key: cord-1039739-7c5ug1u6 authors: Martin-Vicente, M.; Almansa, R.; Martinez, I.; Tedim, A. P.; Bustamante, E.; Tamayo, L.; Aldecoa, C.; Gomez, J. M.; Renedo, G.; Berezo, J. A.; Cedeno, J. A.; Mamolar, N.; Garcia Olivares, P.; Herran, R.; Cicuendez, R.; Enriquez, P.; Ortega, A.; Jorge, N.; de la Fuente, A.; Bustamante-Munguira, J.; Munoz-Gomez, M. J.; Gonzalez-Rivera, M.; Puertas, C.; Mas, V.; Vazquez, M.; Perez-Garcia, F.; Rico-Feijoo, J.; Martin, S.; Motos, A.; Fernandez-Barat, L.; Eiros, J. M.; Dominguez-Gil, M.; Ferrer, R.; Barbe, F.; Kelvin, D.; Bermejo-Martin, J. F.; Resino, S.; Torres, A. title: Absent or insufficient anti-SARS-CoV-2 S antibodies at ICU admission are associated to higher viral loads in plasma, antigenemia and mortality in COVID-19 patients date: 2021-03-08 journal: nan DOI: 10.1101/2021.03.08.21253121 sha: 3b640372d24859cdccef09eb0468d6dfee3f7b9e doc_id: 1039739 cord_uid: 7c5ug1u6 Purpose: to evaluate the association between anti-SARS-CoV-2 S IgM and IgG antibodies with viral RNA load in plasma, the frequency of antigenemia and with the risk of mortality in critically ill patients with COVID-19. Methods: anti-SARS-CoV-2 S antibodies levels, viral RNA load and antigenemia were profiled in plasma of 92 adult patients in the first 24 hours following ICU admission. The impact of these variables on 30-day mortality was assessed by using Kaplan-Meier curves and multivariate Cox regression analysis. Results: non survivors showed more frequently absence of anti-SARS-CoV-2 S IgG and IgM antibodies than survivors (26.3% vs 5.6% for IgM and 18.4% vs 5.6% for IgG), and a higher frequency of antigenemia (47.4% vs 22.2%) (p <0.05). Non survivors showed lower concentrations of anti-S IgG and IgM and higher viral RNA loads in plasma, which were associated to increased 30-day mortality and decreased survival mean time. [Adjusted HR (CI95%), p]: [S IgM (AUC [≥]60): 0.48 (0.24; 0.97), 0.040]; [S IgG (AUC [≥]237): 0.47 (0.23; 0.97), 0.042]; [Antigenemia (+): 2.45 (1.27; 4.71), 0.007]; [N1 viral load ([≥] 2.156 copies/mL): 2.21 (1.11; 4.39),0.024]; [N2 viral load ([≥] 3.035 copies/mL): 2.32 (1.16; 4.63), 0.017]. Frequency of antigenemia was >2.5-fold higher in patients with absence of antibodies. Levels of anti-SARS-CoV-2 S antibodies correlated inversely with viral RNA load. Conclusion: absence / insufficient levels of anti-SARS-CoV-2 S antibodies following ICU admission is associated to poor viral control, evidenced by increased viral RNA loads in plasma, higher frequency of antigenemia, and also to increased 30-day mortality. Take-home message: absent or low levels of antibodies against the S protein of SARS-CoV-2 at ICU admission is associated to an increased risk of mortality, higher frequency of antigenemia and higher viral RNA loads in plasma. Profiling anti-SARS-CoV-2 s antibodies at ICU admission could help to predict outcome and to better identify those patients potentially deserving replacement treatment with monoclonal or polyclonal antibodies. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Anti-SARS-CoV-2 S antibodies bind to the viral spike protein, inhibiting virus attachment to cell surface receptors [1] . Therefore, during infection with SARS-CoV-2, the development of anti-S antibodies could reduce viral replication by interfering with virus entry into a cell. From the clinical point of view, recent evidence suggests that anti-SARS-CoV-2 S antibodies could play a role in protecting against severe disease in patients with COVID-19 [2] . Nonetheless, the impact of these antibodies on patients' survival with COVID-19 admitted to the ICU has not been sufficiently addressed to the present moment. Recent works from our group and others have evidenced the importance of SARS-CoV-2 RNAemia [3] [4] [5] and antigenemia as markers of severity in COVID-19 [6] . The presence of viral material in plasma could represent a surrogated marker of poor viral control by patient`s immune response. Whether anti-SARS-CoV-2 S antibodies could have any influence on the dissemination of viral genomic material or viral proteins at the systemic level has yet to be properly studied. In this work, we profiled levels of anti-SARS-CoV-2 S IgM and IgG antibodies in plasma of 92 patients with COVID-19 in the first 24 hours following admission to the ICU, and evaluated their association with mortality. In parallel, we quantified viral RNA load in plasma and tested the presence or absence of antigenemia in these patients, correlating them with the levels of antibodies and outcome. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 8, 2021. ; https://doi.org/10.1101/2021.03.08.21253121 doi: medRxiv preprint Study design: 92 critically ill adult patients with a positive nasopharyngeal swab polymerase chain reaction (PCR) test for SARS-CoV-2 performed at participating hospitals were recruited during the first pandemic wave in Spain from March 16 th to April 15 th 2020. Blood samples: Plasma from blood collected in EDTA tubes samples was obtained in the first 24 hours following admission to the ICU, following proper centrifugation. Samples were stored at -80ºC until quantification of antibodies, viral RNA load and antigenemia evaluation. Detection and quantification of SARS-CoV-2 RNA in plasma: RNA was extracted from 100 µl of plasma using an automated system, eMAG® from bioMérieux® (Marcy l'Etoile, France). Detection and quantification of SARS-CoV-2 RNA was performed in five µl of the eluted solution using the Bio-Rad SARS-CoV-2 ddPCR kit according to manufacturer's specifications on a QX-200 droplet digital PCR platform from the same provider. This PCR targets the N1 and N2 regions of the viral nucleoprotein gene and also the human ribonuclease (RNase) P gene using the primers and probes sets detailed in the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel [7] . Samples were considered positive for SARS-CoV-2 when N1 and/or N2 presented values ≥ 0.1 copies/µL in a given reaction. RNase P gene was considered positive when it presented values ≥ 0.2 copies/µL, following manufacturer`s indications. The test was only considered valid when RNase P gene was positive. Final results were given in copies of cDNA / mL of plasma. Immunoassay for antibody quantification: a specific immunoassay was developed to quantify anti-SARS-CoV-2 S IgG and IgM antibodies in plasma. The plasmid pαH coding for the S protein ectodomain (residues 1-1208) of the SARS-CoV-2 2019-nCOV (GenBank: MN908947) was kindly provided by Dr. Jason McLellan (the University of Texas at Austin-USA) [8] . Mutagenesis was carried out to obtain a HexaPro construct that allowed a high-. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 8, 2021. ; https://doi.org/10.1101/2021.03.08.21253121 doi: medRxiv preprint yield production of a stabilized prefusion spike protein [9] . The following substitutions were included at the ectodomain: glycine at residue 614 (D614G), a "GSAS" substitution at the furin cleavage site (residues 682-685), and proline at residues 817, 892, 899, 942, 986, and 987. For trimerization and purification, the C-terminal end of the S protein ectodomain was fused to the T4 fibritin trimerization motif (foldon), an HRV3C protease cleavage site, and an 8XHisTag. The expression vector coding for the SARS-CoV-2 S protein ectodomain was used to transiently transfect FreeStyle 293F cells (Thermo Fisher, Waltham, MA, USA) using polyethylenimine. The S protein domain was purified from filtered cell supernatants using Ni-NTA resins (Sigma Aldrich, San Luis, MO, USA) and subjected to an additional purification step by size-exclusion chromatography using a Superose 6 10/300 column (GE Healthcare, Chicago, IL, USA). Antibody titration: Antibody titers against the S protein were determined by incubating serial dilutions of serum samples (starting at a 1:50 dilution) with the purified S protein ectodomain. Ninety-six well plates were coated with 200 ng per well of the S protein ectodomain. The following day, serum samples were added, and the binding to the S protein was determined by successive incubations with a secondary peroxidase-conjugated antihuman IgG or IgM (Jackson Immunoresearch, West Grove, PA, USA) antibody and the OPD substrate (Sigma Aldrich, San Luis, MO, USA). The area under the curve (AUC) was calculated by using GraphPad Prism 8.0 (GraphPad Sofware, Inc., San Diego, CA, USA) and the following parameters: Baseline Y=0.1; ignore peaks that are less than 10% of the distance from minimum to maximum Y; all peaks must go above the baseline. The AUC is expressed as X units times the Y units. The presence/absence of N antigen of SARS-CoV-2 in plasma was evaluated by using the Panbio™ COVID-19 Ag Rapid Test Device from Abbott (Chicago, IL, USA). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. For descriptive analysis of patient characteristics, the differences between independent groups were assessed using the Chi-square test or Fisher's Exact Test for categorical variables. Differences for continuous variables were evaluated by using the Mann-Whitney U test. The ability of the factor variables to differentiate survivors from non-survivors was evaluated using the receiver-operating characteristic (ROC) curve. The cut-off of those factor variables for 30-day mortality prediction was obtained by calculating the optimal operating point (OOP) in the ROC, namely, the point on the ROC that had the minimum distance to the upper left corner calculated by Pythagorean theorem. For association analysis, survival analysis was carried out to evaluate 30-day mortality. According to presence/absence and OOP for the ROC curve, these factor variables were stratified into categorical variables. Kaplan-Meier product-limit method was used to calculate survival probabilities and the log-rank test to compare groups. We also used Cox proportional-hazards models to estimate the risk of dying, adjusted by the significant covariates at baseline (see Table 1 ). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 8, 2021. ; https://doi.org/10.1101/2021.03.08.21253121 doi: medRxiv preprint Baseline characteristics of the patients according to 30-day mortality are shown in Table 1 . Patients who died were older than those who survived (p<0.001). Besides, patients who died had more frequently arterial hypertension (p=0.012) and type-2 diabetes (p=0.031), higher values of glucose (p=0.027) and creatinine (p=0.005), lower values of platelets (p<0.001) and monocytes (p<0.001), and higher APACHE (p<0.001) and SOFA (p=0.013) scores. Eight and thirteen patients had no detectable levels in the plasma of anti-SARS-CoV-2 S IgG or anti-SARS-CoV-2 S IgM respectively. Patients who died were more frequently treated with beta interferon (p=0.044) and had a shorter hospital stay (p<0.001) than those who survived the first 30 days in ICU. CoV-2 S IgM / N1 (copies/mL) (-0.34, < 0.001); anti-SARS-CoV-2 S IgM / N2 (copies/mL) (-0.37, < 0.001) (Figure 3 ). Our study demonstrates that those critically ill COVID-19 patients with absent or insufficient levels of specific IgM or IgG antibodies against the S protein of SARS-CoV-2 following ICU admission show an increased risk of mortality. Li et al found that the production of antibodies is delayed in severe COVID-19 patients as compared to non-severe ones [10] , although the former seem to exhibit higher antibody concentrations than patients with milder forms of the disease [10] [11] . The impact of antibody levels on mortality risk in COVID-19 is controversial. In the study from Röltgen K et al, antibody responses in acute illness were not related with patients` outcomes [2] . In contrast, Hashem AM et al reported significantly higher levels of anti-S1 and -N IgG and IgM antibodies in patients with fatal outcomes [11] . Nonetheless, these studies include a mixture of mild, moderate and severe patients, with only a limited representation of critically ill patients. Previous work coming from our group evidence that the biological response of critically ill patients to SARS-CoV-2 infection differs from that of patients with milder forms of the disease [3] . Consequently, to study the association between levels of antibodies and mortality, it is important to analyze a homogenous population, as we do here, by considering exclusively critically ill COVID-19 patients. Results from Li K et al seem to confirm this notion. By studying only severe/critically ill COVID-19 patients, they found similar results to ours: patients nonsurviving to the disease had significantly lower levels of both IgM and IgG compared to those who survived [10] . Nonetheless, in contrast to our work, the study of Li K et al lacks a multivariate analysis to confirm the association between antibody levels and mortality risk, which constitutes a major strength of our study. In turn, Asif S et al found that at both early and late timepoints following ICU admission, plasma concentrations of IgA, IgG and IgM . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 8, 2021. ; https://doi.org/10.1101/2021.03.08.21253121 doi: medRxiv preprint antibodies tend to be higher in COVID-19 patients who survived compared to those who had died at 30 days, but their study involved only 19 critically ill patients [12] . Our findings suggest that the potential protective role of anti-SARS-CoV-2 S antibodies in critically patients with COVID-19 could be related to an improved control of viral replication and of dissemination of viral material at the systemic level. Patients with absent /low levels of these antibodies show higher viral RNA loads in plasma and present antigenemia more frequently, which translates into an increased risk of mortality. Although we could not determine if the presence of lower antibody levels also correlated with the presence of live virus in plasma, systemic spread of viral RNA or viral antigens can drive unspecific stimulation of the innate immune response [13] , which could contribute to induce immunopathological events. Results from Li et al also support the role of antibodies in controlling SARS-CoV-2 replication, since they found higher frequencies of anti-S receptorbinding domain (RBD)-specific IgG levels in those recovered patients who were SARS CoV-2 RNA negative than those who were RNA positive in respiratory samples [10] . In agreement with our results, Röltgen K et al found that increases in plasma anti-SARS-CoV-2 S antibodies correlated with decreases in viral RNAemia along the course of COVID-19 [2] . In a small study with 39 patients (critically and non-critically ill), Ogata AF et al found a correlation between high concentrations of S1 antigen in plasma and ICU admission [6] . For viral-antigen positive patients, full antigen clearance in plasma was observed 5+1 days after seroconversion [6] . Recent findings in a mixed cohort also of ICU and non-ICU patients from Hingrat QL et al also suggest that clearance of antigenemia seems to be linked with the apparition of specific anti-SARS-CoV-2 antibodies [14] . As far as we know, our work is pioneer in evidencing the direct association between low antibody titers and the presence of antigenemia, and also in demonstrating the impact of antigenemia in the mortality risk of those COVID-19 patients admitted to the ICU. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 8, 2021. ; https://doi.org/10.1101/2021.03.08.21253121 doi: medRxiv preprint In conclusion, a higher concentration of anti-SARS-CoV-2 S antibodies following ICU admission is associated with improved survival, lower antigenemia rates and lower viral RNA loads in plasma. Whether anti-SARS-CoV-2 S antibodies mediate a direct protective effect against the virus and/or reflect a broader immunological response also involving a more efficient cellular response remains to be elucidated. Further works shedding light on this regard will help to clarify the potential role of polyclonal or monoclonal antibodies against the SARS-CoV-2 S protein as treatment of critically ill COVID-19 patients [15] [16] [17] . Stratifying patients by levels of antibodies at ICU admission could help to optimize patients` inclusion criteria, by selecting those with absence or low levels of anti-SARS-CoV-2 S IgM and IgG antibodies as those who could potentially benefit the most from these treatments. In addition, quantifying levels of anti-SARS-CoV-2 S antibodies at ICU admission could help to identify those individuals at higher risk of dying from COVID-19. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 8, 2021. ; https://doi.org/10.1101/2021.03.08.21253121 doi: medRxiv preprint Ethics approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Committee for Ethical Research of the coordinating institution, "Comite de Etica de la Investigacion con Medicamentos del Area de Salud de Salamanca", code PI 2020 03 452. Consent to participate: Informed consent was obtained orally when clinically possible. In the remaining cases, the informed consent waiver was authorized by the Ethics committee. Acknowledgements: we thank the IBSAL and CIBER administrative support to run this study. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 8, 2021 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 8, 2021. ; https://doi.org/10.1101/2021.03.08.21253121 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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