Background
The Coronavirus Disease 2019 (COVID-19) pandemic has claimed the lives of over 5.7 million people worldwide, with close to 402 million cases of the disease reported to date [
1]. The medical and scientific communities continue to investigate the systemic, inflammatory nature of COVID-19 infection as a prognostic measure of disease severity, in order to fully comprehend the virulence of the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These investigations have been expedited by the emergence and prevalence of viral variants [
2‐
4] and have prompted the rapid development and analysis of targeted therapeutic treatment options for patients suffering from advanced stages of COVID-19 infection and the resultant dysregulated immune responses [
5‐
10].
Type I Interferons (IFN-1) play a crucial role in the host immune response to viral infections [
11]. IFN-1 production is triggered by host recognition of viral nucleic acids [
12], which in turn induces an antiviral signaling cascade and the expression of hundreds of genes involved in functions ranging from inhibition of viral replication to activation of immune cells [
13,
14]. However, SARS-CoV viruses have the ability to delay and inhibit IFN-1 responses and IFN-activated genes, thus interfering with the normal immune response to viral infection [
15‐
20]. This interference in IFN-mediated responses is reflected in the immunopathology of infection, ultimately leading to an exaggerated immune response in the form of large-scale pro-inflammatory cytokine production, often referred to as a cytokine storm [
21,
22]. The dysregulated immune response and hyperinflammation associated with a cytokine storm contribute to disease severity in COVID-19 [
23,
24]. In fact, recent studies have shown that individuals with errors in the IFN-1 pathway or neutralizing autoantibodies against IFN-1s had a greater chance of developing life-threatening COVID-19 [
25,
26]. Therefore, a thorough understanding of the functional consequences of the endogenous immune response to SARS-CoV-2 infection is crucial throughout the disease process and during recovery.
Treatment with monoclonal antibodies (mAbs) represents a targeted and fast-acting therapeutic approach to diminish viral dissemination in patients with COVID-19. Bamlanivimab is a potent, neutralizing mAb that binds to the receptor binding domain (RBD) of the COVID-19 spike protein, blocking viral entry to host cells, thus preventing infection and viral replication [
27]. Neutralizing mAbs, such as bamlanivimab, offer immediate passive immunity to patients, provided treatment is administered within 10 days of COVID-symptom onset [
28,
29].
In the current study, we sought to detect and track the immune response to SARS-CoV-2 in patients hospitalized with COVID-19, in the presence or absence of bamlanivimab treatment, by following changes in immune-relevant parameters over the course of 1 month. We present herein, various longitudinal immunological measures within the blood of patients with COVID-19 and demonstrate the connectivity of these measurements to RNA profiles in the nasopharyngeal cavity.
Discussion
The uncontrolled release of pro-inflammatory cytokines and the cytokine storm which ensues has been recognized as the primary cause of death in patients infected with SARS-CoV-2 [
32]. Proteomic analysis highlighted a cytokine storm in the serum of patients with COVID-19, with elevated expression levels of IL-6, CXCL10, CXCL11, IFNγ, MCP-2, and MCP-3 detected in infected patients relative to healthy controls. Many inflammatory markers identified using this analysis were associated with IFN signaling, and an interesting correlation was observed between ferritin and CRP levels with IL-6 and CXCL10. Elevated levels of CRP, ferritin, and IL-6 concentrations have been shown to be higher in cytokine storm disorders [
33], patients with severe COVID-19, and in non-survivors [
34,
35] of COVID-19 compared to discharged patients. In addition, previous studies support our observations that concentrations of these biomarkers decreased as patients recover [
36]. Lastly, these same biomarkers may be critically important indicators of patient outcome as elevated clinical inflammatory markers were inversely correlated with OXYSAT levels and tracked with lymphopenia responses observed in non-survivors of COVID-19 [
37]. This first-in-man study of bamlanivimab was not powered to detect any effects of treatment on longitudinal changes in immune biomarkers and no significant effects were observed.
As we looked to better understand the lymphocyte responses over time in these patients, we focused on serological assays, which demonstrate a high degree of sensitivity and specificity, and are highly favored for the detection and monitoring of anti-SARS-CoV-2 antibodies in patients who present with COVID-19. The accuracy of these assays is significant to clinical management and public health decision-making. In the current study, we demonstrate use of the Luminex serology assay to detect quantitative seroconversion in clinical specimens from patients with COVID-19 with greater sensitivity than the Elecsys® Anti-SARS-CoV-2 assay. While the Elecsys® Anti-SARS-CoV-2 assay is rapid [
38] and has received FDA EUA approval [
39], the Luminex method holds many advantages due to the ability to ascertain broad serological conversion toward multiple viral proteins, including drug-tolerant epitopes, at the same time. A robust antibody response to the drug-tolerant, NCP and NTD of the SARS-CoV-2 spike protein was observed using this assay, with high titers of IgA, IgG, and IgM sharing a similar time course of induction. While IgA and IgM titers peaked around 7 days post-dose, IgG titers remained high, even after 28 days. These findings are in accordance with previous observations made across a number of studies [
40‐
42], and highlight the utility of the Luminex assay for detection and monitoring of anti-SARS-CoV-2 antibodies.
