key: cord-0720413-xiyy5bvc
authors: Vacharathit, Vimvara; Srichatrapimuk, Sirawat; Manopwisedjaroen, Suwimon; Kirdlarp, Suppachok; Srisaowakarn, Chanya; Setthaudom, Chavachol; Inrueangsri, Nanthicha; Pisitkun, Prapaporn; Kunakorn, Mongkol; Hongeng, Suradej; Sungkanuparph, Somnuek; Thitithanyanont, Arunee
title: SARS-CoV-2 neutralizing antibodies decline after one year and patients with severe COVID-19 pneumonia display a unique cytokine profile
date: 2021-09-15
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.09.021
sha: 3fbaec6b8ce78d9e6d2d89402a46b8d826b3d8a4
doc_id: 720413
cord_uid: xiyy5bvc

Objectives As COVID-19 rages on worldwide, there is an urgent need to characterize immune correlates of protection from SARS-CoV-2 infection and to identify immune determinants of COVID-19 severity. Methods We present longitudinal profiles of neutralizing antibody titers in hospitalized COVID-19 patients clinically diagnosed with mild symptoms, pneumonia, and severe pneumonia up to 12 months after illness onset, using live-virus neutralization. Multiplex, correlation, and network analyses were used to characterize serum-derived inflammatory cytokine profiles in all severity groups. Results Peak NAb titers correlated with disease severity, and NAb titers declined over the course of 12 months regardless of severity. Multiplex analyses revealed that IP-10, IL-6, IL-7, and VEGF-α were significantly elevated in severe pneumonia cases compared to mild symptoms and pneumonia cases. Correlation and network analyses further suggested that cytokine network formation was distinct in different COVID-19 severity groups. Conclusions Our findings inform on the long-term kinetics of naturally acquired serological immunity against SARS-CoV-2 and highlight the importance of identifying key cytokine networks for potential therapeutic immunomodulation.

Background "Coronavirus Disease 2019" , caused by the highly transmissible and pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has so far infected about 220 million people worldwide, leading to more than 4.6 million deaths within a period of 21 months. In Thailand, approximately 1.3 million cases and over 13,000 deaths have been confirmed at the time of writing (2021). The ongoing COVID-19 pandemic has taken a significant toll on global public health and economy, calling for a deeper understanding of immune correlates of protection against SARS-CoV-2 that may be vital for implementation of mitigation strategies and development of treatments and vaccines.

The clinical spectrum of COVID-19 include asymptomatic infection, mild, self-limiting disease, and severe critical illness resulting primarily from pulmonary inflammation and diffuse alveolar damage which can lead to acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation (Guan et al., 2020) . Mechanisms underlying severe clinical manifestations of COVID-19 are still unclear, although epidemiological studies suggest that older age, male sex, high body mass index, comorbidities, and raceparticularly Black, Asian and Minority Ethnic (BAME) individualsare associated with elevated disease severity and/or increased risk of in-hospital death from COVID-19 , Tian et al., 2020 .

Neutralizing antibodies (NAbs) are an important immune correlate of protection against SARS-CoV-2. Studies in mouse and non-human primate models suggest that passive transfer of NAbs against SARS-CoV-2 can help mitigate the disease (Cross et al., 2021 , Zost et al., 2020 , while clinical trials have shown partial success of early treatment with high-titer convalescent plasma (Liu and Aberg) . Importantly, NAb titers correlated with protection against SARS-CoV-2 reinfection (Addetia et al., 2020 , Khoury et al., 2021 , Lumley et al., 2021 Thus, an understanding of the long-term kinetics and durability of anti-SARS-CoV-2 NAbs would help inform on the host"s natural response to infection. Several studies have shown robust NAb titers persist over at least 5-8 months, although data on the longevity of these protective antibodies beyond 8 months are still scarce, and the majority of these studies do not use live-virus neutralization assays (Dan et al., 2021 , Gaebler et al., 2021 , Wajnberg et al., 2020 .

The role of specific cytokine networks in COVID-19 severity is still unclear. Studies suggest either no or even inverse (Argyropoulos et al., 2020) correlation between SARS-CoV-2 viral load and COVID-19 severity, suggesting that clinical deterioration may be immune-mediated, independent of viral replication in at least certain subsets of patients.

