key: cord-0969870-8ti6r0eh
authors: Bruni, M.; Cecatiello, V.; Diaz-Basabe, A.; Lattanzi, G.; Mileti, E.; Monzani, S.; Pirovano, L.; Rizzelli, F.; Visintin, C.; Bonizzi, G.; Giani, M.; Lavitrano, M.; Faravelli, S.; Forneris, F.; Caprioli, F.; Pelicci, P. G.; Natoli, G.; Pasqualato, S.; Mapelli, M.; Facciotti, F.
title: Persistence of anti-SARS-CoV-2 antibodies in non-hospitalized COVID-19 convalescent health care workers
date: 2020-08-01
journal: nan
DOI: 10.1101/2020.07.30.20164368
sha: 63cdcb12af7f5212b6b2f5525eadd307d34bc1e3
doc_id: 969870
cord_uid: 8ti6r0eh

Background. Coronavirus disease-19 (COVID-19) is a respiratory illness caused by the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), a novel beta-coronavirus. Although antibody response to SARS-CoV-2 can be detected early during the infection, several outstanding questions remain to be addressed regarding magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response, as measured by serum levels of pro-inflammatory mediators. Methods. An ELISA assay has been developed by expressing and purifying the recombinant SARS-CoV-2 Spike Receptor Binding Domain (RBD), Soluble Ectodomain (Spike), and full length nucleocapsid protein (N protein). Sera from healthcare workers affected by non-severe COVID-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in Intensive Care Units (ICU) and SARS-CoV-2-negative subjects for the presence of IgM, IgG and IgA antibodies as well as soluble pro-inflammatory mediators in the sera. Results. Specificity and sensitivity of the ELISA assays were high for anti-RBD IgG and IgA (92-97%) and slightly lower for IgM and the Spike and N proteins (70-85%). The ELISA allowed quantification of IgM, IgG and IgA antibody responses against all the viral antigens tested and showed a correlation between magnitude of the antibody response and disease severity. Non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles as compared to patients in Intensive Care Units (ICU), irrespective of the antibodies tested. Noteworthy, in non-severe COVID-19 infections, antibody titers against RBD and Spike, but not against the N protein, as well as pro-inflammatory cytokines decreased within a month after viral clearance. Conclusions. Rapid decline in antibody titers and in pro-inflammatory cytokines may be a common feature of non-severe SARS-CoV-2 infection, suggesting that antibody-mediated protection against re-infection with SARS-CoV-2 is of short duration. These results suggest caution in use serological testing to estimate the prevalence of SARS-CoV-2 infection in the general population.

Syndrome Coronavirus 2 (SARS-CoV-2), a novel beta-coronavirus firstly described in Wuhan city, China, on December 2019 [1] . SARS-CoV-2 spreading has been declared pandemic in mid-March 2020 by WHO [2] . At present the virus has infected more than 12 million people worldwide with an associated case fatality rate of 1 to 15%, depending on the country [3] .

COVID-19 is associated with a broad range of mild-to-severe symptoms, potentially leading to hospitalization in Intensive Care Units (ICU) for the most severe cases. The respiratory tract is initially involved with possible development of severe interstitial pneumonia [4, 5] , albeit the gastrointestinal tract can also significantly participate in disease pathogenesis as a consequence of the expression of the ACE2 receptor, that mediates SARS-CoV-2 viral entry [6] , on both alveolar and enteric epithelial cells [7] . Infected subjects manifest a complex clinical pattern appearing as early as two days post exposure and lasting several weeks [1] .

Infection with SARS-CoV-2 induces a prompt activation of the immune system, finalized to the clearance of infected cells [8] . Innate and adaptive immune cells accumulate at the site of infection, where production of cytokines and inflammatory mediators may result in patient recovery or, in case of ineffective viral clearance, in hyperactivation of the immune system and development of severe complications, such as acute respiratory distress syndrome ARDS [4, 9] . Overexpression of pro-inflammatory cytokines (i.e. IL-1 beta, IL-2, IL-6, IL-17, TNFα etc.) and impairment of humoral immunity have been described in patients with the most severe form of disease [5] . Antibodies against SARS-CoV-2 proteins are produced as a consequence of the activation of the humoral arm of the immune system. Virus-specific IgM antibodies are secreted as first class of immunoglobulins, followed by the more specific IgG [10] . Among the latter, those specific for the viral Spike Receptor Binding Domain (RBD) when expressed at higher titer manifest direct neutralizing activity towards the viral entry into cells, as they prevent effective engagement of surface ACE2 receptors by the Spike protein [11, 12] . The IgA response against SARS-CoV-2 has been shown to be rapid and persistent [13, 14] and is associated with mucosal responses, including the respiratory and gastrointestinal ones.

