ABSTRACT
The majority of pathogenic mutations in the neurofibromatosis type I (NF1) gene reduce total neurofibromin protein expression through premature truncation or microdeletion, but it is less well understood how loss-of-function missense variants drive NF1 disease. We have found that patient variants in codons 844 to 848, which correlate with a severe phenotype, cause protein instability and exert an additional dominant-negative action whereby wild-type neurofibromin also becomes destabilized through protein dimerization. We have used our neurofibromin cryogenic electron microscopy structure to predict and validate other patient variants that act through a similar mechanism. This provides a foundation for understanding genotype-phenotype correlations and has important implications for patient counseling, disease management, and therapeutics.
Subject(s)
Neurofibromatosis 1 , Neurofibromin 1 , Humans , Neurofibromin 1/metabolism , Neurofibromatosis 1/genetics , Dimerization , Mutation , Mutation, MissenseABSTRACT
Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.
Subject(s)
Proteomics , Proto-Oncogene Proteins p21(ras) , Proto-Oncogene Proteins p21(ras)/genetics , Mutation , Binding SitesABSTRACT
The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.
Subject(s)
Monomeric GTP-Binding Proteins , Monomeric GTP-Binding Proteins/metabolism , Nucleotides , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Sepharose , Guanosine Triphosphate/metabolism , Guanosine Diphosphate/metabolismABSTRACT
Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110α/p85α and a 3-fold increase in p110α. We also used these protocols to produce comparable constructs of the ß and δ isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases.
ABSTRACT
The SHOC2-MRAS-PPP1CA (SMP) complex is a holoenzyme that plays a vital role in the MAP kinase signaling pathway. Previous attempts to produce this challenging three-protein complex have relied on co-infection with multiple viruses and the use of affinity tags to attempt to isolate functional recombinant protein complexes. Leucine-rich repeat containing proteins have been historically challenging to express, and we hypothesized that co-expression of appropriate chaperones may be necessary for optimal production. We describe here how the SUGT1 chaperone can, in conjunction with polycistronic protein expression in baculovirus-infected insect cells, dramatically enhance production yield and quality of recombinant SHOC2, the SMP complex, and other leucine-rich repeat proteins.
Subject(s)
Baculoviridae , Leucine-Rich Repeat Proteins , Baculoviridae/genetics , MAP Kinase Signaling System , Recombinant Proteins/geneticsABSTRACT
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.
Subject(s)
COVID-19/diagnosis , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/blood , COVID-19/immunology , COVID-19/virology , Cross Reactions , Enzyme-Linked Immunosorbent Assay/methods , Humans , Mutation , SARS-CoV-2/classification , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
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.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , Protein Binding , Protein Domains , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Tandem Mass SpectrometryABSTRACT
Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS-mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.
Subject(s)
Neurofibromin 1/chemistry , Neurofibromin 1/metabolism , Protein Multimerization , HEK293 Cells , Humans , Neurofibromin 1/ultrastructure , Protein Domains , Structure-Activity Relationship , ras GTPase-Activating Proteins/metabolismABSTRACT
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.
Subject(s)
Betacoronavirus/immunology , SARS-CoV-2/immunology , Adolescent , Adult , Antibodies, Viral/blood , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Humans , Middle Aged , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/immunology , Young AdultABSTRACT
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.
Subject(s)
COVID-19/blood , Protein Domains/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/isolation & purification , Antibodies, Viral/immunology , COVID-19/genetics , COVID-19/virology , Enzyme-Linked Immunosorbent Assay , Humans , Protein Binding/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
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.
Subject(s)
Betacoronavirus/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/isolation & purification , Betacoronavirus/genetics , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Infections/blood , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Gene Expression , HEK293 Cells , Humans , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , TransfectionABSTRACT
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.
ABSTRACT
SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of the SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS, and PP1C form a stable ternary complex in which all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex forms only when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation and how mutations found in Noonan syndrome enhance complex formation, and reveal new avenues for therapeutic interventions.
Subject(s)
Noonan Syndrome , Humans , Intracellular Signaling Peptides and Proteins/metabolism , MAP Kinase Signaling System/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Noonan Syndrome/genetics , Noonan Syndrome/metabolism , Phosphoserine/metabolism , Protein Phosphatase 1 , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , raf Kinases/genetics , raf Kinases/metabolism , ras Proteins/metabolismABSTRACT
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.
Subject(s)
Antibodies, Viral/blood , COVID-19 Testing , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/standards , SARS-CoV-2/immunology , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/epidemiology , COVID-19/immunology , COVID-19 Nucleic Acid Testing , COVID-19 Serological Testing/methods , COVID-19 Serological Testing/standards , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Pandemics , Reference Standards , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
COVID-19 , Pandemics , Adult , Antibodies, Viral , Female , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , United States/epidemiologyABSTRACT
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.
ABSTRACT
Reports of adverse effects associated with proton pump inhibitors (PPIs) are concerning because of high usage and over-the-counter availability. We sought to determine the awareness of PPI adverse effects among our patient population, which is medically underserved, low-income, and racially diverse. A 21-item survey was administered to gastroenterology-clinic outpatients. It collected information about age, gender, education, race, specialty of the prescriber, specific PPI, indication, knowledge of dose, adherence, duration of use and awareness of any risks. Medical records were reviewed to verify survey responses pertaining to indication, dosing, and adherence. A vast majority (96%) of 101 participants were not aware of PPI adverse effects. In total, 63% of the patients completed a high school education or less, which was associated with a higher risk of long-term PPI use than completion of at least an undergraduate degree (p = 0.05). In contrast to other studies, the shockingly low patient awareness about PPI adverse effects in our patient population is particularly concerning, especially as it is tied to their demographic attributes. This may lead to long-term and high-dose PPI use. Our study highlights the need for effective provider-driven education regarding medication risks, especially in the communities with significant health disparities.
ABSTRACT
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.