# Time Intervals in Sequence Sampling, Not Data Modifications, Have a Major Impact on Estimates of HIV Escape Rates

^{1}

^{2}

## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

**Data**. Experimental data used in this paper are from previous publications [16,25]. In short, individuals with recent HIV-1 infection were recruited into the study. Patients donated blood at regular time intervals, and viral RNA sequences were obtained using single genome amplification (SGA) techniques. Three patients from the Center for HIV/AIDS Vaccine Immunology (CHAVI) were analyzed: CH40, CH77, and CH58. These patients were infected with a single transmitted/founder virus, and changes in the viral genome were mapped to the HIV-specific cytotoxic T lymphocyte (CTL) responses. In viral sequences, there were also changes that had signatures of viral escape from CTL responses, but no CTL responses specific to proteins in these specific regions have been detected [16]. We re-analyzed viral sequence data for all detected escapes and restricted some analyses to only escapes from detected CTL responses. Subjects are controlled in accordance with the tenets of the Declaration of Helsinki.

**Model**. We used a previously suggested mathematical model of viral escape from a single CTL response [21,22,23,25]. The model was fit to experimental data using likelihood method based on binomial distribution (Equation (2) [31]).

**Statistics**. When fitting mathematical models to experimental data, it is important to estimate confidence intervals for the model parameters. One widely-used approach is bootstrap, in which either experimental data or residuals (difference between model prediction and the data) are sampled with replacement [32]. Two types of bootstraps exit: non-parametric and parametric bootstrap. In nonparametric bootstrap, data are sampled with replacement into a bootstrap sample and then the model is fitted to the sample dataset. For example, one could resample with replacement residuals from the model fit of the data, add resampled residuals to the model predictions to generate a sampled dataset, and fit the model to these sampled data. In contrast, in parameteric bootstrap, one assumes a particular distribution underlying the data and generates samples using this distribution. For example, residuals resulting from a given model fit may follow a normal distribution with zero mean and variance ${\sigma}^{2}$. Therefore, using normal distribution $N(0,{\sigma}^{2})$, one could generate another bootstrap sample by adding randomly generated residuals to predictions of the model best fit [32]. In our analysis, we use a version of the parametric bootstrap whereby we generate samples of data assuming that escape variant frequency follows a beta distribution with m mutant and w wild-type sequences. A beta distribution is a continuous approximation of the binomial distribution, and the beta distribution has been used to calculate confidence intervals for proportions [33].

**Programming**. Model fits of the data were done in Mathematica 11 using a routine FindMinimum. All codes are available from the author upon request. An example of a Mathematica notebook illustrating the strategy of the sampling method to estimate escape rates is available as a supplement to this paper.

