Search

Toxin-antitoxin (TA) systems are abundant genetic modules in bacterial chromosomes and on mobile elements. They are often patchily distributed and their physiological functions remain poorly understood. Here, we characterize a TA system inLegionella pneumophilathat is highly conserved acrossLegionellaspecies. This system is distantly related toEscherichia coliHipBST and we demonstrate that it is a functional tripartite TA system (denoted HipBSTLp). We identify HipBSTLphomologs in diverse taxa, yet in the Gammaproteobacteria these are almost exclusively found inLegionellaspecies. Notably, the toxin HipTLpwas previously reported to be a pathogenic effector protein that is translocated byL. pneumophilainto its eukaryotic hosts. Contrary to this, we find no signal of HipTLptranslocation beyond untranslocated control levels and make several observations consistent with a canonical role as a bacterial toxin. We present structural and biochemical insights into the regulation and neutralization of HipBSTLp, and identify key variations between this system and HipBSTEc. Finally, we show that the target of HipTLpis likely not conserved with any characterized HipA or HipT toxin. This work serves as a unique comparison of a TA system across bacterial species and illustrates the molecular diversity that exists within a single TA family.

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.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.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.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.

Background: Kidney transplant recipients (KTR) have a diminished response to SARS-CoV-2 vaccination in comparison to immunocompetent individuals. Deeper understanding of the antibody response in KTRs following third-dose vaccination would enable identification of those who remain unprotected against Omicron and require additional treatment strategies. Methods: We profiled antibody responses in KTRs pre- and at one and three months post-third-dose SARS-CoV2 mRNA-based vaccine. Anti-spike and anti-RBD IgG 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: 44 KTRs were analysed at 1 and 3 months (n=26) post-third-dose. At one month, 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 months, but the proportion of KTRs with a robust antibody response was unchanged. 38.5% KTRs maintained Omicron-specific neutralization at 3 months. No clinical variables were significantly associated with detectable Omicron neutralizing antibodies, but anti-RBD titres appeared to identify those with Omicron-specific neutralizing capacity. Conclusion: Over 50% of KTRs lack an Omicron-specific neutralization response 1 month following a third mRNA-vaccine dose. Among responders, binding and neutralizing antibody responses were well preserved at 3 months. Anti-RBD antibody titres may be a useful identifier of patients with detectable Omicron neutralizing antibody response. Trial registration: Clinical Trials Ontario: ID 3604

Antibody testing against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable solution to detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction.We developed methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD) and nucleocapsid (N). We automated a surrogate neutralisation (sn)ELISA that measures inhibition of ACE2-spike or -RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard.Our single-point IgG-based ELISAs accurately distinguished non-infected and infected individuals. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for ≥ 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. We present detailed protocols for serum/plasma or dried blood spots analysis performed manually and on automated platforms. The snELISA can be performed automatically at single points, increasing its scalability.Measuring antibodies to three viral antigens and identify neutralising antibodies capable of disrupting spike-ACE2 interactions in high-throughput enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The reagents are available to enable scaling up of standardised serological assays, permitting inter-laboratory data comparison and aggregation.

Pseudoalteromonas is a globally distributed marine-associated genus that can be found in a broad range of aquatic environments, including in association with macroalgal surfaces where they may take advantage of these rich sources of polysaccharides. The metabolic systems that confer the ability to metabolize this abundant form of photosynthetically fixed carbon, however, are not yet fully understood. Through genomics, transcriptomics, microbiology, and specific structure-function studies of pathway components we address the capacity of newly isolated marine pseudoalteromonads to metabolize the red algal galactan carrageenan. The results reveal that the κ/ι-carrageenan specific polysaccharide utilization locus (CarPUL) enables isolates possessing this locus the ability to grow on this substrate. Biochemical and structural analysis of the enzymatic components of the CarPUL promoted the development of a detailed model of the κ/ι-carrageenan metabolic pathway deployed by pseudoalteromonads, thus furthering our understanding of how these microbes have adapted to a unique environmental niche., Hettle et al. investigate the ability of marine Pseudoalteromonas sp. to metabolise carrageenan, a polysaccharide abundant in red algae. They isolate and characterise previously unstudied strains and find that the recently identified κ/ι-carrageenan specific polysaccharide utilization locus (CarPUL) is required for growth on carrageenan, and biochemically map out many of the steps.

