Rapid Emergence and Evolution of SARS-CoV-2 Variants in Advanced HIV Infection.

Previous studies have linked the evolution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic variants to persistent infections in people with immunocompromising conditions1-4, but the evolutionary processes underlying these observations are incompletely understood. Here we used high-throughput, single-genome amplification and sequencing (HT-SGS) to obtain up to ~103 SARS-CoV-2 spike gene sequences in each of 184 respiratory samples from 22 people with HIV (PWH) and 25 people without HIV (PWOH). Twelve of 22 PWH had advanced HIV infection, defined by peripheral blood CD4 T cell counts (i.e., CD4 counts) <200 cells/µL. In PWOH and PWH with CD4 counts ≥200 cells/µL, most single-genome spike sequences in each person matched one haplotype that predominated throughout the infection. By contrast, people with advanced HIV showed elevated intra-host spike diversity with a median of 46 haplotypes per person (IQR 14-114). Higher intra-host spike diversity immediately after COVID-19 symptom onset predicted longer SARS-CoV-2 RNA shedding among PWH, and intra-host spike diversity at this timepoint was significantly higher in people with advanced HIV than in PWOH. Composition of spike sequence populations in people with advanced HIV fluctuated rapidly over time, with founder sequences often replaced by groups of new haplotypes. These population-level changes were associated with a high total burden of intra-host mutations and positive selection at functionally important residues. In several cases, delayed emergence of detectable serum binding to spike was associated with positive selection for presumptive antibody-escape mutations. Taken together, our findings show remarkable intra-host genetic diversity of SARS-CoV-2 in advanced HIV infection and suggest that adaptive intra-host SARS-CoV-2 evolution in this setting may contribute to the emergence of new variants of concern (VOCs).

Continuous-flow macromolecular sieving in slanted nanofilter array: stochastic model and coupling effect of electrostatic and steric hindrance.

Microfabricated slanted nanofilter arrays are a promising technology for integrated biomolecule analysis systems such as online monitoring and point-of-care quality validation, due to their continuous-flow and one-step operation capability. However, an incomplete understanding of the system limits the performance and wider applications of slanted nanofilter arrays. In this paper, we present rigorous theoretical and experimental studies on macromolecule sieving in a slanted nanofilter array. From both stochastic and kinetic models, an explicit theoretical solution describing size-dependent molecule sieving was derived, which was validated using experimental sieving results obtained for various sieving conditions. Our results not only detail the relationship between sieving conditions and sieving efficiency but also demonstrate that sieving is affected by multiple hindrance effects (electrostatic hindrance), not steric hindrance alone. There is an optimal sieving condition for achieving the greatest separation efficiency for DNAs of a certain size range. Small DNA has great size selectivity in small nanofilters and in weak electric fields, whereas large DNA is present in large nanofilters and in strong electric fields. This study provides insights into designing a slanted nanofilter array for particular target applications and understanding the sieving principles in the nanofilter array.

Continuous Online Titer Monitoring in CHO Cell Culture Supernatant Using a Herringbone Nanofluidic Filter Array.

Online monitoring of monoclonal antibody product titers throughout biologics process development and production enables rapid bioprocess decision-making and process optimization. Conventional analytical methods, including high-performance liquid chromatography and turbidimetry, typically require interfacing with an automated sampling system capable of online sampling and fractionation, which suffers from increased cost, a higher risk of failure, and a higher mechanical complexity of the system. In this study, a novel nanofluidic system for continuous direct (no sample preparation) IgG titer measurements was investigated. Tumor necrosis factor α (TNF-α), conjugated with fluorophores, was utilized as a selective binder for adalimumab in the unprocessed cell culture supernatant. The nanofluidic device can separate the bound complex from unbound TNF-α and selectively concentrate the bound complex for high-sensitivity detection. Based on the fluorescence intensity from the concentrated bound complex, a fluorescence intensity versus titer curve can be generated, which was used to determine the titer of samples from filtered, unpurified Chinese hamster ovary cell cultures continuously. The system performed direct monitoring of IgG titers with nanomolar resolution and showed a good correlation with the biolayer interferometry assays. Furthermore, by variation of the concentration of the indicator (TNF-α), the dynamic range of the system can be tuned and further expanded.

