Construction, rescue, amplification, and purification of AdC68-based vaccine

AdC68 is an E1- and E3-deleted, replication-deficient adenoviral vector with a partial deletion in E4, replaced by the corresponding E4 region from human adenovirus 5 (AdHu5). Codon-optimized genes encoding the wild-type (WT) fusion glycoprotein (F) of RSV A2 strain (GenBank accession no: ACO83301.1), along with structure-based designer DS2, DS-Cav1, and SC-TM prefusion F glycoproteins39,40,41, were synthesized by GenScript Biotech Corporation, China. The coding sequences for each of these F glycoproteins, along with the original signal peptide (SP), transmembrane (TM) domain, and cytoplasmic tail (CT) from the RSV A2 strain, were inserted into the E1-deleted region of the pAdC68 vector by isothermal assembly. The resulting vectors, designated pAdC68-DS2, pAdC68-DS-Cav1, pAdC68-SC-TM, and pAdC68-WT, were generated, with an empty pAdC68 vector serving as a negative control. Then, the adenoviruses were rescued by the E1 protein expressed and supplied by the HEK 293A cells. Specifically, HEK 293A cells were seeded on a 6-well plate 1 day before transfection. Cells were cultured overnight to 80–85% confluency at 37 °C with 5% CO2. pAdC68-DS2, pAdC68-DS-Cav1, pAdC68-SC-TM and pAdC68-WT were linearized with PacI and then transfected into HEK 293A cells using Lipofectamine 2000 according to the manufacturer’s instructions (Life Technologies, USA). The rescued replication deficient recombinant chimpanzee adenoviruses AdC68-DS2, AdC68-DS-Cav1, AdC68-SC-TM and AdC68-WT were further amplified in HEK 293A cells which were stably integrated with the E1 expression genes. Adenoviruses were purified by CsCl gradient ultracentrifugation followed by dialysis desalting, and then stored in preservation solution containing 50 g/L sucrose, 0.4060 g/L magnesium chloride hexahydrate and 10 mM Tris-HCl (pH = 8). Similarly, the empty AdC68 was generated without any target gene inserted into the pAdC68 vector and purified as described above.

Production and purification of trimeric recombinant pre-F glycoprotein of RSV and monoclonal antibodies

Genes encoding the ectodomain of the wild-type (WT) and designer DS2, DS-Cav1 and SC-TM pre-F glycoproteins of the RSV A2 strain were engineered to include a foldon trimerization motif, an 8×His tag, and two Strep tags at the C-terminus. These proteins were expressed in HEK 293F cells and purified using Ni-NTA resin (Qiagen, Germany), followed by gel filtration chromatography using Superdex™ 200 Increase columns (Cytiva, UK). A panel of anti-F monoclonal antibodies (mAbs) targeting six predefined antigenic sites was produced based on published data. This panel included D25, 5C4, RSD5, and Nirsevimab for site Ø; CR9501, AM14, and RSB1 for site V; 101F for site IV; MPE8 and ADI-19425 for site III; Motavizumab and Palivizumab for site II; and ADI-14359 and 4D7 for site I (Supplementary Fig. 1)6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23. The heavy and light chain variable genes of these mAbs were cloned into human IgG1 Fc expression vectors. The mAbs were then expressed in HEK 293F cells and purified from the supernatant using AmMag™ Protein A Magnetic Beads (GenScript, USA).

Detection of F glycoprotein expression on the cell surface

HEK 293T cells cultured in 6-well plates were infected with AdC68-DS2, AdC68-DS-Cav1, AdC68-SC-TM, AdC68-WT, or AdC68 at a dose of 1010 viral particles (vp) per well. After 24 h, the cells were harvested, and the expression of F glycoprotein was analyzed by flow cytometry using the panel of monoclonal antibodies (mAbs) targeting six pre-defined antigenic sites on the F glycoprotein. These mAbs can differentiate between pre-fusion and post-fusion conformations of the F glycoprotein by recognizing pre-fusion-specific antigenic sites (site Ø: D25, 5C4, and RSD5; site V: CR9501, AM14, and RSB1) and shared antigenic sites (site IV: 101F; site III: MPE8 and ADI-19425; site II: Motavizumab and Palivizumab; and site I: 4D7). Cells were incubated with each mAb at a final concentration of 10 μg/mL at 4 °C for 30 min, followed by extensive washing. The cells were then incubated with anti-human IgG-PE (BioLegend, USA) at a 1:200 dilution and analyzed using the LSRFortessa™ Cell Analyzer (BD, USA). The percentage of cells positive for the PE signal was calculated relative to the total number of live cells. The results were analyzed by FlowJo 10.8.1.