The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.

BACKGROUND: Testing for antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable "Made-in-Canada" solution that can detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction. METHODS: We developed a set of methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD), and the nucleocapsid (N) protein. These antigens are complemented by a detection antibody (human anti-IgG fused to horseradish peroxidase (HRP)) and a positive control reference antibody (recombinant IgG against the RBD), permitting intra- and inter-laboratory comparisons. Using this toolkit and commercial reagents, we optimized automated ELISAs on two different high throughput platforms to measure antibody responses to SARS-CoV-2 antigens. The assays were calibrated to a reference standard from the World Health Organization. We also automated a surrogate neutralization (sn)ELISA that measures inhibition of ACE2-Spike or -RBD interactions by antibodies using biotinylated ACE2. RESULTS: Our individual IgG-based ELISAs measure antibody levels in single-point measurements in reference to a standard antibody curve to accurately distinguish non-infected and infected individuals (area under the curve > 0.96 for each assay). Positivity thresholds can be established in individual assays using precision-recall analysis (e.g., by fixing the false positive rate), or more stringently, by scoring against the distribution of the means of negative samples across multiple assays performed over several months. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for at least 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. Here, we present detailed protocols to perform these assays using either serum/plasma or dried blood spots both manually and on two automated platforms, and to express the results in international units to facilitate data harmonization and inter-study comparisons. We also demonstrate that the snELISA can be performed automatically at single points, increasing the scalability of this functional assay for large seroprevalence studies. INTERPRETATION: The ability to measure antibodies to three viral antigens and identify neutralizing antibodies capable of disrupting spike-ACE2 interactions in high-throughput assays enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The "Made-in-Canada" set of protein reagents, produced at the National Research Council of Canada are publicly available to enable the up-scaling of standardized serological assays, permitting nationwide data comparison and aggregation.

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 employed, 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, USA) against 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. This article is protected by copyright. All rights reserved.

Prioritizing Ontario’s long-term care home (LTCH) residents for vaccination against severe acute respiratory syndrome coronavirus 2 has drastically reduced their disease burden; however, recent LTCH outbreaks of variants of concern (VOCs) have raised questions regarding their immune responses. In 198 residents, mRNA vaccine dose 1 elicited partial spike and receptor binding domain antibody responses, while the second elicited a response at least equivalent to convalescent individuals in most residents. Residents administered mRNA-1273 (Moderna) mounted stronger total and neutralizing antibody responses than those administered BNT162b2 (Pfizer-BioNTech). Two to four weeks after dose 2, residents (n= 119, median age 88) produced 4.8–6.3-fold fewer neutralizing antibodies than staff (n= 78; median age 47) against wild-type (with D614G) pseudotyped lentivirus, and residents administered BNT162b2 produced 3.89-fold fewer neutralizing antibodies than those who received mRNA-1273. These effects were exacerbated upon serum challenge with pseudotyped VOC spike, with up to 7.94-fold reductions in B.1.351 (Beta) neutralization. Cumulatively, weaker vaccine stimulation, age/comorbidities, and the VOC produced an ∼130-fold reduction in apparent neutralization titers in LTCH residents and 37.9% of BNT162b2-vaccinated residents had undetectable neutralizing antibodies to B.1.351. Continued immune response surveillance and additional vaccine doses may be required in this population with known vulnerabilities.

