Publications by authors named "Mars Stone"

85 Publications

Distinct SARS-CoV-2 antibody reactivity patterns in coronavirus convalescent plasma revealed by a coronavirus antigen microarray.

Sci Rep 2021 04 6;11(1):7554. Epub 2021 Apr 6.

Vaccine Research and Development Center, Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA, USA.

A coronavirus antigen microarray (COVAM) was constructed containing 11 SARS-CoV-2, 5 SARS-1, 5 MERS, and 12 seasonal coronavirus recombinant proteins. The array is designed to measure immunoglobulin isotype and subtype levels in serum or plasma samples against each of the individual antigens printed on the array. We probed the COVAM with COVID-19 convalescent plasma (CCP) collected from 99 donors who recovered from a PCR+ confirmed SARS-CoV-2 infection. The results were analyzed using two computational approaches, a generalized linear model (glm) and random forest (RF) prediction model, to classify individual specimens as either Reactive or non-reactive against the SARS-CoV-2 antigens. A training set of 88 pre-COVID-19 specimens (PreCoV) collected in August 2019 and102 positive specimens from SARS-CoV-2 PCR+ confirmed COVID-19 cases was used for these analyses. Results compared with an FDA emergency use authorized (EUA) SARS-CoV2 S1-based total Ig chemiluminescence immunoassay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and with a SARS-CoV-2 S1-S2 spike-based pseudovirus micro neutralization assay (SARS-CoV-2 reporter viral particle neutralization titration (RVPNT) showed high concordance between the three assays. Three CCP specimens that were negative by the VITROS CoV2T immunoassay were also negative by both COVAM and the RVPNT assay. Concordance between VITROS CoV2T and COVAM was 96%, VITROS CoV2T and RVPNT 93%, and RVPNT and COVAM 91%. The discordances were all weakly reactive samples near the cutoff threshold of the VITROS CoV2T immunoassay. The multiplex COVAM allows CCP to be grouped according to antibody reactivity patterns against 11 SARS-CoV-2 antigens. Unsupervised K-means analysis, via the gap statistics, as well as hierarchical clustering analysis revealed three main clusters with distinct reactivity intensities and patterns. These patterns were not recapitulated by adjusting the VITROS CoV2T or RVPNT assay thresholds. Plasma classified by COVAM reactivity patterns offers potential to improve CCP therapeutic efficacy CoV2T alone. The use of a SARS-CoV-2 antigen array can qualify CCP for administration as a treatment for acute COVID-19, and interrogate vaccine immunogenicity and performance in preclinical, clinical studies, and routine vaccination to identify antibody responses predictive of protection from infection and disease.
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http://dx.doi.org/10.1038/s41598-021-87137-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024395PMC
April 2021

SARS-CoV-2 antibody magnitude and detectability are driven by disease severity, timing, and assay.

medRxiv 2021 Mar 5. Epub 2021 Mar 5.

Serosurveillance studies are critical for estimating SARS-CoV-2 transmission and immunity, but interpretation of results is currently limited by poorly defined variability in the performance of antibody assays to detect seroreactivity over time in individuals with different clinical presentations. We measured longitudinal antibody responses to SARS-CoV-2 in plasma samples from a diverse cohort of 128 individuals over 160 days using 14 binding and neutralization assays. For all assays, we found a consistent and strong effect of disease severity on antibody magnitude, with fever, cough, hospitalization, and oxygen requirement explaining much of this variation. We found that binding assays measuring responses to spike protein had consistently higher correlation with neutralization than those measuring responses to nucleocapsid, regardless of assay format and sample timing. However, assays varied substantially with respect to sensitivity during early convalescence and in time to seroreversion. Variations in sensitivity and durability were particularly dramatic for individuals with mild infection, who had consistently lower antibody titers and represent the majority of the infected population, with sensitivities often differing substantially from reported test characteristics (e.g., amongst commercial assays, sensitivity at 6 months ranged from 33% for ARCHITECT IgG to 98% for VITROS Total Ig). Thus, the ability to detect previous infection by SARS-CoV-2 is highly dependent on the severity of the initial infection, timing relative to infection, and the assay used. These findings have important implications for the design and interpretation of SARS-CoV-2 serosurveillance studies.
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http://dx.doi.org/10.1101/2021.03.03.21251639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941652PMC
March 2021

Selecting COVID-19 convalescent plasma for neutralizing antibody potency using a high-capacity SARS-CoV-2 antibody assay.

Transfusion 2021 04 18;61(4):1160-1170. Epub 2021 Feb 18.

Vitalant Research Institute, Denver, Colorado, USA.

Background: Efficacy of COVID-19 convalescent plasma (CCP) is hypothesized to be associated with the concentration of neutralizing antibodies (nAb) to SARS-CoV-2. High capacity serologic assays detecting binding antibodies (bAb) have been developed; nAb assays are not adaptable to high-throughput testing. We sought to determine the effectiveness of using surrogate bAb signal-to-cutoff ratios (S/Co) in predicting nAb titers using a pseudovirus reporter viral particle neutralization (RVPN) assay.

Methods: CCP donor serum collected by three US blood collectors was tested with a bAb assay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and a nAb RVPN assay. Prediction effectiveness of various CoV2T S/Co criteria was evaluated for RVPN nAb NT titers using receiver operating characteristics.

Results: Seven hundred and fifty-three CCPs were tested with median CoV2T S/Co and NT of 71.2 of 527.5. Proportions of donors with NT over target nAb titers were 86% ≥1:80, 76% ≥1:160, and 62% ≥1:320. Increasing CoV2T S/Co criterion reduced the sensitivity to predict NT titers, while specificity to identify those below increased. As target NT50 titers increase, the CoV2T assay becomes less accurate as a predictor with a decline in positive predictive value and rise in negative predictive value.

