Ongoing HIV replication in lymph node sanctuary sites in treated individuals contributes to the total latent HIV at a very slow rate.

Lymph nodes (LNs) serve as a sanctuary site for HIV viruses due to the heterogeneous distribution of the antiretrovirals (ARVs) inside the LNs. There is an ongoing debate whether this represents ongoing cycles of viral replication in the LNs or merely residual virus production by latently infected cells. Previous work has claimed that the measured levels of genetic variation in proviruses sampled from the blood were inconsistent with ongoing replication. However, it is not clear what rate of variation is consistent with ongoing replication in small sanctuary sites. In this study, we used a spherically symmetric compartmental ODE model to track the HIV viral dynamics in the LN and predict the contribution of ongoing replication within the LN to the whole-body proviral pool in an ARV-suppressed person living with HIV. This model tracks the reaction-diffusion dynamics of uninfected, actively infected, and latently infected T-cells as well as free virus within the LN parenchyma and the blood, and distinguishes between latently infected cells created before ARV therapy and during ARV therapy. We simulated suppressive therapy beginning in year 5 post-infection. Each LN sanctuary site had a volume of 1 ml, and we considered cases of 1 ml, 30 ml, and 250 ml total volume, which represent a single active sanctuary site, moderate systemic involvement, and involvement of the total lymphoid tissue. Viral load in the blood rapidly dropped and remained below the limit of detection in all cases but remained high in the LN sanctuary sites. Novel latent cells increased systemically over time but very slowly, taking between 25 and 50 years to reach 5 % of the total latent pool, depending on the volume of lymphoid tissue involvement. Putative sanctuary sites in LNs are limited in volume and produce novel latent cells slowly. Assays to detect genetic drift due to such sites would require very deep sequencing if sampling only from the blood. Previous studies showing a lack of genetic drift are consistent with the expected contribution of ongoing replication in lymph node sanctuary sites.

Next-Generation Sequencing in a Direct Model of HIV Infection Reveals Important Parallels to and Differences from In Vivo Reservoir Dynamics.

Next-generation sequencing (NGS) represents a powerful tool to unravel the genetic make-up of the HIV reservoir, but limited data exist on its use in vitro Moreover, most NGS studies do not separate integrated from unintegrated DNA, even though selection pressures on these two forms should be distinct. We reasoned we could use NGS to compare the infection of resting and activated CD4 T cells in vitro to address how the metabolic state affects reservoir formation and dynamics. To address these questions, we obtained HIV sequences 2, 4, and 8 days after NL4-3 infection of metabolically activated and quiescent CD4 T cells (cultured with 2 ng/ml interleukin-7). We compared the composition of integrated and total HIV DNA by isolating integrated HIV DNA using pulsed-field electrophoresis before performing sequencing. After a single-round infection, the majority of integrated HIV DNA was intact in both resting and activated T cells. The decay of integrated intact proviruses was rapid and similar in both quiescent and activated T cells. Defective forms accumulated relative to intact ones analogously to what is observed in vivo Massively deleted viral sequences formed more frequently in resting cells, likely due to lower deoxynucleoside triphosphate (dNTP) levels and the presence of multiple restriction factors. To our surprise, the majority of these deleted sequences did not integrate into the human genome. The use of NGS to study reservoir dynamics in vitro provides a model that recapitulates important aspects of reservoir dynamics. Moreover, separating integrated from unintegrated HIV DNA is important in some clinical settings to properly study selection pressures.IMPORTANCE The major implication of our work is that the decay of intact proviruses in vitro is extremely rapid, perhaps as a result of enhanced expression. Gaining a better understanding of why intact proviruses decay faster in vitro might help the field identify strategies to purge the reservoir in vivo When used wisely, in vitro models are a powerful tool to study the selective pressures shaping the viral landscape. Our finding that massively deleted sequences rarely succeed in integrating has several ramifications. It demonstrates that the total HIV DNA can differ substantially in character from the integrated HIV DNA under certain circumstances. The presence of unintegrated HIV DNA has the potential to obscure selection pressures and confound the interpretation of clinical studies, especially in the case of trials involving treatment interruptions.

Episomal HIV-1 DNA and its relationship to other markers of HIV-1 persistence.

