Advertisement
  • Loading metrics

Estimating the Fitness Cost of Escape from HLA Presentation in HIV-1 Protease and Reverse Transcriptase

  • Rafal Mostowy ,

    Contributed equally to this work with: Rafal Mostowy, Roger D. Kouyos

    rafal.mostowy@gmail.com

    Affiliation Institute for Integrative Biology, ETH Zurich, Zurich, Switzerland

  • Roger D. Kouyos ,

    Contributed equally to this work with: Rafal Mostowy, Roger D. Kouyos

    Affiliation Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey, United States of America

  • Ilka Hoof,

    Affiliation Theoretical Biology/Bioinformatics, Utrecht University, Utrecht, The Netherlands

  • Trevor Hinkley,

    Affiliation Institute for Integrative Biology, ETH Zurich, Zurich, Switzerland

  • Mojgan Haddad,

    Affiliation Monogram Biosciences, South San Francisco, California, Unites States of America

  • Jeannette M. Whitcomb,

    Affiliation Monogram Biosciences, South San Francisco, California, Unites States of America

  • Christos J. Petropoulos,

    Affiliation Monogram Biosciences, South San Francisco, California, Unites States of America

  • Can Keşmir,

    Affiliation Theoretical Biology/Bioinformatics, Utrecht University, Utrecht, The Netherlands

  • Sebastian Bonhoeffer

    Affiliation Institute for Integrative Biology, ETH Zurich, Zurich, Switzerland

Estimating the Fitness Cost of Escape from HLA Presentation in HIV-1 Protease and Reverse Transcriptase

  • Rafal Mostowy, 
  • Roger D. Kouyos, 
  • Ilka Hoof, 
  • Trevor Hinkley, 
  • Mojgan Haddad, 
  • Jeannette M. Whitcomb, 
  • Christos J. Petropoulos, 
  • Can Keşmir, 
  • Sebastian Bonhoeffer
PLOS
x

Abstract

Human immunodeficiency virus (HIV-1) is, like most pathogens, under selective pressure to escape the immune system of its host. In particular, HIV-1 can avoid recognition by cytotoxic T lymphocytes (CTLs) by altering the binding affinity of viral peptides to human leukocyte antigen (HLA) molecules, the role of which is to present those peptides to the immune system. It is generally assumed that HLA escape mutations carry a replicative fitness cost, but these costs have not been quantified. In this study, we assess the replicative cost of mutations which are likely to escape presentation by HLA molecules in the region of HIV-1 protease and reverse transcriptase. Specifically, we combine computational approaches for prediction of in vitro replicative fitness and peptide binding affinity to HLA molecules. We find that mutations which impair binding to HLA-A molecules tend to have lower in vitro replicative fitness than mutations which do not impair binding to HLA-A molecules, suggesting that HLA-A escape mutations carry higher fitness costs than non-escape mutations. We argue that the association between fitness and HLA-A binding impairment is probably due to an intrinsic cost of escape from HLA-A molecules, and these costs are particularly strong for HLA-A alleles associated with efficient virus control. Counter-intuitively, we do not observe a significant effect in the case of HLA-B, but, as discussed, this does not argue against the relevance of HLA-B in virus control. Overall, this article points to the intriguing possibility that HLA-A molecules preferentially target more conserved regions of HIV-1, emphasizing the importance of HLA-A genes in the evolution of HIV-1 and RNA viruses in general.

Author Summary

Our immune system can recognize and kill virus-infected cells by distinguishing between self and virus-derived protein fragments, called peptides, displayed on the surface of each cell. One requirement for a successful recognition is that those peptides bind to the human leukocyte antigen (HLA) class I molecules, which present them to the immune system. As a counter-strategy, human immunodeficiency virus type 1 (HIV-1) can acquire mutations that prevent this binding, thereby helping the virus to escape the surveillance of T-lymphocytes. It is likely that the virus pays a replicative cost for such escape mutations, but the magnitude of this cost has remained elusive. Here, we quantified this fitness cost in HIV-1 protease and reverse transcriptase by combining two computational systems biology approaches: one for prediction of in vitro replicative fitness, and one for the prediction of the efficiency of peptide binding to HLA. We found that in viral proteins targeted by HLA-A molecules, mutations which disrupt binding to those molecules carry a lower replicative fitness than mutations which do not have such an effect. We argue that these results are consistent with the hypothesis that our immune systems might have evolved to target genetic regions of RNA viruses which are costly for the pathogen to alter.

Introduction

The evolutionary dynamics of viral infections are often characterized by the opposing forces of immune control and viral escape. These forces shape both the within-host dynamics of infections as well as the dynamics of spread on an epidemiological level. At the within-host level, the role of the opposing forces are manifest in chronic infections such as HIV, SIV, and HCV, where it has been shown that the virus population frequently escapes immune control by B- or T-cell responses [1][8]. Moreover, in HIV/SIV, slow disease progression is associated with efficient immune control via protective human leukocyte antigen (HLA) genes; see [9][11], and escape events can precipitate the loss of immune control [3], [12], [13]. At the epidemiological level, the selection for escape imposed by the host population can be traced in the genetic structure of viral infections [14][17].

The balance between immune control and escape often affects the success of vaccines. Vaccines against viral infections characterized by complete absence of escape, such as smallpox, measles, mumps and rubella, are typically highly protective [18], whereas vaccines against viruses which periodically escape immune control, such as influenza, require periodic updating [19][21]. In HIV-1, antigenic diversity, often stemming from escape at the within-host level, represents a formidable obstacle for any potential vaccine to overcome [22][24].

Deeper insight into the coevolutionary dynamics of immune control and viral escape requires an improved quantitative understanding of the benefits and costs of escape from the immune responses. The literature on HIV-1 and SIV escape clearly documents the existence of costs and benefits for viral escape as well as the necessity of quantifying them. A good example is the escape from the presentation by major histocompatibility complex (MHC) class I molecules, in humans encoded by HLA genes [3]. Furthermore, escape mutations often revert in HLA-mismatched patients [25][27] and they are often followed by compensatory mutations [28][30], suggesting that escape mutations are costly. Large variability in the cost of escape mutations has been demonstrated experimentally [31], and mathematical models have shown that the dynamics of immune escape strongly depend on the cost of escape mutations both at the within-host and the epidemiological level [32][34]. Taken together these studies underline the importance of assessing the replicative cost of escape mutations.

In this study, we quantified the fitness cost of those mutations in HIV-1 protease (PR) and reverse transcriptase (RT) which are likely to escape the presentation of HLA molecules using an in silico approach, focusing on escape from presentation by HLA-A and HLA-B molecules. To this end, we analyzed a dataset of more than 70'000 genetic sequences of HIV-1 PR and RT from the North American population. Specifically, we combined two computational approaches: First, we used the results of a ridge-regression approach to quantify the impact of single amino acid substitutions in the region of interest on viral replicative capacity in vitro [35]. Second, the impact of these mutations on binding affinity to various HLA molecules was predicted using a neural network approach, as implemented in [36], [37]. Combining these two methods we examined whether mutations which are more likely to disrupt binding to HLA molecules carry a higher replicative cost in vitro than other mutations.

Materials and Methods

Data

Over 70'000 virus sequences from HIV-1 subtype B were assayed for the replicative capacity of the Pol gene, specifically the entire protease (PR) and positions 1 to 305 of the reverse transcriptase (RT). The assay is described in detail in ref. [38]. In brief, the region of interest (404 amino acids) was inserted into a viral backbone derived from the NL4-3 molecular clone. The NL4-3 clone was modified so that it can go through a single round of replication. Replicative capacity for each genetic variant was then assessed in vitro by measuring the total infectious progeny virus produced after a single round of replication.

