key: cord-0752831-v6i2afp5
authors: Warner, Bryce M.; Santry, Lisa A.; Leacy, Alexander; Chan, Mable; Pham, Phuc H.; Vendramelli, Robert; Pei, Yanlong; Tailor, Nikesh; Valcourt, Emelissa; Leung, Anders; He, Shihua; Griffin, Bryan D.; Audet, Jonathan; Willman, Marnie; Tierney, Kevin; Albietz, Alixandra; Frost, Kathy L.; Yates, Jacob G.E.; Mould, Robert C.; Chan, Lily; Mehrani, Yeganeh; Knapp, Jason P.; Minott, Jessica A.; Banadyga, Logan; Safronetz, David; Wood, Heidi; Booth, Stephanie; Major, Pierre P.; Bridle, Byram W.; Susta, Leonardo; Kobasa, Darwyn; Wootton, Sarah K.
title: Intranasal vaccination with a Newcastle disease virus-vectored vaccine protects hamsters from SARS-CoV-2 infection and disease
date: 2021-10-06
journal: iScience
DOI: 10.1016/j.isci.2021.103219
sha: c3b5705083eb43fca7a0a2633e7378f18ec035c5
doc_id: 752831
cord_uid: v6i2afp5

The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19). Worldwide efforts are being made to develop vaccines to mitigate this pandemic. We engineered two recombinant Newcastle disease virus (NDV) vectors expressing either the full-length SARS-CoV-2 spike protein (NDV-FLS) or a version with a 19 amino acid deletion at the carboxy terminus (NDV-Δ19S). Hamsters receiving two doses (prime-boost) of NDV-FLS developed a robust SARS-CoV-2-neutralizing antibody response, with elimination of infectious virus in the lungs and minimal lung pathology at five days post-challenge. Single-dose vaccination with NDV-FLS significantly reduced SARS-CoV-2 replication in the lungs, but only mildly decreased lung inflammation. NDV-Δ19S-treated hamsters had a moderate decrease in SARS-CoV-2 titers in lungs and presented with severe microscopic lesions, suggesting that truncation of the spike protein was a less effective strategy. In summary, NDV-vectored vaccines represent a viable option for protection against COVID-19.

Taken together, these data demonstrate that NDV can be engineered to express the SARS-147

CoV-2 spike protein, and that the full-length spike protein is incorporated into the NDV virion 148 more efficiently than the Δ19 truncated version. 149

To test whether the spike protein incorporation into the NDV virion would increase NDV 151 infectivity, we conducted virus neutralization assays in HEK 293T cells over-expressing human 

To determine whether immune responses in animals receiving a single dose or a 195 homologous prime-boost would result in protection from disease, each of the groups of hamsters 196 described above, which were vaccinated with 10 7 PFU NDV-FLS, NDV-Δ19S, or NDV-GFP, 197 were challenged with 10 5 fifty-percent tissue culture infective dose (TCID50) of SARS-CoV-2 on 198 day 56 post-vaccination. An additional experiment was carried out by infecting hamsters at 28 199 days after receiving the prime only, using the same methodology. Animals were weighed and 200 monitored daily for signs of disease throughout the course of infection. Out of ten animals in each 201 group, four were kept for 28 days to examine any differences in weight loss and long-term outcome 202 of infection, while six were euthanized on day five post-infection to examine pathology and viral 203 loads in the tissues during acute infection. 204

In the single-dose group, NDV-FLS-vaccinated hamsters had significantly less weight loss 205 on day 4 post-infection compared to the NDV-GFP and NDV-Δ19S-vaccinated hamsters. The 206 NDV-Δ19S-vaccinated hamsters showed similar weight loss to controls, including several animals 207 that lost greater than 10% of their initial weight within five days ( Figure 4A ). In the prime-boost 208 group, the mean weight loss of the NDV-FLS-treated hamsters was significantly less than NDV-209 GFP-treated hamsters on days 3, 4 and 5 post-infection ( Figure 4B) . Surprisingly, in the NDV-210 GFP-vaccinated animals, we did not see the weight loss that is typically seen in our hamster model, 211 and that has been reported by other groups (Imai et al., 2020) . Of the four animals that were not 212 euthanized, none reached greater than five percent weight loss following infection throughout the 213 28 days, while five of six of the euthanized animals had as high as 8% weight loss and were 214 trending downward. These animals were likely euthanized before reaching peak weight loss, 215 thereby artificially skewing the mean weight loss of that group. 216

