Co-immunization with spike and nucleocapsid based DNA vaccines for long-term protective immunity against SARS-CoV-2 Omicron

Study design

The goal of this study was to investigate the immunogenicity and efficacy of S and N DNA vaccines (alone or combined) against SARS-CoV-2 VOCs. In vitro S and N antigen expression driven by S and N DNA vaccines was confirmed in transfected cells. Immunogenicity and protection were assessed in single- or co-vaccinated K18-hACE2 mice later infected with Beta or Omicron BA.2 VOCs. Short- and long-term protocols were conducted to assess durability. Similar numbers of male and female mice were assigned to experimental groups (N = 3 to 6). Sample sizes for each mouse group were estimated based on previous efficacy and viral challenge experiments in our laboratory and in the literature, aiming to balance the numbers required for statistical rigor while minimizing animal use. Each animal experiment was conducted at least twice. Studies were not randomized or blinded. The animal experiments were performed in strict accordance with recommendations outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the La Jolla Institute for Immunology ABSL2 and ABSL3 facilities (protocol number AP00001242). Tissue samples were collected from immunized and/or infected mice to evaluate Ab and T cell immunity in addition to SARS-CoV-2 RNA levels. All in vitro assays (ELISA, pVNT50, qRT-PCR) included experimental samples and controls in duplicate.

Mice

K18-hACE2 transgenic mice were obtained from The Jackson Laboratory or bred at the La Jolla Institute for Immunology and maintained under pathogen-free conditions. K18-hACE2 transgenic mice express human ACE2 (primary cell receptor for SARS-CoV-2) under the control of the human keratin-18 promoter. In K18-hACE2 mice, human ACE2 is expressed in epithelial cells, including the mucosal epithelium lining the airways; as a result, these mice are highly susceptible to SARS-CoV-2 infection⁴⁰.

S and N DNA vaccines

S and N DNA vaccines⁴¹ were designed to express full-length wild-type SARS-CoV-2 S and N proteins with sequences from SARS-CoV-2/human/USA/WA-CDC-WA1/2020 (GenBank accession: MN985325.1). Coding sequences were codon-optimized for human expression by GenScript and cloned into pVAX1 plasmid vector (Thermo Fisher Scientific) under the control of human cytomegalovirus immediate-early promoter. To enhance translational initiation and performance, optimized DNA sequences were preceded by Kozak and IgE leader sequences. In vitro antigen expression driven by S and N DNA vaccines was confirmed as previously described⁴¹.

Immunization and tissue collection for immunogenicity experiments

K18-hACE2 mice were immunized via the right or left quadriceps (see below) with 25 µg of S and/or N DNA vaccines diluted in 50 µL of Tris-EDTA pH 8.0 (Invitrogen, 15568025), followed by minimally invasive electroporation⁷³. The intramuscular (IM) injections used 30-gauge ultra-fine insulin syringes (BD Bioscience, BD-25150) and electroporation used the BTX AgilePulse IM System (47-0500 N) with a 4 x 4 x 5 mm needle array (47-0045). At 21 days post-priming, mice were boosted in the same manner (boost omitted for the one-dose vaccination protocol). For single immunization with either S or N vaccines, mice were primed in one muscle and boosted ipsilaterally. For co-immunization with both vaccines, mice were primed with S vaccine in the right hind limb and with N vaccine in the left hind limb, and then boosted in the contralateral limb. In all experiments, control mice received commercial vector pVAX1.

For tissue collection, mice were either anesthetized with isoflurane or euthanized with CO₂. In short-term experiments, blood was collected on days 20 (1 day before boost) and 28 (7 days post-boost) into serum-gel collection tubes (Sarstedt, 41.1378.005) from the facial vein or via cardiac puncture, and lungs and spleens were harvested on day 28 (7 days after the boost) in sterile RPMI 1640 media (Gibco, 1187519) supplemented with 10% fetal bovine serum (FBS, Gibco, 1187519), 1% penicillin–streptomycin (Gibco, 15140-122), 1% HEPES (Gibco, 15630130). In long-term experiments, blood samples were collected on days 60, 115, and 140.