Conclusion: Selection of a clinically effective nAb titer will impact availability of CCP. Product release with CoV2T assay S/Co criterion must balance the risk of releasing products below target nAb titers with the cost of false negatives. A two-step testing scheme may be optimal, with nAb testing on CoV2T samples with S/Cos below criterion.
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http://dx.doi.org/10.1111/trf.16321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013397PMC
April 2021

Serosurveillance for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Incidence Using Global Blood Donor Populations.

Clin Infect Dis 2021 01;72(2):254-256

Vitalant Research Institute, Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA.

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http://dx.doi.org/10.1093/cid/ciaa1116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454349PMC
January 2021

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses.

Cell Rep Med 2021 Jan;2(1):100189

The Translational Genomics Research Institute (TGen), Phoenix and Flagstaff, AZ, USA.

The SARS-CoV-2 proteome shares regions of conservation with endemic human coronaviruses (CoVs), but it remains unknown to what extent these may be cross-recognized by the antibody response. Here, we study cross-reactivity using a highly multiplexed peptide assay (PepSeq) to generate an epitope-resolved view of IgG reactivity across all human CoVs in both COVID-19 convalescent and negative donors. PepSeq resolves epitopes across the SARS-CoV-2 Spike and Nucleocapsid proteins that are commonly targeted in convalescent donors, including several sites also recognized in some uninfected controls. By comparing patterns of homologous reactivity between CoVs and using targeted antibody-depletion experiments, we demonstrate that SARS-CoV-2 elicits antibodies that cross-recognize pandemic and endemic CoV antigens at two Spike S2 subunit epitopes. We further show that these cross-reactive antibodies preferentially bind endemic homologs. Our findings highlight sites at which the SARS-CoV-2 response appears to be shaped by previous CoV exposures and which have the potential to raise broadly neutralizing responses.
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http://dx.doi.org/10.1016/j.xcrm.2020.100189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816965PMC
January 2021

SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques.

Nat Commun 2021 01 22;12(1):541. Epub 2021 Jan 22.

Center for Immunology and Infectious Diseases, UC Davis, Davis, CA, USA.

CD4 T follicular helper (T) cells are important for the generation of durable and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates T cells and stimulates the germinal center (GC) response is an important question as we investigate vaccine induced immunity against COVID-19. Here, we report that SARS-CoV-2 infection in rhesus macaques, either infused with convalescent plasma, normal plasma, or receiving no infusion, resulted in transient accumulation of pro-inflammatory monocytes and proliferating T cells with a T1 profile in peripheral blood. CD4 helper cell responses skewed predominantly toward a T1 response in blood, lung, and lymph nodes. SARS-CoV-2 Infection induced GC T cells specific for the SARS-CoV-2 spike and nucleocapsid proteins, and a corresponding early appearance of antiviral serum IgG antibodies. Collectively, the data show induction of GC responses in a rhesus model of mild COVID-19.
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http://dx.doi.org/10.1038/s41467-020-20642-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822826PMC
January 2021

No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells.

BMC Biol 2021 Jan 20;19(1):10. Epub 2021 Jan 20.

Department of Medicine, Division of HIV/AIDS, UCSF, San Francisco, CA, USA.

Background: Single-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures.

Results: Here, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30 min at 4 °C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets.

Conclusions: We demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.
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http://dx.doi.org/10.1186/s12915-020-00941-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816397PMC
January 2021

Relationship between CD4 T cell turnover, cellular differentiation and HIV persistence during ART.

PLoS Pathog 2021 Jan 19;17(1):e1009214. Epub 2021 Jan 19.

Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, California, United States of America.

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.
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http://dx.doi.org/10.1371/journal.ppat.1009214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846027PMC
January 2021

Analysis of SARS-CoV-2 antibodies in COVID-19 convalescent blood using a coronavirus antigen microarray.

Nat Commun 2021 01 4;12(1). Epub 2021 Jan 4.

Division of Infectious Diseases, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.
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http://dx.doi.org/10.1038/s41467-020-20095-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782488PMC
January 2021

Streamlined Subpopulation, Subtype, and Recombination Analysis of HIV-1 Half-Genome Sequences Generated by High-Throughput Sequencing.

mSphere 2020 10 14;5(5). Epub 2020 Oct 14.

Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA

High-throughput sequencing (HTS) has been widely used to characterize HIV-1 genome sequences. There are no algorithms currently that can directly determine genotype and quasispecies population using short HTS reads generated from long genome sequences without additional software. To establish a robust subpopulation, subtype, and recombination analysis workflow, we amplified the HIV-1 3'-half genome from plasma samples of 65 HIV-1-infected individuals and sequenced the entire amplicon (∼4,500 bp) by HTS. With direct analysis of raw reads using HIVE-hexahedron, we showed that 48% of samples harbored 2 to 13 subpopulations. We identified various subtypes (17 A1s, 4 Bs, 27 Cs, 6 CRF02_AGs, and 11 unique recombinant forms) and defined recombinant breakpoints of 10 recombinants. These results were validated with viral genome sequences generated by single genome sequencing (SGS) or the analysis of consensus sequence of the HTS reads. The HIVE-hexahedron workflow is more sensitive and accurate than just evaluating the consensus sequence and also more cost-effective than SGS. The highly recombinogenic nature of human immunodeficiency virus type 1 (HIV-1) leads to recombination and emergence of quasispecies. It is important to reliably identify subpopulations to understand the complexity of a viral population for drug resistance surveillance and vaccine development. High-throughput sequencing (HTS) provides improved resolution over Sanger sequencing for the analysis of heterogeneous viral subpopulations. However, current methods of analysis of HTS reads are unable to fully address accurate population reconstruction. Hence, there is a dire need for a more sensitive, accurate, user-friendly, and cost-effective method to analyze viral quasispecies. For this purpose, we have improved the HIVE-hexahedron algorithm that we previously developed with short sequences to analyze raw HTS short reads. The significance of this study is that our standalone algorithm enables a streamlined analysis of quasispecies, subtype, and recombination patterns from long HIV-1 genome regions without the need of additional sequence analysis tools. Distinct viral populations and recombination patterns identified by HIVE-hexahedron are further validated by comparison with sequences obtained by single genome sequencing (SGS).
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http://dx.doi.org/10.1128/mSphere.00551-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565892PMC
October 2020