The magnitude of microscopic lesions in the lungs was assessed semi-quantitatively, in 217 order to evaluate the efficacy of the vaccine candidates to decrease the severity of lesions 218 associated with SARS-CoV-2 infection. For both the prime and prime-boost experiments, nominal 219 reporting of lesion categories for each hamster is summarized in Supplemental Table 1 and 2, 220 while the extent of affected lung tissue area is reported in Supplemental Table 3 . 221

In hamsters treated with the prime only, lesions in the control group were similar to those 222 described in the prime-boost group (Supplemental Table 2 ; see below for a detailed description of 223 histopathology). When the compound score of nominal categories was considered, NDV-FLS-224 treated hamsters had significantly lower scores compared to the NDV-Δ19S-, but not the NDV-225 GFP-treated group ( Figure 4C ). Similarly, NDV-FLS-treated hamsters had significantly less 226 extensive areas of lung pathology compared to the NDV-Δ19S-, but not the NDV-GFP-treated 227 group ( Figure 4D ). The mean compound nominal score was significantly lower in NDV-FLS-228 treated hamsters compared to those in the NDV-GFP group; differences were not significant 229 between the scores of the NDV-Δ19S-and the NDV-GFP-or NDV-FLS-treated groups ( Figure  230 4E). Lastly, NDV-GFP-and NDV-Δ19S-treated hamsters had the highest extent of lesions, with 231 five of six and three of six animals showing > 50% of lung tissue affected, respectively; while in 232 the NDV-FLS-treated group lesions were present in > 50% of lung sections only in one hamster 233 (Supplemental Table 3 ). The average affected area scores were significantly different between 234 NDV-FLS-treated hamsters and those in the NDV-GFP-treated group ( Figure 4F) . 235  In the prime-boost experiment, all hamsters treated with NDV-GFP presented with severe  236   exudative lesions characterized by accumulation of sloughed cells, macrophages and neutrophils  237 within the alveolar spaces, variably admixed with multifocal areas of hemorrhage and edema 238 ( Figures 5A, 5D, 5G ). In every hamster of this group, most bronchioles were filled with cellular 239 debris and neutrophils. The connective tissues surrounding vessels and bronchioles was markedly 240 expanded by edema and populated by inflammatory cells, such as macrophages, lymphocytes, 241 fewer plasma cells and scattered neutrophils. In all six hamsters, medium-size vessels showed 242 segmental hyperplasia of the endothelial cells and sub-intimal accumulation of inflammatory cells, 243 although no thrombosis was observed ( Figure 5J ). As animals were euthanized at day five pi, 244 subacute changes were also observed, which included type II cell hyperplasia ( Figure 5M ), and 245 presence of hemosiderin-laden macrophages mainly around the terminal bronchioles. In hamsters 246 vaccinated with NDV-Δ19S (Figs. 5B, 5E, 5H, 5K, 5N), lesions were similar to the NDV-GFP-247 treated group, although the exudative changes were less prominent, noticeably with decreased 248 amounts of desquamated cells in the alveolar spaces and bronchioles. In this group, most hamsters 249 presented with numerous hemosiderin-laden macrophages (suggesting resolving haemorrhage) 250 either in the alveoli or around bronchioles, type II cell hyperplasia (five of six hamsters), and 251 marked hyperplasia of the bronchiolar epithelium in three of six hamsters ( Figure 5N ). In hamsters 252 vaccinated with NDV-FLS, only two of six hamsters showed mild exudation of neutrophils in the 253 alveoli, and only two presented with multifocal haemorrhages. Most of the lung parenchyma was 254 unaffected (Figs. 5C, 5F, 5I, 5L, 5O). The other changes were subacute, including mild hyperplasia 255 of type II cells (four of six hamsters), epithelial bronchial hyperplasia (one of six hamsters), and 256 accumulation of hemosiderin-laden macrophages in four hamsters. 257 J o u r n a l P r e -p r o o f At the time of euthanasia, we also evaluated a series of hematological and serum chemistry 258 parameters to test whether prime-boost vaccination could prevent any hematological changes upon 259 infection of hamsters with SARS-CoV-2. All parameters were within normal limits and no clear 260 trends emerged between vaccinated and control hamsters. Of note, NDV-GFP-vaccinated hamsters 261

showed an elevated neutrophils count, and a higher neutrophil:lymphocyte ratio compared to those 262 that were vaccinated with NDV-FLS, which has been correlated with disease severity in SARS-263

