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1.
PLoS One ; 17(1): e0262868, 2022.
Article in English | MEDLINE | ID: covidwho-1643287

ABSTRACT

A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination.


Subject(s)
Antibodies, Viral/chemistry , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/epidemiology , COVID-19 Serological Testing/standards , Coronavirus Nucleocapsid Proteins/chemistry , Epidemiological Monitoring , Female , Humans , Immunoglobulin A/chemistry , Immunoglobulin A/immunology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Immunoglobulin M/chemistry , Immunoglobulin M/immunology , Male , Middle Aged , New York/epidemiology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , SARS-CoV-2/classification , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/chemistry
2.
MAbs ; 14(1): 2005507, 2022.
Article in English | MEDLINE | ID: covidwho-1585297

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a serious public health crisis worldwide, and considering the novelty of the disease, preventative and therapeutic measures alike are urgently needed. To accelerate such efforts, the development of JS016, a neutralizing monoclonal antibody directed against the SARS-CoV-2 spike protein, was expedited from a typical 12- to 18-month period to a 4-month period. During this process, transient Chinese hamster ovary cell lines are used to support preclinical, investigational new drug-enabling toxicology research, and early Chemistry, Manufacturing and Controls development; mini-pool materials to supply Phase 1 clinical trials; and a single-clone working cell bank for late-stage and pivotal clinical trials were successively adopted. Moreover, key process performance and product quality investigations using a series of orthogonal and state-of-the-art techniques were conducted to demonstrate the comparability of products manufactured using these three processes, and the results indicated that, despite observed variations in process performance, the primary and high-order structures, purity and impurity profiles, biological and immunological functions, and degradation behaviors under stress conditions were largely comparable. The study suggests that, in particular situations, this strategy can be adopted to accelerate the development of therapeutic biopharmaceuticals and their access to patients.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/therapeutic use , Antibody Affinity/immunology , Antibody Specificity/immunology , CHO Cells , COVID-19/prevention & control , COVID-19/virology , Chromatography, High Pressure Liquid/methods , Circular Dichroism , Clone Cells , Cricetinae , Cricetulus , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Immunoglobulin G/therapeutic use , Isoelectric Point , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism
3.
ACS Synth Biol ; 10(12): 3595-3599, 2021 12 17.
Article in English | MEDLINE | ID: covidwho-1517598

ABSTRACT

The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutations.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , CRISPR-Cas Systems , DNA, Viral/genetics , Mutation , SARS-CoV-2/genetics , Biosensing Techniques , COVID-19/virology , DNA/analysis , Genetic Techniques , HEK293 Cells , Humans , Immunoglobulin G/chemistry , Peptide Library , Polymers/chemistry , Spain/epidemiology , Surface Plasmon Resonance
4.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Article in English | MEDLINE | ID: covidwho-1493344

ABSTRACT

Here, we expressed two neutralizing monoclonal antibodies (Abs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; H4 and B38) in three formats: IgG1, IgA1 monomers (m), and IgA1 dimers (d) in glycoengineered Nicotiana benthamiana plants. All six Ab variants assembled properly and exhibited a largely homogeneous glycosylation profile. Despite modest variation in antigen binding between Ab formats, SARS-CoV-2 neutralization (NT) potency significantly increased in the following manner: IgG1 < IgA1-m < IgA1-d, with an up to 240-fold NT increase of dimers compared to corresponding monomers. Our results underscore that both IgA's structural features and multivalency positively impact NT potency. In addition, they emphasize the versatile use of plants for the rapid expression of complex human proteins.


