key: cord-0842995-7wp1uvbe
authors: Maruggi, Giulietta; Mallett, Corey P.; Westerbeck, Jason W.; Chen, Tiffany; Lofano, Giuseppe; Friedrich, Kristian; Qu, Lin; Sun, Jennifer Tong; McAuliffe, Josie; Kanitkar, Amey; Arrildt, Kathryn T.; Wang, Kai-Fen; McBee, Ian; McCoy, Deborah; Terry, Rebecca; Rowles, Alison; Abrahim, Maia Araujo; Ringenberg, Michael A.; Gains, Malcolm J.; Spickler, Catherine; Xie, Xuping; Zou, Jing; Shi, Pei-Yong; Dutt, Taru; Henao-Tamayo, Marcela; Ragan, Izabela; Bowen, Richard A.; Johnson, Russell; Nuti, Sandra; Luisi, Kate; Ulmer, Jeffrey B.; Steff, Ann-Muriel; Jalah, Rashmi; Bertholet, Sylvie; Stokes, Alan H.; Yu, Dong
title: A self-amplifying mRNA SARS-CoV-2 vaccine candidate induces safe and robust protective immunity in preclinical models
date: 2022-01-03
journal: Mol Ther
DOI: 10.1016/j.ymthe.2022.01.001
sha: db348f0a7a358c04a301ef616bf25678bbea2caa
doc_id: 842995
cord_uid: 7wp1uvbe

RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans and the technology, is being leveraged for rapid emergency response. In this report, we assessed immunogenicity, and, for the first time, toxicity, biodistribution and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion stabilized Spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNP). In mice, one immunization with the SAM vaccine elicited a robust Spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of Spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to Phase-1 clinical evaluation (NCT04758962).

On March 11 th , 2020 the World Health Organization declared the coronavirus disease 2019 47 to the cell membrane ( Figure 1D ). Importantly, the surface-expressed SpikeFL-2P protein directly 115 bound to the target hACE2 receptor, as shown by flow cytometry of live, unpermeabilized 116 transfected cells incubated with soluble hACE2 and stained with an anti-ACE2 antibody (Figure 117 1E). Incubation with a Golgi-mediated protein transport inhibitor such as Brefeldin A (BFA) 118 substantially reduced surface expression of the Spike proteins and its binding to the soluble hACE2 119 receptor. Therefore, the SpikeFL-2P expressed from SAM was antigenically functional, and 120 consequently the SAM encoding SpikeFL-2P was formulated with LNP for further evaluation in 121 animal studies. 122

To evaluate vaccine immunogenicity in BALB/c mice, Spike-specific serum antibodies as 124 well as B and T cell responses were characterized after one or two intramuscular (i.m.) injections 125 of SARS-CoV-2 SAM (LNP), given 3 weeks apart, at doses of 0.015 μg, 0.15 μg, or 1.5 μg, or 126 injection of saline as a mock control ( Figure S2A ). 127 SARS-CoV-2 Spike-specific serum antibodies induced by the SARS-CoV-2 SAM (LNP) 128 vaccine candidate were measured 3 weeks after the first vaccination (3wp1, Day 21) and 2 weeks 129 after the second vaccination (2wp2, Day 35). A single immunization induced high titers of anti-130

Spike-IgG binding antibodies in a dose-dependent manner, which were further boosted after the 131 second vaccination ( Figure S2B ). The neutralizing activity was measured by a vesicular stomatitis 132 virus (VSV)-based SARS-CoV-2 (Wuhan strain) pseudovirus neutralization assay on Day 21 and 133

Day 35. At all three doses (0.015 µg, 0.15 µg, and 1.5 µg), the SAM vaccine candidate induced 134 neutralizing antibody titers after the first immunization, which were then boosted ~ 30-fold by the 135 second immunization (Figure 2A ). There was a clear and statistically significant dose response in 136 antibody levels elicited by different SAM vaccine dosage levels (p < 0.0001). This robust, dose-137 J o u r n a l P r e -p r o o f dependent neutralizing antibody response was also confirmed by a SARS-CoV-2 live virus assay 138 (VNT50, live virus 50% neutralization titers) 19 ( Figure 2B ). Importantly, both pseudovirus and live 139 virus-based neutralization Geometric Mean Titers (GMTs) following the second vaccination at all 140 three dose levels were greater than the GMT of COVID-19 convalescent human sera (~100-fold 141 at the 1.5 µg SAM dose) (Figures 2A-2B ). There was a strong correlation (r = 0.7803, p <0.001) 142 between the two neutralization assays (pVNT50 (pseudovirus 50% neutralization titer) and VNT50) 143

indicating that the two assays were comparable in terms of measuring neutralizing antibody titers 144 ( Figure S2C ). 145

