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. 2024 Jul 16;121(29):e2310421121.
doi: 10.1073/pnas.2310421121. Epub 2024 Jul 8.

Recombinant OC43 SARS-CoV-2 spike replacement virus: An improved BSL-2 proxy virus for SARS-CoV-2 neutralization assays

Zhe Hu  1 , Alberto Domingo López-Muñoz  1 , Ivan Kosik  1 , Tiansheng Li  1 , Victoria Callahan  2 , Kelsie Brooks  3 , Debra S Yee  3 , Jaroslav Holly  1 , Jefferson J S Santos  1 , Ayslan Castro Brant  4 , Reed F Johnson  5 , Kazuyo Takeda  6 , Zhi-Ming Zheng  4 , Jason M Brenchley  3 , Jonathan W Yewdell  1 , Julie M Fox  2
Affiliations

Recombinant OC43 SARS-CoV-2 spike replacement virus: An improved BSL-2 proxy virus for SARS-CoV-2 neutralization assays

Zhe Hu et al. Proc Natl Acad Sci U S A. .

Abstract

We generated a replication-competent OC43 human seasonal coronavirus (CoV) expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in place of the native spike (rOC43-CoV2 S). This virus is highly attenuated relative to OC43 and SARS-CoV-2 in cultured cells and animals and is classified as a biosafety level 2 (BSL-2) agent by the NIH biosafety committee. Neutralization of rOC43-CoV2 S and SARS-CoV-2 by S-specific monoclonal antibodies and human sera is highly correlated, unlike recombinant vesicular stomatitis virus-CoV2 S. Single-dose immunization with rOC43-CoV2 S generates high levels of neutralizing antibodies against SARS-CoV-2 and fully protects human ACE2 transgenic mice from SARS-CoV-2 lethal challenge, despite nondetectable replication in respiratory and nonrespiratory organs. rOC43-CoV2 S induces S-specific serum and airway mucosal immunoglobulin A and IgG responses in rhesus macaques. rOC43-CoV2 S has enormous value as a BSL-2 agent to measure S-specific antibodies in the context of a bona fide CoV and is a candidate live attenuated SARS-CoV-2 mucosal vaccine that preferentially replicates in the upper airway.

