key: cord-0724312-lot4oy23
authors: Tang, Juanjie; Grubbs, Gabrielle; Lee, Youri; Golding, Hana; Khurana, Surender
title: Impact of convalescent plasma therapy on SARS CoV-2 antibody profile in COVID-19 patients
date: 2021-04-16
journal: Clin Infect Dis
DOI: 10.1093/cid/ciab317
sha: 8549b18855178ce88a92a09aa58e8369addbb290
doc_id: 724312
cord_uid: lot4oy23

Convalescent plasma (CP) have been used for treatment of COVID-19, but their effectiveness varies significantly. Moreover, the impact of CP treatment on the composition of SARS-CoV-2 antibodies in COVID-19 patients and antibody markers that differentiate between those who survive and those who succumb to the COVID-19 disease are not well understood. Herein, we performed longitudinal analysis of antibody profile on 115 sequential plasma samples from 16 hospitalized COVID-19 patients treated with either CP or standard of care, only half of them survived. Differential antibody kinetics was observed for antibody binding, IgM/IgG/IgA distribution, and affinity maturation in ‘survived’ vs. ‘fatal’ COVID-19 patients. Surprisingly, CP treatment did not predict survival. Strikingly, marked decline in neutralization titers was observed in the fatal patients prior to death, and convalescent plasma treatment did not reverse this trend. Furthermore, irrespective of CP treatment, higher antibody affinity to the SARS-CoV-2 prefusion spike was associated with survival outcome, while sustained elevated IgA response was associated with fatal outcome in these COVID-19 patients. These findings propose that treatment of COVID-19 patients with convalescent plasma should be carefully targeted, and effectiveness of treatment may depend on the clinical and immunological status of COVID-19 patients as well as the quality of the antibodies in the convalescent plasma.

An expedited access to treatment of COVID-19 patients with convalescent plasma (CP) was issued by FDA under Emergency Use Authorization on August 23 2020. Early studies supported the safety of CP transfusions [1] , but their effectiveness remains an area of intense investigation. Li et al, reported no significant difference in clinical improvement or mortality between CP treated group vs. control group [2] . More recent reports suggested that convalescent plasma with pre-determined high titers of neutralizing antibodies (> 1:640) may improve clinical symptoms but did not change the mortality rates [1, [3] [4] [5] [6] . However, the impact of CP on the quality of antibody profile of treated COVID-19 patients is not known and the antibody markers that predict COVID-19 outcome are still not fully understood.

Therefore, there is need to perform longitudinal evaluation of the antibody profile in COVID- 19 patients treated with/without CP following SARS-CoV-2 infection to identify the impact of antibody therapy and hopefully to identify antibody markers associated with resolution vs. (Table 1) . We designed the study to collect samples for age-matched adults (age >25 years) and at least one matched co-morbidity (hypertension, obesity or diabetes), half of them received CP and another half did not. In a strict clinical trial design nomenclature, this study was not a planned matched case-control study during the early phase of pandemic. All patients were hospitalized in intensive care unit (ICU) with supplementary oxygen and mechanical ventilation. No other immunoglobulin preparations were given to these patients. Even though the planned study was to recruit adults (age >25 years), with equal numbers of males and females, however, during the collection timeframe, most of the patients admitted with COVID-19 at the hospital were males. So, this study observations interpretation may not be commutable to females (Table 1) . Eight patients (all males) were treated with CP administered on days 2-26 post-onset of symptoms. Four patients treated with plasma survived [S-81(P), S-83(P), S-85(P) and S-94(P)], while four succumbed to disease [F-00(P), F-36(P), F-46(P) and F-61(P)]. Similarly, four of the eight patients (total 7/8 males) not treated with plasma died (F-27, F-89, F-92 and F-94), while four survived (S-81, S-84, S-93 and S-95). The patients in the sentinel groups of survived vs. fatal cases were matched for sex (mostly males), age, and co-morbidities (hypertension, diabetes, obesity). We were not unblinded for CP and did not get complete information on which convalescent plasma lot was used for treatment of each specific patient. The neutralization titers for the CP lots ranged between 160-640. This study was approved by Food and Drug Administration's Research Involving Human Subjects Committee (RIHSC #2020-04-02).

A c c e p t e d M a n u s c r i p t 6 Samples were evaluated blindly in SARS-CoV-2 pseudovirus neutralization assay and surface plasmon resonance for antibody titers, isotype analysis, and antibody off-rate constants against SARS-CoV-2 prefusion spike (from Barney Graham, NIH). Methods were described in detail previously [7, 8] .