Quantification of fitness costs

The quantification of fitness costs was based on a method described in detail elsewhere [35]. In short, the data described above were fitted to a fitness model of single and double mutations to estimate the effect of these mutations on the viral replicative capacity in vitro using a machine-learning approach (generalized kernel ridge regression). This fitness prediction model had previously been trained on a data set of 65,000 sequences and had been cross-validated on 5,000 sequences, where it explained between 35% and 66% of the variance in fitness depending on the environment in which replication was measured. Specifically, the model used here is based on the estimated main effects and pairwise epistatic effects of the 1'848 amino acid variants found in these sequences, and therefore can predict the fitness of all 1'848 single non-synonymous mutations which naturally occurred in the viral population derived from North American patients.

Here we used this predictive model to calculate the fitness effects of single mutants, which were generated by substituting a single amino acid in the wild-type NL4-3 sequence. The relative fitness of such a single mutant was then given by its predicted replicative capacity relative to the predicted replicative capacity of the NL4-3 sequence. The predicted fitness of the NL4-3 sequence was therefore equal to 1.

Epitope prediction

In order to achieve broad HLA coverage, we used the pan-specific method NetMHCpan2.4 [36], [37] to predict binding affinities of peptides to HLA class I molecules. This machine-learning method (neural network approach) had been trained on a large set of quantitative MHC-peptide binding data for more than 80 human and non-human MHC class I molecules (90% human). The method extrapolated the “rules” of MHC-peptide binding from the training set to predict the binding affinity of MHC:peptide combinations that were not part of the training process. In this study we focused on two of three HLA class I loci: HLA-A and HLA-B. In a benchmark study based on these two genes, NetMHCpan was shown to outperform other pan-specific prediction methods [39]. Binding predictions to HLA-C were ignored because of performance issues.

To utilize NetMHCpan to predict epitopes in PR (amino acids 1–99) and RT (amino acids 1–305), we cleaved the corresponding amino acid sequence of NL4-3 into all possible peptides of length 9 (9mers). We quantified the binding affinity for each peptide:HLA combination by predicting a binding affinity of the complex, which was characterized by an value (concentration at which half-maximal inhibition in the assay was achieved). A low score translated into a high binding affinity, and a high score translated into a low binding affinity. A peptide was then considered an epitope if its predicted binding affinity for a given HLA molecule exceeded either a relative or an absolute threshold. According to the absolute criterion, a peptide was an epitope for a given HLA allele if the obtained score was lower than 500 nM, as suggested experimentally [40]. According to the relative criterion, a peptide was an epitope for a given HLA allele if the obtained score fell within the bottom 1% of all scores obtained for this HLA allele based on a large set () of random natural peptides. Both binding definitions are widely used in the literature [41][43].

Impairment of binding affinity in mutated epitopes

The degree to which a single mutation in a peptide impairs its binding to an HLA allele is considered here only if the peptide was an epitope for the given allele based either on the relative or the absolute criterion. Hence, assessing binding impairment depends on the criterion used to predict epitopes. The impairment of binding affinity due to single mutation in epitopes was defined as(1)where is the binding threshold to a HLA molecule in question, is the score of the mutated peptide to this HLA molecule, is the score of the wild-type (non-mutated) peptide to this HLA molecule, and denotes the value of 50,000 nM considered a maximal experimental sensitivity threshold. If multiple HLA molecules were affected by a single mutation, the strongest impairment was taken into account. Note that for the absolute criterion used here, the multiplication factor in the front reduces to 1, as for each HLA molecule.

Frequency of HLA-alleles and escape

The frequency of a given HLA allele in the US population was estimated from a HLA haplotype study in the US population stratified by ethnicity [44]. The HLA alleles used for this study consisted of all alleles for which we obtained frequency data and which featured in netMHCpan2.4, giving overall 98 HLA-A alleles and 184 HLA-B alleles. An expected frequency of each given HLA allele was calculated as the mean ethnicity-stratified frequency, weighted by the HIV-1 prevalence estimates in the US population for different ethnicities [45]. The list of all HLA alleles used in this study, together with the calculated frequency values are given in Table S1.

To investigate the role of the selective pressure at the population level, we defined a ‘mutation impact’ and a ‘population-weighted mutation impact’, given by(2)where is an impairment of binding HLA molecule due to mutation , is the expected frequency of HLA in the population, and is the number of HLA molecules binding to the wild-type; all values characterized by were ignored.

We classified HLA alleles that occur at frequencies of 0.5% or lower in the host population as infrequent, assuming that these HLA alleles impose negligible selection pressure for escape in the host population. In the analysis of population-independent effects we removed all frequent HLA alleles from the analysis.

Protective and non-protective alleles

To identify protective and non-protective HLA alleles, we assigned a relative hazard for progression to AIDS (RH) to each HLA molecule, based on a survival analysis of HIV-1 positive patients from an earlier study [46]. HLA alleles with a low RH value tend to be associated with long-term non-progression to AIDS, while HLA alleles with a high RH value tend to be associated with rapid progression to AIDS. As the relative hazard values in this study had been associated with low resolution HLA molecules (two-digit, e.g., HLA-A*02) rather than with high-resolution HLA molecules (four-digit, e.g., HLA-A*02:01), in the analysis we assigned the relative hazard values to all HLA alleles of a particular group. For example, the , associated with the allele HLA-A*26, was assigned to HLA-A*26:01, -A*26:02, -A*26:03, -A*26:07, -A*26:08, -A*26:09, and -A*26:12. In our analysis, protective HLA alleles were defined as the 10 alleles (according to the two-digit classification) with the lowest RH value (HLA-B*58, -B*27, -B*57, -A*26, -B*51, -A*11, -A*32, -B*13, -B*14, -A*31), and non-protective HLA alleles were defined as the 10 alleles (according to the two-digit classification) with the highest RH value (HLA-A*66, -A*74, -B*35, -B*53, -B*45, -B*50, -A*33, -B*39, -A*68, -B*56). According to the four-digit classification this corresponds to 54 protective HLA alleles and 60 non-protective alleles.

Statistical analysis

The non-parametric Mann-Whitney or Wilcoxon signed-rank test was used for statistical comparisons of data samples with measured replicative fitness. The non-parametric Spearman rank test was used to calculate correlation coefficients between fitness and impairment of binding, with the confidence intervals calculated using the Fisher transformation. Results with or less were considered significant; results with were considered as showing a trend of significance. All correlations and data analyses were performed using the R software package [47].

Results

In order to test whether the fitness cost of single mutations is associated with their impact on HLA presentation, we analyzed the correlation between the predicted relative fitness of single mutations and their effect on HLA binding affinity. The in vitro fitness of mutations (replicative capacity) was predicted via ridge regression [35] and their impact on HLA presentation was predicted via a neural-networks approach [37]; see Materials & Methods. We first assessed the relation between the fitness of mutations and their effect on HLA binding. To that end, we quantified the impairment of binding caused by each mutation as a log of the ratio of binding affinity prior to the mutation and after the mutation, as given by equation (1). The analysis was performed once for HLA-A molecules, and once for HLA-B molecules. Specifically, in the analysis for HLA-A we considered all mutations in epitopes restricted by HLA-A molecules, and in the analysis for HLA-B we considered all mutations in epitopes restricted by HLA-B molecules (even though these sets of mutations may overlap). If, for a given HLA locus (A or B), a mutation was part of more than one epitope, the strongest effect was considered for that HLA locus, ensuring that one mutation gave rise to a single data point per HLA locus. Fig. 1 shows a correlation between the predicted fitness of single mutations and their impact on binding affinity to HLA-A and HLA-B molecules. The disruptive effect of mutations on binding between HIV-1 peptides and HLA-A molecules was found to correlate significantly with the fitness of those mutations, independent of the epitope definition employed (absolute criterion assumes same binding threshold for each HLA molecule; relative criterion assumes that each HLA molecule binds a similar number of peptides; see Materials & Methods), however no such correlation was observed for HLA-B molecules (HLA-A: absolute criterion: , ; relative criterion: , ; HLA-B: absolute criterion: ; relative criterion: ). Thus, mutations which disrupt binding to HLA-A molecules seem to carry a higher fitness cost than mutations that do not.

thumbnail
Figure 1. Cost of mutations which impair the HLA-binding.