Subject(s)
Antibodies, Monoclonal/chemistry , COVID-19/virology , Immunoglobulin A/chemistry , Immunoglobulin G/chemistry , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/immunology , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Humans , Neutralization Tests , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
5.
Nature ; 599(7885): 465-470, 2021 11.
Article in English | MEDLINE | ID: covidwho-1428880

ABSTRACT

Monoclonal antibodies with neutralizing activity against SARS-CoV-2 have demonstrated clinical benefits in cases of mild-to-moderate SARS-CoV-2 infection, substantially reducing the risk for hospitalization and severe disease1-4. Treatment generally requires the administration of high doses of these monoclonal antibodies and has limited efficacy in preventing disease complications or mortality among hospitalized patients with COVID-195. Here we report the development and evaluation of anti-SARS-CoV-2 monoclonal antibodies with optimized Fc domains that show superior potency for prevention or treatment of COVID-19. Using several animal disease models of COVID-196,7, we demonstrate that selective engagement of activating Fcγ receptors results in improved efficacy in both preventing and treating disease-induced weight loss and mortality, significantly reducing the dose required to confer full protection against SARS-CoV-2 challenge and for treatment of pre-infected animals. Our results highlight the importance of Fcγ receptor pathways in driving antibody-mediated antiviral immunity and exclude the possibility of pathogenic or disease-enhancing effects of Fcγ receptor engagement of anti-SARS-CoV-2 antibodies upon infection. These findings have important implications for the development of Fc-engineered monoclonal antibodies with optimal Fc-effector function and improved clinical efficacy against COVID-19 disease.


Subject(s)
Antibodies, Monoclonal/therapeutic use , COVID-19/drug therapy , COVID-19/immunology , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/therapeutic use , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Neutralizing/therapeutic use , Cricetinae , Disease Models, Animal , Female , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/pharmacology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Male , Mice , Pre-Exposure Prophylaxis , Receptors, IgG/chemistry , Receptors, IgG/immunology , Treatment Outcome
6.
Science ; 372(6546): 1108-1112, 2021 06 04.
Article in English | MEDLINE | ID: covidwho-1388437

ABSTRACT

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/chemistry , Antibodies, Viral/blood , Antibodies, Viral/chemistry , Antibody Affinity , COVID-19/prevention & control , Epitopes/immunology , Humans , Immune Evasion , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Variable Region/immunology , Mice , Mice, Inbred BALB C , Mutation , Protein Domains , Proteomics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
7.
Immunity ; 54(10): 2385-2398.e10, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1370548

ABSTRACT

Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.


Subject(s)
Broadly Neutralizing Antibodies/immunology , Cross Reactions/immunology , SARS-CoV-2/immunology , Animals , Betacoronavirus/immunology , Binding Sites, Antibody , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/therapeutic use , COVID-19/prevention & control , COVID-19/therapy , COVID-19/virology , Cricetinae , Humans , Immunoglobulin Class Switching , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Mice , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
8.
Adv Mater ; 33(37): e2103221, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1321675

ABSTRACT

Liquid-phase electron microscopy (LP-EM) is an exciting new area in the materials imaging field, providing unprecedented views of molecular processes. Time-resolved insights from LP-EM studies are a strong complement to the remarkable results achievable with other high-resolution techniques. Here, the opportunities to expand LP-EM technology beyond 2D temporal assessments and into the 3D regime are described. The results show new structures and dynamic insights of human viruses contained in minute volumes of liquid while acquired in a rapid timeframe. To develop this strategy, adeno-associated virus (AAV) is used as a model system. AAV is a well-known gene therapy vehicle with current applications involving drug delivery and vaccine development for COVID-19. Improving the understanding of the physical properties of biological entities in a liquid state, as maintained in the human body, has broad societal implications for human health and disease.


Subject(s)
Cryoelectron Microscopy/methods , Dependovirus , Particle Size , COVID-19 , COVID-19 Vaccines , Drug Delivery Systems , Equipment Design , Genetic Therapy , HEK293 Cells/virology , Humans , Hydrogen-Ion Concentration , Immunoglobulin G/chemistry , Materials Testing , SARS-CoV-2
9.
Sci Transl Med ; 13(596)2021 06 02.
Article in English | MEDLINE | ID: covidwho-1225692