CoV-2-infected people (Karimi Shahri et al., 2020) . This is consistent with the results of 264 microscopic pathology, which showed numerous neutrophils into the lung of NDV-GFP-265 vaccinated hamsters. 266

To evaluate the extent of virus replication in tissues, we quantified the presence of SARS-269

CoV-2 genomic RNA and infectious titers in the tissues of hamsters euthanized on day five pi. In 270 the single-dose group, SARS-CoV-2 genome copy numbers were reduced significantly in the 271 proximal and distal lungs of the NDV-FLS-treated group, while genome copies remained high in 272 the hamsters treated with NDV-Δ19S, and were not significantly different compared to controls 273 ( Figure 6A ). Viral RNA levels in the nasal turbinates, small intestine, and blood did not differ 274 between groups that received a single vaccine dose. Similarly, a single dose of NDV-FLS 275 significantly reduced the titers of infectious SARS-CoV-2 in the nasal turbinates, proximal, and 276 distal lungs, compared to the NDV-GFP control. The NDV-Δ19S-vaccinated hamsters only had 277 significantly reduced viral titers in the distal lung ( Figure 6B ). This may be due to some partial 278 protection that prevented the spread of virus into the lower lung. 279

In animals receiving two vaccine doses, the NDV-FLS group had significantly reduced 280 SARS-CoV-2 genome copies in all tissues examined, except for blood. While the NDV-Δ19S-281 vaccinated hamsters only had significantly reduced viral RNA levels in the small intestine ( Figure  282 6C), there was a clear trend toward lower viral RNA levels in both the proximal and distal lung 283 ( Figure 6C ). Hamsters in both prime-boost vaccine groups did not have any infectious virus in the 284 lungs, and only two animals had low levels of virus in the nasal turbinates, suggesting protection 285 of both the upper and lower airway is provided by two vaccine doses ( Figure 6D ). 286

We also examined SARS-CoV-2 mucosal shedding following infection with SARS-CoV-287 2, since a key question regarding the protective efficacy of vaccination and whether vaccination 288 can effectively prevent virus transmission. Oral and rectal swabs were sampled day 2 pi to test 289 whether vaccination may prevent acute virus shedding by these routes. Viral RNA was detected in 290 oral and rectal swabs in all hamsters regardless of the vaccine regimen, without differences in the 291 magnitude of shedding between groups (Supplemental Figure 1A) . Quantification of infectious 292 titers in swabs showed that most hamsters shed at very low levels (< 10 2 TCID50 / ml) in both 293 groups, and no differences were observed in the magnitude of shedding (Supplemental Figure 1B ) 294 or the proportion of shedding compared to non-shedding animals (Fisher's exact test, data not 295 show). This suggests that vaccination did not prevent infection and that virus shedding occurred 296 in the early phases of infection, despite protection from disease. Whether the low levels of 297 infectious virus in the oral and rectal swabs are sufficient to infect other hamsters is a question that 298 remains to be answered. Our data suggest that while vaccination prevented disease and 299 significantly reduced viral growth in the tissues, infected animals may shed virus acutely after 300 infection. 301

To characterize the molecular drivers of inflammation and immune response in vaccinated 303 and non-vaccinated hamsters, we examined the mRNA expression of various immune response-304 related genes (n = 11) in the lungs of hamsters in the prime-boost experimental groups, at day 5 305 pi. While several examined genes showed significant difference between vaccinated and control 306 groups, only expression of interleukin (IL)-1β was significantly upregulated in both vaccine groups 307 compared to the control (Supplemental Figure S3 ). Vascular endothelial growth factor (VEGF) 308 expression was the other and only gene to be upregulated in the NDV-FLS-treated group compared 309 to the NDV-GFP control (Supplemental Figure S3 ). The NDV-Δ19S-treated group showed 310 differential expression of five additional genes compared to the NDV-GFP-treated control group: 311 IL-6, FoxP3, IL-4, transforming growth factor (TGF)-, and tumor necrosis factor alpha(TNF)-α 312 ( Figure S6 ). Upregulation of cytokine gene expression in vaccinated hamsters may account also 313 for activation of the immune response, rather than tissue damage or inflammation, as pathology 314

shows more severe lesions in the control (NDV-GFP) group. 315

We also examined relative expression levels of interferon gamma (IFN-γ) and IL-4, to 316 attempt to determine whether a T helper type 1 (Th1) or Th2-associated bias in immune responses 317 between groups may be driving susceptibility to infection. Both vaccine groups had higher, albeit 318 not statistically significant, median IFN-γ:IL-4, ratios and it is possible that inducing a Th1-319 associated immune response via vaccination may lead to improved infection outcomes (Jeyanathan 320 et al., 2020) . 321 322

