Evolutionary Escape Research Articles

QuickFit: A High-Throughput RT-qPCR-Based Assay to Quantify Viral Growth and Fitness In Vitro.

Quantifying viral growth rates is key to understanding evolutionary dynamics and the potential for mutants to escape antiviral drugs. Defining evolutionary escape paths and their impact on viral fitness allows for the development of drugs that are resistant to escape. In the case of HIV, combination antiretroviral therapy can successfully prevent or treat infection, but it relies on strict adherence to prevent escape. Here, we present a method termed QuickFit that enables the quantification of viral fitness by employing large numbers of parallel viral cultures to measure growth rates accurately. QuickFit consistently recapitulated HIV growth measurements obtained by traditional approaches, but with significantly higher throughput and lower rates of error. This method represents a promising tool for rapid and consistent evaluation of viral fitness.

When perfection isn't enough: host egg signatures are an effective defence against high-fidelity African cuckoo mimicry.

Most mimicry systems involve imperfect mimicry, whereas perfect and high-fidelity mimicry are rare. When the fidelity of mimicry is high, mimics might be expected to have the upper hand against their antagonists. However, in coevolving systems, diversification of model phenotypes may provide an evolutionary escape, because mimics cannot simultaneously match all model individuals in the population. Here we investigate high-fidelity mimicry in a highly specialized, Afrotropical brood parasite-host system: the African cuckoo and fork-tailed drongo. Specifically, we test whether host egg polymorphisms are an effective defence against such mimicry. We show, using a combination of image analysis, field experiments and simulations, that: (1) egg colour and pattern mimicry of fork-tailed drongo eggs by African cuckoos is near-perfect on average; (2) drongos show fine-tuned rejection of foreign eggs, exploiting unpredictable pattern differences between parasitic eggs and their own; and (3) the high degree of interclutch variation (polymorphic egg 'signatures') exhibited by drongos gives them the upper hand in the arms race, with 93.7% of cuckoo eggs predicted to be rejected, despite cuckoos mimicking the full range of drongo egg phenotypes. These results demonstrate that model diversification is a highly effective defence against mimics, even when mimicry is highly accurate.

A Structurally Characterized Staphylococcus aureus Evolutionary Escape Route from Treatment with the Antibiotic Linezolid.

ABSTRACTMethicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment. Clonal isolates which had evolved a linezolid-resistant phenotype were characterized by whole-genome sequencing. Linezolid-resistant mutants were identified which had accumulated mutations in the ribosomal protein uL3. Multiple clones which had two mutations in uL3 exhibited resistance to linezolid, 2-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single-particle cryo-electron microscopic analysis and compared to the ribosomes from the parent strain. We found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix 90, propagating a structural change over 15 Å away. This distal change swings nucleotide U2504 into the binding site of the antibiotic, causing linezolid resistance.IMPORTANCE Antibiotic resistance poses a critical problem to human health and decreases the utility of these lifesaving drugs. Of particular concern is the “superbug” methicillin-resistant Staphylococcus aureus (MRSA), for which treatment of infection requires the use of last-line antibiotics, including linezolid. In this paper, we characterize the atomic rearrangements which the ribosome, the target of linezolid, undergoes during its evolutionary journey toward becoming drug resistant. Using cryo-electron microscopy, we describe a particular molecular mechanism which MRSA uses to become resistant to linezolid.

Solving multi-objective pickup and delivery with time windows using mediocre evolutionary distributed microservices re-optimization algorithm

The pickup and delivery problem with time windows (PDPTW) is an important problem to be solved. This is due to its resemblance to real-life services such as food delivery, courier service, curbside pickup service, and dial-a-ride, among others. However, the research on multi-objective pickup and delivery problems with time windows (MOPDPTW) remains scarce even after many years. The MOPDPTW objective is to achieve the optimal solution on both the number of vehicle and total travelled distance. It is challenging to obtain optimal results, the recurring optimal results that has least difference in magnitude and broader Pareto sets at the same time. Hence, an algorithm that can fulfil these requirements are highly sought after. This paper presents a mediocre evolutionary distributed microservices re-optimization algorithm (MEDMRA) to solve the MOPDPTW. The combination of the re-optimize algorithm and mediocre evolutionary algorithm escape the local optimal, optimized results and produce broader Pareto value. We compare our results using Li & Lim instances. Our results outperform the latest published hybrid algorithms and competitive with the best-known solutions.

Development of antibacterial compounds that constrain evolutionary pathways to resistance.