ABSTRACT

Patients diagnosed with coronavirus disease 2019 (COVID-19) become critically ill primarily around the time of activation of the adaptive immune response. Here, we provide evidence that antibodies play a role in the worsening of disease at the time of seroconversion. We show that early-phase severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) spike protein-specific immunoglobulin G (IgG) in serum of critically ill COVID-19 patients induces excessive inflammatory responses by human alveolar macrophages. We identified that this excessive inflammatory response is dependent on two antibody features that are specific for patients with severe COVID-19. First, inflammation is driven by high titers of anti-spike IgG, a hallmark of severe disease. Second, we found that anti-spike IgG from patients with severe COVID-19 is intrinsically more proinflammatory because of different glycosylation, particularly low fucosylation, of the antibody Fc tail. Low fucosylation of anti-spike IgG was normalized in a few weeks after initial infection with SARS-CoV-2, indicating that the increased antibody-dependent inflammation mainly occurs at the time of seroconversion. We identified Fcγ receptor (FcγR) IIa and FcγRIII as the two primary IgG receptors that are responsible for the induction of key COVID-19-associated cytokines such as interleukin-6 and tumor necrosis factor. In addition, we show that anti-spike IgG-activated human macrophages can subsequently break pulmonary endothelial barrier integrity and induce microvascular thrombosis in vitro. Last, we demonstrate that the inflammatory response induced by anti-spike IgG can be specifically counteracted by fostamatinib, an FDA- and EMA-approved therapeutic small-molecule inhibitor of Syk kinase.


Subject(s)
Antibodies, Viral/chemistry , COVID-19/immunology , Immunoglobulin G/chemistry , Macrophages, Alveolar/immunology , Glycosylation , Humans , Inflammation , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology
10.
Science ; 372(6546): 1108-1112, 2021 06 04.
Article in English | MEDLINE | ID: covidwho-1216772

ABSTRACT

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/chemistry , Antibodies, Viral/blood , Antibodies, Viral/chemistry , Antibody Affinity , COVID-19/prevention & control , Epitopes/immunology , Humans , Immune Evasion , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Variable Region/immunology , Mice , Mice, Inbred BALB C , Mutation , Protein Domains , Proteomics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
11.
Sci Rep ; 11(1): 7880, 2021 04 12.
Article in English | MEDLINE | ID: covidwho-1180278

ABSTRACT

Since the pandemic outbreak of Covid-19 in December 2019, several lateral flow assay (LFA) devices were developed to enable the constant monitoring of regional and global infection processes. Additionally, innumerable lateral flow test devices are frequently used for determination of different clinical parameters, food safety, and environmental factors. Since common LFAs rely on non-biodegradable nitrocellulose membranes, we focused on their replacement by cellulose-composed, biodegradable papers. We report the development of cellulose paper-based lateral flow immunoassays using a carbohydrate-binding module-fused to detection antibodies. Studies regarding the protein binding capacity and potential protein wash-off effects on cellulose paper demonstrated a 2.7-fold protein binding capacity of CBM-fused antibody fragments compared to the sole antibody fragment. Furthermore, this strategy improved the spatial retention of CBM-fused detection antibodies to the test area, which resulted in an enhanced sensitivity and improved overall LFA-performance compared to the naked detection antibody. CBM-assisted antibodies were validated by implementation into two model lateral flow test devices (pregnancy detection and the detection of SARS-CoV-2 specific antibodies). The CBM-assisted pregnancy LFA demonstrated sensitive detection of human gonadotropin (hCG) in synthetic urine and the CBM-assisted Covid-19 antibody LFA was able to detect SARS-CoV-2 specific antibodies present in serum. Our findings pave the way to the more frequent use of cellulose-based papers instead of nitrocellulose in LFA devices and thus potentially improve the sustainability in the field of POC diagnostics.


