Intranasal vaccination of hamsters with a Newcastle disease virus vector expressing the S1 subunit protects animals against SARS-CoV-2 disease

Abstract

The coronavirus disease-19 (COVID-19) pandemic has already claimed millions of lives and remains one of the major catastrophes in the recorded history. While mitigation and control strategies provide short term solutions, vaccines play critical roles in long term control of the disease. Recent emergence of potentially vaccine-resistant and novel variants necessitated testing and deployment of novel technologies that are safe, effective, stable, easy to administer, and inexpensive to produce. Here we developed three recombinant Newcastle disease virus (rNDV) vectored vaccines and assessed their immunogenicity, safety, and protective efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in mice and hamsters. Intranasal administration of rNDV-based vaccine candidates elicited high levels of neutralizing antibodies. Importantly, the nasally administrated vaccine prevented lung damage, and significantly reduced viral load in the respiratory tract of vaccinated animal which was compounded by profound humoral immune responses. Taken together, the presented NDV-based vaccine candidates fully protected animals against SARS-CoV-2 challenge and warrants evaluation in a Phase I human clinical trial as a promising tool in the fight against COVID-19.

Figure 1

The insertion of the expression cassette into the non-coding region between the P/M genes of NDV genome was verified by RT-PCR using the primers NDV-3LS1-2020-F1 and NDV-3LS1-2020-R1.

Figure 2

Expression of SARS-CoV-2 RBD and S1 proteins in infected Vero-E6 cells and NDV particles. (A) Western blot detection for the HN-RBD and S1-F proteins expression. Vero-E6 cells were infected with the rLS1, rLS1-HN-RBD, and rLS1-S1-F viruses at an MOI of 1.0. After 48 hpi, the cells were lysed and analyzed by Western blotting. (B) To verify the incorporation of the HN-RBD and S1-F proteins into rLS1-HN-RBD, and rLS1-S1-F viruses, the viral particles in allantoic fluid of infected SPF chicken embryonated eggs with the recombinant viruses and rLS1, was concentrated by ultracentrifugation, and partially purified on a 25% sucrose cushion. Western blot analysis was carried out using partially purified viruses and lysate from infected cells, using a rabbit antibody specific to SARS-CoV-2 RBD protein and Anti-rabbit IgG conjugated to HRP. The beta-actin protein was used as a loading control in lysate cells. The black arrow indicates the expected protein band. The gels shown were run under identical conditions. (C) Vero-E6 cells infected with the rLS1, rLS1-HN-RBD, and rLS1-S1-F at an MOI of 0.5. After 48 hpi, the expression of RBD and S1 proteins was detected by immunofluorescence assay using a rabbit antibody specific to SARS-CoV-2 RBD protein, and a Donkey Anti-rabbit IgG H&L-Alexa Fluor 594. Therefore, the NDV was detected using a chicken antiserum specific to the NDV, and a Goat Anti-chicken IgY H&L-Alexa Fluor 488. Cell nuclei were stained with DAPI. A scale bar of 50-µm. Image magnification 200×. (D) Detection of S1 or RBD proteins on the viral surface of rLS1-S1-F and rLS1-HN-RBD viruses’ attachment to Vero-E6 cells was performed in two independent experiments. The cells were incubated with purified viruses rLS1-HN-RBD or rLS1-S1-F, for 30 min. Subsequently, the cells were labeled with rabbit monoclonal antibody anti-SARS-CoV-2 S1 as the primary antibody, followed by secondary antibody goat anti-rabbit IgG Alexa Fluor 488. The cells were then analyzed by a flow cytometer. The percentage of positive cells indicates the detection of S1 or RBD proteins on the viral surface of viruses bound to Vero-E6 and is shown in the dot plot for rLS1-S1-F virus and rLS1-HN-RBD virus; including negative controls for each assay determined by cells incubated with DPBS or rLS1 virus.

Figure 3

Genetic stability of the recombinants. The genetic stability of the rLS1-HN-RBD, and rLS1-S1-F viruses was evaluated at the 3rd and 6th passages by (A) Western blot analysis using a rabbit polyclonal antibody specific to SARS-CoV-2 RBD protein, and (B) RT-PCR using the primers NDV-3LS1-2020-F1 and NDV-3LS1-2020-R1 to amplify the complete inserts. P3: 3rd passage, P6: 6th passage. Black arrow indicates the expected molecular mass.

Figure 4

Stability of the lyophilized NDV vaccine. The expression of S1-F and HN-RBD proteins in Vero-E6 cells infected with the lyophilized NDV vaccine was confirmed at day 1, 30, and 50 days post-lyophilization by Western blot assay using a rabbit polyclonal antibody specific to SARS-CoV-2 RBD protein. Black arrow indicates the expected protein band.