In addition to the SARS-CoV-2 Wuhan strain, 2wp2 sera from SAM-immunized mice were 146 also tested against the Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617.2) variants of concern 20 147 using pVNT50 assay. GMTs of neutralization activity of mouse sera against B.1.1.7, B.1.351 and 148 B.1.617.2 variants were reduced (p < 0.01) compared to the GMTs to the Wuhan strain (Figures 149 2C, D and E) . However, compared to the GMT of human convalescent sera, GMTs elicited by 150 SAM vaccine candidate at the dose of 0.015 µg, 0.15 µg, or 1.5 µg were 5.5-, 28-, or 40-fold higher 151 against B.1.1.7, 5.4-, 27-, or 37-fold higher against B.1.351, and 2-, 3.2-, or 10-fold higher against 152 B.1.617.2 respectively. This result further demonstrates that in mice the SAM vaccine candidate 153 elicited robust immunity in both magnitude and breadth. Spike-specific B cells were observed in the draining lymph nodes that consisted predominantly 163 (over 80% in the 1.5 µg and 0.15 μg groups) of Spike-specific GC B cells (identified as live CD3-164

CD19+IgM-IgD-Spike+GL7 + CD95 + cells) ( Figure S3B ). Conversely, in the spleens, most of the 165 Spike-specific B cells from SAM vaccinated groups had a memory phenotype (identified as live 166 CD3-CD19+IgM-IgD-Spike+CD95 -CD38 + cells) ( Figure S3F Further, vaccine induced T cell mediated immune responses against the Spike antigen were 172 analysed in mice spleens at 2wp2 by intracellular-cytokine-staining and multi-parametric flow 173 cytometry, after ex vivo restimulation with full-length Spike, S1, S2, or receptor binding domain 174 (RBD) peptide mixes (Figures 3 and S4 ). The analysis measured the frequencies of total Spike-175 specific CD4 + and CD8 + T cells, as well as the phenotype of various polyfunctional T cell subsets 176 within the Spike-specific CD4 + (Th0, Th1, Th2 or Th17) and CD8 + (Tc0, Tc1, Tc2 and Tc17 T-177 cytotoxic (Tc)) compartments. The SARS-CoV-2 SAM (LNP) vaccine induced robust dose-178 dependent total Spike-specific cytotoxic CD8 + and Th1 CD4 + T cell responses even at the very 179 low dose of 0.015 µg RNA ( Figure 3A and 3B). The CD8 + responses were higher than CD4 + 180 responses and were characterized by high expression of CD107a degranulation marker, Interferon-181 γ (IFN-γ), Tumor Necrosis Factor-α (TNF-α), and interleukin-2 (IL-2) cytokines ( Figure S4A ) 182 with a polyfunctional cytotoxic Tc1/Tc0 phenotype ( Figure 3A ) consisting primarily of 183 J o u r n a l P r e -p r o o f CD107a + IFN-γ + TNF-α + triple positive and CD107a + IFN-γ + double positive CD8 + T cells ( Figure  184 3C) at all doses. The CD4 + responses were characterized by expression of IFN-γ, TNF-α and IL-185 2 cytokines ( Figure S4B ) as triple positive or various double positive polyfunctional combinations 186 ( Figure 3D ), giving predominantly a Th1/Th0 CD4 + T-helper phenotype at all doses ( Figure 3B ). 187