Subject(s)
Antibodies, Viral/analysis , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Carbohydrates/chemistry , Collodion/chemistry , Immunoassay/methods , Biocompatible Materials , Chorionic Gonadotropin/chemistry , Clostridium thermocellum/immunology , Humans , Immunoglobulin Fragments/chemistry , Immunoglobulin G/chemistry , Point-of-Care Systems , Protein Binding , SARS-CoV-2/immunology , Urinalysis
12.
J Mol Biol ; 433(13): 166983, 2021 06 25.
Article in English | MEDLINE | ID: covidwho-1174385

ABSTRACT

Recombinant antibodies (Abs) against the SARS-CoV-2 virus hold promise for treatment of COVID-19 and high sensitivity and specific diagnostic assays. Here, we report engineering principles and realization of a Protein-fragment Complementation Assay (PCA) detector of SARS-CoV-2 antigen by coupling two Abs to complementary N- and C-terminal fragments of the reporter enzyme Gaussia luciferase (Gluc). Both Abs display comparably high affinities for distinct epitopes of viral Spike (S)-protein trimers. Gluc activity is reconstituted when the Abs are simultaneously bound to S-protein bringing the Ab-fused N- and C-terminal fragments close enough together (8 nm) to fold. We thus achieve high specificity both by requirement of simultaneous binding of the two Abs to the S-protein and also, in a steric configuration in which the two Gluc complementary fragments can fold and thus reconstitute catalytic activity. Gluc activity can also be reconstituted with virus-like particles that express surface S-protein with detectable signal over background within 5 min of incubation. Design principles presented here can be readily applied to develop reporters to virtually any protein with sufficient available structural details. Thus, our results present a general framework to develop reporter assays for COVID-19, and the strategy can be readily deployed in response to existing and future pathogenic threats and other diseases.


Subject(s)
Antibodies, Viral/chemistry , Antigens, Viral/immunology , COVID-19/diagnosis , COVID-19/virology , SARS-CoV-2/isolation & purification , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antigens, Viral/chemistry , Antigens, Viral/isolation & purification , Epitopes/immunology , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Luciferases , Luminescent Measurements/methods , Protein Engineering , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
13.
Cell Res ; 31(5): 517-525, 2021 05.
Article in English | MEDLINE | ID: covidwho-1139736

ABSTRACT

Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical intervention against coronavirus disease 2019 (COVID-19). We isolated a large number of nAbs from SARS-CoV-2-infected individuals capable of disrupting proper interaction between the receptor binding domain (RBD) of the viral spike (S) protein and the receptor angiotensin converting enzyme 2 (ACE2). However, the structural basis for their potent neutralizing activity remains unclear. Here, we report cryo-EM structures of the ten most potent nAbs in their native full-length IgG-form or in both IgG-form and Fab-form bound to the trimeric S protein of SARS-CoV-2. The bivalent binding of the full-length IgG is found to associate with more RBDs in the "up" conformation than the monovalent binding of Fab, perhaps contributing to the enhanced neutralizing activity of IgG and triggering more shedding of the S1 subunit from the S protein. Comparison of a large number of nAbs identified common and unique structural features associated with their potent neutralizing activities. This work provides a structural basis for further understanding the mechanism of nAbs, especially through revealing the bivalent binding and its correlation with more potent neutralization and the shedding of S1 subunit.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/immunology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antibodies, Viral/ultrastructure , Host-Pathogen Interactions , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/ultrastructure , Models, Molecular , Protein Conformation , Protein Multimerization , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure
14.
Science ; 371(6532)2021 02 26.
Article in English | MEDLINE | ID: covidwho-1066801

ABSTRACT

Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Adult , Aged , Antibodies, Viral/blood , Antibodies, Viral/chemistry , COVID-19/physiopathology , Cells, Cultured , Critical Illness , Cytomegalovirus/immunology , Female , Fucose/analysis , Glycosylation , HIV/immunology , Hepatitis B Vaccines/immunology , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Inflammation , Interleukin-6/biosynthesis , Interleukin-6/immunology , Macrophages/immunology , Male , Middle Aged , Parvovirus B19, Human/immunology , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Subunit/immunology , Young Adult
15.
Viruses ; 13(1)2021 Jan 15.
Article in English | MEDLINE | ID: covidwho-1038679