Patients with severe COVID-19 may experience cytokine-release syndrome (CRS), also colloquially referred to as a "cytokine storm", systemic hyperinflammation that can lead to ARDS, secondary hemophagocytic lymphohistiocytosis (sHLH) (Mehta et al., 2020) , pulmonary edema, multiple-organ failure, and death (de la Rica et al., 2020) . Use of adjunctive cytokine-targeted therapy, including the FDA-approved monoclonal antibody IL-6 receptor antagonist tocilizumab, has been reported to confer clinical improvement in COVID-19 patients (Rubin et al., 2021) , suggesting that intervention of specific inflammatory networks may help attenuate disease.

Herein, we studied antibody and cytokine profiles of COVID-19 patients clinically diagnosed with mild symptoms, pneumonia, and severe pneumonia. We conducted a longitudinal study of SARS-CoV-2-directed NAbs in these patients up to 12 months post-infection, using in vitro neutralization of live virusthe "gold standard" method for NAb assessment that is often circumvented due to its time-consuming nature and the need for a BSL-3 facility. We also provide a snapshot of inflammatory cytokine profiles in these groups. We profiled patients with pneumonia and severe pneumonia separately in acknowledgment of the gamut of COVID-19 clinical manifestations, as there is still a paucity of information on the distinct immune-mediated pathologies underlying various classifications of SARS-CoV-2-induced pneumonia.

All experiments involving live SARS-CoV-2 were performed in a certified Biosafety Level 3 (BSL-3) facility at the Department of Microbiology, Faculty of Science, Mahidol University.

The experimental protocols were approved by Mahidol University, and all methods were performed following standard protocols approved by the institutional review committee.

A total of 75 COVID-19 patients hospitalized from March 2020 to May 2020 were enrolled in this study. Sequential sera samples were collected through the Chakri Naruebodindra Medical Institute (CNMI), Faculty of Medicine Ramathibodi Hospital, Mahidol University, Samut Prakan, Thailand between March 2020 and March 2021. Patients were confirmed to be infected with SARS-CoV-2 by RT-PCR on nasopharyngeal and throat swab specimens through amplification of SARS-COV-2 ORF1AB and N target gene fragments (Sansure Biotech Inc, Changsha, PR China). Sera were stored at -80ºC until use.

Pneumonia was defined as clinical symptoms of respiratory tract infection together with abnormal lung imaging compatible with pneumonia. Patients with pneumonia were classified as severe pneumonia based on the following criteria: respiratory rate >30 breaths/min, severe respiratory distress, or an oxygen saturation ≤93% on room air [World Health

Organization. Clinical management of severe acute respiratory infection when Novel coronavirus (nCoV) infection is suspected: interim guidance. January 28, 2020. Available from:

https://www.who.int/publications-detail/clinical-management-of-severe-acuterespiratory-infection-when-novel-coronavirus-(ncov)-infection-is-suspected]. Patients who had no symptoms, uncomplicated (mild) symptoms, or non-severe pneumonia were described as having mild to moderate disease.

IgG and IgM antibodies directed against SARS-CoV-2 spike (S) and nucleocapsid (N) were detected using the fully automated MAGLUMI TM analyzers (Snibe, Shenzhen, China) according to the manufacturer"s protocols. SARS-CoV-2 S1-targeted IgG and IgA were detected by ELISA (Euroimmun, Medizinische Labordiagnostika AG The average optical density (O.D.) at 450 and 620 nm were determined for virus and cell control wells, and the neutralizing endpoint was determined by 50% specific signal calculation. The virus neutralizing endpoint titer of each serum was expressed as the reciprocal of the highest serum dilution with an OD value less than X (Organization, 2011) , which was calculated as follows:

X=[(average A450−A620 of 100×TCID50 virus control wells)−average A450−A620 of cell control wells)]/2+average A450−A620 of cell control wells).

Sera which tested negative at 1:10 dilution were assigned a titer of <10. Sera were considered positive if NAb titer was ≥20. Live SARS-CoV-2 viruses at passage 3 or 4 and

Vero E6 cells at 20 maximum passages were employed.

Viruses in serum samples were inactivated with 10% Triton X-100 for 1h at room temp. The onset at the time of study enrollment was 12 (7-12.5) for mild, 10 (7-14) for pneumonia, and 14 (9.5-17.5) for severe pneumonia cases. Age was significantly higher in severe pneumonia cases compared to mild symptoms or pneumonia cases (Supplemental Figure 1) .

