A proximity-dependent biotinylation map of a human cell.

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.

Systematic Examination of Antigen-Specific Recall T Cell Responses to SARS-CoV-2 versus Influenza Virus Reveals a Distinct Inflammatory Profile.

There is a pressing need for an in-depth understanding of immunity to SARS-CoV-2. In this study, we investigated human T cell recall responses to fully glycosylated spike trimer, recombinant N protein, as well as to S, N, M, and E peptide pools in the early convalescent phase and compared them with influenza-specific memory responses from the same donors. All subjects showed SARS-CoV-2-specific T cell responses to at least one Ag. Both SARS-CoV-2-specific and influenza-specific CD4+ T cell responses were predominantly of the central memory phenotype; however SARS-CoV-2-specific CD4+ T cells exhibited a lower IFN-γ to TNF ratio compared with influenza-specific memory responses from the same donors, independent of disease severity. SARS-CoV-2-specific T cells were less multifunctional than influenza-specific T cells, particularly in severe cases, potentially suggesting exhaustion. Most SARS-CoV-2-convalescent subjects also produced IFN-γ in response to seasonal OC43 S protein. We observed granzyme B+/IFN-γ+, CD4+, and CD8+ proliferative responses to peptide pools in most individuals, with CD4+ T cell responses predominating over CD8+ T cell responses. Peripheral T follicular helper (pTfh) responses to S or N strongly correlated with serum neutralization assays as well as receptor binding domain-specific IgA; however, the frequency of pTfh responses to SARS-CoV-2 was lower than the frequency of pTfh responses to influenza virus. Overall, T cell responses to SARS-CoV-2 are robust; however, CD4+ Th1 responses predominate over CD8+ T cell responses, have a more inflammatory profile, and have a weaker pTfh response than the response to influenza virus within the same donors, potentially contributing to COVID-19 disease.

Resistance of SARS-CoV-2 beta and gamma variants to plasma collected from Canadian blood donors during the spring of 2020.

BACKGROUND: This pilot study assesses the ability of plasma collected from Canadian blood donors in the first wave of the SARS-CoV-2 pandemic to neutralize later SARS-CoV-2 variants of concern (VOCs). STUDY DESIGN AND METHODS: A repeated cross-sectional design was used, and a random cross-sectional sample of all available Canadian Blood Services retention samples (n = 1500/month) was drawn monthly for April and May of 2020. Qualitative IgG analysis was performed on aliquots of specimens using anti-spike, anti-receptor binding domain, and anti-nucleocapsid protein enzyme-linked immunosorbent assays as well as the Abbott Architect SARS CoV-2 IgG assay (Abbott Laboratories) against the anti-nucleocapsid protein. Selected plasma specimens were then assessed for neutralization against VOCs using pseudotyped lentivirus inhibition assays as well as plaque reduction neutralization test 50% (PRNT50 ). RESULTS: Six specimens with a high neutralizing titer against wild-type SARS-CoV-2 and three specimens with a low neutralizing titer against wild-type SARS-CoV-2 were chosen for further analysis against VOCs. Four of six high neutralizing titer specimens had a reduced neutralizing capacity against beta VOCs by both neutralization methods. Three of six high neutralizing titer specimens had reduced neutralization capacity against gamma VOCs. CONCLUSIONS: This preliminary data can be used as a justification for limiting the use of first wave plasma products in upcoming clinical trials but cannot be used to speculate on general trends in the immunity of Canadian blood donors to SARS-CoV-2.

Differences in mRNA-1273 (Moderna) and BNT162b2 (Pfizer-BioNTech) SARS-CoV-2 vaccine immunogenicity among patients undergoing dialysis.