10-year analysis of human immunodeficiency virus incidence in first-time and repeat donors in Brazil.

Vox Sang 2021 Feb 30;116(2):207-216. Epub 2020 Sep 30.

Departamento de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.

Background And Objectives: Incidence in first-time and repeat blood donors is an important measure of transfusion-transmitted HIV infection (TT-HIV) risk. This study assessed HIV incidence over time at four large blood centres in Brazil.

Materials And Methods: Donations were screened and confirmed using serological assays for HIV from 2007 to 2016, and additionally screened by nucleic acid testing from 2011 forward. Limiting antigen (LAg) avidity testing was conducted on HIV seroreactive samples from first-time donors to classify whether an infection was recently acquired. We calculated incidence in first-time donors using the mean duration of recent infection and in repeat donors using classical methods. Time and demographic trends were assessed using Poisson regression.

Results: Over the 10-year period, HIV incidence in first-time donors was highest in Recife (45·1/100 000 person-years (10 py)) followed by São Paulo (32·2/10 py) and then Belo Horizonte (23·3/10 py), and in repeat donors was highest in Recife (33·2/10 py), Belo Horizonte (27·5/10 py) and São Paulo (17·0/10 py). Results from Rio de Janeiro were available from 2013 to 2016 with incidence in first-time donors of 35·9/10 py and repeat donors from 2011 to 2016 of 29·2/10 py. Incidence varied by other donor demographics. When incidence was considered in 2-year intervals, no significant trend was evident. Overall residual risk of TT-HIV was 5·46 and 7·41 per million units of pRBC and FFP transfused, respectively.

Conclusion: HIV incidence in both first-time and repeat donors varied by region in Brazil. Clear secular trends were not evident.
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http://dx.doi.org/10.1111/vox.13002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019535PMC
February 2021

ReScan, a Multiplex Diagnostic Pipeline, Pans Human Sera for SARS-CoV-2 Antigens.

Cell Rep Med 2020 Oct 24;1(7):100123. Epub 2020 Sep 24.

Weill Institute for Neurosciences, Department of Neurology, University of California, San Francisco, San Francisco, CA, USA.

Comprehensive understanding of the serological response to SARS-CoV-2 infection is important for both pathophysiologic insight and diagnostic development. Here, we generate a pan-human coronavirus programmable phage display assay to perform proteome-wide profiling of coronavirus antigens enriched by 98 COVID-19 patient sera. Next, we use ReScan, a method to efficiently sequester phage expressing the most immunogenic peptides and print them onto paper-based microarrays using acoustic liquid handling, which isolates and identifies nine candidate antigens, eight of which are derived from the two proteins used for SARS-CoV-2 serologic assays: spike and nucleocapsid proteins. After deployment in a high-throughput assay amenable to clinical lab settings, these antigens show improved specificity over a whole protein panel. This proof-of-concept study demonstrates that ReScan will have broad applicability for other emerging infectious diseases or autoimmune diseases that lack a valid biomarker, enabling a seamless pipeline from antigen discovery to diagnostic using one recombinant protein source.
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http://dx.doi.org/10.1016/j.xcrm.2020.100123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513813PMC
October 2020

Replicate Aptima Assay for Quantifying Residual Plasma Viremia in Individuals on Antiretroviral Therapy.

J Clin Microbiol 2020 11 18;58(12). Epub 2020 Nov 18.

Vitalant Research Institute, San Francisco, California, USA.

Detection of residual plasma viremia in antiretroviral therapy (ART)-suppressed HIV-infected individuals is critical for characterizing the latent reservoir and evaluating the impact of cure interventions. Ultracentrifugation-based single-copy assays are sensitive but labor intensive. Fully automated replicate testing using a standard clinical viral load assay was evaluated as a high-throughput alternative for the quantification of low-level viremia. Four plasma samples from blood donors with acute HIV-1 infection and one viral culture supernatant were serially diluted into 25-ml samples to nominal viral loads ranging from 39 to <0.5 copies (cp)/ml. Each dilution was tested with 45 replicates (reps) using 0.5 ml/rep with the Aptima HIV-1 Quant assay. The nominal and estimated viral loads based on the single-hit Poisson model were compared, and a hybrid Poisson digital model for calibrated viral load estimation was derived. Testing performed using 45 reps on longitudinal plasma samples from 50 ART-suppressed individuals in the Reservoir Assay Validation and Evaluation Network (RAVEN) study cohort (range of 1 to 19 years of continuous ART suppression) showed a median viral load of 0.54 cp/ml (interquartile range [IQR], 0.22 to 1.46 cp/ml) and a 14% (95% confidence interval [CI], 9% to 19%) decline in viral load for each additional year in duration suppressed. Within the RAVEN cohort, the expected false-negative rate for detection at lower rep numbers using 9 and 18 reps was 26% and 14%, respectively. Residual plasma viremia levels positively correlated with cell-associated HIV RNA and DNA. The performance characteristics of the replicate Aptima assay support its use for quantifying residual plasma viremia to study the latent HIV reservoir and cure interventions.
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http://dx.doi.org/10.1128/JCM.01400-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685884PMC
November 2020

Automated Multireplicate Quantification of Persistent HIV-1 Viremia in Individuals on Antiretroviral Therapy.