Reverse transcription of HIV-1 results in the generation of a linear cDNA that serves as the precursor to the integrated provirus. Other classes of extrachromosomal viral cDNA molecules can be found in acutely infected cells including the 1-LTR and 2-LTR circles of viral DNA, also referred as episomal HIV-1 DNA. Circulating CD4+ T-cells of treatment-naïve individuals contain significant levels of unintegrated forms of HIV-1 DNA. However, the importance of episomal HIV-1 DNA in the study of viral persistence during antiviral therapy (ART) is debatable. 2-LTR circles are preferentially observed in the effector memory CD4+ T cell subset of long-term treated subjects. Treatment intensification of standard regimens has been used to determine if more potent ART can impact viral reservoir activity. Adding a potent antiretroviral drug to a stable triple-drug regimen has no measurable impact on plasma HIV-1 RNA levels, suggesting that ongoing cycles of HIV-1 replication are not a major mechanism driving persistent plasma viremia during triple-drug ART. However, in randomized clinical trials of HIV-1-infected adults on apparently effective ART, the addition of an integrase inhibitor (raltegravir) to stable regimens resulted in a transient increase in 2-LTR circles in some patients, suggesting a pre-intensification steady-state in which the processes of virion generation and de novo infection were occurring. Mathematical modeling of 2-LTR production during integrase inhibitor intensification suggests the coexistence, at different levels, of ongoing de novo infection and de novo replication mechanisms, specifically in inflamed lymphoid drug sanctuaries. Most reports looking into potential changes in 2-LTR circles in interventional clinical studies have simultaneously assessed other potential surrogate markers of viral persistence. Transient increases in 2-LTR circles have been correlated to decreases in CD8+ T-cell activation, transient CD45RA-CD4+ T-cell redistribution, and decreases in the hypercoagulation biomarker D-dimer in ART-intensified individuals. It is difficult, however, to establish a systematic association because the level of correlation with different types of markers differs significantly among studies. In conclusion, despite suppressive ART, a steady-state of de novo infection may persist in some infected individuals and that this may drive immune activation and inflammation changes reflecting residual viral reservoir activity during otherwise apparently suppressive ART.

Synaptic transmission may provide an evolutionary benefit to HIV through modulation of latency.

Transmission of HIV is known to occur by two mechanisms in vivo: the free virus pathway, where viral particles bud off an infected cell before attaching to an uninfected cell, and the cell-cell pathway, where infected cells form virological synapses through close contact with an uninfected cell. It has also been shown that HIV replication includes a positive feedback loop controlled by the viral protein Tat, which may act as a stochastic switch in determining whether an infected cell enters latency. In this paper, we introduce a simple mathematical model of HIV replication containing both the free virus and cell-cell pathways. Using this model, we demonstrate that the high multiplicity of infection in cell-cell transmission results in a suppression of latent infection, and that this modulation of latency through balancing the two transmission mechanisms can provide an evolutionary benefit to the virus. This benefit increases with decreasing overall viral fitness, which may provide a within-host evolutionary pressure toward more cell-cell transmission in late-stage HIV infection.

Long-term antiretroviral treatment initiated at primary HIV-1 infection affects the size, composition, and decay kinetics of the reservoir of HIV-1-infected CD4 T cells.

UNLABELLED: Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCE: Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy, levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.

Spatial modeling of HIV cryptic viremia and 2-LTR formation during raltegravir intensification.

Combination Antiretroviral Therapy (cART) can suppress plasma HIV below the limit of detection in normal assays. Recently reported results suggest that viral replication may continue in some patients, despite undetectable levels in the blood. It has been suggested that the appearance of the circularized episomal HIV DNA artifact 2-LTR following treatment intensification with the integrase inhibitor raltegravir is a marker of ongoing viral replication. Other work has suggested that lymphoid organs may be a site of reduced antiviral penetration and increased viral production. In this study we model the hypothesis that this ongoing replication occurs in lymphoid follicle sanctuary sites and investigate the patterns of 2-LTR formation expected after raltegravir application. Experimental data is used to estimate the reaction and diffusion parameters in the model, and Monte-Carlo simulations are used to explore model behavior subject to variation in these rates. The results suggest that conditions for the formation of an observed transient peak in 2-LTR formation following raltegravir intensification include a sanctuary site diameter larger than 0.2mm, a viral basic reproductive ratio within the site larger than 1, and a total volume of active sanctuary sites above 20mL. Significant levels of uncontrolled replication can occur in the sanctuary sites without measurable changes in the plasma viral load. By contrast, subcritical replication (where the basic reproductive ratio of the virus is less than 1 in all sites) always results in monotonic increases of measured 2-LTR following raltegravir intensification, occurring at levels below the limit of detection.

