Binding and neutralizing anti-AAV antibodies: Detection and implications for rAAV-mediated gene therapy.

Assessment of anti-adeno-associated virus (AAV) antibodies in patients prior to systemic gene therapy administration is an important consideration regarding efficacy and safety of the therapy. Approximately 30%-60% of individuals have pre-existing anti-AAV antibodies. Seroprevalence is impacted by multiple factors, including geography, age, capsid serotype, and assay type. Anti-AAV antibody assays typically measure (1) transduction inhibition by detecting the neutralizing capacity of antibodies and non-antibody neutralizing factors, or (2) total anti-capsid binding antibodies, regardless of neutralizing activity. Presently, there is a paucity of head-to-head data and standardized approaches associating assay results with clinical outcomes. In addition, establishing clinically relevant screening titer cutoffs is complex. Thus, meaningful comparisons across assays are nearly impossible. Although complex, establishing screening assays in routine clinical practice to identify patients with antibody levels that may impact favorable treatment outcomes is achievable for both transduction inhibition and total antibody assays. Formal regulatory approval of such assays as companion diagnostic tests will confirm their suitability for specific recombinant AAV gene therapies. This review covers current approaches to measure anti-AAV antibodies in patient plasma or serum, their potential impact on therapeutic safety and efficacy, and investigative strategies to mitigate the effects of pre-existing anti-AAV antibodies in patients.

Reduced Exercise Capacity, Chronotropic Incompetence, and Early Systemic Inflammation in Cardiopulmonary Phenotype Long Coronavirus Disease 2019.

BACKGROUND: Mechanisms underlying persistent cardiopulmonary symptoms after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (postacute sequelae of coronavirus disease 2019 [COVID-19; PASC] or "long COVID") remain unclear. This study sought to elucidate mechanisms of cardiopulmonary symptoms and reduced exercise capacity. METHODS: We conducted cardiopulmonary exercise testing (CPET), cardiac magnetic resonance imaging (CMR) and ambulatory rhythm monitoring among adults >1 year after SARS-CoV-2 infection, compared those with and those without symptoms, and correlated findings with previously measured biomarkers. RESULTS: Sixty participants (median age, 53 years; 42% female; 87% nonhospitalized; median 17.6 months after infection) were studied. At CPET, 18/37 (49%) with symptoms had reduced exercise capacity (<85% predicted), compared with 3/19 (16%) without symptoms (P = .02). The adjusted peak oxygen consumption (VO2) was 5.2 mL/kg/min lower (95% confidence interval, 2.1-8.3; P = .001) or 16.9% lower percent predicted (4.3%-29.6%; P = .02) among those with symptoms. Chronotropic incompetence was common. Inflammatory markers and antibody levels early in PASC were negatively correlated with peak VO2. Late-gadolinium enhancement on CMR and arrhythmias were absent. CONCLUSIONS: Cardiopulmonary symptoms >1 year after COVID-19 were associated with reduced exercise capacity, which was associated with earlier inflammatory markers. Chronotropic incompetence may explain exercise intolerance among some with "long COVID."

Higher Levels of Cerebrospinal Fluid and Plasma Neurofilament Light in Human Immunodeficiency Virus-Associated Distal Sensory Polyneuropathy.