J Clin Microbiol 2020 11 18;58(12). Epub 2020 Nov 18.

Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

Clearance of low-level viremia that persists in most HIV-1-positive individuals on antiretroviral therapy (ART) is an important milestone for efforts to cure HIV-1 infection. The level of persistent viremia on ART is generally below the lower limit of quantification (LOQ) of current FDA-cleared plasma HIV-1 RNA assays (20 to 40 copies/ml) but can be quantified by reverse transcriptase PCR (RT-PCR) assays with single-copy sensitivity. Such assays require multistep manual methods, and their low throughput limits the capacity to monitor the effects of interventions on persistent viremia. Recently, S. Bakkour, X. Deng, P. Bacchetti, E. Grebe, et al. (J Clin Microbiol 58:e01400-20, 2020, https://doi.org/10.1128/JCM.01400-20), reported the use of multiple replicates and Poisson statistics to infer HIV-1 RNA concentrations below the commercial LOQ of an automated platform (Hologic Panther Aptima). Here, we evaluate the detection and quantitation of low-level viremia using the following two adaptions of the automated platform: a multireplicate strategy (9×) and a concentrated single-replicate strategy in which 5 ml of plasma is concentrated by centrifugation (1×, concentrated). We compare these new methods to a recently reported manual -targeting single-copy assay version 2 (iSCA v2). Using laboratory-generated HIV-1 RNA plasma samples at known concentrations, all three methods had similar sensitivity for HIV-1 RNA detection, although iSCA v2 was most sensitive (95% LOD, 2.3 copies/ml), 9× was marginally less sensitive (95% LOD, 3.0 copies/ml), and 1×, concentrated was least sensitive (95% LOD, 3.9 copies/ml). In contrast, for clinical plasma samples, 9× had greater sensitivity than iSCA v2 (82% of samples were quantifiable compared with 62% of samples by iSCA v2). These results support 9× as an acceptable high-throughput alternative to iSCA v2 for quantifying low-level viremia in individuals on ART.
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http://dx.doi.org/10.1128/JCM.01442-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685899PMC
November 2020

SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood.

Nat Commun 2020 09 17;11(1):4698. Epub 2020 Sep 17.

Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA.

Given the limited availability of serological testing to date, the seroprevalence of SARS-CoV-2-specific antibodies in different populations has remained unclear. Here, we report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seroreactivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors in early April 2020. We additionally describe the longitudinal dynamics of immunoglobulin-G (IgG), immunoglobulin-M (IgM), and in vitro neutralizing antibody titers in COVID-19 patients. The median time to seroconversion ranged from 10.3-11.0 days for these 3 assays. Neutralizing antibodies rose in tandem with immunoglobulin titers following symptom onset, and positive percent agreement between detection of IgG and neutralizing titers was >93%. These findings emphasize the importance of using highly accurate tests for surveillance studies in low-prevalence populations, and provide evidence that seroreactivity using SARS-CoV-2 anti-nucleocapsid protein IgG and anti-spike IgM assays are generally predictive of in vitro neutralizing capacity.
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http://dx.doi.org/10.1038/s41467-020-18468-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499171PMC
September 2020

SARS-CoV-2 infection induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques.

Res Sq 2020 Aug 14. Epub 2020 Aug 14.

University of California Davis School of Veterinary Medicine and California National Primate Research Center.

CD4 T follicular helper (T ) cells are important for the generation of durable and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates T cells and stimulates the germinal center response is an important question as we investigate vaccine options for the current pandemic. Here we report that SARS-CoV-2 infection resulted in transient accumulation of pro-inflammatory monocytes and proliferating T cells with a T 1 profile in peripheral blood. CD4 helper cell responses were skewed predominantly toward a T 1 response in blood, lung, and lymph nodes. We observed the generation of germinal center T cells specific for the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, and a corresponding early appearance of antiviral serum IgG antibodies. Our data suggest that a vaccine promoting T 1-type T responses that target the S protein may lead to protective immunity.
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http://dx.doi.org/10.21203/rs.3.rs-51545/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430596PMC
August 2020

Proteome-Wide Zika Virus CD4 T Cell Epitope and HLA Restriction Determination.

Immunohorizons 2020 08 4;4(8):444-453. Epub 2020 Aug 4.

Department of Medicine, University of Washington, Seattle, WA 98195;

Zika virus (ZIKV) is a mosquito-borne pathogen that caused an epidemic in 2015-2016. ZIKV-specific T cell responses are functional in animal infection models, and helper CD4 T cells promote avid Abs in the vaccine context. The small volumes of blood available from field research limit the determination of T cell epitopes for complex microbes such as ZIKV. The goal of this project was efficient determination of human ZIKV CD4 T cell epitopes at the whole proteome scale, including validation of reactivity to whole pathogen, using small blood samples from convalescent time points when T cell response magnitude may have waned. Polyclonal enrichment of candidate ZIKV-specific CD4 T cells used cell-associated virus, documenting that T cells in downstream peptide analyses also recognize whole virus after Ag processing. Sequential query of bulk ZIKV-reactive CD4 T cells with pooled/single ZIKV peptides and molecularly defined APC allowed precision epitope and HLA restriction assignments across the ZIKV proteome and enabled discovery of numerous novel ZIKV CD4 T cell epitopes. The research workflow is useful for the study of emerging infectious diseases with a very limited human blood sample availability.
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http://dx.doi.org/10.4049/immunohorizons.2000068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839664PMC
August 2020

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with an endemic human CoV.

bioRxiv 2020 Jul 27. Epub 2020 Jul 27.

