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2.
J Immunol Methods ; 510: 113328, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-1977497

ABSTRACT

Monocytes are highly versatile innate immune cells responsible for pathogen clearance, innate immune coordination, and induction of adaptive immunity. Monocytes can directly and indirectly integrate pathogen-destructive instructions and contribute to disease control via pathogen uptake, presentation, or the release of cytokines. Indirect pathogen-specific instructions are conferred via Fc-receptor signaling and triggered by antibody opsonized material. Given the tremendous variation in polyclonal humoral immunity, defining the specific antibody-responses able to arm monocytes most effectively remains incompletely understood. While monocyte cell line-based assays have been used previously, cell lines may not faithfully recapitulate the full biology of monocytes. Thus, here we describe a multifaceted antigen-specific method for probing antibody-dependent primary monocyte phagocytosis (ADMP) and secondary responses. The assay not only reliably captures phagocytic uptake of immune complexes, but also detects unique changes in surface markers and cytokine secretions profiles, poorly detected by monocytic cell lines. The assay captures divergent polyclonal-monocyte recruiting activity across subjects with varying SARS-CoV-2 disease severity and also revealed biological nuances in Fc-mutant monoclonal antibody activity related to differences in Fc-receptor binding. Thus, the ADMP assay is a flexible assay able to provide key insights into the role of humoral immunity in driving monocyte phenotypic transitions and downstream functions across many diseases.


Subject(s)
COVID-19 , Monocytes , Antibodies, Monoclonal , Antigen-Antibody Complex , Antigens , Cytokines , Humans , Immunoglobulin Fc Fragments , Phagocytosis , SARS-CoV-2
3.
Nat Biotechnol ; 40(9): 1336-1340, 2022 09.
Article in English | MEDLINE | ID: covidwho-1815566

ABSTRACT

We designed a protein biosensor that uses thermodynamic coupling for sensitive and rapid detection of neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in serum. The biosensor is a switchable, caged luciferase-receptor-binding domain (RBD) construct that detects serum-antibody interference with the binding of virus RBD to angiotensin-converting enzyme 2 (ACE-2) as a proxy for neutralization. Our coupling approach does not require target modification and can better distinguish sample-to-sample differences in analyte binding affinity and abundance than traditional competition-based assays.


Subject(s)
Biosensing Techniques , COVID-19 , Antibodies, Neutralizing/chemistry , Antibodies, Viral/genetics , COVID-19/diagnosis , Humans , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
4.
JCI Insight ; 7(10)2022 05 23.
Article in English | MEDLINE | ID: covidwho-1794308

ABSTRACT

BACKGROUNDMeasuring the immune response to SARS-CoV-2 enables assessment of past infection and protective immunity. SARS-CoV-2 infection induces humoral and T cell responses, but these responses vary with disease severity and individual characteristics.METHODSA T cell receptor (TCR) immunosequencing assay was conducted using small-volume blood samples from 302 individuals recovered from COVID-19. Correlations between the magnitude of the T cell response and neutralizing antibody (nAb) titers or indicators of disease severity were evaluated. Sensitivity of T cell testing was assessed and compared with serologic testing.RESULTSSARS-CoV-2-specific T cell responses were significantly correlated with nAb titers and clinical indicators of disease severity, including hospitalization, fever, and difficulty breathing. Despite modest declines in depth and breadth of T cell responses during convalescence, high sensitivity was observed until at least 6 months after infection, with overall sensitivity ~5% greater than serology tests for identifying prior SARS-CoV-2 infection. Improved performance of T cell testing was most apparent in recovered, nonhospitalized individuals sampled > 150 days after initial illness, suggesting greater sensitivity than serology at later time points and in individuals with less severe disease. T cell testing identified SARS-CoV-2 infection in 68% (55 of 81) of samples with undetectable nAb titers (<1:40) and in 37% (13 of 35) of samples classified as negative by 3 antibody assays.CONCLUSIONThese results support TCR-based testing as a scalable, reliable measure of past SARS-CoV-2 infection with clinical value beyond serology.TRIAL REGISTRATIONSpecimens were accrued under trial NCT04338360 accessible at clinicaltrials.gov.FUNDINGThis work was funded by Adaptive Biotechnologies, Frederick National Laboratory for Cancer Research, NIAID, Fred Hutchinson Joel Meyers Endowment, Fast Grants, and American Society for Transplantation and Cell Therapy.


