Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate

Abstract

Background: Due to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus.

Methods: Here, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated.

Findings: We report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.

Interpretation: The results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2.

Funding: This work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants.

Keywords: Coronavirus vaccine; Intramuscular administration; Live COVID-19 vaccine; Mouse-adapted SARS-CoV-2; Neutralizing antibodies; Viral vector vaccine.

Conflict of interest statement

Declaration of Competing Interests The Icahn School of Medicine at Mount Sinai has filed patent applications entitled “RECOMBINANT NEWCASTLE DISEASE VIRUS EXPRESSING SARS-COV-2 SPIKE PROTEIN AND USES THEREOF” (63/057,267), in which W.S., F.K., A.G.S and P.P. were listed as inventors. A.G.S and P.P. also declared COI as consultants for AviMex and patents entitled “USE OF RECOMBINANT NDV EXPRESSING CHIMERIC ANTIGENS FOR VETERINARIAN VACCINE” (9387,242) and “USE OF RECOMBINANT NDV FOR ONCOLYTIC THERAPIES” (10,251,922). S.R.L, S.M., Y.L, S.S., J.O, F.A., A.S, K.H.D. and R.S.B. have nothing to declare

Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1

NDV vectors expressing the spike protein of SARS-CoV-2. (a) Two forms of spike proteins expressed by NDV. Spike (S) has the wild type amino acid sequence. The Spike-F chimera (S-F) consists of the ectodomain of S without the polybasic cleavage site and the transmembrane domain (TM) and cytoplasmic tail (CT) of the F protein from NDV. (b) Illustration of genome structures of wild type NDV LaSota (WT NDV_LS), NDV expressing the S or S-F in the wild type LaSota backbone (NDV_LS_S or NDV_LS_S-F) or NDV expressing the S-F in the L289A mutant backbone (NDV_LS/L289A_S-F). The L289A mutation supports the HN-independent fusion of the F protein. (c) Titers of NDV vectors grown in embryonated chicken eggs. The rescued viruses were grown in 10-day old embryonated chicken eggs for 2 or 3 days at 37 °C at limited dilutions. The peak titers of each virus were determined by immunofluorescence assay (IFA). The error bars represent standard deviation (SD).

Fig. 2

Expression of spike protein in infected cells and NDV particles. (a) Expression of the S and S-F protein in infected cells. Vero E6 cells were infected with three NDV vectors encoding the S or S-F for 16 to 18 h. A WT NDV control was included. The next day, cells were fixed with methanol-free paraformaldehyde. Surface proteins were stained with anti-NDV rabbit serum or a spike receptor-binding domain (RBD)-specific monoclonal antibody CR3022. A scale bar of 200 µM were shown. (b) Incorporation of S and S-F into NDV particles. Three NDV vectors expressing the S or S-F including the NDV_LS_S (green), NDV_LS_S-F (red) and NDV_LS/L289A_S-F (blue) were concentrated through a 20% sucrose cushion. Two clones were shown for NDV_LS_S and NDV_LS_S-F. The concentrated WT NDV expressing no transgenes was used as a control. Two micrograms of each concentrated virus were resolved on a 4–20% SDS-PAGE, the spike protein and NDV HN protein were detected by western blot using an anti-spike 2B3E5 mouse monoclonal antibody and an anti-HN 8H2 mouse monoclonal antibody.

Fig. 3

NDV vector vaccines elicit high titers of binding and neutralizing antibodies in mice. (a) Vaccination groups and regimen. A prime-boost vaccination regimen was used with a three-week interval. Mice (n = 5) were bled pre-boost and 8 days after the boost. Mice were challenged with a mouse-adapted SARS-CoV-2 MA strain 11 days after the boost. A total of ten groups of mice were used in a vaccination and challenge study. Group 1 (10 µg) and 2 (50 µg) received the WT NDV; Group 3 (10 µg) and 4 (50 µg) received the NDV_LS_S; Group 5 (10 µg) and 6 (50 µg) received NDV_LS_S-F; Group 7 (10 µg) and 8 (50 µg) received NDV_LS/L289A_S-F; Group 9 received PBS as negative controls. An age-matched healthy control group 10 was provided upon challenge. (b) Spike-specific serum IgG titers measured by ELISAs. Sera from animals at 3 weeks after-prime (patterned bars) and 8 days after-boost (solid bars) were isolated. Serum IgG was measured against a recombinant trimeric spike protein by ELISAs. The endpoint titers (mean with SD) were calculated as the readout for ELISAs. (c) Neutralization titers of serum antibodies. Sera from 3 weeks after-prime and 8 days after-boost were pooled within each group. Technical duplicates were performed to measure neutralization activities of serum antibodies using a USA-WA1/2020 SARS-CoV-2 strain. The ID50 value was calculated as the readout of the neutralization assay. For the samples (WT NDV and PBS groups) showing no neutralizing activity in the assay, an ID50 of 10 was given as the starting dilution of the sera is 1:20 (LoD: limit of detection). The error bars represent SD.

Fig. 4

NDV vector vaccines protected mice from the SARS-CoV-2 challenge. (a) Viral titers in the lungs. All mice were infected intranasally with 104 PFU SARS-CoV-2 MA strain except the healthy control group, which was mock infected with PBS. At day 4 post-challenge, lungs (n = 3) were collected and homogenized in PBS. Viral titers in the lung homogenates were determined by plaque assay. Plaque-forming units (PFU) per lung lobe was calculated. Geometric mean titer was shown for all the groups (LoD: limit of detection). Statistical analysis was performed using one-way analysis of variance (ANOVA), and corrected for multiple comparisons using Dunnett's test (ns, not significant; **, p<0.01). The error bars represent SD. (b) Immunohistochemistry (IHC) staining of lungs. A SARS-CoV-2 NP specific antibody was used for IHC to detect viral antigens. Slides were counterstained with hematoxylin. A presentative image was shown for each group. The brown staining indicates the presence of NP protein of SARS-CoV-2. A scale bar of 100 µm is shown.