The ribosomal S6 kinase 2 (RSK2)-SPRED2 complex regulates the phosphorylation of RSK substrates and MAPK signaling.

Sprouty-related EVH-1 domain-containing (SPRED) proteins are a family of proteins that negatively regulate the RAS-Mitogen-Activated Protein Kinase (MAPK) pathway, which is involved in the regulation of the mitogenic response and cell proliferation. However, the mechanism by which these proteins affect RAS-MAPK signaling has not been elucidated. Patients with mutations in SPRED give rise to unique disease phenotypes; thus, we hypothesized that distinct interactions across SPRED proteins may account for alternative nodes of regulation. To characterize the SPRED interactome and evaluate how members of the SPRED family function through unique binding partners, we performed affinity purification mass spectrometry. We identified 90-kDa ribosomal S6 kinase 2 (RSK2) as a specific interactor of SPRED2 but not SPRED1 or SPRED3. We identified that the N-terminal kinase domain of RSK2 mediates the interaction between amino acids 123 to 201 of SPRED2. Using X-ray crystallography, we determined the structure of the SPRED2-RSK2 complex and identified the SPRED2 motif, F145A, as critical for interaction. We found that the formation of this interaction is regulated by MAPK signaling events. We also find that this interaction between SPRED2 and RSK2 has functional consequences, whereby the knockdown of SPRED2 resulted in increased phosphorylation of RSK substrates, YB1 and CREB. Furthermore, SPRED2 knockdown hindered phospho-RSK membrane and nuclear subcellular localization. We report that disruption of the SPRED2-RSK complex has effects on RAS-MAPK signaling dynamics. Our analysis reveals that members of the SPRED family have unique protein binding partners and describes the molecular and functional determinants of SPRED2-RSK2 complex dynamics.

Unexpected tobacco etch virus (TEV) protease cleavage of recombinant human proteins.

The tobacco etch virus (TEV) protease is a commonly used reagent for removal of solubility and purification tags from recombinant proteins and is cited as being highly specific for its canonical cleavage site. Flexibility in some amino acids within this recognition sequence has been described in the literature but researchers generally assume few native human proteins will carry off-target sequences for TEV cleavage. We report here the aberrant cleavage of three human proteins with non-canonical TEV protease cleavage sites and identify broader sequence specificity rules that can be used to predict unwanted cleavage of recombinant proteins. Using these rules, 456 human proteins were identified that could be substrates for unwanted TEV protease cleavage.

Effect of D614G Spike Variant on Immunoglobulin G, M, or A Spike Seroassay Performance.

Emergence of a new spike protein variant (D614G) with increased infectivity has prompted many to analyze its role in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. There is concern regarding whether an individual exposed to one variant of a virus will have cross-reactive memory to the second variant. Accordingly, we analyzed the serologic reactivity of both variants, and we found that antibodies from 88 donors from a high-incidence population reacted toward both the original spike and the D614 spike variant. These data suggest that patients who are exposed to either variant have cross-responsive humoral immunity. This represents an important finding both for SARS-CoV-2 disease biology and for therapeutics.

Refining the N-Termini of the SARS-CoV-2 Spike Protein and Its Discrete Receptor-Binding Domain.

Previous work employing five SARS-CoV-2 spike protein receptor-binding domain (RBD) constructs, comprising versions originally developed by Mt. Sinai or the Ragon Institute and later optimized in-house, revealed potential heterogeneity which led to questions regarding variable seropositivity assay performance. Each construct was subjected to N-deglycosylation and subsequent intact mass analysis, revealing significant deviations from predicted theoretical mass for all five proteins. Complementary tandem MS/MS analysis revealed the presence of an additional pyroGlu residue on the N-termini of the two Mt. Sinai RBD constructs, as well as on the N-terminus of the full-length spike protein from which they were derived, thus explaining the observed mass shift and definitively establishing the spike protein N-terminal sequence. Moreover, the observed mass additions for the three Ragon Institute RBD constructs were identified as variable N-terminal cleavage points within the signal peptide sequence employed for recombinant expression. To resolve this issue and minimize heterogeneity for further seropositivity assay development, the best-performing RBD construct was further optimized to exhibit complete homogeneity, as determined by both intact mass and tandem MS/MS analysis. This new RBD construct has been validated for seropositivity assay performance, is available to the greater scientific community, and is recommended for use in future assay development.

Serologic Cross-Reactivity of SARS-CoV-2 with Endemic and Seasonal Betacoronaviruses.