## 3. Results

#### 3.1. A Sampling Method to Estimate Escape Rates without Data Modification

#### 3.2. The Rate of HIV Escape Declines with Time since Infection

#### 3.3. Time Frequency of Sampling Biases Estimates of the Escape Rate

## 4. Discussion

## Supplementary Materials

## Acknowledgments

## Author Contributions

## Conflicts of Interest

## References

- Mansky, L.M.; Temin, H.M. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J. Virol.
**1995**, 69, 5087–5094. [Google Scholar] [PubMed] - Mansky, L.M. Forward mutation rate of human immunodeficiency virus type 1 in a T lymphoid cell line. AIDS Res. Hum. Retroviruses
**1996**, 12, 307–314. [Google Scholar] [CrossRef] [PubMed] - Mansky, L.M. Retrovirus mutation rates and their role in genetic variation. J. Gen. Virol.
**1998**, 79 Pt 6, 1337–1345. [Google Scholar] [CrossRef] - SanjuÃn, R.; Nebot, M.R.; Chirico, N.; Mansky, L.M.; Belshaw, R. Viral mutation rates. J. Virol.
**2010**, 84, 9733–9748. [Google Scholar] [CrossRef] [PubMed] - Geller, R.; Domingo-Calap, P.; Cuevas, J.M.; Rossolillo, P.; Negroni, M.; Sanjuán, R. The external domains of the hiv-1 envelope are a mutational cold spot. Nat. Commun.
**2015**, 6, 8571. [Google Scholar] [CrossRef] [PubMed] - Cuevas, J.M.; Geller, R.; Garijo, R.; López-Aldeguer, J.; Sanjuán, R. Extremely high mutation rate of HIV-1 in vivo. PLoS Biol.
**2015**, 13, e1002251. [Google Scholar] [CrossRef] [PubMed] - Perelson, A.S. Modelling viral and immune system dynamics. Nat. Rev. Immunol.
**2002**, 2, 28–36. [Google Scholar] [CrossRef] [PubMed] - Haase, A.T. Population biology of HIV-1 infection: Viral and CD4+ T cell demographics and dynamics in lymphatic tissues. Annu. Rev. Immunol.
**1999**, 17, 625–656. [Google Scholar] [CrossRef] [PubMed] - Estes, J.D.; Kityo, C.; Ssali, F.; Swainson, L.; Makamdop, K.N.; Del Prete, G.Q.; Deeks, S.G.; Luciw, P.A.; Chipman, J.G.; Beilman, G.J.; et al. Defining total-body aids-virus burden with implications for curative strategies. Nat. Med.
**2017**, 23, 1271–1276. [Google Scholar] [CrossRef] [PubMed] - Walker, B.D.; Korber, B.T. Immune control of HIV: the obstacles of HLA and viral diversity. Nat. Immunol.
**2001**, 2, 473–475. [Google Scholar] [CrossRef] [PubMed] - Barouch, D.; Kunstman, J.; Kuroda, M.; Schmitz, J.; Santra, S.; Peyerl, F.; Krivulka, G.; Beaudry, K.; Lifton, M.; Gorgone, D.A.; et al. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature
**2002**, 415, 335–339. [Google Scholar] [CrossRef] [PubMed] - Mullins, J.I.; Rolland, M.; Allen, T.M. Viral evolution and escape during primary human immunodeficiency virus-1 infection: Implications for vaccine design. Curr. Opin. HIV AIDS
**2008**, 3, 60–66. [Google Scholar] [CrossRef] [PubMed] - Rolland, M.; Tovanabutra, S.; Decamp, A.C.; Frahm, N.; Gilbert, P.B.; Sanders-Buell, E.; Heath, L.; Magaret, C.A.; Bose, M.; Bradfield, A.; et al. Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial. Nat. Med.
**2011**, 17, 366–371. [Google Scholar] [CrossRef] [PubMed] - Nowak, M.A.; Anderson, R.M.; McLean, A.R.; Wolfs, T.F.; Goudsmit, J.; May, R.M. Antigenic diversity thresholds and the development of AIDS. Science
**1991**, 254, 963–969. [Google Scholar] [CrossRef] [PubMed] - Alizon, S.; Magnus, C. Modelling the course of an hiv infection: insights from ecology and evolution. Viruses
**2012**, 4, 1984–2013. [Google Scholar] [CrossRef] [PubMed] - Goonetilleke, N.; Liu, M.K.; Salazar-Gonzalez, J.F.; Ferrari, G.; Giorgi, E.; Ganusov, V.V.; Keele, B.F.; Learn, G.H.; Turnbull, E.L.; Salazar, M.G.; et al. The first T cell response to transmitted/founder virus contributes to the control of acute viremia in HIV-1 infection. J. Exp. Med.
**2009**, 206, 1253–1272. [Google Scholar] [CrossRef] [PubMed] - McMichael, A.J.; Borrow, P.; Tomaras, G.D.; Goonetilleke, N.; Haynes, B.F. The immune response during acute HIV-1 infection: Clues for vaccine development. Nat. Rev. Immunol.
**2010**, 10, 11–23. [Google Scholar] [CrossRef] [PubMed] - Bar, K.J.; Tsao, C.-Y.; Iyer, S.S.; Decker, J.M.; Yang, Y.; Bonsignori, M.; Chen, X.; Hwang, K.-K.; Montefiori, D.C.; Liao, H.-X.; et al. Early Low-Titer Neutralizing Antibodies Impede HIV-1 Replication and Select for Virus Escape. PLoS Pathog.
**2012**, 8, e1002721. [Google Scholar] [CrossRef] [PubMed] - Kijak, G.H.; Sanders-Buell, E.; Chenine, A.