HIV silencing and cell survival signatures in infected T cell reservoirs.

Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1-3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.

SARS-CoV-2 infection and persistence in the human body and brain at autopsy.

Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction1-3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4,5). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain3,6-14. Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.

High-throughput, single-copy sequencing reveals SARS-CoV-2 spike variants coincident with mounting humoral immunity during acute COVID-19.

Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH2-terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting.

High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19.

Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH 2 -terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting. AUTHOR SUMMARY: Mutant sequences of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) arising during any individual case of coronavirus disease 2019 (COVID-19) could theoretically enable the virus to evade immune responses or antiviral therapies that target the predominant infecting virus sequence. However, commonly used sequencing technologies are not optimally designed to detect variant virus sequences within each sample. To address this issue, we developed novel technology for sequencing large numbers of individual SARS-CoV-2 genomic RNA molecules across the region encoding the virus surface proteins. This technology revealed extensive genetic diversity in cultured viruses from a clinical isolate of SARS-CoV-2, but lower diversity in samples from 7 individuals with COVID-19. Importantly, concurrent analysis of paired serum samples in selected individuals revealed relatively low levels of antibody binding to the SARS-CoV-2 spike protein at the time of initial sequencing. With increased serum binding to spike protein, we detected multiple SARS-CoV-2 variants bearing independent mutations in a single epitope, as well as a transient increase in virus burden. These findings suggest that SARS-CoV-2 replication creates sufficient virus genetic diversity to allow immune-mediated selection of variants within the time frame of acute COVID-19. Large-scale studies of SARS-CoV-2 variation and specific immune responses will help define the contributions of intra-individual SARS-CoV-2 evolution to COVID-19 clinical outcomes and antiviral drug susceptibility.

Fc-mediated effector function contributes to the in vivo antiviral effect of an HIV neutralizing antibody.

Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal antibodies (NAbs) leads to a reduction in HIV plasma virus. Both ARVs and NAbs prevent new rounds of viral infection, but NAbs may have the additional capacity to accelerate the loss of virus-infected cells through Fc gamma receptor (FcγR)-mediated effector functions, which should affect the kinetics of plasma-virus decline. Here, we formally test the role of effector function in vivo by comparing the rate and timing of plasma-virus clearance in response to a single-dose treatment with either unmodified NAb or those with either reduced or augmented Fc function. When infused into viremic simian HIV (SHIV)-infected rhesus macaques, there was a 21% difference in slope of plasma-virus decline between NAb and NAb with reduced Fc function. NAb engineered to increase FcγRIII binding and improve antibody-dependent cellular cytotoxicity (ADCC) in vitro resulted in arming of effector cells in vivo, yet led to viral-decay kinetics similar to NAbs with reduced Fc function. These studies show that the predominant mechanism of antiviral activity of HIV NAbs is through inhibition of viral entry, but that Fc function can contribute to the overall antiviral activity, making them distinct from standard ARVs.

VRC34-Antibody Lineage Development Reveals How a Required Rare Mutation Shapes the Maturation of a Broad HIV-Neutralizing Lineage.

Rare mutations have been proposed to restrict the development of broadly neutralizing antibodies against HIV-1, but this has not been explicitly demonstrated. We hypothesized that such rare mutations might be identified by comparing broadly neutralizing and non-broadly neutralizing branches of an antibody-developmental tree. Because sequences of antibodies isolated from the fusion peptide (FP)-targeting VRC34-antibody lineage suggested it might be suitable for such rare mutation analysis, we carried out next-generation sequencing (NGS) on B cell transcripts from donor N123, the source of the VRC34 lineage, and functionally and structurally characterized inferred intermediates along broadly neutralizing and poorly neutralizing developmental branches. The broadly neutralizing VRC34.01 branch required the rare heavy-chain mutation Y33P to bind FP, whereas the early bifurcated VRC34.05 branch did not require this rare mutation and evolved less breadth. Our results demonstrate how a required rare mutation can restrict development and shape the maturation of a broad HIV-1-neutralizing antibody lineage.