ABSTRACT

Adenovirus (AdV) infection elicits a strong immune response with the production of neutralizing antibodies and opsonization by complement and coagulation factors. One anti-hexon neutralizing antibody, called 9C12, is known to activate the complement cascade, resulting in the deposition of complement component C4b on the capsid, and the neutralization of the virus. The mechanism of AdV neutralization by C4b is independent of downstream complement proteins and involves the blockage of the release of protein VI, which is required for viral escape from the endosome. To investigate the structural basis underlying how C4b blocks the uncoating of AdV, we built a model for the complex of human adenovirus type-5 (HAdV5) with 9C12, together with complement components C1 and C4b. This model positions C4b near the Arg-Gly-Asp (RGD) loops of the penton base. There are multiple amino acids in the RGD loop that might serve as covalent binding sites for the reactive thioester of C4b. Molecular dynamics simulations with a multimeric penton base and C4b indicated that stabilizing interactions may form between C4b and multiple RGD loops. We propose that C4b deposition on one RGD loop leads to the entanglement of C4b with additional RGD loops on the same penton base multimer and that this entanglement blocks AdV uncoating.


Subject(s)
Adenoviridae/immunology , Complement C4/chemistry , Complement C4/immunology , Models, Molecular , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Binding Sites , Capsid/chemistry , Capsid/metabolism , Capsid/ultrastructure , Capsid Proteins/chemistry , Capsid Proteins/immunology , Capsid Proteins/metabolism , Capsid Proteins/ultrastructure , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Structure-Activity Relationship
16.
Cell ; 182(4): 828-842.e16, 2020 08 20.
Article in English | MEDLINE | ID: covidwho-1027977

ABSTRACT

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.


Subject(s)
Antibodies, Neutralizing/chemistry , Betacoronavirus/chemistry , Coronavirus Infections/immunology , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin G/chemistry , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/immunology , Antibodies, Viral/isolation & purification , Betacoronavirus/immunology , COVID-19 , Coronavirus Infections/blood , Coronavirus Infections/therapy , Cross Reactions , Cryoelectron Microscopy , Epitope Mapping , Epitopes , Humans , Immunization, Passive , Immunoglobulin Fab Fragments/blood , Immunoglobulin Fab Fragments/isolation & purification , Immunoglobulin Fab Fragments/ultrastructure , Immunoglobulin G/blood , Immunoglobulin G/isolation & purification , Immunoglobulin G/ultrastructure , Middle East Respiratory Syndrome Coronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/immunology , Models, Molecular , Pandemics , Pneumonia, Viral/blood , SARS Virus/chemistry , SARS Virus/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology
17.
J Biol Chem ; 295(36): 12814-12821, 2020 09 04.
Article in English | MEDLINE | ID: covidwho-1005807

ABSTRACT

There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS- coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)-interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.


Subject(s)
Antibody Affinity , Single-Chain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2 , Binding Sites , Epitopes/chemistry , Epitopes/immunology , HEK293 Cells , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Molecular Docking Simulation , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Single-Chain Antibodies/chemistry , Spike Glycoprotein, Coronavirus/chemistry
18.
Bioanalysis ; 13(1): 13-28, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-977787

ABSTRACT

Aim: Coronavirus disease 2019 antibody testing often relies on venous blood collection, which is labor-intensive, inconvenient and expensive compared with finger-stick capillary dried blood spot (DBS) collection. The purpose of our work was to determine if two commercially available anti-severe acute respiratory syndrome coronavirus 2 enzyme-linked immunosorbent assays for IgG antibodies against spike S1 subunit and nucleocapsid proteins could be validated for use with DBS. Materials & methods: Kit supplied reagents were used to extract DBS, and in-house DBS calibrators were included on every run. Results: Positive/negative concordance between DBS and serum was 100/99.3% for the spike S1 subunit assay and 100/98% for the nucleocapsid assay. Conclusion: Validation of the DBS Coronavirus disease 2019 IgG antibody assays demonstrated that serum and DBS can produce equivalent results with minimal kit modifications.