BACKGROUND: Differences in immunogenicity between mRNA SARS-CoV-2 vaccines have not been well characterized in patients undergoing dialysis. We compared the serologic response in patients undergoing maintenance hemodialysis after vaccination against SARS-CoV-2 with BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna). METHODS: We conducted a prospective observational cohort study at 2 academic centres in Toronto, Canada, from Feb. 2, 2021, to July 20, 2021, which included 129 and 95 patients who received the BNT162b2 and mRNA-1273 SARS-CoV-2 vaccines, respectively. We measured SARS-CoV-2 immunoglobulin G antibodies to the spike protein (anti-spike), receptor binding domain (anti-RBD) and nucleocapsid protein (anti-NP) at 6-7 and 12 weeks after the second dose of vaccine and compared those levels with the median convalescent serum antibody levels from 211 controls who were previously infected with SARS-CoV-2. RESULTS: At 6-7 weeks after 2-dose vaccination, we found that 51 of 70 patients (73%) who received BNT162b2 and 83 of 87 (95%) who received mRNA-1273 attained convalescent levels of anti-spike antibody (p < 0.001). In those who received BNT162b2, 35 of 70 (50%) reached the convalescent level for anti-RBD compared with 69 of 87 (79%) who received mRNA-1273 (p < 0.001). At 12 weeks after the second dose, anti-spike and anti-RBD levels were significantly lower in patients who received BNT162b2 than in those who received mRNA-1273. For anti-spike, 70 of 122 patients (57.4%) who received BNT162b2 maintained the convalescent level versus 68 of 71 (96%) of those who received mRNA-1273 (p < 0.001). For anti-RBD, 47 of 122 patients (38.5%) who received BNT162b2 maintained the anti-RBD convalescent level versus 45 of 71 (63%) of those who received mRNA-1273 (p = 0.002). INTERPRETATION: In patients undergoing hemodialysis, mRNA-1273 elicited a stronger humoral response than BNT162b2. Given the rapid decline in immunogenicity at 12 weeks in patients who received BNT162b2, a third dose is recommended in patients undergoing dialysis as a primary series, similar to recommendations for other vulnerable populations.

Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29.

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

KdgF, the missing link in the microbial metabolism of uronate sugars from pectin and alginate.

Uronates are charged sugars that form the basis of two abundant sources of biomass-pectin and alginate-found in the cell walls of terrestrial plants and marine algae, respectively. These polysaccharides represent an important source of carbon to those organisms with the machinery to degrade them. The microbial pathways of pectin and alginate metabolism are well studied and essentially parallel; in both cases, unsaturated monouronates are produced and processed into the key metabolite 2-keto-3-deoxygluconate (KDG). The enzymes required to catalyze each step have been identified within pectinolytic and alginolytic microbes; yet the function of a small ORF, kdgF, which cooccurs with the genes for these enzymes, is unknown. Here we show that KdgF catalyzes the conversion of pectin- and alginate-derived 4,5-unsaturated monouronates to linear ketonized forms, a step in uronate metabolism that was previously thought to occur spontaneously. Using enzyme assays, NMR, mutagenesis, and deletion of kdgF, we show that KdgF proteins from both pectinolytic and alginolytic bacteria catalyze the ketonization of unsaturated monouronates and contribute to efficient production of KDG. We also report the X-ray crystal structures of two KdgF proteins and propose a mechanism for catalysis. The discovery of the function of KdgF fills a 50-y-old gap in the knowledge of uronate metabolism. Our findings have implications not only for the understanding of an important metabolic pathway, but also the role of pectinolysis in plant-pathogen virulence and the growing interest in the use of pectin and alginate as feedstocks for biofuel production.

Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of ß-1,3-glucan.

BH0236 from Bacillus halodurans is a multimodular ß-1,3-glucanase comprising an N-terminal family 81 glycoside hydrolase catalytic module, an internal family 6 carbohydrate-binding module (CBM) that binds the nonreducing end of ß-1,3-glucan chains, and an uncharacterized C-terminal module classified into CBM family 56. Here, we determined that this latter CBM, BhCBM56, bound the soluble ß-1,3-glucan laminarin with a dissociation constant (Kd ) of ∼26 µm and displayed higher affinity for insoluble ß-1,3-glucans with Kd values of ∼2-10 µm but lacked affinity for ß-1,3-glucooligosaccharides. The X-ray crystal structure of BhCBM56 and NMR-derived chemical shift mapping of the binding site revealed a ß-sandwich fold, with the face of one ß-sheet possessing the ß-1,3-glucan-binding surface. On the basis of the functional and structural properties of BhCBM56, we propose that it binds a quaternary polysaccharide structure, most likely the triple helix adopted by polymerized ß-1,3-glucans. Consistent with the BhCBM56 and BhCBM6/56 binding profiles, deletion of the CBM56 from BH0236 decreased activity of the enzyme on the insoluble ß-1,3-glucan curdlan but not on soluble laminarin; additional deletion of the CBM6 also did not affect laminarin degradation but further decreased curdlan hydrolysis. The pseudo-atomic solution structure of BH0236 determined by small-angle X-ray scattering revealed structural insights into the nature of avid binding by the BhCBM6/56 pair and how the orientation of the active site in the catalytic module factors into recognition and degradation of ß-1,3-glucans. Our findings reinforce the notion that catalytic modules and their cognate CBMs have complementary specificities, including targeting of polysaccharide quaternary structure.