Given that hamsters vaccinated with NDV-FLS were protected from clinical signs and 324 lesions following SARS-CoV-2 challenge, we sought to investigate whether it would be feasible 325 to lyophilize this promising vaccine candidate thereby greatly simplifying its storage and 326 distribution requirements. Aliquots of NDV-FLS stock containing the same number of PFU were 327 adjusted to a final concentration of 5% sucrose, 5% sucrose/5% iodixanol, or mixed 1:1 with a 328 solution containing 10% lactose, 2% peptone, 10mM Tris-HCl, pH 7.6 and lyophilized for 16h at 329 -52°C. Two days later, samples were reconstituted in phosphate-buffered saline with 5% sucrose 330 at the same volume and virus titer determined. As shown in Figure 7A , there was a ~2 fold loss of 331 infectivity when NDV-FLS was lyophilized in 10% lactose, 2% peptone, 10mM Tris-HCl, pH 7.6 332 compared to virus frozen at -70°C. Further, as shown by western blot analysis, the reconstituted 333 vaccine preparation contained S protein at amounts comparable with the purified virus stock 334 maintained at ultracold temperature, and was able to infect and induce expression of S protein in 335 DF-1 cells ( Figure 7B in production capacity of developed vaccines (Khamsi, 2020) , and the need for evidence of 348 efficacy for multiple platforms moving forward, there will be a need for a diverse set of vaccine 349 candidates to advance through all stages of development. Here we tested two live vaccine 350 candidates based on an NDV vector expressing SARS-CoV-2 spike protein or a truncated version 351 of the same protein in a hamster model to assess their potential for prevention of COVID-19. 352

The Syrian hamster has been used by our and other groups as a robust model for SARS-353

CoV-2 infection, and it is used to examine viral pathogenesis as well as testing vaccine efficacy 354 (Griffin et al., 2021; Muñoz-Fontela et al., 2020). We vaccinated groups of ten hamsters with either 355 NDV vaccine candidate, or NDV expressing GFP as a negative control. The vaccines were 356 administered either as a single dose or as two homologous doses 28 days apart. Both vaccines 357 expressing SARS-CoV-2 S protein were immunogenic as assessed by ELISA for anti-SARS-CoV-358 2 IgG. Neither vaccine induced significantly high IgG titers after a single dose; however, following 359 homologous booster immunization, both vaccine groups had significantly higher antibody titers 360 compared to controls. When evaluating a prime-boost vaccination schedule, the vaccine expressing 361 full-length S protein induced neutralizing antibody responses that were significantly greater than 362 those seen in the group that received the vaccine with the truncated S protein. It is possible the low 363 incorporation of the Δ19S into the NDV virion might have compromised an effective immune 364 response due to lack of surface antigen presentation, despite the fact that NDV-Δ19S appeared to 365 express higher amounts of S protein in vitro. Nonetheless, even the vaccine expressing the full-366 length S protein induced only modest PRNT90 titers, suggesting that efficacy to protect from 367 clinical disease and microscopic pathology may not be entirely dependent upon the magnitude of 368 neutralizing antibody titers. Similarly, hamsters in the prime-boost NDV-Δ19S group appeared to 369 be nearly fully protected from SARS-CoV-2 replication in the lungs, where no infectious titers 370 were evident, despite barely detectable neutralizing antibodies and modest IgG serum titers. It is 371 possible that protection following vaccination with NDV may also rely on T cell responses, which 372 might have been more strongly activated by the NDV-Δ19S vaccine, as it expresses higher level 373 of S protein upon infection. However, this was not examined in our study due to a lack of 374 appropriate reagents, such as hamster-specific antibodies for flow cytometry and depletion studies. 375

Overall, these data suggest that some protection against disease and decreased viral replication can 376 be afforded in the absence of high titers of neutralizing antibodies and further characterization of 377 the immune responses generated by NDV-vectored vaccines and others will be critical for 378 enhanced immunization strategies. importance to curtail circulation of SARS-CoV-2 in a partially immune population, and therefore 531 limit the development of vaccine escape variants. Notably, in mice, complete protection against 532 SARS-CoV-1 challenge is afforded only upon intranasal, but not subcutaneous, vaccine 533 administration (Zhao et al., 2016) . Although in our study we did not test development of SARS-534