The IL-4/IL-13 (Th2) and IL-17F (Th17) cytokine responses were negligible in both CD4 + and 188 CD8 + compartments (Figures 3 and S4 ). The majority of the Spike-specific total CD8+ T cell 189 responses were detected against the S1 domain with relatively lower levels of CD8 + responses to 190 the S2 and RBD domains ( Figure S4C ), whereas similar levels of spike-specific CD4 + responses 191 to S1 and S2 domains with some RBD-specific CD4 + responses were induced ( Figure S4D ). 192

Overall, these data indicate a strong induction of SARS-CoV-2 anti-Spike IgG binding and 193 broadly neutralizing antibody titers, systemic polyfunctional cytotoxic CD8 + cell response, and 194

Th1-driven CD4 + T cell response by the SARS-CoV-2 SAM (LNP) vaccine candidate in mice. 195

The polarized T cell phenotype is particularly critical for the development of a safe vaccine against 196 SARS-CoV-2, given the potential risk of enhanced lung immunopathology associated with Th2 197 responses observed after Coronavirus vaccine administration in animal models. 17,18 198 Immunogenicity, protective efficacy, and enhanced respiratory disease assessments of 199

To determine protective efficacy of the SAM vaccine candidate against SARS-CoV-2 201 infection, we measured clinical and virologic endpoints of SARS-CoV-2 infection in Golden 202 Syrian hamsters after vaccination and then followed by viral challenge. Hamsters are susceptible 203 to SARS-CoV-2, given the homology between hamster and human ACE2 receptors, supporting 204 high levels of virus replication and transmission and leading to weight loss and severe pneumonia 205 similar to 23 206 J o u r n a l P r e -p r o o f

The SARS-CoV-2 SAM (LNP) vaccine candidate was injected i.m. into hamsters at a dose 207 of 0.03 µg (low dose) or 3 µg (high dose) on Day 0 and Day 21; saline was used as a mock control 208 ( Figure S5A ). Serum neutralizing antibody titers, measured by a plaque reduction neutralization 209 test (PRNT90), were readily detected at 3wp1 in all the SAM-vaccinated animals ( Figure S5B ). 210

After the second immunization, titers were boosted significantly in both vaccinated groups (p ≤ 211 0.0178). For the low dose vaccine, neutralizing antibody titers were generally higher in female 212 than in male hamsters after both immunizations, whereas with the high dose vaccine both genders 213 responded comparably after both immunizations. 214

Three weeks after the second dose, animals in all treatment groups were challenged 215 intranasally with 10 4 plaque forming units (PFU) of SARS-CoV-2 (isolate USA-WA1/2020). 216

Oral-pharyngeal swabs were taken at 1, 2, 3, and 7 days post infection (dpi) and assayed by plaque 217 assay to monitor viral load in the upper respiratory tract. At 1 dpi, infectious virus was detected 218 in all groups, but viral loads in the SAM high and low dose groups were 156-fold and 49-fold 219 (female hamsters) or 149-fold and 3-fold (male hamsters) lower than those in the mock control 220 ( Figures 4A-B) . Interestingly, at 1 dpi, viral loads in male hamsters were 50-fold higher than those 221 in female hamsters in the SAM low dose group ( Figures 4A-B) , possibly due to the lower 222 neutralizing antibody titers elicited in males ( Figure S5B ). At 3dpi, viral loads were reduced in all 223 SAM groups compared to mock vaccinated animals (p < 0.0002), approaching the limit of 224 detection of the assay. No virus was detected at 7 dpi in SAM and mock vaccinated animals. 225

At 3 dpi, 8 animals from each group (4 female and 4 male hamsters) were euthanized to 226 measure the viral loads in nasal turbinates, cranial right and caudal right lung lobes. In nasal 227 turbinates, a vaccine dose-dependent reduction in viral load was apparent in SAM vaccinated 228 groups. At 0.03 µg dose, the SAM vaccine reduced viral loads by 1,000-fold, but only in female 229 hamsters ( Figure 4C ). At 3 µg dose there was no detectable virus in both female and male 230 vaccinated animals. In the cranial and lung lobes, SAM vaccine at either dose markedly reduced 231 levels of virus (p < 0.001), with no detectable virus in 4 of the 8 animals from the 0.03 µg SAM 232 group and in all animals from the 3 µg SAM group ( Figures 4D and 4E) . 233

