Intramuscular administration of recombinant Newcastle disease virus expressing SARS-CoV-2 spike protein protects hACE-2 TG mice against SARS-CoV-2 infection

Coronavirus disease 2019 (Covid-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) became a pandemic, causing significant burden on public health worldwide. Although the timely development and production of mRNA and adenoviral vector vaccines against SARS-CoV-2 have been successful, issues still exist in vaccine platforms for wide use and production. With the potential for proliferative capability and heat stability, the Newcastle disease virus (NDV)-vectored vaccine is a highly economical and conceivable candidate for treating emerging diseases. In this study, a recombinant NDV-vectored vaccine expressing the spike (S) protein of SARS-CoV-2, rK148/beta-S, was developed and evaluated for its efficacy against SARS-CoV-2 in K18-hACE-2 transgenic mice. Intramuscular vaccination with low dose (106.0 EID50) conferred a survival rate of 76 % after lethal challenge of a SARS-CoV-2 beta (B.1.351) variant. When administered with a high dose (107.0 EID50), vaccinated mice exhibited 100 % survival rate and reduced lung viral load against both beta and delta variants (B.1.617.2). Together with the protective immunity, rK148/beta-S is an accessible and cost-effective SARS-CoV-2 vaccine.


Introduction
In 2019, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) had an outbreak in Wuhan, China and was declared a pandemic by the World Health Organization (WHO) in March 2020, causing devastating consequences for the world's population, resulting in >6 million deaths worldwide and emerging as the most significant global health crisis worldwide until 2022 [1]. The outcome of SARS-CoV-2 infection ranges from asymptomatic to severe pneumonia, potentially leading to intensive care unit admission or death [2]. SARS-CoV-2 belongs to the Betacoronavirus genus under the Coronaviridae family and is a non-segmented, single-stranded, positive-sense RNA virus with a genome size of 29.8-29.9 kb. SARS-CoV-2 has spike (S), nucleoprotein (N), membrane (M), and envelope (E) structural proteins [3]. The SARS-CoV-2 S protein plays a critical role in viral attachment to angiotensin-converting enzyme 2 (ACE-2) on the cell surface, membrane fusion, and internalization [4]. The receptor-binding domain (RBD) in the S1 subunit of the S protein plays a crucial role in interacting with the ACE-2 receptor of the host cell. This specific region is widely recognized as the main target for antibodies against SARS-CoV-2. [5,6]. Since the emergence of SARS-CoV-2 in 2019, many variants have been identified until 2022. WHO has designated alpha (B. 1.1.7), beta (B. 1.351), gamma (P. 1), delta (B. 1.617.2), and omicron (B. 1.1.529) as variants of concern [7]. Three SARS-CoV-2 vaccines have been supplied worldwide, including two mRNA vaccines; BNT162b2 from Pfizer [8] and mRNA-1273 from Moderna [9], and an adenovirus-based vaccine, ChAdOx1 from AstraZeneca [10]. In the meantime, various viral vector vaccines are under clinical trials or development against SARS-CoV-2; measles vaccine strain, vesicular stomatitis virus, horsepox, and Newcastle disease virus (NDV) [11].
Newcastle disease virus belongs to the genus orthoavulavirus family of Paramyxoviridae and is a non-segmented, singlestranded, negative-sense RNA virus with a genome size of 15.2 kb. The NDV genome encodes six structural proteins (and at least two non-structural proteins): nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and large protein (L) [12]. For veterinary purposes, NDVs are categorized into lentogenic (avirulent), mesogenic (moderately virulent), and velogenic (severe virulent) based on pathogenicity testing in chickens. While mesogenic or velogenic NDV causes serious diseases in poultry [13], human infection manifests as conjunctivitis only when the infection occurs through a wound in the eyes and rarely exhibits very mild respiratory symptoms [14]. With the ability of NDV to propagate to a high titer in eggs and cells [15], it has been tested for developing viral vector vaccines through reverse genetics [15,16]. In our previous study, a lentogenic NDV, K148/08 strain was found to have the advantages of high propagation and heat resistance [17].
To further explore the potential of the K148/08 strain as a viral vector vaccine, a recombinant NDV K148/08 vector SARS-CoV-2 vaccine expressing the S protein of the SARS-CoV-2 beta variant (rK148/beta-S) was developed, and vaccine efficacy was evaluated in mice against lethal infections caused by two different SARS-CoV-2 variants.