ImportanceHemodialysis patients have an exceptionally high mortality from COVID-19 and this patient population often has a poor response to vaccinations. Randomized controlled trials for COVID-19 vaccines included few patients with kidney disease, therefore vaccine immunogenicity is uncertain in this population.ObjectiveEvaluate the SARS-CoV-2 antibody response in chronic hemodialysis patients following one versus two doses of BNT162b2 COVID-19 vaccination compared to health care worker controls and convalescent serum.DesignProspective observational cohort study.SettingSingle centre study in Toronto, Ontario, Canada.Participants142 in-centre hemodialysis patients and 35 health care worker controls.ExposureBNT162b2 (Pfizer-BioNTech) COVID-19 vaccine.Main Outcomes and MeasuresSARS-CoV-2 IgG antibodies to the spike protein (anti-spike), receptor binding domain (anti-RBD), and nucleocapsid protein (anti-NP) were measured in 66 hemodialysis patients receiving one vaccine dose following a public health policy change, 76 patients receiving two vaccine doses, and 35 health care workers receiving two vaccine doses.ResultsDetectable anti-NP suggestive of natural SARS-CoV-2 infection was detected in 15/142 (11%) of patients at baseline while only three patients had prior RT-PCR confirmed COVID-19. Two additional patients contracted COVID-19 after receiving two doses of vaccine. In patients receiving a single BNT162b2 dose, seroconversion occurred in 53/66 (80%) for anti-spike and 35/66 (55%) for anti-RBD by 28 days post dose, but only 15/66 (23%) and 4/66 (6%), respectively attained a robust response as defined by reaching the median level of anti-spike and anti-RBD in convalescent serum from COVID-19 survivors. In patients receiving two doses of BNT162b2 vaccine, seroconversion occurred in 69/72 (96%) for anti-spike and 63/72 (88%) for anti-RBD by 2 weeks following the second dose while 52/72 (72%) and 43/76 (41%) reached the median convalescent serum level of anti-spike and anti-RBD, respectively. In contrast, 35/35 (100%) of health care workers exceeded the median level of anti-spike and anti-RBD found in convalescent serum 2-4 weeks after the second dose.Conclusions and RelevanceThis study confirms poor immunogenicity 28 days following a single dose of BNT162b2 vaccine in the hemodialysis population, supporting adherence to recommended vaccination schedules, and avoiding delay of the second dose in these at-risk individuals.Key PointsQuestionWhat is the serologic response to the BNT162b2 COVID-19 vaccine in hemodialysis patients?FindingsIn this prospective observational study, humoral response was compared in 66 hemodialysis patients sampled 28 days after receipt of one dose of vaccine to 76 patients who received two doses of vaccine sampled 14 days after the second dose. Among those receiving one dose, 6% had anti-RBD response above the median level of convalescent serum versus 41% who received two doses.MeaningGiven that hemodialysis patients exhibit a poor humoral response to a single dose of BNT162b2 vaccine, the second dose should not be delayed.

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.

Saliva is an alternative biofluid to serum for detecting and monitoring IgG to SARS-CoV-2 spike and RBD antigens in COVID-19., While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the antibody response in saliva and its relationship to systemic antibody levels. Here, we profiled by enzyme-linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor-binding domain (RBD) in serum and saliva of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3–115 days post-symptom onset (PSO), compared to negative controls. Anti-SARS-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16–30 days PSO. Longitudinal analysis revealed that anti-SARS-CoV-2 IgA and IgM antibodies rapidly decayed, while IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that serum and saliva IgG antibodies to SARS-CoV-2 are maintained in the majority of COVID-19 patients for at least 3 months PSO. IgG responses in saliva may serve as a surrogate measure of systemic immunity to SARS-CoV-2 based on their correlation with serum IgG responses.

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector–based assay., An ELISA-type competition assay serves as a neutralization surrogate for SARS-CoV-2 infection and was validated against standard neutralization assays on cohorts of convalescent patients.