Continuous Online Protein Quality Monitoring during Perfusion Culture Production Using an Integrated Micro/Nanofluidic System.

We demonstrate a new micro/nanofluidic system for continuous and automatic monitoring of protein product size and quantity directly from the culture supernatant during a high-cell-concentration CHO cell perfusion culture. A microfluidic device enables clog-free cell retention for a bench-scale (350 mL) perfusion bioreactor that continuously produces the culture supernatant containing monoclonal antibodies (IgG1). A nanofluidic device directly monitors the protein size and quantity in the culture supernatant. The continuous-flow and fully automated operation of this nanofluidic protein analytics reduces design complexity and offers more detailed information on protein products than offline and batch-mode conventional analytics. Moreover, chemical and mechanical robustness of the nanofluidic device enables continuous monitoring for several days to a week. This continuous and online protein quality monitoring could be deployed at different steps and scales of biomanufacturing to improve product quality and manufacturing efficiency.

Effects of a mental fitness positive psychology intervention program on inpatients with schizophrenia in South Korea: A feasibility study.

PURPOSE: This study investigated the effects of a mental fitness positive psychology intervention program on the self-esteem and interpersonal relationship ability of inpatients with schizophrenia. DESIGN AND METHODS: A pretest-posttest nonequivalent control group quasi-experimental design was used. Participants (N = 60) completed scales measuring self-esteem and interpersonal relationship ability. FINDINGS: The program effectively improved participants' self-esteem and interpersonal relationship ability. PRACTICE IMPLICATIONS: Psychiatric nurses can use this program as a nursing intervention to enhance the self-esteem and interpersonal skills of inpatients with schizophrenia in mental health facilities.

Nanofluidic device for continuous multiparameter quality assurance of biologics.

Process analytical technology (PAT) is critical for the manufacture of high-quality biologics as it enables continuous, real-time and on-line/at-line monitoring during biomanufacturing processes. The conventional analytical tools currently used have many restrictions to realizing the PAT of current and future biomanufacturing. Here we describe a nanofluidic device for the continuous monitoring of biologics' purity and bioactivity with high sensitivity, resolution and speed. Periodic and angled nanofilter arrays served as the molecular sieve structures to conduct a continuous size-based analysis of biologics. A multiparameter quality monitoring of three separate commercial biologic samples within 50 minutes has been demonstrated, with 20 µl of sample consumption, inclusive of dead volume in the reservoirs. Additionally, a proof-of-concept prototype system, which integrates an on-line sample-preparation system and the nanofluidic device, was demonstrated for at-line monitoring. Thus, the system is ideal for on-site monitoring, and the real-time quality assurance of biologics throughout the biomanufacturing processes.

Microfluidic Platform for Assessment of Therapeutic Proteins Using Molecular Charge Modulation Enhanced Electrokinetic Concentration Assays.

Therapeutic proteins (TPs) are critical in modern medicine, yet shortage of TPs in disaster situations and remote areas remains a worldwide challenge. Manufacturing and real-time release of TPs on demand at the point-of-care is considered the key to this issue, which requires reliable and rapid analytics techniques for quality assurance. Herein we report a microfluidic platform that could be implemented in-line and at the point-of-care for real-time decision-making about the quality of a TP. The in vivo efficacy and duration of efficacy of TPs were assessed by the equilibrium and kinetics of TP and TP receptor (TPR) binding, using electrokinetic concentration (EC) and molecular charge modulation (MCM). EC can simultaneously concentrate and separate bound and unbound species in an assay based on electrical mobility, allowing for the quantification of binding. MCM enables the application of EC to arbitrary TPs by enhancing the mobility differences between TPs, TPRs, and TP-TPR complexes. This technology is homogeneous and overcomes many practical challenges of conventional heterogeneous assays. We developed various formats of assays for equilibrium and kinetic analysis and rapid determination of degradation of TPs, obtaining results comparable to state-of-the-art technologies with significantly less time (<1 h) and simpler setup. Finally, we demonstrated that the results of MCM-EC based assays correlated well with those from mass spectrometry and cell-based assay, which are the industrial standards for quality testing of TPs.