SARS-CoV-2 S and N IgG ELISA

SARS-CoV-2-binding Abs were detected by endpoint ELISA assays using recombinant wild-type S, RBD, and N proteins, and commercial RBD from the Omicron BA.1 (B.1.1.529.1) variant (Sino Biological, 40592-V08H121) as coating antigens. Recombinant wild-type SARS-CoV-2 S, N, and RBD proteins were generated from synthetic codon-optimized DNA of Wuhan-Hu-1 isolate (GenBank accession MN908947.3) by sub-cloning into the pHCMV3 expression vector. Site-directed mutagenesis was performed to generate sequences of the Beta (B.1.351) and Omicron BA.1 (B.1.1.529.1) variants, and positive clones were fully sequenced to ensure that no additional mutations were introduced. Protein purification was previously described⁷⁴.

High-binding flat-bottom plates (Corning, 9018) were coated overnight (4 °C) with 0.1 µg/mL of S, RBD, or N proteins in phosphate buffered saline (PBS, Corning, 21040). The following steps were carried out at room temperature. Plates were washed 3 times with 1% BSA (Sigma-Aldrich, A3059) in PBS (PBS/BSA), and blocked with 5% Blotting Grade Blocker (Bio-Rad, 1706404) in PBS for 2 h. Serial dilutions of mouse sera were prepared in PBS/BSA (5-fold dilutions; 1:50 to 1:156.250) and added to the plates for 1.5 h. After 3 washes, plates were incubated with HRP-labeled anti-mouse IgG (Jackson ImmunoResearch, 115-035-008) in PBS/BSA for 1.5 h, washed, developed in the dark for 10 min with 1 Step TMB (Thermo Fisher Scientific, 34028), and the reaction was then stopped by addition of an equal volume of 2 N sulfuric acid (Fisher Chemical, A468-2). Control wells were treated identically except without the serum incubation. Optical density was immediately measured at 450 nm using the SpectraMax M2 microplate reader (Molecular Devices). The universal cut-off value for all ELISA assays was 0.2 (3 standard deviations above the mean of the control wells). Endpoint titers were calculated based on the interpolation from the cut-off value in a 4-parameter logistic curve fit of each test sample.

Pseudovirus neutralization (pVNT) assay

SARS-CoV-2-specific nAbs in mouse sera were measured using a pVNT assay based on recombinant replication-deficient vesicular stomatitis virus (VSV) vectors encoding GFP pseudotyped with SARS-CoV-2 S protein derived from the Wuhan-Hu-1 isolate reference strain (GenBank: MN908947.3), or the Beta (B.1.351), Omicron BA.2 (B.1.1.529.2), or Omicron XBB1.5 variants generated by site-directed mutagenesis, as previously described in ref. ⁷⁴. Briefly, Vero cells (ATCC CCL81) were seeded (2.5 × 10⁴ cells/well) in flat-bottom 96-well black plates (Corning, CLS3603) to achieve 80% to 90% confluence at the time of infection. Mouse sera were heat-inactivated (30 min, 56 °C), serially diluted in PBS (3-fold dilutions; 1:25 to 1:54,675), incubated with pre-titrated amounts of rVSV-SARS-CoV-2 (1.5 h, 5% CO₂), and then added to confluent Vero monolayers for 16 to 18 h (37 °C, 5% CO₂). After infection, cells were washed with PBS, fixed with 4% paraformaldehyde (Electron Microscopy Science, 15700), stained in the dark with 1 µg/mL of Hoechst (Thermo Fisher Scientific, 62249) in PBS for (30 min, room temperature), and washed twice with PBS. Pseudovirus titers were quantified as the number of focus forming units (FFU/mL) using a Cell Insight CX5 imager (Thermo Fisher Scientific). Neutralizing Ab titers were computed using a 4-parameter logistic curve fitting regression.