Serological testing is a valuable tool to monitor viral spreading throughout the population [15] .

Furthermore, serological assays allow the identification of past infection in individuals with viral RNA levels undetectable by RT-PCR for epidemiological purposes [16] . Various commercial and inhouse assays that utilize distinct viral antigens and detect different antibody classes are currently available. However, SARS-CoV-2 serological tests available on the market do not always allow systematic simultaneous detection of a wide antibody spectrum for several antigens in a reliable manner, and this may hamper a proper population testing for clinical or epidemiological purposes [17] . Conversely, serological enzyme-linked immunosorbent assay (ELISA) to detect Immunoglobulins raised against the viral Spike Soluble Ectodomain (Spike) and its highly immunogenic receptor binding domain (RBD), or against the Nucleocapsid protein (N), are providing promising results in terms of accuracy and reproducibility [11] . Recently, these ELISA assays have been used to show that neutralizing antibodies (nAb) against different viral antigens may decline after 20-30 days post symptoms onset, and that the magnitude of nAb response may be associated with disease severity in COVID-19 patients [18] .

In order to measure the presence and evolution of antibody responses against different viral proteins, we set up and validated an in-house direct ELISA assay based on three distinct SARS-CoV-2 viral antigens, i.e. eukaryotically-expressed RBD and Spike and bacterially-expressed Nucleocapsid protein. Using this assay, we simultaneously measured IgM, IgG and IgA anti-viral antibodies titers in the sera of COVID-19 patients, as well as levels of pro-inflammatory cytokines. In addition, we longitudinally collected the sera of 16 convalescent healthcare workers who tested positive for SARS-CoV-2 by nasopharyngeal (NF) swabs, and were symptomatic but not hospitalized. Our data show that humoral immune responses against SARS-CoV-2 correlated with disease severity in terms of both antibody titers, persistence over time and serum levels of pro-inflammatory cytokines.

Notably, 90% of COVID-19 mildly symptomatic patients halved their anti-RBD IgG titers after 4 weeks from viral negativization, thus confirming the short lifespan of humoral immune responses against SARS-CoV-2.

To evaluate the antibody response of individuals infected by SARS-CoV-2, ELISA assays were developed in-house by producing and purifying recombinant RBD, Spike and Nucleocapsid proteins of the SARS-CoV-2 virus following the protocols published in [19] ( Figure 1A ).

The performances of these ELISA assays were assessed for the different viral antigens and classes of antibodies by determining ROC curves using i) a cohort of 56 sera from COVID-19 patients collected between April and June 2020 and tested positive for nasopharyngeal swabs, and ii) 436 pre-COVID-19 sera, collected between 2012 and 2015 (Supplementary Table 1 and Figure S1 ). Anti-SARS-CoV2 IgG showed the highest specificity and sensitivity irrespective of the antigen used (Supplementary Figures 1A,B) . Anti-RBD IgG showed specificity and sensitivity of 97% and 95%, respectively, while the assay performed with the Spike ectodomain reached values of 98.5% and 77% and the one with the N protein values of 91% and 95% (Supplementary Table 1 and Figure S1 ).

These performances are in line with those published for both in-house and commercial assays approved for emergency use by the FDA [20, 21]. Performance of IgA detection was high for the RBD assay (91.5% specificity and 95% sensitivity), while it was slightly lower for the N protein (85% and 69%) and for the Spike (73% and 71%). The performance of the IgM assay was comparatively lower for all the viral proteins tested (Supplementary Figures 1A,B) .

The validated ELISA assays were then used to systematically test the antibody titers of different classes of SARS-CoV-2 specific antibodies in sera from the following groups of patients: i) 24 severe COVID-19 patients admitted to ICUs; ii) 16 health care workers from two hospitals in Milan, exposed to the virus between February and March 2020 and confirmed positive to SARS-CoV-2 RNA by RT-qPCR on nasopharyngeal swabs. 58 SARS-CoV-2-negative subjects collected between April and June 2020 were used as negative controls (Supplementary Table 1 ). Sera of the 16 health care workers were collected in the convalescence phase of the disease after two consecutively negative nasopharyngeal swab tests. Time between the first detection of the virus and the first negative swab ranged from 14 to 35 days from onset of symptoms to disappearance of viral RNA (Supplementary Table 2 ). These subjects all manifested clinical symptoms strongly related to SARS-CoV-2 infection, including fever, ageusia, anosmia, fatigue, myalgia, diarrhea, coryza and cough [5] . Two of them manifested a more severe disease course with episodes of dyspnea. None of the patients required hospitalization and they all recovered from the disease (Supplementary Table 2 ).