The Translational Genomics Research Institute (TGen), Phoenix and Flagstaff, AZ, USA.

A high-resolution understanding of the antibody response to SARS-CoV-2 is important for the design of effective diagnostics, vaccines and therapeutics. However, SARS-CoV-2 antibody epitopes remain largely uncharacterized, and it is unknown whether and how the response may cross-react with related viruses. Here, we use a multiplexed peptide assay ('PepSeq') to generate an epitope-resolved view of reactivity across all human coronaviruses. PepSeq accurately detects SARS-CoV-2 exposure and resolves epitopes across the Spike and Nucleocapsid proteins. Two of these represent recurrent reactivities to conserved, functionally-important sites in the Spike S2 subunit, regions that we show are also targeted for the endemic coronaviruses in pre-pandemic controls. At one of these sites, we demonstrate that the SARS-CoV-2 response strongly and recurrently cross-reacts with the endemic virus hCoV-OC43. Our analyses reveal new diagnostic and therapeutic targets, including a site at which SARS-CoV-2 may recruit common pre-existing antibodies and with the potential for broadly-neutralizing responses.
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http://dx.doi.org/10.1101/2020.07.27.222943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386487PMC
July 2020

HIV incidence in US first-time blood donors and transfusion risk with a 12-month deferral for men who have sex with men.

Blood 2020 09;136(11):1359-1367

Vitalant Research Institute, San Francisco, CA.

In 2015, the US Food and Drug Administration published revised guidance that recommended a change in blood donor deferral of men who have sex with men (MSM) from an indefinite to a 12-month deferral since the donor last had sex with a man. We assessed whether HIV incidence in first-time blood donors or associated transfusion risk increased. Donations in 4 major blood collection organizations were monitored for 15 months before and 2 years after implementation of the 12-month MSM deferral policy. HIV-positive donations were classified as recently acquired or long-term using a recent infection testing algorithm and incidence in both periods estimated. Residual transfusion transmission risk was estimated by multiplying incidence by the length of the infectious window period. The latter was estimated using a model based on infectious dose and the sensitivity of nucleic acid testing. Factors associated with incident infection in each period were assessed using Poisson regression. Overall HIV incidence in first-time donors before implementation of the 12-month MSM deferral was estimated at 2.62 cases per 100 000 person-years (105 PY) (95% credible interval [CI], 1.53-3.93 cases/105 PY), and after implementation at 2.85 cases/105 PY (95% CI, 1.96-3.93 cases/105 PY), with no statistically significant change. In male first-time donors, the incidence difference was 0.93 cases/105 PY (95% CI, -1.74-3.58 cases/105 PY). The residual risk of HIV transfusion transmission through components sourced from first-time donors was estimated at 0.32 transmissions per million (106) packed red blood cell transfusions (95% CI, 0.29-0.65 transmissions/106 transfusions) before and 0.35 transmissions/106 transfusions (95% CI, 0.31-0.65 transmissions/106 transfusions) after implementation. The difference was not statistically significant. Factors associated with incident infection were the same in each period. We observed no increase in HIV incidence or HIV transfusion transmission risk after implementation of a 12-month MSM deferral policy.
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http://dx.doi.org/10.1182/blood.2020007003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483431PMC
September 2020

SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques.

bioRxiv 2020 Jul 8. Epub 2020 Jul 8.

CD4 T follicular helper (T ) cells are important for the generation of long-lasting and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates T cells and stimulates the germinal center response is an important question as we investigate vaccine options for the current pandemic. Here we report that, following infection with SARS-CoV-2, adult rhesus macaques exhibited transient accumulation of activated, proliferating T cells in their peripheral blood on a transitory basis. The CD4 helper cell responses were skewed predominantly toward a T 1 response in blood, lung, and lymph nodes, reflective of the interferon-rich cytokine environment following infection. We also observed the generation of germinal center T cells specific for the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, and a corresponding early appearance of antiviral serum IgG antibodies but delayed or absent IgA antibodies. Our data suggest that a vaccine promoting Th1-type Tfh responses that target the S protein may lead to protective immunity.
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http://dx.doi.org/10.1101/2020.07.07.191007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359530PMC
July 2020

Evolving viral and serological stages of Zika virus RNA-positive blood donors and estimation of incidence of infection during the 2016 Puerto Rican Zika epidemic: an observational cohort study.

Lancet Infect Dis 2020 12 13;20(12):1437-1445. Epub 2020 Jul 13.

Vitalant Research Institute, San Francisco, CA, USA.

Background: Puerto Rico began screening blood donations for Zika virus RNA with nucleic acid amplification tests (NAATs) on April 3, 2016, because of an emerging Zika virus outbreak. We followed up positive donors to assess the dynamics of viral and serological markers during the early stages of Zika virus infection and update the estimate of infection incidence in the Puerto Rican population during the outbreak.

