key: cord-0779619-7id8tonh authors: Prévost, Jérémie; Gasser, Romain; Beaudoin-Bussières, Guillaume; Richard, Jonathan; Duerr, Ralf; Laumaea, Annemarie; Anand, Sai Priya; Goyette, Guillaume; Benlarbi, Mehdi; Ding, Shilei; Medjahed, Halima; Lewin, Antoine; Perreault, Josée; Tremblay, Tony; Gendron-Lepage, Gabrielle; Gauthier, Nicolas; Carrier, Marc; Marcoux, Diane; Piché, Alain; Lavoie, Myriam; Benoit, Alexandre; Loungnarath, Vilayvong; Brochu, Gino; Haddad, Elie; Stacey, Hannah D.; Miller, Matthew S.; Desforges, Marc; Talbot, Pierre J.; Gould Maule, Graham T.; Côté, Marceline; Therrien, Christian; Serhir, Bouchra; Bazin, Renée; Roger, Michel; Finzi, Andrés title: Cross-sectional evaluation of humoral responses against SARS-CoV-2 Spike date: 2020-09-30 journal: Cell Rep Med DOI: 10.1016/j.xcrm.2020.100126 sha: af787baece1faaff34abd51f3e64828d60b16410 doc_id: 779619 cord_uid: 7id8tonh The SARS-CoV-2 virus is responsible for the coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoproteins of SARS-CoV-2 mediates viral entry and is the main target for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic and public health interventions. Here we perform a cross-sectional study on 106 different SARS-CoV-2-infected individuals to evaluate humoral responses against SARS-CoV-2 Spike. The vast majority of infected individuals elicits anti-Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding domain (RBD)-specific IgG persist overtime, while the levels of anti-RBD IgM decrease after symptoms resolution. While most of individuals develop neutralizing antibodies within two weeks of infection, the level of neutralizing activity is significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection. The SARS-CoV-2 virus is responsible for the coronavirus disease 2019 simultaneously. Antibody levels targeting the SARS-CoV-2 Spike significantly increased from 112 T1 to T2/T3 and remained relatively stable thereafter. As expected, the levels of antibodies 113 recognizing the full Spike correlated with the presence of both RBD-specific IgG and IgM 114 ( Figure S1 ). We also evaluated potential cross-reactivity against the closely related SARS-CoV 115 Spike. None of the COVID-19 negative samples recognized the SARS-CoV Spike. While the 116 reactivity of COVID-19+ samples to SARS-CoV S was lower than for SARS-CoV-2 S, it 117 followed a similar progression and significantly correlated with their reactivity to SARS-CoV-2 118 full Spike or RBD protein (Figure 2 and S1). This indicates that SARS-CoV-2-elicited antibodies 119 cross-react with human Sarbecoviruses. This was also observed with another Betacoronavirus 120 (OC43) but not with Alphacoronavirus (NL63, 229E) S glycoproteins, suggesting a genus-121 restrictive cross-reactivity ( Figure 2C and S1). Of note, anti-OC43 RBD antibodies did not 122 fluctuate upon SARS-CoV-2 infection ( Figure S2 ). Therefore, this differential cross-reactivity 123 could be explained by the high degree of conservation in the S protein fusion machinery, 124 particularly in the S2 subunit among Betacoronaviruses 17-19 . 125 126 We next measured the capacity of patient samples to neutralize pseudoparticles bearing 127 SARS-CoV-2 S, SARS-CoV S or VSV-G glycoproteins using 293T cells stably expressing 128 ACE2 as target cells (Figure 3 and S3). Neutralizing activity, as measured by the neutralization 129 half-maximum inhibitory dilution (ID 50 ) or the neutralization 80% inhibitory dilution (ID 80 ), was 130 detected in most patients within 2 weeks after the onset of symptoms (T2, T3, T4 and 131 neutralize SARS-CoV-2 S-pseudotyped particles significantly correlated with the presence of 134 RBD-specific IgG/IgM and anti-S antibodies ( Figure S4 ). While the percentage of patients 135 eliciting neutralizing antibodies against SARS-CoV-2 Spike remained relatively stable 2 weeks 136 after disease symptom onset (T2, T3, T4 and Convalescent patients), neutralizing antibody titers 137 significantly decreased after 1 month of infection (T4) or after the complete resolution of 138 symptoms as observed in the convalescent patients ( Figure 3G and 3H). Similarly to RBD-139 specific IgM, levels of RBD-specific IgA were also found to peak at T2 and decrease over time. 140 However, RBD-specific IgM levels displayed a stronger correlation with neutralization activity 141 compared to RBD-specific IgG and IgA, suggesting a more prominent role for IgM, but the 142 decrease in IgA could also contribute to the loss of neutralization activity as recently suggested 143 20 . Cross-reactive neutralizing antibodies against SARS-CoV S protein ( Figure 2B ) were also 144 detected in some SARS-CoV-2-infected individuals, but with significantly lower potency and 145 waned over time. We note that around 40% of convalescent patients did not exhibit any 146 neutralizing activity. This suggests that the production of neutralizing antibodies is not a 147 prerequisite to the resolution of the infection and that other arms of the immune system could be 148 sufficient to control the infection in an important proportion of the population. 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R Foundation 575 for Statistical Computing. Vienna, Austria2013. 576 38. R Studio Team. RStudio: Integrated Development for R RAWGraphs: A Visualisation 579 Platform to Create Open Outputs COVID-19 -T1 **** *** * **** ** secondary antibodies to detect sera binding in ELISA experiments. Alexa Fluor-647-conjugated 391 goat anti-human IgG (H+L) Abs (Invitrogen) were used as secondary antibodies to detect sera 392 binding in flow cytometry experiment. Polyclonal goat anti-ACE2 (R&D systems) and Fluor-conjugated donkey anti-goat IgG Abs (Invitrogen) were used to detect cell-surface 394 expression of human ACE2. 395 396 Recombinant SARS-CoV-2 S RBD proteins (or OC43 S RBD proteins) (2.5 µg/ml), or bovine 398 serum albumin (BSA) (2.5 µg/ml) as a negative control, were prepared in PBS and were 399 adsorbed to plates (MaxiSorp; Nunc) overnight at 4°C. Coated wells were subsequently blocked 400 with blocking buffer (Tris-buffered saline [TBS] containing 0.1% Tween20 and 2% BSA) for 1h 401 at room temperature. Wells were then washed four times with washing buffer (Tris-buffered 402 saline [TBS] containing 0.1% Tween20). CR3022 mAb (50ng/ml) or sera from SARS-CoV-2-403 infected or uninfected donors (1/100; 1/250; 1/500; 1/1000; 1/2000; 1/4000) were diluted in 404 blocking buffer and incubated with the RBD-coated wells for 1h at room temperature. Plates 405 were washed four times with washing buffer followed by incubation with secondary Abs (diluted 406 in blocking buffer) for 1h at room temperature, followed by four washes. HRP enzyme activity 407 was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol 408 reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB942 TriStar 409 luminometer (Berthold Technologies). Signal obtained with BSA was subtracted for each serum 410 and were then normalized to the signal obtained with CR3022 mAb present in each plate.