Antibiotic resistance is a worldwide challenge. A potential approach to block resistance is to simultaneously inhibit WT and known escape variants of the target bacterial protein. Here, we applied an integrated computational and experimental approach to discover compounds that inhibit both WT and trimethoprim (TMP) resistant mutants of E. coli dihydrofolate reductase (DHFR). We identified a novel compound (CD15-3) that inhibits WT DHFR and its TMP resistant variants L28R, P21L and A26T with IC50 50-75 µM against WT and TMP-resistant strains. Resistance to CD15-3 was dramatically delayed compared to TMP in in vitro evolution. Whole genome sequencing of CD15-3-resistant strains showed no mutations in the target folA locus. Rather, gene duplication of several efflux pumps gave rise to weak (about twofold increase in IC50) resistance against CD15-3. Altogether, our results demonstrate the promise of strategy to develop evolution drugs - compounds which constrain evolutionary escape routes in pathogens.

Risk of rapid evolutionary escape from biomedical interventions targeting SARS-CoV-2 spike protein.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is the molecular target for many vaccines and antibody-based prophylactics aimed at bringing COVID-19 under control. Such a narrow molecular focus raises the specter of viral immune evasion as a potential failure mode for these biomedical interventions. With the emergence of new strains of SARS-CoV-2 with altered transmissibility and immune evasion potential, a critical question is this: how easily can the virus escape neutralizing antibodies (nAbs) targeting the spike RBD? To answer this question, we combined an analysis of the RBD structure-function with an evolutionary modeling framework. Our structure-function analysis revealed that epitopes for RBD-targeting nAbs overlap one another substantially and can be evaded by escape mutants with ACE2 affinities comparable to the wild type, that are observed in sequence surveillance data and infect cells in vitro. This suggests that the fitness cost of nAb-evading mutations is low. We then used evolutionary modeling to predict the frequency of immune escape before and after the widespread presence of nAbs due to vaccines, passive immunization or natural immunity. Our modeling suggests that SARS-CoV-2 mutants with one or two mildly deleterious mutations are expected to exist in high numbers due to neutral genetic variation, and consequently resistance to vaccines or other prophylactics that rely on one or two antibodies for protection can develop quickly -and repeatedly- under positive selection. Predicted resistance timelines are comparable to those of the decay kinetics of nAbs raised against vaccinal or natural antigens, raising a second potential mechanism for loss of immunity in the population. Strategies for viral elimination should therefore be diversified across molecular targets and therapeutic modalities.

Co-dominant neutralizing epitopes make anti-measles immunity resistant to viral evolution

Munoz-Alia and colleagues1 demonstrate that neutralizing antibody immunity to measles resists viral evolutionary escape because it targets numerous distinct viral epitopes. Their work contributes to our understanding of what determines whether a virus can evolve to evade immunity.

Positive feedback in coordination games: Stochastic evolutionary dynamics and the logit choice rule

We study the problem of stochastic stability for evolutionary dynamics under the logit choice rule. We consider general classes of coordination games, symmetric or asymmetric, with an arbitrary number of strategies, which satisfies the marginal bandwagon property (i.e., there is positive feedback to coordinate). Our main result is that the most likely evolutionary escape paths from a status quo convention consist of a series of identical mistakes. As an application of our result, we show that the Nash bargaining solution arises as the long run convention for the evolutionary Nash demand game under the usual logit choice rule. We also obtain a new bargaining solution if the logit choice rule is combined with intentional idiosyncratic plays. The new bargaining solution is more egalitarian than the Nash bargaining solution, demonstrating that intentionality implies equality under the logit choice model.

The evolution of coexistence from competition in experimental co-cultures of Escherichia coli and Saccharomyces cerevisiae.

Microbial communities are comprised of many species that coexist on small spatial scales. This is difficult to explain because many interspecies interactions are competitive, and ecological theory predicts that one species will drive the extinction of another species that competes for the same resource. Conversely, evolutionary theory proposes that natural selection can lead to coexistence by driving competing species to use non-overlapping resources. However, evolutionary escape from extinction may be slow compared to the rate of competitive exclusion. Here, we use experimental co-cultures of Escherichia coli and Saccharomyces cerevisiae to study the evolution of coexistence in species that compete for resources. We find that while E. coli usually outcompetes S. cerevisiae in co-culture, a few populations evolved stable coexistence after ~1000 generations of coevolution. We sequenced S. cerevisiae and E. coli populations, identified multi-hit genes, and engineered alleles from these genes into several genetic backgrounds, finding that some mutations modified interactions between E. coli and S. cerevisiae. Together, our data demonstrate that coexistence can evolve, de novo, from intense competition between two species with no history of coevolution.