Ongoing HIV replication in lymph node sanctuary sites in treated patients contributes to the total latent HIV at a very slow rate.

Lymph nodes (LNs) serve as a sanctuary site for HIV viruses due to the heterogeneous distribution of the antiretrovirals (ARVs) inside the LNs. There is an ongoing debate whether this represents ongoing cycles of viral replication in the LNs or merely residual virus production by latently infected cells. Previous work has claimed that the measured levels of genetic variation in proviruses sampled from the blood were inconsistent with ongoing replication. However, it is not clear what rate of variation is consistent with ongoing replication in small sanctuary sites. In this study, we used a spherically symmetric compartmental ODE model to track the HIV viral dynamics in the LN and predict the contribution of ongoing replication within the LN to the wholebody proviral pool in an ARV-suppressed patient. This model tracks the reaction-diffusion dynamics of uninfected, actively infected, and latently infected T-cells as well as free virus within the LN parenchyma and the blood, and distinguishes between latently infected cells created before ARV therapy and during ARV therapy. We simulated suppressive therapy beginning in year 5 post-infection. Each LN sanctuary site had a volume of 1 ml, and we considered cases of 1ml, 30ml, and 250ml total volume, which represent a single active sanctuary site, moderate systemic involvement, and involvement of the total lymphoid tissue. Viral load in the blood rapidly dropped and remained below the limit of detection in all cases but remained high in the LN sanctuary sites. Novel latent cells increased systemically over time but very slowly, taking between 25 and 50 years to reach 5% of the total latent pool, depending on the volume of lymphoid tissue involvement. Putative sanctuary sites in LNs are limited in volume and produce novel latent cells slowly. Assays to detect genetic drift due to such sites would require very deep sequencing if sampling only from the blood. Previous studies showing a lack of genetic drift are consistent with the expected contribution of ongoing replication in lymph node sanctuary sites.

Modeling uncertainty in single-copy assays for HIV.

We present a simple computational model of measurement accuracy for single-copy sensitivity assays (SCA) of HIV RNA that was developed from first principles. The model shows that the SCA is significantly right-skewed. Measured virus concentrations of 1 and 10 virions/ml had overlapping 95% confidence intervals and were statistically indistinguishable.

Nonlinear observer output-feedback MPC treatment scheduling for HIV.

BACKGROUND: Mathematical models of the immune response to the Human Immunodeficiency Virus demonstrate the potential for dynamic schedules of Highly Active Anti-Retroviral Therapy to enhance Cytotoxic Lymphocyte-mediated control of HIV infection. METHODS: In previous work we have developed a model predictive control (MPC) based method for determining optimal treatment interruption schedules for this purpose. In this paper, we introduce a nonlinear observer for the HIV-immune response system and an integrated output-feedback MPC approach for implementing the treatment interruption scheduling algorithm using the easily available viral load measurements. We use Monte-Carlo approaches to test robustness of the algorithm. RESULTS: The nonlinear observer shows robust state tracking while preserving state positivity both for continuous and discrete measurements. The integrated output-feedback MPC algorithm stabilizes the desired steady-state. Monte-Carlo testing shows significant robustness to modeling error, with 90% success rates in stabilizing the desired steady-state with 15% variance from nominal on all model parameters. CONCLUSIONS: The possibility of enhancing immune responsiveness to HIV through dynamic scheduling of treatment is exciting. Output-feedback Model Predictive Control is uniquely well-suited to solutions of these types of problems. The unique constraints of state positivity and very slow sampling are addressable by using a special-purpose nonlinear state estimator, as described in this paper. This shows the possibility of using output-feedback MPC-based algorithms for this purpose.

Controlling the Evolution of Resistance.

Evolution has long been understood as the driving force for many problems of medical interest. The evolution of drug resistance in HIV and bacterial infections is recognized as one of the most significant emerging problems in medicine. In cancer therapy, the evolution of resistance to chemotherapeutic agents is often the differentiating factor between effective therapy and disease progression or death. Interventions to manage the evolution of resistance have, up to this point, been based on steady-state analysis of mutation and selection models. In this paper, we review the mathematical methods applied to studying evolution of resistance in disease. We present a broad review of several classical applications of mathematical modeling of evolution, and review in depth two recent problems which demonstrate the potential for interventions which exploit the dynamic behavior of resistance evolution models. The first problem addresses the problem of sequential treatment failures in HIV; we present a review of our recent publications addressing this problem. The second problem addresses a novel approach to gene therapy for pancreatic cancer treatment, where selection is used to encourage optimal spread of susceptibility genes through a target tumor, which is then eradicated during a second treatment phase. We review the recent in Vitro laboratory work on this topic, present a new mathematical model to describe the treatment process, and show why model-based approaches will be necessary to successfully implement this novel and promising approach.