(Top row) Effect of mutations on binding affinity to HLA-A molecules according to two alternative epitope definitions (absolute criterion: , relative criterion: ). In both cases we observed a significant correlation between the fitness of single mutants and the impairment of binding to HLA-A (absolute criterion: , ; relative criterion: , ). Each datapoint corresponds to a single amino acid substitution in the genetic region restricted by HLA alleles of the corresponding locus, A or B. Note, that if multiple HLA molecules were affected by a single mutation at a given locus, the strongest impairment was plotted here. (Bottom row) Effect of mutations on binding affinity to HLA-B molecules for the two alternative epitope definitions (absolute criterion: , relative criterion: ). Here, no significant correlation between the quantities in question was found (absolute criterion: ; relative criterion: ). For the sake of illustration, the blue line shows the best fit of a linear regression and the 95% confidence interval.

https://doi.org/10.1371/journal.pcbi.1002525.g001

To assess whether this association holds independently of the binding criterion employed, we examined the association between fitness and change in binding for a published list of experimentally defined optimal CTL epitopes and the corresponding HLA molecules [48], which defined the used peptide:HLA complexes. We observed a trend of a negative correlation between fitness and binding impairment when mutations in peptides restricted by both HLA-A and HLA-B were considered (, , ), however no signal was detected for individual correlations in HLA-A (, ) or HLA-B (, ). Although the first result seems to support the notion of a negative correlation between replicative fitness and binding impairment to HLA molecules in the HIV-1 region examined, it remains not fully clear whether the lack of a strong signal is due to a low number of HLA:peptide pairs provided in the list of best-defined CTL epitopes (81 instead of 1850 and 796 predicted ones for the absolute and relative criterion, respectively), or due to any problems associated with the classification of optimal epitopes.

The association between the effect of a mutation on fitness and its predicted effect on HLA-A binding could be due to two, mutually not exclusive, factors. First, there might be a population effect caused by selection for escape at the host population level, as shown in Fig. 2. Since our data are not based on random mutations but mutations as they are observed in viral populations sampled from the infected host population, we would expect mutations with a high replicative cost (i.e., low replicative capacity in vitro) to be underrepresented in our dataset. However, for escape mutations such replicative cost could be offset by the benefit of conferring escape, especially if the mutant confers escape in a large fraction of the host population. For this reason, two mutations of the same replicative capacity, one escaping a large fraction of the host population and another escaping none, would not spread evenly in the host population because the escape mutation would have a higher in vivo fitness than the non-escape mutation, and consequently the escape mutation would be present at a higher frequency in the population than the non-escape mutation. The resulting excess of escape mutations with high replicative cost would manifest itself in the data as a difference in relative fitness in vitro between the two types of mutations (see also Fig. 2). We expect this effect to be the most dramatic for mutations with high replicative cost because mutations with a low replicative cost will most likely occur in the dataset even if they carry no additional advantage against the immune response. Second, there could be an intrinsic effect stemming from intrinsic differences between mutations that reduce HLA-A binding and mutations that do not.

thumbnail
Figure 2. Population effect in the observed cost of escape from HLA presentation.

The distribution of replicative fitness effects (DFE) of random mutations in HIV-1 (green) will differ from the corresponding distribution of mutations derived from the patient population (blue/orange) as the latter will feature only mutations which have undergone selection. Mutations with a particularly high replicative cost (i.e., low replicative capacity) will have a low probability of being present in the patient-derived data because they will persist at very low frequencies in the virus population. However, escape mutations carry an additional benefit of avoiding being killed by the immune system and therefore are expected to have a higher in vivo fitness, which allows for persistence in spite of a lower replicative capacity. For this reason, even if the DFE of escape and non-escape mutations are identical, the escape mutations derived from the patient population (blue) may appear to have on average a lower replicative capacity than the non-escape mutations derived from the patient population (orange).

https://doi.org/10.1371/journal.pcbi.1002525.g002

To test the first hypothesis, we examined whether the observed dependence of the binding effect of single mutations on their fitness becomes more prominent if the frequency of HLA-A alleles are taken into account. To this end we defined a ‘mutation impact’, , which calculates the unweighted binding impairment to all HLA molecules which bind to the wild-type, and a ‘population-weighted mutation impact’, , which calculates the same effect but weighted by the frequencies of particular HLA alleles (see Materials & Methods). A significant correlation between and fitness was observed (absolute criterion: , ; relative criterion: , ), as well as between and fitness (absolute criterion: , ; relative criterion: , ), however the two correlation coefficients were not significantly different (absolute criterion: , ; relative criterion: , ). Furthermore, a partial correlation between fitness and corrected for , was found to be non-significant (absolute criterion: ; relative criterion: ). Hence, our analysis shows that the lower replicative fitness of mutations which impair binding to HLA-A molecules is unlikely to be caused by the population effect explained above.

To test the second hypothesis (intrinsic effect), we considered only escape mutations from rare HLA-A alleles (less frequent than 0.5%) because such rare alleles are likely to impose a negligible selective pressure on the virus population. Fig. 3 shows a correlation between fitness and impairment of binding due to single mutations in regions targeted by such infrequent HLA-A alleles, in analogy to Fig. 1. For both definitions of escape, we saw a significant correlation between fitness and impairment of binding affinity (absolute criterion: , ; relative criterion: , ). Our analysis thus suggests that the association between replicative cost and escape from HLA-A-binding in HIV-1 may be due to intrinsic differences between the mutational effects of escape versus non-escape mutations.

thumbnail
Figure 3. Cost of escape from rare HLA-A alleles.

Correlation between fitness of single mutations and impairment of binding to rare (lower than 0.5%, see Materials & Methods) HLA-A molecules (absolute criterion: , relative criterion: ). We observed a significant correlation for both epitope definitions (absolute criterion: , ; relative criterion: , ). For the sake of illustration, the blue line shows the best fit of a linear regression and the 95% confidence interval.

https://doi.org/10.1371/journal.pcbi.1002525.g003

Finally, we examined whether escape mutations from the presentation by protective HLA alleles are more costly than escape mutations from the presentation by non-protective HLA alleles. We defined protective and non-protective HLA alleles based on the associated relative hazard of progression to AIDS reported in an earlier study [46] (see also Materials & Methods). Fig. 4 shows a correlation between fitness and binding, in analogy to Fig. 1, for the 10 most protective and the 10 most non-protective HLA allele groups. A significant correlation is observed in the case of protective HLA alleles (absolute criterion: , ; relative criterion: , ) but not in the case of non-protective HLA alleles (absolute criterion: ; relative criterion: ). Furthermore, we observed that the correlations for protective HLA alleles became stronger when performed on the subset of protective HLA-A alleles (absolute criterion: , ; relative criterion: , ), and non-significant when performed on the subset of protective HLA-B alleles (both criteria: , ). An analysis based on a dataset from a different study by Gao et al. [49] revealed qualitatively identical results. Our results thus suggest that escape from protective HLA alleles (particularly HLA-A alleles) may be more costly than escape from non-protective HLA alleles.

thumbnail
Figure 4. Cost of escape from the protective and the non-protective HLA alleles.