Humoral Response Following 3 Doses of mRNA COVID-19 Vaccines in Patients With Non-Dialysis-Dependent CKD: An Observational Study.

Background: Chronic kidney disease (CKD) is associated with a lower serologic response to vaccination compared to the general population. There is limited information regarding the serologic response to coronavirus disease 2019 (COVID-19) vaccination in the non-dialysis-dependent CKD (NDD-CKD) population, particularly after the third dose and whether this response varies by estimated glomerular filtration rate (eGFR). Methods: The NDD-CKD (G1-G5) patients who received 3 doses of mRNA COVID-19 vaccines were recruited from renal clinics within British Columbia and Ontario, Canada. Between August 27, 2021, and November 30, 2022, blood samples were collected serially for serological testing every 3 months within a 9-month follow-up period. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike, anti-receptor binding domain (RBD), and anti-nucleocapsid protein (NP) levels were determined by enzyme-linked immunosorbent assay (ELISA). Results: Among 285 NDD-CKD patients, the median age was 67 (interquartile range [IQR], 52-77) years, 58% were men, 48% received BNT162b2 as their third dose, 22% were on immunosuppressive treatment, and COVID-19 infection by anti-NP seropositivity was observed in 37 of 285 (13%) patients. Following the third dose, anti-spike and anti-RBD levels peaked at 2 months, with geometric mean levels at 1131 and 1672 binding antibody units per milliliter (BAU/mL), respectively, and seropositivity rates above 93% and 85%, respectively, over the 9-month follow-up period. There was no association between eGFR or urine albumin-creatinine ratio (ACR) with mounting a robust antibody response or in antibody levels over time. The NDD-CKD patients on immunosuppressive treatment were less likely to mount a robust anti-spike response in univariable (odds ratio [OR] 0.43, 95% confidence interval [CI]: 0.20, 0.93) and multivariable (OR 0.52, 95% CI: 0.25, 1.10) analyses. An interaction between age, immunoglobulin G (IgG) antibody levels, and time was observed in both unadjusted (anti-spike: P = .005; anti-RBD: P = .03) and adjusted (anti-spike: P = .004; anti-RBD: P = .03) models, with older individuals having a more pronounced decline in antibody levels over time. Conclusion: Most NDD-CKD patients were seropositive for anti-spike and anti-RBD after 3 doses of mRNA COVID-19 vaccines and we did not observe any differences in the antibody response by eGFR.

Evolution of chromosome-arm aberrations in breast cancer through genetic network rewiring.

The basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and displays consistent large chromosomal deletions. Here, we characterize evolution and maintenance of chromosome 4p (chr4p) loss in basal breast cancer. Analysis of The Cancer Genome Atlas data shows recurrent deletion of chr4p in basal breast cancer. Phylogenetic analysis of a panel of 23 primary tumor/patient-derived xenograft basal breast cancers reveals early evolution of chr4p deletion. Mechanistically we show that chr4p loss is associated with enhanced proliferation. Gene function studies identify an unknown gene, C4orf19, within chr4p, which suppresses proliferation when overexpressed-a member of the PDCD10-GCKIII kinase module we name PGCKA1. Genome-wide pooled overexpression screens using a barcoded library of human open reading frames identify chromosomal regions, including chr4p, that suppress proliferation when overexpressed in a context-dependent manner, implicating network interactions. Together, these results shed light on the early emergence of complex aneuploid karyotypes involving chr4p and adaptive landscapes shaping breast cancer genomes.

Functional diversification despite structural congruence in the HipBST toxin-antitoxin system of Legionella pneumophila.

IMPORTANCE: Toxin-antitoxin (TA) systems are parasitic genetic elements found in almost all bacterial genomes. They are exchanged horizontally between cells and are typically poorly conserved across closely related strains and species. Here, we report the characterization of a tripartite TA system in the bacterial pathogen Legionella pneumophila that is highly conserved across Legionella species genomes. This system (denoted HipBSTLp) is a distant homolog of the recently discovered split-HipA system in Escherichia coli (HipBSTEc). We present bioinformatic, molecular, and structural analyses of the divergence between these two systems and the functionality of this newly described TA system family. Furthermore, we provide evidence to refute previous claims that the toxin in this system (HipTLp) possesses bifunctionality as an L. pneumophila virulence protein. Overall, this work expands our understanding of the split-HipA system architecture and illustrates the potential for undiscovered biology in these abundant genetic elements.

Evaluation of commercial assays for the assessment of SARS-CoV-2 antibody response in hemodialysis patients.