Naive infection predicts reservoir diversity and is a formidable hurdle to HIV eradication.

Historically, naive cells have been considered inconsequential to HIV persistence. Here, we compared the contributions of naive and memory cells to the reservoirs of individuals with a spectrum of reservoir sizes and variable immunological control. We performed proviral sequencing of approximately 6000 proviruses from cellular subsets of 5 elite controllers (ECs) off antiretroviral therapy (ART) and 5 chronic progressors (CPs) on ART. The levels of naive infection were barely detectable in ECs and approximately 300-fold lower compared with those in CPs. Moreover, the ratio of infected naive to memory cells was significantly lower in ECs. Overall, the naive infection level increased as reservoir size increased, such that naive cells were a major contributor to the intact reservoir of CPs, whose reservoirs were generally very diverse. In contrast, the reservoirs of ECs were dominated by proviral clones. Critically, the fraction of proviral clones increased with cell differentiation, with naive infection predicting reservoir diversity. Longitudinal sequencing revealed that the reservoir of ECs was less dynamic compared with that of CPs. Naive cells play a critical role in HIV persistence. Their infection level predicts reservoir size and diversity. Moreover, the diminishing diversity of the reservoir as cellular subsets mature suggests that naive T cells repopulate the memory compartment and that direct infection of naive T cells occurs in vivo.

An Integrated Spatial Dynamics-Pharmacokinetic Model Explaining Poor Penetration of Anti-retroviral Drugs in Lymph Nodes.

Although combined anti-retroviral therapy (cART) suppresses plasma HIV viremia below the limit of detection in a majority of HIV patients, evidence is emerging that the distribution of the anti-retroviral drugs is heterogeneous in tissue. Clinical studies measuring antiretroviral drug concentrations in lymph nodes (LNs) revealed lower concentrations compared to peripheral blood levels suggesting poor drug penetration properties. Our current study is an attempt to understand this poor anti-retroviral drug penetration inside lymph node lobules through integrating known pharmacokinetic and pharmacodynamic (PK/PD) parameters of the anti-retroviral drugs into a spatial model of reaction and transport dynamics within a solid lymph node lobule. Simulated drug penetration values were compared against experimental results whenever available or matched with data that is available for other drugs in a similar class. Our integrated spatial dynamics pharmacokinetic model reproduced the experimentally observed exclusion of antivirals from lymphoid sites. The strongest predictor of drug exclusion from the lymphoid lobule, independent of drug class, was lobule size; large lobules (high inflammation) exhibited high levels of drug exclusion. PK/PD characteristics associated with poor lymphoid penetration include high cellular uptake rates and low intracellular half-lives. To determine whether this exclusion might lead to ongoing replication, target CD4+ T cell, infected CD4+ T cell, free virus, and intracellular IC50 values of anti-retroviral drugs were incorporated into the model. Notably, for median estimates of PK/PD parameters and lobule diameters consistent with low to moderate inflammation, the model predicts no ongoing viral replication, despite substantial exclusion of the drugs from the lymphoid site. Monte-Carlo studies drawn from the prior distributions of the PK/PD parameters predicts increases in site-specific HIV replication in a small fraction of the patient population for lobule diameters greater than 0.2 mm; this fraction increases as the site diameter/ inflammation level increases. The model shows that cART consisting of two nRTIs and one PI is the most likely treatment combination to support formation of a sanctuary site, a finding that is consistent with clinical observations.

Persistence of an intact HIV reservoir in phenotypically naive T cells.

Despite the efficacy of antiretroviral therapy (ART), HIV persists in a latent form and remains a hurdle to eradication. CD4+ T lymphocytes harbor the majority of the HIV reservoir, but the role of individual subsets remains unclear. CD4+ T cells were sorted into central, transitional, effector memory, and naive T cells. We measured HIV DNA and performed proviral sequencing of more than 1900 proviruses in 2 subjects at 2 and 9 years after ART initiation to estimate the contribution of each subset to the reservoir. Although our study was limited to 2 subjects, we obtained comparable findings with publicly available sequences. While the HIV integration levels were lower in naive compared with memory T cells, naive cells were a major contributor to the intact proviral reservoir. Notably, proviral sequences isolated from naive cells appeared to be unique, while those retrieved from effector memory cells were mainly clonal. The number of clones increased as cells differentiated from a naive to an effector memory phenotype, suggesting naive cells repopulate the effector memory reservoir as previously shown for central memory cells. Naive T cells contribute substantially to the intact HIV reservoir and represent a significant hurdle for HIV eradication.