Virus and cells
Hep-2 (CCL-81; American Type Culture Collection, USA) and Vero-E6 (CRL-1586; American Type Culture Collection) cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 8 % fetal bovine serum (Biowest, France) and antibiotics at 37°C in a 5 % CO 2 incubator. SARS-CoV-2 beta and delta variants were obtained from the Korea Disease Control and Prevention Agency. SARS-CoV-2 was propagated in the Vero-E6 cell line. The modified vaccinia Ankara T7 recombinant virus (MVA-T7) and K148/08 were prepared as previously described [18]. All experiments using viable SARS-CoV-2 viruses were conducted in a biosafety level (BSL)-3 facility (Konkuk University) in accordance with the procedures approved by KU22050.

RNA extraction and reverse transcription
The viral RNA of the SARS-CoV-2 beta variant, and rK148/beta-S were extracted using a RNeasy kit (Qiagen, Germany). The complementary DNA (cDNA) of the SARS-CoV-2 beta variant S, and rK148/ beta-S were synthesized using the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific, USA).

Construction of rK148/beta-S plasmid
The construction of the full-genome plasmid of the K148 NDV vector (pK148) was described in a previous study [18]. pK148 was linearized to insert the SARS-CoV-2 beta variant S gene and an additional cDNA fragment containing the noncoding region (NCR) between the P and M genes of the full K148/08 genome. To enhance the expression of the S gene, two nucleotides at the 3 0 end of the NCR were substituted with the Kozak sequence (GCC ACC) using a Muta-direct TM site-directed mutagenesis kit (iNtRON Biotechnology, Korea). The cDNA of the NCR and S gene were ligated and inserted between the P and M genes of pK148 using an In-fusion PCR cloning kit (Clontech Laboratories Inc., USA), forming a pK148/beta-S plasmid. The pK148/beta-S plasmid was transformed into HIT-DH5a competent cells (RBC Bioscience, Taiwan), incubated at 37°C for 18 h, and purified using the PureLink TM Hipure plasmid midiprep kit (Thermo Fisher Scientific).

Recombinant virus rescue and propagation
The pK148 or pK148/beta-S plasmids with three supporting plasmids were cotransfected into Hep-2 cells. Hep-2 cells seeded in six-well plates were infected with MVA-T7 virus to utilize T7 transcriptase. A mixture of 8.8 lg of pK148 or pK148/beta-S, 1 lg of pSupporting NP, 0.1 lg of pSupporting P, and 0.1 lg pSupporting L plasmids were transfected into Hep-2 cells using Lipofectamine 3000 (Thermo Fisher Scientific). After 1 h of transfection, the cells were washed thrice with phosphate-buffered saline (PBS) and incubated in Opti-MEM (1.5 mL) at 37°C for 72 h. After incubation, the six-well plates were frozen and thawed to release the rK148 and rK148/beta-S viruses from the cells, which were then inoculated into 10-day-old specific pathogen-free (SPF) embryonated chicken eggs and incubated at 37°C. After 3 d of incubation, the inoculated eggs were chilled at 4°C for 2 h, and allantoic fluids were used for the hemagglutination assay (HA) to detect the rescued virus [19]. Hemagglutination assay-positive samples were filtered through a 0.45 lm syringe filter (Minisart RC15; Sartorius, Germany) and subsequently passaged in 10-dayold SPF embryonated chicken eggs [18]. Allantoic fluid was harvested, aliquoted, and stored at À80°C for vaccination and growth kinetics. The same batch of the vaccine virus for inoculation was also validated by western blot, conformational PCR, and sequencing.