BACKGROUND: Neurofilament light (NFL) chain concentrations, reflecting axonal damage, are seen in several polyneuropathies but have not been studied in human immunodeficiency virus (HIV) distal sensory polyneuropathy (DSP). We evaluated NFL in cerebrospinal fluid (CSF) and plasma in relation to DSP in people with HIV (PWH) from 2 independent cohorts and in people without HIV (PWoH). METHODS: Cohort 1 consisted of PWH from the CHARTER Study. Cohort 2 consisted of PWH and PWoH from the HIV Neurobehavioral Research Center (HNRC). We evaluated DSP signs and symptoms in both cohorts. Immunoassays measured NFL in CSF for all and for plasma as well in Cohort 2. RESULTS: Cohort 1 consisted of 111 PWH, mean ± SD age 56.8 ± 8.32 years, 15.3% female, 38.7% Black, 49.6% White, current CD4+ T-cells (median, interquartile range [IQR]) 532/µL (295, 785), 83.5% with plasma HIV RNA ≤50 copies/mL. Cohort 2 consisted of 233 PWH of similar demographics to PWH in Cohort 1 but also 51 PWoH, together age 58.4 ± 6.68 years, 41.2% female, 18.0% Black, Hispanic, non-Hispanic White 52.0%, 6.00% White. In both cohorts of PWH, CSF and plasma NFL were significantly higher in both PWH with DSP signs. Findings were similar, albeit not significant, for PWoH. The observed relationships were not explained by confounds. CONCLUSIONS: Both plasma and CSF NFL were elevated in PWH and PWoH with DSP. The convergence of our findings with others demonstrates that NFL is a reliable biomarker reflecting peripheral nerve injury. Biomarkers such as NFL might provide, validate, and optimize clinical trials of neuroregenerative strategies in HIV DSP.

The breadth of the neutralizing antibody response to original SARS-CoV-2 infection is linked to the presence of Long COVID symptoms.

The associations between longitudinal dynamics and the breadth of SARS-CoV-2 neutralizing antibody (nAb) response with various Long COVID phenotypes before vaccination are not known. The capacity of antibodies to cross-neutralize a variety of viral variants may be associated with ongoing pathology and persistent symptoms. We measured longitudinal neutralizing and cross-neutralizing antibody responses to pre- and post-SARS-CoV-2 Omicron variants in participants infected early in the COVID-19 pandemic, before widespread rollout of SARS-CoV-2 vaccines. Cross-sectional regression models adjusted for clinical covariates and longitudinal mixed-effects models were used to determine the impact of the breadth and rate of decay of neutralizing responses on the development of Long COVID symptoms, as well as Long COVID phenotypes. We identified several novel relationships between SARS-CoV-2 antibody neutralization and the presence of Long COVID symptoms. Specifically, we show that, although nAb responses to the original, infecting strain of SARS-CoV-2 were not associated with Long COVID in cross-sectional analyses, cross-neutralization ID50 levels to the Omicron BA.5 variant approximately 4 months following acute infection was independently and significantly associated with greater odds of Long COVID and with persistent gastrointestinal and neurological symptoms. Longitudinal modeling demonstrated significant associations in the overall levels and rates of decay of neutralization capacity with Long COVID phenotypes. A higher proportion of participants had antibodies capable of neutralizing Omicron BA.5 compared with BA.1 or XBB.1.5 variants. Our findings suggest that relationships between various immune responses and Long COVID are likely complex but may involve the breadth of antibody neutralization responses.

Characterization of Human Immunodeficiency Virus (HIV) Infections in Women Who Received Injectable Cabotegravir or Tenofovir Disoproxil Fumarate/Emtricitabine for HIV Prevention: HPTN 084.

BACKGROUND: HIV Prevention Trials Network 084 demonstrated that long-acting injectable cabotegravir (CAB) was superior to daily oral tenofovir (TFV) disoproxil fumarate (TDF)/emtricitabine (FTC) for preventing human immunodeficiency virus (HIV) infection in sub-Saharan African women. This report describes HIV infections that occurred in the trial before unblinding. METHODS: Testing was performed using HIV diagnostic assays, viral load testing, a single-copy RNA assay, and HIV genotyping. Plasma CAB, plasma TFV, and intraerythrocytic TFV-diphosphate concentrations were determined by liquid chromatography-tandem mass spectrometry. RESULTS: Forty HIV infections were identified (CAB arm, 1 baseline infection, 3 incident infections; TDF/FTC arm, 36 incident infections). The incident infections in the CAB arm included 2 with no recent drug exposure and no CAB injections and 1 with delayed injections; in 35 of 36 cases in the TDF/FTC arm, drug concentrations indicated low or no adherence. None of the cases had CAB resistance. Nine women in the TDF/FTC arm had nonnucleoside reverse-transcriptase inhibitor resistance; 1 had the nucleoside reverse-transcriptase inhibitor resistance mutation, M184V. CONCLUSIONS: Almost all incident HIV infections occurred in the setting of unquantifiable or low drug concentrations. CAB resistance was not detected. Transmitted nonnucleoside reverse-transcriptase inhibitor resistance was common; 1 woman may have acquired nucleoside reverse-transcriptase inhibitor resistance from study drug exposure.