Significant Unresolved Questions and Opportunities for Bioengineering in Understanding and Treating COVID-19 Disease Progression.

COVID-19 is a disease that manifests itself in a multitude of ways across a wide range of tissues. Many factors are involved, and though impressive strides have been made in studying this novel disease in a very short time, there is still a great deal that is unknown about how the virus functions. Clinical data has been crucial for providing information on COVID-19 progression and determining risk factors. However, the mechanisms leading to the multi-tissue pathology are yet to be fully established. Although insights from SARS-CoV-1 and MERS-CoV have been valuable, it is clear that SARS-CoV-2 is different and merits its own extensive studies. In this review, we highlight unresolved questions surrounding this virus including the temporal immune dynamics, infection of non-pulmonary tissue, early life exposure, and the role of circadian rhythms. Risk factors such as sex and exposure to pollutants are also explored followed by a discussion of ways in which bioengineering approaches can be employed to help understand COVID-19. The use of sophisticated in vitro models can be employed to interrogate intercellular interactions and also to tease apart effects of the virus itself from the resulting immune response. Additionally, spatiotemporal information can be gleaned from these models to learn more about the dynamics of the virus and COVID-19 progression. Application of advanced tissue and organ system models into COVID-19 research can result in more nuanced insight into the mechanisms underlying this condition and elucidate strategies to combat its effects.

Optimal control modulation of HIV reservoir formation rate by antigen infusion.

The Human Immunodeficiency Virus (HIV) infects helper-T cells, and takes advantage of the naturally occurring quiescent phenotype of T cells to persist even under effective treatment conditions. If an infected cell does not produce virus and enters this quiescent state, it forms a natural reservoir that is not targeted by either the existing antiretroviral drugs or the immune system. These quiescent cells intermittently switch to an activated phenotype and begin to produce virus, and are the primary source of viral rebound following treatment cessation. Recent experimental results have shown that, despite this reservoir having a years-long half-life under treatment, most of the cells in the reservoir were infected in a few weeks prior to the start of treatment. This can only be explained by assuming that this reservoir has a short half-life off treatment and a very long half-life on treatment. In this paper, we introduce a novel model of reservoir formation and turnover explaining this difference as a result of antigen-dependent activation. We introduce a second control input through infusion of HIV antigen, mimicking the non-infection pseudovirus (PV) produced by protease inhibitor therapy. This model is coupled to an existing model of immune response to HIV. We fit the parameters of this model to the existing clinical observations of latency. We show that the use of antigen infusion therapy can result in order-of-magnitude decrease in the size of the quiescent reservoir, and that this may provide a way to rapidly stabilize a post-treatment control state in treated HIV infected individuals.

Positive feedback through inflammation creates bistable behavior in HIV tissue sanctuaries.

Combination Antiretroviral Therapy (cART) consists of a cocktail of drugs administered to HIV-infected patients that can suppress the amount of HIV in the patient's blood plasma to an undetectable level. Our previous work has suggested that some HIV-infected patients, despite being placed on cART, can still have ongoing viral replication occurring in self-sustaining inflamed lymph node follicle sanctuary sites. Spatial models of the putative sites show that inflammation is a necessary condition for ongoing HIV replication. In this study, we model the hypothesis that ongoing HIV replication may provide a sufficiently strong pro-inflammatory signal to maintain inflammation levels consistent with continued HIV replication. A system of ordinary differential equations integrated with a reactive-diffusion system is used to model the HIV dynamics and the diameter of a lymph node follicle as a function of time and external influence. The estimates of the parameters in our model come from prior data when available. The results of our study show that these dynamics have two stable steady-state solutions, one with low inflammation and no ongoing HIV replication in the site, and one with high inflammation and high levels of ongoing HIV replication in the site. We furthermore show that the system can transition between the two outcomes in response to a transient exogenous addition of pro-inflammatory signaling, consistent with the antigenic stimulus of a secondary infection. The spatial isolation of the sites results in a low viral load in the blood plasma for both conditions.