Virus purification for western blot
rK148 and rK148/beta-S viruses in allantoic fluid were harvested and clarified using centrifugation. The supernatant was inactivated with 0.2 % formalin for 48 h at 25°C. After inactivation, the supernatant was pelleted using centrifugation at 30,000 Â g for 1 h. The pellets were then resuspended in PBS, loaded on a 20-50 % (w/v) sucrose density gradient, and centrifuged at 150,000 Â g for 1.5 h. The layer between the two sucrose gradients containing viruses was collected, and de sucrose was performed via centrifugation at 150,000 Â g for 1.5 h using PBS. The concentration of the resuspended pellet was measured using a Bradford protein assay plus kit (GenDEPOT, USA) according to the manufacturer's instructions. The purified samples were analyzed using western blotting.

Virus western blot
To detect the SARS-CoV-2 S protein in the sample, SARS-CoV-2 S protein S2 monoclonal antibody (1A9) (Invitrogen, USA) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Komabio, Republic of Korea) were used as primary and secondary antibodies, respectively. To detect NDV HN protein in the sample, anti-NDV HN mouse IgG antibody (BioNote, Republic of Korea) and HRP-conjugated goat anti-mouse IgG antibody (Komabio) were used as primary and secondary antibodies, respectively.

Vaccine virus characterization
The complementary DNA (cDNA) of the rK148/beta-S was used to amplify genome of insertion used PfuUltra Fusion HS DNA polymerase (Agilent, USA). The amplified DNA was compared with the original SARS-CoV-2 beta variant spike gene by sanger sequencing (Macrogen, Korea). rK148/08 and rK148/beta-S were diluted to 10 2 D.-H. Kim, J. Lee, S. Youk et al.
Vaccine xxx (xxxx) xxx 50 % egg infective dose (EID 50 )/mL using PBS. The virus was inoculated into specific-pathogen free (SPF) embryonated chicken eggs, and 0.3 mL of allantoic fluid was collected using a 1 cc syringe every 12 h for 96 h. The collected allantoic fluid was serially diluted ten-fold with PBS and titered into SPF eggs. After 72 h of incubation at 37°C, HA was performed using 10 % chicken red blood cells. EID 50 /mL was calculated using the Reed-Muench method [20].

Mouse immunization and challenge
Female K18-hACE-2 mice (7-week-old) were purchased from Jackson Laboratory (USA). The mice were held for one week at a BSL-2 facility. The low-dose experiment was divided into two groups (n = 7): group 1 was vaccinated with 100 lL of live rK148/beta-S 10 7 EID 50 /mL via intramuscular injection, and group 2 was vaccinated with 100 lL PBS via intramuscular injection ( Table 1). The high-dose experiment was divided into five groups: G1 (rK148/beta-S 10 7.0 EID 50 ; n = 18), G2 (NDV K148/08; n = 10), G3 (control: PBS; n = 18), G4 (rK148/beta 10 7.0 EID 50 ; n = 13), and G5 (control: PBS; n = 13). One hundred microliters of each vaccine were intramuscularly administered with 100 lL of live virus 10 8.0 EID 50 /mL ( Table 2). All groups received the secondary vaccine 4 weeks after the primary vaccination. Blood was collected 4, 6, and 8 weeks after the primary vaccination, and body weight was measured every week. To observe adverse effect on body weight gain by high dose of vaccine, the number of mice per group was increased for high dose animal study. The SARS-CoV-2 beta and delta variants were challenged at 4 weeks after the second vaccination. For the challenge, 50 lL each of both beta and delta (10 6.0 EID 50 /mL) were inoculated intranasally. Clinical signs, body weight, and mortality were observed and measured daily for 14 d after the challenge (Figs. 2A, 3A). Vaccination was performed in the BSL-2 facility, and infection was performed in the animal BSL-3 facility at Konkuk University. Immunization and infection studies using animals were reviewed, approved, and supervised by Konkuk University institutional animal care and used committee KU22050.

Serological analysis
For each group, blood was collected at 4, 6, and 8 weeks after the primary vaccine. Serum was mixed with a receptordestroying enzyme (Denka Seiken, Japan) at a ratio of 1:3 and treated at 37°C for 18 h and 56°C for 30 min to eliminate nonspecific hemagglutination inhibition (HI) factors. The inactivated serum was diluted two-fold with PBS and incubated with 4 hemagglutination units of the K148/08 antigen for 40 min. The incubated samples were mixed with 1 % turkey red blood cells in 96-well Vbottom plates. The HI titer of each sample was determined using a standard protocol [19].