The incidence of infection during SARS-CoV-2 viral waves, the factors associated with infection, and the durability of antibody responses to infection among Canadian adults remain undocumented.To assess the cumulative incidence of SARS-CoV-2 infection during the first 2 viral waves in Canada by measuring seropositivity among adults.The Action to Beat Coronavirus study conducted 2 rounds of an online survey about COVID-19 experience and analyzed immunoglobulin G levels based on participant-collected dried blood spots (DBS) to assess the cumulative incidence of SARS-CoV-2 infection during the first and second viral waves in Canada. A sample of 19 994 Canadian adults (aged ≥18 years) was recruited from established members of the Angus Reid Forum, a public polling organization. The study comprised 2 phases (phase 1 from May 1 to September 30, 2020, and phase 2 from December 1, 2020, to March 31, 2021) that generally corresponded to the first (April 1 to July 31, 2020) and second (October 1, 2020, to March 1, 2021) viral waves.SARS-CoV-2 immunoglobulin G seropositivity (using a chemiluminescence assay) by major geographic and demographic variables and correlation with COVID-19 symptom reporting.Among 19 994 adults who completed the online questionnaire in phase 1, the mean (SD) age was 50.9 (15.4) years, and 10 522 participants (51.9%) were female; 2948 participants (14.5%) had self-identified racial and ethnic minority group status, and 1578 participants (8.2%) were self-identified Indigenous Canadians. Among participants in phase 1, 8967 had DBS testing. In phase 2, 14 621 adults completed online questionnaires, and 7102 of those had DBS testing. Of 19 994 adults who completed the online survey in phase 1, fewer had an educational level of some college or less (4747 individuals [33.1%]) compared with the general population in Canada (45.0%). Survey respondents were otherwise representative of the general population, including in prevalence of known risk factors associated with SARS-CoV-2 infection. The cumulative incidence of SARS-CoV-2 infection among unvaccinated adults increased from 1.9% in phase 1 to 6.5% in phase 2. The seropositivity pattern was demographically and geographically heterogeneous during phase 1 but more homogeneous by phase 2 (with a cumulative incidence ranging from 6.4% to 7.0% in most regions). The exception was the Atlantic region, in which cumulative incidence reached only 3.3% (odds ratio [OR] vs Ontario, 0.46; 95% CI, 0.21-1.02). A total of 47 of 188 adults (25.3%) reporting COVID-19 symptoms during phase 2 were seropositive, and the OR of seropositivity for COVID-19 symptoms was 6.15 (95% CI, 2.02-18.69). In phase 2, 94 of 444 seropositive adults (22.2%) reported having no symptoms. Of 134 seropositive adults in phase 1 who were retested in phase 2, 111 individuals (81.8%) remained seropositive. Participants who had a history of diabetes (OR, 0.58; 95% CI, 0.38-0.90) had lower odds of having detectable antibodies in phase 2.The Action to Beat Coronavirus study found that the incidence of SARS-CoV-2 infection in Canada was modest until March 2021, and this incidence was lower than the levels of population immunity required to substantially reduce transmission of the virus. Ongoing vaccination efforts remain central to reducing viral transmission and mortality. Assessment of future infection-induced and vaccine-induced immunity is practicable through the use of serial online surveys and participant-collected DBS.

There is a pressing need for an in-depth understanding of immunity to SARS-CoV-2. Here we investigated 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. All subjects showed SARS-CoV-2-specific T cell responses to at least one antigen. SARS-CoV-2-specific CD4+ T cells were primarily of the central memory phenotype and exhibited a lower IFN-γ to TNF-α ratio compared to influenza-specific responses of 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. High IL-10 production was noted in response to N protein, possibly contributing to immunosuppression, with potential implications for vaccine design. We observed granzyme B+/IFN-γg+ CD4+ and CD8+ proliferative responses to peptide pools in most individuals, with CD4+ responses predominating over CD8+ responses. Peripheral T follicular helper responses to S or N strongly correlated with serum neutralization assays as well as RBD-specific IgA. Overall, T cell responses to SARS-CoV-2 are robust, however, CD4+ Th1 responses predominate over CD8+ responses and are more inflammatory with a weaker Tfh response than influenza-specific CD4+ responses, potentially contributing to COVID-19 disease.