Contrasting Effects of Climate Change on Alpine Chamois

ABSTRACTGlobal climate change can affect animal ecology in numerous ways, but researchers usually emphasize undesirable consequences. Temperature increases, for instance, can induce direct physiological costs and indirect effects via mismatches in resource needs and availability. Species living in mountainous regions, however, could experience beneficial effects because winters might become less severe. We examined the potentially opposing effects of climate change during spring, summer, and winter on recruitment in Alpine chamois (Rupicapra rupicapra). We examined initial recruitment (i.e., the ratio of kids to adult females) and net recruitment (i.e., the ratio of yearlings to adult females) of Alpine chamois through the use of linear mixed effects models and data from block count censuses performed across a 1,500‐km2 study area in the Italian Alps during summer from 2001 to 2015. Initial recruitment was relatively resistant to the effects of climate change, declining slightly over the study period. We suggest that the effects of increased forage availability and lower snow cover in winter may benefit the reproductive output of adult females, compensating for any negative effects of trophic mismatch and higher temperatures during summer. By contrast, net recruitment strongly declined throughout the study period, consistent with the slight decline of initial recruitment and the negative effects of increasing summer temperatures on the survival of kids during their first winter. These negative effects seemed to outweigh positive effects of climate change, even in a species strongly challenged by winter conditions. These findings provide important information for hunted populations; setting more appropriate hunting bags for yearling chamois should be considered. The ecological plasticity of the chamois, which also inhabits low altitudes, may allow a possible evolutionary escape for the species. © 2020 The Wildlife Society.

Selective Maintenance of Multiple CRISPR Arrays Across Prokaryotes.

Prokaryotes are under nearly constant attack by viral pathogens. To protect against this threat of infection, bacteria and archaea have evolved a wide array of defense mechanisms, singly and in combination. While immune diversity in a single organism likely reduces the chance of pathogen evolutionary escape, it remains puzzling why many prokaryotes also have multiple, seemingly redundant, copies of the same type of immune system. Here, we focus on the highly flexible CRISPR adaptive immune system, which is present in multiple copies in a surprising 28% of the prokaryotic genomes in RefSeq. We use a comparative genomics approach looking across all prokaryotes to demonstrate that on average, organisms are under selection to maintain more than one CRISPR array. Given this surprising conclusion, we consider several hypotheses concerning the source of selection and include a theoretical analysis of the possibility that a trade-off between memory span and learning speed could select for both "long-term memory" and "short-term memory" CRISPR arrays.

Trojan Females and Judas Goats: Evolutionary Traps as Tools in Wildlife Management

Evolutionary traps occur when rapid environmental change causes animals to prefer poor-quality resources (e.g., habitats) or behaviors over higher-quality ones that lead to greater survival or reproductive success. Here, we bring together science from the pest-control, eco-evolutionary, and conservation communities to outline how evolutionary traps can be repurposed to eliminate or control pest species. We highlight case studies and devise strategies for the selection of appropriate cues to manipulate, traits to target, and mechanisms to use in setting evolutionary traps that will most rapidly reduce animal abundance while preventing evolutionary escape. Approaches to setting evolutionary traps have been diverse but uneven with respect to principles that would make them more effective, such as strengthening resource preference, triggering out-of-context behaviors, and targeting underexploited sensory modalities. We find that evolutionary traps are demonstrably effective and unique tools with high target-species specificity that are deployable in concert with more traditional approaches.

Second-Order Free-Riding on Antisocial Punishment Restores the Effectiveness of Prosocial Punishment

Economic experiments have shown that punishment can increase public goods game contributions over time. However, the effectiveness of punishment is challenged by second-order freeriding and antisocial punishment. The latter implies that non-cooperators punish cooperators, while the former implies unwillingness to shoulder the cost of punishment. Here we extend the theory of cooperation in the spatial public goods game by considering four competing strategies, which are traditional cooperators and defectors, as well as cooperators who punish defectors and defectors who punish cooperators. We show that if the synergistic effects are high enough to sustain cooperation based on network reciprocity alone, antisocial punishment does not deter public cooperation. Conversely, if synergistic effects are low and punishment is actively needed to sustain cooperation, antisocial punishment does act detrimental, but only if the cost-to-fine ratio is low. If the costs are relatively high, cooperation again dominates as a result of spatial pattern formation. Counterintuitively, defectors who do not punish cooperators, and are thus effectively second-order freeriding on antisocial punishment, form an active layer around punishing cooperators, which protects them against defectors that punish cooperators. A stable three-strategy phase that is sustained by the spontaneous emergence of cyclic dominance is also possible via the same route. The microscopic mechanism behind the reported evolutionary outcomes can be explained by the comparison of invasion rates that determine the stability of subsystem solutions. Our results reveal an unlikely evolutionary escape from adverse effects of antisocial punishment, and they provide a rationale for why second-order freeriding is not always an impediment to the evolutionary stability of punishment.