Methods: Blood donations from volunteer donors in Puerto Rico were screened for the presence of Zika virus RNA using the cobas Zika NAAT. Positive donations were further tested to confirm infection, estimate viral load, and identify Zika virus-specific IgM antibodies. Individuals with positive blood donations were invited to attend follow-up visits. Donations with confirmed infection (defined as detection of Zika virus RNA or IgM on additional testing of index or follow-up samples) were assessed for stage of infection according to Zika virus RNA detectability in simulated minipools, viral load, and Zika virus IgM status. A three-step process was used to estimate the mean duration of NAAT reactivity of Zika virus in human plasma from individuals identified pre-seroconversion with at least one follow up visit and to update the 2016 incidence estimate of Zika virus infection.

Findings: Between April 3 and Dec 31, 2016, 53 112 blood donations were screened for Zika virus, of which 351 tested positive, 339 had confirmed infections, and 319 could be staged. Compared with IgM-positive index donations (n=110), IgM-negative index donations (n=209) had higher mean viral loads (1·1 × 10vs 8·3 × 10 international units per mL) and were more likely to be detected in simulated minipools (93% [n=194] vs 26% [n=29]). The proportions of donations with confirmed infections that had viral RNA detected only in individual-donation NAATs (ie, not in simulated minipools) and were IgM positive increased as the epidemic evolved. The estimated mean duration of NAAT detectability in the 140 donors included in the follow-up study was 11·70 days (95% CI 10·06-14·36). Applying this detection period to the observed proportion of donations that were confirmed NAAT positive yielded a Zika virus seasonal incidence estimate of 21·1% (95% CI 18·1-24·1); 768 101 infections in a population of 3 638 773 in 2016.

Interpretation: Characterisation of early Zika virus infection has implications for blood safety because infectivity of blood donations and utility of screening methods likely correlate with viral load and serological stage of infection. Our findings also have implications for diagnostic testing, public health surveillance, and epidemiology, and we estimate that around 21% of the Puerto Rican population was infected during the 2016 outbreak.

Funding: Biomedical Advanced Research and Development Authority, National Heart, Lung, and Blood Institute.
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http://dx.doi.org/10.1016/S1473-3099(19)30706-6DOI Listing
December 2020

Zika virus RNA and IgM persistence in blood compartments and body fluids: a prospective observational study.

Lancet Infect Dis 2020 12 13;20(12):1446-1456. Epub 2020 Jul 13.

Vitalant Research Institute, San Francisco, CA, USA; Department of Laboratory Medicine, University of California, San Francisco, CA, USA.

Background: Characterisation of the dynamics of Zika virus persistence following acute infection is needed to inform blood donor and diagnostic testing policies and understand the natural history of Zika virus infection. We aimed to characterise the natural history, persistence, and clinical outcomes of Zika virus infection through a prospective study in initially asymptomatic Zika virus RNA-positive blood donors.

Methods: Zika virus-infected blood donors identified through Zika virus nucleic acid amplification test (NAAT) screening at three blood collection organisations in the USA were enrolled into a 1-year follow-up study, with blood and body fluid samples and detailed symptom data collected at up to seven visits. All samples were tested for Zika virus RNA by real-time PCR (rtPCR); follow-up plasma, whole blood, and urine were also tested by replicate NAAT. Plasma was tested for flavivirus-specific IgM and IgG by ELISA. Zika virus RNA persistence for each assay or sample type and plasma antibody persistence from estimated date of plasma NAAT-detectable infection were calculated from follow-up data using survival statistical methods.

Findings: Between July 6, 2016 and March 7, 2017, we enrolled 53 participants. From the estimated date of plasma NAAT-detectable infection, Zika virus RNA was detectable in plasma for 9·9 days (95% CI 8·1-12·0), in red blood cells for 95·4 days (62·8-129·1), and in whole blood for 73·5 days (39·8-107·5). Replicate NAATs (one or more of eight replicates positive) extended detection of Zika virus RNA in plasma to 34·8 days (19·9-56·2) and in whole blood (at least one of two tests positive) to 104·8 days (76·7-129·9). Urine was rtPCR reactive up to 14·5 days (10·5-20·3) and saliva up to 26·4 days (19·7-38·7). Zika virus IgM persisted for 237·7 days (128·7-459·5) from estimated time since plasma NAAT-detectable infection. Zika virus RNA fell below detectable limits more rapidly in the saliva of participants with pre-existing dengue virus IgG than in those without. Of 25 donors identified pre-seroconversion with symptom data at the first or second study visit, 16 (64%) developed multiple Zika virus-related symptoms after asymptomatic index donations, compared with nine (36%) of 25 donors detected after seroconversion.

Interpretation: Determination of viral marker persistence is enhanced by follow-up of blood donors who are pre-symptomatic or asymptomatic, Zika virus RNA-positive, and antibody negative. Zika virus RNA persists in red blood cells for several months following clearance from plasma and body fluids, and replicate, highly sensitive NAATs extend RNA detection in all compartments. Whole blood testing can extend detection of acute infection for diagnostics and monitoring of pregnant women, sexual partners, and travellers.

Funding: National Heart, Lung, and Blood Institute, Biomedical Advanced Research and Development Authority.
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http://dx.doi.org/10.1016/S1473-3099(19)30708-XDOI Listing
December 2020

HIV antiretroviral therapy and prevention use in US blood donors: a new blood safety concern.

Blood 2020 09;136(11):1351-1358

Vitalant Research Institute, San Francisco, CA.

