key: cord-0711011-p3g84z3p
authors: Ai, Jingwen; Zhang, Haocheng; Zhang, Qiran; Zhang, Yi; Lin, Ke; Fu, Zhangfan; Song, Jieyu; Zhao, Yuanhan; Fan, Mingxiang; Wang, Hongyu; Qiu, Chao; Zhou, Yang; Zhang, Wenhong
title: Recombinant protein subunit vaccine booster following two-dose inactivated vaccines dramatically enhanced anti-RBD responses and neutralizing titers against SARS-CoV-2 and Variants of Concern
date: 2021-11-23
journal: Cell Res
DOI: 10.1038/s41422-021-00590-x
sha: 6169e50b553bdcb806cd08741bea4dafdfc2c0a7
doc_id: 711011
cord_uid: p3g84z3p

nan

Grade 1 0 (0.0%) 0 (0.0%) 1 (1.4%) Anorexia 0 (0.0%) 0 (0.0%) 1 (1.4%) Grade 1 0 (0.0%) 0 (0.0%) 1 (1.4%) Arthralgia 0 (0.0%) 0 (0.0%) 0 (0.0%) Dyspnea 0 (0.0%) 0 (0.0%) 0 (0.0%) Nausea 0 (0.0%) 0 (0.0%) 0 (0.0%) Pharyngalgia 0 (0.0%) 0 (0.0%) 0 (0.0%) Syncope 0 (0.0%) 0 (0.0%) 0 (0.0%) Vertigo 0 (0.0%) 0 (0.0%) 0 (0.0%) Vomiting 0 (0.0%) 0 (0.0%) 0 (0.0%)

Any (%) 0 (0.0%) 0 (0.0%) 0 (0.0%) Grade 1 0 (0.0%) 0 (0.0%) 0 (0.0%)

Any (%) 5 (7.0%) 0 (0.0%) 0 (0.0%) Grade 1 5 (7.0%) 0 (0.0%) 0 (0.0%) 

The study was conducted complying the following study protocol. Written informed consent was obtained before the enrollment. The study protocol and informed consent form were approved by the Ethics Committee of Huashan Hospital.

Inclusion criteria:

1. Healthy adults, or adults with pre-existing but stable medical conditions (Participants didn't require significant change in therapy or hospitalization within 3 months before enrollment).

whole-virion vaccines (CoronaVac or BBIBP-CorV) within 4 to 8 months.

3. Participants that are willingly to comply with the study procedures and provide written informed consent.

Exclusion criteria:

1. SARS-CoV-2 infection confirmed by positive reverse transcription-polymerase-chainreaction (RT-PCR) assay. to the day of screening.

The primary endpoint was the increased geometric mean titer (GMT) of pseudovirus neutralizing antibodies against SARS-CoV-2 and VOCs on day 14 after the boosting dose.

The secondary outcomes included increased anti-RBD responses and T cell-mediated responses on day 14 and day 28 after the boosting dose, as well as the occurrence of adverse reactions within 28 days after the boosting dose.

Solicited systemic and local adverse reactions were recorded by the participants on designed electronic questionnaires. Questionnaires were distributed and collected by trained site personnel at day 3, day 14 and day 28 after the boosting dose. According to the manufactory brochures (www.perkinelmer.com), we used superparamagnetic microparticles together with direct chemiluminescence technology to detect antibody in plasma samples. Plasma was serial diluted before detection, 50μl diluted sample was added to sample wells, and then mixed with 50ul SARS-CoV-2 receptor binding domain (RBD) protein labeled with acridinium ester. Signals were captured using PerkinElmer SuperFlex automatic chemiluminescence immunoassay analyzer.

To measure the neutralizing titer, the signals were converted to sVNT titer using a reference standard curve plotted with kit-suppled reagents. The sVNT titer was determined by the reciprocal of the last dilution that resulted in >50% reduction of chemiluminescence signal.

To measure the anti-RBD antibody, s sample was added to a sample well, and then To measure the anti-RBD IgG, a specimen was added to a specimen well, and then bounded with the magnetic particles coated with SARS-CoV-2 antigen. After washing, acridinium ester labeled anti-human IgG antibody was added to form an immunocomplex. 

Blood samples were taken from participants for serology tests at days 0 and 14.

Pseudovirus incorporating with spike protein from either SARS-CoV-2, variants (Alpha, Beta, Gamma and Delta) were constructed using a procedure described by Nie et al 1 pseudovirus was determined using a single-use aliquot from the pseudovirus bank to avoid inconsistencies resulted from repeated freezing-thawing cycles.

The 96-well flat-bottom culture plates were used to titration the pseudovirus. A 2-fold initial dilution with six replicates was made, followed by serial 3-fold dilutions, and the last column was used as the cells control without pseudovirus. Subsequently, cells were adjusted to 2x10 5 cells/mL with DMEM complete medium, and 100μl of cell suspension was added to each well. After 2-min incubation in darkness at room temperature, the cell completed lysis, and 150μl of lysate was transferred to 96-well chemiluminescence detection plates (PerkinElmer, Ensight) for detection. Positive was determined to be ten-fold higher than the negative (cells only) in terms of relative luminescence unit (RLU) values. TCID50 was calculated using the Reed-Muench method.

The virus neutralization assay was conducted as follows: 100 μl serial dilutions of human sera or monoclonal antibody preparations were added into 96-well plates 1 . After that, 50 μl pseudoviruses with concentration of 1300 TCID50/mL were added into the plates, followed by incubation at 37°C for 1 hour. Afterwards, HuH-7 cells were added into the plates (2×104 cells/100 μl cells per well), followed by incubation at 37°C in a humidified atmosphere with 5% CO2. Chemiluminescence detection was performed after 24 hours incubation. The

Reed-Muench method was used to calculate the virus neutralization titer. The results are based on 3-5 replicates unless specified. In order to validate the test operation process, the Coefficient of Variance (CV) control of replicates is set within 30% of six wells, so is the CV for the duplicate sample wells.

ELISpot assays were performed to evaluate SARS-CoV-2 specific T-cell response using

Human IFN-gamma ELISpot kit (Fosun Pharma, Shanghai, China). The peripheral blood mononuclear cells (PBMC) were stimulated by S1, S2 and N peptide pools for 20 hours at 37℃ with 5% CO2. Phytohemagglutinin was added as a positive control, and cells cultured without stimulations were used as a negative control. After incubation, detection antibody was added according to the manufactures' s instruction. We counted IFN-γ producing spots by ELISpot Reader (version 7.0). Then the IFN-γ spot-forming units (SFU) per million PBMC was calculated.

We present summary statistics for individuals as median with inter-quartile ranges (IQRs), or geometric mean with 95% confidence intervals (CIs were used for statistical analysis. 

Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2