Immunization with Recombinant Accessory Protein-Deficient SARS-CoV-2 Protects against Lethal Challenge and Viral Transmission

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a worldwide coronavirus disease 2019 (COVID-19) pandemic. Despite the high efficacy of the authorized vaccines, there may be uncertain and unknown side effects or disadvantages associated with current vaccination approaches. Live-attenuated vaccines (LAVs) have been shown to elicit robust and long-term protection by the induction of host innate and adaptive immune responses. In this study, we sought to verify an attenuation strategy by generating 3 double open reading frame (ORF)-deficient recombinant SARS-CoV-2s (rSARS-CoV-2s) simultaneously lacking two accessory ORF proteins (ORF3a/ORF6, ORF3a/ORF7a, and ORF3a/ORF7b). We report that these double ORF-deficient rSARS-CoV-2s have slower replication kinetics and reduced fitness in cultured cells compared with their parental wild-type (WT) counterpart. Importantly, these double ORF-deficient rSARS-CoV-2s showed attenuation in both K18 hACE2 transgenic mice and golden Syrian hamsters. A single intranasal dose vaccination induced high levels of neutralizing antibodies against SARS-CoV-2 and some variants of concern and activated viral component-specific T cell responses. Notably, double ORF-deficient rSARS-CoV-2s were able to protect, as determined by the inhibition of viral replication, shedding, and transmission, against challenge with SARS-CoV-2 in both K18 hACE2 mice and golden Syrian hamsters. Collectively, our results demonstrate the feasibility of implementing the double ORF-deficient strategy to develop safe, immunogenic, and protective LAVs to prevent SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Live-attenuated vaccines (LAVs) are able to induce robust immune responses, including both humoral and cellular immunity, representing a very promising option to provide broad and long-term immunity. To develop LAVs for SARS-CoV-2, we engineered attenuated recombinant SARS-CoV-2 (rSARS-CoV-2) that simultaneously lacks the viral open reading frame 3a (ORF3a) in combination with either ORF6, ORF7a, or ORF7b (Δ3a/Δ6, Δ3a/Δ7a, and Δ3a/Δ7b, respectively) proteins. Among them, the rSARS-CoV-2 Δ3a/Δ7b was completely attenuated and able to provide 100% protection against an otherwise lethal challenge in K18 hACE2 transgenic mice. Moreover, the rSARS-CoV-2 Δ3a/Δ7b conferred protection against viral transmission between golden Syrian hamsters.

Keywords: SARS-CoV-2; coronavirus; immune protection; live-attenuated vaccine; viral shedding; viral transmission.

FIG 1

Generation of the double ORF-deficient rSARS-CoV-2 strain. (A) Schematic representations of the double ORF-deficient rSARS-CoV-2 genomes (not drawn to scale). Fragments in the shuttle plasmids that contain a single deletion of the viral ORF6, ORF7a, and ORF7b were released using PpuMI and XhoI restriction enzymes and then were ligated to the PpuMI-XhoI-linearized vector backbone that contains the ORF3a deletion. The fragments in the resultant shuttle plasmids that contain a double deletion of ORF3a/ORF6, ORF3a/ORF7a, and ORF3a/ORF7b were released using BamHI and RsrII digestion and reassembled into the BAC that was linearized with the same restriction enzymes. PCR-positive BAC plasmid colonies were prepared and transfected into Vero E6 cells for virus rescue. (B) Confirmation of the double ORF-deficient rSARS-CoV-2 strain by RT-PCR amplification of ORF3a, ORF6, ORF7a, ORF7b, and N. (C) Deep sequencing analysis of the double ORF-deficient rSARS-CoV-2 P1 genome. Nonreference alleles present in less than 10% of reads are not shown. Amino acid changes respective to rSARS-CoV-2 WT are indicated. (D) Deep sequencing analysis of the double ORF-deficient rSARS-CoV-2 P10 genome. Nonreference alleles present in less than 10% of reads are not shown. Amino acid changes respective to rSARS-CoV-2 WT are indicated.

FIG 2

In vitro characterization of the double ORF-deficient rSARS-CoV-2 strain. (A) Plaque phenotype. Plaque phenotypes of the WT and double ORF-deficient rSARS-CoV-2 strains in Vero E6 cells. Plaques were visualized by immunostaining with a monoclonal antibody (1C7C7) against the viral N protein. (B) Viral plaque size analysis. Six plaques were randomly selected and measured using a standard ruler (millimeters, mm). Data are presented as mean ± SD, and comparisons of diameter means between indicated groups were performed by one-way ANOVA. *, P < 0.05; **, P < 0.01; and ns, not significant. (C) Growth kinetics. Viral growth kinetics of the WT and double ORF-deficient rSARS-CoV-2 strains in Vero E6 (left) and A549-hACE2 (right) cells were measured in triplicate by plaque assay. Dotted lines indicate the limit of detection (LOD). Data are presented as mean ± SEM, and viral titer means of the supernatant collected from the double ORF-deficient rSARS-CoV-2-infected cells are compared with that of the rSARS-CoV-2 WT-infected cells by one-way ANOVA. *, P < 0.05; and **, P < 0.01.

