Convergent evolution and targeting of diverse E2 epitopes by human broadly neutralizing antibodies are associated with HCV clearance.

The early appearance of broadly neutralizing antibodies (bNAbs) in serum is associated with spontaneous hepatitis C virus (HCV) clearance, but to date, the majority of bNAbs have been isolated from chronically infected donors. Most of these bNAbs use the VH1-69 gene segment and target the envelope glycoprotein E2 front layer. Here, we performed longitudinal B cell receptor (BCR) repertoire analysis on an elite neutralizer who spontaneously cleared multiple HCV infections. We isolated 10,680 E2-reactive B cells, performed BCR sequencing, characterized monoclonal B cell cultures, and isolated bNAbs. In contrast to what has been seen in chronically infected donors, the bNAbs used a variety of VH genes and targeted at least three distinct E2 antigenic sites, including sites previously thought to be non-neutralizing. Diverse front-layer-reactive bNAb lineages evolved convergently, acquiring breadth-enhancing somatic mutations. These findings demonstrate that HCV clearance-associated bNAbs are genetically diverse and bind distinct antigenic sites that should be the target of vaccine-induced bNAbs.

Neutralizing antibodies evolve to exploit vulnerable sites in the HCV envelope glycoprotein E2 and mediate spontaneous clearance of infection.

Individuals who clear primary hepatitis C virus (HCV) infections clear subsequent reinfections more than 80% of the time, but the mechanisms are poorly defined. Here, we used HCV variants and plasma from individuals with repeated clearance to characterize longitudinal changes in envelope glycoprotein E2 sequences, function, and neutralizing antibody (NAb) resistance. Clearance of infection was associated with early selection of viruses with NAb resistance substitutions that also reduced E2 binding to CD81, the primary HCV receptor. Later, peri-clearance plasma samples regained neutralizing capacity against these variants. We identified a subset of broadly NAbs (bNAbs) for which these loss-of-fitness substitutions conferred resistance to unmutated bNAb ancestors but increased sensitivity to mature bNAbs. These data demonstrate a mechanism by which neutralizing antibodies contribute to repeated immune-mediated HCV clearance, identifying specific bNAbs that exploit fundamental vulnerabilities in E2. The induction of bNAbs with these specificities should be a goal of HCV vaccine development.

Challenge Inoculum for Hepatitis C Virus Controlled Human Infection Model.

For any controlled human infection model (CHIM), a safe, standardized, and biologically relevant challenge inoculum is necessary. For hepatitis C virus (HCV) CHIM, we propose that human-derived high-titer inocula of several viral genotypes with extensive virologic, serologic, and molecular characterizations should be the most appropriate approach. These inocula should first be tested in human volunteers in a step-wise manner to ensure safety, reproducibility, and curability prior to using them for testing the efficacy of candidate vaccines.

Corrigendum: Convergent antibody responses are associated with broad neutralization of hepatitis C virus.

[This corrects the article DOI: 10.3389/fimmu.2023.1135841.].

Convergent antibody responses are associated with broad neutralization of hepatitis C virus.

Introduction: Early development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known. Methods: To identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects. Results: We found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing. Discussion: Together, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.

Factors associated with resistance to SARS-CoV-2 infection discovered using large-scale medical record data and machine learning.

There have been over 621 million cases of COVID-19 worldwide with over 6.5 million deaths. Despite the high secondary attack rate of COVID-19 in shared households, some exposed individuals do not contract the virus. In addition, little is known about whether the occurrence of COVID-19 resistance differs among people by health characteristics as stored in the electronic health records (EHR). In this retrospective analysis, we develop a statistical model to predict COVID-19 resistance in 8,536 individuals with prior COVID-19 exposure using demographics, diagnostic codes, outpatient medication orders, and count of Elixhauser comorbidities in EHR data from the COVID-19 Precision Medicine Platform Registry. Cluster analyses identified 5 patterns of diagnostic codes that distinguished resistant from non-resistant patients in our study population. In addition, our models showed modest performance in predicting COVID-19 resistance (best performing model AUROC = 0.61). Monte Carlo simulations conducted indicated that the AUROC results are statistically significant (p < 0.001) for the testing set. We hope to validate the features found to be associated with resistance/non-resistance through more advanced association studies.

Lens Free Holographic Imaging for Urinary Tract Infection Screening.