Surrogate SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA)
Surrogate SARS-CoV-2 ELISA (BioNote, Korea) was used to confirm the presence of antibodies against SARS-CoV-2 in the serum according to the manufacturer's instructions. Briefly, 60 lL of 1/10-diluted serum, 30 lL of the working enzyme conjugate, and 30 lL of the working ACE-2 solution were mixed in a flat-bottom 96-well plate and incubated at 37°C for 30 min. Then, 100 lL of the mixture was transferred to a capture antibody-coated plate and incubated at 37°C for 30 min. After washing five times with the wash buffer, 100 lL of 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) was added to the wells. After 15 min, 100 lL of stop solution was added. The optical density (OD) of each well was measured at 450 nm wavelength using a microplate reader. The PI value was calculated using the formula OD ((1-(sample OD/negative OD) Â 100).

Splenocyte and IFN-c enzyme-linked immunospot (ELISpot)
Spleen was isolated from the mice of each vaccinated individual. The spleens were placed in Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich, USA) supplemented with 10 % FBS (R10 RPMI 1640). The spleens were separated into single cells by mincing using a 70 lm cell strainer (SPL Life Sciences, Korea) in a petri dish. After adding 10 mL of R10 RPMI 1640, the cells were transferred to a 15 mL conical tube and centrifuged at 1500 rpm for 5 min, after which the supernatants were discarded and the cells were resuspended in 2 mL of Red Blood Cell Lysis Buffer (Sigma-Aldrich). After incubation at room temperature for 10 min, 5 mL of R10 RPMI1640 was added, followed by centrifugation at 1500 rpm for 5 min. After removing the supernatant, 2 mL of R10 RPMI 1640 medium was added to resuspend the cells. The number of cells was measured on an automated cell counter (NanoEntek, Korea) using an Accuchip kit (NanoEntek). IFN-c ELI-Spot was measured using a 3321-4APW-10 kit (Mabtech, Sweden).
Cells were seeded in IFN-c ELISpot pre-coated microwells (50 lL each) at a density of 5 Â 10 5 cells/well. As a negative control, 50 lL of R10 RPMI 1640 was used, and as the positive control, 50 lL was used to a final concentration of 2.5 lg/mL of ConA.
PepTivator SARS-CoV-2 Prot_S1 (130-127-041) (Miltenyi Biotec, Germany) was used as the antigen and was dissolved in 200 lL (stock solution) and diluted 1/100 (working solution), from which 50 lL volumes were obtained and added to each well. The 96well ELISpot plates were incubated at 37°C in 5 % CO 2 for 24 h, after which all media were discarded and the cells were washed with PBS. The cells were then incubated with anti-mouse IFN-c monoclonal antibody (mAb R4-6A2) for 2 h. After washing with PBS, the cells were incubated at room temperature for 1 h, after which the secondary antibody was added. After washing with PBS, the cells were treated with 100 lL of TMB and incubated in the dark for 15 min. After washing with PBS, the samples were dried at room temperature for 2 h. The number of spots was measured using an AID iSpot (AID Autoimmun Diagnostika GmbH, Germany).

Lung viral titer
Animals from each group were euthanized at 3 and 6 d post infection to harvest the lungs. Each lung was homogenized by pestle and mortar, and stored in 10 % (w/v) PBS. After centrifugation at

Statistical analysis
Statistical analyses were performed using GraphPad prism 8.0 (Insightful Science, USA). Statistical significance was determined using one-way analysis of variance with Tukey's correction. To compare two groups, a one-tail t-test was used, and log-rank test was used for the survival rate. A P-value below 0.05 was considered significant (*p < 0.05, **p < 0.01, and ***p < 0.001).