BACKGROUND: Hemodialysis patients exhibit variable immunogenicity following administration of the SARS-CoV-2 mRNA vaccine. The aim of the current study was to evaluate the use of two commercial assays in the assessment of SARS-CoV-2 antibody response in hemodialysis patients and to compare their utility to commonly used SARS-CoV-2 serological assays developed in Canada. METHODS: We evaluated serologic antibody response in 85 hemodialysis patients up to 6 months after receiving both doses of the Pfizer-BioNTech BNT162b2 COVID-19 mRNA vaccine. In addition, antibody response was assessed in 46 chronic kidney disease patients and 40 COVID-19 naïve health care workers (HCW) up to 3 months and 9 months, respectively. Anti-spike (S) and anti-nucleocapsid (N) levels were measured using Elecsys anti-SARS-CoV-2 immunoassays on the Roche analyzer and compared to ELISA-based detection of anti-S, anti-receptor binding domain (RBD), and anti-N. RESULTS: The Elecsys anti-N immunoassay showed 93 % concordance with the anti-N ELISA. The Elecsys anti-S immunoassay showed 97 % concordance with the anti-S ELISA and 89 % concordance with the anti-RBD ELISA. HCWs exhibited significantly higher anti-S levels relative to hemodialysis patients. Anti-S levels decreased significantly over a 6-month period (p < 0.001) in patients receiving maintenance hemodialysis. In addition, anti-S levels decreased significantly over a 9-month (p < 0.001) and 3-month period (p < 0.001) in HCWs and CKD patients, respectively. CONCLUSIONS: There is high concordance between commercial SARS-CoV-2 serological assays and SARS-CoV-2 serological assays developed in Canada. Hemodialysis patients exhibited varying immunogenicity following two doses of the COVID-19 mRNA vaccine with anti-S levels decreasing over time.

Humoral Responses in the Omicron Era Following 3-Dose SARS-CoV-2 Vaccine Series in Kidney Transplant Recipients.

Kidney transplant recipients (KTRs) have a diminished response to SARS-CoV-2 vaccination compared with immunocompetent individuals. Deeper understanding of antibody responses in KTRs following third-dose vaccination would enable identification of those who remain unprotected against Omicron. Methods: We profiled antibody responses in KTRs pre- and at 1 and 3 mo post-third-dose SARS-CoV-2 mRNA-based vaccine. Binding antibody levels were determined by ELISA. Neutralization against wild type, Beta, Delta, and Omicron (BA.1) variants was determined using a SARS-CoV-2 spike-pseudotyped lentivirus assay. Results: Forty-four KTRs were analyzed at 1 and 3 mo (n = 26) post-third dose. At 1 mo, the proportion of participants with a robust antibody response had increased significantly from baseline, but Omicron-specific neutralizing antibodies were detected in just 45% of KTRs. Median binding antibody levels declined at 3 mo, but the proportion of KTRs with a robust antibody response was unchanged; 38.5% KTRs maintained Omicron-specific neutralization at 3 mo. No clinical variables were significantly associated with Omicron-neutralizing antibodies, but antireceptor binding domain titers appeared to identify those with Omicron-specific neutralizing capacity. Conclusions: Over 50% of KTRs lack Omicron-specific neutralization capacity 1 mo post-third mRNA-vaccine dose. Antibody levels of responders were well preserved at 3 mo. Anti receptor binding domain antibody titers may identify patients with a detectable Omicron-neutralizing antibody response.

Diminished Neutralization Capacity of SARS-CoV-2 Omicron BA.1 in Donor Plasma Collected from January to March 2021.