OBJECTIVE: The diagnosis of urinary tract infection (UTI) currently requires precise specimen collection, handling infectious human waste, controlled urine storage, and timely transportation to modern laboratory equipment for analysis. Here we investigate holographic lens free imaging (LFI) to show its promise for enabling automatic urine analysis at the patient bedside. METHODS: We introduce an LFI system capable of resolving important urine clinical biomarkers such as red blood cells, white blood cells, crystals, and casts in 2 mm thick urine phantoms. RESULTS: This approach is sensitive to the particulate concentrations relevant for detecting several clinical urine abnormalities such as hematuria and pyuria, linearly correlating to ground truth hemacytometer measurements with R 2 = 0.9941 and R 2 = 0.9973, respectively. We show that LFI can estimate E. coli concentrations of 10 3 to 10 5 cells/mL by counting individual cells, and is sensitive to concentrations of 10 5 cells/mL to 10 8 cells/mL by analyzing hologram texture. Further, LFI measurements of blood cell concentrations are relatively insensitive to changes in bacteria concentrations of over seven orders of magnitude. Lastly, LFI reveals clear differences between UTI-positive and UTI-negative urine from human patients. CONCLUSION: LFI is sensitive to clinically-relevant concentrations of bacteria, blood cells, and other sediment in large urine volumes. SIGNIFICANCE: Together, these results show promise for LFI as a tool for urine screening, potentially offering early, point-of-care detection of UTI and other pathological processes.

Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) exposure investigations using genomic sequencing among healthcare workers and patients in a large academic center.

Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) transmissions among healthcare workers and hospitalized patients are challenging to confirm. Investigation of infected persons often reveals multiple potential risk factors for viral acquisition. We combined exposure investigation with genomic analysis confirming 2 hospital-based clusters. Prolonged close contact with unmasked, unrecognized infectious, individuals was a common risk.

Assessing Associations Between COVID-19 Symptomology and Adverse Outcomes After Piloting Crowdsourced Data Collection: Cross-sectional Survey Study.

BACKGROUND: Crowdsourcing is a useful way to rapidly collect information on COVID-19 symptoms. However, there are potential biases and data quality issues given the population that chooses to participate in crowdsourcing activities and the common strategies used to screen participants based on their previous experience. OBJECTIVE: The study aimed to (1) build a pipeline to enable data quality and population representation checks in a pilot setting prior to deploying a final survey to a crowdsourcing platform, (2) assess COVID-19 symptomology among survey respondents who report a previous positive COVID-19 result, and (3) assess associations of symptomology groups and underlying chronic conditions with adverse outcomes due to COVID-19. METHODS: We developed a web-based survey and hosted it on the Amazon Mechanical Turk (MTurk) crowdsourcing platform. We conducted a pilot study from August 5, 2020, to August 14, 2020, to refine the filtering criteria according to our needs before finalizing the pipeline. The final survey was posted from late August to December 31, 2020. Hierarchical cluster analyses were performed to identify COVID-19 symptomology groups, and logistic regression analyses were performed for hospitalization and mechanical ventilation outcomes. Finally, we performed a validation of study outcomes by comparing our findings to those reported in previous systematic reviews. RESULTS: The crowdsourcing pipeline facilitated piloting our survey study and revising the filtering criteria to target specific MTurk experience levels and to include a second attention check. We collected data from 1254 COVID-19-positive survey participants and identified the following 6 symptomology groups: abdominal and bladder pain (Group 1); flu-like symptoms (loss of smell/taste/appetite; Group 2); hoarseness and sputum production (Group 3); joint aches and stomach cramps (Group 4); eye or skin dryness and vomiting (Group 5); and no symptoms (Group 6). The risk factors for adverse COVID-19 outcomes differed for different symptomology groups. The only risk factor that remained significant across 4 symptomology groups was influenza vaccine in the previous year (Group 1: odds ratio [OR] 6.22, 95% CI 2.32-17.92; Group 2: OR 2.35, 95% CI 1.74-3.18; Group 3: OR 3.7, 95% CI 1.32-10.98; Group 4: OR 4.44, 95% CI 1.53-14.49). Our findings regarding the symptoms of abdominal pain, cough, fever, fatigue, shortness of breath, and vomiting as risk factors for COVID-19 adverse outcomes were concordant with the findings of other researchers. Some high-risk symptoms found in our study, including bladder pain, dry eyes or skin, and loss of appetite, were reported less frequently by other researchers and were not considered previously in relation to COVID-19 adverse outcomes. CONCLUSIONS: We demonstrated that a crowdsourced approach was effective for collecting data to assess symptomology associated with COVID-19. Such a strategy may facilitate efficient assessments in a dynamic intersection between emerging infectious diseases, and societal and environmental changes.