Mice immunization and sample collection

Fifty 6-week-old female BALB/c mice were purchased from Charles River and randomly divided into five groups of ten. Each group was prime-boost immunized with a total of 2 × 1010 viral particles (vp) of AdC68-DS2, AdC68-DS-Cav1, AdC68-SC-TM, AdC68-WT, or the empty AdC68 via intramuscular (i.m.) injection. Serum samples were collected every two weeks up to 10 weeks post-priming, heat-inactivated at 56 °C for 30 min, and stored at −80 °C prior to analysis for RSV F-specific binding and neutralizing antibodies.

Binding antibodies measured by enzyme-linked immunosorbent assay (ELISA)

To measure serum binding antibodies in immunized mice, 96-well plates were coated overnight at 4 °C with 100 ng/well of recombinant F trimers from RSV designer DS2, DS-Cav1, SC-TM, or WT, produced in HEK 293 F cells. The plates were then blocked with phosphate-buffered saline (PBS) containing 3% BSA at 37 °C for 3 h, followed by the addition of serially diluted serum samples. After a 1-h incubation at 37 °C, the plates were washed three times with PBS containing 0.06% Tween 20 (PBST) and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody against mouse IgG (1:4000, Promega, USA) at 37 °C for 45 min. After additional washes with PBST, TMB substrate was added, and the reaction was stopped with 1 M H2SO4. Absorbance at 450 nm was measured using an iMark™ Microplate Absorbance Reader (Bio-Rad, USA).

To assess the binding activity of humanized monoclonal antibodies (mAbs) derived from antibody-secreting cells (ASCs) in the bone marrow or previously published control mAbs6,7,8,9,11,12,15,16,17,19,20,23, serial dilutions were applied to 96-well ELISA plates coated with recombinant F trimers from DS2. After extensive washing with PBST, bound mAbs were detected using an HRP-conjugated anti-human IgG (1:4000, Promega, USA), following the same protocol as for serum binding antibodies. ED50 or EC50 values for serum samples or mAbs were calculated based on binding curves generated using Prism 8.0.2 software (GraphPad, USA), respectively.

Virus neutralization assay

The virus neutralization assay was conducted using the Long strain (GenBank accession no: ACO83302.1) and 9671 strain (GenBank accession no: MW582529.1) of live respiratory syncytial virus (RSV) expressing green fluorescent protein, kindly provided by Dr. Qiang Ding at the Center for Infection Biology, Tsinghua University. The experiments were performed in a Biosafety Level 2 (BSL-2) facility at Tsinghua University. Briefly, serial dilutions of serum samples or monoclonal antibodies (mAbs) were prepared in 96-well plates, mixed with 100 units of the 50% tissue culture infectious dose (TCID50) of live RSV per well, and incubated for 1 h at 37 °C with 5% CO2. Subsequently, 104 Hep2 target cells were added to each well, and green fluorescence intensity, indicating levels of viral replication, was monitored daily until day five using a fluorescence microscope (Olympus, Japan). On the fifth day, fluorescence intensity was quantified using the Opera Phenix™ High-Content Screening System (PerkinElmer, USA). The 50% inhibitory dose (ID50 or IC50) for each serum sample or mAb was calculated using the Reed-Muench method.

Isolation of DS2-specific antibody-secreting cells from bone marrow

Bone marrow antibody-secreting cells (ASCs) specific for the DS2 trimer were isolated using the Modular Superhydrophobic Microwell Array Chip (MoSMAR) platform (Homemade unpublished) from a mouse six months after initial immunization with AdC68-DS2. ASCs were first enriched using the CD138+ Plasma Cell Isolation Kit (Miltenyi Biotec, Germany) and then incubated with DS2 trimer-coated polystyrene particles (Spherotech, USA) and Alexa Fluor™ 488-labeled anti-mouse IgG secondary antibody (Invitrogen, USA) at 37 °C for 2 h on the MoSMAR platform. DS2-specific ASCs were identified by the strong circular green fluorescence signal observed under a fluorescence microscope (Olympus, Japan). Individual DS2-specific ASCs were then isolated using a microneedle for subsequent single-cell amplification of the antibody heavy and light chain variable regions by polymerase chain reaction (PCR).