Keywords: HCoV-OC43; SARS-CoV-2; coronavirus; mucosal vaccine; virus-neutralizing antibodies.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Recovery of rOC43-eGFP and rOC43-CoV2 S. (A) Schematic of CoV genomes and strategy for insertion of SARS-CoV-2 S to rOC43-eGFP genome. The hOC43 genome comprises nine main ORFs: ORF1a, ORF1b, ns2 (nonstructural protein 2), HE (hemagglutinin-esterase), S (spike), ns12.9 (nonstructural protein of 12.9 kDa), E (envelope), M (membrane), and N (nucleocapsid). The replicase gene includes both ORF1a and ORF1b. The eGFP gene is inserted in the 3′ of ORF1b followed by T2A sequence. For rOC43-CoV2 S, the codon-optimized full-length S of SARS-CoV-2 WT (hCoV-19/USA_WA1/2020) was amplified by PCR and replaced with S of the rOC43-eGFP. (B) Representative plaque morphologies in crystal violet-stained RD cells infected with hOC43-WT and rOC43-eGFP at 2 dpi. (C) Growth kinetics of hOC43-WT, rOC43 (recombinant hOC43 virus without eGFP), and rOC43-eGFP over a 2-d time course in RD cells infected at a MOI = 0.01, n = 3 independent experiments with three replicates in each. Statistical analysis to compare each time point for each of the recombinant viruses against hOC43-WT or rOC43-eGFP virus was performed by two-way ANOVA with Tukey’s multiple comparisons test. Mean values of the three independent experiments ± SD of the mean are shown. (D) Infection of Vero_hTMPRSS2 cells with supernatant from cells transfected with the eGFP reporter rOC43-CoV2 S. Images were acquired 72 hpi using a fluorescence microscope. (E) Detection of SARS-CoV-2 S protein in cells lysate by western blot. SARS-CoV-2 S and S2 was detected using anti-SARS-CoV-2 S2 mAb. Anti-OC43-N antibody was used as a loading control (data are representative of two independent experiments). (F) Maximum intensity projections of laser confocal microscopy z-stack images of infected Vero cells and BHK-21_hACE2 cells with rOC43-CoV2 S, stained live at 24 hpi (MOI = 1). (Scale bar, 20 μm.) Images are representative of at least three independent experiments with similar results. DAPI, 4′,6-diamidino-2-phenylindole. (G) Flow cytometry analyses of Vero CCL81 cells inoculated with rOC43-CoV2 S, rOC43-eGFP, or eGFP-expressing SARS-CoV-2 (MOI = 1), stained live at 24 hpi against SARS-CoV-2 S proteins. Representative dot plots of flow cytometry analyses showing double staining of eGFP and surface S, indicating the percentage of the gated cell population for each quadrant of the double staining. Representative histogram of flow cytometry analyses showing staining of surface S proteins, as well as the mean fluorescent intensity (MFI) is plotted showed mean ± SEM (n = 3). Data are representative of at least three independent experiments, each performed with triplicate samples. Data were analyzed using one-way ANOVA (****P < 0.0001).
Fig. 2.
Fig. 2.
Characterization of an infectious rOC43-CoV2 S chimera in cells. (A) Growth kinetics of hOC43-WT and rOC43-CoV2 S over 3 d with cells infected at an MOI = 0.1. Three independent experiments with three replicates each. Statistical analysis comparing each time point was performed by two-way ANOVA with Tukey’s multiple comparisons test (*P < 0.05; **P < 0.01; ***P < 0.001). Mean values of the three independent experiments ± SD of the mean are shown. (B) Stable RD cells with inducible expression of hACE2 induced with the displayed concentration of doxycycline (RD_hACE2_dox) for 48 h, then infected with rOC43-CoV2 S at MOI = 0.01 (in triplicate). The percentage of eGFP-positive cells was quantified by flow cytometry 1 and 2 dpi. (C and D) SARS-CoV-2 RBD-specific and NonRBD mAbs were tested for their capacity to neutralize rOC43-CoV2 S (C) or hOC43-WT (D) (n = 2 and 2, respectively). (E–H) Growth kinetics of SARS-CoV-2 (black) and rOC43-CoV2 S (green) over a 3-d time course in Vero E6 (E), Vero E6_TMPRSS2 (F), A549_hACE2 (G), and Calu-3 cells (H) infected at MOI = 0.01, n = 3 independent experiments with three replicates in each, statistical analysis was performed by two-way ANOVA. (*P < 0.05; **P < 0.01; ***P < 0.001), the bar graph is shown as mean values ± SD. Grid line, limit of detection.
Fig. 3.
Fig. 3.