Repeated exposure to heterologous hepatitis C viruses associates with enhanced neutralizing antibody breadth and potency.

A prophylactic hepatitis C virus (HCV) vaccine that elicits neutralizing antibodies could be key to HCV eradication. However, the genetic and antigenic properties of HCV envelope (E1E2) proteins capable of inducing anti-HCV broadly neutralizing antibodies (bNAbs) in humans have not been defined. Here, we investigated the development of bNAbs in longitudinal plasma of HCV-infected persons with persistent infection or spontaneous clearance of multiple reinfections. By measuring plasma antibody neutralization of a heterologous virus panel, we found that the breadth and potency of the antibody response increased upon exposure to multiple genetically distinct infections and with longer duration of viremia. Greater genetic divergence between infecting strains was not associated with enhanced neutralizing breadth. Rather, repeated exposure to antigenically related, antibody-sensitive E1E2s was associated with potent bNAb induction. These data reveal that a prime-boost vaccine strategy with genetically distinct, antibody-sensitive viruses is a promising approach to inducing potent bNAbs in humans.

IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function.

BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38-42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6-21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891).

B cell overexpression of FCRL5 and PD-1 is associated with low antibody titers in HCV infection.

Antibodies targeting the hepatitis C virus (HCV) envelope glycoprotein E2 are associated with delayed disease progression, and these antibodies can also facilitate spontaneous clearance of infection in some individuals. However, many infected people demonstrate low titer and delayed anti-E2 antibody responses. Since a goal of HCV vaccine development is induction of high titers of anti-E2 antibodies, it is important to define the mechanisms underlying these suboptimal antibody responses. By staining lymphocytes with a cocktail of soluble E2 (sE2) glycoproteins, we detected HCV E2-specific (sE2+) B cells directly ex vivo at multiple acute infection timepoints in 29 HCV-infected subjects with a wide range of anti-E2 IgG titers, including 17 persistently infected subjects and 12 subjects with spontaneous clearance of infection. We performed multi-dimensional flow cytometric analysis of sE2+ and E2-nonspecific (sE2-) class-switched B cells (csBC). In sE2+ csBC from both persistence and clearance subjects, frequencies of resting memory B cells (rMBC) were reduced, frequencies of activated MBC (actMBC) and tissue-like MBC (tlMBC) were increased, and expression of FCRL5, an IgG receptor, was significantly upregulated. Across all subjects, plasma anti-E2 IgG levels were positively correlated with frequencies of sE2+ rMBC and sE2+ actMBC, while anti-E2 IgG levels were negatively correlated with levels of FCRL5 expression on sE2+ rMBC and PD-1 expression on sE2+ actMBC. Upregulation of FCRL5 on sE2+ rMBC and upregulation of PD-1 on sE2+ actMBC may limit anti-E2 antibody production in vivo. Strategies that limit upregulation of these molecules could potentially generate higher titers of protective antibodies against HCV or other pathogens.

An Update on Severe Acute Respiratory Syndrome Coronavirus 2 Diversity in the US National Capital Region: Evolution of Novel and Variants of Concern.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants concerning for enhanced transmission, evasion of immune responses, or associated with severe disease have motivated the global increase in genomic surveillance. In the current study, large-scale whole-genome sequencing was performed between November 2020 and the end of March 2021 to provide a phylodynamic analysis of circulating variants over time. In addition, we compared the viral genomic features of March 2020 and March 2021. METHODS: A total of 1600 complete SARS-CoV-2 genomes were analyzed. Genomic analysis was associated with laboratory diagnostic volumes and positivity rates, in addition to an analysis of the association of selected variants of concern/variants of interest with disease severity and outcomes. Our real-time surveillance features a cohort of specimens from patients who tested positive for SARS-CoV-2 after completion of vaccination. RESULTS: Our data showed genomic diversity over time that was not limited to the spike sequence. A significant increase in the B.1.1.7 lineage (alpha variant) in March 2021 as well as a transient circulation of regional variants that carried both the concerning S: E484K and S: P681H substitutions were noted. Lineage B.1.243 was significantly associated with intensive care unit admission and mortality. Genomes recovered from fully vaccinated individuals represented the predominant lineages circulating at specimen collection time, and people with those infections recovered with no hospitalizations. CONCLUSIONS: Our results emphasize the importance of genomic surveillance coupled with laboratory, clinical, and metadata analysis for a better understanding of the dynamics of viral spread and evolution.