The 50% plaque reduction neutralization assay (PRNT50) has been previously used to assess the neutralization capacity of donor plasma against wild-type and variant of concern (VOC) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Emerging data suggest that plasma with an anti-SARS-CoV-2 level of ≥2 × 104 binding antibody units/mL (BAU/mL) protects against SARS-CoV-2 Omicron BA.1 infection. Specimens were collected using a cross-sectional random sampling approach. For PRNT50 studies, 63 previously analyzed specimens by PRNT50 versus SARS-CoV-2 wild-type, Alpha, Beta, Gamma, and Delta were analyzed by PRNT50 versus Omicron BA.1. The 63 specimens plus 4,390 specimens (randomly sampled regardless of serological evidence of infection) were also tested using the Abbott SARS-CoV-2 IgG II Quant assay (anti-spike [S]; Abbott, Chicago, IL, USA; Abbott Quant assay). In the vaccinated group, the percentages of specimens with any measurable PRNT50 versus wild-type or VOC were wild type (21/25 [84%]), Alpha (19/25 [76%]), Beta (18/25 [72%]), Gamma (13/25 [52%]), Delta (19/25 [76%]), and Omicron BA.1 (9/25 [36%]). In the unvaccinated group, the percentages of specimens with any measurable PRNT50 versus wild type or VOC were wild-type SARS-CoV-2 (16/39 [41%]), Alpha (16/39 [41%]), Beta (10/39 [26%]), Gamma (9/39 [23%]), Delta (16/39 [41%]), and Omicron BA.1 (0/39) (Fisher's exact tests, vaccinated versus unvaccinated for each variant, P < 0.05). None of the 4,453 specimens tested by the Abbott Quant assay had a binding capacity of ≥2 × 104 BAU/mL. Vaccinated donors were more likely than unvaccinated donors to neutralize Omicron when assessed by a PRNT50 assay. IMPORTANCE SARS-CoV-2 Omicron emergence occurred in Canada during the period from November 2021 to January 2022. This study assessed the ability of donor plasma collected earlier (January to March 2021) to generate any neutralizing capacity against Omicron BA.1 SARS-CoV-2. Vaccinated individuals, regardless of infection status, were more likely to neutralize Omicron BA.1 than unvaccinated individuals. This study then used a semiquantitative binding antibody assay to screen a larger number of specimens (4,453) for individual specimens that might have high-titer neutralizing capacity against Omicron BA.1. None of the 4,453 specimens tested by the semiquantitative SARS-CoV-2 assay had a binding capacity suggestive of a high-titer neutralizing capacity against Omicron BA.1. These data do not imply that Canadians lacked immunity to Omicron BA.1 during the study period. Immunity to SARS-CoV-2 is complex, and there is still no wide consensus on correlation of protection to SARS-CoV-2.

BNT162b2 versus mRNA-1273 Third Dose COVID-19 Vaccine in Patients with CKD and Maintenance Dialysis Patients.

BACKGROUND: There is a lack of randomized controlled trial data regarding differences in immunogenicity of varying coronavirus disease 2019 (COVID-19) mRNA vaccine regimens in CKD populations. METHODS: We conducted a randomized controlled trial at three kidney centers in Toronto, Ontario, Canada, evaluating the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody response after third dose vaccination. Participants ( n =273) with CKD not on dialysis or receiving dialysis were randomized 1:1 to third dose 30- µ g BNT162b2 (Pfizer-BioNTech) or 100- µ g mRNA-1273 (Moderna). The primary outcome of this study was SARS-CoV-2 IgG-binding antibodies to the receptor-binding domain (anti-RBD). Spike protein (antispike), nucleocapsid protein, and vaccine reactogenicity were also evaluated. Serology was measured before third dose and 1, 3, and 6 months after third dose. A subset of participants ( n =100) were randomly selected to assess viral pseudovirus neutralization against wild-type D614G, B.1.617.2 (Delta), and B.1.1.529 (Omicron BA.1). RESULTS: Among 273 participants randomized, 94% were receiving maintenance dialysis and 59% received BNT162b2 for initial two dose COVID-19 vaccination. Third dose of mRNA-1273 was associated with higher mean anti-RBD levels (1871 binding antibody units [BAU]/ml; 95% confidence interval [CI], 829 to 2988) over a 6-month period in comparison with third dose BNT162b2 (1332 BAU/ml; 95% CI, 367 to 2402) with a difference of 539 BAU/ml (95% CI, 139 to 910; P = 0.009). Neither antispike levels nor neutralizing antibodies to wild-type, Delta, and Omicron BA.1 pseudoviruses were statistically different. COVID-19 infection occurred in 10% of participants: 15 (11%) receiving mRNA-1273 and 11 (8%) receiving BNT162b2. Third dose BNT162b2 was not associated with a significant different risk for COVID-19 in comparison with mRNA-1273 (hazard ratio, 0.78; 95% CI, 0.27 to 2.2; P = 0.63). CONCLUSIONS: In patients with CKD, third dose COVID-19 mRNA vaccination with mRNA-1273 elicited higher SARS-CoV-2 anti-RBD levels in comparison with BNT162b2 over a 6-month period. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: COVID-19 Vaccine Boosters in Patients With CKD (BOOST KIDNEY), NCT05022329 .

Determining the Longitudinal Serologic Response to COVID-19 Vaccination in the Chronic Kidney Disease Population: A Clinical Research Protocol.

