key: cord-0878498-0m2hqvxj
authors: Sun, Weina; McCroskery, Stephen; Liu, Wen-Chun; Leist, Sarah R.; Liu, Yonghong; Albrecht, Randy A.; Slamanig, Stefan; Oliva, Justine; Amanat, Fatima; Schäfer, Alexandra; Dinnon, Kenneth H.; Innis, Bruce L.; García-Sastre, Adolfo; Krammer, Florian; Baric, Ralph S.; Palese, Peter
title: A Newcastle disease virus (NDV) expressing membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine
date: 2020-07-31
journal: bioRxiv
DOI: 10.1101/2020.07.30.229120
sha: a9668fccfa6f826eea6de30514e49bfa9200a93a
doc_id: 878498
cord_uid: 0m2hqvxj

A successful SARS-CoV-2 vaccine must be not only safe and protective but must also meet the demand on a global scale at 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 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 or significantly attenuated SARS-CoV-2 induced disease. 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.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine is urgently needed to mitigate 41 the current coronavirus disease 2019 (COVID-19) pandemic worldwide. Numerous vaccine approaches 42 are being developed (1) (2) (3) (4) , however, many of them are not likely to be cost-effective and affordable by 43 low-income countries and under-insured populations. This could be of concern in the long run, as it is 44 crucial to vaccinate a larger population than the high-income minority to establish herd immunity and 

We have previously reported the construction of Newcastle disease virus (NDV)-based viral vectors 61 expressing a pre-fusion spike protein, whose transmembrane domain and cytoplasmic tail were replaced 62 with those from the NDV fusion (F) protein (S-F chimera) (10). We have shown that these NDV vector 63 vaccines grow well in embryonated chicken eggs, and that the SARS-CoV-2 spike (S) proteins are 64 abundantly incorporated into the NDV virions. The NDV vector, based on a vaccine virus strain against 65 an avian pathogen, overcomes the abovementioned limitation for viral vector vaccines and allows the 66 manufacturing of the vaccine prior to its inactivation under BSL-2 conditions. In this study, we 67 investigated an attenuated recombinant NDV expressing the membrane-anchored S-F chimera (NDV-S) 68 as an inactivated SARS-CoV-2 vaccine candidate with and without an adjuvant in mice and hamsters. We 69 found that the S-F chimera expressed by the NDV vector is very stable with no antigenicity loss after 3 70 weeks of 4°C storage in allantoic fluid. The beta-propiolactone (BPL) inactivated NDV-S vaccine is 71 immunogenic, inducing high titers of S-specific antibodies in both animal models. Furthermore, the 72 effects of a clinical-stage investigational liposomal suspension adjuvant (R-enantiomer of the cationic 73 lipid DOTAP, R-DOTAP) (11) (12) (13) (14) , as well as an MF-59 like oil-in-water emulsion adjuvant (AddaVax) 74 were also evaluated in mice. Both adjuvants were shown to achieve dose sparing (>10 fold) in mice. The 75 vaccinated animals were protected from SARS-CoV-2 infection or SARS-CoV-2 induced disease. This is 76 encouraging as the existing global egg-based production capacity for inactivated influenza virus vaccines 77 could be utilized immediately to rapidly produce egg-based NDV-S vaccine with minimal modifications 78 to their production pipelines. Most importantly, this class of products is amenable to large-scale 79 production at low cost and has an excellent safety profile in infants, pregnant women and the elderly (15-80 17). Alternatively, the NDV-S and other chimeric NDV vaccines can also be manufactured in cultured 81 cells such as Vero cells (18).

The construction of NDV_LS/L289A_S-F rescue plasmid has been described in a previous study (10). was concentrated immediately after centrifugation as described above through a 20% sucrose cushion.

The pelleted virus was re-suspended in 300 µL phosphate buffered saline (PBS) and stored at -80°C. The 155 other three aliquots of the allantoic fluid were maintained at 4°C to test the stability of the S-F construct. 156 Wk 1, 2 and 3 samples were collected consecutively on a weekly basis, and concentrated virus was 157 prepared in 300 µL PBS using the same method. The protein content of the concentrated virus from wk 0, 158 1, 2, and 3 was determined using BCA assay after one free-thaw from -80°C. One microgram of each 159 concentrated virus preparation was resolved on a 4-20% sodium dodecyl sulfate polyacrylamide gel 

This approach should be suited to safely induce spike-specific protective antibodies (Fig. 1B) .

The spike protein expressed on NDV virions is stable in allantoic fluid

The stability of the antigen could be of concern as the vaccine needs to be purified and inactivated 278 through a temperature-controlled (~4°C) process. The final product is often formulated and stored in 279 liquid buffer at 4°C. To examine the stability of the S-F protein, allantoic fluid containing the NDV-S live 280 virus was aliquoted into equal volumes (15 ml), and stored at 4°C. Samples were collected weekly (wk 0, 281 1, 2, 3) and concentrated through a 20% sucrose cushion. The concentrated virus was re-suspended in 282 equal amounts of PBS. The total protein content of the 4 aliquots was comparable among the preparations 283 (wk 0: 0.94 mg/ml; wk 1: 1.04 mg/ml; wk 2: 0.9 mg/ml; wk 3: 1.08 mg/ml). The cold stability of the S-F 284 construct was evaluated by Western blot with the anti-S monoclonal antibody, 2B3E5. As compared to 285 the stability of the NDV HN protein, the Spike protein remained stable when kept in allanotic fluid at 286 4 °C ( Fig. 2A) . Moreover, the inactivation procedure using 0.05% BPL did not cause any loss of 287 antigenicity of the S-F, as evaluated by Western blot (Fig. 2B) . These observations demonstrate that the 288 membrane anchored S-F chimera expressed by the NDV vector is very stable without degradation at 4°C 289 for 3 weeks or when treated with BPL for inactivation. (Fig. 3A) . Mice were bled pre-boost (2 weeks after prime) and 11 days post-boost to 301 examine antibody responses by ELISA using a trimeric full-length S protein as the substrate (19), and 302 micro-neutralization assay using the USA-WA1/2020 strain of SARS-CoV-2 (Fig.3A) . After one 303 immunization, all vaccination groups developed S-specific antibodies. The boost greatly increased the 304 antibody titers of all NDV-S immunization groups. Immunization with R-DOTAP combined with 5 µg of 305 vaccine induced the highest antibody titer. Immunization with one microgram of vaccine formulated with 306 R-DOTAP or AddaVax and 5 µg of vaccine with AddaVax induced comparable levels of binding 307 antibody, which is also similar to the titers induced by 20 µg of vaccine without an adjuvant. As expected, 308 immunization with the inactivated wild type NDV virus did not induce S-specific antibody responses 309 (Fig. 3B) . We performed microneutralization assays to determine the neutralizing activity of serum 

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We thank Dr. Benhur Lee to kindly share the BSRT7 cells. We also thank Dr. Thomas Moran for the 2B3E5 408 and 1C7 antibodies. This work was partially supported by an NIAID funded Center of Excellence for