National Landscape of Human Immunodeficiency Virus-Positive Deceased Organ Donors in the United States.

BACKGROUND: Organ transplantation from donors with human immunodeficiency virus (HIV) to recipients with HIV (HIV D+/R+) presents risks of donor-derived infections. Understanding clinical, immunologic, and virologic characteristics of HIV-positive donors is critical for safety. METHODS: We performed a prospective study of donors with HIV-positive and HIV false-positive (FP) test results within the HIV Organ Policy Equity (HOPE) Act in Action studies of HIV D+/R+ transplantation (ClinicalTrials.gov NCT02602262, NCT03500315, and NCT03734393). We compared clinical characteristics in HIV-positive versus FP donors. We measured CD4 T cells, HIV viral load (VL), drug resistance mutations (DRMs), coreceptor tropism, and serum antiretroviral therapy (ART) detection, using mass spectrometry in HIV-positive donors. RESULTS: Between March 2016 and March 2020, 92 donors (58 HIV positive, 34 FP), representing 98.9% of all US HOPE donors during this period, donated 177 organs (131 kidneys and 46 livers). Each year the number of donors increased. The prevalence of hepatitis B (16% vs 0%), syphilis (16% vs 0%), and cytomegalovirus (CMV; 91% vs 58%) was higher in HIV-positive versus FP donors; the prevalences of hepatitis C viremia were similar (2% vs 6%). Most HIV-positive donors (71%) had a known HIV diagnosis, of whom 90% were prescribed ART and 68% had a VL <400 copies/mL. The median CD4 T-cell count (interquartile range) was 194/µL (77-331/µL), and the median CD4 T-cell percentage was 27.0% (16.8%-36.1%). Major HIV DRMs were detected in 42%, including nonnucleoside reverse-transcriptase inhibitors (33%), integrase strand transfer inhibitors (4%), and multiclass (13%). Serum ART was detected in 46% and matched ART by history. CONCLUSION: The use of HIV-positive donor organs is increasing. HIV DRMs are common, yet resistance that would compromise integrase strand transfer inhibitor-based regimens is rare, which is reassuring regarding safety.

Higher cerebrospinal fluid biomarkers of neuronal injury in HIV-associated neurocognitive impairment.

We evaluated whether biomarkers of age-related neuronal injury and amyloid metabolism are associated with neurocognitive impairment (NCI) in people with and without HIV (PWH, PWoH). This was a cross-sectional study of virally suppressed PWH and PWoH. NCI was assessed using a validated test battery; global deficit scores (GDS) quantified overall performance. Biomarkers in cerebrospinal fluid (CSF) were quantified by immunoassay: neurofilament light (NFL), total Tau (tTau), phosphorylated Tau 181 (pTau181), amyloid beta (Aß)42, and Aß40. Factor analysis was used to reduce biomarker dimensionality. Participants were 256 virally suppressed PWH and 42 PWoH, 20.2% female, 17.1% Black, 7.1% Hispanic, 60.2% non-Hispanic White, and 15.6% other race/ethnicities, mean (SD) age 56.7 (6.45) years. Among PWH, the best regression model for CSF showed that higher tTau (ß = 0.723, p = 3.79e-5) together with lower pTau181 (ß = -0.510, p = 0.0236) best-predicted poor neurocognitive performance. In univariable analysis, only higher tTau was significantly correlated with poor neurocognitive performance (tTau r = 0.214, p = 0.0006; pTau181 r = 0.00248, p = 0.969). Among PWoH, no CSF biomarkers were significantly associated with worse NCI. Predicted residual error sum of squares (PRESS) analysis showed no evidence of overfitting. Poorer neurocognitive performance in aging PWH was associated with higher CSF tTau, a marker of age-related neuronal injury, but not with biomarkers of amyloid metabolism. The findings suggest that HIV might interact with age-related neurodegeneration to contribute to cognitive decline in PWH.

