COVID-19-neutralizing antibodies predict disease severity and survival.

Coronavirus disease 2019 (COVID-19) exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, pro-inflammatory cytokines, and high anti-receptor binding domain (RBD) antibody levels. Although anti-RBD immunoglobulin G (IgG) levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting cross-protection from reinfection by either strain. However, SARS-CoV-2 sera generally lacked cross-neutralization to a highly homologous pre-emergent bat coronavirus, WIV1-CoV, which has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies.

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

A sensitive assay for measuring whole-blood responses to type I IFNs.

Human inborn errors of the type I IFN response pathway and auto-Abs neutralizing IFN-α, -ß, and/or -ω can underlie severe viral illnesses. We report a simple assay for the detection of both types of condition. We stimulate whole blood from healthy individuals and patients with either inborn errors of type I IFN immunity or auto-Abs against type I IFNs with glycosylated human IFN-α2, -ß, or -ω. As controls, we add a monoclonal antibody (mAb) blocking the type I IFN receptors and stimulated blood with IFN-γ (type II IFN). Of the molecules we test, IP-10 (encoded by the interferon-stimulated gene (ISG) CXCL10) is the molecule most strongly induced by type I and type II IFNs in the whole blood of healthy donors in an ELISA-like assay. In patients with inherited IFNAR1, IFNAR2, TYK2, or IRF9 deficiency, IP-10 is induced only by IFN-γ, whereas, in those with auto-Abs neutralizing specific type I IFNs, IP-10 is also induced by the type I IFNs not neutralized by the auto-Abs. The measurement of type I and type II IFN-dependent IP-10 induction therefore constitutes a simple procedure for detecting rare inborn errors of the type I IFN response pathway and more common auto-Abs neutralizing type I IFNs.

KcsA-Kv1.x chimeras with complete ligand-binding sites provide improved predictivity for screening selective Kv1.x blockers.

Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.

m6A-ELISA, a simple method for quantifying N6-methyladenosine from mRNA populations.

N6-methyladenosine (m6A) is a widely studied and abundant RNA modification. The m6A mark regulates the fate of RNAs in various ways, which in turn drives changes in cell physiology, development, and disease pathology. Over the last decade, numerous methods have been developed to map and quantify m6A sites genome-wide through deep sequencing. Alternatively, m6A levels can be quantified from a population of RNAs using techniques such as liquid chromatography-mass spectrometry or thin layer chromatography. However, many methods for quantifying m6A levels involve extensive protocols and specialized data analysis, and often only a few samples can be handled in a single experiment. Here, we developed a simple method for determining relative m6A levels in mRNA populations from various sources based on an enzyme-linked immunosorbent-based assay (m6A-ELISA). We have optimized various steps of m6A-ELISA, such as sample preparation and the background signal resulting from the primary antibody. We validated the method using mRNA populations from budding yeast and mouse embryonic stem cells. The full protocol takes less than a day, requiring only 25 ng of mRNA. The m6A-ELISA protocol is quick, cost-effective, and scalable, making it a valuable tool for determining relative m6A levels in samples from various sources that could be adapted to detect other mRNA modifications.

Propagating and banking genetically diverse human sapovirus strains using a human duodenal cell line: investigating antigenic differences between strains.

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078-32085, 2020, https://10.1073/pnas.2007310117). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1-7, GII.1-5, -8, and GV.1-2) and accomplishing preparation of viral stocks containing 1.0 × 109 to 3.4 × 1011 viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection. IMPORTANCE: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

Efficient overexpression and purification of severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins in Escherichia coli.

The fundamental biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.

trans-Interacting Plasma Membrane Proteins and Binding Partner Identification.