Antiretroviral therapy (ART) to treat and pre-exposure prophylaxis (PrEP) to prevent HIV infection are effective tools to help end the HIV epidemic. However, their use could affect HIV transfusion-transmission risk. Three different ART/PrEP prevalence analyses in blood donors were conducted. First, blood samples from HIV-positive and a comparison group of infection-nonreactive donors were tested under blind using liquid chromatography-tandem mass spectrometry for ART. Second, blood donor samples from infection-nonreactive, 18- to 45-year-old, male, first-time blood donors in 6 US locations were tested for emtricitabine and tenofovir. Third, in men who have sex with men (MSM) participating in the 2017 Centers for Disease Control and Prevention National HIV Behavioral Surveillance (NHBS) from 5 US cities, self-reported PrEP use proximate to donation was assessed. In blind testing, no ART was detected in 300 infection-nonreactive donor samples, but in 299 HIV confirmed-infected donor samples, 46 (15.4%; 95% confidence interval [CI], 11.5% to 20.0%) had evidence of ART. Of the 1494 samples tested from first-time male donors, 9 (0.6%; 95% CI, 0.03% to 1.1%) had tenofovir and emtricitabine. In the NHBS MSM survey, 27 of 591 respondents (4.8%; 95% CI, 3.2% to 6.9%) reported donating blood in 2016 or 2017 and PrEP use within the same time frame as blood donation. Persons who are HIV positive and taking ART and persons taking PrEP to prevent HIV infection are donating blood. Both situations could lead to increased risk of HIV transfusion transmission if blood screening assays are unable to detect HIV in donations from infected donors.
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http://dx.doi.org/10.1182/blood.2020006890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483439PMC
September 2020

Detection of SARS-CoV-2 neutralizing antibodies with a cell-free PCR assay.

medRxiv 2020 Jun 2. Epub 2020 Jun 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 4 million confirmed infections worldwide and over 300,000 deaths. While Remdesivir has recently received FDA emergency use authorization for treatment of SARS-CoV-2 infection, convalescent plasma (CP) with high titers of SARS-CoV-2 neutralizing antibodies (NAbs) from recovered donors remains a promising and widely accessible method to mitigate severe disease symptoms. Here, we describe the development and validation of a cell-free neutralization PCR assay using SARS-CoV-2 spike protein S1 and human ACE2 receptor-DNA conjugates. By comparing with samples collected prior to the outbreak, we confirmed that NAbs were specifically detected in COVID-19 cases. Using our unique assay, the NAb signals are detectable as early as 10 days after onset of symptoms and continue to rise, plateauing after 18 days. Notably, we showed that the use of licensed pathogen reduction technology to inactivate potentially contaminating infectious pathogens in CP did not alter NAb signals, paving a path to safely administer effective CP therapies. The described neutralization PCR assay can serve as a qualification tool to easily identify suitable CP donors of a potentially lifesaving therapy. In addition, this assay tool is readily deployable in standard laboratories with biosafety level 2 capability, and can yield results within 2-3 hr. This advancement can facilitate research on factors driving diverse COVID-19 disease manifestations, and to evaluate the impact of various CP processing protocols on CP therapeutic efficacy.
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http://dx.doi.org/10.1101/2020.05.28.20105692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302305PMC
June 2020

SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood from the San Francisco Bay Area.

medRxiv 2020 May 25. Epub 2020 May 25.

Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA.

We report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seropositivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors. We additionally describe the longitudinal dynamics of immunoglobulin-G, immunoglobulin-M, and in vitro neutralizing antibody titers in COVID-19 patients. Neutralizing antibodies rise in tandem with immunoglobulin levels following symptom onset, exhibiting median time to seroconversion within one day of each other, and there is >93% positive percent agreement between detection of immunoglobulin-G and neutralizing titers.
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http://dx.doi.org/10.1101/2020.05.19.20107482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273245PMC
May 2020

Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Plasma using a Coronavirus Antigen Microarray.

bioRxiv 2020 Apr 17. Epub 2020 Apr 17.

Division of Infectious Diseases, Department of Medicine, University of California Irvine Health, Orange, CA.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates complete discrimination of these two groups. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.
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http://dx.doi.org/10.1101/2020.04.15.043364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217240PMC
April 2020

Stored RBC metabolism as a function of caffeine levels.

Transfusion 2020 Jun 11;60(6):1197-1211. Epub 2020 May 11.

Vitalant Research Institute, San Francisco, California.

Background: Coffee consumption is extremely common in the United States. Coffee is rich with caffeine, a psychoactive, purinergic antagonist of adenosine receptors, which regulate red blood cell energy and redox metabolism. Since red blood cell (purine) metabolism is a critical component to the red cell storage lesion, here we set out to investigate whether caffeine levels correlated with alterations of energy and redox metabolism in stored red blood cells.

Study Design And Methods: We measured the levels of caffeine and its main metabolites in 599 samples from the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study via ultra-high-pressure-liquid chromatography coupled to high-resolution mass spectrometry and correlated them to global metabolomic and lipidomic analyses of RBCs stored for 10, 23, and 42 days.

Results: Caffeine levels positively correlated with increased levels of the main red cell antioxidant, glutathione, and its metabolic intermediates in glutathione-dependent detoxification pathways of oxidized lipids and sugar aldehydes. Caffeine levels were positively correlated with transamination products and substrates, tryptophan, and indole metabolites. Expectedly, since caffeine and its metabolites belong to the family of xanthine purines, all xanthine metabolites were significantly increased in the subjects with the highest levels of caffeine. However, high-energy phosphate compounds ATP and DPG were not affected by caffeine levels, despite decreases in glucose oxidation products-both via glycolysis and the pentose phosphate pathway.