Human codon-optimized cDNA encoding SARS-CoV-2 S glycoprotein (NC_045512) was synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamHI and XhoI sites. Pseudovirions were produced by co-transfection Lenti-X 293T cells with pMLV-gag-pol, pFBluc, and pcDNA 3.1 SARS-CoV-2 S using Lipofectamine 3000. The supernatants were harvested at 48h and 72h post transfection and filtered through 0.45-mm membranes.

For neutralization assay, 50 µL of SARS-CoV-2 S pseudovirions were pre-incubated with an equal volume of medium containing plasma at varying dilutions at room temperature for 1 h, then virus-antibody mixtures were added to Vero E6 cells in a 96-well plate. After a 12 h incubation, the inoculum was refreshed with fresh medium. Cells were lysed 48 h later, and luciferase activity was measured using luciferin-containing substrate.

SARS-CoV-2 genetically-stabilized prefusion spike ectodomain (aa 1-1208), lacking the cytoplasmic and transmembrane domains (delta CT-TM), fused to His tag at C-terminus, was produced in FreeStyle293F mammalian cells. Steady-state equilibrium binding of post-SARS-CoV-2 infected human polyclonal plasma was monitored at 25°C using a ProteOn surface plasmon resonance (BioRad). The purified recombinant SARS-CoV-2 prefusion A c c e p t e d M a n u s c r i p t 7 spike protein was captured via a His-tag to a Ni-NTA sensor chip with 200 resonance units (RU) in the test flow channels. The protein density on the chip was optimized such as to measure monovalent interactions independent of the antibody isotype [8] . Serial dilutions (10-, 50-and 250-fold) of freshly prepared plasma in BSA-PBST buffer (PBS pH 7.4 buffer with Tween-20 and BSA) were injected at a flow rate of 50 µL/min (120 sec contact duration) for association, and disassociation was performed over a 600-second interval.

Responses from the protein surface were corrected for the response from a mock surface and for responses from a buffer-only injection. SPR was performed with serially diluted plasma of each individual time point in this study. Antibody isotype analysis for the SARS-CoV-2 spike protein bound antibodies in the polyclonal plasma was performed using SPR to determine the relative contribution of each antibody isotype: IgM, IgG (including subclasses) and IgA in plasma antibody bound to spike protein. Total antibody binding and antibody isotype analysis were calculated with BioRad ProteOn manager software (version 3.1). The resonance units for each antibody isotype was divided by the total resonance units for all the antibody isotypes combined to calculate the percentage of each antibody isotype. All SPR experiments were performed twice, and the researchers performing the assay were blinded to sample identity. Under these optimized SPR conditions, the variation for each sample in duplicate SPR runs was <5%. The maximum resonance units (Max RU) data shown in the figures was the calculated RU signal for the 10-fold diluted plasma sample. In addition to spike-specific binding, total IgM, IgG subtypes, and IgA in serum was measured for each individual during the peak neutralization titers (Supplementary Table 1 ).

Antibody off-rate constants, which describe the stability of the antigen-antibody complex, i.e., the fraction of complexes that decays per second in the dissociation phase, were determined directly from the human polyclonal plasma sample interaction with recombinant purified SARS CoV-2 prefusion spike ectodomain using SPR in the dissociation A c c e p t e d M a n u s c r i p t 8 phase only for the sensorgrams with Max RU in the range of 10-100 RU and calculated using the BioRad ProteOn manager software for the heterogeneous sample model as described before [7, 9] . Off-rate constants were determined from two independent SPR runs. The variation of off-rate between the two SPR runs was <4.8%.

Statistical differences were performed using GraphPad prim version 8 (Graph Pad software Inc, San Diego, CA). The statistical significances between the groups were determined by non-parametric (Kruskal-Wallis) statistical test using Dunn's multiple comparisons analysis in GraphPad prism. The differences were considered statistically significant with a 95% confidence interval when the p value was less than 0.05. (Table 1) .

Despite the small cohort, the patients in the two groups (survived and fatal) were well matched for sex (mainly males) and comorbidities (Table 1) . We identified a very heterogenous neutralizing antibody responses among the 16 hospitalized COVID-19 patients.