The inverse correlation observed between the levels of antibodies produced against the NCP and NTD of the SARS-CoV-2 spike protein and the levels of inflammatory mediators present in serum samples is indicative of the power of humoral immunity to resolve infection. The ability of the Luminex serology method to detect both bamlanivimab as well as the endogenous immune response towards spike protein has already been documented [
43]. There were no observed changes in the endogenous antibody responses in bamlanivimab-treated patients vs placebo in this small study, although larger studies are ongoing. The lack of distinguishable biomarker differentiation between placebo and bamlanivimab arms was not unexpected based on subsequent studies demonstrating that passive immunization was not an effective treatment for patients hospitalized with COVID-19 [
44‐
46].
Importantly, our study revealed a remarkably similar pattern in gene expression profiles observed in nasopharyngeal swab material and the inflammatory, COVID-19-related responses observed in the serum of infected patients. While responses were not different between bamlanivimab- treated individuals and those who received placebo,
CCL2,
CCL19, CCL20, CXCL8 and
CXCL10 were upregulated in samples from the nasopharyngeal cavity and the circulating blood of patients with COVID-19. Many of these chemokines are strongly linked to macrophage and neutrophil function.
CCL2 is vital for monocyte recruitment [
29], is expressed at higher levels in lung macrophages of patients with severe COVID-19 [
30] and upregulated in response to increases in SARS-CoV-2 viral load levels in infected patients [
31].
IL-8/CXCL8 plays a key role in the recruitment and activation of neutrophils during inflammation [
33]. Given that neutrophilia is frequently observed in patients with COVID-19 [
47‐
49], it is possible that
CXCL8 contributes to the pathophysiology of the disease. The elevated expression levels of
CXCL8, which we observed in the serum of patients with COVID-19, reflected the findings of Del Valle et al. in their study examining predictive biomarkers of SARS-CoV-2-related inflammation [
50]. Our study aligns with the authors observations that high levels of
CXCL8 were associated with worse patient outcomes and may be an important predictive biomarker of patient survival. Similarly, Li et al. revealed that high levels of
CXCL8 in the serum of patients with COVID-19 were consistent with severity of disease [
51].
In line with recent bioinformatic analysis of COVID-19 sequencing data we determined elevated expression of IFNγ -linked biomarkers, such as CXCL10, in both serum and nasopharyngeal swab material collected from patients with COVID-19. While this study was small and failed to identify pharmacodynamic biomarkers for bamlanivimab, the rapid decrease of key inflammatory mediators (e.g., IFNγ, IL-6, IL-10, MCP-2, MCP-3, and CXCL10), beginning as early as Day 3, emphasizes the need for normalization of key immune analytes for favorable outcomes in the course of COVID-19 infection. These observations add to our understanding of core biomarkers of COVID-19 severity, including recent observations in children suffering from multisystem inflammatory syndrome, and further our knowledge around key immune pathways that therapeutic options must target early in infection to best promote favorable patient outcomes [
52]. This could be most valuable when evaluating the pathology of SARS-CoV-2 variants or when performing combination studies of multiple neutralizing mAbs. Future studies could be focused on monoclonal-resistant SARS-CoV-2 variants, with the current knowledge that serum biomarkers adequately reflect the nasal compartment.
Acknowledgements
The authors would like to thank healthcare workers throughout the world for their efforts and continued dedication in treating patients. We thank Mary Zuniga, B.Sc., of Eli Lilly for assistance with sample management. We thank Angie Fulford, M.Sc, Gary Heady, and Erin Wray, B.Sc., of Eli Lilly and Company for their assistance with Next-Generation Sequencing. We thank Katherine Birt, B.Sc., Elizabeth Parker, B.Sc., and Jeff Fill, B.Sc, M.B.A., of Eli Lilly and Company for Cobas sample analysis. Bamlanivimab emerged from the collaboration between Eli Lilly and Company and AbCellera Inc. to create antibody therapies for the prevention and treatment of COVID-19. Lilly developed the antibody after it was discovered by AbCellera and scientists at the National Institute of Allergy and Infectious Diseases (NIAID) Vaccine Research Center.
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