FIG 3

Characterization of double ORF-deficient rSARS-CoV-2 in K18 hACE2 transgenic mice. (A) Schematic representation of the experimental timeline used to infect K18 hACE2 transgenic mice with the WT and double ORF-deficient rSARS-CoV-2 strains. (B) Pathological lesions in the lung surface of K18 hACE2 transgenic mice mock infected or infected (2 × 10⁵ PFU/mouse) with the indicated rSARS-CoV-2 strain at 2 and 4 dpi (n = 4/group). (C) Gross pathological lesion scoring on lung images in B using NIH ImageJ. Data are presented as mean ± SD, and comparisons of means between indicated groups are analyzed by one-way ANOVA. *, P < 0.05; **, P < 0.01; and ns, not significant. (D) Viral titers in the clarified homogenate of lungs (left) and nasal turbinate (right) of K18 hACE2 transgenic mice infected in B at 2 and 4 dpi. The viral titers in the supernatant of the homogenate were determined in triplicate by plaque assay. Data are presented as mean ± SEM, and comparisons of the means between indicated groups were analyzed by one-way ANOVA. *, P < 0.05; and **, P < 0.01. (E) Body weight changes in K18 hACE2 transgenic mice mock infected or infected (2 × 10⁵ PFU/mouse, n = 5/group) with the indicated WT or double ORF-deficient rSARS-CoV-2. (F) Survival curves of K18 hACE2 transgenic mice infected in E were calculated and plotted using daily observations for 21 days. The Kaplan-Meier survival analysis with a log rank (Mantel-Cox) test was applied to compare overall survival time. *, P < 0.05; and **, P < 0.01. (G) The IgG (sera) and IgA (BALF) against the full-length S glycoprotein in mice that survived in F were tested in triplicate by ELISA at 21 dpi. Sera and BALF collected from the two surviving K18 hACE2 transgenic mice infected with rSARS-CoV-2 WT (10³ PFU/mouse, n = 5) for 21 days were included as a positive control. Data are presented as mean ± SEM, and means of the double ORF-deficient rSARS-CoV-2 groups are compared with that of the rSARS-CoV-2 WT group by one-way ANOVA. *, P < 0.05; **, P < 0.01; and ns, not significant. (H) Splenocytes were isolated from the mice that survived in F at 21 dpi, and IFN-γ-specific spot-forming cells (SFCs) were counted (duplicate) after stimulation with peptide pools of S1, S2, and N using flow cytometry. The splenocytes isolated from the two surviving K18 hACE2 mice infected with rSARS-CoV-2 WT (10³ PFU/mouse, n = 5) for 21 days were included as a positive control. Data are presented as mean ± SEM, and comparisons of the means between indicated groups are analyzed by one-way ANOVA. *, P < 0.05; **, P < 0.01; and ns, not significant.

FIG 4

Protection efficacy of rSARS-CoV-2 Δ3a/Δ7b-vaccinated K18 hACE2 transgenic mice against lethal challenge with SARS-CoV-2. (A) Schematic representation of the experimental timeline used for the protection studies with rSARS-CoV-2 Δ3a/Δ7b in K18 hACE2 transgenic mice challenged with rSARS-CoV-2 mCherryNluc. (B) In vivo imaging of K18 hACE2 transgenic mice mock vaccinated or vaccinated with rSARS-CoV-2 Δ3a/Δ7b at 2 and 4 days postchallenge with rSARS-CoV-2 mCherryNluc (n = 4/group). Mock-vaccinated and mock-challenged K18 hACE2 transgenic mice were used as controls. (C) Quantitative analysis of Nluc expression in K18 hACE2 transgenic mice from B. Data are presented as mean ± SD, and comparisons of the means between indicated groups are analyzed by one-way ANOVA. **, P < 0.01; and ns, not significant. (D) Viral replication in the lungs and nasal turbinate of K18 hACE2 transgenic mice at 2 and 4 days postchallenge with rSARS-CoV-2 mCherryNluc. The viral titers in the supernatant of tissue homogenates were determined in triplicate by plaque assay. Data are presented as mean ± SEM, and comparisons of the means between indicated groups are analyzed by Student’s t test. **, P < 0.01. (E) Nluc activity in the clarified lung and nasal turbinate homogenates of the K18 hACE2 transgenic mice was determined at 2 and 4 days postchallenge with rSARS-CoV-2 mCherryNluc. Nluc activities in the supernatant of the tissue homogenates were determined in triplicate under a multiplate reader. Data are presented as mean ± SEM, and comparisons of the means between indicated groups are analyzed by Student’s t test. **, P < 0.01. (F) Body weight changes of mock-vaccinated or rSARS-CoV-2 Δ3a/Δ7b-vaccinated K18 hACE2 transgenic mice were monitored for 15 days after challenge with rSARS-CoV-2 mCherryNluc (n = 5/group). Mock-vaccinated and mock-challenged K18 hACE2 transgenic mice were used as controls (n = 5/group). (G) Survival curves of mock-vaccinated or rSARS-CoV-2 Δ3a/Δ7b-vaccinated K18 hACE2 transgenic mice after challenge with rSARS-CoV-2 mCherryNluc (n = 5/group). Mock-vaccinated and mock-challenged K18 hACE2 transgenic mice were used as controls (n = 5/group). The Kaplan-Meier survival analysis with a log rank (Mantel-Cox) test was applied to compare overall survival time. **, P < 0.01; and ns, not significant.