In our cohort, patients who had severe pneumonia were mostly male (80%) compared to those with pneumonia (45.9%) and mild symptoms (34.8%). Within the cohort, 20% of the patients had comorbidities, and severe pneumonia cases were more likely to present with comorbidities (60%) compared to pneumonia (16.2%) and mild symptoms (0%) cases. The most common comorbidities in our cohort were dyslipidemia (8.0%) and diabetes mellitus ( 

The spike (S) and nucleocapsid (N) proteins are key targets for vaccine design (Ahmed et al., 2020) . We compared levels of S-and N-specific IgG and IgM, as well as subunit 1 (S1)-IgG and IgA in the 3 severity groups. Severe pneumonia cases had the highest seropositivity rates across all Ig targets, followed by pneumonia and mild symptoms cases. Levels of all Ig targets were significantly higher in severe pneumonia cases compared to mild symptoms cases. S and N-IgG was also significantly higher in severe cases compared to pneumonia cases (Figures 1A-D) . We then defined associations of Ig and NAb titers across clinical disease severity levels. S and N-IgG, S1-IgG and S1-IgA showed strong positive correlations with NAbs in all severity groups, while S and N-IgM titers were positively correlated with NAbs in pneumonia and severe pneumonia cases, but not significantly so in mild cases ( Figures 1E-H) .

We then performed longitudinal profiling of NAb titers against live SARS-CoV-2 in sera from all 3 groups. We arbitrarily stratified NAb titers into low, medium and high levels based on criteria for COVID-19 convalescent plasma (CCP) donation (Wendel et al.) and observed that a majority of the patients" NAb titers dropped to medium and low titers within 1 year regardless of disease severity ( Figure 1I) (62.9 [29.7-132.9 ]) within the first month after illness onset. All 3 groups exhibited decline in NAb titer between 2 to 12 months after illness onset regardless of disease severity. The biggest drop in mean peak NAb titer occurred between 6-12 months for all 3 groups; severe pneumonia cases experienced an approximate 10-fold decrease, pneumonia 5-fold, and mild symptoms cases experienced a 6-fold decline between 6 and 12 months. At the 12month mark NAb titers were no longer significantly different between severity groups, and all 3 groups retained lower NAb titers than were produced during the first month post-illness onset, although only those with severe pneumonia experienced a NAb titer decline that was statistically significant in this regard. Compared to the peak geometric mean titers for each group, after 12 months mild symptoms and pneumonia cases retained approximately 20% antibody concentrations (geometric mean titers of 40 and 116.2, respectively), while those with severe pneumonia retained about 5% (geometric mean titer of 160) ( Figure 1J) .

We next assessed 25 key cytokines associated with inflammation and cytokine storms (Fajgenbaum and June, 2020, Ragab et al., 2020) in serum from a sub-cohort of 48 patients with mild symptoms, pneumonia, and severe pneumonia using a multiplex assay. Our results showed that IP-10, IL-6, IL-7, and VEGF-α concentrations were significantly elevated in severe pneumonia cases compared to mild symptoms and pneumonia cases. Meanwhile, MCP-1, IL-1RA, and IL-8 concentrations were also significantly higher in severe pneumonia cases compared to mild symptoms cases, and TNF-α was significantly upregulated in severe pneumonia cases compared to pneumonia cases. MIP-1α was higher in healthy controls compared to mild and pneumonia cases (Figure 2A) . GM-CSF, IL-2, IL-3, IL-10, IL- Severe pneumonia cases in Cluster A had a median age of 54.8 years and comprised of 62.5% males, while those in Cluster B had a median age of 54.7 years and were 100% male ( Figure 2B ).

Although patients with severe pneumonia were significantly associated with older age compared to other groups (Supplemental Figure 1) , and the majority of our patients with severe pneumonia were male (12/15), there was no statistically significant correlation between target cytokine levels and patient age or sex within each severity group. Cytokine levels and days after illness onset were also not significantly correlated, with the exception of VEGF-α, which displayed positive correlation (data not shown).

To determine the strength of association between target cytokines in the context of clinical disease severity, we performed separate hierarchical correlation matrix analyses for each group. All groups displayed differential patterns of pairwise positive and negative correlations ( Figure 3A-C) .