Confirmation of gene and protein expression and propagation of rK148/beta-S
The S gene of the SARS-CoV-2 beta variant (B.1.351) was inserted between the P and M genes of the pK148 plasmid, as described in a previous study (Fig. 1A). After transfection, rK148/ beta-S virus obtained from Hep-2 cells were passaged in 10-dayold SPF embryonated chicken eggs. After two consecutive passages, the viral genome was extracted from the allantoic fluid to identify the S gene in the viral genome. Primers were designed to flank the S gene inside the rK148/beta-S genome, and consecutive sequencings of virus from each passages revealed there were no changes in the inserted SARS-CoV-2 spike gene. A band was identified at 4,416 bp, indicating that the transgene was inserted between the P and M genes of K148 (Fig. 1B). Western blotting was performed to observe the expression of the SARS-CoV-2 S protein in rK148/ beta-S (Fig. 1C). Growth kinetics were examined in embryonated eggs. The titer of rK148/beta-S increased at a slower rate than that of rK148 until 36 h but reached a similar titer (10 9.5 EID 50 /mL) to rK148 starting at 48 h (Fig. 1D).

Immunity and survival rate of mice intramuscularly vaccinated with low-dose rK148/beta-S
Mice in the intramuscular vaccination group were vaccinated with a low dose (10 6.0 EID 50 ) of live K148/beta-S ( Fig. 2A, Table 1). No significant differences in body weights were observed between the groups. The average body weight of both the inoculated and control groups increased by approximately 15 % at 8 weeks post vaccination (wpv), suggesting no vaccine-associated weight loss (Fig. 2B). Hemagglutination inhibition for NDV and surrogate SARS-CoV-2 ELISA was performed with serum collected at 4, 6, and 8 wpv. The HI titer started to be detected in the vaccine group at 6 wpv (4.0 ± 1.3 Log 2 ) and remained at a similar level at 8 wpv (4.71 ± 2.8 Log 2 ), as shown in Fig. 2C. The surrogate ELISA titer for SARS-CoV-2 became detectable in the vaccine group at 6 wpv 56.2 (±7.5) and decreased at 8 wpv to 48.9 (±14.9), as shown in Fig. 2D.
To evaluate the efficacy of the rK148/beta-S vaccine, infection with SARS-CoV-2 beta variants was performed 8 wpv (4 weeks after the second vaccine; Table 1). Body weight and mortality were measured daily until 14 days post challenge (dpc). The control group exhibited 100 % mortality at the 9 dpc, whereas one mouse died at 8 dpc and another at 12 dpc in the vaccination group, resulting in a survival rate of 72 % (Fig. 2E). The control group showed rapid weight loss at 4 dpc (Fig. 2F).

Survival rate and viral load of mice intramuscularly vaccinated with high-dose rK148/beta-S after challenge with beta and delta variants
To evaluate the efficacy of rK148/beta-S as a vaccine, infection with SARS-CoV-2 beta variant was performed at 8 wpv (Table 2). Body weight and mortality were measured daily until 14 dpc. In the control group, the NDV (K148/08) group showed 100 % mortality at 9 dpc. In the rK148/beta-S group, all mice survived until 14 dpc (Fig. 4A). The rK148/beta-S group exhibited no change in body weight until 14 dpc, but the NDV (K148/08) and control groups showed a sharp decrease in body weight starting at 4 dpc (Fig. 4B). At 3 dpc with the beta variant, three mice in the rK148/ beta-S, NDV (K148/08), and control groups were sacrificed to measure lung viral load. The NDV (K148/08) and control groups showed high viral loads of 10 5.7 TICD 50 /mL and 10 5.5 TICD 50 /mL,    whereas the rK148/beta-S group showed a significantly low viral load of 10 2.3 TCID 50 /mL (Fig. 4C). At 6 dpc, three mice in the rK148/beta-S and control groups were sacrificed to measure the lung viral load. The lung viral titer in the rK148/beta-S group was below 10 2.0 TCID 50 /mL, while that in the control group was at 10 3.5 TICD 50 /mL (Fig. 4D).
To evaluate the efficacy of rK148/beta-S as a vaccine, challenge with the SARS-CoV-2 delta variant was performed at 8 wpv   (Table 2). Body weight and mortality were measured daily until 14 dpc. The control group showed 100 % mortality by 7dpc, while the rK148/beta-S group survived until 14 dpc (Fig. 4E). The rK148/ beta-S group showed no change in body weight until 14 d, but the control group showed a sharp decrease in body weight starting at 4 dpc (Fig. 4F). At 3 dpc with the delta variant, three mice in the rK148/beta-S, NDV (K148/08), and control groups were sacrificed to measure lung viral load. The rK148/beta-S group showed a significantly low titer of 10 2.6 TCID 50 /mL, whereas the control group showed a high titer of 10 4.6 TCID 50 /mL (Fig. 4G). At 6 dpc, three mice in the rK148/beta-S and control groups were autopsied to measure the lung viral load titer. The rK148/beta-S group showed a lung viral load below 10 2.0 TCID 50 /mL, while the control group had a viral load of 10 3.1 TCID 50 /mL (Fig. 4H).

