Longitudinal characterisation of phagocytic and neutralisation functions of anti-Spike antibodies in plasma of patients after SARS-CoV-2 infection
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Abstract
Phagocytic responses by effector cells to antibody or complement-opsonised viruses have been recognized to play a key role in anti-viral immunity. These include antibody dependent cellular phagocytosis mediated via Fc-receptors, phagocytosis mediated by classically activated complement-fixing IgM or IgG1 antibodies and antibody independent phagocytosis mediated via direct opsonisation of viruses by complement products activated via the mannose-binding lectin pathway. Limited data suggest these phagocytic responses by effector cells may contribute to the immunological and inflammatory responses in SARS-CoV-2 infection, however, their development and clinical significance remain to be fully elucidated. In this cohort of 62 patients, acutely ill individuals were shown to mount phagocytic responses to autologous plasma-opsonised SARS-CoV-2 Spike protein-coated microbeads as early as 10 days post symptom onset. Heat inactivation of the plasma prior to use as an opsonin caused 77-95% abrogation of the phagocytic response, and pre-blocking of Fc-receptors on the effector cells showed only 18-60% inhibition. These results suggest that SARS-CoV-2 can provoke early phagocytosis, which is primarily driven by heat labile components, likely activated complements, with variable contribution from anti-Spike antibodies. During convalescence, phagocytic responses correlated significantly with anti-Spike IgG titers. Older patients and patients with severe disease had significantly higher phagocytosis and neutralisation functions when compared to younger patients or patients with asymptomatic, mild, or moderate disease. A longitudinal study of a subset of these patients over 12 months showed preservation of phagocytic and neutralisation functions in all patients, despite a drop in the endpoint antibody titers by more than 90%. Interestingly, surface plasmon resonance showed a significant increase in the affinity of the anti-Spike antibodies over time correlating with the maintenance of both the phagocytic and neutralisation functions suggesting that improvement in the antibody quality over the 12 months contributed to the retention of effector functions.
Author Summary
Limited data suggest antibody dependent effector functions including phagocytosis may contribute to the immunological and inflammatory responses in SARS CoV-2 infection, however, their development, maintenance, and clinical significance remain unknown. In this study we show:
Patients with acute SARS CoV-2 infection can mount phagocytic responses as early as 10 days post symptom onset and these responses were primarily driven by heat labile components of the autologous plasma. These results indicate that the current approach of studying phagocytosis using purified or monoclonal antibodies does not recapitulate contribution by all components in the plasma.
In convalescent patients, high phagocytic responses significantly correlated with increasing age, increasing disease severity, high neutralisation functions and high anti-Spike antibody titers, particularly IgG1.
Longitudinal study of convalescent patients over a 12-month period showed maintenance of phagocytic and neutralisation functions, despite a drop in the anti-Spike endpoint antibody titers by more than 90%. However, we found significant increase in the affinity of the anti-Spike antibodies over the 12-month period and these correlated with the maintenance of functions suggesting that improvement in the antibody quality over time contributed to the retention of effector functions. Clinically, measuring antibody titers in sera but not the quality of antibodies is considered a gold standard indicator of immune protection following SARS-CoV 2 infection or vaccination. Our results challenge this notion and recommends change in the current clinical practice.
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SciScore for 10.1101/2021.12.21.473774: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The study protocol was approved by the Human Research Ethics Committees of the Northern Sydney Local Health District, the University of New South Wales, NSW Australia (ETH00520), CALHN Human Research Ethics Committee, Adelaide, Australia
Consent: Written informed consent was obtained from all participants before enrolment.Sex as a biological variable Archival serum or plasma collected from 25 healthy donors prior to the pandemic with an age range of 24-73 years, and a male to female ratio of 1:2.4 was used as controls. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Expression of … SciScore for 10.1101/2021.12.21.473774: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The study protocol was approved by the Human Research Ethics Committees of the Northern Sydney Local Health District, the University of New South Wales, NSW Australia (ETH00520), CALHN Human Research Ethics Committee, Adelaide, Australia