Single cell polymerase chain reaction (PCR), cloning and expression of monoclonal antibodies

The heavy and light chain variable genes of mouse-derived mAbs were amplified by nested PCR and cloned into human linear expression cassettes to generate full-length IgG1 antibodies, following previously established protocols55,56,57. Specifically, second-round PCR primers containing tag sequences were utilized to produce linear immunoglobulin expression cassettes via overlapping PCR. Paired heavy and light chain expression cassettes were co-transfected into HEK 293T cells and mAbs secreted into the supernatant were used for initial binding screening. For pairs demonstrating positive binding, the heavy and light chain variable regions were sequenced and subsequently cloned into antibody expression vectors containing the constant regions of human IgG1. Large-scale mAb production was achieved through the transient transfection of HEK 293F cells with equal amounts of paired heavy and light chain expression plasmids. The resulting antibodies were purified from culture supernatants using AmMag™ Protein A Magnetic Beads (GenScript, USA).

Genetic analysis of isolated monoclonal antibodies

Germline gene usage, somatic hypermutation, framework regions, and complementarity-determining region 3 (CDR3) loop lengths of each antibody were analyzed using the IMGT/V-QUEST program (http://www.imgt.org/IMGT_vquest/vquest). Genetic clustering analysis and neighbor-joining phylogenetic trees were generated using MEGA version 11.0.13 and refined using iTOL (https://itol.embl.de/).

Surface plasmon resonance (SPR) analysis

Equilibrium dissociation constants (KD) and epitope mapping of the isolated mAbs were analyzed using the Biacore™ 8 K SPR system (Cytiva, UK). For KD determination, mAbs were injected at gradient concentrations onto a CM5 chip immobilized with recombinant DS2 trimer for 120 s, followed by a dissociation phase of 1200 s. Kinetic parameters, including the on-rate (Ka) and off-rate (Kd), were used to calculate the KD (KD = Kd/Ka). For epitope mapping, pairs of mAbs were sequentially injected to evaluate whether prior binding of the first mAb affected the binding of the second mAb, thus determining if the two mAbs recognized distinct or overlapping epitopes. Specifically, control mAbs (D25, CR9501, 101F, MPE8, Motavizumab, and 4D7), which target the six major antigenic sites (Ø, V, IV, III, II, and I) on the F glycoprotein, were injected onto the CM5 chip immobilized with recombinant DS2 trimer for 240 s, followed by the injection of the test mAbs for 90 s. The level of competition between the test and control mAbs was used to assess binding specificity. Biacore Insight Evaluation 5.0.18.22102 software was employed for KD determination and epitope mapping.

Vaccine and antibody protection in BALB/c mice

Animal protection assays were performed in the Animal Biosafety Level 2 (ABSL-2) facility at Tsinghua University. For vaccine protection analysis, 6-week-old female BALB/c mice (Charles River) were intramuscularly immunized with AdC68-DS2. Ten weeks later, the mice were challenged intranasally with 3 × 105 plaque-forming units (PFU) of live RSV Long strain (GenBank accession no: ACO83302.1). Mice were anesthetized using an isoflurane anesthesia machine before being intranasally challenged with live RSV in the Animal Biosafety Level 2 (ABSL-2) facility at Tsinghua University. The mice were placed in an induction box for 1 min with an oxygen flow rate of 1 L/min and 3% isoflurane concentration, while maintaining an O2 pressure of 0.1 MPa. Hind limb reflexes were tested with hemostatic forceps to assess anesthesia depth and ensure readiness of unconsciousness for subsequent procedures. Afterward, the mice were allowed to recover from anesthesia. Body weights were monitored daily for 7 days. For antibody protection analysis, 4-week-old female BALB/c mice (Charles River) received either mAb60 (10 mg/kg) or an equal volume of phosphate-buffered saline via intraperitoneal injection one day before intranasal challenge with 3 × 105 PFU of live RSV Long strain under the same anesthesia conditions.

In both the vaccine and antibody protection studies, half of the mice from the treated and untreated control groups were euthanized on day 2 post-challenge to collect lung tissues for viral load measurement and histopathological analysis. Euthanasia was conducted using CO2 in the ABSL-2 facility. CO2 flow was gradually increased by 30%–70% per minute to fill the euthanasia chamber. After observing the loss of consciousness due to hypoxia, CO2 aeration was continued for an additional 3–4 min to ensure painless death. Respiratory and cardiac arrest, along with pupil dilation for 1–3 min, were observed to confirm death.