(Top row) Effect of mutations on binding affinity to 10 most protective HLA molecules according to two alternative epitope definitions (absolute criterion: , relative criterion: ). Protectiveness of alleles was characterized based on the relative hazard for AIDS progression (see Materials & Methods). A significant correlation between the fitness of single mutants and the impairment of binding to the most protective HLA molecules was observed (absolute criterion: , ; relative criterion: , ). (Bottom row) Effect of mutations on binding affinity to 10 most non-protective HLA molecules according to two alternative epitope definitions (absolute criterion: , relative criterion: ). In this case, no significant correlation between the fitness of single mutants and the impairment of binding to the most non-protective HLA molecules was found (absolute criterion: ; relative criterion: ). For the sake of illustration, the blue line shows the best fit of a linear regression and the 95% confidence interval.

https://doi.org/10.1371/journal.pcbi.1002525.g004

Discussion

This study provides, to our knowledge, the first quantitative assessment of the fitness cost of escape mutations which disrupt presentation by HLA-A and HLA-B molecules. Our results indicate that the fitness cost of mutations increases with the resulting impairment of binding strength to HLA-A, suggesting that a higher tendency to escape HLA binding is more costly in terms of replicative fitness.

Our analysis suggests that the increased replicative cost of those mutations which tend to impair binding to HLA-A molecules is due to an intrinsic property of the virus, where HLA molecules preferentially bind peptides in which escape mutations are particularly costly to change, and this is not biased by the fact the analysis was done on clinically derived sequences (cf. Fig. 2). This intrinsic property of HLA binding is reflected as a significant difference between fitness cost and change in binding to rare HLA-A alleles, which are unlikely to exert any selective pressure at the population level (see Fig. 3). Furthermore, we found that mutations which help escape the protective HLA alleles (particularly HLA-A alleles) are associated with a higher fitness cost, unlike mutations which help escape the non-protective HLA alleles (see Fig. 4). This result also points to an intrinsic cost of escape as the protective HLA alleles used in our analysis are not only on average less frequent than the non-protective alleles, but also less frequent than all other alleles used in this study (protective: 0.56%; non-protective: 0.61%; all: 0.74%). We tested whether certain biochemical properties of the peptides, such as hydrophobicity [50], [51], amino acid similarity [52] or GC content [53] could potentially underlie the observed intrinsic property of MHC binding. However, even though such properties may partially explain changes in binding to HLA molecules (e.g., change in hydrophobicity according to [54], [55]; see Fig. S1), none of them significantly correlates with changes in fitness (). Therefore, what mechanism could explain that mutations which help to escape HLA-A binding are associated with a replicative cost, and whether intrinsic costs occur in other regions of the HIV-1 genome, remain open questions.

One of the most puzzling outcomes of this study is that no significant fitness costs have been observed for escape mutations from HLA-B molecules. This is surprising because HLA-B molecules have been shown to have the strongest impact on the outcome of HIV-1 infection: some HLA-B alleles have been associated with long-term non-progression to AIDS [16], [56], [57], and HLA-B restricted CTL responses have been shown to exert the strongest selective pressure on the virus [11], [58], [59]. In line with these observations, it has been previously shown that HLA-B alleles target more conserved genetic regions of HIV-1 than HLA-A alleles [60], [61]. However, the fact that we do not observe a significant cost of escape mutations from HLA-B can be potentially attributed to several, mostly methodological factors. First, a general feature of the currently available prediction software is that binding predictions are more accurate for HLA-A than for HLA-B, and this is also the case for netMHCpan used here [37]. Second, the differences in fitness costs between HLA-A and HLA-B could be due to an unknown property of the HIV-1 genomic region considered (PR and RT). Third, because the mutations analyzed here are extracted from clinically derived sequences, it is conceivable that the lack of a significant difference between fitness of escape and non-escape mutations for HLA-B is due to the differences between the distributions of HLA-A and HLA-B alleles in the North American population. This topic, however, warrants further investigation. For the reasons outlined above, our results should not be used to suggest that escape from HLA-A presentation is more costly than escape from HLA-B presentation. Rather, our study emphasizes an important role of HLA-A alleles (particularly the protective ones) in the evolution of HIV-1, and RNA viruses in general, as suggested previously [62][65].

One of the limitations of this study is that we have consistently ignored all mutations outside the regions predicted to be restricted by any HLA alleles used in the analysis (for the list see Table S1). Interestingly, when we compared the fitness of mutations restricted by HLA-A molecules to those not predicted to be restricted by any of the HLA-A molecules used in this study, we found that mutations in the restricted regions are on average less costly than mutations in the non-restricted regions (absolute criterion: , relative criterion: ), and a similar effect was found for HLA-B molecules (absolute criterion: , relative criterion: ). The difference in fitness cost of the restricted versus non-restricted regions may seem to contradict our earlier conclusion that HLA-A molecules target regions which are more costly for the virus to change. However, as the extent of those regions strongly depends on the number of HLA molecules considered in the analysis, as well as the binding threshold considered, this result needs to be interpreted with caution. For example, in the case of the relative criterion, we find that HLA-A molecules span 60% and HLA-B span 67% of the considered HIV-1 PR and RT, and for the absolute criterion these numbers are even larger (85% and 98%, respectively). Even though we used the largest subset of HLA molecules for which the employed binding predictor works and for which frequency data in the US population were obtained, these numbers constitute only a fraction of the actual numbers of HLA alleles in the human population (98 out of known 486 HLA-A alleles and 184 out of known 817 HLA-B alleles) [16]. It therefore seems likely that the non-restricted regions here (i.e., those regions in which no peptide binds to any of the HLA molecules included in the study) do bind to other HLA molecules not included in this analysis, however this hypothesis remains to be tested.

Another caveat of this study is that the viral fitness has been assessed in an in vitro assay. This may be partially responsible for the seemingly counter-intuitive fact that many single mutations increase viral replicative capacity, as seen e.g. in Fig. 1. Nevertheless, a recent study has shown that in vitro fitness measures in PR and RT are indicative of in vivo HIV-1 virus load, suggesting that our fitness measures are justified as a proxy for the virus fitness in vivo [66].

It should also be noted that the correlations we observe throughout this study are generally weak, suggesting that escape from HLA-A presentation explains around 10% of the variance in viral fitness. This may seem surprising as HLA molecules have been shown as a potent factor in the evolution of HIV-1 [16], [17], [67], [68]. However, there are several reasons to expect such a result. First, HLAs are probably not the sole driver of the evolution of the virus. Many of the mutations predicted to be non-escape mutations could actually be escape mutations from other elements of the antigen-processing and presentation pathway (e.g., TAP, proteasome, MHC class II), or from the recognition by the T cell receptors [3]. Second, many epitopes are known to vary in their immunogenicity, and some will be more likely to elicit CTL responses than others [69]. Third, the signature of evolution in our dataset will be strongly influenced by the population structure from which the data was obtained, e.g., the distribution of HLA allele frequencies. Fourth, the Pol gene analyzed here plays an important role in the evolution of drug resistance, leading to a potential interaction between immune- and treatment-mediated selection (see below). Finally, the dataset used cannot provide any insight into the distribution of the impairment of binding of mutations with extremely high replicative fitness costs, because such mutations will only be found at very low frequencies in the patient population and may therefore be completely absent in the dataset underlying our analysis. Overall, it is plausible that even if CTL-mediated pressure is as strong as suggested previously [26], [70], [71], many other factors (in addition to escape from HLA class I) have influenced the evolution of HIV-1, resulting in a weak correlation between replicative fitness and change in binding to HLA molecules.

