Raji (Burkitt’s lymphoma/B lymphoblasts), THP-1 (human acute monocytic leukemia cells), and K562 (human chronic myelogenous leukemia cells) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (BD, USA) supplemented with 10% v/v fetal bovine serum (FBS, Biological Industries, Israel) and 100 U/mL penicillin-streptomycin (Thermo Fisher, USA). Vero (African green monkey kidney epithelial cells) and KMB17 (human embryonic lung fibroblast-like cells) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, BD, USA) supplemented with 10% v/v FBS and 100 U/mL penicillin-streptomycin. All cells were obtained from the Conservation Genetics Chinese Academy of Sciences Kunming Cell Bank and maintained at 37°C in an environment with 5% CO₂ before use.

Human sera from a phase II clinical trial (Clinical trials registration number: NCT04412538) of an inactivated SARS-CoV-2 vaccine adjuvant with aluminum and SARS-CoV-2-infected human convalescent serum (HCS) were supplied by Professor Qihan Li from the Institute of Medical Biology, Chinese Academy of Medical Sciences (IMB, CAMS) (39). Specifically, healthy volunteers aged 18-59 years were intramuscularly inoculated twice with the KMS-1 SARS-CoV-2 strain (GenBank accession number MT226610.1) that was double inactivated by formaldehyde plus β-propiolactone and adjuvanted with aluminum hydroxide at medium doses (containing 100 enzyme-linked immunosorbent assay units (EUs) of viral antigen) or high doses (containing 150 EUs of viral antigen). Fourteen or 28 days after boost immunization, immune serum was collected to determine the authentic SARS-CoV-2 neutralization titers. Briefly, diluted serum samples (1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256) were incubated at 37°C for 2 h with a virus at a titer 100 times higher than the 50% cell culture infectious dose (CCID50). The mixture was then added to Vero cells in 96-well plates and incubated at 37°C. After 1 week, the viral cytopathic effect (CPE) was observed and assessed with an inverted microscope (Nikon, Tokyo, Japan). The neutralization titers were defined as the highest dilution at which no CPE was observed, and neutralization titers under 4 were defined as 1 for calculation. Typical serum (N=5) with positive seroconversion, including neutralization titers equal to 4, 8, 32, 128, and 256, was collected randomly for this study.

Specific pathogen-free (SPF) female BALB/c mice at 6 weeks of age (14-17 g) were supplied and maintained by the Central Services of the IMB, CAMS. The animals were randomly divided into 3 groups, and each group consisted of 6 mice (N=6). SARS-CoV-2 S1 proteins expressed by HEK293 cells were purchased from Sino Biological Inc. (China). The purity of S1 was >90% as determined by SDS–PAGE and >95% as determined by size-exclusion chromatography high-performance liquid chromatography. S1 was diluted to 5 μg/mouse/dose in 25 µL of phosphate-buffered saline (PBS, pH 7.40) and mixed with the same volume of aluminum (Thermo Fisher, USA) to induce a typical Th2 response or nucleic acid immunostimulant mixtures that have been proven to induce a typical Th1 response (40). The nucleic acid immunostimulant mixtures contained 20 µg/mouse/dose oligodeoxynucleotide containing CpG motifs (CpG ODN 2395, from In vivoGen, USA) and 25 µg/mouse/dose low-molecular-weight polyinosinic-polycytidylic acid (poly(I:C), from In vivoGen, USA). Fifty microliters of immunogens or PBS (sham group) were administered intramuscularly to the thigh muscle three times at 2-week intervals.

Two weeks after the final immunization, mice were anesthetized by an intraperitoneal injection of tribromoethanol, and blood was collected via cardiac puncture. After overnight clotting at 4°C, serum was collected by centrifugation at 1000 g for 10 min and pooled for further analysis. S1-specific IgG/IgG1/IgG2a titers were detected by ELISA (41, 42), and the IgG1-to-IgG2a titer ratio was calculated to evaluate the Th1-Th2 balance described previously (43, 44). Specifically, HEK293 cells expressing SARS-CoV-2 S1 protein (Sino Biological Inc., China) at 2 µg/mL were coated on 96-well plates overnight at 4°C. The plates were washed with wash buffer (0.05% (v/v) polysorbate 20 in PBS) once and then blocked with 5% (v/v) skim milk dissolved in wash buffer for 1 h at 37°C. The plates were washed four times and incubated with serially diluted mouse sera for 1 h at 37°C. After five washes, the plates were incubated with goat anti-mouse IgG/IgG1/IgG2a HRP-conjugated secondary antibodies (Thermo Fisher Scientific, USA) for 1 h at 37°C. After five washes, 3,3’,5,5’-tetramethylbenzidine (TMB, BD Bioscience, USA) substrate was added, the plates were incubated in the dark at room temperature for 10 min. The reactions were stopped by adding 2 M sulfuric acid, and the absorbance at 450 nm was detected using a microplate reader (Bio-Tek Instruments, Inc., USA). The antibody titers were defined by end-point dilution with a cutoff signal of OD450 = 0.1.