-L.; Eller, M.A.; Goonetilleke, N.; Thomas, R.; Leviyang, S.; Harbolick, E.A.; Bose, M.; Pham, P.; et al. Rare hiv-1 transmitted/founder lineages identified by deep viral sequencing contribute to rapid shifts in dominant quasispecies during acute and early infection. PLoS Pathog.
**2017**, 13, e1006510. [Google Scholar] [CrossRef] - Liu, M.K.P.; Hawkins, N.; Ritchie, A.J.; Ganusov, V.V.; Whale, V.; Brackenridge, S.; Li, H.; Pavlicek, J.W.; Cai, F.; Rose-Abrahams, M.; et al. Vertical T cell immunodominance and epitope entropy determine HIV-1 escape. J. Clin. Investig.
**2013**, 123, 380–393. [Google Scholar] [CrossRef] [PubMed] - Fernandez, C.; Stratov, I.; De Rose, R.; Walsh, K.; Dale, C.; Smith, M.; Agy, M.; Hu, S.; Krebs, K.; Watkins, D.I.; et al. Rapid viral escape at an immunodominant simian-human immunodeficiency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost. J. Virol.
**2005**, 79, 5721–5731. [Google Scholar] [CrossRef] [PubMed] - Asquith, B.; Edwards, C.; Lipsitch, M.; McLean, A. Inefficient cytotoxic T lymphocyte-mediated killing of HIV-1-infected cells in vivo. PLoS Biol.
**2006**, 4, e90. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Ganusov, V.V.; De Boer, R.J. Estimating costs and benefits of CTL escape mutations in SIV/HIV Infection. PLoS Comput. Biol.
**2006**, 2, e24. [Google Scholar] [CrossRef] [PubMed] - Fischer, W.; Ganusov, V.V.; Giorgi, E.E.; Hraber, P.T.; Keele, B.F.; Leitner, T.; Han, C.S.; Gleasner, C.D.; Green, L.; Lo, CC.; et al. Transmission of single HIV-1 genomes and dynamics of early immune escape revealed by ultra-deep sequencing. PLoS ONE
**2010**, 5, e12303. [Google Scholar] [CrossRef] [PubMed] - Ganusov, V.V.; Goonetilleke, N.; Liu, M.K.P.; Ferrari, G.; Shaw, G.M.; McMichael, A.J.; Borrow, P.; Korber, B.T.; Perelson, A.S. Fitness Costs and Diversity of the Cytotoxic T Lymphocyte (CTL) Response Determine the Rate of CTL Escape during Acute and Chronic Phases of HIV Infection. J. Virol.
**2011**, 85, 10518–10528. [Google Scholar] [CrossRef] [PubMed] - Kessinger, T.A.; Perelson, A.S.; Neher, R.A. Inferring HIV Escape Rates from Multi-Locus Genotype Data. Front. Immunol.
**2013**, 4, 252. [Google Scholar] [CrossRef] [PubMed] - Leviyang, S. Constructing lower-bounds for ctl escape rates in early siv infection. J. Theor. Biol.
**2014**, 352, 82–91. [Google Scholar] [CrossRef] [PubMed] - Asquith, B.; McLean, A. In vivo CD8+ T cell control of immunodeficiency virus infection in humans and macaques. Proc. Natl. Acad. Sci. USA
**2007**, 104, 6365–6370. [Google Scholar] [CrossRef] [PubMed] - Leviyang, S.; Ganusov, V.V. Broad CTL Response in Early HIV Infection Drives Multiple Concurrent CTL Escapes. PLoS Comput. Biol.
**2015**, 11, e1004492. [Google Scholar] [CrossRef] [PubMed] - Garcia, V.; Feldman, M.W.; Regoes, R.R. Investigating the Consequences of Interference between Multiple CD8+ T Cell Escape Mutations in Early HIV Infection. PLoS Comput. Biol.
**2016**, 12, e1004721. [Google Scholar] [CrossRef] [PubMed] - Ganusov, V.V.; Neher, R.A.; Perelson, A.S. Modeling HIV escape from cytotoxic T lymphocyte responses. J. Stat. Mech.
**2013**, 2013, P01010. [Google Scholar] [CrossRef] [PubMed] - Efron, B.; Tibshirani, R. An Introduction to the Bootstrap; Chapman & Hall: New York, NY, USA, 1993. [Google Scholar]
- Brown, L.; Cai, T.; DasGupta, A.; Agresti, A.; Coull, B.; Casella, G.; Corcoran, C.; Mehta, C.; Ghosh, M. Interval estimation for a binomial proportion. Stat. Sci.
**2001**, 16, 101–133. [Google Scholar] - Yang, Y.; Ganusov, V.V. Kinetics of HIV-specic CTL responses plays a minimal role in determining HIV escape dynamics. Front. Immunol.
**2018**, 9, 1–15. [Google Scholar] [CrossRef] [PubMed] - Geldmacher, C.; Currier, J.R.; Herrmann, E.; Haule, A.; Kuta, E.; McCutchan, F.; Njovu, L.; Geis, S.; Hoffmann, O.; Maboko, L.; et al. CD8 T-cell recognition of multiple epitopes within specific Gag regions is associated with maintenance of a low steady-state viremia in human immunodeficiency virus type 1-seropositive patients. J. Virol.
**2007**, 81, 2440–2448. [Google Scholar] [CrossRef] [PubMed] - Streeck, H.; Jolin, J.S.; Qi, Y.; Yassine-Diab, B.; Johnson, R.C.; Kwon, D.S.; Addo, M.M.; Brumme, C.; Routy, J.-P.; Little, S.; et al. Human immunodeficiency virus type 1-specific cd8+ t-cell responses during primary infection are major determinants of the viral set point and loss of cd4+ t cells. J. Virol.
**2009**, 83, 7641–7648. [Google Scholar] [CrossRef] [PubMed]