Plasma membrane proteins (PMPs) play critical roles in a myriad of physiological and disease conditions. A unique subset of PMPs functions through interacting with each other in trans at the interface between two contacting cells. These trans-interacting PMPs (tiPMPs) include adhesion molecules and ligands/receptors that facilitate cell-cell contact and direct communication between cells. Among the tiPMPs, a significant number have apparent extracellular binding domains but remain orphans with no known binding partners. Identification of their potential binding partners is therefore important for the understanding of processes such as organismal development and immune cell activation. While a number of methods have been developed for the identification of protein binding partners in general, very few are applicable to tiPMPs, which interact in a two-dimensional fashion with low intrinsic binding affinities. In this review, we present the significance of tiPMP interactions, the challenges of identifying binding partners for tiPMPs, and the landscape of method development. We describe current avidity-based screening approaches for identifying novel tiPMP binding partners and discuss their advantages and limitations. We conclude by highlighting the importance of developing novel methods of identifying new tiPMP interactions for deciphering the complex protein interactome and developing targeted therapeutics for diseases.

Low expression of TGF-ß2 and matrilin2 in human aqueous humour with acute primary angle closure.

Primary angle-closure glaucoma (PACG) is the leading cause of irreversible blindness in the world. Angle closure induced by pupil block and secondary iris synechia is the fundamental pathology of the PACG. The molecular mechanisms of angle closure have not yet been clearly illustrated. This study was designed to investigate the protein difference in the aqueous humour and explore new biomarker of the PACG. Aqueous humour (AH) was collected from patients with acute primary angle closure (APAC) and cataract (n = 10 in APAC group) and patients with cataract only (n = 10 in control group). Samples were pooled and measured using label-free proteome technology. Then, the differentially expressed proteins (DEPs) were verified by ELISA using independent AH samples (n = 20 each group). More than 400 proteins were revealed in both groups through proteomics. Comparing the two groups, there were 91DEPs. These proteins participate in biological activities such as inflammation, fibrosis, nerve growth and degeneration and metabolism. We found that the expression of transforming growth factor-ß2 and matrilin2 was downregulated in the APAC group. The two proteins are related to inflammation and extracellular matrix formation, which might be involved in angle closure. This study characterized DEPs in AH of the APAC and found a downregulated protein matrilin2.

Identification of Babesia microti immunoreactive antigens by phage display cDNA screen.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development.

Development of new NGLY1 assay systems - toward developing an early screening method for NGLY1 deficiency.

Cytosolic peptide: N-glycanase (PNGase/NGLY1 in mammals) is an amidase (EC:3.5.1.52) widely conserved in eukaryotes. It catalyzes the removal of N-glycans on glycoproteins, converting N-glycosylated Asn into Asp residues. This enzyme also plays a role in the quality control system for nascent glycoproteins. Since the identification of a patient with an autosomal recessive genetic disorder caused by NGLY1 gene dysfunction, known as NGLY1 deficiency or NGLY1 congenital disorder of deglycosylation (OMIM: 615273), in 2012, more than 100 cases have been reported worldwide. NGLY1 deficiency is characterized by a wide array of symptoms, such as global mental delay, intellectual disability, abnormal electroencephalography findings, seizure, movement disorder, hypolacrima or alacrima, and liver dysfunction. Unfortunately, no effective therapeutic treatments for this disease have been established. However, administration of adeno-associated virus 9 (AAV9) vector harboring human NGLY1 gene to an NGLY1-deficient rat model (Ngly1-/- rat) by intracerebroventricular injection was found to drastically improve motor function defects. This observation indicated that early therapeutic intervention could alleviate various symptoms originating from central nervous system dysfunction in this disease. Therefore, there is a keen interest in the development of facile diagnostic methods for NGLY1 deficiency. This review summarizes the history of assay development for PNGase/NGLY1 activity, as well as the recent progress in the development of novel plate-based assay systems for NGLY1, and also discusses future perspectives.

Preliminary Findings From the Dynamics of the Immune Responses to Repeat Influenza Vaccination Exposures (DRIVE I) Study: A Randomized Controlled Trial.