FIG 5

Double ORF-deficient rSARS-CoV-2 vaccination prevents replication and shedding of SARS-CoV-2 in hamsters. (A) Schematic representation of the experimental timeline used for the protection studies with the double ORF-deficient rSARS-CoV-2 strain in hamsters. (B) Representative images of the H&E-stained lungs of the double ORF-deficient rSARS-CoV-2-infected hamsters (n = 4/group). Scale bars = 1 mm. (C) Quantitative analysis (% inflammation area) of the extent of bronchointerstitial pneumonia in all of the H&E-stained sections was performed using HALO v3.4 software (n = 4/group). Data are presented as mean ± SD, and comparisons of the means between indicated groups are analyzed by one-way ANOVA. *, P < 0.05; and ns, not significant. (D) Viral titers in the clarified homogenate of lungs (left) and nasal turbinate (right) of double ORF-deficient rSARS-CoV-2-infected hamsters at 2 and 4 dpi (n = 4/group). Viral titers in the supernatant of the tissue homogenates were determined in triplicate by plaque assay. Data are presented as mean ± SEM, and comparisons of the means between indicated groups are analyzed by one-way ANOVA. **, P < 0.01. (E) Body weight changes of mock-infected and WT or double ORF-deficient rSARS-CoV-2-infected hamsters were monitored for 21 days (n = 4/group). Data are presented as mean ± SD, and the means of the virus-infected groups were compared with that of the mock control group at 6 dpi by one-way ANOVA. ns, not significant. (F) In vivo imaging of the rSARS-CoV-2 mCherryNluc replication in hamsters at 2 and 4 days postchallenge. (G) Quantitative analysis of Nluc expression in hamsters from C at 2 (left) and 4 (right) days postchallenge with rSARS-CoV-2 mCherryNluc by Aura program. Data are presented as mean ± SD, and comparisons of the means between the indicated groups are analyzed by one-way ANOVA. *, P < 0.05; and **, P < 0.01. (H) Replication of rSARS-CoV-2 mCherryNluc in the lungs and nasal turbinate of challenged and contact hamsters. Viral titers in the supernatant of the tissue homogenates were determined in triplicate by plaque assay. Data are presented as mean ± SEM, and comparisons of the means between indicated groups are analyzed by one-way ANOVA. **, P < 0.01. (I) Nluc activity in the clarified lung and nasal turbinate homogenates of infected and contact hamsters. Nluc activity in the supernatant of the tissue homogenates was determined in triplicate under a multiplate reader. Data are presented as mean ± SEM, and comparisons of the means between indicated groups are analyzed by ANOVA. *, P < 0.05; and **, P < 0.01.

FIG 6

Double ORF-deficient rSARS-CoV-2 vaccination prevents transmission in hamsters. (A) Schematic representation for the experimental timeline used to test the prevention of transmission by double ORF-deficient rSARS-CoV-2 in hamsters. (B) Sera collected at 18 days postvaccination were evaluated for the neutralizing capacity against SARS-CoV-2 WA1/2020, Alpha (α), Beta (β), Delta (δ), and Omicron (ο) VOC by PRMNT assay in quadruplicate (n = 4/group). Data are presented as mean ± SEM. (C) Summary of NT₅₀ values of sera against the different SARS-CoV-2 VOC. n.d., not determined. (D) In vivo imaging of the rSARS-CoV-2 mCherryNluc replication in hamsters at 2 and 4 dpi. (E) Quantitative analysis of Nluc expression in hamsters from D at 2 (left) and 4 (right) dpi by the Aura program. Data are presented as mean ± SD, and comparisons of the means between indicated groups are analyzed by one-way ANOVA. *, P < 0.05; and **, P < 0.01. (F) Replication of rSARS-CoV-2 mCherryNluc in the lungs and nasal turbinate of infected donor and vaccinated contact hamsters. Viral titers in the supernatant of the tissue homogenates were determined in triplicate by plaque assay. Data are presented as mean ± SEM, and comparisons of the means between indicated groups are analyzed by One-way ANOVA. *, P < 0.05. (G) Nluc activity in the clarified lung and nasal turbinate tissue homogenates of infected donor and vaccinated contact hamsters. Nluc activity in the supernatant of the tissue homogenates was determined in triplicate under a multiplate reader. Data are presented as mean ± SEM, and comparisons of the means between indicated groups are analyzed by one-way ANOVA. *, P < 0.05; and **, P < 0.01.