Subject(s)
COVID-19 , Antibodies, Neutralizing , Antibodies, Viral , Humans , Receptors, Antigen, T-Cell/genetics , SARS-CoV-2 , Severity of Illness Index , United States
6.
JCI Insight ; 7(6)2022 03 22.
Article in English | MEDLINE | ID: covidwho-1673605

ABSTRACT

SARS-CoV-2 provokes a robust T cell response. Peptide-based studies exclude antigen processing and presentation biology, which may influence T cell detection studies. To focus on responses to whole virus and complex antigens, we used intact SARS-CoV-2 and full-length proteins with DCs to activate CD8 and CD4 T cells from convalescent people. T cell receptor (TCR) sequencing showed partial repertoire preservation after expansion. Resultant CD8 T cells recognize SARS-CoV-2-infected respiratory tract cells, and CD4 T cells detect inactivated whole viral antigen. Specificity scans with proteome-covering protein/peptide arrays show that CD8 T cells are oligospecific per subject and that CD4 T cell breadth is higher. Some CD4 T cell lines enriched using SARS-CoV-2 cross-recognize whole seasonal coronavirus (sCoV) antigens, with protein, peptide, and HLA restriction validation. Conversely, recognition of some epitopes is eliminated for SARS-CoV-2 variants, including spike (S) epitopes in the Alpha, Beta, Gamma, and Delta variant lineages.

7.
PLoS Pathog ; 17(12): e1010203, 2021 12.
Article in English | MEDLINE | ID: covidwho-1594501

ABSTRACT

Class II tetramer reagents for eleven common DR alleles and a DP allele prevalent in the world population were used to identify SARS-CoV-2 CD4+ T cell epitopes. A total of 112, 28 and 42 epitopes specific for Spike, Membrane and Nucleocapsid, respectively, with defined HLA-restriction were identified. Direct ex vivo staining of PBMC with tetramer reagents was used to define immunodominant and subdominant T cell epitopes and estimate the frequencies of these T cells in SARS-CoV-2 exposed and naïve individuals. Majority of SARS-CoV-2 epitopes identified have <67% amino acid sequence identity with endemic coronaviruses and are unlikely to elicit high avidity cross-reactive T cell responses. Four SARS-CoV-2 Spike reactive epitopes, including a DPB1*04:01 restricted epitope, with ≥67% amino acid sequence identity to endemic coronavirus were identified. SARS-CoV-2 T cell lines for three of these epitopes elicited cross-reactive T cell responses to endemic cold viruses. An endemic coronavirus Spike T cell line showed cross-reactivity to the fourth SARS-CoV-2 epitope. Three of the Spike cross-reactive epitopes were subdominant epitopes, while the DPB1*04:01 restricted epitope was a dominant epitope. Frequency analyses showed Spike cross-reactive T cells as detected by tetramers were present at relatively low frequency in unexposed people and only contributed a small proportion of the overall Spike-specific CD4+ T cells in COVID-19 convalescent individuals. In total, these results suggested a very limited number of SARS-CoV-2 T cells as detected by tetramers are capable of recognizing ccCoV with relative high avidity and vice versa. The potentially supportive role of these high avidity cross-reactive T cells in protective immunity against SARS-CoV-2 needs further studies.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , COVID-19/immunology , Cross Reactions , SARS-CoV-2/immunology , COVID-19/epidemiology , Convalescence , Epitopes , Epitopes, T-Lymphocyte/immunology , Humans , Pandemics , Spike Glycoprotein, Coronavirus/immunology
8.
J Transl Autoimmun ; 4: 100114, 2021.
Article in English | MEDLINE | ID: covidwho-1479662