Markers of Immune Activation and Inflammation in Individuals With Postacute Sequelae of Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: The biological processes associated with postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (PASC) are unknown. METHODS: We measured soluble markers of inflammation in a SARS-CoV-2 recovery cohort at early (<90 days) and late (>90 days) timepoints. We defined PASC as the presence of 1 or more coronavirus disease 2019 (COVID-19)-attributed symptoms beyond 90 days. We compared fold-changes in marker values between those with and without PASC using mixed-effects models with terms for PASC and early and late recovery time periods. RESULTS: During early recovery, those who went on to develop PASC generally had higher levels of cytokine biomarkers including tumor necrosis factor-α (1.14-fold higher mean ratio [95% confidence interval {CI}, 1.01-1.28]; P = .028) and interferon-γ-induced protein 10 (1.28-fold higher mean ratio [95% CI, 1.01-1.62]; P = .038). Among those with PASC, there was a trend toward higher interleukin 6 levels during early recovery (1.29-fold higher mean ratio [95% CI, .98-1.70]; P = .07), which became more pronounced in late recovery (1.44-fold higher mean ratio [95% CI, 1.11-1.86]; P < .001). These differences were more pronounced among those with a greater number of PASC symptoms. CONCLUSIONS: Persistent immune activation may be associated with ongoing symptoms following COVID-19. Further characterization of these processes might identify therapeutic targets for those experiencing PASC.

Characterization of Human Immunodeficiency Virus (HIV) Infection in Cisgender Men and Transgender Women Who Have Sex With Men Receiving Injectable Cabotegravir for HIV Prevention: HPTN 083.

BACKGROUND: The HIV Prevention Trials Network (HPTN) 083 trial demonstrated that long-acting cabotegravir (CAB-LA) was more effective than tenofovir disoproxil fumarate-emtricitabine (TDF/FTC) in preventing human immunodeficiency virus (HIV) in cisgender men and transgender women who have sex with men. We characterized HIV infections that occurred in the blinded phase of HPTN 083. METHODS: Retrospective testing included HIV testing, viral load testing, quantification of study drugs, and HIV drug resistance testing. RESULTS: Fifty-eight infections were evaluated, including 51 incident infections (12 in CAB arm and 39 in TDF/FTC arm). In many cases (5 in CAB arm and 37 in TDF/FTC arm), infection was associated with low or unquantifiable study drug concentrations. In 4 cases, infection occurred with on-time CAB-LA injections and expected plasma CAB concentrations. CAB exposure was associated with prolonged viral suppression and delayed antibody expression. In some cases, delayed HIV diagnosis resulted in CAB provision to participants with undetected infection, delayed antiretroviral therapy, and emergence of drug resistance; most of these infections would have been detected earlier with viral load testing. CONCLUSIONS: Early detection of HIV infection and prompt antiretroviral therapy initiation could improve clinical outcomes in persons who become infected despite CAB-LA prophylaxis. Further studies are needed to elucidate the correlates of HIV protection in persons receiving CAB-LA.

Impact of HIV-1 Resistance-Associated Mutations on Susceptibility to Doravirine: Analysis of Real-World Clinical Isolates.

Clinical management of human immunodeficiency virus type-1 (HIV-1) infection may be negatively impacted by either acquired or transmitted drug resistance. Here, we aim to extend our understanding of the impact of resistance-associated mutations (RAMs) on the susceptibility of clinical isolates to the nonnucleoside reverse transcriptase inhibitor (NNRTI) doravirine. Clinical isolates from people living with HIV-1 undergoing routine testing for susceptibility to doravirine and other approved NNRTIs (etravirine, rilpivirine, efavirenz, and nevirapine) were collected from August 2018 to August 2019. Susceptibility in the presence/absence of NNRTI and nucleos(t)ide reverse transcriptase inhibitor (NRTI) mutations was determined using cutoffs for relative fold change in inhibition (ratio of the 50% inhibitory concentration [IC50] of patient virus compared with the IC50 of a wild-type reference strain). Biological cutoffs of 3- to 15-fold change were investigated for doravirine, with preestablished cutoffs used for the other NNRTIs. Of 4,070 clinical isolates, 42.9% had ≥1 NNRTI RAM. More isolates were susceptible to doravirine (92.5-96.7%) than to etravirine (91.5%), rilpivirine (89.5%), efavirenz (81.5%), or nevirapine (77.5%). Based on a 3-fold cutoff, doravirine susceptibility was retained in 44.7-65.8% of isolates resistant to another NNRTI and 28.5% of isolates resistant to all other tested NNRTIs. The presence of NRTI RAMs, including thymidine analog mutations, was associated with doravirine hypersusceptibility in some isolates, particularly in the absence of NNRTI RAMs. These results support the favorable resistance profile of doravirine and are of particular importance given the challenge posed by both acquired and transmitted resistance.