In order to properly understand the spread of SARS-CoV-2 infection and development of humoral immunity, researchers have evaluated the presence of serum antibodies of people worldwide experiencing the pandemic. These studies rely on the use of recombinant proteins from the viral genome in order to identify serum antibodies that recognize SARS-CoV-2 epitopes. Here, we discuss the cross-reactivity potential of SARS-CoV-2 antibodies with the full spike proteins of four other betacoronaviruses that cause disease in humans, MERS-CoV, SARS-CoV, HCoV-OC43, and HCoV-HKU1. Using enzyme-linked immunosorbent assays (ELISAs), we detected the potential cross-reactivity of antibodies against SARS-CoV-2 towards the four other coronaviruses, with the strongest cross-recognition between SARS-CoV-2 and SARS /MERS-CoV antibodies, as expected based on sequence homology of their respective spike proteins. Further analysis of cross-reactivity could provide informative data that could lead to intelligently designed pan-coronavirus therapeutics or vaccines.

Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection.

Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays.

The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots.

FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry.

Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.

Long-range enhancers on 8q24 regulate c-Myc.

Recent genomewide association studies have found multiple genetic variants on chromosome 8q24 that are significantly associated with an increased susceptibility to prostate, colorectal, and breast cancer. These risk loci are located in a "gene desert," a few hundred kilobases telomeric to the Myc gene. To date, the biological mechanism(s) underlying these associations remain unclear. It has been speculated that these 8q24 genetic variant(s) might affect Myc expression by altering its regulation or amplification status. Here, we show that multiple enhancer elements are present within this region and that they can regulate transcription of Myc. We also demonstrate that one such enhancer element physically interacts with the Myc promoter via transcription factor Tcf-4 binding and acts in an allele specific manner to regulate Myc expression.

Dynamics of SARS-CoV-2 Seroprevalence in a Large US population Over a Period of 12 Months.

Due to a combination of asymptomatic or undiagnosed infections, the proportion of the United States population infected with SARS-CoV-2 was unclear from the beginning of the pandemic. We previously established a platform to screen for SARS-CoV-2 positivity across a representative proportion of the US population, from which we reported that almost 17 million Americans were estimated to have had undocumented infections in the Spring of 2020. Since then, vaccine rollout and prevalence of different SARS-CoV-2 variants have further altered seropositivity trends within the United States population. To explore the longitudinal impacts of the pandemic and vaccine responses on seropositivity, we re-enrolled participants from our baseline study in a 6- and 12- month follow-up study to develop a longitudinal antibody profile capable of representing seropositivity within the United States during a critical period just prior to and during the initiation of vaccine rollout. Initial measurements showed that, since July 2020, seropositivity elevated within this population from 4.8% at baseline to 36.2% and 89.3% at 6 and 12 months, respectively. We also evaluated nucleocapsid seropositivity and compared to spike seropositivity to identify trends in infection versus vaccination relative to baseline. These data serve as a window into a critical timeframe within the COVID-19 pandemic response and serve as a resource that could be used in subsequent respiratory illness outbreaks.

Standardization of ELISA protocols for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling.

The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is key to avoiding medically costly diagnostic errors, as well as to assuring properly informed public health decisions. Here, we present an optimized ELISA-based serology protocol, from antigen production to data analyses, that helps define thresholds for IgG and IgM seropositivity with high specificities. Validation of this protocol is performed using traditionally collected serum as well as dried blood on mail-in blood sampling kits. Archival (pre-2019) samples are used as negative controls, and convalescent, PCR-diagnosed COVID-19 patient samples serve as positive controls. Using this protocol, minimal cross-reactivity is observed for the spike proteins of MERS, SARS1, OC43 and HKU1 viruses, and no cross reactivity is observed with anti-influenza A H1N1 HAI. Our protocol may thus help provide standardized, population-based data on the extent of SARS-CoV-2 seropositivity, immunity and infection.

A majority of uninfected adults show preexisting antibody reactivity against SARS-CoV-2.