We next visualized the relationship between pairs of target cytokines in different severity groups using network analyses. The 3 groups were found to have distinct cytokine network topologies characterized by differential groups of closely associated inflammatory mediators ( Figure 3D-F) . Notably, there was a significant positively correlated group comprising of IL-6, IL-1RA, MIP-1β, TNF-α, MCP-1, GCSF, and IP-10 in severe pneumonia cases ( Figure   3F ), some of which were significantly upregulated in severe cases compared to mild and pneumonia cases (Figure 2A ).

The kinetics and duration of NAb titers in response to viral infection are not always accurately predictable from the early phases of infection (Sallusto et al., 2010) , although many valuable studies have extrapolated the trajectory of SARS-CoV-2 NAb production through applications of machine learning algorithms (Chia et al., Legros et al., 2021 . Moreover, most longitudinal studies forego use of live SARS-CoV-2 clinical isolates in neutralization tests, which require BSL-3 certified laboratories, often opting for safer alternatives such as pseudotyped SARS-CoV-2-based neutralization assays instead (Dan et al., 2021 , Gaebler et al., 2021 , Nie et al., 2020 . In the present study, we monitored temporal changes in NAb titers in hospitalized COVID-19 patients with varying disease severity over a period of up to 1 year after illness onset using live-virus neutralization.

The decline of NAb titers over time, regardless of COVID-19 severity, may be due to transient plasmablast expansion, which show signs of decay fewer than 10 days after the COVID-19 symptom onset (Laing et al., 2020) . NAb decline may also stem from a biphasic shift between antibodies produced by short-lived plasma cells during the acute phase to those subsequently generated by the 10-20% that differentiate into long-lived memory plasma cells (Turner et al., 2021) . It is still unclear why antibody levels correlate with COVID-19 severity; high viral loads may result in higher disease severity in some patients (Fajnzylber et al., 2020) , which in turn may result in robust production of antibodies in response to extended antigen exposure. Unfortunately, cycle threshold (Ct) values that semi-quantitatively assess SARS-CoV-2 viral load were not recorded for our present cohort, but correlations between viral load and cytokine levels would be an important issue to address in the future. Alternatively, theories of antibody-dependent enhancement (ADE) in COVID-19 are emerging, but so far no definitive role for ADE in human coronaviruses has been established (Lee et al., 2020) . Nevertheless, a recent study suggests that NAbs may expand coronavirus cell tropism by binding to Fc receptor-expressing immune cells and guiding viral entry (Wan et al., 2020) , thus the possibility of ADE in COVID-19 exacerbation

should not be ruled out.

It has become clear that SARS-CoV-2 infection leads to immune imbalance and dysregulation (Blanco-Melo et al., 2020 , Mathew et al., 2020 and that unbridled cytokine storms are key to COVID-19 immunopathology (de la Rica et al., 2020) . We found that several cytokines, including IP-10 (CXCL10), IL-6, IL-7, and VEGF-α were distinctly upregulated in severe pneumonia cases compared to mild symptoms and pneumonia cases;

these may serve as key prognostic markers of COVID-19 severity. We also found that the formation of cytokine networks was dependent on clinical disease severity. Our results, along with multiple studies showing that tocilizumab and corticosteroid use conferred varying levels of success, suggest a need for personalized therapies in the context of SARS-CoV-2driven immunopathogenesis and hyperinflammation.

Several caveats to our study should be pointed out. Firstly, cytokine levels may correlate with severity but this does not necessarily imply their pathogenic roles. Our results also provide a mere snapshot of the cytokine/chemokine/growth factor interactions in the early phase of COVID-19 and do not capture the spatiotemporal dynamics of these mediator expressions. Longitudinal profiling of pertinent cytokines in larger cohorts of COVID-19

patients would ideally provide a more complete picture of inflammatory networks during distinct stages of disease. Other aspects of the immune response, including T cell-and innate immune cell-mediated responses should also be further investigated.

The ongoing COVID-19 pandemic has brought the whole world to a standstill. Case numbers have continued to surge in persistent "waves" across the globe (Karagiannidis et al., 2021 , Kuehn, 2021 . Our results highlight the need to maintain protective measures in the face of a potentially transient serological immunity against SARS-CoV-2 and the emergence of unusually divergent viral strains (Cohen and Burbelo, 2020, Frampton et al.) . There is also a need for further in-depth studies of inflammatory cytokine networks linked to severity that may lead to new prognostic and/or therapeutic avenues.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Information in this article have not been presented in any meeting.