Prognostic significance of MEOX2 in gliomas.

Gliomas are the most common malignant primary tumors in the central nervous system and have variable predictive clinical courses. Glioblastoma, the most aggressive form of glioma, is a complex disease with unsatisfactory therapeutic solutions and a very poor prognosis. Some processes at stake in gliomagenesis have been discovered but little is known about the role of homeobox genes, even though they are highly expressed in gliomas, particularly in glioblastoma. Among them, the transcription factor Mesenchyme Homeobox 2 (MEOX2) had previously been associated with malignant progression and clinical prognosis in lung cancer and hepatocarcinoma but never studied in glioma. The aim of our study was to investigate the clinical significance of MEOX2 in gliomas. We assessed the expression of MEOX2 according to IDH1/2 molecular profile and patient survival among three different public datasets: The Cancer Genome Atlas (TCGA), The Chinese Glioma Genome Atlas (CGGA) and the US National Cancer Institute Repository for Molecular Brain Neoplasia Data (Rembrandt). We then evaluated the prognostic significance of MEOX2 protein expression on 112 glioma clinical samples including; 56 IDH1 wildtype glioblastomas, 7 IDH1 wild-type lower grade gliomas, 49 IDH1 mutated lower grade gliomas. Survival rates were estimated by the Kaplan-Meier method followed by uni/multivariate analyses. We demonstrated that MEOX2 was one of the transcription factors most closely associated with overall survival in glioma. Moreover, MEOX2 expression was associated with IDH1/2 wildtype molecular subtype and was significantly correlated with overall survival of all gliomas and, more interestingly, in lower grade glioma. To conclude, our results may be the first to provide insight into the clinical significance of MEOX2 in gliomas, which is a factor closely related to patient outcome. MEOX2 could constitute an interesting prognostic biomarker, especially for lower grade glioma.

Fatal correlation between YAP1 expression and glioma aggressiveness: clinical and molecular evidence.

During the last decade, large-scale genomic analyses have clarified the somatic alterations in gliomas, providing new molecular classification based on IDH1/2 mutations and 1p19q codeletion with more accurate patient prognostication. The Hippo pathway downstream effectors, YAP1 and TAZ, have recently emerged as major determinants of malignancy by inducing proliferation, chemoresistance, and metastasis in solid tumors. In this study, we investigated the expression of YAP1 in 117 clinical samples of glioma described according to the WHO 2016 classification. We showed for the first time that YAP1 was tightly associated with glioma molecular subtypes and patient outcome. We validated our results in an independent cohort from the TCGA database. More interestingly, we found that YAP1 may have prognostic significance for predicting patient survival, especially in low-grade gliomas. Using patient-derived glioblastoma stem cell cultures, we demonstrated that YAP1 was activated and that it controlled cell proliferation. Transcriptome analysis revealed lower expression of YAP1 in the proneural GBM subtype. Furthermore, we found that overexpression of YAP1 was sufficient to inhibit the OLIG2 proneural marker, suggesting its involvement in maintenance of the GBM phenotype. Taken together, our results showed that YAP1 could be a relevant prognostic biomarker and a potential therapeutic target in glioma. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epistasis and Pleiotropy Affect the Modularity of the Genotype-Phenotype Map of Cross-Resistance in HIV-1.