Fate-Regulating Circuits in Viruses: From Discovery to New Therapy Targets.

Current antivirals effectively target diverse viruses at various stages of their life cycles. Nevertheless, curative therapy has remained elusive for important pathogens, such as human immunodeficiency virus type 1 (HIV-1) and herpesviruses, in large part due to viral latency and the evolution of resistance to existing therapies. Here, we review the discovery of viral master circuits: virus-encoded autoregulatory gene networks that autonomously control viral expression programs (i.e., between active, latent, and abortive fates). These circuits offer the opportunity for a new class of antivirals that could lead to intrinsic combination-antiviral therapies within a single molecule-evolutionary escape from such circuit-disrupting antivirals would require simultaneous evolution of both the viral cis regulatory element (e.g., the DNA-binding site) and the trans element (e.g., the transcription factor) in order for the virus to recapitulate a circuit that would not be disrupted. We review the architectures of these fate-regulating master circuits in HIV-1 and the human herpesvirus cytomegalovirus along with potential circuit-disruption strategies that may ultimately enable escape-resistant antiviral therapies.

Modeling cancer evolution: evolutionary escape under immune system control

It can be expected that adequate immune response should be able to annihilate cancer at a very early stage of its appearance. However, in some cases cancer is able to persist avoiding immune response. One can conject that cancer is able to avoid immune response control due to a succession of mutations leading to the development of immune-resistant cells. In order to illustrate this possibility, in this paper we present a 2n–dimensional mathematical model that describes interaction of n subtypes of tumor cells with corresponding genotype-specific immune response. The model postulates that there is a probability for tumor cells of each of n subtype to produce offsprings of other types. Each of the subtypes activates the genotype-specific immune response with a possibility of suppressing cancer cells of other genotypes (the cross-immunity). Numerical simulations show that if cancer cells are able to mutate comparatively fast and if immune response is not strong enough, then, despite immune system pressure, cancer is able to persist.

Venom Insulins of Cone Snails Diversify Rapidly and Track Prey Taxa.

A specialized insulin was recently found in the venom of a fish-hunting cone snail, Conus geographus. Here we show that many worm-hunting and snail-hunting cones also express venom insulins, and that this novel gene family has diversified explosively. Cone snails express a highly conserved insulin in their nerve ring; presumably this conventional signaling insulin is finely tuned to the Conus insulin receptor, which also evolves very slowly. By contrast, the venom insulins diverge rapidly, apparently in response to biotic interactions with prey and also possibly the cones’ own predators and competitors. Thus, the inwardly directed signaling insulins appear to experience predominantly purifying sele\\ction to target an internal receptor that seldom changes, while the outwardly directed venom insulins frequently experience directional selection to target heterospecific insulin receptors in a changing mix of prey, predators and competitors. Prey insulin receptors may often be constrained in ways that prevent their evolutionary escape from targeted venom insulins, if amino-acid substitutions that result in escape also degrade the receptor’s signaling functions.

Initiating a watch list for Ebola virus antibody escape mutations.

The 2014 Ebola virus (EBOV) outbreak in West Africa is the largest in recorded history and resulted in over 11,000 deaths. It is essential that strategies for treatment and containment be developed to avoid future epidemics of this magnitude. With the development of vaccines and antibody-based therapies using the envelope glycoprotein (GP) of the 1976 Mayinga strain, one important strategy is to anticipate how the evolution of EBOV might compromise these efforts. In this study we have initiated a watch list of potential antibody escape mutations of EBOV by modeling interactions between GP and the antibody KZ52. The watch list was generated using molecular modeling to estimate stability changes due to mutation. Every possible mutation of GP was considered and the list was generated from those that are predicted to disrupt GP-KZ52 binding but not to disrupt the ability of GP to fold and to form trimers. The resulting watch list contains 34 mutations (one of which has already been seen in humans) at six sites in the GP2 subunit. Should mutations from the watch list appear and spread during an epidemic, it warrants attention as these mutations may reflect an evolutionary response from the virus that could reduce the effectiveness of interventions such as vaccination. However, this watch list is incomplete and emphasizes the need for more experimental structures of EBOV interacting with antibodies in order to expand the watch list to other epitopes. We hope that this work provokes experimental research on evolutionary escape in both Ebola and other viral pathogens.

