Human subjects

We conducted a cross-sectional study to investigate the effect of vaccination and breakthrough infection on the cross-variant neutralization capacity of human sera after vaccination or infection. We enrolled SARS-CoV-2 naïve individuals with two or three doses of BBIBP-CorV and two doses of BBIBP-CorV boosted by ZF2001, as well as previously vaccinated individuals with Delta or BA.1 breakthrough infection. Vaccine sera of 36 (22 females/14 males with a median age of 44.5 years; interquartile range [IQR], 36.3–49.8), 36 (19 females/17 males with a median age of 42.0 years; IQR, 31.5–52.8), and 31 (20 females/11 males with a median age of 45.0 years; IQR, 39.0–50.0) individuals who had two doses of BBIBP-CorV were collected a median of 20.5 (one month), 91.0 (three months), and 221.0 (seven months) days after the second dose, respectively. Vaccine sera of 25 (12 females/13 males with a median age of 46.0 years; IQR, 38.5–52.0) and 30 (19 females/11 males with a median age of 39.0 years; IQR, 34.3–47.3) individuals who were boosted with the third dose of BBIBP-CorV or ZF2001 were collected a median of 21.0 and 28.0 days after the third dose vaccination, respectively.

Sera of 30 individuals (15 females/15 males with a median age of 39.0 years; IQR, 34.0–48.8) with Delta breakthrough infections were collected when they were discharged from the Fifth Hospital of Shijiazhuang, Hebei Medical University (Shijiazhuang, China), with a median of 41.0 days post-symptom onset or PCR positive result. Sera of 26 individuals (7 females/19 males with a median age of 32.5 years; IQR, 16.0–36.8) with BA.1 breakthrough infections were collected when they were discharged from the hospital, with a median of 15.5 days post-symptom onset or PCR positive result. Sera were separated by centrifugation at 2000 rpm for 10 min, aliquoted into three cryovials, and cryopreserved at − 80 °C until use. Sera were heat inactivated at 56 °C for 60 min prior to use in neutralization assays. Serum-neutralizing capability was characterized using pseudovirus neutralization assays. The details of the demographic information (e.g., age and sex) and sample collection time points of different cohorts are summarized in Additional file 1: Tables S1 and S2.

Breakthrough infection was defined as fully vaccinated individuals being diagnosed with SARS-CoV-2 infection [23, 24]. Full vaccination was defined as when the second shot of the BBIBP-CorV vaccination was administered at least 14 days before symptom onset or a positive PCR test for SARS-CoV-2 [23, 24]. All individuals with breakthrough infection had sequence-confirmed Delta and BA.1 infection or PCR-confirmed symptomatic disease occurring while in isolation and direct contact with Delta or BA.1 sequence-confirmed cases.

Cell lines

Human embryonic kidney 293T (HEK-293T, CRL-3216, ATCC) cells were cultured at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco) and supplemented with 1% penicillin–streptomycin (Gibco). Cells were disrupted at the confluence with 0.25% trypsin in 1 mM EDTA (Solarbio) every 48–72 h. HEK-293T cells over-expressing human angiotensin-converting enzyme 2 (293T-ACE2) and HeLa-ACE2 cells kindly provided by Dr. Lin-Qi Zhang at Tsinghua University were cultured under the same conditions.

Monoclonal antibody

Bebtelovimab (LY-CoV1404) was purchased from AtaGenix (Wuhan, China).

Spike plasmid pseudovirus production

Codon-optimized cDNA encoding the SARS-CoV-2 S glycoprotein of D614G, Delta, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/5, or BA.3 was synthesized by GenScript and cloned into pCDNA3.1. Compared to the Wuhan-Hu-1 strain S protein amino acid sequence, the following changes were included in the respective expression plasmids: D614G (D614G), Delta (T19R, G142D, Δ156–157, R158G, L452R, T478K, D614G, P681R and D950N), BA.1 (A67V, Δ69–70, T95I, G142D, Δ143-145, Δ211, L212I, 214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F), BA.1.1 (A67V, Δ69–70, T95I, G142D, Δ143-145, Δ211, L212I, 214EPE, G339D, R346K, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F), BA.2 (T19I, Δ24–26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K), BA.2.12.1 (BA.2+L452Q+S704L), BA.4/5 (T19I, Δ24–26, A27S, Δ69–70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K), and BA.3 (A67V, Δ69–70, T95I, G142D, Δ143–145, Δ211, L212I, G339D, S371F, S373P, S375F, D405N, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K). All plasmid spike sequences were verified by Sanger sequencing.

Pseudovirus particles were generated by co-transfecting HEK-293T cells (ATCC) with human immunodeficiency virus backbones expressing firefly luciferase (pNL4-3-R-E-luciferase) and the pcDNA3.1 vector encoding either D614G or mutated S protein (Delta and Omicron subvariants) plasmids. The medium was replaced with fresh medium at 24 h, and the supernatants were harvested at 48 h post-transfection and clarified by centrifugation at 300×g for 10 min before being aliquoted and stored at − 80 °C until use.