The genotype-phenotype (GP) map is a central concept in evolutionary biology as it describes the mapping of molecular genetic variation onto phenotypic trait variation. Our understanding of that mapping remains partial, especially when trying to link functional clustering of pleiotropic gene effects with patterns of phenotypic trait co-variation. Only on rare occasions have studies been able to fully explore that link and tend to show poor correspondence between modular structures within the GP map and among phenotypes. By dissecting the structure of the GP map of the replicative capacity of HIV-1 in 15 drug environments, we provide a detailed view of that mapping from mutational pleiotropic variation to phenotypic co-variation, including epistatic effects of a set of amino-acid substitutions in the reverse transcriptase and protease genes. We show that epistasis increases the pleiotropic degree of single mutations and provides modularity to the GP map of drug resistance in HIV-1. Moreover, modules of epistatic pleiotropic effects within the GP map match the phenotypic modules of correlated replicative capacity among drug classes. Epistasis thus increases the evolvability of cross-resistance in HIV by providing more drug- and class-specific pleiotropic profiles to the main effects of the mutations. We discuss the implications for the evolution of cross-resistance in HIV.

Elucidation of the Molecular Mechanism Driving Duplication of the HIV-1 PTAP Late Domain.

UNLABELLED: HIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6(Gag)), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6(Gag) significantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6(Gag) confers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy. IMPORTANCE: Resistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients.

Persistence of transmitted HIV-1 drug resistance mutations associated with fitness costs and viral genetic backgrounds.

Transmission of drug-resistant pathogens presents an almost-universal challenge for fighting infectious diseases. Transmitted drug resistance mutations (TDRM) can persist in the absence of drugs for considerable time. It is generally believed that differential TDRM-persistence is caused, at least partially, by variations in TDRM-fitness-costs. However, in vivo epidemiological evidence for the impact of fitness costs on TDRM-persistence is rare. Here, we studied the persistence of TDRM in HIV-1 using longitudinally-sampled nucleotide sequences from the Swiss-HIV-Cohort-Study (SHCS). All treatment-naïve individuals with TDRM at baseline were included. Persistence of TDRM was quantified via reversion rates (RR) determined with interval-censored survival models. Fitness costs of TDRM were estimated in the genetic background in which they occurred using a previously published and validated machine-learning algorithm (based on in vitro replicative capacities) and were included in the survival models as explanatory variables. In 857 sequential samples from 168 treatment-naïve patients, 17 TDRM were analyzed. RR varied substantially and ranged from 174.0/100-person-years;CI=[51.4, 588.8] (for 184V) to 2.7/100-person-years;[0.7, 10.9] (for 215D). RR increased significantly with fitness cost (increase by 1.6[1.3,2.0] per standard deviation of fitness costs). When subdividing fitness costs into the average fitness cost of a given mutation and the deviation from the average fitness cost of a mutation in a given genetic background, we found that both components were significantly associated with reversion-rates. Our results show that the substantial variations of TDRM persistence in the absence of drugs are associated with fitness-cost differences both among mutations and among different genetic backgrounds for the same mutation.

Recombination accelerates adaptation on a large-scale empirical fitness landscape in HIV-1.

Recombination has the potential to facilitate adaptation. In spite of the substantial body of theory on the impact of recombination on the evolutionary dynamics of adapting populations, empirical evidence to test these theories is still scarce. We examined the effect of recombination on adaptation on a large-scale empirical fitness landscape in HIV-1 based on in vitro fitness measurements. Our results indicate that recombination substantially increases the rate of adaptation under a wide range of parameter values for population size, mutation rate and recombination rate. The accelerating effect of recombination is stronger for intermediate mutation rates but increases in a monotonic way with the recombination rates and population sizes that we examined. We also found that both fitness effects of individual mutations and epistatic fitness interactions cause recombination to accelerate adaptation. The estimated epistasis in the adapting populations is significantly negative. Our results highlight the importance of recombination in the evolution of HIV-I.

Evolution of a novel pathway leading to dolutegravir resistance in a patient harbouring N155H and multiclass drug resistance.