Smoking Cessation Failure Among Korean Adolescents.

The aim of this study was to identify smoking cessation failure subgroups among Korean adolescents. Participants were 379 smoking adolescents who joined a smoking cessation program. A questionnaire and a cotinine urine test were administered before the program began. Three months after the program ended, the cotinine urine test was repeated. A decision-tree model identified seven subgroups with low or high smoking cessation rates. The predictors of smoking cessation were intention to stop smoking, initiation of smoking, amount of cigarette use, self-efficacy, and paternal smoking status. The subgroup with the lowest smoking cessation rate included adolescents who did not have any intention to stop smoking and who had started smoking after eighth grade, and none of the participants in this group stopped smoking. The results of this study provide crucial information for tailored smoking cessation programs.

Reliability and Validity of the Korean Version of the Dimensions of Tobacco Dependence Scale for Adolescents.

This study assessed the reliability and validity of the Korean version of the Dimensions of Tobacco Dependence Scale (DTDS) for adolescents in Korea. The DTDS, Modified Fagerström Tolerance Questionnaire (M-FTQ), and urine nicotine test were administered to 360 Korean adolescents. The collected data were analyzed using SPSS 21.0 and AMOS 21.0. The construct validity, criterion validity, test-retest reliability, internal consistency reliability, and the area under the curve (AUC) of the Korean version of the DTDS were evaluated. The 4-subscale model of the DTDS (with social, emotional, physical, and sensory subscales) was validated using confirmatory factor analysis, and criterion validity was demonstrated with the M-FTQ. Furthermore, the AUC of the DTDS was 83.1. The Cronbach's α coefficient for internal consistency was .96, demonstrating sufficient test-retest reliability. The Korean version of the DTDS is a reliable and valid measure of tobacco dependence among Korean adolescents.

Lifetime prevalence of and risk factors for suicidal ideation and suicide attempts in a Korean community sample.

OBJECTIVES: Our study evaluated the lifetime prevalence of and risk factors for suicidal ideation and suicide attempts in Jeollabuk-do Province, Korea. METHOD: Participants were selected from the population of individuals aged 13-100 years living Jeollabuk-do Province, Korea. A total of 2,964 subjects provided information about lifetime suicidal behavior and sociodemographic and psychological characteristics, completing the Zung Depression Scale, the Scale for Suicidal Ideation, the Multidimensional Anger Inventory, and the Rosenberg Self-Esteem Scale. RESULTS: The lifetime prevalence of suicidal ideation and suicide attempts, 24.8% and 6.2%, respectively, were higher than in previous studies. Multivariate regression revealed that family harmony had the highest odds ratio of all variables, including psychological factors. Along with depression and self-esteem, anger--which is the basic symptom of the Korean culture-related anger syndrome, Hwa-byung--was significantly associated with lifetime suicidal behavior. CONCLUSIONS: Lifetime suicidal behavior was highly prevalent in Jeollabuk-do Province. The most significant risk factors were found to be social support, family disharmony, anger, depression, and low self-esteem in Koreans.

Predictors of suicidal ideation in a community sample: roles of anger, self-esteem, and depression.