Plasma virome and the risk of blood-borne infection in persons with substance use disorder.

There is an urgent need for innovative methods to reduce transmission of bloodborne pathogens like HIV and HCV among people who inject drugs (PWID). We investigate if PWID who acquire non-pathogenic bloodborne viruses like anelloviruses and pegiviruses might be at greater risk of acquiring a bloodborne pathogen. PWID who later acquire HCV accumulate more non-pathogenic viruses in plasma than matched controls who do not acquire HCV infection. Additionally, phylogenetic analysis of those non-pathogenic virus sequences reveals drug use networks. Here we find first in Baltimore and confirm in San Francisco that the accumulation of non-pathogenic viruses in PWID is a harbinger for subsequent acquisition of pathogenic viruses, knowledge that may guide the prioritization of the public health resources to combat HIV and HCV.

Decreased Activated CD4+ T Cell Repertoire Diversity After Antiretroviral Therapy in HIV-1/HCV Coinfection Correlates with CD4+ T Cell Recovery.

Dysfunctional immune activation accumulates during chronic viral infection and contributes to disease pathogenesis. In HIV-1, immune activation is exacerbated by concurrent infection with hepatitis C virus (HCV), accelerating depletion of CD4+ T cells. HIV-1 suppression with antiretroviral therapy (ART) generally reconstitutes CD4+ T cell counts, while also reducing the proportion that is activated. Whether this immune reconstitution also reduces the complexity of the CD4+ T cell population is unknown. We sought to characterize the relationship between activated CD4+ T cell repertoire diversity and immune reconstitution following ART in HIV-1/HCV coinfection. We extracted T cell receptor (TCR) sequences from RNA sequencing data obtained from activated CD4+ T cells of HIV-1/HCV coinfected individuals before and after treatment with ART (clinical trial NCT01285050). There was notable heterogeneity in both the extent of CD4+ T cell reconstitution and in the change in activated CD4+ TCR repertoire diversity following ART. Decreases in activated CD4+ TCR repertoire diversity following ART were predictive of the degree of CD4+ T cell reconstitution. The association of decreased activated CD4+ TCR repertoire diversity and improved CD4+ T cell reconstitution may represent loss of nonspecifically activated TCR clonotypes, and possibly selective expansion of specifically activated CD4+ clones. These results provide insight into the dynamic relationship between activated CD4+ TCR diversity and CD4+ T cell recovery of HIV-1/HCV coinfected individuals after suppression of HIV-1 viremia.

Interleukin-18 and tumor necrosis factor-α are elevated in solid organ transplant recipients with possible cytomegalovirus end-organ disease.

End-organ cytomegalovirus (CMV) disease can be life threatening to solid organ transplant recipients. Diagnosis is often complicated by variation in amount of CMV DNA in plasma and the need for an invasive procedure to obtain a biopsy of the suspected affected organ, which can delay recognition and treatment. Several inflammatory cytokines are elevated in CMV disease, and the purpose of this study was to determine if they could be used to distinguish solid organ transplant recipients with CMV DNAemia alone from those with possible end-organ CMV disease. We found that regardless of pre-transplant CMV serostatus, plasma interleukin (IL)-18, tumor necrosis factor-α (TNF-α), and amount of CMV DNA in plasma were increased in possible end-organ CMV disease, with elevated IL-18 associated with increased odds of possible end-organ CMV disease even after adjusting for amount of CMV DNA. These findings highlight IL-18 and TNF-α as potential non-invasive markers of possible end-organ CMV disease regardless of transplanted organ or serostatus in solid organ transplant recipients.

Design and Synthesis of Pyrrolo[2,3-d]pyrimidine-Derived Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors Using a Checkpoint Kinase 1 (CHK1)-Derived Crystallographic Surrogate.

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3-d]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2R)-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S cKi 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18/CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino] derivative 32. Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45, which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.