Discussion
Since SARS-CoV-2 occurred in December 2019, there has been a lot of effort to overcome SARS-CoV-2. One of them is a way to overcome pathogenic viruses by developing vaccines. We developed SARS-CoV-2 vaccine using NDV vector, one of the viral vector vaccine platforms. SARS-CoV-2 S full gene was inserted between P and M of NDV, where foreign gene was mainly inserted, and beta variant of SARS-CoV-2 selected at the time of the study. As experimental animal, the survival rate and lung viral load was observed using K18-hACE2 mice, and the cross protection against delta variant was evaluated. Previous studies also showed protection against SARS-CoV-2 after administration with NDV-vectored SARS-CoV-2 vaccine [21][22][23][24]. In this study, we evaluated the protectivity of rK148/beta-s as a live vaccine and observed the T cell immunity through SARS-CoV-2 specific IFN-c ELISpot.
Various platforms such as inactivated virus, mRNA, recombinant subunit, and viral vector vaccines have been employed to develop the SARS-CoV-2 vaccine [11,25]. SARS-CoV-2 inactivated vaccines have been reported to have potential epitope alteration by the inactivation process [26] and requires large quantity of antigen to deliver protective immunity [27]. mRNA vaccines also have issues in that they must be stored and transported at a low temperature because of the instability of the mRNA [28]. Subunit vaccines require the use of adjuvant for a proper immune response [29]. On the other hand, viral vector vaccines deliver antigens of pathogens using a vector virus; thus, they are safe because they do not directly use pathogens, and they induce both T cell and B cell immunity. However, an issue with viral vector vaccines is that they are less effective because of the immune response to the viral vector when repeatedly administered. In addition, nonreplicating viral vectors have difficulty in mass production [30,31].
Unlike adenovirus, which is a non-replicating viral vector, NDV is easily propagated as a replication viral vector [32]. Since rK148/ beta-S is an egg-based vaccine that proliferates to a high titer, it can take advantage of repurposing the pre-existing infrastructure for influenza vaccine manufacturers. rK148/beta-S is easy to mass-produce through existing manufacturing techniques without special equipment, and as it is a live viral vector, it can be used as a vaccine without additional inactivation or adjuvant [33]. Genetic stability of transgene incorporated in recombinant NDV vector is crucial for vaccine production. According to study by Yu Q et al., recombinant NDV expressing the laryngotracheitis virus glycoprotein D was passaged eight times in embryonated chicken eggs. In three separate repetition, neither single-nucleotide polymorphism nor insertion or deletion was detected in the ILTV gD insert region, suggesting that, NDV vector vaccine is a genetically stable vaccine candidate [34]. Safety is of utmost importance for vaccines used in humans [35]. Newcastle disease virus, the backbone of the NDV vector vaccine, is a host range-restricted virus that causes disease only in poultry [36]. It is also used as an oncolytic virus and has been shown to be safe even when intravenously administered to humans [37]. With the safety profile, NDV has also been developed as a viral vector vaccine against influenza, Ebola virus, human immunodeficiency virus (HIV), and Nipah henipavirus, among others [38]. The NDV vector vaccine platform is not only limited to laboratory studies, but there are also case that have entered clinical trials. As of 17 May 2023, six clinical trials are situated in different stages (NCT numbers; NCT05205746, NCT04871737, NCT05181709, NCT05710783, NCT04993209, NCT05354024). Leading one is about to enter Phase 2/3 in Brazil and Mexico based on promising immunogenicity result from Phase 1 [39].