Pathogenicity of rOC43-CoV2 S in hACE2 transgenic mice and wild-type mice. (A and B) 8-wk-old K18-hACE2 transgenic mice were challenged intranasally with 5 × 103 TCID50 of SARS-CoV-2 WT or rOC43-CoV2 S. Mock-inoculated hACE2 mice were used as control. Weight loss (A) and survival (B) were recorded for 14 d. SARS-CoV-2 infected hACE2 mice displayed significant weight loss compared to rOC43-CoV2 S or mock-inoculated hACE2 mice (n = 8 to 10/group; male and female; two independent experiments). (C and D) Indicated organs were collected at 1, 4, and 7 dpi and infectious virus (C) or viral RNA titers (D) were determined by TCID50 or RT-qPCR, respectively. (n = 8/group at each time point; male and female; two independent experiments; one-way ANOVA with Dunnett’s test was used to determine significance: ***P < 0.001, *P < 0.05; asterisks indicate statistical significance as compared with rOC43-CoV2 S infection; symbols represent means ± SD. (E) Histopathology of rOC43-CoV2 S and SARS-CoV-2 infection of K18-hACE2 mouse, lung tissue of mice killed at 4 and 7 dpi after challenge is shown. Micrographs with 100× (or 400× for inserts) magnification of a representative lung section from each group are shown. (F) Survival curves of 7-d-old C57BL/6 mice after 2 × 104 TCID50 hOC43-WT and rOC43-CoV2 S infection (n = 6 to 8/group; male and female; one independent experiment).
Fig. 4.
Fig. 4.
Correlation analysis of SARS-CoV-2 and rOC43-CoV2 S VN. (A–C) SARS-CoV S mAbs were tested for flow-based VN activity against SARS-CoV-2 (A), rOC43-CoV2 S (B), or rVSV-CoV2 S (C), n = 3. (D) IC50 values of mAbs tested for neutralization of SARS-CoV-2, rOC43-CoV2 S, and rVSV-CoV2 S viruses on Vero E6 cells. (E–G) Human immune sera were tested for VN activity against SARS-CoV-2 (E), rOC43-CoV2 S (F), or rVSV-CoV2 S (G) on Vero E6 cells. (H) 50% neutralizing titer (NT50) values of all human immune sera tested for neutralization of SARS-CoV-2, rOC43-CoV2 S, and rVSV-CoV2 S. (I and J) IC50/NT50 values determined in Fig. 3 A–D and E–H were used to determine correlation between neutralization assays. Spearman’s correlation r and P values are indicated.
Fig. 5.
Fig. 5.
Immunogenicity of rOC43-CoV2 S protects mice against SARS-CoV-2 infection. (A) Scheme depicting vaccination, bleeding, and SARS-CoV-2 challenge. (B–D) IgG serum responses of vaccinated mice were evaluated 4 wk after priming by ELISA for binding to SARS-CoV-2 S (B and D) or RBD (C). Panel D shows IgG heavy chain class of anti-S Abs. n = 3 to 9 per group; Mann–Whitney test: ***P < 0.001. Bars indicate median values. (E) Neutralizing antibody titers were determined by the focus reduction neutralization test (FRNT), n = 3 to 9 per group; Mann–Whitney test: ***P < 0.001. Bars indicate median values. Weights (F) and survival (G) curves of SARS-CoV-2 challenge in vaccinated mice (n = 3 to 9 per group; male and female; two independent experiments).
Fig. 6.
Fig. 6.
Timeline of the rhesus macaque study, rOC43-CoV2 S replication following intranasal immunization of rhesus macaques. (A) Experimental timing for the immunization of four macaques with the rOC43-CoV2 S and sampling schedules are shown. (B) Genomic SARS-CoV-2 S total RNA in upper airway was quantified by RT-qPCR (limit of detection: 2.5 log10 copies/mL). (C and D) Replication of rOC43-CoV2 S in Upper (C) and Lower (D) airways of macaques. Four macaques were immunized intranasally with 6.0 log10 PFU of rOC43-CoV2 S. Nasopharyngeal swabs (UA; daily from days 1 to 9 pi and 14 pi, without day 5, 6 pi), and BALs (LA; day 3 and 14 pi) were performed as described in Materials and Methods. Vaccine virus titers were determined by TCID50; expressed as log10 TCID50/mL (limit of detection: 0.7 log10 TCID50/mL).
Fig. 7.
Fig. 7.
rOC43-CoV2 S induces humoral and mucosal immunity in macaques. (A–C) An ELISA measured anti-S and RBD serum IgG (A), IgA (B), and IgM (C) levels. (D–F) Serum neutralizing titers to SARS-CoV-2 WA1/2020 (D), B.1.617.2/Delta (E), or B.1.1.529/Omicron (F). IC50 titers of sera were determined. The detection limit is 1.0 log10. (G and H) S- and RBD-specific mucosal IgA and IgG titers on indicated days pi in the Upper (G) and Lower (H) airways by ELISA. Endpoint ELISA titers of IgG, IgA, and IgM expressed as log10 values.
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