Viral loads measurement by qPCR and plaque assay

To quantify viral loads in the lungs, total RNA was extracted from homogenized lung tissues using the RNA Easy Fast Total RNA Extraction Kit (TIANGEN, China). First-strand cDNA was synthesized using the SuperScript™ IV First-Strand Synthesis System (Invitrogen, USA). Quantitative real-time PCR (qPCR) was then performed using the PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, USA) on a QuantStudio™ 6 Pro Real-Time PCR System (Thermo Fisher Scientific, USA) to measure RSV-specific transcripts (N, NS1, and M). The primers used for qPCR were obtained from previously published studies58. Relative quantification was employed for data analysis, and results were presented as the number of RSV-specific transcripts per gram of lung tissue.

For determining live virus titers, supernatants from homogenized lung tissues were serially diluted in DMEM and added to Hep2 cells in 6-well plates to allow viral adsorption for 1 h. The Hep2 cells were then washed with phosphate-buffered saline and overlaid with 1% methylcellulose-DMEM medium. The plates were incubated at 37 °C with 5% CO2 for 5 days to enable plaque formation. The cells were subsequently fixed with 4% formaldehyde and stained with crystal violet to calculate the plaque-forming units (PFU) per gram of lung tissue.

Histopathological analysis of lung tissues

For histopathological analysis, lung tissues were fixed in 4% formaldehyde, processed, and embedded in paraffin blocks (Leica Biosystems, Germany). The embedded tissues were sectioned at a thickness of 4 µm and stained with hematoxylin and eosin (Thermo Fisher Scientific, USA) following standard protocols. The stained slides were examined using CaseViewer 2.4.

Cryo-EM sample preparation

To identify the binding epitope of mAb60, single-particle cryo-electron microscopy (cryo-EM) was employed to determine the structure of the DS2 pre-fusion F-mAb60 complex. Purified DS2 pre-fusion F trimer was mixed with mAb60 Fab at a molar ratio of 1:1.2 and incubated overnight at 4 °C. The DS2 pre-fusion F-mAb60 Fab complex was then separated using Superdex™ 200 Increase columns (Cytiva, UK) and concentrated to 1.5 mg/mL for cryo-EM sample preparation. Data collection was performed using an FEI Titan Krios microscope.

Cryo-EM structural determination

Five microliters of the DS2 pre-fusion F-mAb60 Fab complex in a buffer containing 20 mM Tris (pH = 8.0) and 150 mM NaCl were applied to glow-discharged holey carbon grids (Quantifoil grid, Cu 300 mesh, R1.2/1.3). Grids were blotted for 3 s and immediately plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific, USA). Cryo-EM data were collected using an FEI Titan Krios microscope (Thermo Fisher Scientific, USA) at 300 kV with a Gatan K3 Summit direct electron detector (Gatan) at Tsinghua University. A total of 2132 movies were collected using AutoEMation2 at a magnification of 81,000×, with a pixel size of 1.0742 Å and a defocus range of −1.2 to −1.8 µm. Each movie had a total exposure of 50 e/Ų, fractionated into 32 frames over 2.13 s. Data collection statistics are summarized in Supplementary Table 1. Motion correction was performed automatically using MotionCor2 v.1.2.6 via the TsingTitan.py program. Data processing was carried out with cryoSPARC v.3.3.159. Initial models were constructed using cryoNet (https://cryonet.ai/), developed by Cliff Zhang’s Lab. Model refinement was performed with COOT v.0.9.260 and PHENIX v.1.18.261. The final model quality was assessed using PHENIX v.1.18.2, and structural figures were generated with ChimeraX v.1.6.162.

Ethics statement

All experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals of the People’s Republic of China and approved by the Committee on the Ethics of Animal Experiments at Tsinghua University. Mouse immunization, characterization, animal protection assays, and viral neutralization assays involving the live RSV were conducted in the ABSL-2 or BSL-2 facilities at Tsinghua University.

Statistical analysis

Statistical analysis and graph generation were performed using GraphPad Prism 8.0.2 and IBM SPSS Statistics 23. Data are presented as median ± interquartile range. The Mann–Whitney U test was used for comparisons between two independent groups, and the Kruskal–Wallis H test was applied for multiple group comparisons, as the data was not uniformly normally distributed. All tests were two-sided, with a 5% significance level. p-values are indicated in the graphs.