HIV 2-LTR experiment design optimization.

Clinical trials are necessary in order to develop treatments for diseases; however, they can often be costly, time consuming, and demanding to the patients. This paper summarizes several common methods used for optimal design that can be used to address these issues. In addition, we introduce a novel method for optimizing experiment designs applied to HIV 2-LTR clinical trials. Our method employs Bayesian techniques to optimize the experiment outcome by maximizing the Expected Kullback-Leibler Divergence (EKLD) between the a priori knowledge of system parameters before the experiment and the a posteriori knowledge of the system parameters after the experiment. We show that our method is robust and performs equally well if not better than traditional optimal experiment design techniques.

Experiment Design for Early Molecular Events in HIV Infection.

The recent introduction of integrase inhibitors to the HIV antiviral repertoire permits us to create in vitro experiments that reliably terminate HIV infection at the point of chromosomal integration. This allows us to isolate the dynamics of a single round of infection, without needing to account for the influence of multiple overlapping rounds of infection. By measuring the various nucleic acid concentrations in a population of infected target cells at multiple time points, we can infer the rates of these molecular events with great accuracy, which allows us to compare the rates between target cells with different functional phenotypes. This information will help in understanding the behavior of the various populations of reservoir cells such as active and quiescent T-cells which maintain HIV infection in treated patients. In this paper, we introduce a family of models of the early molecular events in HIV infection, with either linear dynamics or age-structured delays at each step. We introduce an experimental design metric based on the delta AIC (Akaike Information Criteria) between a model fit for simulated data from a matching model vs a mismatched model, which allows us to determine a candidate experiment design's ability to discriminate between models. Using parameters values drawn from experimentally-derived priors corrupted with appropriate measurement noise, we confirm that a proposed sampling schedule at different time points allows us to consistently discriminate between candidate models.

Implications of Measurement Assay Type in Design of HIV Experiments.

Time series measurements of circular viral episome (2-LTR) concentrations enable indirect quantification of persistent low-level Human Immunodeficiency Virus (HIV) replication in patients on Integrase-Inhibitor intensified Combined Antiretroviral Therapy (cART). In order to determine the magnitude of these low level infection events, blood has to be drawn from a patients at a frequency and volume that is strictly regulated by the Institutional Review Board (IRB). Once the blood is drawn, the 2-LTR concentration is determined by quantifying the amount of HIV DNA present in the sample via a PCR (Polymerase Chain Reaction) assay. Real time quantitative Polymerase Chain Reaction (qPCR) is a widely used method of performing PCR; however, a newer droplet digital Polymerase Chain Reaction (ddPCR) method has been shown to provide more accurate quantification of DNA. Using a validated model of HIV viral replication, this paper demonstrates the importance of considering DNA quantification assay type when optimizing experiment design conditions. Experiments are optimized using a Genetic Algorithm (GA) to locate a family of suboptimal sample schedules which yield the highest fitness. Fitness is defined as the expected information gained in the experiment, measured by the Kullback-Leibler Divergence (KLD) between the prior and posterior distributions of the model parameters. We compare the information content of the optimized schedules to uniform schedules as well as two clinical schedules implemented by researchers at UCSF and the University of Melbourne. This work shows that there is a significantly greater gain information in experiments using a ddPCR assay vs. a qPCR assay and that certain experiment design considerations should be taken when using either assay.

Increased inflammation in sanctuary sites may explain viral blips in HIV infection.

Combined antiretroviral therapy (cART) suppress HIV-1 viral replication, such that viral load in plasma remains below the limit of detection in standard assays. However, intermittent episodes of transient viremia (blips) occur in a set of HIV-patients. Given that follicular hyperplasia occurs during lymphoid inflammation as a normal response to infection, it is hypothesised that when the diameter of the lymph node follicle (LNF) increases and crosses a critical size, a viral blip occurs due to cryptic viremia. To study this hypothesis, a theoretical analysis of a mathematical model is performed to find the conditions for virus suppression in all compartments and different scenarios of LNF size changes are simulated. According to the analysis, blips with duration of around 30 days arise when the diameter rise rate is between 0.02 and 0.03 days(-1). Moreover, the final diameter of the site is directly related to the steady states of the virus load after the occurrence of a blip. When the value of R0 is around 2.1, to have a steady-state below the limit of detection after the viral blip, the maximum final diameters should be greater than 0.7 mm so that there is a relative loss of connection between compartments.