Consent: Written informed consent was obtained from all participants before enrolment.Sex as a biological variable Archival serum or plasma collected from 25 healthy donors prior to the pandemic with an age range of 24-73 years, and a male to female ratio of 1:2.4 was used as controls. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Expression of surface Fc-receptors was assessed by flow cytometry using monoclonal antibodies (mAbs) against Fcγ receptor RI (CD64)-FITC, FcγRIII (CD16)-PE, CD14-PerCP CD64)-FITCsuggested: NoneEndpoint titre and Isotyping of anti-SARS-CoV-2 Spike and anti-RBD antibodies in sera of patients with SARS-CoV-2 infection: Anti-SARS-CoV-2 Spike and RBD IgG antibody in sera was quantified using a modified direct ELISA [27]. anti-SARS-CoV-2suggested: NoneRBD IgGsuggested: NoneFor the endpoint titre of total anti-Spike or anti-RBD antibodies, 50μL of horseradish peroxidase (HRP)-conjugated mouse anti-human detection antibody added per well (Jackson ImmunoResearch, USA) (1:5000 dilution in 5% skim milk). anti-Spikesuggested: Noneanti-RBDsuggested: Noneanti-human detectionsuggested: NoneFor isotyping of the anti-Spike or anti-RBD antibodies, 50μL/well of HRP-conjugated immunoglobulin subtype or IgG subclass specific detection antibodies were added. IgG subclass specific detection antibodiessuggested: NoneThese include 1:6000 dilution of anti-total human IgG (Jackson Immunoresearch), 1:3000 dilution of anti-human IgA (α-chain-specific) (Sigma) or 1:3000 dilution of anti-human IgM (μ-chain-specific) (Sigma) antibody subtypes and 1:6000 dilution of anti-human IgG1, IgG2, IgG3 or IgG4 (Southern Biotech, USA). anti-total human IgGsuggested: (Cell Signaling Technology Cat# 13123, RRID:AB_2617178)anti-human IgA ( α-chain-specific ) ( Sigma )suggested: Noneanti-human IgM ( μ-chain-specific ) ( Sigma )suggested: Noneanti-human IgG1suggested: NoneIgG2suggested: NoneIgG3suggested: NoneIgG4suggested: NoneTo assess whether the uptake of the microbeads opsonised with the plasma of patients with acute disease was via Fc-receptor (antibody-dependent), and other heat labile opsonins such as complements (eg C3b), either the Fc-receptors on the THP-1 cells were pre-blocked using the universal Fc receptor blocking agent (Miltenyi, USA) [23], or the plasma heat inactivated at 56°C for 30 minutes, as described [28]. antibody-dependent) ,suggested: NoneSurface plasmon resonance: Surface plasmon resonance (SPR) was performed using a Biacore T200 (Cytiva, USA) to determine the binding characteristics of the anti-Spike polyclonal antibodies in patient plasma to SARS-CoV-2 Spike antigen. SARS-CoV-2 Spike antigen .suggested: NoneFriedman’s test with pairwise Dunn’s tests were used to compare the repeated measures of Spike p-score, Spike and RBD end point titre, neutralisation titre and antibody affinity across the timepoints V1, V2 and V3. V3suggested: NoneTo rank the variables of statistical importance that associated with Spike p-score (dependent variable), multiple linear regression analysis was performed using disease severity, age, gender, DPS, anti-Spike/RBD endpoint antibody titre, anti-Spike/RBD antibody subtypes and neutralisation titre as independent variables. anti-Spike/RBDsuggested: NoneExperimental Models: Cell Lines Sentences Resources To assess whether the uptake of the microbeads opsonised with the plasma of patients with acute disease was via Fc-receptor (antibody-dependent), and other heat labile opsonins such as complements (eg C3b), either the Fc-receptors on the THP-1 cells were pre-blocked using the universal Fc receptor blocking agent (Miltenyi, USA) [23], or the plasma heat inactivated at 56°C for 30 minutes, as described [28]. THP-1suggested: NoneRetroviral SARS-CoV-2 Spike pseudovirus were generated in 293T cells by co-transfecting expression plasmids containing SARS-CoV-2 Spike and MLV gag/pol and luciferase vectors using Calphos transfection kit (Takara Bio, USA) as described [20]. 293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)Briefly, pseudovirus were incubated for one hour with heat inactivated (56 °C for 30 minutes) patient serum prior to infecting 293T-ACE2 cells (kindly provided by A/Prof Jesse Bloom) by a two hour spinoculated at 800xg in 96-well white flat bottom plates in triplicates (Sigma-Aldrich, USA) 293T-ACE2suggested: NoneRecombinant DNA Sentences Resources Biotinylated recombinant SARS-CoV-2 Spike and RBD proteins: SARS-CoV-2 Wuhan-Hu-1 (GenPept: QJE37812) RBD protein (amino acid residues 319–541) and Spike protein (amino acid residue 15-1213) were cloned into pCEP4 mammalian expression vector containing N-terminal human Ig kappa leader sequence and C-terminal Avi-tag and His-tag (Invitrogen, USA) pCEP4suggested: RRID:Addgene_16479)Software and Algorithms Sentences Resources After a two hours incubation, cells were washed once with 1mL of cold PBS containing 0.5% FBS and 0.005% of sodium azide and gentle centrifugation at 335xg for five minutes at 4°C, fixed in 400μL of 1% paraformaldehyde and kept at 4°C in the dark until acquisition of data using BD FACSCalibur™ Flow cytometer. BD FACSCalibur™suggested: (BD FACSCalibur Flow Cytometry System, RRID:SCR_000401)A total of 2×104 events were acquired and the proportions of cells that phagocytosed the beads (% of cells that took up the beads) and their fluorescent intensities (amounts of beads taken up per cell) were analysed using BD FlowJo version 10.5.0 software. FlowJosuggested: (FlowJo, RRID:SCR_008520)All the images were Airyscan processed with Zen Black Edition (Zeiss Software). Zensuggested: NoneRelative Luminescence Unit (RLU) in cell lysates was measured using CLARIOstar microplate reader (BMG Labtech, Australia), percentage neutralisation of Spike pseudovirus determined and the fifty percent inhibitory (ID50) calculated using non-linear regression model (GraphPad Prism version 9.0) [29] GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Kinetic constants, including association constant (Ka), equilibrium constant (KD) (affinity) and dissociation constant (Ka) (avidity), were calculated using BIAcore evaluation software version 4.1 [30]. BIAcoresuggested: (Biacore T100 System, RRID:SCR_019679)Statistical analysis: All data were analysed with Prism Software (version 9.0, GraphPad, USA) Prismsuggested: (PRISM, RRID:SCR_005375)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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