BACKGROUND: Studies have reported that repeated annual vaccination may influence influenza vaccination effectiveness in the current season. METHODS: We established a 5-year randomized placebo-controlled trial of repeated influenza vaccination (Flublok; Sanofi Pasteur) in adults 18-45 years of age. In the first 2 years, participants were randomized to receive vaccine or saline placebo as follows: placebo-placebo (P-P), placebo-vaccine (P-V), or vaccine-vaccine (V-V). Serum samples were collected each year just before vaccination and after 30 and 182 days. A subset of serum samples collected at 5 time points from 95 participants were tested for antibodies against vaccine strains. RESULTS: From 23 October 2020 through 11 March 2021 we enrolled and randomized 447 adults. Among vaccinated individuals, antibody titers increased between days 0 and 30 against each of the vaccine strains, with smaller increases for repeat vaccinees who on average had higher prevaccination titers in year 2. There were statistically significant differences in the proportions of participants achieving ≥4-fold rises in antibody titer for the repeat vaccinees for influenza A(H1N1), B/Victoria, and B/Yamagata, but not for A(H3N2). Among participants who received vaccination in year 2, there were no significant differences between the P-V and V-V groups in geometric mean titers at day 30 or the proportions of participants with antibody titers ≥40 at day 30 for any of the vaccine strains. CONCLUSIONS: In the first 2 years, during which influenza did not circulate, repeat and first-time vaccinees had similar postvaccination geometric mean titers to all 4 vaccine strains, indicative of similar levels of clinical protection. Clinical Trials Registration. NCT04576377.

Serologic Immunity to Tetanus in the United States, National Health and Nutrition Examination Survey, 2015-2016.

BACKGROUND: Tetanus, a life-threatening infection, has become rare in the United States since introduction of tetanus toxoid-containing vaccines (TTCVs), recommended as a childhood series followed by decennial boosters beginning at age 11-12 years; vaccination uptake is high in children but suboptimal in adults. The objective of this study was to estimate the prevalence of sero-immunity to tetanus among persons aged ≥6 years in the United States and to identify factors associated with tetanus sero-immunity. Understanding population protection against tetanus informs current and future vaccine recommendations. METHODS: Anti-tetanus toxoid antibody concentrations were measured for participants of the 2015-2016 National Health and Nutrition Examination Survey (NHANES) aged ≥6 years for whom surplus serum samples were available using a microsphere-based multiplex antibody capture assay. Prevalence of sero-immunity, defined as ≥0.10 IU/mL, was estimated overall and by demographic characteristics. Factors associated with tetanus sero-immunity were examined using multivariable regression. RESULTS: Overall, 93.8% of the US population aged ≥6 years had sero-protection against tetanus. Prevalence of sero-immunity was above 90% across racial/ethnic categories, sex, and poverty levels. By age, ≥ 90% had protective sero-immunity through age 69 years, but prevalence of sero-immunity declined thereafter, with 75.8% of those aged ≥80 years having protective sero-immunity. Older age (adjusted prevalence ratio [aPR]: 0.89, 95% confidence interval [CI]: .85-.92) and being born outside the United States (aPR: 0.96, 95% CI: .93-.98) were significantly associated with lower prevalence of sero-immunity. CONCLUSIONS: The majority of the US population has vaccine-induced sero-immunity to tetanus, demonstrating the success of the vaccination program.

Usefulness of Galectin-3 as a Biochemical Marker to Detect Ventricular and Supraventricular Arrhythmias in Children.

Galectin-3 (Gal-3) has been demonstrated to play a pivotal role in the pathogenesis of several fibrotic disorders. A number of studies have examined the relationship between galectin-3 levels and cardiac fibrosis in heart failure. Nevertheless, the role of galectin-3 in the etiology of supraventricular (SVa) and ventricular (Va) arrhythmias remains largely unexamined. The objective of this prospective study was to investigate the potential correlation between galectin concentration and the occurrence of idiopathic cardiac arrhythmias in pediatric patients. Biochemistry analysis was performed on 30 children (11-18 years; 14 boys and 16 girls). The control group consisted of 20 children. Cardiac arrhythmia was confirmed by a 24 h Holter ECG recording. Serum galectin-3 levels were measured via enzyme-linked immunosorbent assay (ELISA). Statistical analysis of the data showed significant associations between creatinine kinase (CK) and Gal-3 in patients with SVa (SVT-supraventricular tachycardia) arrhythmias, suggesting a potential effect of CK on Gal-3 levels. However, no correlation was identified between Gal-3 concentration and the occurrence of cardiac arrhythmias under investigation. We concluded that galectin-3 does not have the potential to be a biomarker in the diagnosis of idiopathic arrhythmias in pediatric patients.