Background: People living with chronic kidney disease (CKD) have been disproportionately affected by the coronavirus disease 2019 (COVID-19) pandemic, including higher rates of infection, hospitalization, and death. Data on responsiveness to COVID-19 vaccination strategies and immunogenicity are limited, yet required to inform vaccination strategies in this at-risk population. Objective: The objective of this study is to characterize the longitudinal serologic response to COVID-19 vaccination. Design: This is a prospective observational cohort study. Setting: Participating outpatient kidney programs within Ontario and British Columbia. Patients: Up to 2500 participants with CKD G3b-5D receiving COVID-19 vaccination, including participants receiving dialysis and kidney transplant recipients (CKD G1T-5T). Measurements: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies (anti-spike, anti-receptor binding domain, anti-nucleocapsid) will be detected by ELISA (enzyme-linked immunosorbent assay) from serum or dried blood spot testing. In a subset of participants, neutralizing antibodies against novel variants of concern will be evaluated. Peripheral blood mononuclear cells will be collected for exploratory immune profiling of SARS-CoV-2 specific cellular immunity. Methods: Participants will be recruited prior to or following any COVID-19 vaccine dose and have blood sampled for serological testing at multiple timepoints: 1, 3, 6, 9, and 12 months post vaccination. When possible, samples will be collected prior to a dose or booster. Participants will remain in the study for at least 1 year following their last COVID-19 vaccine dose. Strengths and limitations: The adaptive design of this study allows for planned modification based on emerging evidence or rapid changes in public health policy surrounding vaccination. Limitations include incomplete earlier timepoints for blood collection due to rapid vaccination of the population. Conclusions: This large multicenter serologic study of participants living with kidney disease will generate data on the kinetics of SARS-CoV-2 immune response to vaccination across the spectrum of CKD, providing insights into the amplitude and duration of immunity conferred by COVID-19 vaccination and allowing for characterization of factors associated with immune response. The results of this study may be used to inform immunization guidelines and public health recommendations for the 4 million Canadians living with CKD.

Contexte: Les personnes atteintes d'insuffisance rénale chronique (IRC) ont été touchées de façon disproportionnée par la pandémie de COVID-19 ayant notamment présenté des taux plus élevés d'infection, d'hospitalisation et de décès. Les données sur la réactivité aux stratégies de vaccination de la COVID-19 et à l'immunogénicité sont limitées, mais elles sont nécessaires pour développer des stratégies de vaccination dans cette population à risque. Objectif: Caractériser la réponse sérologique longitudinale à la vaccination contre la COVID-19. Conception: Étude de cohorte observationnelle prospective. Cadre: Les programmes ambulatoires de santé rénale participants en Ontario et en Colombie-Britannique. Sujets: Jusqu'à 2 500 personnes atteintes d'IRC G3B-5D recevant un vaccin contre la COVID-19, y compris des patients suivant des traitements de dialyse et des receveurs d'une greffe rénale (IRC G1T-5T). Mesures: Les anticorps IgG anti-SARS-CoV-2 (anti-spike, anti-domaine de liaison au récepteur, anti-nucléocapside) seront détectés par ELISA à partir du sérum ou de taches de sang séché. Un sous-groupe de sujets participera également à l'évaluation d'anticorps neutralisants dirigés contre les nouveaux variants préoccupants. Des cellules mononuclées de sang périphérique seront prélevées pour établir un profil immunitaire exploratoire de l'immunité cellulaire spécifique au SARS-CoV-2. Méthodologie: Les sujets seront recrutés avant ou après toute dose du vaccin contre la COVID-19 et se soumettront à des prélèvements sanguins pour les tests sérologiques à 1, 3, 6, 9 et 12 mois post-vaccination. Lorsque possible, des échantillons seront prélevés avant l'administration d'une dose ou d'un rappel. Les sujets demeureront dans l'étude pendant au moins un an après leur dernière dose de vaccin contre la COVID-19. Points forts et limites: La conception adaptative de l'étude permet d'apporter des modifications planifiées fondées sur de nouvelles données ou des changements rapides dans les politiques de santé publique entourant la vaccination. Les résultats sont limités par l'absence de certains prélèvements sanguins antérieurs (point temporels) en raison de la vaccination rapide de la population. Conclusion: Cette vaste étude sérologique multicentrique menée auprès de personnes atteintes de néphropathie fournira des données sur la cinétique de la réponse immunitaire à la vaccination contre le SARS-CoV-2 dans l'ensemble du spectre de l'IRC. Elle fournira des informations sur l'amplitude et la durée de l'immunité conférée par la vaccination contre la COVID-19 et permettra de caractériser les facteurs associés à la réponse immunitaire. Ces résultats serviront à orienter les recommandations de santé publique et les lignes directrices en matière d'immunisation pour les quatre millions de Canadiens et Canadiennes qui vivent avec l'IRC.