Can the implications of this analysis be extrapolated to the entire HIV-1 genome? Previous studies showed that the Pol gene, even though not as immunodominant as the Gag gene, can still play an important role in the interactions with CTL-mediated immunity [11], [72][74], and many optimal CTL epitopes have been identified in the genetic region examined here [48]. Therefore, it is conceivable that the effects observed here may be even stronger in the most immunodominant HIV-1 proteins, like p24 Gag. Related to this point is the fact that the gene analyzed here gives rise to many known drug resistance mutations. Even though our results were qualitatively identical when all such known mutations were excluded [75], we observed an interesting interplay between in vitro fitness and binding impairment for the subset of known drug resistance mutations. In particular, we found a significant negative correlation between fitness and binding impairment of those mutations to HLA-A molecules for one of the criteria (absolute criterion: , ; relative criterion: ), and a trend of a positive correlation between fitness and binding impairment of those mutations to HLA-B molecules (absolute criterion: , ; relative criterion: , ). This could point to an interesting interplay between the evolution of resistance and the evolution of escape in HIV-1, as emphasized previously [76]. However, the exact character of this relation remains unclear.

Assessing the fitness cost of immune escape mutations presents a crucial step towards a quantitative understanding of the dynamics of infectious diseases and their interactions with the immune system. To our knowledge, this study represents the first attempt to quantify the cost of mutations on a large scale and to compare it with the cost of mutations which do not affect the interaction with the immune system. The fact that a relation between fitness cost of mutations and their propensity to confer escape is not caused by a population effect in the analyzed data suggests that MHC class I molecules might have evolved to bind the genetic regions of RNA viruses which are costly to change.

Supporting Information

Figure S1.

Hydrophobicity vs. impairment of binding to HLA alleles. Change in hydrophobicity (hydrophobicity of the mutant amino acid minus hydrophobicity of the wild-type amino acid) was correlated with the maximal impairment of binding to both HLA-A and HLA-B alleles (see main text). A significant negative correlation was found for all three measures of hydrophobicity and for both binding criteria used (absolute criterion: , [measure 1], , [measure 2], , [measure 3]; relative criterion: , [measure 1], , [measure 2], , [measure 3]). The new consensus hydrophobicity scale was used as measure 1 (Tossi et al., 2002), the pH 7.4 hydrophobicity scale was used as measure 2, and the pH 2.1 hydrophobicity scale was used as measure 3 (Meek, 1980). Even though the results shown here are obtained for both HLA-A and HLA-B, the results for only HLA-A and only HLA-B were qualitatively identical. For the sake of visibility, the blue line shows the best fit of a linear regression with 95% confidence interval.

https://doi.org/10.1371/journal.pcbi.1002525.s001

(TIF)

Table S1.

The table lists all HLA alleles (A and B) used in the analysis. The first row lists the HLA alleles in the four-digit precision. The second row gives their expected frequency in the US population (see also Materials & Methods).

https://doi.org/10.1371/journal.pcbi.1002525.s002

(XLS)

Acknowledgments

We thank Rob de Boer, and Christian Althaus for helpful comments and discussions, Morten Nielsen for technical help with the epitope prediction software, and Katrina Lythgoe for helping us improve the manuscript at the final stage.

Author Contributions

Conceived and designed the experiments: RM RDK IH CK SB. Performed the experiments: RM RDK IH. Analyzed the data: RM RDK IH CK SB. Contributed reagents/materials/analysis tools: MH JMW CJP SB. Wrote the paper: RM RDK SB. Developed software used in the analysis: TH IH.