The objective of this cross-sectional study was to investigate the relationships of anger, self-esteem, and depression with suicidal ideation. A survey was conducted in a wide range of community areas across Jeollabuk-do Province, Korea. A total of 2964 subjects (mean age=44.4yr) participated in this study. Hierarchical regression was used to investigate predictors of suicidal ideation in terms of their sociodemographic characteristics, depression, self-esteem, and anger. Hierarchical regression analyses revealed that anger and self-esteem were significantly associated with suicidal ideation regardless of age and after controlling for depression. Moderation analysis showed that the impact of anger on suicidal ideation was significantly greater among females than males in adolescents, but not in other age groups. Additionally, there were some differences in sociodemographic predictors of suicidal ideation among age groups. Predictors included gender and family harmony in adolescents, marital status and family harmony in middle-aged individuals, and economic status and family harmony in elderly individuals. Our results revealed that anger and self-esteem play important roles in suicidal ideation beyond the effect of depression. Development and implementation of preventive strategies, including management of anger and self-esteem, could possibly reduce suicidal ideation and subsequent suicide attempts.

Predictors of suicidal ideation in older individuals receiving home-care services.

OBJECTIVES: Despite the importance of tending to older individuals who are vulnerable to suicide, little is known about suicidal ideation in the portion of this population receiving home-care services in Asian countries. The objective of this cross-sectional study was to examine predictors of suicidal ideation in older individuals using home-care service. METHOD: Participants were randomly selected from the individuals 50 years old and over using home-care services across Jeollabuk-do Province, Korea. A total of 697 subjects participated in this study. Each participant completed the short version of the Geriatric Depression Scale, the Scale for Suicidal Ideation, the Multidimensional Scale of Perceived Social Support, and the World Health Organization Disability Assessment Schedule II. RESULTS: Hierarchical regression analyses revealed that depression, perceived social support, and disability were significant predictors of suicidal ideation, whereas the roles of subjective health status and fish consumption remained ambiguous in this regard. In terms of social support, we also found that less perceived social support from family members was related to higher levels of suicidal ideation. The associations between various categories of disability and suicidal ideation disappeared after controlling for depression. Our investigation of the mediating effect of depression on the relationship between disability and suicidal ideation revealed that depression was either a complete (disability related to cognition, self-care, getting along with others, and life activities) or partial (disability related to participation) mediator. CONCLUSIONS: Preventive strategies focusing on depression, social support, and disability should be emphasized during encounters with older people receiving home-care services.

Multi-vortical flow inducing electrokinetic instability in ion concentration polarization layer.

In this work, we investigated multiple vortical flows inside the ion concentration polarization (ICP) layer that forms due to a coupling of applied electric fields and the semipermeable nanoporous junction between microchannels. While only a primary vortex near perm-selective membrane is traditionally known to lead to electrokinetic instability, multiple vortexes induced by the primary vortex were found to play a major role in the electrokinetic instability. The existence of multiple vortexes was directly confirmed by experiments using particle tracers and interdigitated electrodes were used to measure the local concentration profile inside the ICP layer. At larger applied electric fields, we observed aperiodic fluid motion due to electrokinetic instabilities which develop from a coupling of applied electric fields and electrical conductivity gradients induced by the ICP. The electrokinetic instability at micro-nanofluidic interfaces is important in the development of various electro-chemical-mechanical applications such as fuel cells, bio-analytical preconcentration methods, water purification/desalination and the fundamental study of ion electromigration through nanochannels and nonporous perm-selective membranes.

Nanofluidic preconcentration device in a straight microchannel using ion concentration polarization.

In this paper, we introduce a simple, straight microchannel design for a nanofluidic protein concentration device. Compared with concentration devices previously developed, the anode channel and cathode channel in this new concentration scheme are both integrated into a straight microchannel, with one inlet and one outlet. Most of the functions of a conventional two-channel concentration device can be achieved by this concentration device, and the efficiency of sample accumulation can be controlled by the dimension of the Nafion membrane. Also, the operating mechanism of this device was tested on various material combinations such as PDMS (polydimethyl-siloxane) channel-glass substrate and silicon channel-PDMS substrate. Using a combined PDMS-silicon device which was sealed reversibly without plasma bonding, surface based immunoassay for concentrator-enhanced detection of clinically relevant samples such as C-reactive protein (CRP) was demonstrated. As a result, it was possible to enhance the detection sensitivity of the immunoassay by more than 500 folds compared to the immunoassay without preconcentration process.