Cells were cultured in 6-well plates until the concentration reached 1×10⁶ cells/mL for protein expression analysis. After three washes with PBS, radioimmunoprecipitation (RIPA) lysis buffer (Sigma, USA) supplemented with 1% protease inhibitor cocktail (MedChemExpress, USA) was added for the extraction of cellular protein. After quantification with a bicinchoninic acid (BCA) protein assay kit (Beyotime, China), 10 μg of total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% milk, antibodies (Abcam, USA) against angiotensin-converting enzyme 2 (anti-ACE2, 1:5 000), FcγR1 (anti-CD64, 1:10 000) and FcγR2 (anti-CD32A+CD32B+CD32C, 1:10 000) were used for assessing the protein expression of cells. A mouse monoclonal antibody against β-actin (Multi Sciences Biotech, China) was used to identify the quality of the protein extracted. Detection was performed using the enhanced chemiluminescence (ECL) reagent (Multi Sciences Biotech, China).

Cells were cultured in 6-well plates at a concentration of 1×10⁶ cells/mL for gene transcription analysis. Total RNA of cells was isolated with TRIzol™ Reagent (Thermo Fisher Scientific, USA) and stored at -80°C until use. According to the manufacturer’s instructions, cDNAs were constructed with a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, China). Briefly, 2 μL of 5×gDNA Eraser Buffer, 1 μL of gDNA Eraser, and 7 μL of RNA dissolved in RNase-free water were mixed to obtain a total volume of 10 μL. After incubation at 42°C for 2 min, the mixture was then added to the reaction solution, which contained 1 μL of PrimeScript RT Enzyme Mix I, 1 μL of RT Primer Mix, 4 μL of 5× PrimeScript Buffer, and 4 μL of RNase-free water. cDNAs were synthesized using the following PCR procedure: 37°C for 15 min, 85°C for 5 s, and maintained at 4°C.

RT–PCR was performed using previously described primer pairs to examine the expression of the genes encoding human ACE2, human FcγR1A, human FcγR2A, human FcγR2B, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5). The PCRs were initialized using a general procedure of 94°C for 5 min, 30 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 45 s, and a final extension step of 72°C for 7 min. The PCR products were verified by 2% agarose gel electrophoresis.

SARS-CoV-2 S pseudotyped virus was used for neutralization assays in biosafety level 2 facilities. This pseudovirus was based on vesicular stomatitis virus (VSV) with the G gene replaced by the firefly luciferase reporter gene and the spike protein from SARS-CoV-2 incorporated as the membrane protein (45). Specifically, the pseudoviruses were diluted to 1.3×10⁴ 50% tissue culture infectious dose (TCID50)/mL with complete DMEM before use. Fifty microliters of diluted SARS-CoV-2 S pseudovirus and 50 μL of immune serum in serial dilutions were coincubated at 37°C in an environment with 5% CO₂ for 60 min. Subsequently, 100 μL of Vero-E6 cells (2×10⁵ cells/mL) was seeded in each mixture for another 24 h at 37°C in an atmosphere with 5% CO₂. Finally, 100 μL of supernatant was discarded before testing. A cell control (CC), in which only cells with culture medium were added, and a virus control (VC), in which only pseudovirus and cells but no serum were added, were established separately. According to the manual protocol, the luciferase activity was assayed using a Britelite Plus Ultra-High Sensitivity Luminescence Reporter Gene Assay System (PerkinElmer, USA) and monitored using an EnSight™ Multimode Plate Reader (PerkinElmer, USA) (45).

The ADE of infection was evaluated in vitro using immune cell lines with different FcR expression patterns. Briefly, 25 μL of serially diluted serum and 25 μL of SARS-CoV-2 S pseudovirus (containing 250 TCID50 pseudoviruses) were incubated at 37°C in an atmosphere with 5% CO₂ for 60 min. Then, 100 μL of cells (2×10⁵ cells/mL) was added to the mixtures for an additional 24 h of incubation. Afterward, the plates were centrifuged for 10 min at 300 ×g, and 50 μL of cell supernatant was discarded before testing. Cell control (CC), virus control (VC), and luciferase activity assays were performed as described above (37).

The data were processed with GraphPad Prism 7.0 (San Diego, CA, USA) and are shown as the means and standard deviations.