Non-hospitalized COVID-19 subjects manifested a lower antibody titer as compared to severe ICU patients for all the tested antibody classes and viral antigens ( Figure 1B-D) . This finding is in accordance to what published for asymptomatic [22] and paucisymptomatic [14] patients whose antibody titers were detected using commercial ELISA or chemiluminescence assays against either the Spike or the N-protein. When comparing the presence of the different classes of antibodies, all the COVID-19 positive subjects resulted positive for the presence of IgG antibodies against all the viral antigens tested ( Figure 1E ). Interestingly, a few of them were IgM negative or with an antibody concentration close to the detection limit of the Spike and RBD assay, as compared to the N protein.

The observation that all of them instead showed N-specific IgM antibodies may be a genuine persistence of anti-N protein IgM or the consequence of a lower specificity of the N assay, possibly reflecting the high conservation of the N proteins among beta-coronaviruses other than SARS-CoV2 [23] . Interestingly, 25% of the non-hospitalized COVID-19 patients did not develop RBD-specific IgA, and only 1 out of 4 developed N-specific IgA antibodies, a percentage that was instead above 80% for the hospitalized ones ( Figure 1E) . Notably, IgA production has been associated with disease severity, suggesting that IgA production might occur locally at the mucosal sites, possibly correlating with the viral load, the duration of the viral exposure and the virus entry route [13, 24] . Consistently, a recent communication [14] confirmed that the highest levels of IgG and IgA antibodies against the Spike S1 domain, encompassing the N-terminal half of the protein with the RBD, were associated with severe disease [13, 14] Since severe COVID-19 is associated with a strong release of pro-inflammatory cytokines [8, 25] Table 3 ). ICU patients, whose sera were collected in the acute phase of the disease, showed a sustained production of pro-inflammatory mediators, among which IL-6, IL-17A, IL-12p70, IL-1b, IL-4, IL-5 and IL-13, all associated with the "cytokine storm" observed in very severe COVID-19 patients, were the most abundantly detected (Figure 2A ). On the contrary, even in the early convalescent phase, those cytokines were undetectable in the sera of non-hospitalized It is tempting to speculate that as a consequence of the higher conservation of the N protein compared to the Spike protein across different coronavirus species [23] , antibodies produced against previous common cold coronaviruses (and cross-reacting with the SARS-CoV2 antigens) might still be present in the sera at high levels, and therefore be detectable at the same titers, while the antibodies specific to SARS-CoV-2 decline.

Interestingly, similarly to the antibody titers, the presence of proinflammatory mediators in the sera of convalescent patients also decreased over time and became almost undetectable one month after a negative PCR for viral RNA, a finding that mirrors the successful control of the infection and the consequent switch off of the immune response ( Figure 3F, Supplementary Figure 4) .

Overall, we suggest that the decline in antibody titer and pro-inflammatory cytokines is a common characteristic of SARS-CoV-2 infection. This study has therefore important implications for the use of serological testing for the monitoring of infection outbreaks against re-infection with SARS-CoV-2. Our results indicate that the detection of antibodies with serological assays for epidemiological and monitoring purposes in non-hospitalized seroconverted COVID19+ subjects, who most likely represent the majority of people who encountered the virus, is highly reliable only within a limited window of time after viral clearance.

Human Subjects.

infection (by NF swab), not hospitalized but with manifested COVID-19 symptoms (Supplementary Table 2 ) were monitored for seroconversion by IgM, IgG and IgA serum levels at two time points after viral clearance between April and June 2020. The study has been conducted in accordance with the Standards of Good Clinical Practice, with the ethical principles deriving from the Helsinki Declaration and the current legislation on observational studies. Clearance from the Ethical Committee has been obtained (IEO1271). Additional study populations were ICU hospitalized severe COVID-19 patients (n=24) and (n=58) COVID-19 negative subjects whose sera were collected between April and June 2020. 436 pre-COVID subjects enrolled in IEO studies between 2009 and 2015 were used to calculate the ROC curves for the assays.