References

  1. 1. O'Connor D, Friedrich T, Hughes A, Allen TM, Watkins D (2001) Understanding cytotoxic T-lymphocyte escape during simian immunodeficiency virus infection. Immunol Rev 183: 115–126.D. O'ConnorT. FriedrichA. HughesTM AllenD. Watkins2001Understanding cytotoxic T-lymphocyte escape during simian immunodeficiency virus infection.Immunol Rev183115126
  2. 2. Wei X, Decker JM, Wang S, Hui H, Kappes JC, et al. (2003) Antibody neutralization and escape by HIV-1. Nature 422: 307–312.X. WeiJM DeckerS. WangH. HuiJC Kappes2003Antibody neutralization and escape by HIV-1.Nature422307312
  3. 3. Goulder PJ, Watkins DI (2004) HIV and SIV CTL escape: implications for vaccine design. Nat Rev Immunol 4: 630–640.PJ GoulderDI Watkins2004HIV and SIV CTL escape: implications for vaccine design.Nat Rev Immunol4630640
  4. 4. Bowen DG, Walker CM (2005) Mutational escape from CD8+ T cell immunity: HCV evolution, from chimpanzees to man. J Exp Med 201: 1709–1714.DG BowenCM Walker2005Mutational escape from CD8+ T cell immunity: HCV evolution, from chimpanzees to man.J Exp Med20117091714
  5. 5. Guglietta S, Garbuglia AR, Pacciani V, Scotta C, Perrone MP, et al. (2005) Positive selection of cytotoxic T lymphocyte escape variants during acute hepatitis C virus infection. Eur J Immunol 35: 2627–2637.S. GugliettaAR GarbugliaV. PaccianiC. ScottaMP Perrone2005Positive selection of cytotoxic T lymphocyte escape variants during acute hepatitis C virus infection.Eur J Immunol3526272637
  6. 6. Davenport MP, Loh L, Petravic J, Kent SJ (2008) Rates of HIV immune escape and reversion: implications for vaccination. Trends Microbiol 16: 561–566.MP DavenportL. LohJ. PetravicSJ Kent2008Rates of HIV immune escape and reversion: implications for vaccination.Trends Microbiol16561566
  7. 7. Thimme R, Neumann-Haefelin C, Boettler T, Blum HE (2008) Adaptive immune responses to hepatitis C virus: from viral immunobiology to a vaccine. Biol Chem 389: 457–467.R. ThimmeC. Neumann-HaefelinT. BoettlerHE Blum2008Adaptive immune responses to hepatitis C virus: from viral immunobiology to a vaccine.Biol Chem389457467
  8. 8. Liu Y, McNevin JP, Holte S, McElrath MJ, Mullins JI (2011) Dynamics of viral evolution and CTL responses in HIV-1 infection. PLoS ONE 6: e15639.Y. LiuJP McNevinS. HolteMJ McElrathJI Mullins2011Dynamics of viral evolution and CTL responses in HIV-1 infection.PLoS ONE6e15639
  9. 9. Draenert R, Verrill CL, Tang Y, Allen TM, Wurcel AG, et al. (2004) Persistent recognition of autologous virus by high-avidity CD8 T cells in chronic, progressive human immunodeficiency virus type 1 infection. J Virol 78: 630–641.R. DraenertCL VerrillY. TangTM AllenAG Wurcel2004Persistent recognition of autologous virus by high-avidity CD8 T cells in chronic, progressive human immunodeficiency virus type 1 infection.J Virol78630641
  10. 10. Frater AJ, Brown H, Oxenius A, Gunthard HF, Hirschel B, et al. (2007) Effective T-cell responses select human immunodeficiency virus mutants and slow disease progression. J Virol 81: 6742–6751.AJ FraterH. BrownA. OxeniusHF GunthardB. Hirschel2007Effective T-cell responses select human immunodeficiency virus mutants and slow disease progression.J Virol8167426751
  11. 11. Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, et al. (2007) CD8+ T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med 13: 46–53.P. KiepielaK. NgumbelaC. ThobakgaleD. RamduthI. Honeyborne2007CD8+ T-cell responses to different HIV proteins have discordant associations with viral load.Nat Med134653
  12. 12. Goulder PJ, Phillips RE, Colbert RA, McAdam S, Ogg G, et al. (1997) Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS. Nat Med 3: 212–217.PJ GoulderRE PhillipsRA ColbertS. McAdamG. Ogg1997Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS.Nat Med3212217
  13. 13. Oxenius A, Price DA, Trkola A, Edwards C, Gostick E, et al. (2004) Loss of viral control in early HIV-1 infection is temporally associated with sequential escape from CD8+ T cell responses and decrease in HIV-1-specific CD4+ and CD8+ T cell frequencies. J Infect Dis 190: 713–721.A. OxeniusDA PriceA. TrkolaC. EdwardsE. Gostick2004Loss of viral control in early HIV-1 infection is temporally associated with sequential escape from CD8+ T cell responses and decrease in HIV-1-specific CD4+ and CD8+ T cell frequencies.J Infect Dis190713721
  14. 14. Poon AF, Kosakovsky Pond SL, Bennett P, Richman DD, Leigh Brown AJ, et al. (2007) Adaptation to human populations is revealed by within-host polymorphisms in HIV-1 and hepatitis C virus. PLoS Pathog 3: e45.AF PoonSL Kosakovsky PondP. BennettDD RichmanAJ Leigh Brown2007Adaptation to human populations is revealed by within-host polymorphisms in HIV-1 and hepatitis C virus.PLoS Pathog3e45
  15. 15. Cobey S, Koelle K (2008) Capturing escape in infectious disease dynamics. Trends Ecol Evol (Amst) 23: 572–577.S. CobeyK. Koelle2008Capturing escape in infectious disease dynamics.Trends Ecol Evol (Amst)23572577
  16. 16. Goulder PJ, Watkins DI (2008) Impact of MHC class I diversity on immune control of immunodeficiency virus replication. Nat Rev Immunol 8: 619–630.PJ GoulderDI Watkins2008Impact of MHC class I diversity on immune control of immunodeficiency virus replication.Nat Rev Immunol8619630
  17. 17. Kawashima Y, Pfafferott K, Frater J, Matthews P, Payne R, et al. (2009) Adaptation of HIV-1 to human leukocyte antigen class I. Nature 458: 641–645.Y. KawashimaK. PfafferottJ. FraterP. MatthewsR. Payne2009Adaptation of HIV-1 to human leukocyte antigen class I.Nature458641645
  18. 18. Lipsitch M, O'Hagan JJ (2007) Patterns of antigenic diversity and the mechanisms that maintain them. J R Soc Interface 4: 787–802.M. LipsitchJJ O'Hagan2007Patterns of antigenic diversity and the mechanisms that maintain them.J R Soc Interface4787802
  19. 19. Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF, et al. (2004) Mapping the antigenic and genetic evolution of inuenza virus. Science 305: 371–376.DJ SmithAS LapedesJC de JongTM BestebroerGF Rimmelzwaan2004Mapping the antigenic and genetic evolution of inuenza virus.Science305371376
  20. 20. Smith DJ (2006) Predictability and preparedness in inuenza control. Science 312: 392–394.DJ Smith2006Predictability and preparedness in inuenza control.Science312392394
  21. 21. Russell CA, Jones TC, Barr IG, Cox NJ, Garten RJ, et al. (2008) Inuenza vaccine strain selection and recent studies on the global migration of seasonal inuenza viruses. Vaccine 26: Suppl 4D31–34.CA RussellTC JonesIG BarrNJ CoxRJ Garten2008Inuenza vaccine strain selection and recent studies on the global migration of seasonal inuenza viruses.Vaccine26Suppl 4D3134
  22. 22. Barouch DH, Kunstman J, Kuroda MJ, Schmitz JE, Santra S, et al. (2002) Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature 415: 335–339.DH BarouchJ. KunstmanMJ KurodaJE SchmitzS. Santra2002Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes.Nature415335339
  23. 23. Barouch DH (2008) Challenges in the development of an HIV-1 vaccine. Nature 455: 613–619.DH Barouch2008Challenges in the development of an HIV-1 vaccine.Nature455613619
  24. 24. Picker LJ, Hansen SG, Lifson JD (2012) New paradigms for hiv/aids vaccine development. Annu Rev Med 63: 1–17.LJ PickerSG HansenJD Lifson2012New paradigms for hiv/aids vaccine development.Annu Rev Med63117
  25. 25. Leslie AJ, Pfafferott KJ, Chetty P, Draenert R, Addo MM, et al. (2004) HIV evolution: CTL escape mutation and reversion after transmission. Nat Med 10: 282–289.AJ LeslieKJ PfafferottP. ChettyR. DraenertMM Addo2004HIV evolution: CTL escape mutation and reversion after transmission.Nat Med10282289
  26. 26. Fernandez CS, Stratov I, De Rose R, Walsh K, Dale CJ, et al. (2005) Rapid viral escape at an immunodominant simian-human immunode_ciency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost. J Virol 79: 5721–5731.CS FernandezI. StratovR. De RoseK. WalshCJ Dale2005Rapid viral escape at an immunodominant simian-human immunode_ciency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost.J Virol7957215731