Two weeks after the third immunization ( Figure 1A ), vaccines with nucleic acid adjuvants (i.e., 20 μg of CpG ODN 2395 + 25 μg of poly(I:C)/mouse/dose) induced total S1-specific IgG antibody titers that were twofold higher (32,000 compared with 16,000) than those obtained with alum adjuvants ( Figure 1B ) and substantially higher production of S1-specific IgG2a subtype antibody (64,000 versus 500) ( Figure 1C ). Although the S1-specific IgG1/IgG2a ratio in nucleic acid-adjuvanted immunized serum was 0.25, the IgG1/IgG2a ratio in alum-adjuvanted immunized serum was as high as 64, which is a typical Th2-oriented immune response ( Figure 1D ).

Both expressed proteins ( Figure 2A ) and transcribed genes ( Figure 2B ) of ACE2 (the main receptor for SARS-CoV-2 infection) but not any type of FcγR were detected in Vero cells that were used for the proliferation of SARS-CoV-2 for inactivated vaccines, which makes Vero cells suitable for detection of the FcγR-independent enhancement of infection by serum (4, 46).

The protein level of FcγR1 in THP-1 and KMB17 cells was detected ( Figure 2A ). According to a detailed subtype analysis at the gene transcription level ( Figure 2B ), FcγR1A was expressed in THP-1 cells, and the other type of FcγR1, i.e., FcγR1B, was expressed in KMB17 cells, possibly as a pseudogene.

FcγR2 was detected at the protein level in Raji and THP-1 cells ( Figure 2A ). According to a detailed subtype analysis at the transcribed gene level ( Figure 2B ), FcγR2B was expressed in Raji cells, and FcγR2A was expressed in THP-1 cells. Although the gene transcription of FcγR2A was also detected in K562 cells, a Western blot assay indicated that the corresponding protein was not expressed ( Figure 2A ).

For the pseudovirus-based neutralization assay, although no luminescence was detected in the Vero cell control (CC), the luminescence of the virus control (VC) group was as high as 6×10⁵, which implied successful infection of Vero cells by SARS-CoV-2 S pseudotyped virus. Although the serum of PBS-administered mice (Sham in Figure 3A ) showed no influence on pseudovirus infection, serum from S1-immunized mice blocked pseudovirus entry. If a luminescence of 1×10⁵ was taken as the cutoff value (which represents a more than 80% reduction in luminescence compared with that of the VC control), the virus neutralization titer (VNT) for serum from the nucleic acid-adjuvanted S1 subunit vaccine group was approximately 1150, which was higher than that for serum from the alum-adjuvanted S1 subunit vaccine group (VNT=700).

In the in vitro assays of the pseudovirus-based ADE of infection, even luminescence as low as 1×10⁴ (1/10 of that in the pseudovirus-based neutralization assay in Figure 3A , and background values around those detected in the VC control groups for each of the four cells) was taken as the cutoff value, and serum from neither the nucleic acid-adjuvanted S1 subunit vaccine group nor the alum-adjuvanted S1 subunit vaccine group showed ADE of infection in any of the four tested cell lines ( Figures 3B–E ). These cell lines included Raji cells that were confirmed to exhibit FcγR2B expression, which contributes to the enhancement of SARS-CoV-2 infection (37, 46), and THP-1 cells were confirmed to exhibit FcγR2A expression, which contributes to enhancement of MERS-CoV (47) and SARS-CoV-1 (5, 48) infection.

Considering that differences exist between the Fc fragment of mouse IgG and human IgG and that antibodies targeting other parts of the SARS-CoV-2 antigen except for the S1 segment may also show potential to promote infection, human sera after an inactivated SARS-CoV-2 vaccine adjuvant with alum were also tested in the abovementioned cell lines.

For the pseudovirus-based neutralization assay ( Figure 4A ), none of the five sera from the SARS-CoV-2 inactivated vaccine groups, nor the six HCSs efficiently blocked the entrance of pseudovirus at a dilution of 1:1000, as reflected by corresponding luminescence reads that were higher than the cutoff value of 1×10⁵ established in the previous section ( Figure 3A ).

In the in vitro assay of the pseudovirus-based ADE of infection, none of the five sera from the SARS-CoV-2 inactivated vaccine groups, nor the six HCSs showed ADE of infection (shown as luminescence lower than 1×10⁴, the cutoff value for ADE discussed in the above subsection) in all four cell lines with serum dilutions from 1:1000 to 1:4000 ( Figures 4B–E ), as previously reported for the dilutions to detect ADE of infection with the SARS-CoV-1 subunit and inactivated vaccines adjuvanted with alum (5).