Intracellular cytokine staining (ICS) assay

Single-cell suspensions of leukocytes and splenocytes were prepared from the lungs and spleens, respectively, and then seeded into 96-well round-bottom plates (Corning, 38018) in complete RPMI media (2 × 10⁶ cells/well). Cells were stimulated with individual or pooled SARS-CoV-2 S- or N-derived peptides (2 µg of each peptide/well, 5 h, 37 °C, 5% CO₂; Table 1). Prediction of peptide-MHC class I or II binding affinity was performed using tools from the Immune Epitope Data Base website (www.iedb.org) and selection of the “IEDB-recommended” method, as described previously⁴⁴. The selected peptides were identified as immunodominant in previous INF-γ -ELISPOT assays (data not shown) for CD8⁺ and CD4⁺ T cells in C57BL/6 mice (the genetic background of K18-hACE2 mice). After 1 h incubation, brefeldin A (BioLegend, 420601) and anti-CD107a PE (clone eBio1D4B, eBioscience, 12-1071-83) were added and the incubation was continued for 4 h. Positive and negative control cells were incubated with a commercial stimulation cocktail containing PMA and ionomycin (eBioscience, 00-4970-93) or RPMI 1640 medium alone, respectively. Peptide-stimulated cells were then labeled with viability dye efluor 455 UV, (eBioscience, 65-0868-18), blocked with Fc Block (CD16/CD32 mAb 2.4G2; BD Biosciences, 553142) and stained with fluorophore-conjugated Abs: anti-CD3e PE-Cy7 (clone 145-2C11, eBioscience, 25-0031-82), anti-CD4 BUV395 (clone GK1.5, BD Bioscience, 565974), anti-CD8a BV510 (clone 53-6.7, BioLegend, 100751), anti-CD44 BV785 (clone IM7, BioLegend, 103041) and anti-CD62-L APC efluor 780 (clone MEL-14, eBioscience, 47-0621-82). Cells were then fixed, permeabilized with Cytofix/Cytoperm commercial kit (BD Bioscience, 554722), and stained with anti-IFN-γ FITC (clone XMG1.2, Tonbo Bioscience, 35-7311-U100), anti-TNF-α APC (clone MP6-XT22, eBioscience, 17-7321-82), and anti-IL-2 BV711 (clone JES6-5H4, BioLegend, 503837). Data were acquired on an LSRFortessa flow cytometer (BD Bioscience) and analyzed using FlowJo software v10.8.1 (Tree Star).

Viruses and in vivo challenge

Vaccine efficacy was assessed against SARS-CoV-2 Beta (hCoV-19/South Africa/KRISP-K005325/2020, BEI NR-54009) and Omicron BA.2 (CoV-19/Japan/UT-NCD1288-2N/2022, NCD1288), generously provided by Ralph Baric (University of North Carolina) and Yoshihiro Kawaoka (University of Wisconsin; via Michael Diamond at Washington University in St. Louis), respectively. The SARS-CoV-2 Beta strain was propagated in Vero cells (ATCC, CCL81), and the Omicron BA.2 strain in Vero/TMPRSS2 cells (kindly donated by Michael Diamond, Washington University) for 3 days in DMEM (Corning, 10-013-CV) supplemented with 10% FBS, 1% penicillin–streptomycin, 1% HEPES, and 1% non-essential amino acids (Gibco, 11140050), and the supernatants were then harvested and frozen. The genetic homogeneity of both virus stocks was confirmed by deep-sequencing analysis (La Jolla Institute for Immunology Sequencing Core). The viral stocks were titrated by plaque assay. Briefly, viral supernatants were serially diluted 10-fold and added to confluent Vero E6 cells (ATCC, CRL-1587) in 24-well plates for 1 h (8 × 10⁴ cells/well); the medium was then switched (DMEM, 1% carboxymethylcellulose, 2% FBS) and the cells incubated for 3 days (all incubations were at 37 °C, 5% CO₂). Cells were then fixed with 10% formaldehyde (1 h, room temperature), washed, and stained with 0.1% crystal violet in methanol (20 min, room temperature), and plaque-forming units (PFU) per mL quantified. All SARS-CoV-2 propagation and titration were performed in a BSL-3 facility.

For the viral challenge protocol, immunized K18-hACE2 mice were inoculated intranasally (IN, 15 µL per nostril, 30 µL total) with Beta (10³ PFU) or Omicron BA.2 (10⁴ PFU) variants on days 35 or 140 (14- or 119-days post-boost, respectively). At 3 days post-challenge, mice were euthanized by CO₂ inhalation. Lungs and nasal turbinates were harvested in 1 mL RNA/DNA shield (ZYMO Research, R1100-250) and spleens were harvested in complete RPMI medium for isolation of splenocytes used in ICS assays. All SARS-CoV-2 infections and tissue harvesting were performed in an ABSL-3 facility.