Evolutionary escape on complex genotype–phenotype networks

We study the problem of evolutionary escape that is the process whereby a population under sudden changes in the selective pressures acting upon it try to evade extinction by evolving from previously well-adapted phenotypes to those that are favoured by the new selective pressure. We perform a comparative analysis between results obtained by modelling genotype space as a regular hypercube (H-graphs), which is the scenario considered in previous work on the subject, to those corresponding to a complex genotype–phenotype network (B-graphs). In order to analyse the properties of the escape process on both these graphs, we apply a general theory based on multi-type branching processes to compute the evolutionary dynamics and probability of escape. We show that the distribution of distances between phenotypes in B-graphs exhibits a much larger degree of heterogeneity than in H-graphs. This property, one of the main structural differences between both types of graphs, causes heterogeneous behaviour in all results associated to the escape problem. We further show that, due to the heterogeneity characterising escape on B-graphs, escape probability can be underestimated by assuming a regular hypercube genotype network, even if we compare phenotypes at the same distance in H-graphs. Similarly, it appears that the complex structure of B-graphs slows down the rate of escape.

Surviving evolutionary escape on complex genotype–phenotype networks

We study the problem of evolutionary escape and survival of cell populations with a genotype-phenotype structure. We refer to evolutionary escape as the process where a cell of a given ill-adapted population to reach a well-adapted phenotype. Similarly, survival refers to the dynamics of the population once the escape phenotype has been reached. The aim of this paper is to analyse the influence of topological properties associated to robustness and evolvability on the probability of escape and on the probability of survival. In order to explore these issues, we formulate a population dynamics model, consisting of a multi-type time-continuous branching process, where types are associated to genotypes and their birth and death probabilities depend on the associated phenotype (non-escape or escape). We exploit the separation of time scales introduced by the the difference in reproductive ratios between the ill-adapted phenotypes and the escape phenotype. Two dynamical regimes emerge: a fast-decaying regime associated to the escape process itself, and a slow regime which corresponds to the survival dynamics of the population once the escape phenotype has been reached. We exploit this separation of time scales to analyse the topological factors which determine escape and survival probabilities. We show that, while the escape probability depends on the degree of escape phenotype, the probability of survival is essentially determined by its robustness, measured in terms of a weighted clustering coefficient.

141. Combinatorial Aptamer Transcripts (CATs) for HIV RT Suppression

The Human Immunodeficiency Virus (HIV) Reverse Transcriptase (RT) is a DNA polymerase encoded by the viral genome and it is preferentially targeted by current therapeutics due to its critical role in the viral life cycle. High affinity RNA aptamers that bind RT out-compete viral genome for access to the active site and thereby inhibit replication. Aptamer binding to RT during viral formation is believed to drive aptamer encapsidation into the budding virus, which leads to significantly reduced infectivity. We reasoned that co-transcribing multiple aptamer modules as combinatorial transcripts would increase avidity and packaging, resulting in greater net viral suppression. To test this hypothesis, we built a series of Combinatorial Aptamer Transcripts (CATs) carrying multiple, co-transcribed aptamer modules and compared their inhibitory capabilities as a function of valency. Specifically, aptamers from two different structural types (representing (6/5)AL and UCAA structural motifs) were combined into homodimeric and homotrimeric CATs.Two designs were explored for increasing valency; in the first we generated threads of homo-aptamer modules. In general, transcripts with more modules exhibited moderately increased RT inhibition in Primer Extension Assays and increased net binding affinity in Electrophoretic Mobility Shift Assays (EMSAs). The EMSAs do not show evidence of binding multiple RT per transcript, and preliminary cell-based assays do not indicate that this approach to multivalency improves net viral suppression. In contrast, an alternative design appears to be more promising, in which individual aptamer modules are incorporated into a stable3-Way Junction (3WJ) to increase modularity. The 3WJ structural core is expected both to isolate the individual aptamer structures from each other (and thereby reduce misfolding) and to allow sufficient separation to prevent steric hindrance in the binding of multiple RT. Other design features simplify the operational requirements of replacing individual modules with other aptamers or with libraries of aptamers. Initial data based on this design are encouraging.Combinatorial transcripts are expected to block evolutionary escape by forcing the virus to acquire multiple simultaneous mutations, especially for designs that include hetero-multimers. It is well established that combinations of small molecule drugs or of siRNA improves their targeting, delivery, and potency, while reducing their susceptibility to escape mutations. By demonstrating the benefits of multimerization we will inform downstream utilization of CATs as a therapeutic design in the treatment of HIV.