The recombinant Spike SARS-CoV2 glycoprotein receptor binding domain (RBD) and the soluble fulllength trimeric ectodomain have been produced in mammalian HEK293F cells as glycosylated proteins by transient transfection with pCAGGS vectors generated in Prof. Krammer's laboratory [11] . The constructs were synthesized using the genomic sequence of the isolated virus, Wuhan-Hi-1 released in January 2020, and contain codons optimized for expression in mammalian cells.

Briefly, HEK293F cells were seeded at a final concentration of 0.5 million/ml in freestyle medium (Thermo Fisher Scientific), incubated at 37 °C, 5% CO2 at 120 rpm O/N in an Eppendorf New Brunswick s41i incubator. The day after HEK293F cells were transfected using 1 µg of DNA per 1x10 6 cells and a DNA:PEI MAX ratio of 1:5 in OptiMEM medium. 4h post-transfection, the medium was supplemented with Peptone Primatone RL (Merck) to a final concentration of 0.6% w/v. Cells were then incubated for 6 days, checking cell viability daily if needed (a mortality higher than 30% is indicative of a toxic protein). 

The ELISA assay to detect Immunoglobulins (Ig) uses fragment of the SARS-CoV2 Spike glycoprotein (S-protein) and the Nucleocapsid (N protein) as antigens based on the protocol published in [11, 19] . After binding of the proteins (RBD and N proteins) to a Nunc Maxisorp ELISA plate, And blocking aspecific bindings with PBS-BSA 3%, patients' sera to be analyzed were applied to the plate to allow for emergency use approval. The assay has been validated with a cohort of n=56 COVID-19 + subjects (severe, moderate and mild disease) and n=463 (subjects collected in pre-COVID era (between 2012 and 2015)). ROC curves have been implemented to determine sensitivity and specificity of the assay (Supplementary Figure 1) Multiplexing analysis of sera cytokines.

Quantification of soluble biomarkers was performed in sera of patients collected immediately after virus clearance (2 consecutive negative NF swabs) and one month post virus clearance using a Luminex Immunoassay (Human Cytokine/Chemokine/GF ProcartaPlex 45plex, Thermo Fisher) with MAP technology according to manufacturer's protocol. Samples were acquired on a Luminex 200SD and analyzed with Xponent software 4.2. The sera of healthy subjects (n=20) collected between April and June 2020 as well as ICU COVID-19+ patients (n=24) were used as control groups.

The categorical variables were described as absolute frequency and percentage. The continuous variables with normal distribution were described as median ± standard deviation (SD), whereas the continuous variables without normal distribution were given as median and range. Normality of continuous variables was checked with D'Agostino-Pearson omnibus normality test. The Mann-Whitney test or Student's t-test for continuous variables, and the Chi-square or Fisher's exact tests for categorical variables, were used to associate clinical variables with the result of SARS-CoV-2 serological test (positive or negative). The p values lower than 0.05, two-tailed, will be considered statistically significant. Prism software was used for all statistical analyses. Cytokines not significantly different between ICU (dark blue symbols) and non-hospitalized (blue symbols) COVID+ patients (C) Chemokines levels in sera of patients (ICU, dark blue, not hospitalized blue symbols, healthy subjects light blue). P < 0.05 (*), P < 0.01 (**) P < 0.001 (***), P < 0.0001 (****) were regarded as statistically significant. ns, not significant (E) Cumulative fold decrease between T1 and T2 antibody titers in ELISA assays against the RBD (squares), the Spike ectodomain (circles) and the N (triangles) SARS-CoV2 viral proteins. (F) Longitudinal variation of serum cytokines and chemokines in non-hospitalized COVID-19+ patients. Statistical significance was calculated using Kruskal-Wallis nonparametric test for multiple comparisons. P < 0.05 (*), P < 0.01 (**) P < 0.001 (***) were regarded as statistically significant. ns, not significant. Figure 2 : Cytokine levels in sera of COVID19+ patients. Cytokines not significantly different between hospitalized (dark blue) and non-hospitalized (blue) COVID+ patients. Statistical significance was calculated using Kruskal-Wallis nonparametric test for multiple comparisons. P < 0.05 (*) were regarded as statistically significant.

Supplementary Figure 3 : (A) Growth factors present in ICU hospitalized (dark blue) but not in non-hospitalized (blue) COVID+ patients (B) Growth factors not significantly different between ICU hospitalized (dark blue) and non-hospitalized (blue) COVID+ patients. Statistical significance was calculated using Kruskal-Wallis nonparametric test for multiple comparisons. P < 0.05 (*), P < 0.01 (**) were regarded as statistically significant.

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