CoV-2-specific mucosal immunity due to lack of reagents, in the prime-boost schedule NDV-FLS-535 treated hamsters had no detectable infectious SARS-CoV-2 in the turbinates or the upper and lower 536 lungs day 5 pi, suggesting development of sterilizing immunity. However, considering that low-537 level shedding of infectious virus was observed at day 2 pi even in vaccinated groups, vaccination 538 may not protect against the early phases of SARS-CoV-2 replication. It is unclear if such low 539 amounts of infectious virus would be able to infect other animals, or even if the detected virus may 540 -at least in partrepresent left-over inoculum. Lastly, as the prime-boost group in our study 541 showed a clear increase of S-specific antibodies after the second dose, it is likely that immunity 542 against the vector backbone did not impact the efficacy of a booster shot. 543

Many COVID-19 vaccine candidates are in various stages of clinical development, with 544 some currently being given emergency-use approval in several countries. However, due to the 545 relative advantages and disadvantages of different vaccine platforms, there is an ongoing need to 546 develop and test novel vaccine platforms and strategies. This will also be critical in the case of 547 potential future pandemics and emerging and re-emerging infections, which will require swift 548 development of vaccine candidates. The prospect of having several different platforms available 549 for rapid development should a novel pathogen arise, is of critical importance. Live viral vectors 550 are particularly advantageous due to their generally high immunogenicity, ability to induce both 551 humoral and cellular immune responses, and the lack of a need for adjuvants (Vrba et al., 2020) . 552

This work provides evidence that this platform can provide substantial protection against SARS-553

CoV-2 infection and could be a viable option for further clinical development. 554

Oral and rectal swabs were taken on day two post-challenge with SARS-CoV-2 primarily to 556 confirm viral replication in our hamster infection model; therefore, we did not take swabs on day 557 five post-challenge. As these swabs were taken at such an acute time point, we cannot know for 558 certain whether the virus or RNA detected was from leftover inoculum, at least in the oral swabs. 559

Additionally, although the vaccine was administered to hamsters intranasally, the method of We worked to ensure sex balance in the selection of non-human subjects. One or more of the 595 authors of this paper self-identifies as an underrepresented ethnic minority in science. One or more 596 of the authors of this paper self-identifies as a member of the LGBTQ+ community. One or more 597 of the authors of this paper self-identifies as living with a disability. While citing references 598 scientifically relevant for this work, we also actively worked to promote gender balance in our 599 reference list. 600 J o u r n a l P r e -p r o o f 

Further information and requests for resources and reagents should be directed to and will be 713 fulfilled by the lead contact, Dr. Sarah Wootton, University of Guelph (kwootton@uoguelph.ca). 714

Plasmids generated in this study are available upon request following execution of a material 716 transfer agreement (MTA). 717

 All data reported in this paper will be shared by the lead contact upon request. 719  This paper does not report original code. 720  Any additional information required to reanalyze the data reported in this paper is 721 available from the lead contact upon request. 722

Ethics Statement 725

The animal experiments described were carried out at the National Microbiology into 15mL conical tubes in 1mL volumes. Aliquots were either left untreated or adjusted to a final 868 concentration of 5% sucrose, 5% sucrose/5% Iodixanol or mixed 1:1 with a solution containing 869 10% lactose, 2% peptone, 10mM Tris-HCl, pH 7.6. Using a LABCONCO Freeze Dry system 870 Freezone®4.5, samples were immediately lyophilized at 44 x 10-3 MBAR and -52°C for 16 hours. 871

Lyophilized samples were stored at 4°C for 48 hours before being resuspended in 1 mL of 5% 872 sucrose/PBS and titered. Three 1 mL aliquots of allantoic fluid containing NDV-FLS were 873 adjusted to 5% sucrose and frozen at -70°C before titering. An additional three 1 mL aliquots were 874 used to titer NDV-FLS in allantoic fluid immediately following harvest from eggs. All samples 875 were titered by TCID50 on DF-1 cells as described above. 876

