IgG antibody levels against wild type spike from SARS-CoV-2 (A) and NDV virus preps (B) were measured by ELISA in sera from vaccinated rats at days 0, 15 and 30 after the first vaccine dose administration. induced against the spike of B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variants of concern (VOCs). Importantly, robust levels of serum antibodies with neutralizing activity against the authentic SARS-CoV-2 USA\WA1/2020 isolate were detected Pungiolide A after the boost. Overall, our study expands the pre-clinical safety and immunogenicity characterization of NDV-HXP-S and reinforces previous findings in other animal models about its high immunogenicity. Clinical testing of this vaccination approach is ongoing in different countries including Thailand, Vietnam, Brazil and Mexico. Keywords: COVID-19, SARS-CoV-2, newcastle disease virus, vaccine, immunogenicity, safety, rat model Introduction The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has afflicted over 250 million people worldwide and has caused the death of at least 5 million people (WHO, 2021). To mitigate the burden caused by this virus, diverse countermeasures have been established worldwide, including social distancing, use of face masks, and more recently the rollout of different vaccines (1). Currently, in the United States three vaccines have been authorized to prevent the coronavirus disease 2019 (COVID-19): two mRNA-based approaches, including BNT162b2 (from Pfizer) and mRNA-1273 (from Moderna), and the viral vector-based approach JNJ-78436735 (from Johnson & Johnson) (2C4). Recently, BNT162b2 was fully licensed by the U.S. Food and Drug Administration as the first vaccine to prevent COVID-19 (5). The generation of these vaccines in such a record time and their rollout in the population – with an initial Emergency Use Authorization (EUA) – represents a milestone for vaccine development and science in general. Overall, COVID-19 vaccines CSPG4 have proven to be effective and display a robust safety profile, with very few people experiencing severe adverse effects (6). Moreover, as stated by the World Health Organization (WHO), the potential benefits of vaccination outweigh potential risks (7). Importantly, vaccination has been directly associated with prevention of viral infection (8, 9), reduced virus transmission (10), attenuation of severe disease (11, 12), and reduced viral load during breakthrough infections (13). Mechanisms of vaccine-induced protection involve the induction of neutralizing antibodies, mostly directed against the viral spike protein (14, 15), as well as cellular responses (16, 17). While there are a handful of vaccines widely available in the United States and many regions worldwide, the Pungiolide A number of vaccine doses available and the vaccination rates in places with limited resources is still suboptimal. Particularly, large parts of Africa, Asia, and some countries of Latin-America display poor vaccination rates (18, 19). Moreover, specific populations, such as immunocompromised or transiently immunosuppressed individuals, might require additional vaccine doses to elicit robust immune responses (20C22). Even healthy individuals at high risk, such as healthcare workers and elderly might be in need of multiple vaccine doses (23, 24). Importantly, the current vaccines available display limited induction of mucosal immunity that prevents infection in the upper respiratory tract, as compared to natural infection (25). Hence, expansion of the existing vaccination platforms and development of novel vaccination approaches that elicit robust immune responses – especially in the upper respiratory tract – is imperative. Different COVID-19 vaccines are being currently developed, including: a) additional/alternative mRNA vaccines, b) additional whole virus inactivated vaccines, c) live virus Pungiolide A attenuated vaccines, d) recombinant protein (RBD or spike) based vaccines, e) virus like particle (VLP) based vaccines, f) DNA-based vaccines, and g) additional vector-based vaccines. Within this last category, replication competent, replication incompetent and inactivated vector-based vaccines are in development and replication incompetent vectors are authorized already (26). Here we report the safety and immunogenicity profile of a vaccine candidate (NDV-HXP-S) based on the Newcastle Disease Virus (NDV) – LaSota strain – expressing a modified version of the spike protein on its surface. The altered spike lacks the polybasic cleavage site and includes six prolines to maintain the pre-fusion conformation of the protein. The transmembrane and C-terminal domains of the spike protein were swapped with the corresponding domains of the NDV fusion protein to allow the integration of the spike into NDV particles. We evaluated the local reactogenicity and the systemic toxicity, including post-mortem.