Pseudovirus neutralization assay

A SARS-CoV-2 pseudovirus neutralization assay (pVNT) was performed as described [25], with the target cell line HeLa over-expressing hACE2 orthologs. All viruses were first titrated to normalize the viral input between assays. Duplicate 3-fold 8-point serial dilutions of heat-inactivated sera (starting at 1:30) or LY-CoV1404 (1.0 μg ml−1) were incubated with 500–1000 TCID50 of SARS-CoV-2 pseudotyped virus for 1 h at 37 °C and 5% CO2. Subsequently, 1 × 104 HeLa-ACE2 cells per well were added and incubated at 37 °C and 5% CO2 for 48 h. Afterward, the supernatant was removed, and the cells were lysed using passive lysis buffer (Vazyme) for 3 min at room temperature. The lysates were transferred to an opaque white 96-well plate, and reconstituted luciferase assay buffer (Vazyme) was added and mixed with each lysate. Luminescence was measured immediately after mixing using a GloMax 96 Microplate Luminometer (Promega). The neutralization titer (NT50) was determined by luciferase activity with a four-parameter non-linear regression inhibitor curve in GraphPad Prism 8.4.2 (GraphPad Software). NT50 was defined as the highest reciprocal serum dilution causing a 50% reduction in relative light units. A sample with NT50 values no more than 30 (the detectable limit) was considered negative for neutralizing antibodies and was assigned a nominal value of 10 in geometric mean titer (GMT) calculations, which is the lowest serum dilution factor used in the pseudovirus neutralization assay.

Cell–cell fusion assay

The cell-fusion activity of SARS-CoV-2 S variants was evaluated with a cell–cell fusion reporting assay based on split mNeonGreen and NanoLuc proteins. Briefly, HEK-293T cells at 80% confluence were co-transfected with pNGJS (NG-bJun-SmBiT expression) and pLVX-hACE2-IRES-tdTomato plasmids at a 1:1 ratio for 24 h at 37 °C and 5% CO2, for preparing target cells. HEK-293T cells co-transfected with pCGFL (CG-bFos-LgBiT expression) and S plasmids at a 1:1 ratio for 24 h at 37 °C and 5% CO2 to prepare effector cells, and a no-S expression plasmid was co-transfected as the negative control. Following 24 h of transfection, the cells were detached and resuspended in fresh DMEM containing 5% FBS. Subsequently, 1.0 × 104 target cells per well were added to a 96-well plate, and 1.0 × 104 effector cells per well were added to the 96-well plate with target cells. The mNeonGreen and tdTomato fluorescent proteins were confirmed by an IX71 fluorescence microscope (Olympus), and the luciferase activity was measured 6 h after co-culturing using the Nano-Light Luciferase Assay Kit (Meilunbio, Dalian, China).

Western blot analysis

Western blotting was performed as previously described. HEK-293T cells co-transfected with the S variant plasmids and pCGFL were used. Briefly, cells were lysed in RIPA buffer containing protease inhibitors (Solarbio, Beijing, China). The protein concentration was measured using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Polyacrylamide gel electrophoresis and protein transfer to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA) were carried out following standard protocols. Rabbit anti-SARS-CoV-2 S IgG antibody (Sino Biological, Beijing, China) or mouse anti-β-actin monoclonal antibody (Solarbio, Beijing, China) was used for immunodetection according to the manufacturer’s instructions. Protein bands were visualized using a Novex™ ECL substrate reagent kit (Thermo Fisher Scientific, Inc.) with the ECL imaging system (Tanon, Shanghai, China).

Pseudovirus infection assay

For analysis of pseudovirus infection ability, 293T-hACE2 cells were added to 96-well plates and infected with the indicated pseudoviruses collected between 36 and 48 h post-transfection. The medium was replaced with fresh medium at 12 h post-infection. Luciferase activity was detected at 60 h post-infection with a Steady-Lumi™ II Firefly Luciferase Assay Kit (Beyotime, Shanghai, China). The pseudovirus infection ability was calculated based on the relative luciferase activity and normalized to the pseudovirus RNA genome copy numbers determined by real-time reverse transcription-polymerase chain reaction (RT–PCR).

Quantitation of pseudovirus by RT–PCR

To determine the genome copy numbers of pseudoviruses, lentiviral RNA was extracted with the TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (TaKaRa, Beijing, China). After DNase I digestion, the relative genome RNA copy numbers of pseudoviruses were analyzed with RT–PCR using the One-Step TB Green PrimeScript PLUS RT–PCR kit (Takara, Beijing, China) according to the manufacturer’s instructions. The primers were as follows: LV-F 5′-TAGTAGGAGGCTTGGTAGGT-3′ and LV-R 5′-GTGGGTCTGAAACGATAATGG-3′. The following cycling conditions were used: 42 °C for 5 min, 95 °C for 10 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s, 95 °C for 15 s and 60°C for 1 min, and 95°C for 15 s.

Statistical analysis

Data and statistical analyses were performed using GraphPad Prism 8.4.2 (La Jolla, CA, USA) and R v4.0.5. Fold changes in serum-neutralizing activity were measured by comparing GMT. Fold changes in monoclonal antibodies were determined by comparing individual IC50 or IC90 values and then averaging the individual fold changes for reporting. Categorical and continuous variables in supplementary tables were analyzed using the chi-square test, Fisher’s exact test, or Kruskal–Wallis test. The Friedman test with the false discovery rate method was used for multiple comparisons for paired groups, while the Kruskal–Wallis test with the false discovery rate method was used for multiple comparisons for unpaired groups. The Wilcoxon rank-sum test was used for unpaired comparisons between the two groups. All statistical tests were 2-sided with a significance level of 0.05.