  27. 27. Kearney M, Maldarelli F, Shao W, Margolick JB, Daar ES, et al. (2009) Human immunodeficiency virus type 1 population genetics and adaptation in newly infected individuals. J Virol 83: 2715–2727.M. KearneyF. MaldarelliW. ShaoJB MargolickES Daar2009Human immunodeficiency virus type 1 population genetics and adaptation in newly infected individuals.J Virol8327152727
  28. 28. Kelleher AD, Long C, Holmes EC, Allen RL, Wilson J, et al. (2001) Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses. J Exp Med 193: 375–386.AD KelleherC. LongEC HolmesRL AllenJ. Wilson2001Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses.J Exp Med193375386
  29. 29. Brockman MA, Schneidewind A, Lahaie M, Schmidt A, Miura T, et al. (2007) Escape and compensation from early HLA-B57-mediated cytotoxic T-lymphocyte pressure on human immunodeficiency virus type 1 Gag alter capsid interactions with cyclophilin A. J Virol 81: 12608–12618.MA BrockmanA. SchneidewindM. LahaieA. SchmidtT. Miura2007Escape and compensation from early HLA-B57-mediated cytotoxic T-lymphocyte pressure on human immunodeficiency virus type 1 Gag alter capsid interactions with cyclophilin A.J Virol811260812618
  30. 30. Crawford H, Prado JG, Leslie A, Hue S, Honeyborne I, et al. (2007) Compensatory mutation partially restores fitness and delays reversion of escape mutation within the immunodominant HLA-B*5703-restricted Gag epitope in chronic human immunodeficiency virus type 1 infection. J Virol 81: 8346–8351.H. CrawfordJG PradoA. LeslieS. HueI. Honeyborne2007Compensatory mutation partially restores fitness and delays reversion of escape mutation within the immunodominant HLA-B*5703-restricted Gag epitope in chronic human immunodeficiency virus type 1 infection.J Virol8183468351
  31. 31. Troyer RM, McNevin J, Liu Y, Zhang SC, Krizan RW, et al. (2009) Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response. PLoS Pathog 5: e1000365.RM TroyerJ. McNevinY. LiuSC ZhangRW Krizan2009Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response.PLoS Pathog5e1000365
  32. 32. Althaus CL, De Boer RJ (2008) Dynamics of immune escape during HIV/SIV infection. PLoS Comput Biol 4: e1000103.CL AlthausRJ De Boer2008Dynamics of immune escape during HIV/SIV infection.PLoS Comput Biol4e1000103
  33. 33. Fryer HR, Frater J, Duda A, Roberts MG, SPARTAC Trial Investigators (2010) Modelling the evolution and spread of HIV immune escape mutants. PLoS Pathog 6: e1001196.HR FryerJ. FraterA. DudaMG RobertsSPARTAC Trial Investigators2010Modelling the evolution and spread of HIV immune escape mutants.PLoS Pathog6e1001196
  34. 34. Mostowy R, Kouyos RD, Fouchet D, Bonhoeffer S (2011) The role of recombination for the coevolutionary dynamics of HIV and the immune response. PLoS ONE 6: e16052.R. MostowyRD KouyosD. FouchetS. Bonhoeffer2011The role of recombination for the coevolutionary dynamics of HIV and the immune response.PLoS ONE6e16052
  35. 35. Hinkley T, Martins J, Chappey C, Haddad M, Stawiski E, et al. (2011) A systems analysis of mutational effects in HIV-1 protease and reverse transcriptase. Nat Genet 43: 487–489.T. HinkleyJ. MartinsC. ChappeyM. HaddadE. Stawiski2011A systems analysis of mutational effects in HIV-1 protease and reverse transcriptase.Nat Genet43487489
  36. 36. Nielsen M, Lundegaard C, Blicher T, Lamberth K, Harndahl M, et al. (2007) NetMHCpan, a method for quantitative predictions of peptide binding to any HLA-A and -B locus protein of known sequence. PLoS ONE 2: e796.M. NielsenC. LundegaardT. BlicherK. LamberthM. Harndahl2007NetMHCpan, a method for quantitative predictions of peptide binding to any HLA-A and -B locus protein of known sequence.PLoS ONE2e796
  37. 37. Hoof I, Peters B, Sidney J, Pedersen LE, Sette A, et al. (2009) NetMHCpan, a method for MHC class I binding prediction beyond humans. Immunogenetics 61: 1–13.I. HoofB. PetersJ. SidneyLE PedersenA. Sette2009NetMHCpan, a method for MHC class I binding prediction beyond humans.Immunogenetics61113
  38. 38. Petropoulos CJ, Parkin NT, Limoli KL, Lie YS, Wrin T, et al. (2000) A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrob Agents Chemother 44: 920–928.CJ PetropoulosNT ParkinKL LimoliYS LieT. Wrin2000A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1.Antimicrob Agents Chemother44920928
  39. 39. Zhang H, Lundegaard C, Nielsen M (2009) Pan-specific MHC class I predictors: a benchmark of HLA class I pan-specific prediction methods. Bioinformatics 25: 83–89.H. ZhangC. LundegaardM. Nielsen2009Pan-specific MHC class I predictors: a benchmark of HLA class I pan-specific prediction methods.Bioinformatics258389
  40. 40. Ruppert J, Sidney J, Celis E, Kubo RT, Grey HM, et al. (1993) Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 74: 929–937.J. RuppertJ. SidneyE. CelisRT KuboHM Grey1993Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules.Cell74929937
  41. 41. Rapin N, Hoof I, Lund O, Nielsen M (2008) MHC motif viewer. Immunogenetics 60: 759–765.N. RapinI. HoofO. LundM. Nielsen2008MHC motif viewer.Immunogenetics60759765
  42. 42. MacNamara A, Kadolsky U, Bangham CR, Asquith B (2009) T-cell epitope prediction: rescaling can mask biological variation between MHC molecules. PLoS Comput Biol 5: e1000327.A. MacNamaraU. KadolskyCR BanghamB. Asquith2009T-cell epitope prediction: rescaling can mask biological variation between MHC molecules.PLoS Comput Biol5e1000327
  43. 43. Rao X, Costa AI, van Baarle D, Keşmir C (2009) A comparative study of HLA binding affinity and ligand diversity: implications for generating immunodominant CD8+ T cell responses. J Immunol 182: 1526–1532.X. RaoAI CostaD. van BaarleC. Keşmir2009A comparative study of HLA binding affinity and ligand diversity: implications for generating immunodominant CD8+ T cell responses.J Immunol18215261532
  44. 44. Maiers M, Gragert L, Klitz W (2007) High-resolution HLA alleles and haplotypes in the United States population. Hum Immunol 68: 779–788.M. MaiersL. GragertW. Klitz2007High-resolution HLA alleles and haplotypes in the United States population.Hum Immunol68779788
  45. 45. CDC (2008) HIV prevalence estimates–United States, 2006. MMWR Morb Mortal Wkly Rep 57: 1073–1076.CDC2008HIV prevalence estimates–United States, 2006.MMWR Morb Mortal Wkly Rep5710731076
  46. 46. O'Brien SJ, Gao X, Carrington M (2001) HLA and AIDS: a cautionary tale. Trends Mol Med 7: 379–381.SJ O'BrienX. GaoM. Carrington2001HLA and AIDS: a cautionary tale.Trends Mol Med7379381
  47. 47. R Development Core Team (2008) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. R Development Core Team2008R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.URL http://www.R-project.org. ISBN 3-900051-07-0. URL http://www.R-project.org. ISBN 3-900051-07-0.
  48. 48. Yusim K, Korber BTM, Brander C, Haynes BF, Koup R, et al. (2009) HIV Molecular Immunology 2009. Los Alamos, NM: Los Alamos National Laboratory, Theoretical Biology and Biophysics. pp. 3–24.K. YusimBTM KorberC. BranderBF HaynesR. Koup2009HIV Molecular Immunology 2009Los Alamos, NMLos Alamos National Laboratory, Theoretical Biology and Biophysics324
  49. 49. Gao X, Nelson GW, Karacki P, Martin MP, Phair J, et al. (2001) Effect of a single amino acid change in MHC class I molecules on the rate of progression to AIDS. N Engl J Med 344: 1668–1675.X. GaoGW NelsonP. KarackiMP MartinJ. Phair2001Effect of a single amino acid change in MHC class I molecules on the rate of progression to AIDS.N Engl J Med34416681675
  50. 50. Hughes AL, Hughes MK (1995) Self peptides bound by HLA class I molecules are derived from highly conserved regions of a set of evolutionarily conserved proteins. Immunogenetics 41: 257–262.AL HughesMK Hughes1995Self peptides bound by HLA class I molecules are derived from highly conserved regions of a set of evolutionarily conserved proteins.Immunogenetics41257262
  51. 51. Lucchiari-Hartz M, Lindo V, Hitziger N, Gaedicke S, Saveanu L, et al. (2003) Differential proteasomal processing of hydrophobic and hydrophilic protein regions: contribution to cytotoxic T lymphocyte epitope clustering in HIV-1-Nef. Proc Natl Acad Sci U S A 100: 7755–7760.M. Lucchiari-HartzV. LindoN. HitzigerS. GaedickeL. Saveanu2003Differential proteasomal processing of hydrophobic and hydrophilic protein regions: contribution to cytotoxic T lymphocyte epitope clustering in HIV-1-Nef.Proc Natl Acad Sci U S A10077557760