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin converting-enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of two viral based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus, and a spike pseudotyped viral-vector-based assay.

Compartmentalization is a defining characteristic of eukaryotic cells, partitioning cellular processes into discrete subcellular locations. High throughput microscopy1 and biochemical fractionation coupled with mass spectrometry2–6 have helped to define the proteomes of a variety of organelles and macromolecular structures. However, many other intracellular compartments have remained refractory to such approaches, due for example to difficulty in purifying non-membrane bound structures. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach for defining the composition of cellular compartments in living cells7–10. Here we present a BioID-based map of a human cell based on 192 markers from 32 different subcellular compartments, comprising 35,902 high confidence proximity interactions, and defining the intracellular locations of 4,145 unique proteins in HEK 293 cells. Our localization predictions meet or exceed previous-approaches, with higher specificity, and enabled the discovery of proteins at the mitochondrial outer membrane-endoplasmic reticulum (ER) interface that are critical for mitochondrial homeostasis. Based on this dataset, we have established 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 better understand BioID datasets.

Key Points Question What is the serologic response to the BNT162b2 COVID-19 vaccine in patients undergoing hemodialysis? Findings In this cohort study of 142 patients receiving hemodialysis, humoral response was compared in 66 patients sampled 28 days after receipt of 1 dose of vaccine with 76 patients who received 2 doses of vaccine sampled 14 days after the second dose. Among those receiving 1 dose, 6% had antireceptor binding domain response above the median level of convalescent serum vs 41% of those who received 2 doses at 1 week, increasing to 60% by 2 weeks. Meaning The findings of this study suggest that, given that patients receiving hemodialysis appeared to exhibit a poor humoral response to a single dose of BNT162b2 vaccine, the second dose should not be delayed., Importance Patients undergoing hemodialysis have a high mortality rate associated with COVID-19, and this patient population often has a poor response to vaccinations. Randomized clinical trials for COVID-19 vaccines included few patients with kidney disease; therefore, vaccine immunogenicity is uncertain in this population. Objective To evaluate the SARS-CoV-2 antibody response in patients undergoing chronic hemodialysis following 1 vs 2 doses of BNT162b2 COVID-19 vaccination compared with health care workers serving as controls and convalescent serum. Design, Setting, and Participants A prospective, single-center cohort study was conducted between February 2 and April 17, 2021, in Toronto, Ontario, Canada. Participants included 142 patients receiving in-center hemodialysis and 35 health care worker controls. Exposures BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine. Main Outcomes and Measures SARS-CoV-2 IgG antibodies to the spike protein (anti-spike), receptor binding domain (anti-RBD), and nucleocapsid protein (anti-NP). Results Among the 142 participants undergoing maintenance hemodialysis, 94 (66%) were men; median age was 72 (interquartile range, 62-79) years. SARS-CoV-2 IgG antibodies were measured in 66 patients receiving 1 vaccine dose following a public health policy change, 76 patients receiving 2 vaccine doses, and 35 health care workers receiving 2 vaccine doses. Detectable anti-NP suggestive of natural SARS-CoV-2 infection was detected in 15 of 142 (11%) patients at baseline, and only 3 patients had prior COVID-19 confirmed by reverse transcriptase polymerase chain reaction testing. Two additional patients contracted COVID-19 after receiving 2 doses of vaccine. In 66 patients receiving a single BNT162b2 dose, seroconversion occurred in 53 (80%) for anti-spike and 36 (55%) for anti-RBD by 28 days postdose, but a robust response, defined by reaching the median levels of antibodies in convalescent serum from COVID-19 survivors, was noted in only 15 patients (23%) for anti-spike and 4 (6%) for anti-RBD in convalescent serum from COVID-19 survivors. In patients receiving 2 doses of BNT162b2 vaccine, seroconversion occurred in 69 of 72 (96%) for anti-spike and 63 of 72 (88%) for anti-RBD by 2 weeks following the second dose and median convalescent serum levels were reached in 52 of 72 patients (72%) for anti-spike and 43 of 72 (60%) for anti-RBD. In contrast, all 35 health care workers exceeded the median level of anti-spike and anti-RBD found in convalescent serum 2 to 4 weeks after the second dose. Conclusions and Relevance This study suggests poor immunogenicity 28 days following a single dose of BNT162b2 vaccine in the hemodialysis population, supporting adherence to recommended vaccination schedules and avoiding delay of the second dose in these at-risk individuals., This cohort study examines the immune response of patients receiving maintenance hemodialysis to vaccination with 1 vs 2 doses of the BNT162b2 vaccine.