Development of an Indirect ELISA to Distinguish between Porcine Sapelovirus-Infected and -Vaccinated Animals Using the Viral Nonstructural Protein 3AB.

Porcine sapelovirus (PSV) is a new pathogen that negatively impacts the pig industry in China. Affected pigs experience severe diarrhea and even death. Vaccination is used to control disease outbreaks, and sensitive diagnostic methods that can distinguish infected animals from vaccinated animals (DIVA) are essential for monitoring the effectiveness of disease control programs. Tests based on the detection of the nonstructural protein (NSP) 3AB are reliable indicators of viral replication in infected and vaccinated animals. In this study, the recombinant PSV 3AB protein was expressed by a prokaryotic expression system, and an indirect ELISA method was established. Serum samples from healthy animals, immunized animals, and infected animals were evaluated. The ELISA method identified 3AB with high sensitivity (99.78%) and specificity (100.0%), and no cross-reaction was observed with serum antibodies against porcine reproductive and respiratory syndrome virus (PRRSV), infection with classical swine fever virus (CSFV), pseudorabies virus (PRV), bovine viral diarrhea virus (BVDV), porcine epidemic diarrhea virus (PEDV), or foot-and-mouth disease virus (FMDV). The ELISA method described here can effectively distinguish infected and vaccinated animals and is an important inexpensive tool for monitoring serum and controlling PSV.

Exploring the Gene Expression and Plasma Protein Levels of HSP90, HSP60, and GDNF in Multiple Sclerosis Patients and Healthy Controls.

Multiple sclerosis (MS) is a chronic neurodegenerative disease characterized by immune-mediated inflammation and neurodegeneration in the central nervous system (CNS). In this study; we aimed to investigate the gene expression and plasma protein levels of three neuroprotective genes-heat shock proteins (HSP90 and HSP60) and glial cell line-derived neurotrophic factor (GDNF)-in MS patients compared to healthy controls. Forty patients with relapsing-remitting MS and 40 healthy volunteers participated in this study. Gene expression was measured using reverse transcription quantitative real-time PCR, and protein levels were assessed via ELISA. The results showed a significant increase in HSP90 (1.7-fold) and HSP60 (2-fold) gene expression in MS patients compared to controls, along with corresponding increases in protein levels (1.5-fold for both HSP90 and HSP60). In contrast, GDNF gene expression and protein levels were significantly reduced in MS patients, with a 7-fold decrease in gene expression and a 1.6-fold reduction in protein levels. Notably, a non-linear relationship between GDNF gene expression and protein concentration was observed in MS patients, suggesting complex regulatory mechanisms influencing GDNF in the disease. The upregulation of HSP90 and HSP60 in MS highlights their roles in immune regulation and stress responses, while the reduction in GDNF indicates impaired neuroprotection. These findings suggest that HSP90, HSP60, and GDNF could serve as biomarkers for disease progression and as potential therapeutic targets in MS, offering promising avenues for future research and treatment development.

Serum ITIH5 as a novel diagnostic biomarker in cholangiocarcinoma.

Cholangiocarcinoma often remains undetected until advanced stages due to the lack of reliable diagnostic markers. Our goal was to identify a unique secretory protein for cholangiocarcinoma diagnosis and differentiation from other malignancies, benign hepatobiliary diseases, and chronic liver conditions. We conducted bulk RNA-seq analysis to identify genes specifically upregulated in cholangiocarcinoma but not in most other cancers, benign hepatobiliary diseases, and chronic liver diseases focusing on exocrine protein-encoding genes. Single-cell RNA sequencing examined subcellular distribution. Immunohistochemistry and enzyme-linked immunosorbent assays assessed tissue and serum expression. Diagnostic performance was evaluated via receiver-operating characteristic (ROC) analysis. Inter-alpha-trypsin inhibitor heavy chain family member five (ITIH5), a gene encoding an extracellular protein, is notably upregulated in cholangiocarcinoma. This elevation is not observed in most other cancer types, benign hepatobiliary diseases, or chronic liver disorders. It is specifically expressed by malignant cholangiocytes. ITIH5 expression in cholangiocarcinoma tissues exceeded that in nontumorous bile duct, hepatocellular carcinoma, and nontumorous hepatic tissues. Serum ITIH5 levels were elevated in cholangiocarcinoma compared with controls (hepatocellular carcinoma, benign diseases, chronic hepatitis B, and healthy individuals). ITIH5 yielded areas under the ROC curve (AUCs) from 0.839 to 0.851 distinguishing cholangiocarcinoma from controls. Combining ITIH5 with carbohydrate antigen 19-9 (CA19-9) enhanced CA19-9's diagnostic effectiveness. In conclusion, serum ITIH5 may serve as a novel noninvasive cholangiocarcinoma diagnostic marker.