Most patients (apart from F-92, F-94 and S-95) developed neutralizing antibodies that peaked around 2-weeks post-symptoms onset ( Fig. 1 A-B) . CP (PsVNA50 titers of 160-640) administration did not remarkably change neutralizing antibody titers for 7/8 patients [except S-81 (P)] that were transfused before the start of sample collection (Fig. 1A) . Strikingly, despite high neutralization titers prior to CP infusion, a decline in neutralization titers was observed in all four CP-treated fatal patients prior to their death. In the four CP-treated patients who survived, the neutralizing antibody titers prior to CP infusion varied between 

Since neutralizing antibodies represent fraction of total antibodies targeting SARS-CoV-2, we evaluated antibody kinetics to measure antibody profile to SARS-CoV-2 prefusion spike, as previously described [7, 10] . Quantitative and qualitative analyses of IgM, IgG & IgA antibodies were performed on longitudinal human plasma collected frequently from SARS-CoV-2 infected hospitalized patients with COVID-19 disease during acute illness, prior to disease resolution or death, during the entire duration of their hospital stay (1 -38 days). In several fatal cases, the antibody binding titers (that account for total binding of all antibody isotypes) were low on the day of admittance to hospital (Fig. 1C-D, red curves) .

Following treatment with CP, antibody binding to prefusion spike increased in most patients ( Fig. 1C) . However, they declined after few days in the fatal patients. In the survivors, the SARS-CoV-2 prefusion spike binding antibodies increase overtime, and CP infusion did not result in appreciable increase of prefusion spike binding antibodies.

In addition to total binding antibodies, it was important to determine if SARS-CoV-2 infection induced antibody affinity maturation against the native prefusion spike. Technically, A c c e p t e d M a n u s c r i p t 11 since antibodies are bivalent, the proper term for their binding to multivalent antigens like viruses is avidity, but here we use the term affinity throughout, since we measured primarily monovalent interactions [7, 10] . Antibody off-rate constants, which describe the stability of the antigen-antibody complex, i.e., the fraction of complexes that decays per second in the dissociation phase, were determined directly from the serial dilutions (10-, 50-and 250-fold) of human plasma interaction with SARS CoV-2 prefusion spike using SPR [7] . The plasma antibody avidity against the prefusion spike was stronger (i.e., slower dissociation rates; 0.01-0.001/sec) in survivors compared with fatal patients (~0.1/sec in untreated patient and 0.1-0.01 per sec for CP treated patients) (Fig. 1C-D black curves) . CP treatment did not remarkably impact the SARS-CoV-2 prefusion spike antibody affinity in these of COVID-19 patients (Fig. 1 C) .

Isotyping analysis revealed that all immunoglobulin isotypes contributed to antibody binding to prefusion spike ( Fig. 2A-B) . CP treatment resulted in increase of IgG subclasses of spike-binding antibodies in some COVID-19 patients ( Fig. 2A) . Most CP-untreated COVID-19 patients contained prefusion spike antibody that consisted of 40-50% IgM isotype during the time of hospitalization (Fig. 2B) . Interestingly, percent contribution of IgA isotype to spike binding was significantly higher in the fatal COVID-19 patients (Fig. 2C) . The elevated IgA was sustained throughout the hospitalization period of fatal patients ( Fig. 2A-B) compared with survivors ( Fig. 2 A-C) . Following CP treatment, the percentage of anti-spike IgG isotypes was not significantly different between survivors and fatal patients (Fig. 2 A-C) .

We also measured the total IgM, IgG subtypes, and IgA concentrations during the peak neutralization titers (Supplementary Table 1 ). No statistically significant differences were A c c e p t e d M a n u s c r i p t 12 identified between CP treated and untreated patients or between patients that dies or survived (Supplementary Table 1 ).

The total anti-prefusion spike antibody binding (AUC of RU values for total antibody binding of all antibody isotypes) was significantly higher in the CP-treated individuals that did not survive compared with the other three subgroups (Fig. 2D) . Therefore, the total antibody binding to prefusion spike negatively associated with survival in this study. Together, these data underscore the limited impact of CP treatment on antibody profile and clinical outcome of severe hospitalized COVID-19 patients.

Based on historical reports on the potential use of CP in treatment of acute infectious diseases in hospitalized patients, there was hope that severe COVID-19 patients can benefit from infusion with CP from recovered individuals. At the same time some investigators expressed concerns about potential of enhanced respiratory disease after CP infusions [11, 12] . The Our study suggests that CP transfusion had minimal or transient impact on the composition of antibodies in hospitalized COVID-19 patients in terms of SARS-CoV-2 neutralization, prefusion spike antibody binding/isotype distribution, and antibody affinity.

Positive clinical outcome correlated with high avidity antibodies but not neutralizing antibody titers or level of spike-binding antibodies in most survivors. These findings are in agreement with previous reports on discordance between serum neutralization titers and recovery from COVID-19 and evidence of prefusion spike-specific antibody affinity maturation in COVID-19 survivors [13, 14] [15, 16] .