In this study, K18-hACE2 mice were chosen to evaluate the vaccine efficacy of rK148/beta-S. K18-hACE2 mouse and Syrian golden hamster has commonly used to animal models for SARS-CoV-2 infection. As described in a study by Jeong H et al., when infected with SASR-CoV-2, Syrian golden hamsters lose 10 % of their weight until 6dpc and then increase again, which renders it hard to examine the protectivity of vaccine after the challenge. On the other hand, K18-hACE2 mice lose weight from 4dpc and have a 100 % mortality rate by 8dpc, makes it more suitable for observing the efficacy of vaccination after the challenge [40]. Therefore, the K18-hACE2 mouse model was chosen to evaluate the efficacy of the rK148/beta-S vaccine in a manifest manner. According to previous studies, both T and B cell immunities are important in the case of coronavirus to which SARS-CoV-2. Stimulated memory T cells perform faster T-cell immunity upon reinfection [41,42]. rK148/beta-S vaccine increased SARS-CoV-2 specific IFN-c spots in splenocytes. Neutralizing antibodies are also important for viral clearance from the body and to have an important immune function that distinguishes mild and severe cases [43]. In this study, we measured blood total immunoglobulin using a surrogate SARS-CoV-2 ELISA. Regardless of the dose inoculated and immune response to NDV vector itself or SARS-Cov-2 spike protein, relative antibody level significantly increased after boost vaccination. Similar trend was also observed in the other studies [44] when the mouse was immunized by NDV-vectored vaccine. It has been reported that the viral load of SARS-CoV-2 in the mouse lung at the early stage of the challenge is associated not only with survival but also with the transmission between individuals and wounding of the lung tissue [45][46][47]. The rK148/beta-S vaccinated mice showed a low viral load at the initial stage of infection (3 dpc), and then no viral load was observed in the vaccine group during the middle stage of infection (6 dpc). And the viral load was observed only in the control group. Other study using live recombinant NDV as intranasal and intramuscular SARS-CoV-2 vaccine also showed a slight increment in lung viral load on the 2dpc, but the lung viral load was not detected in the vaccinated group on the 5dpc [21].
Due to the nature of RNA virus rapid mutation rates, SARS-CoV-2 have exhibited a very high mutation rate [48]. The RBD mutations in the S gene of the SARS-CoV-2 beta variant are K417N, E484K, and N501Y, whereas the delta variant contains L452R, E484Q, and T478K mutations [49]. The beta variant possesses the lowest binding rate to the antibody generated by a vaccine designed using the Wuhan strain [50]. Although beta variant has various amino acid changes that cause immune evasion compared to delta variant, the rK148/beta-S expressing the spike protein of the beta variant conferred 100 % survival rate with potent humoral and cellular immunity against delta variant when used as live viral vector vaccine.
To further investigate the potential of rK148/beta-s, it is necessary to assess its thermostability since the K148/08 is known as a thermostable NDV strain [17]. Additionally, even though the significant reduction in lung viral load was found in the vaccination group challenged with the delta variant, the cross-immunity of rK148/beta-S against other variants of concern or emerging variants needs to be validated. As part of a further study, the use of rK148/beta-S as an intranasal vaccine and the IgA response in lungs will be evaluated. Unlike the Intramuscular vaccine, the intranasal vaccination can provide mucosal antibodies in the upper respiratory tract, offering protection against infection, blocking virus transmission, and reducing virus invasion of lungs [51]. Hence, the NDV vector vaccine platform shows a promise as a mucosal vaccine that can block infection and transmission through IgA, making it a potential candidate for a wide range of vaccines for respiratory diseases such as RSV, Parainfluenza, Adenovirus, and Influenza. Also, as seen it other studies that used NDV as a vector virus against SARS-CoV-2, a follow up study should be conducted using pre-fusion form of S as a protein of interest [21,22]. In conclusion, the NDV vector vaccine holds a potential as an emergency vaccine candidate for future pandemics.

Data availability
Data will be made available on request.