To determine presence of S protein in the reconstituted preparations, lyophilized (10% 877 lactose and 2% peptone group, only) and reconstituted NDV-FLS was compared to NDV-FLS 878 stored at -70°C (purified preparation frozen at -70°C in sucrose). Equal amounts of virus 879 preparations (10 7 PFU, as determined by post-reconstitution titers) were loaded on SDS-PAGE 880 gel, transferred to a nitrocellulose membrane and immunoblotted for the S protein using a rabbit 881 anti-SARS-CoV-2 S2 subunit (dilution: 1:1000; NB100-56578; Novus Biologicals), as described 882 in the previous section. To determine the ability of lyophilized and reconstituted virus to express 883 S protein in infected cells, DF-1 cells were infected with the same amounts of reconstituted or 884 frozen virus (MOI = 0.5). Whole cell lysates were harvested 24 hrs post-infection, immunoblotting 885 for the S protein was conducted as above. 886

For initial immunization and booster immunization of hamsters, groups of ten Syrian 888

Golden hamsters (five male and five female, four to six weeks of age; Charles River) were 889 anaesthetized with inhalation isoflurane and administered 1 x 10 7 PFU of recombinant NDV-GFP, 890 hamsters were scruffed and vaccines were delivered in a 100 µL volume (q.s. with PBS) through 892 the nares (50 µL per nare). Animals had their mouths held closed to ensure inhalation through the 893 nose. After recovery from anesthesia hamsters were monitored daily for any adverse signs 894 following vaccine administration. 895

For SARS-CoV-2 infection following immunization, hamsters were moved into a CL-4 896 facility and then anaesthetized with inhaled isoflurane. Hamsters were then infected with 10 5 897 TCID50 of SARS-CoV-2 via the same IN method described above. After recovery from anesthetic 898 hamsters were monitored daily throughout the course of infection. Body weights and temperatures 899 of hamsters were recorded daily. 900

At day five post-challenge, six hamsters per group were euthanized, and the proximal and 902 distal lobes of the lung from each hamster were sampled and fixed in 10% buffered formalin, 903 followed by routine paraffin embedding, sectioning, and staining with hematoxylin and eosin 904 (HE). The magnitude of microscopic lesions caused by SARS-CoV-2 in the lungs of vaccinated 905 and control mice was evaluated histologically using two semi-quantitative scoring systems based 906 on the presence of nominal categories (Supplemental Tables 1 and 2) (Meyerholz and Beck, 2020) 907 and extent of the pulmonary parenchyma affected (Supplemental Table 3 Step Multiplex Master Mix kit (Applied Biosystems) and was carried out on a QuantStudio 5 real-922 time PCR system (Appiled Biosystems), as per the manufacturer's instructions. RNA was reverse 923 transcribed and amplified using the primers reported by the WHO and include E_Sarbeco_F1 (5′-924 ACAGGTACGTTAATAGTTAATAGCGT-3′) and E_Sarbeco_R2 (5′-ATATTGCAGCAGTA 925 CGCACACA-3′) and probe E_Sarbeco_P1 (5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG 926 -BBQ-3′). A standard curve produced with synthesized target DNA was run with every plate and 927 used for the interpolation of viral genome copy numbers. 928

For infectious virus assays, thawed tissue samples were weighed and placed in 1 mL of 930 minimum essential medium supplemented with 1% heat-inactivated fetal bovine serum (FBS) and 931 1x L-glutamine, then homogenized in a Bead Ruptor Elite Bead Mill Homogenizer (Omni 932

International) at 4 m/s for 30 seconds then clarified by centrifugation at 1,500 xg for 10 minutes. 933

Prior to titration procedures, oropharyngeal and rectal swabs stored in MEM + 2% penicillin-934 streptomycin were vortexed and centrifuged briefly. Samples were serially diluted 10-fold in 935 media and dilutions were then added to 96-well plates of 95% confluent Vero cells containing 50 936 L of the same medium in replicates of three and incubated for five days at 37 °C with 5% CO2. 937

Plates were scored for the presence of cytopathic effect on day five after infection. Titers were 938 calculated using the Reed-Muench method, and reported as TCID50 units. 939

For detection of anti-SARS-CoV-2-specific antibody responses, all hamsters were bled via 941 jugular vein bleeds for serum on days 21, 29, 49, and 56 post-first vaccination. For ELISAs for 942 detection of total IgG detection, SARS-CoV-2 spike-and nucleoprotein-specific responses were 943 assessed using an in-house assay. A 1:400 dilution of serum was carried out in duplicate and added 944 to plates pre-coated with both spike and nucleoprotein in the same assay wells. IgG was detected 945 with a peroxidase-labeled polyclonal goat anti-hamster IgG (H+L) (KPL). 946