  52. 52. Henikoff S, Henikoff JG (1992) Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A 89: 10915–10919.S. HenikoffJG Henikoff1992Amino acid substitution matrices from protein blocks.Proc Natl Acad Sci U S A891091510919
  53. 53. Calis JJ, Sanchez-Perez GF, Keşmir C (2010) MHC class I molecules exploit the low G+C content of pathogen genomes for enhanced presentation. Eur J Immunol 40: 2699–2709.JJ CalisGF Sanchez-PerezC. Keşmir2010MHC class I molecules exploit the low G+C content of pathogen genomes for enhanced presentation.Eur J Immunol4026992709
  54. 54. Meek JL (1980) Prediction of peptide retention times in high-pressure liquid chromatography on the basis of amino acid composition. Proc Natl Acad Sci U S A 77: 1632–1636.JL Meek1980Prediction of peptide retention times in high-pressure liquid chromatography on the basis of amino acid composition.Proc Natl Acad Sci U S A7716321636
  55. 55. Tossi A, Sandri L, Giangaspero A (2002) New consensus hydrophobicity scale extended to non-proteinogenic amino acids. Peptides 416–417.A. TossiL. SandriA. Giangaspero2002New consensus hydrophobicity scale extended to non-proteinogenic amino acids.Peptides416417
  56. 56. Carrington M, O'Brien SJ (2003) The inuence of HLA genotype on AIDS. Annu Rev Med 54: 535–551.M. CarringtonSJ O'Brien2003The inuence of HLA genotype on AIDS.Annu Rev Med54535551
  57. 57. Kiepiela P, Leslie AJ, Honeyborne I, Ramduth D, Thobakgale C, et al. (2004) Dominant inuence of HLA-B in mediating the potential co-evolution of HIV and HLA. Nature 432: 769–775.P. KiepielaAJ LeslieI. HoneyborneD. RamduthC. Thobakgale2004Dominant inuence of HLA-B in mediating the potential co-evolution of HIV and HLA.Nature432769775
  58. 58. Bihl F, Frahm N, Di Giammarino L, Sidney J, John M, et al. (2006) Impact of HLA-B alleles, epitope binding affinity, functional avidity, and viral coinfection on the immunodominance of virus-specific CTL responses. J Immunol 176: 4094–4101.F. BihlN. FrahmL. Di GiammarinoJ. SidneyM. John2006Impact of HLA-B alleles, epitope binding affinity, functional avidity, and viral coinfection on the immunodominance of virus-specific CTL responses.J Immunol17640944101
  59. 59. Matthews PC, Prendergast A, Leslie A, Crawford H, Payne R, et al. (2008) Central role of reverting mutations in HLA associations with human immunodeficiency virus set point. J Virol 82: 8548–8559.PC MatthewsA. PrendergastA. LeslieH. CrawfordR. Payne2008Central role of reverting mutations in HLA associations with human immunodeficiency virus set point.J Virol8285488559
  60. 60. Fontaine Costa AI, Rao X, Lechenadec E, van Baarle D, Keşmir C (2010) HLA-B molecules target more conserved regions of the HIV-1 proteome. AIDS 24: 211–215.AI Fontaine CostaX. RaoE. LechenadecD. van BaarleC. Keşmir2010HLA-B molecules target more conserved regions of the HIV-1 proteome.AIDS24211215
  61. 61. Hertz T, Nolan D, James I, John M, Gaudieri S, et al. (2011) Mapping the landscape of host-pathogen coevolution: HLA class I binding and its relationship with evolutionary conservation in human and viral proteins. J Virol 85: 1310–1321.T. HertzD. NolanI. JamesM. JohnS. Gaudieri2011Mapping the landscape of host-pathogen coevolution: HLA class I binding and its relationship with evolutionary conservation in human and viral proteins.J Virol8513101321
  62. 62. Iversen AK, Stewart-Jones G, Learn GH, Christie N, Sylvester-Hviid C, et al. (2006) Conicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope. Nat Immunol 7: 179–189.AK IversenG. Stewart-JonesGH LearnN. ChristieC. Sylvester-Hviid2006Conicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope.Nat Immunol7179189
  63. 63. Pereyra F, Jia X, McLaren PJ, Telenti A, de Bakker PI, et al. (2010) The major genetic determinants of HIV-1 control affect HLA class I peptide presentation. Science 330: 1551–1557.F. PereyraX. JiaPJ McLarenA. TelentiPI de Bakker2010The major genetic determinants of HIV-1 control affect HLA class I peptide presentation.Science33015511557
  64. 64. Fitzmaurice K, Petrovic D, Ramamurthy N, Simmons R, Merani S, et al. (2011) Molecular footprints reveal the impact of the protective HLA-A*03 allele in hepatitis C virus infection. Gut 60: 1563–1571.K. FitzmauriceD. PetrovicN. RamamurthyR. SimmonsS. Merani2011Molecular footprints reveal the impact of the protective HLA-A*03 allele in hepatitis C virus infection.Gut6015631571
  65. 65. Matthews PC, Adland E, Listgarten J, Leslie A, Mkhwanazi N, et al. (2011) HLA-A*7401-mediated control of HIV viremia is independent of its linkage disequilibrium with HLA-B*5703. J Immunol 186: 5675–5686.PC MatthewsE. AdlandJ. ListgartenA. LeslieN. Mkhwanazi2011HLA-A*7401-mediated control of HIV viremia is independent of its linkage disequilibrium with HLA-B*5703.J Immunol18656755686
  66. 66. Kouyos RD, von Wyl V, Hinkley T, Petropoulos CJ, Haddad M, et al. (2011) Assessing predicted HIV-1 replicative capacity in a clinical setting. PLoS Pathog 7: e1002321.RD KouyosV. von WylT. HinkleyCJ PetropoulosM. Haddad2011Assessing predicted HIV-1 replicative capacity in a clinical setting.PLoS Pathog7e1002321
  67. 67. Moore CB, John M, James IR, Christiansen FT, Witt CS, et al. (2002) Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level. Science 296: 1439–1443.CB MooreM. JohnIR JamesFT ChristiansenCS Witt2002Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level.Science29614391443
  68. 68. Schellens IM, Navis M, van Deutekom HW, Boeser-Nunnink B, Berkhout B, et al. (2011) Loss of HIV-1-derived cytotoxic T lymphocyte epitopes restricted by protective HLA-B alleles during the HIV-1 epidemic. AIDS 25: 1691–1700.IM SchellensM. NavisHW van DeutekomB. Boeser-NunninkB. Berkhout2011Loss of HIV-1-derived cytotoxic T lymphocyte epitopes restricted by protective HLA-B alleles during the HIV-1 epidemic.AIDS2516911700
  69. 69. Yewdell JW, Bennink JR (1999) Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol 17: 51–88.JW YewdellJR Bennink1999Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses.Annu Rev Immunol175188
  70. 70. Asquith B, Edwards CT, Lipsitch M, McLean AR (2006) Inefficient cytotoxic T lymphocyte-mediated killing of HIV-1-infected cells in vivo. PLoS Biol 4: e90.B. AsquithCT EdwardsM. LipsitchAR McLean2006Inefficient cytotoxic T lymphocyte-mediated killing of HIV-1-infected cells in vivo.PLoS Biol4e90
  71. 71. Asquith B, McLean AR (2007) In vivo CD8+ T cell control of immunodeficiency virus infection in humans and macaques. Proc Natl Acad Sci U S A 104: 6365–6370.B. AsquithAR McLean2007In vivo CD8+ T cell control of immunodeficiency virus infection in humans and macaques.Proc Natl Acad Sci U S A10463656370
  72. 72. Addo MM, Yu XG, Rathod A, Cohen D, Eldridge RL, et al. (2003) Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J Virol 77: 2081–2092.MM AddoXG YuA. RathodD. CohenRL Eldridge2003Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load.J Virol7720812092
  73. 73. Borghans JA, Mølgaard A, de Boer RJ, Keşmir C (2007) HLA alleles associated with slow progression to AIDS truly prefer to present HIV-1 p24. PLoS ONE 2: e920.JA BorghansA. MølgaardRJ de BoerC. Keşmir2007HLA alleles associated with slow progression to AIDS truly prefer to present HIV-1 p24.PLoS ONE2e920
  74. 74. Vider-Shalit T, Almani M, Sarid R, Louzoun Y (2009) The HIV hide and seek game: an immunogenomic analysis of the HIV epitope repertoire. AIDS 23: 1311–1318.T. Vider-ShalitM. AlmaniR. SaridY. Louzoun2009The HIV hide and seek game: an immunogenomic analysis of the HIV epitope repertoire.AIDS2313111318
  75. 75. Johnson VA, Calvez V, Gunthard HF, Paredes R, Pillay D, et al. (2011) 2011 update of the drug resistance mutations in HIV-1. Top Antivir Med 19: 156–164.VA JohnsonV. CalvezHF GunthardR. ParedesD. Pillay20112011 update of the drug resistance mutations in HIV-1.Top Antivir Med19156164
  76. 76. Mueller SM, Schaetz B, Eismann K, Bergmann S, Bauerle M, et al. (2007) Dual selection pressure by drugs and HLA class I-restricted immune responses on human immunodeficiency virus type 1 protease. J Virol 81: 2887–2898.SM MuellerB. SchaetzK. EismannS. BergmannM. Bauerle2007Dual selection pressure by drugs and HLA class I-restricted immune responses on human immunodeficiency virus type 1 protease.J Virol8128872898