Subject(s)
COVID-19 Testing/standards , COVID-19/diagnosis , Dried Blood Spot Testing/standards , Enzyme-Linked Immunosorbent Assay/standards , SARS-CoV-2/immunology , Antibodies, Viral/chemistry , Antigens, Viral/blood , Antigens, Viral/immunology , COVID-19/blood , COVID-19/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/blood , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Immunoglobulin G/chemistry , Male , Middle Aged , Phosphoproteins/blood , Phosphoproteins/immunology , Reagent Kits, Diagnostic/standards , Reproducibility of Results , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/immunology
19.
Med Hypotheses ; 144: 110247, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-741428

ABSTRACT

Type-2 diabetes (T2D) is a major comorbidity of COVID-19, and poorly controlled diabetes is associated with high mortality rate, emphasizing the necessity to improve glycemic control. Angiotensin-converting enzyme 2 (ACE2) is the receptor responsible for SARS-CoV-2 access to human cells, and ACE2 expression is increased in patients with diabetes and hypertension treated with ACE-inhibitors or angiotensin II receptor blockers. We hypothesize that an upregulation of ACE2 due to its non-enzymatic glycation could be considered, as well as a change of the protein tertiary structure in terms of amino acid (mostly lysine) available to be glycated. In fact, in a single ACE2 molecule, 34 lysine residues are present in the extracellular portion, and at least one of these is co-involved in a fundamental hydrogen-bond interaction with the SARS-CoV-2 receptor binding domain (RBD). The worse outcome of COVID-19 in people with diabetes could be related to the non-enzymatic glycation that triggers the activity of ACE2. Moreover, DNA methylation of genes regulating islet beta-cell function, as well as in insulin resistance of peripheral tissues such as liver, muscle, and adipose tissue may be involved, as already demonstrated for cancer conditions. DNA methylation, besides being considered as a biomarker to predict the risk of obesity and T2D, has been suggested also as a target for dietary and pharmacological treatments. The present observations may suggest further interventions in order to improve the outcome of COVID-19 in people affected by diabetes.


Subject(s)
COVID-19/complications , COVID-19/metabolism , Diabetes Complications , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/metabolism , Adipose Tissue , Angiotensin-Converting Enzyme 2/metabolism , Biomarkers/metabolism , COVID-19/immunology , Comorbidity , DNA Methylation , Diabetes Mellitus, Type 2/immunology , Glycosylation , Humans , Immunoglobulin G/chemistry , Insulin Resistance , Methylation , Multicenter Studies as Topic , Neoplasms/metabolism , Protein Domains , Renin-Angiotensin System , Retrospective Studies , SARS-CoV-2 , Up-Regulation
20.
Nat Commun ; 11(1): 2070, 2020 04 24.
Article in English | MEDLINE | ID: covidwho-116533

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019, and there are currently no specific antiviral treatments or vaccines available. SARS-CoV-2 has been shown to use the same cell entry receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In this report, we generate a recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. A fusion protein containing an ACE2 mutant with low catalytic activity is also used in this study. The fusion proteins are then characterized. Both fusion proteins have a high binding affinity for the receptor-binding domains of SARS-CoV and SARS-CoV-2 and exhibit desirable pharmacological properties in mice. Moreover, the fusion proteins neutralize virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they have potential applications in the diagnosis, prophylaxis, and treatment of SARS-CoV-2.


Subject(s)
Betacoronavirus/drug effects , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Neutralization Tests , Peptidyl-Dipeptidase A/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/metabolism , Binding, Competitive/drug effects , Cross Reactions , Drug Design , Humans , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin Fc Fragments/pharmacology , Immunoglobulin G/metabolism , Immunoglobulin G/pharmacology , In Vitro Techniques , Inhibitory Concentration 50 , Membrane Fusion/drug effects , Mice , Mice, Inbred BALB C , Mutation , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/pharmacokinetics , Peptidyl-Dipeptidase A/pharmacology , Protein Domains/genetics , Protein Stability , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/chemistry , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacokinetics , SARS Virus/drug effects , SARS Virus/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
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