Figure 5

The intranasal vaccine elicits specific antibodies against RBD and S1 protein and neutralizing antibodies against SARS-CoV-2 in hamsters. (A) Immunization regimen. To evaluate the immunogenicity of the NDV vaccines, five- week-old female and male golden Syrian hamsters were used in this study. The hamsters were randomly divided into four groups. The hamsters were vaccinated by intranasal route with live NDV vaccine, following a prime-boost- regimen with a two-week interval. Group 1 received rLS1-HN-RBD (n = 12), Group 2 received the rLS1-S1-F (n = 12), Group 3 received the mixture of rLS1-HN-RBD/rLS1-S1-F (n = 12), and Group 4 was the non-vaccinated control group (n = 12) One booster immunization with the same concentration of each vaccine was applied in all vaccinated groups at the second week. (B) ELISA assay to measure SARS-CoV-2 RBD-specific serum IgG antibody, and (C) S1 subunit-specific serum IgG antibody. Sera from hamsters at pre-boost and 15 days after boost were evaluated. SARS-CoV-2 RBD purified recombinant protein was used for ELISA. The cutoff was set at 0.06. (D). Immunized hamsters were bled pre-boost and 15 days after boost. All sera were isolated by low-speed centrifugation. Serum samples were processed to evaluate the neutralizing antibody titers against SARS-CoV-2 RBD protein using the sVNT. The positive cut-off and negative cut-off for SARS-CoV-2 neutralizing antibody detection were interpreted as the inhibition rate. The cut-off interpretation of results: result positive ≥ 20% (neutralizing antibody detected), result negative < 20% (neutralizing antibody not detectable. (E) Figure depicts titers of PRNT of SARS-CoV-2 on Vero cells with pooled serum from hamsters immunized with rLS1-S1-F, rLS1-HN-RBD, and the mixture of both. (F) Plaque reduction (%) curves using pooled serum from the different groups of hamsters. Two-way ANOVA and Tukey’s post hoc were performed. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 6

Efficacy of live NDV vaccines against SARS-CoV-2 infection in hamsters. Golden Syrian hamster groups vaccinated with rLS1-S1-F, rLS1-HN-RBD, the mixture rLS1-S1-F/rLS1-HN-RBD, or non-vaccinated (positive control) were challenged 30 days after the boost with SARS-CoV-2, along with a mock group that was non-vaccinated nor challenged (negative control). (A) Viral isolation (% of animals that were positive for SARS-CoV-2) was done from the lung of each hamster group (n = 4) at days 2, 5, and 10 post-challenge. Two-way ANOVA and Tukey’s post hoc were performed. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (B) Detection by RT-qPCR of SARS-CoV-2 in culture supernatant of Vero cells, inoculated with lung homogenates of immunized and challenged hamster groups. The data show a significant difference in the viral copy number *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant. (C) Lung histopathology of each hamster group (n = 4) was euthanized at different days post-infection (DPI). Hemorrhagic and infiltrated areas are indicated by a yellow and black arrow, respectively. Image amplitude: 20×. Scale-bar: 100 µm.

Figure 7

Body weight and mobility analysis of SARS-CoV-2 challenged golden Syrian hamsters. (A) Changes in body weight (percent weight change compared to day 0) of hamsters inoculated with SARS-CoV-2 and Mock group, at days 2, 5, and 10 post-challenged. Mobility assessment results shown (B) average velocity, (C) average acceleration, and (D) average displacement. Mean ± SD are shown. Asterisks indicate that results were statistically significant compared to the control group (*P < 0.05).

Figure 8

The strategy used for the generation of the recombinant NDVs expressing SARS-CoV-2 RBD and S1. (A) The schematic representation of the strategy of construction of the recombinant NDVs. Two transcriptional cassettes were designed for expressing RBD and S1: 1) HN-RBD was fused with the complete transmembrane domain (TM) and the cytoplasmic tail (CT) of the haemagglutinin–neuraminidase (HN) gene, 2) S1-F was fused with the TM/CT of the fusion (F) gene from the full-length pFLC-LS1. (B) The full-length anti-genome of NDV strain LaSota clone (pFLC-LS1) was used as a backbone clone, the pFLC-LS1-HN-RBD and pFLC-LS1-S1-F were generated from cassettes expressing RBD and S1 genes inserted into NDV genome under control of transcriptional gene end (GE) and gene start (GS) signals. The names, position, and direction of the primers used are shown with arrows (blacks) indicating the size products.