SARS-CoV-2 incidence, seroprevalence, and antibody dynamics in a rural, population-based cohort: March 2020 - July 2022.

Studies of SARS-CoV-2 incidence are important for response to continued transmission and future pandemics. We followed a rural community cohort with broad age representation with active surveillance for SARS-CoV-2 identification from November 2020 through July 2022. Participants provided serum specimens at regular intervals and following SARS-CoV-2 infection or vaccination. We estimated the incidence of SARS-CoV-2 infection identified by study RT-PCR, electronic health record documentation or self-report of a positive test, or serology. We also estimated the seroprevalence of SARS-CoV-2 spike and nucleocapsid antibodies measured by ELISA. Overall, 65% of the cohort had ≥1 SARS-CoV-2 infection by July 2022, and 19% of those with primary infection were reinfected. Infection and vaccination contributed to high seroprevalence, 98% (95% CI: 95%, 99%) of participants were spike or nucleocapsid seropositive at the end of follow-up. Among those seropositive, 82% were vaccinated. Participants were more likely to be seropositive to spike than nucleocapsid following infection. Infection among seropositive individuals could be identified by increases in nucleocapsid, but not spike, ELISA optical density values. Nucleocapsid antibodies waned more quickly after infection than spike antibodies. High levels of SARS-CoV-2 population immunity, as found in this study, are leading to changing epidemiology necessitating ongoing surveillance and policy evaluation.

Increased α2,3-sialyl N-glycosylated prostate-specific membrane antigen (PSMA) in post-DRE urine is associated with high grade group prostate cancer.

INTRODUCTION: Aberrant glycosylation of proteins is an important hallmark in multiple cancers. Prostate-specific membrane antigen (PSMA), a highly glycosylated protein with 10 N-linked glycosylation sites, is an Food and Drug Administration approved theranostic for prostate cancer. However, glycosylation changes in PSMA that are associated with prostate cancer disease progression have not been fully characterized. METHODS: We investigated whether urinary PSMA sialylation correlate with high-grade prostate cancer. Urine samples were collected from men after digital rectal examination (DRE) before prostate biopsy. Lectin-antibody enzyme-linked immunoassay was used to quantify α2,3-sialyl PSMA in post-DRE urine samples from subjects with benign prostate tumors, Grade Group 1 prostate cancer and those with Grade Group ≥2 disease. RESULTS: There are significant increases in α2,3-sialylated PSMA in patients with Grade Group ≥2 disease compared to benign (p = 0.0009) and those with Grade Group 1 disease (p = 0.0063). There were no significant differences in α2,3-sialyl PSMA levels between Grade Group 1 and benign prostate tumors (p = 0.7947). CONCLUSIONS: Our study shows that there are significant differences in the abundance of α2,3-sialylated PSMA in post-DRE urines from disease stratified prostate cancer patients, and the increase is correlated with progression and disease severity. The detection of increased PSMA sialyation in post-DRE urines from patients with higher Grade Group ≥2 disease states provides novel untapped potential for the development of prognostic biomarkers for prostate cancer. Specifically, quantitation of α2,3-sialylated PSMA shows potential for discriminating between benign to intermediate grade disease, which is a significant clinical challenge in staging and risk stratification of prostate cancer.

ELISA-based highly sensitive assay system for the detection of endogenous NGLY1 activity.

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 µg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.