**Figure 1.**A sampling method accurately recovers virus escape rate for data when both wild-type and escape variant are detected at multiple sequential time points. We simulated viral escape using a logistic equation with two escape rates: $\epsilon =0.22$/day (bullets) and $\epsilon =0.022$/day (triangles). Three data points at which the escape variant was present at 10%, 50%, and 90% out of $N=10$ sequences were generated (panel

**A**). These simulated data were fitted by the simple logistic model (Equation (1)) using a likelihood approach, leading to expected escape rates (lines in panel A). These data were then resampled using the beta distribution (panel

**B**, black solid line is for $\epsilon =0.22$/day and red dashed line is for $\epsilon =0.022$/day) and fitted by the same model using the likelihood method. Resulting averages and their 95% confidence intervals match well with the numbers estimated using actual data (panel

**C**). Dashed lines in panel (

**C**) show estimates of the escape rates found in panel (

**A**), and 95% confidence intervals in panel (

**B**) are predicted using Jeffrey’s intervals [33].

**Figure 2.**A sampling method which does not involve data modification still predicts a decline in the estimated escape rate with the time since infection. We used a novel approach of data sampling and estimated the rate of human immunodeficiency virus (HIV) escape from CD8 T cell responses for three Center for HIV/AIDS Vaccine Immunology (CHAVI) patients using previously published data [16,25]. Rates ($\epsilon $) and time to 50% of the escape variant (${t}_{50}$) and associated errors are shown for three different patients in different panels (panel

**A**: CH40, panel

**B**: CH77, panel

**C**: CH58); bullets indicate all escapes (in black) and triangles are for confirmed escapes (in red) [16]. Confirmed escapes were defined as fixed changes in HIV genome with a clear signature of viral escape with detected epitope-specific T cell response. Other escapes had a well-defined signature of escape but no epitope-specific T cell response was detected [16]. Resulting p values from the Spearman rank correlation test are indicated on individual panels. Panel (

**B**) does not show an estimate for one escape due to very low escape rate $\epsilon $ and long ${t}_{50}$.

**Figure 3.**A sampling method provides higher estimates of the escape rates. We plot estimates of HIV escape rates in three patients (panel

**A**: CH40, panel

**B**: CH77, panel

**C**: CH58) as was found a previous study [25] employing data modifications (“Old” analysis) and estimates found using sampling method (“New” analysis). Different markers on plots indicate escape rate estimates for different escapes in our previous analysis [25]. Comparison of the escape rates in two analyses was done using Wilcoxon signed rank test and p values from these tests are indicated on individual panels.

**Figure 4.**Time interval between measurements has a strong bias on the estimate of the escape rate and leads to a correlation between the escape rate ($\epsilon $) and time of escape (${t}_{50}$). We generated artificial data in which viral sequences were measured in three time points, resulting in 0%, 50%, and 100% of the escape variant with $N=10$ sequences per time point (panel

**A**). In all cases, escape can be described by the logistic equation (Equation (1)) assuming a delayed immune response and escape rate $\epsilon =0.4$/day. We resampled these simulated “data” using a beta distribution (panel

**B**, different lines indicate different escapes as in panel

**A**) and estimated the average escape rate (panel

**C**). We show the prediction of the logistic curve obtained using $\epsilon =0.4$/day (panel

**A**), sampled data (panel

**B**), and estimates of the escape rate obtained using parametric bootstrap (panel

**C**). The horizontal dashed line in (panel

**C**) denotes the theoretical escape rate of 0.4/day. The 95% confidence intervals in (panel

**B**) are predicted using Jeffrey’s intervals [33], and in (panel

**C**) CIs were calculated from bootstrapped samples.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## Share and Cite

**MDPI and ACS Style**

Ganusov, V.V.
Time Intervals in Sequence Sampling, Not Data Modifications, Have a Major Impact on Estimates of HIV Escape Rates. *Viruses* **2018**, *10*, 99.
https://doi.org/10.3390/v10030099

**AMA Style**

Ganusov VV.
Time Intervals in Sequence Sampling, Not Data Modifications, Have a Major Impact on Estimates of HIV Escape Rates. *Viruses*. 2018; 10(3):99.
https://doi.org/10.3390/v10030099

**Chicago/Turabian Style**

Ganusov, Vitaly V.
2018. "Time Intervals in Sequence Sampling, Not Data Modifications, Have a Major Impact on Estimates of HIV Escape Rates" *Viruses* 10, no. 3: 99.
https://doi.org/10.3390/v10030099