key: cord-1046506-s1flhjhz
authors: Chen, Cheng-Pin; Huang, Kuan-Ying A.; Shih, Shin-Ru; Lin, Yi-Chun; Cheng, Chien-Yu; Huang, Yhu-Chering; Lin, Tzou-Yien; Cheng, Shu-Hsing
title: Anti-spike antibody response to natural infection with SARS-CoV-2 and its activity against emerging variants
date: 2022-03-07
journal: bioRxiv
DOI: 10.1101/2022.03.07.481737
sha: b5ba3a27550e56cedeb5a0f9259a9cca84fd8e1b
doc_id: 1046506
cord_uid: s1flhjhz

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has substantially impacted human health globally. Spike-specific antibody response plays a major role in protection against SARS-CoV-2. Here, we demonstrated that acute SARS-CoV-2 infection elicits rapid and robust spike-binding and ACE2-blocking antibody responses, which wane approximately 11 months after infection. Serological responses were found to be correlated with the frequency of spike-specific memory B cell responses to natural infections. Further, significantly higher spike-binding, ACE2-blocking, and memory B cell responses were detected in patients with fever and pneumonia. Spike-specific antibody responses were found to be greatly affected by spike mutations in emerging variants, especially the Beta and Omicron variants. These results warrant continued surveillance of spike-specific antibody responses to natural infections and highlight the importance of maintaining functional anti-spike antibodies through immunization. Importance As spike protein-specific antibody responses play a major role in protection against SARS-CoV-2, we examined the spike-binding and ACE2-blocking antibody responses in SARS-CoV-2 infection at different time points. We found robust responses following acute infection, which waned approximately 11 months after infection. Further, the serological responses were correlated with the frequency of spike-specific memory B cell responses to natural infections. Patients with fever and pneumonia showed significantly stronger spike-binding, ACE2-blocking antibody, and memory B cell responses. Moreover, the spike-specific antibody responses were substantially affected by the emerging variants, especially the Beta and Omicron variants. These results warrant continued surveillance of spike-specific antibody responses to natural infections and highlight the importance of maintaining functional anti-spike antibodies through immunization.

Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll lymphocyte 152 separation medium. Resuspended PBMCs were cultured in complete medium 153 containing pokeweed mitogen (PWM), Staphylococcus aureus Cowan I (SAC), and 154

CpG at 37 °C for 5 days. Cultured PBMCs were collected, washed, and resuspended for 155 the ELISpot assay. The ELISpot assay was used to detect spike-binding IgG-, IgM-, and 156

IgA-secreting cells. Briefly, a 96-well Millipore plate was coated with SARS-CoV-2 157 spike or anti-human IgG at 4 °C overnight. After washing, plates were blocked with 2% 158 dry skim milk at 37 °C for 2 h. After washing, resuspended cultured cells were added to 159 the wells and incubated at 37 °C for 16 h. After washing, the wells were incubated with 160 anti-human IgG, IgM, or IgA, secondary antibodies conjugated with alkaline 161 phosphatase at room temperature for 2 h. After washing, the wells were developed using 162 an alkaline phosphatase substrate kit at room temperature for 2-5 minutes. The spot-163 forming cells were counted using an automated ELISpot plate reader [21] . 164

Statistical analyses were performed using GraphPad Prism or Excel. The unpaired t-test 166 was used to determine the differences between two independent groups. One-way 167 analysis of variance was used to determine the differences among three or more 168 independent groups, and Tukey's test was used for post-hoc analysis. The chi-squared 169 test was used to analyze the relationships between categorical variables. Linear 170 regression was used to evaluate the correlation between variables. Statistical 171 significance was set at p < 0.05.

In total, 25 patients were enrolled. Their mean age was 38.68 (SD, 13.4) years. The 175 male-to-female ratio was 12:13, and most patients were from other countries/regions. 176

Among the subjects, five developed pneumonia, as confirmed by chest radiography or 177 computed tomography. The mean age of patients with and without pneumonia was 47.4 178 and 36.5 years respectively (p = 0.105). Fever (60%) and cough (60%) were the 179 frequently reported symptoms. Patients with pneumonia had higher level of ALT (46.8 180 U/L vs 26.6 U/L, p = 0.018) than those without pneumonia. Patients with and without 181 pneumonia did not have remarkable differences in white blood cell count, C-reactive 182 protein, creatinine, and lactate dehydrogenase, and received similar therapeutic 183 regimens (Table 1) . 184

In total of 21 (84%) patients showed a detectable spike-binding antibody response in the 186 serum at day 21 ± 8 (6-33) after the onset of illness. Patients negative for the anti-spike 187 antibody response (n = 4) presented mild illness, and three of them had no fever ( Figure  188 1A, Supplemental Table 1) . 189

The spike-binding antibody response was detected as early as the first week after onset, 190 continued to rise between the second and third weeks, and reached a plateau at three 191 weeks after onset during the convalescence period ( Figure 1B, 1C) . In most cases, the 192 RBD-binding antibody response was detectable at the same time point, whereas the 193 spike-binding antibodies were elicited (Figure 1 ). 194

We also examined the level of functional anti-spike antibodies in the serum using hemagglutination inhibition and ACE2-blocking assays. Nineteen (90%) patients with 196 positive spike-binding antibody responses showed detectable hemagglutination-197 inhibition titers, and 15 (71%) patients showed ACE2-RBD blocking serological 198 activities ( Figure 1A) . 199

The magnitude of the spike-binding antibody response was significantly correlated with 1.2% of peripheral B cells, and were detected on day 19 ± 7 (6-33) after the onset of 211 illness. The memory B cell response was detectable within two weeks (n = 5, 0.6 ± 212 1.0%, day 6-13) and increased three weeks after the onset of illness (n = 15, 1.5 ± 1.2%, 213 day 14-33), but the difference was not statistically significant (p = 0.12, unpaired two-214 tailed t-test). Three patients (60%) had no detectable memory B cell response in the first 215 two weeks after onset; in contrast, one patient (7%) failed to develop a memory B cell 216 response even at three weeks after onset. Although SARS-CoV-2 infection elicited a 217 spike-specific B cell response, IgM memory B cells were predominantly induced, 218 followed by IgG and IgA B cells ( Figure 2B ). to 16-fold reduction in titer was noted in the remaining patients. An average of 2.6 ± 1.0 245

(1.0-3.5) fold reduction in the spike-binding antibody response was also detected, 246 which was in accordance with the decline in the functional serological titer. 247

The neutralization activity of convalescence and follow-up sera was then tested against 

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We acknowledge Alain Townsend, Tiong Kit Tan, Pramila Rijal and Lisa Schimanski 340 for the support of VHH(IH4)-RBD. We also thank the patients and the care team of the ** ** *** ns