SARS-CoV-2 Virus-Like Particle Neutralizing Capacity in Blood Donors Depends on Serological Profile and Donor-Declared SARS-CoV-2 Vaccination History.

This study attempted to understand the levels of neutralizing titers and the breadth of antibody protection against wild-type and variant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Canadian blood donors during the first 3 months of 2021. During this period, it is unlikely that many of the blood donors had received a second dose, since vaccine rollout had not yet ramped up, and less than 2% of the Canadian population had received a second dose of vaccine. A repeated cross-sectional design was used. A random cross-sectional sampling of all available Canadian Blood Services retention samples (n = 1,500/month) was drawn monthly for January, February, and March 2021. A tiered testing approach analyzed 4,500 Canadian blood donor specimens for potential evidence of a signal for anti-spike (anti-S), anti-receptor-binding domain (anti-RBD), and anti-nucleocapsid protein (anti-N). Specimens were stratified based on donor-declared vaccination history and then stratified on the presence or absence of anti-N as follows: (i) "vaccinated plus anti-N" (n = 5), (ii) "vaccinated and no anti-N" (n = 20), (iii) "unvaccinated plus anti-N" (n = 20), and (iv) "unvaccinated and no anti-N" (n = 20). Randomized specimens were then characterized for neutralizing capacity against wild-type as well as SARS-CoV-2 variants of concern (VOCs) (Alpha [B.1.1.7], Beta [B.1.351], Gamma [P.1], and Delta [B.1.617.2]) using S-pseudotyped virus-like particle (VLP) neutralization assays. There was no neutralizing capacity against wild-type and VOC VLPs within the "no vaccine and no anti-N" group. Neutralization of Beta VLPs was less than wild-type VLPs within "vaccinated plus anti-N," "vaccinated and no anti-N", and "unvaccinated plus anti-N" groups. IMPORTANCE In the first 3 months of 2021 as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination was in the initial stages of a mass rollout, Canadian blood donors had various levels of humoral protection against wild-type and variant of concern (VOC) SARS-CoV-2. Very few Canadians would have received a second dose of a SARS-CoV-2 vaccine. In this study, we identified elevated levels of neutralizing capacity, albeit with reduced neutralization capacity against one or more SARS-CoV-2 strains (wild type and VOCs) in vaccinated blood donors. This broad neutralizing response we present regardless of evidence of natural SARS-CoV-2 infection. Neutralizing capacity against wild type and VOCs varied significantly within the unvaccinated group, with one subset of unvaccinated plasma specimens (unvaccinated and no anti-N) having no measurable wild type- nor variant-neutralizing capacity. The study is important because it indicates that vaccination can be associated with a broad neutralizing antibody capacity of donor plasma against SARS-CoV-2 VOCs.

Investigating the mechanisms of peritoneal metastasis in gastric adenocarcinoma using a novel ex vivo peritoneal explant model.

Gastric adenocarcinoma, commonly known as stomach cancer, has a predilection for metastasis to the peritoneum, which portends limited survival. The peritoneal metastatic cascade remains poorly understood, and existing models fail to recapitulate key elements of the interaction between cancer cells and the peritoneal layer. To explore the underlying cellular and molecular mechanisms of peritoneal metastasis, we developed an ex vivo human peritoneal explant model. Fresh peritoneal tissue samples were suspended, mesothelial layer down but without direct contact, above a monolayer of red-fluorescent dye stained AGS human gastric adenocarcinoma cells for 24 h, then washed thoroughly. Implantation of AGS cells within the explanted peritoneum and invasion beyond the mesothelial layer were examined serially using real-time confocal fluorescence microscopy. Histoarchitecture of the explanted peritoneum was preserved over 5 days ex vivo. Both implantation and invasion were suppressed by restoration of functional E-cadherin through stable transfection of AGS cells, demonstrating sensitivity of the model to molecular manipulation. Thus, our ex vivo human peritoneal explant model permits meaningful investigation of the pathways and mechanism that contribute to peritoneal metastasis. The model will facilitate screening of new therapies that target peritoneal dissemination of gastric, ovarian and colorectal cancer.

A Qualitative Comparison of the Abbott SARS-CoV-2 IgG II Quant Assay against Commonly Used Canadian SARS-CoV-2 Enzyme Immunoassays in Blood Donor Retention Specimens, April 2020 to March 2021.