Preexisting cross-reactivity to SARS-CoV-2 occurs in the absence of prior viral exposure. However, this has been difficult to quantify at the population level due to a lack of reliably defined seroreactivity thresholds. Using an orthogonal antibody testing approach, we estimated that about 0.6% of nontriaged adults from the greater Vancouver, Canada, area between May 17 and June 19, 2020, showed clear evidence of a prior SARS-CoV-2 infection, after adjusting for false-positive and false-negative test results. Using a highly sensitive multiplex assay and positive/negative thresholds established in infants in whom maternal antibodies have waned, we determined that more than 90% of uninfected adults showed antibody reactivity against the spike protein, receptor-binding domain (RBD), N-terminal domain (NTD), or the nucleocapsid (N) protein from SARS-CoV-2. This seroreactivity was evenly distributed across age and sex, correlated with circulating coronaviruses' reactivity, and was partially outcompeted by soluble circulating coronaviruses' spike. Using a custom SARS-CoV-2 peptide mapping array, we found that this antibody reactivity broadly mapped to spike and to conserved nonstructural viral proteins. We conclude that most adults display preexisting antibody cross-reactivity against SARS-CoV-2, which further supports investigation of how this may impact the clinical severity of COVID-19 or SARS-CoV-2 vaccine responses.

SARS-CoV-2 Seroprevalence and Drug Use in Trauma Patients from Six Sites in the United States.

In comparison to the general patient population, trauma patients show higher level detections of bloodborne infectious diseases, such as Hepatitis and Human Immunodeficiency Virus. In comparison to bloodborne pathogens, the prevalence of respiratory infections such as SARS-CoV-2 and how that relates with other variables, such as drug usage and trauma type, is currently unknown in trauma populations. Here, we evaluated SARS-CoV-2 seropositivity and antibody isotype profile in 2,542 trauma patients from six Level-1 trauma centers between April and October of 2020 during the first wave of the COVID-19 pandemic. We found that the seroprevalence in trauma victims 18-44 years old (9.79%, 95% confidence interval/CI: 8.33 - 11.47) was much higher in comparison to older patients (45-69 years old: 6.03%, 4.59-5.88; 70+ years old: 4.33%, 2.54 - 7.20). Black/African American (9.54%, 7.77 - 11.65) and Hispanic/Latino patients (14.95%, 11.80 - 18.75) also had higher seroprevalence in comparison, respectively, to White (5.72%, 4.62 - 7.05) and Non-Latino patients (6.55%, 5.57 - 7.69). More than half (55.54%) of those tested for drug toxicology had at least one drug present in their system. Those that tested positive for narcotics or sedatives had a significant negative correlation with seropositivity, while those on anti-depressants trended positive. These findings represent an important consideration for both the patients and first responders that treat trauma patients facing potential risk of respiratory infectious diseases like SARS-CoV-2.

Antigen-based multiplex strategies to discriminate SARS-CoV-2 natural and vaccine induced immunity from seasonal human coronavirus humoral responses.

Sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 sero-surveillance. With the rollout of SARS-CoV-2 vaccines, such assays must be able to distinguish vaccine from natural immunity to SARS-CoV-2 and related human coronaviruses. Here, we developed and implemented multiplex microsphere-based immunoassay strategies for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2 and the endemic seasonal human coronaviruses (HCoV), enabling high throughout measurement of pre-existing cross-reactive antibodies. We varied SARS-CoV-2 antigen compositions within the multiplex assay, allowing direct comparisons of the effects of spike protein, receptor-binding domain protein (RBD) and nucleocapsid protein (NP) based SARS-CoV-2 antibody detection. Multiplex immunoassay performance characteristics are antigen-dependent, and sensitivities and specificities range 92-99% and 94-100%, respectively, for human subject samples collected as early as 7-10 days from symptom onset. SARS-CoV-2 spike and RBD had a strong correlative relationship for the detection of IgG. Correlation between detectable IgG reactive with spike and NP also had strong relationship, however, several PCR-positive and spike IgG-positive serum samples were NP IgG-negative. This spike and NP multiplex immunoassay has the potential to be useful for differentiation between vaccination and natural infection induced antibody responses. We also assessed the induction of de novo SARS-CoV-2 IgG cross reactions with SARS-CoV and MERS-CoV spike proteins. Furthermore, multiplex immunoassays that incorporate spike proteins of SARS-CoV-2 and HCoVs will permit investigations into the influence of HCoV antibodies on COVID-19 clinical outcomes and SARS-CoV-2 antibody durability.