ABSTRACT

Vaccination can prevent infection and disease due to SARS-CoV-2. Early reports indicate that immune suppressed or immune compromised populations have reduced immune responses to US emergency use authorized (EUA) vaccines. Patients with autoimmune disorders are at risk for severe COVID-19, and are frequently immune suppressed related to therapy, the underlying disease, or both. Myasthenia gravis (MG) is an autoimmune disorder characterized by antibodies that interrupt neuromuscular transmission. Chronic immune suppressive therapy is typically required. We report the case of a 74 year old woman with MG receiving mycophenolate, prednisone, and eculizumab in whom mRNA vaccination failed to elicit detectable circulating vaccine-specific IgG or IFN-γ T cell responses. Eculizumab was discontinued, and repeat vaccination with two doses of an alternative EUA mRNA vaccine led to circulating IgG specific for the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein, and to detectable S-specific T cell responses. While it is not known if these responses will protect against SARS-CoV-2 infection or disease, a repeat course of mRNA vaccination appears to be safe and was broadly immunogenic in this individual.

9.
J Clin Invest ; 131(3)2021 02 01.
Article in English | MEDLINE | ID: covidwho-1124908

ABSTRACT

BACKGROUNDSARS-CoV-2-specific antibodies may protect from reinfection and disease, providing rationale for administration of plasma containing SARS-CoV-2-neutralizing antibodies (nAbs) as a treatment for COVID-19. Clinical factors and laboratory assays to streamline plasma donor selection, and the durability of nAb responses, are incompletely understood.METHODSPotential convalescent plasma donors with virologically documented SARS-CoV-2 infection were tested for serum IgG against SARS-CoV-2 spike protein S1 domain and against nucleoprotein (NP), and for nAb.RESULTSAmong 250 consecutive persons, including 27 (11%) requiring hospitalization, who were studied a median of 67 days since symptom onset, 97% were seropositive on 1 or more assays. Sixty percent of donors had nAb titers ≥1:80. Correlates of higher nAb titers included older age (adjusted OR [AOR] 1.03 per year of age, 95% CI 1.00-1.06), male sex (AOR 2.08, 95% CI 1.13-3.82), fever during illness (AOR 2.73, 95% CI 1.25-5.97), and disease severity represented by hospitalization (AOR 6.59, 95% CI 1.32-32.96). Receiver operating characteristic analyses of anti-S1 and anti-NP antibody results yielded cutoffs that corresponded well with nAb titers, with the anti-S1 assay being slightly more predictive. nAb titers declined in 37 of 41 paired specimens collected a median of 98 days (range 77-120) apart (P < 0.001). Seven individuals (2.8%) were persistently seronegative and lacked T cell responses.CONCLUSIONnAb titers correlated with COVID-19 severity, age, and sex. SARS-CoV-2 IgG results can serve as useful surrogates for nAb testing. Functional nAb levels declined, and a small proportion of convalescent individuals lacked adaptive immune responses.FUNDINGThe project was supported by the Frederick National Laboratory for Cancer Research with support from the NIAID under contract number 75N91019D00024, and was supported by the Fred Hutchinson Joel Meyers Endowment, Fast-Grants, a New Investigator award from the American Society for Transplantation and Cellular Therapy, and NIH contracts 75N93019C0063, 75N91019D00024, and HHSN272201800013C, and NIH grants T32-AI118690, T32-AI007044, K08-AI119142, and K23-AI140918.


Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , Blood Donors , COVID-19/therapy , Immunoglobulin G , SARS-CoV-2 , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/immunology , Female , Humans , Immunization, Passive , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged , SARS-CoV-2/immunology , SARS-CoV-2/metabolism
10.
Sci Rep ; 11(1): 4290, 2021 02 22.
Article in English | MEDLINE | ID: covidwho-1096333

ABSTRACT

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N = 80 samples from persons with RT-PCR confirmed SARS-CoV-2 infection), and a specificity of 97.2% (N = 106 control samples).


Subject(s)
COVID-19/diagnosis , COVID-19/immunology , Enzyme-Linked Immunosorbent Assay/methods , Epitopes, B-Lymphocyte/immunology , SARS-CoV-2/pathogenicity , Humans , Real-Time Polymerase Chain Reaction , SARS-CoV-2/immunology , Signal-To-Noise Ratio
11.
JCI Insight ; 6(6)2021 03 22.
Article in English | MEDLINE | ID: covidwho-1097059

ABSTRACT

Comorbid medical illnesses, such as obesity and diabetes, are associated with more severe COVID-19, hospitalization, and death. However, the role of the immune system in mediating these clinical outcomes has not been determined. We used multiparameter flow cytometry and systems serology to comprehensively profile the functions of T cells and antibodies targeting spike, nucleocapsid, and envelope proteins in a convalescent cohort of COVID-19 subjects who were either hospitalized (n = 20) or not hospitalized (n = 40). To avoid confounding, subjects were matched by age, sex, ethnicity, and date of symptom onset. Surprisingly, we found that the magnitude and functional breadth of virus-specific CD4+ T cell and antibody responses were consistently higher among hospitalized subjects, particularly those with medical comorbidities. However, an integrated analysis identified more coordination between polyfunctional CD4+ T cells and antibodies targeting the S1 domain of spike among subjects who were not hospitalized. These data reveal a functionally diverse and coordinated response between T cells and antibodies targeting SARS-CoV-2, which is reduced in the presence of comorbid illnesses that are known risk factors for severe COVID-19.


Subject(s)
Antibodies, Viral/physiology , CD4-Positive T-Lymphocytes/physiology , COVID-19/virology , Hospitalization , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus , Virion , Adult , Aged , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/physiology , Antibodies, Viral/metabolism , CD4-Positive T-Lymphocytes/metabolism , COVID-19/epidemiology , COVID-19/immunology , Cardiovascular Diseases/epidemiology , Cardiovascular Diseases/immunology , Comorbidity , Diabetes Mellitus/epidemiology , Diabetes Mellitus/immunology , Female , Humans , Immunity, Humoral , Male , Middle Aged , Nucleocapsid , Severity of Illness Index , Viral Envelope , Viral Proteins , Young Adult
12.
medRxiv ; 2020 Nov 30.
Article in English | MEDLINE | ID: covidwho-955709

ABSTRACT

Comorbid medical illnesses, such as obesity and diabetes, are associated with more severe COVID-19, hospitalization, and death. However, the role of the immune system in mediating these clinical outcomes has not been determined. We used multi-parameter flow cytometry and systems serology to comprehensively profile the functions of T cells and antibodies targeting spike, nucleocapsid, and envelope proteins in a convalescent cohort of COVID-19 subjects who were either hospitalized (n=20) or not hospitalized (n=40). To avoid confounding, subjects were matched by age, sex, ethnicity, and date of symptom onset. Surprisingly, we found that the magnitude and functional breadth of virus-specific CD4 T cell and antibody responses were consistently higher among hospitalized subjects, particularly those with medical comorbidities. However, an integrated analysis identified more coordination between polyfunctional CD4 T-cells and antibodies targeting the S1 domain of spike among subjects that were not hospitalized. These data reveal a functionally diverse and coordinated response between T cells and antibodies targeting SARS-CoV-2 which is reduced in the presence of comorbid illnesses that are known risk factors for severe COVID-19. Our data suggest that isolated measurements of the magnitudes of spike-specific immune responses are likely insufficient to anticipate vaccine efficacy in high-risk populations.

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