A Newcastle Disease Virus (NDV) Expressing a Membrane-Anchored Spike as a Cost-Effective Inactivated SARS-CoV-2 Vaccine

Abstract

A successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine must not only be safe and protective, but must also meet the demand on a global scale at a low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent coronavirus disease 2019 (COVID-19) vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine.

Keywords: COVID-19; adjuvant; antigen-sparing; egg-based vaccine; hamster model; mouse-adapted SARS-CoV-2.

Figure 1

Design and concept of an inactivated Newcastle disease virus (NDV)-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. (A) Design of the NDV-S vaccine. The sequence of the spike-fusion (S-F) chimera (green: ectodomain of S, and black: the transmembrane domain and cytoplasmic tail of the NDV F protein) was inserted between the P and M gene of the NDV LaSota (NDV_LS) strain L289A mutant (NDV_LS/L289A). NDV-S: NDV_LS/L289A_S-F. The polybasic cleavage site of the S was removed (⁶⁸²RRAR⁶⁸⁵ to A). (B) The concept overview of an inactivated NDV-based SARS-CoV-2 vaccine. The NDV-S vaccine could be produced using the current global influenza virus vaccine production capacity. Such an NDV-S vaccine displays abundant S proteins on the surface of the virions. The NDV-S vaccine could be inactivated by beta-propiolactone (BPL). The NDV-S vaccine could be administered intramuscularly (i.m.) to elicit protective antibody responses in humans.

Figure 2

The S-F chimera is stable. (A) Stability of the S-F chimera at 4 °C. Allantoic fluid containing the NDV-S virus was aliquoted into equal amounts (15 mL) and stored at 4 °C. Virus from each aliquot was concentrated through a 20% sucrose cushion, re-suspended in an equal amount of phosphate buffered saline (PBS), and then stored at −80 °C for several weeks (wk 0, wk 1, wk 2, and wk 3). One microgram of each concentrated virus was resolved onto 4–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein degradation was evaluated by Western blot using the S-specific mouse monoclonal antibody 2B3E5. The hemagglutinin-neuraminidase (HN) protein of NDV was used as an NDV protein control. (B) Inactivation of the virus by beta-propiolactone (BPL). Viruses in the allantoic fluid were inactivated by 0.05% BPL, as described previously. Clarified allantoic fluids with live and inactivated viruses were diluted in PBS (at 1000-fold dilution) and inoculated into 10-day-old embryonated chicken eggs. The eggs were incubated at 37 °C for 3 days. The loss of infectivity of the inactivated virus was confirmed by the lack of growth of the virus determined by a hemagglutination (HA) assay. (C) Stability of the S-F before and after BPL inactivation. Live or inactivated (using 0.05% BPL) NDV-S virus was concentrated through a 20% sucrose cushion, as described previously. Two micrograms of live or BPL-inactivated virus were loaded onto 4–20% SDS-PAGE. Stability loss of the S-F was evaluated by Western blot, as described in A.

Figure 3

Inactivated NDV-S vaccine elicits high antibody responses in mice. (A) Immunization regimen and groups. BALB/c mice were given two immunizations via the intramuscular administration route with a 2-week interval. Mice were bled pre-boost and 11 days after the boost for in vitro serological assays. Mice were challenged with a mouse-adapted SARS-CoV-2 strain 19 days after the boost. Ten groups described in the table were included in this study. Group 1, 2, and 3 were immunized with 5, 10, and 20 μg of vaccine, respectively; group 4, 5, and 6 were immunized with 0.2, 1, and 5 μg of vaccine formulated with the R-enantiomer of the cationic lipid DOTAP (R-DOTAP), respectively; group 7, 8, and 9 were immunized with 0.2, 1, and 5 μg of vaccine combined with AddaVax, respectively; and group 10 was immunized with 20 μg of WT NDV virus as the vector-only control. (B) Spike-specific serum IgG titers. Serum IgG titers from animals after prime (pattern bars) and boost (solid bars) toward the recombinant trimeric spike protein were measured by an enzyme linked immunosorbent assay (ELISA). Endpoint titers were shown as the readout for ELISA. (C) Neutralization titers of serum antibodies. Microneutralization assays were performed to determine the neutralizing activities of serum antibodies from animals after the boost (D26) using the USA-WA1/2020 SARS-CoV-2 strain. The 50% of inhibitory dilution (ID₅₀) of serum samples showing no neutralizing activity (WT NDV) was set as 10 (LoD: limit of detection).

Figure 4

Inactivated NDV-S vaccine protects mice from SARS-CoV-2 infection. (A) Weight loss of mice infected with SARS-CoV-2. Weight loss of mice challenged with a mouse-adapted SARS-CoV-2 strain was monitored for 4 days. (B) Viral titers in the lung. Lungs of mice were harvested at day 4 post-infection. Viral titers of the lung homogenates were determined by a plaque assay. Geometric mean titer (PFU/lobe) is shown (LoD: limit of detection). Statistical analysis was performed using the Kruskal–Wallis test with Dunn’s correction for multiple comparisons. P-values between groups were shown.

Figure 5

Inactivated NDV-S vaccine attenuates SARS-CoV-2 infection in hamsters. (A) Immunization regimen and groups. Golden Syrian hamsters were vaccinated with inactivated NDV-S following a prime-boost regimen with a 2-week interval. Hamsters were challenged 24 days after the boost with the USA-WA1/2020 SARS-CoV-2 strain. Four groups of hamsters (n = 8) were included in this study. Group 1 received 10 μg of inactivated NDV-S vaccine without any adjuvant. Group 2 received 5 μg of inactivated NDV-S vaccine adjuvanted with AddaVax. Group 3 receiving the 10 μg of inactivated WT NDV was included as the vector-only (negative) control. Group 4 animals receiving no vaccine were mock challenged with PBS as healthy controls. (B) Spike-specific serum IgG titers. Hamsters were bled pre-boost and a subset of hamsters were terminally bled at 2 days post-infection (dpi). Vaccine-induced serum IgG titers towards the trimeric spike protein were determined by ELISA. Endpoint titers are shown as the readout for ELISA. (C) Weight loss of hamsters challenged with SARS-CoV-2. Weight loss of SARS-CoV-2-infected hamsters was monitored for 5 days. (D) Viral titers in the lungs. Viral titers in the upper right (UR) and lower right (LR) lung lobes of the animals at 2 and 5 dpi were measured by a plaque assay (LoD: limit of detection). Statistical analysis was performed using the Kruskal–Wallis test with Dunn’s correction for multiple comparisons. P-values between groups were shown.