Conclusion: Though preliminary, this study is suggestive of a beneficial correlation between the caffeine levels and improved antioxidant capacity of stored red cells.
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http://dx.doi.org/10.1111/trf.15813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990510PMC
June 2020

Ethyl glucuronide, a marker of alcohol consumption, correlates with metabolic markers of oxidant stress but not with hemolysis in stored red blood cells from healthy blood donors.

Transfusion 2020 Jun 8;60(6):1183-1196. Epub 2020 May 8.

Vitalant Research Institute, San Francisco, California.

Background: Red blood cell (RBC) storage in the blood bank is associated with the progressive accumulation of oxidant stress. While the mature erythrocyte is well equipped to cope with such stress, recreative habits like alcohol consumption may further exacerbate the basal level of oxidant stress and contribute to the progress of the storage lesion.

Study Design And Methods: RBC levels of ethyl glucuronide, a marker of alcohol consumption, were measured via ultra-high-pressure liquid chromatography coupled with high-resolution mass spectrometry. Analyses were performed on 599 samples from the recalled donor population at Storage Days 10, 23, and 42 (n = 250), as part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study. This cohort consisted of the 5th and 95th percentile of donors with extreme hemolytic propensity out of the original cohort of 13,403 subjects enrolled in the REDS-III RBC Omics study. Ehtyl glucuronide levels were thus correlated to global metabolomics and lipidomics analyses and RBC hemolytic propensity.

Results: Ethyl glucuronide levels were positively associated with oxidant stress markers, including glutathione consumption and turnover, methionine oxidation, S-adenosylhomocysteine accumulation, purine oxidation, and transamination markers. Decreases in glycolysis and energy metabolism, the pentose phosphate pathway and ascorbate system were observed in those subjects with the highest levels of ethyl glucuronide, though hemolysis values were comparable between groups.

Conclusion: Though preliminary, this study is suggestive that markers of alcohol consumption are associated with increases in oxidant stress and decreases in energy metabolism with no significant impact on hemolytic parameters in stored RBCs from healthy donor volunteers.
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http://dx.doi.org/10.1111/trf.15811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7967801PMC
June 2020

Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers.

Transfusion 2020 Jun 8;60(6):1160-1174. Epub 2020 May 8.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, Colorado.

Background: Cigarette smoking is a frequent habit across blood donors (approx. 13% of the donor population), that could compound biologic factors and exacerbate oxidant stress to stored red blood cells (RBCs).

Study Design And Methods: As part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, a total of 599 samples were sterilely drawn from RBC units stored under blood bank conditions at Storage Days 10, 23, and 42 days, before testing for hemolysis parameters and metabolomics. Quantitative measurements of nicotine and its metabolites cotinine and cotinine oxide were performed against deuterium-labeled internal standards.

Results: Donors whose blood cotinine levels exceeded 10 ng/mL (14% of the tested donors) were characterized by higher levels of early glycolytic intermediates, pentose phosphate pathway metabolites, and pyruvate-to-lactate ratios, all markers of increased basal oxidant stress. Consistently, increased glutathionylation of oxidized triose sugars and lipid aldehydes was observed in RBCs donated by nicotine-exposed donors, which were also characterized by increased fatty acid desaturation, purine salvage, and methionine oxidation and consumption via pathways involved in oxidative stress-triggered protein damage-repair mechanisms.

Conclusion: RBCs from donors with high levels of nicotine exposure are characterized by increases in basal oxidant stress and decreases in osmotic hemolysis. These findings indicate the need for future clinical studies aimed at addressing the impact of smoking and other sources of nicotine (e.g., nicotine patches, snuff, vaping, secondhand tobacco smoke) on RBC storage quality and transfusion efficacy.
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http://dx.doi.org/10.1111/trf.15812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960685PMC
June 2020

Impact of taurine on red blood cell metabolism and implications for blood storage.

Transfusion 2020 Jun 27;60(6):1212-1226. Epub 2020 Apr 27.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus Denver, Aurora, Colorado, USA.

Background: Taurine is an antioxidant that is abundant in some common energy drinks. Here we hypothesized that the antioxidant activity of taurine in red blood cells (RBCs) could be leveraged to counteract storage-induced oxidant stress.

Study Design And Methods: Metabolomics analyses were performed on plasma and RBCs from healthy volunteers (n = 4) at baseline and after consumption of a whole can of a common, taurine-rich (1000 mg/serving) energy drink. Reductionistic studies were also performed by incubating human RBCs with taurine ex vivo (unlabeled or C N-labeled) at increasing doses (0, 100, 500, and 1000 μmol/L) at 37°C for up to 16 hours, with and without oxidant stress challenge with hydrogen peroxide (0.1% or 0.5%). Finally, we stored human and murine RBCs under blood bank conditions in additives supplemented with 500 μmol/L taurine, before metabolomics and posttransfusion recovery studies.

Results: Consumption of energy drinks increased plasma and RBC levels of taurine, which was paralleled by increases in glycolysis and glutathione (GSH) metabolism in the RBC. These observations were recapitulated ex vivo after incubation with taurine and hydrogen peroxide. Taurine levels in the RBCs from the REDS-III RBC-Omics donor biobank were directly proportional to the total levels of GSH and glutathionylated metabolites and inversely correlated to oxidative hemolysis measurements. Storage of human RBCs in the presence of taurine improved energy and redox markers of storage quality and increased posttransfusion recoveries in FVB mice.

Conclusion: Taurine modulates RBC antioxidant metabolism in vivo and ex vivo, an observation of potential relevance to transfusion medicine.
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http://dx.doi.org/10.1111/trf.15810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995806PMC
June 2020