OBJECTIVES: Dolutegravir has been recently approved for treatment-naive and -experienced HIV-infected subjects, including integrase inhibitor (INI)-experienced patients. Dolutegravir is a second-generation INI that can overcome many prior raltegravir and elvitegravir failures. Here, we report the evolution of resistance to dolutegravir in a highly treatment-experienced patient harbouring the major N155H mutation consequent to raltegravir treatment failure. METHODS: Genotypic and phenotypic analyses were done on longitudinal samples to determine viral resistance to INIs. Integrase amino acid sequence interactions with raltegravir and dolutegravir were assessed by molecular modelling and docking simulations. RESULTS: Five mutations (A49P, L68FL, T97A, E138K and L234V) were implicated in emergent dolutegravir resistance, with a concomitant severe compromise in viral replicative capacity. Molecular modelling and docking simulations revealed that dolutegravir binding to integrase was affected by these acquired dolutegravir mutations. CONCLUSIONS: Our findings identify a novel mutational pathway involving integrase mutations A49P and L234V, leading to dolutegravir resistance in a patient with the N155H raltegravir mutation.

Exploring the complexity of the HIV-1 fitness landscape.

Although fitness landscapes are central to evolutionary theory, so far no biologically realistic examples for large-scale fitness landscapes have been described. Most currently available biological examples are restricted to very few loci or alleles and therefore do not capture the high dimensionality characteristic of real fitness landscapes. Here we analyze large-scale fitness landscapes that are based on predictive models for in vitro replicative fitness of HIV-1. We find that these landscapes are characterized by large correlation lengths, considerable neutrality, and high ruggedness and that these properties depend only weakly on whether fitness is measured in the absence or presence of different antiretrovirals. Accordingly, adaptive processes on these landscapes depend sensitively on the initial conditions. While the relative extent to which mutations affect fitness on their own (main effects) or in combination with other mutations (epistasis) is a strong determinant of these properties, the fitness landscape of HIV-1 is considerably less rugged, less neutral, and more correlated than expected from the distribution of main effects and epistatic interactions alone. Overall this study confirms theoretical conjectures about the complexity of biological fitness landscapes and the importance of the high dimensionality of the genetic space in which adaptation takes place.

Predicting HIV-1 broadly neutralizing antibody epitope networks using neutralization titers and a novel computational method.

BACKGROUND: Recent efforts in HIV-1 vaccine design have focused on immunogens that evoke potent neutralizing antibody responses to a broad spectrum of viruses circulating worldwide. However, the development of effective vaccines will depend on the identification and characterization of the neutralizing antibodies and their epitopes. We developed bioinformatics methods to predict epitope networks and antigenic determinants using structural information, as well as corresponding genotypes and phenotypes generated by a highly sensitive and reproducible neutralization assay.282 clonal envelope sequences from a multiclade panel of HIV-1 viruses were tested in viral neutralization assays with an array of broadly neutralizing monoclonal antibodies (mAbs: b12, PG9,16, PGT121 - 128, PGT130 - 131, PGT135 - 137, PGT141 - 145, and PGV04). We correlated IC50 titers with the envelope sequences, and used this information to predict antibody epitope networks. Structural patches were defined as amino acid groups based on solvent-accessibility, radius, atomic depth, and interaction networks within 3D envelope models. We applied a boosted algorithm consisting of multiple machine-learning and statistical models to evaluate these patches as possible antibody epitope regions, evidenced by strong correlations with the neutralization response for each antibody. RESULTS: We identified patch clusters with significant correlation to IC50 titers as sites that impact neutralization sensitivity and therefore are potentially part of the antibody binding sites. Predicted epitope networks were mostly located within the variable loops of the envelope glycoprotein (gp120), particularly in V1/V2. Site-directed mutagenesis experiments involving residues identified as epitope networks across multiple mAbs confirmed association of these residues with loss or gain of neutralization sensitivity. CONCLUSIONS: Computational methods were implemented to rapidly survey protein structures and predict epitope networks associated with response to individual monoclonal antibodies, which resulted in the identification and deeper understanding of immunological hotspots targeted by broadly neutralizing HIV-1 antibodies.

The molecular basis of drug resistance against hepatitis C virus NS3/4A protease inhibitors.

Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors--telaprevir, danoprevir, vaniprevir and MK-5172--in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.