Our group has previously used laboratory and commercially developed assays to understand the IgG responses to SARS-CoV-2 antigens, including nucleocapsid (N), spike (S), and receptor binding domain (RBD), in Canadian blood donors. In this current study, we analyzed 17,428 available and previously characterized retention samples collected from April 2020 to March 2021. The analysis compared the characteristics of the Abbott SARS-CoV-2 IgG II Quant assay (Abbott anti-spike [S], Abbott, Chicago, IL) against four other IgG assays. The Abbott anti-S assay has a qualitative threshold of 50 AU/mL. The four comparator assays were the Abbott anti-nucleocapsid (N) assay and three commonly used Canadian in-house IgG enzyme-linked immunosorbent assays (ELISAs) recognizing distinct recombinant viral antigens, full-length spike glycoprotein, glycoprotein RBD, and nucleocapsid. The strongest qualitative relationship was between Sinai RBD and the Abbott anti-S assay (kappa, 0.707; standard error [SE] of kappa, 0.018; 95% confidence interval, 0.671 to 0.743). We then scored each previously characterized specimen as positive when two anti-SARS-COV-2 assays identified anti-SARS-CoV-2 IgG in the specimen. Using this composite reference standard approach, the sensitivity of the Abbott anti-S assay was 95.96% (95% confidence interval [CI], 93.27 to 97.63%). The specificity of the Abbott anti-S assay was 99.35% (95% CI, 99.21 to 99.46%). Our study provides context on the use of commonly used SARS-CoV-2 serologies in Canada and identifies how these assays qualitatively compare to newer commercial assays. Our next steps are to assess how well the Abbott anti-S assays quantitatively detect wild-type and SARS-CoV-2 variants of concern. IMPORTANCE We describe the qualitative test characteristics of the Abbott SARS-CoV-2 IgG II Quant assay against four other anti-SARS-CoV-2 IgG assays commonly used in Canada. Although there is no gold standard for identifying anti-SARS-CoV-2 seropositivity, aggregate standards can be used to assess seropositivity. In this study, we used a specimen bank of previously well-characterized specimens collected between April 2020 and March 2021. The Abbott anti-S assay showed the strongest qualitative relationship with a widely used laboratory-developed IgG assay for the SARS-CoV-2 receptor binding domain. Using the composite reference standard approach, we also showed that the Abbott anti-S assay was highly sensitive and specific. As new anti-SARS-CoV-2 assays are developed, it is important to compare their test characteristics against other assays that have been extensively used in prior research.

Estimating SARS-CoV-2 Seroprevalence in Canadian Blood Donors, April 2020 to March 2021: Improving Accuracy with Multiple Assays.

We have previously used composite reference standards and latent class analysis (LCA) to evaluate the performance of laboratory assays in the presence of tarnished gold standards. Here, we apply these techniques to repeated, cross-sectional study of Canadian blood donors, whose sera underwent parallel testing with four separate SARS-CoV-2 antibody assays. We designed a repeated cross-sectional design with random cross-sectional sampling of all available retention samples (n = 1500/month) for a 12 -month period from April 2020 until March 2021. Each sample was evaluated for SARS-CoV-2 IgG antibodies using four assays an Abbott Architect assay targeting the nucleocapsid antigen (Abbott-NP, Abbott, Chicago IL) and three in-house IgG ELISAs recognizing distinct recombinant viral antigens: full-length spike glycoprotein (Spike), spike glycoprotein receptor binding domain (RBD) and nucleocapsid (NP). We used two analytic approaches to estimate SAR-CoV-2 seroprevalence: a composite reference standard and LCA. Using LCA to estimate true seropositivity status based on the results of the four antibody tests, we estimated that seroprevalence increased from 0.8% (95% CI: 0.5-1.4%) in April 2020 to 6.3% (95% CI: 5.1-7.6%) in March 2021. Our study provides further support for the use of LCA in upcoming public health crises, epidemics, and pandemics when a gold standard assay may not be available or identifiable. IMPORTANCE Here, we describe an approach to estimating seroprevalence in a low prevalence setting when multiple assays are available and yet no known gold standard exists. Because serological studies identify cases through both diagnostic testing and surveillance, and otherwise silent, unrecognized infections, serological data can be used to estimate the true infection fatality ratio of a disease. However, seroprevalence studies rely on assays with imperfect sensitivity and specificity. Seroreversion (loss of antibody response) also occurs over time, and with the advent of vaccination, distinction of antibody response resulting from vaccination as opposed to antibody response due to infection has posed an additional challenge. Our approach indicates that seroprevalence on Canadian blood donors by the end of March 2021was less than 10%. Our study supports the use of latent class analysis in upcoming public health crises, epidemics, and pandemics when a gold standard assay may not be available or identifiable.