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. A proximity-dependent biotinylation technique defines the location of more than 4,000 proteins in a human cell, and almost 36,000 proximal interactions between proteins, including those at the interface of the mitochondria and ER.

The use of proximity-dependent biotinylation approaches combined with mass spectrometry (e.g. BioID and APEX) has revolutionized the study of protein-protein interactions and organellar proteomics. These powerful techniques are based on the fusion of an enzyme (e.g. a biotin ligase or peroxidase) to a 'bait' protein of interest, which is then expressed in a relevant biological setting. Addition of enzyme substrate enables covalent biotin labeling of proteins in the vicinity of the bait in vivo. These approaches thus allow for the capture and identification of 'neighborhood' proteins in the context of a living cell, and provide data that are complementary to more established techniques such as fractionation or affinity purification. As compared to standard affinity-based purification approaches, proximity-dependent biotinylation (PDB) can help to: first, identify interactions with and amongst membrane proteins, and other polypeptide classes that are less amenable to study by standard pulldown techniques; second, enrich for transient and/or low affinity interactions that are not readily captured using affinity purification approaches; third, avoid post-lysis artefacts associated with standard biochemical purification experiments and; fourth, provide deep insight into the organization of membrane-less organelles and other subcellular structures that cannot be easily isolated or purified. Given the increasing use of these techniques to answer a variety of different types of biological questions, it is important to understand how best to design PDB-MS experiments, what type of data they generate, and how to analyze and interpret the results.

Clostridium perfringens is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31). This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the CpGH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s.

Clostridium perfringens is an opportunistic pathogen of humans and animals whose genome encodes a wide variety of putative carbohydrate-hydrolyzing enzymes that are increasingly being shown to be directed toward the cleavage of host glycans. Among these putative enzymes is a member of glycoside hydrolase family 123. Here we show that the recombinant enzyme (referred to as CpNga123) encoded by the gene cloned from C. perfringens strain ATCC 13124 (locus tag CPF_1473) is a β-N-acetylgalactosaminidase, similar to NgaP from Paenibacillus sp. TS12. Like NgaP, CpNga123 was able to cleave the terminal β-D-GalNAc-(1→4)-D-Gal and β-D-GalNAc-(1→3)-D-Gal motifs that would be found in glycosphigolipids. The X-ray crystal structure of CpNga123 revealed it to have an N-terminal β-sandwich domain and a (β/α)8-barrel catalytic domain with a C-terminal α-helical elaboration. The structures determined in complex with reaction products provide details of the -1 subsite architecture, catalytic residues, and a structural change in the active site that is likely required to enable hydrolysis of the glycosidic bond by promoting engagement of the substrate by the catalytic residues. The features of the active site support the likelihood of a substrate-assisted catalytic mechanism for this enzyme. The structures of an inactive mutant of CpNga123 in complex with intact GA2 and Gb4 glycosphingolipid motifs reveal insight into aglycon recognition and suggest that the kinked or pleated conformation of GA2 caused by the β-1,4-linkage between N-acetylgalactosamine and galactose, and the accommodation of this conformation by the enzyme active site, may be responsible for greater activity on GA2.