In the current and earlier studies, we noticed a drop in virus neutralization titers in majority of ICU-admitted patients within few days of their demise [15, 16] . The cause for this rapid drop is not fully understood. However, autopsy studies on COVID-19 patients described changes to the endothelial cells in the blood vessels lining the lungs as well as distal organs. Altered endothelial cell metabolism was associated both with thrombosis and loss of barrier intactness [17, 18] . Therefore, it is conceivable that fatal COVID-19 patients experience shift of plasma proteins including immunoglobulins from intravascular to extravascular spaces prior to death.

We identified sustained high IgA responses and minimal antibody affinity maturation against the SARS-CoV-2 prefusion spike as key feature of patients that succumb to the COVID-19 disease. A recent study suggested that IgA2 antibodies against SARS-CoV-2 correlate with NET formation and fatal outcome in severely diseased COVID-19 patients [19] .

The treatment of COVID-19 patients with CP should be carefully targeted, and effectiveness may depend on the clinical and immunological status of COVID-19 patients.

The most severe patients are less likely to benefit from CP infusions [5, 6, 20] . Furthermore, A c c e p t e d M a n u s c r i p t 14 the selection of CP should be carefully analyzed with emphasis on antibody neutralizing titers [6, 21] , and based on the current study, antibody affinity against prefusion spike, to provide the optimal clinical impact of antibody therapy.

There are several limitations to our study. The overall patient size was small (only 16 patients), even though we investigated 115 sequential samples from these 16 patients. We characterized the CP used in the study, however, we were not unblinded to match the CP that were used for treatment of each specific patient. However, two groups matched for age, sex and co-morbidities and the percentage of survival vs. fatal outcome were the same.

This study underscores the importance of following COVID-19 patients over the entire hospitalization period and collection of sequential samples for multi-assay analyses.

The interplay between exogenous and endogenous antibodies may provide important information that can help in the management of COVID-19 patients. This approach will promote identification of predictive antibody markers of disease outcome and assist in evaluating the impact of CP or other immunoglobulin preparations (i.e., monoclonal antibodies and hyperimmune CoV-2 immunoglobulins).

A c c e p t e d M a n u s c r i p t

Early safety indicators of COVID-19 convalescent plasma in 5000 patients

Effect of Convalescent Plasma Therapy on Time to Clinical Improvement in Patients With Severe and Life-threatening COVID-19: A Randomized Clinical Trial

Deployment of convalescent plasma for the prevention and treatment of COVID-19

SARS-CoV-2 viral load and antibody responses: the case for convalescent plasma therapy

A Randomized Trial of Convalescent Plasma for COVID-19-Potentially Hopeful Signals

Effect of Convalescent Plasma on Mortality among Hospitalized Patients with COVID-19: Initial Three-Month Experience

Antibody signature induced by SARS-CoV-2 spike protein immunogens in rabbits

Longitudinal Human Antibody Repertoire against Complete Viral Proteome from Ebola Virus Survivor Reveals Protective Sites for Vaccine Design

MF59 adjuvant enhances diversity and affinity of antibody-mediated immune response to pandemic influenza vaccines

AS03-adjuvanted H5N1 vaccine promotes antibody diversity and affinity maturation, NAI titers, cross-clade H5N1 neutralization, but not H1N1 cross-subtype neutralization

Passive antibody therapy in COVID-19

Convalescent plasma as a potential therapy for COVID-19. The Lancet infectious diseases

Discordance between Serum Neutralizing Antibody Titers and the Recovery from COVID-19

SARS-CoV-2 Antibody Avidity Responses in COVID-19 Patients and Convalescent Plasma Donors

Longitudinal antibody repertoire in "mild" versus "severe" COVID-19 patients reveals immune markers associated with disease severity and resolution

Antibody affinity maturation and plasma IgA associate with clinical outcome in hospitalized COVID-19 patients

Hallmarks of Endothelial Cell Metabolism in Health and Disease

Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Replication and Tropism in the Lungs, Airways, and Vascular Endothelium of Patients With Fatal Coronavirus Disease 2019: An Autopsy Case Series

IgA2 Antibodies against SARS-CoV-2 Correlate with NET Formation and Fatal Outcome in Severely Diseased COVID-19 Patients

Convalescent Plasma Antibody Levels and the Risk of Death from Covid-19

The Principles of Antibody Therapy for Infectious Diseases with Relevance for COVID-19

We thank Keith Peden and Marina Zaitseva for their insightful review of the manuscript. We