Mapping a Pandemic: SARS-CoV-2 Seropositivity in the United States.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates. To address this, we analyzed seropositivity in US adults who have not previously been diagnosed with COVID-19. Individuals with characteristics that reflect the US population (n = 11,382) and who had not previously been diagnosed with COVID-19 were selected by quota sampling from 241,424 volunteers (ClinicalTrials.gov NCT04334954). Enrolled participants provided medical, geographic, demographic, and socioeconomic information and 9,028 blood samples. The majority (88.7%) of samples were collected between May 10th and July 31st, 2020. Samples were analyzed via ELISA for anti-Spike and anti-RBD antibodies. Estimation of seroprevalence was performed by using a weighted analysis to reflect the US population. We detected an undiagnosed seropositivity rate of 4.6% (95% CI: 2.6 - 6.5%). There was distinct regional variability, with heightened seropositivity in locations of early outbreaks. Subgroup analysis demonstrated that the highest estimated undiagnosed seropositivity within groups was detected in younger participants (ages 18-45, 5.9%), females (5.5%), Black/African American (14.2%), Hispanic (6.1%), and Urban residents (5.3%), and lower undiagnosed seropositivity in those with chronic diseases. During the first wave of infection over the spring/summer of 2020 an estimate of 4.6% of adults had a prior undiagnosed SARS-CoV-2 infection. These data indicate that there were 4.8 (95% CI: 2.8-6.8) undiagnosed cases for every diagnosed case of COVID-19 during this same time period in the United States, and an estimated 16.8 million undiagnosed cases by mid-July 2020.

Undiagnosed SARS-CoV-2 seropositivity during the first 6 months of the COVID-19 pandemic in the United States.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates in the United States and elsewhere. To address this, we analyzed seropositivity in 9089 adults in the United States who had not been diagnosed previously with COVID-19. Individuals with characteristics that reflected the U.S. population (n = 27,716) were selected by quota sampling from 462,949 volunteers. Enrolled participants (n = 11,382) provided medical, geographic, demographic, and socioeconomic information and dried blood samples. Survey questions coincident with the Behavioral Risk Factor Surveillance System survey, a large probability-based national survey, were used to adjust for selection bias. Most blood samples (88.7%) were collected between 10 May and 31 July 2020 and were processed using ELISA to measure seropositivity (IgG and IgM antibodies against SARS-CoV-2 spike protein and the spike protein receptor binding domain). The overall weighted undiagnosed seropositivity estimate was 4.6% (95% CI, 2.6 to 6.5%), with race, age, sex, ethnicity, and urban/rural subgroup estimates ranging from 1.1% to 14.2%. The highest seropositivity estimates were in African American participants; younger, female, and Hispanic participants; and residents of urban centers. These data indicate that there were 4.8 undiagnosed SARS-CoV-2 infections for every diagnosed case of COVID-19, and an estimated 16.8 million infections were undiagnosed by mid-July 2020 in the United States.

D614G Spike Variant Does Not Alter IgG, IgM, or IgA Spike Seroassay Performance.

Emergence of a new variant of spike protein (D614G) with increased infectivity and transmissibility has prompted many to analyze the potential role of this variant in the SARS-CoV-2 pandemic. When a new variant emerges, there is a concern regarding whether an individual exposed to one variant of a virus will have cross-reactive immune memory to the second variant. Accordingly, we analyzed the serologic reactivity of D614 (original) and G614 variant spike proteins. We found that antibodies from a high-incidence population in New York City reacted both toward the original D614 spike and the G614 spike variant. These data suggest that patients who have been exposed to either SARS-CoV-2 variant have humoral immunity that can respond against both variants. This is an important finding both for SARS-CoV-2 disease biology and for potential antibody-based therapeutics.

Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Highlights: Improved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parametersTwo versions of RBD show different sensitivity in serology assaysYields of greater than 50 mg/l obtained under optimal conditionsMagnetic bead purification technology improves throughput of protein production.

Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays.

The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots.

Serologic cross-reactivity of SARS-CoV-2 with endemic and seasonal Betacoronaviruses.

In order to properly understand the spread of SARS-CoV-2 infection and development of humoral immunity, researchers have evaluated the presence of serum antibodies of people worldwide experiencing the pandemic. These studies rely on the use of recombinant proteins from the viral genome in order to identify serum antibodies that recognize SARS-CoV-2 epitopes. Here, we discuss the cross-reactivity potential of SARS-CoV-2 antibodies with the full spike proteins of four other Betacoronaviruses that cause disease in humans, MERS-CoV, SARS-CoV, HCoV-OC43, and HCoV-HKU1. Using enzyme-linked immunosorbent assays (ELISAs), we detected the potential cross-reactivity of antibodies against SARS-CoV-2 towards the four other coronaviruses, with the strongest cross-recognition between SARS-CoV-2 and SARS /MERS-CoV antibodies, as expected based on sequence homology of their respective spike proteins. Further analysis of cross-reactivity could provide informative data that could lead to intelligently designed pan-coronavirus therapeutics or vaccines.