In a recent study posted to the medRxiv* preprint server, an interdisciplinary team of researchers from the United States (US) conducted a comprehensive and systematic analysis of functional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody profiles in a controlled randomized trial of severe coronavirus disease 2019 (COVID-19) hospitalized individuals treated with COVID-19 convalescent plasma (CCP).
From the start of the COVID-19 pandemic, CCP has been used to treat COVID-19 due to its safety, use, and immediate availability. The attenuation of COVID-19 by antibodies via a wide array of effector functions of the immune system in CCP therapy remains to be elucidated.
In this study, the authors assessed the therapeutic ability of CCP beyond the binding and neutralization of SARS-CoV-2-specific antibodies by applying systematic serology to an open-label randomized clinical trial.
Study design
The study included a clinical cohort of 80 hospitalized SARS-CoV-2-confirmed participants with a median age of 63. All participants had severe COVID-19 between May 2020 and January 2021 and were enrolled at a median of six days following the onset of symptoms. Out of the 80 participants, 41 participants were randomized to the treatment group and received two units of convalescent plasma, and 39 participants were assigned to the control group.
The researchers performed high-throughput quantification of antigen-specific humoral responses and detection of different antigens by multiplexed customized Luminex bead array. They used a SARS-CoV-2 antigen panel including the spike glycoprotein (S), receptor-binding domain (RBD), nucleocapsid (N), S1, S2, and N-terminal domain (NTD). Antigen controls were run with a mix of three proteins of Flu-HA and Ebola glycoprotein.
Antibody titers specific to antigens were analyzed with antibodies coupled with phycoerythrin (PE) against immunoglobulin A1 (IgA1), IgM, IgG1, IgG2, IgG3, IgG4. Biotinylated fragment crystallizable receptors (Fc-R) such as FcR2AH, 2B, 3AV, 3B, FcRn, FCAR, and FCR3AV were coupled to PE to form tetramers to detect antigen-specific Fc-R binding.
Fluorescent yellow and red neutravidin beads were coupled with biotinylated antigens for SARS-CoV-2 to quantify antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent complement deposition (ADCD). The assays were performed using flow cytometry with Intellicyt and an S- Lab plate handling robot (PAA). Further, the researchers measured Fc glycan using capillary electrophoresis.
The visualization of the data was achieved by creating heatmaps with the heatmap function in R. During the clinical trial, polar plots were used for the visualization of individual antibody profiles specific to S-protein in the CCP and control group participants.
Different multivariate models were used, such as the logistic regression model consisting of four parameters. Sixteen models controlled by the combination of four parameters were evaluated by the Akaike information criterion (AIC) to balance the fitness and complexity of the model.
The researchers used the least absolute shrinkage and selection operator (LASSO) feature algorithm selection to spin out important features. The researchers applied the LASSO feature 10 times and picked features that occurred more than seven times on the whole dataset. The average receiver operating characteristic (ROC) curves were visualized through function roc in R.
Findings
The researchers observed a significant difference in clinical severity score (CSC) and mortality of participants treated with CCP (median 7 and 5%, respectively) as compared to the control group participants (median 10 and 25.6%, respectively).
A significant change in SARS-CoV-2-specific humoral responses like IgM, IgA, and its subclasses and Fc-receptor binding against S, the S1 domain of S, RBD of S, NTD of S, and N was observed in CCP-treated and control participants. Moreover, innate immune cell antibodies including ADCD, ADNP, ADCP, and antibody-dependent NK cell activation (ADNK) were also observed. These changes were similar at the start of the study but increased with time post enrolment.
After three weeks of symptoms onset, the team observed a lower SARS-CoV-2 S- specific titers for S-protein, functional activity dependent on Ab, and Fc-receptor binding in the CCP-treated group. In control groups, they also found higher final levels of titers specific to RBD and FcR-binding (FcaR, FcgR2a, and FcgR3b). N-specific titers showed the same plateau level in both groups with slightly higher levels of IgG2, IgM, and FCgR3b binding specific to the N-protein in the CCP group.
The researchers noted an enrichment of six selected AIC features, including S1-specific FcgR2a binding antibody levels, S-specific ADNP, RBD-specific IgG1 levels, RBD-specific FcaR binding levels, S-specific C1q binding levels, and S1-specific FcRn binding in control and severe disease individuals (CSC>20). Using LASSO-selected features specific to S, researchers predicted that these six features were associated with worse clinical outcomes of COVID-19 in control participants.
The researchers observed that the risk of COVID-19 disease severity increased S-specific features in the control group. Features like binding strength to FcgR2B, FcgR3B specific to N, and ADCD were enriched in CCP-treated individuals, while only RBD antibody binding to the IgA Fc-receptor FcaR was enriched in the control group.
There was an association between better clinical outcomes in the studied cohort and features of N-specific antibodies, especially N-specific ADCD. In CCP-treated individuals, N-specific, FcgR3B, FcgR2B, IgM titers, and C1q binding were highly enriched and linked with significant clinical outcomes. However, N-specific macrophage inflammatory protein -1 beta (MIP1b) and natural killer (NK) cluster of differentiation (CD) 107a expression were not enriched in CCP-treated and control group but was associated strongly with better outcomes indicating that ADCC was more beneficial in COVID-19 but was not affected by CCP treatment.
The team formed four different clusters and showed that individuals with reduced SARS-CoV-2 functional antibodies benefited more from CCP treatment. Moreover, the team observed that former humoral functions for anti-SARS-CoV-2 were a stronger indicator of response against therapy than the seronegative status only.
An enrichment of disialylated and diglycosylated peaks specific to spikes like G2S2F, G2S21F, and G2S2B was observed in CCP-treated subjects. Interestingly, the CCP group exhibited increased titers for N-specific antibodies and binding antibodies for Fc-receptor. In contrast, control subjects had increased antibody titers specific for S1 and RBD and Fc receptor binding. Thus CCP benefit in severe COVID-19 was partly due to an immunodominance shift resulting in solid transition in antibody profiles even after months of treatment.
Conclusion
This study demonstrated that treatment of COVID-19 pneumonia with CCP reduced mortality and ameliorated clinical severity.
The study detected a novel pathway for the mechanism of action of CCP and suggested patient parameters optimum for the initiation of CCP therapy. Moreover, it revealed persistent, long-lasting immunomodulatory effects of CCP in COVID-19 patients. Further, it provided a key for the evolution of potential therapeutics focused on N-antibody to treat COVID-19-induced severe hyperinflammation.
*Important notice
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