For virus PRNT (plaque reduction neutralization assays), serum samples were heat-947 inactivated at 56°C for 30 minutes and diluted two-fold from 1:40 to 1:1280 in DMEM 948 supplemented with 2% FBS. Diluted sera were incubated with 50 PFU of SARS-CoV-2 at 37°C 949 and 5% CO2 for 1 hour. The sera-virus mixtures were added to 24-well plates containing Vero E6 950 cells at 100% confluence, followed by incubation at 37 °C and 5% CO2 for 1 hour. After 951 adsorption, 1.5% carboxymethylcellulose diluted in MEM supplemented with 4% FBS, L-952 glutamine, non-essential amino acids, and sodium bicarbonate was added to each well and plates 953 were incubated at 37°C and 5% CO2 for 72 hours. The liquid overlay was removed and cells were 954 fixed with 10% neutral-buffered formalin for 1 hour at room temperature. The monolayers were 955 stained with 0.5% crystal violet for 10 minutes and washed with 20% ethanol. Plaques were 956 enumerated and compared to a 90% neutralization control. The PRNT-90 endpoint titre was 957 defined as the highest serum dilution resulting in a 90% reduction in the number of plaques. PRNT-958 90 titers ≥1:40 were considered positive for neutralizing antibodies. 959

Hematological and biochemical analysis of blood and serum 960

Complete blood counts were carried out using a VetScan HM5 hematology system (Abaxis 961 Veterinary Diagnostics), as per the manufacturer's instructions. Analysis of serum biochemistry 962 was performed with a VetScan VS2 analyzer (Abaxis Veterinary Diagnostics), as per the 963 manufacturer's instructions. 964

Cytokine mRNA analysis 965 RNA was extracted from proximal lung samples as described above using a RNeasy Plus 966

Mini Kit (Qiagen), which includes a genomic DNA elimination step, as per the manufacturer's 967 instructions. Expression of IFNγ, IL-2, IL-4, IL-10, IL-1b, FoxP3, TGF-β, VEGF, IL-6, and TNFα 968 mRNA was analyzed using the one-step TaqPath Master Mix kit using the primer/probe sets 969 described previously (Warner et al., 2017) . Ribosomal protein L18 (RPL18) was used as an 970 internal control. All RT-qPCR assays were performed on a QuantStudio 5 instrument (Applied 971 Biosystems). Expression is reported as Log2 of the fold-change for each gene as calculated using 972 the ΔΔCt method compared with expression of the same genes in sex-matched control tissues that 973 were unvaccinated and uninfected. 974

Mean scores between experimental groups were compared using analysis of variance 976 (ANOVA) with Tukey's post-hoc (lyophilization experiment) or Holm Sidak (neutralization) tests 977 for multiple comparisons, two-way ANOVA (serology), non-parametric Mann-Whitney or 978 Kruskal-Wallis test followed by the Dunn's method for multiple comparisons (all other tests and 979 pathology data), with significance set at p < 0.05 as implemented in GraphPad software version 980 8.0.0 (San Diego, California, USA, www.graphpad.com). Data are represented as scatterplots with 981 median, median with range, or average with standard deviation (see figure legends) . 982 

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Newcastle Disease Virus as a Vaccine Vector for Development 1110 of Human and Veterinary Vaccines

Genetic emergence of B.1.617.2 in COVID-19

Paramyxovirus RNA synthesis and the requirement for hexamer genome length: the rule of six 1115 revisited

New mutations raise specter of 'immune escape

Integrity of membrane 1119 lipid rafts is necessary for the ordered assembly and release of infectious Newcastle disease virus 1120 particles

Early Transmission Dynamics in Wuhan

Predictive values of 1125 neutrophil-to-lymphocyte ratio on disease severity and mortality in COVID-19 patients: a 1126 systematic review and meta-analysis

Community Transmission of Severe Acute Respiratory Syndrome 1129 Coronavirus 2

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for 1133 virus origins and receptor binding

How deadly is the coronavirus? Scientists are close to an answer

Pathology and Pathogenesis of 1138 SARS-CoV-2 Associated with Fatal Coronavirus Disease, United States

Newcastle disease virus: 1141 propagation, quantification, and storage

Histopathologic Evaluation and Scoring of Viral Lung 1144 Infection

Delta variants of SARS-CoV-2 cause 1147 significantly increased vaccine breakthrough COVID-19 cases in

Animal models for 1151 COVID-19

Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) From a Symptomatic Patient

Draft landscape and tracker of COVID-19 candidate vaccines

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-1160 reactivity with SARS-CoV

Engineered viral 1162 vaccine constructs with dual specificity: avian influenza and Newcastle disease

Phase I trial of intravenous administration of 1166 PV701, an oncolytic virus, in patients with advanced solid cancers

What can we expect from first-generation COVID-19 1169 vaccines?