VV wrote the manuscript, carried out experiments and analyzed the data, S Srichatrapimuk, SM, SK, C Srisaowakarn, C Setthaudom, and NI carried out experiments and collected data, PP, MK, SH, S Sungkanuparph supervised the project, and AT conceived the original idea and supervised the project.

The authors would like to thank Dr. Ponpan Matangkasombut Choopong for provision of lab equipment and sharing of protocols, as well as all the study participants and professionals of our COVID-19 medical and research teams.

The experimental protocols were approved by Mahidol University, and all methods were performed following standard protocols approved by the institutional review committee. 

Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with high attack rate

Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies

Association of Initial Viral Load in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Patients with Outcome and Symptoms

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

Convalescent plasma as a potential therapy for COVID-19

Dynamics of SARS-CoV-2 neutralising antibody responses and duration of immunity: a longitudinal study

Reinfection With SARS-CoV-2: Implications for Vaccines

Use of convalescent serum reduces severity of COVID-19 in nonhuman primates

Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection

SARS-CoV-2 viral load is associated with increased disease severity and mortality

Genomic characteristics and clinical effect of the emergent SARS-CoV-2 B.1.1.7 lineage in London, UK: a whole-genome sequencing and hospital-based cohort study. The Lancet Infectious Diseases

A platform for the graph-based visualisation and analysis of complex data

Evolution of antibody immunity to SARS-CoV-2

Mild or Moderate Covid-19

Development of an inactivated vaccine candidate for SARS-CoV-2

Clinical Characteristics of Coronavirus Disease 2019 in China

Temporal dynamics in viral shedding and transmissibility of COVID-19

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Major differences in ICU admissions during the first and second COVID-19 wave in Germany

Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection

Africa Succeeded Against COVID-19's First Wave, but the Second Wave Brings New Challenges

A dynamic COVID-19 immune signature includes associations with poor prognosis

Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies

A longitudinal study of SARS-CoV-2-infected patients reveals a high correlation between neutralizing antibodies and COVID-19 severity

Risk factors for severity and mortality in adult COVID-19 inpatients in Wuhan

Convalescent plasma in patients hospitalised with COVID-19

Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers

Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications

COVID-19: consider cytokine storm syndromes and immunosuppression

Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay

WHO Global Influenza Surveillance Network. Manual for the laboratory diagnosis and virological surveillance of influenza

The COVID-19 Cytokine Storm; What We Know So Far

Tocilizumab in Hospitalized Patients with Severe Covid-19 Pneumonia

Interleukin-6 Receptor Inhibition in Covid-19 -Cooling the Inflammatory Soup

From vaccines to memory and back

Is a "Cytokine Storm" Relevant to COVID-19?

Inflammatory cytokines in vascular dysfunction and vascular disease

Clinical characteristics and risk factors associated with COVID-19 disease severity in patients with cancer in Wuhan, China: a multicentre, retrospective, cohort study

SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans

Robust neutralizing antibodies to SARS-CoV-2 infection persist for months

The impact of COVID-19 and strategies for mitigation and suppression in low-and middle-income countries

Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry

low-dose and short-term application of corticosteroid treatment in patients with severe COVID-19 pneumonia: single-center experience from Wuhan, China

A longitudinal study of convalescent plasma (CCP) donors and correlation of ABO group, initial neutralizing antibodies (nAb), and body mass index (BMI) with nAb and anti-nucleocapsid (NP) SARS-CoV-2 antibody kinetics: Proposals for better quality of CCP collections

Evolution of immune responses to SARS-CoV-2 in mild-moderate COVID-19

lines represent the LLOD for each target. Only targets with significantly different levels between groups are shown here

Clustered heatmap of 25 inflammatory cytokines from hospitalized COVID-19 patients at the time of study enrollment. Values below the LLOD were imputed for each target and cytokine data were log-transformed, scaled, and both rows and columns were subjected to the kmeans clustering algorithm

Columns were annotated with patient characteristics including disease severity

Correlation matrices of cytokine expression in hospitalized COVID-19 patients with (A) mild symptoms (n=12), (B) pneumonia (n=22), and (C) severe pneumonia (n=14) at the time of study enrollment. Only significantly correlated (P≤0.05) mediator interactions are shown. Positive and negative correlations are shown in blue and red, respectively. The size and color intensity of the dots are proportional to the Spearman correlation coefficients (r S ). Pairwise correlation networks showing positive relationships between cytokines in hospitalized COVID-19 patients with (D) mild symptoms (E) pneumonia, and (F) severe pneumonia