Draw a uniformly distributed random variable R i ∈ [0, 1] for each possible rare mutation event.
3. When Λ i reaches R i the appearance of mutant i occurs.
4. If the mutant appears into an environment in which it is fit enough to thrive, pause the simulation.
5. Add one cell of the new rare mutant.
6. Resume running the new deterministic model with = Nd + 1 strains.
This piecewise deterministic model is hardly more complex than a purely deterministic model. The abrupt changes to the evolving process transforms the ODE system into differential equations involving impulse effects (impulsive differential equations) . The difference between our computational approach and previously considered schemes of which we are aware, is that in our scheme, mutation hazards are derived from explicit deterministic model state variables, and also, they depend on mutational pathways, whereas, for example, in Nowak et al , the probability of generating a new mutant is proportional to the total virus population.
This new state variable is just the probability that the relevant mutant genome is absent at time t. The smallest physically feasible value of Δ is the lifetime of an infected cell (as noted above for the case where the rare mutant produces no daughter cells from the seminal mutation) and the largest feasible value is greater than the expected survival time of the infected individual (if the genome is significantly banked into a latently infected cell population) i.e. essentially infinite for practical purposes. It is particularly relevant when we model environmental shifts, such as the start or end of an antiretroviral (ARV) regimen. We will demonstrate scenarios in which the presence or absence of a 'currently unfit' genome, at the moment of initiation of therapy, can have an impact on rates of treatment failure.
Results and discussion
We now apply, to clinically inspired scenarios, the survival analysis of the model presented in the previous section. The particular model implementations are in certain respects simplistic preliminary work, but they demonstrate the kinds of questions that can be seriously investigated within this framework. We use a model with two continuously variable strains (wild-type and common mutant) and a waiting time for the appearance of the third strain (rare mutant). In the absence of treatment, the wild-type strain is dominant and the mutant subpopulation is present in trace quantities, of the order of the mutation rate.
which shows how a rare mutant variant can arise through a number of pathways, such as sequential single-point mutations or simultaneous higher-order mutations.
First, we consider a quasi steady state scenario corresponding to chronic treatment, then we model a short course of monotherapy, followed, after some delay, by initiation of chronic therapy. Important interactions between the two regimens are captured by the newly introduced state variable ΛΔ.
Chronic treatment/Steady state
The less-fit strain is present in trace quantities that will be very difficult to detect by standard clinical assays, even if the fitness difference is marginal. This is a modified version of the usual ecological phenomenon that two species in a single niche do not coexist even with very similar fitness; one dominates and drives the other to extinction. The non-extinction of the less-fit quasispecies observed in this case results from the high mutation rate, which leads to waiting times between mutation events that are very small compared to the lifetimes of productively infected cells, so that the subdominant species persists in significant quantities. Given realistic orders of magnitude for infected cell populations (109) and lifetimes (a day), and the mutation rates between strains that differ by a single base mutation (10-5), certain minority populations (single-point and double-point mutations relative to a dominant wild type) are large enough to be modelled deterministically.
For the chosen parameter values (Table 1), the time required to attain one cell infected by the new strain is of the order of weeks (see figure 1). On the other hand, the explicitly modelled mean waiting time (⟨τ w ⟩) to the occurrence of the new mutation according to the constant Poisson rate is of the order of years. It seems to us that the latter is a reasonable model of rare events and the former is fundamentally flawed.
We now return to the stochastic waiting time model, which at the pre-treatment equilibrium state, has a waiting time to the occurrence of a rare mutant of the order of years for the chosen parameter values given in Table 1. We are interested in the impact of long term treatment on these waiting times. Let treatment efficacy on strain i be denoted by ξ i ∈ [0, 1], so that the infectivity parameter during treatment is = ξ i k i .
respectively. Recalling that in the untreated state, the viral load is strongly dominated by the wild type ((k1, k2)), this notation facilitates comparisons between the treated and untreated states, both in terms of overall viral suppression, and selection between strains.
Disruption of the pre-treatment equilibrium state (F w = 1 and F m ≪ 1) by therapy leads to different possible effects on the "benchmark" (pre-treatment) waiting time. The limiting case scenarios of interest are
1. Therapy suppresses the mutant subpopulation (F m → 0) but allows the dominant wild-type strain to replicate relatively unhindered (F w ≈ 1); this increases the waiting times to the occurrence of a new strain. In other words, even though the total viral load is barely affected, there is a benefit in terms of impaired viral evolution.
where is strain i reproductive ratio. This leads to a dramatic reduction in waiting times to the appearance of rare mutations, i.e. the much more rapid emergence of the rare mutant.
3. Treatment is optimal; that is, treatment that successfully suppresses both viral subpopulations ((F w , F m ) → (0, 0)). This essentially guarantees the non-occurrence of rare mutations.
Transient Nevirapine monotherapy
We have considered a number of more-or-less standard deterministic multi-strain models of in-vivo viral dynamics, which are tunable to produce scenarios like a chronic ARV regimen, and a short course of monotherapy. We have adjoined a stochastic component to these models, in the form of a 'sliding window' survival analysis, which substantially expands the possible analyses of rare strain dynamics within the framework of ordinary differential equations.
We have considered scenarios which capture the concepts of a dominant wild-type strain, a relatively unimpaired 1 base mutant, a number of unviable 2 base mutants, and the possibility of compensatory mutations which lead to a treatment defeating 3 base mutant that is reachable by different pathways, which have different relative importance at various stages during transient dynamics. Different phylogenies, together with different chosen fitness parameter values, will result in different numbers of deterministically and stochastically modelled strains and pathways, all of which can immediately be accommodated into our general model.
The transient increases in common subpopulations of cells infected by mutant genomes produced by the short course of antiviral therapy affects waiting times to the appearance of rare mutations, conceived as differing more from the wild type than from the deterministically modelled primary mutant. Over a range of model choices which produce a transient of the order (size and duration) of that known to occur under nevirapine monotherapy used for PMTCT, there is significant acceleration of viral evolution – even from just the short course alone. This effect is suspected, but not unambiguously observed, from clinical studies.
A further important set of questions arises about the risks associated with initiation of chronic therapy (HAART) a short while after the suboptimal transient regimen like for PMTCT. Our newly proposed additional timescale Δ, representing the persistence of a new genome in an infavourable environment has a substantial impact on the rates of treatment failure.
These models demonstrate that even transient subpopulations of common mutants which appear to fade are associated with accelerated appearance of rarer mutations. Further work which should be performed includes 1) variations on these models which are designed to capture precise genetic differences (and hence realistic pathways and mutation rates) between sets of quasispecies being directly observed in studies utilising highly sensitive assays, and 2) biological investigation into the dynamics of small populations of new mutants, which these models summarise into the timescale ΛΔ.
Appendix: mutation combinatorics
We relate the HIV mutation process parameters ε ij and f to an underlying single-point-mutation process.
This research was supported by South African Department of Science and Technology/National Research Foundation (DST/NRF) Centre of Excellence in Epidemiological Modelling and Analysis (SACEMA), University of Stellenbosch.
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