In vivo T cell depletion and viral challenge

For in vivo T cell depletion, pVAX and S + N-immunized K18-hACE2 mice were inoculated intraperitoneally (IP, 200 µL total) with 250 µg anti-CD4 (clone GK1.5, Bio X Cell, BE0003-1) and anti-CD8 (clone 2.43, Brand, BE0061) monoclonal Abs or isotype control Abs on days 137, 138, and 139. On day 140, mice were inoculated with Omicron BA.2 variant as described above, and at 3 days post-challenge, lungs and nasal turbinates were harvested.

To assess T cell depletion efficiency, mice were anesthetized with isoflurane inhalation and blood collected via the facial vein just before viral challenge. Blood cells were labeled with viability dye efluor 455 UV (eBioscience, 65-0868-18), blocked with Fc Block (CD16/CD32 mAb 2.4G2; BD Biosciences, 553142), and stained with the following 3 fluorophore-conjugated Abs: anti-CD3e PE-Cy7 (clone 145-2C11, eBioscience, 25-0031-82), anti-CD4 BUV395 (clone GK1.5, BD Bioscience, 565974), anti-CD8a BV510 (clone 53-6.7, BioLegend, 100751). Data were acquired on an LSRFortessa flow cytometer (BD Bioscience) and analyzed using FlowJo software v10.8.1 (Tree Star).

Lung histopathology

Histopathological analysis was conducted according to previous methods⁴¹. Briefly, lungs from immunized/challenged mice were harvested and fixed in zinc formalin for 24–48 h at room temperature with gentle agitation. After fixation, samples were transferred to 70% alcohol. Lung tissues were then embedded in paraffin using standard procedures, sectioned into 4-μm slices, stained with H&E using a Leica ST5020 autostainer, and imaged with a Zeiss AxioScan Z1 (40 × 0.95 NA objective). Histopathological evaluation was performed by a certified veterinary pathologist who was blinded to group identities. Sections were scored on a scale of 0–5 based on 10 criteria for SARS-CoV-2-induced lung pathology, as observed in hamsters, monkeys, and COVID-19 patients⁴⁵. Scores for 5 parameters are shown: necrosis of bronchiolar epithelial cells (BEC), cellular debris in bronchioles, inducible bronchus-associated lymphoid tissue (iBALT) hyperplasia, perivascular lymphocytic cuffing, and bronchointerstitial pneumonia.

SARS-CoV-2 quantification in tissues

Lungs and nasal turbinates were harvested from immunized/challenged mice into 1 mL RNA/DNA shield (Zymo Research, R1100-250), and SARS-CoV-2 RNA was extracted using RNeasy Mini Kits (Qiagen, 52904) and then stored at −80 °C. Total SARS-CoV-2 genomic and subgenomic RNA copies were quantified using the qScript One-Step qRT-PCR Kit (Quanta BioSciences, 76047-080). Genomic RNA was quantified using the envelope gene as a target and the following primer sets⁷⁵: Fwd, 5′-ACAGGTACGTTAATAGTTAATAGCGT-3′; Rev, 5′-ATATTGCAGCAGTACGCACACA-3′; and Probe, FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ. Subgenomic RNA copies were quantified using the Orf7a gene as a target and the following primer sets⁷⁶: Fwd, 5′-TCCCAGGTAACAAACCAACCAACT-3′; Rev, 5′-AAATGGTGAATTGCCCTCGT-3′; and Probe, FAM-CAGTACTTTTAAAAGAACCTTGCTCTTCTGGAAC-Tamra-Q. Amplification for genomic and subgenomic RNA was performed using the CFX Real-Time PCR System and the following program: 48 °C for 30 min, 95 °C for 10 min, and then 40 cycles of 95 °C for 15 s and 55 °C for 1 min. Viral RNA concentration was calculated using a standard curve composed of four 100-fold serial dilutions of in vitro-transcribed RNA from SARS-CoV-2 (RNA/human/USA/WA-CDC-WA1/2020, ATCC NR-52347).

Statistical analysis

Statistical analyses were performed using GraphPad Prism v10.0.2 software. Outliers were identified using GraphPad Prism outlier calculator. All data are presented as the mean ± SEM. Differences between group means were analyzed by the non-parametric Kruskal-Wallis test for more than 2 groups, or the non-parametric Mann–Whitney test for 2 groups; P < 0.05 was considered significant.

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• Source: https://www.nature.com/articles/s41541-024-01043-3