Isolation of Ontario aquatic bird 1171 bornavirus 1 and characterization of its replication in immortalized avian cell lines

Shedding of Infectious SARS-CoV-2 Despite Vaccination when the Delta Variant is Prevalent -1176

Cmmid 1178 Covid-Working

Estimating the infection and case fatality ratio for coronavirus disease (COVID-19) using age-1180 adjusted data from the outbreak on the Diamond Princess cruise ship

Production and Purification of High-Titer Newcastle Disease Virus for Use 1184 in Preclinical Mouse Models of Cancer

How the pandemic might play out in 2021 and beyond

A single amino acid change in the 1189

Newcastle disease virus fusion protein alters the requirement for HN protein in fusion

Newcastle Disease Virus as a Vaccine Vector for SARS-1192

CoV-2. Pathogens 9

Pathogenesis and transmission of 1195 SARS-CoV-2 in golden hamsters

Newcastle disease virus (NDV) expressing 1198 the spike protein of SARS-CoV-2 as a live virus vaccine candidate

A Newcastle Disease Virus (NDV) Expressing a 1202 Membrane-Anchored Spike as a Cost-Effective Inactivated SARS-CoV-2 Vaccine. Vaccines 1203 (Basel)

Endothelial cell infection and 1209 endotheliitis in COVID-19

Estimates of the severity of coronavirus 1212 disease 2019: a model-based analysis

Development and Applications 1216 of Viral Vectored Vaccines to Combat Zoonotic and Emerging Public Health Threats

Syrian Hamsters as a Small Animal 1219 Model for Emerging Infectious Diseases: Advances in Immunologic Methods

Observations on the Repeated Administration of Viruses 1222 to a Patient with Acute Leukemia. A Preliminary Report

Factors associated with COVID-19-related 1226 death using OpenSAFELY

Airway Memory CD4(+) T Cells Mediate 1229 Protective Immunity against Emerging Respiratory Coronaviruses

P and M gene junction is the optimal insertion 1232 site in Newcastle disease virus vaccine vector for foreign gene expression

A pneumonia outbreak associated with a new coronavirus of probable bat 1236 origin

SARS-CoV-2 Receptor ACE2 Is an Interferon-1239

Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across 1240 Tissues

NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1X protease inhibitor 801 cocktail (0087785, ThermoFisher)]. Cell lysates were centrifuged at 10,000 ×g for 15 min at 4 °C, 802 supernatants collected and used to quantify the protein concentration using the Pierce BCA Protein 803 Assay Kit (ThermoFisher). For SDS-PAGE, purified virus (1x10 7 PFU) or virus infected cell 804 lysates (mixed with 6x loading dye containing and 30% β-mercaptoethanol) were heated at 95 °C 805 for 10 min to denature proteins, followed by cooling on ice. The same PFU or protein amounts of 806 each sample (ranging from 5 to 70 μg depending on the experiment) were loaded into wells of 4% 807 stacking / 12% resolving gels, and proteins were resolved at 120 V for 1.5 h in running buffer 808 ThermoFisher), rabbit anti-SARS-CoV-2 S1 (dilution: 1:1000; PA5-81795; ThermoFisher) or S2 816 (dilution: 1:1000; NB100-56578; Novus Biologicals) subunits, or mouse anti-beta actin (diluted 817 1:1000; MA5-15739; ThermoFisher). The secondary antibodies were either goat anti-rabbit (G-818 21234) or goat anti-mouse IgG (G-21040) conjugated to horseradish peroxidase (diluted 1:2000; 819 ThermoFisher), and incubated for 1 to 3 h at RT. Protein was detected using the Pierce SuperSignal 820West Pico PLUS Chemiluminescent Substrate (ThermoFisher) and the BioRad ChemiDoc MP 821Imaging System (BioRad Image Lab 6.0.1. software). 822Neutralization of pseudotyped lentiviruses and NDV recombinant vaccines 823