An additional 3 females from each group were euthanized at 3 dpi to evaluate resident lung 234 cells secreting IFN-γ, TNF-α, and IL-4. Data showed increased levels of IFN-γ and TNF-α, but 235 no IL-4 in the vaccinated groups, consistent with the Th1-biased immune response elicited by the 236 SAM vaccine in mice, and indicating a shift of the immune response away from a Th2 response 237 that is associated with the risk of enhanced lung immunopathology ( Figure S5C ). 238

The remaining hamsters were observed until the study's endpoint at 21 dpi for body weight, 239 body temperature, and clinical sign changes. Both the low and high dose SAM vaccinations 240 prevented significant weight loss in both male and female hamsters as compared to the mock 241 vaccinated animals by 21 dpi (p < 0.01) ( Figures S6A and S6B ). The maximum percentage loss 242 in body weight between the low and high dose SAM vaccinated groups was not statistically 243 different (4.2% low dose and 2.1% high dose groups). Mock vaccinated hamsters lost an average 244 maximum body weight of 11% by 21 dpi. No significant changes in body temperature, as 245 measured by thermal microchips, were observed in any of the challenged groups (Figures S6C and 246 S6D) . 247

To further evaluate the efficacy of the SARS-CoV-2 SAM (LNP) vaccine candidate and 248 any potential vaccine-mediated enhanced respiratory disease following challenge with SARS-249

CoV-2, hematoxylin and eosin (H&E) stained sections of the lungs (left lung and median lobe of 250 the right lung, Figure 5 ), trachea, brain, liver, kidney, spleen, thymus, heart, and adrenal gland 251 were microscopically evaluated at 3, 7 and 21 dpi. Naïve non-infected hamsters (4 females and 4 252 males) were euthanized on at 21 dpi and included as a baseline control ( Figure 5C ). 253

Histopathological examination of samples of the brain, liver, kidney, spleen, thymus, heart and 254 adrenal gland revealed no notable vaccine-or virus-related changes in SARS-CoV-2 SAM ( PMNs were present within the airway epithelium and lumen and surrounding large blood vessels. 263

The inflammatory infiltrate was associated with varying levels of necrosis of alveolar pneumocytes 264 and/or capillary endothelia, hemorrhage and edema. A dose-dependent reduction in the incidence 265 and severity of inflammation was observed in vaccinated animals ( Figure 5 ). Most of the 0.03 µg 266 SARS-CoV-2 SAM (LNP) vaccinated animals had minimal or mild changes. Among the hamsters 267

given the 3 µg dose, one male had minimal inflammation in the lung, whereas all females and 268 other three males exhibited normal lung morphology. By 7 dpi, mild to marked lung inflammation 269 was observed in the mock vaccinated animals, similar to that observed at 3 dpi ( Figure 5) . A 270 marked reduction in both the incidence and severity of the inflammatory and epithelial changes 271 was observed in lungs of animals from both vaccine-treated groups when compared with the mock 272 vaccinated animals. In the 0.03 µg dose group, minimal inflammation and epithelial 273 hypertrophy/hyperplasia were observed in one male, with no changes noted in the other 7/8 274 animals in this treatment group. For the high dose group (3 µg), the lungs of all animals (8/8) were 275 morphologically normal. By 21 dpi, inflammatory changes in the lung of the mock vaccinated 276 animals had a similar distribution to that seen at 3 and 7 dpi, but were notably reduced in severity, 277 with minimal or mild changes observed in all animals. Multifocal mild to marked 278 hypertrophy/hyperplasia of the goblet cells of the bronchial epithelium was observed in all 279 unvaccinated animals, reflecting an expected metaplastic response to chronic cell injury following 280 SARS-CoV-2 viral infection. None of these findings were present in animals given either the 0.03 281 µg or 3 µg dose of the SAM vaccine candidate prior to SARS-CoV-2 exposure ( Figure 5B ). 282

In the trachea at Day 3, minimal to moderate multifocal mixed inflammatory cell infiltrates 283 were observed within the lamina propria in 7/8 mock-vaccinated animals and were associated with 284 focal hemorrhage in some instances. A similar level of inflammatory infiltrate was observed in 285 the trachea from the SAM low dose group animals, with all (8/8) animals having minimal or mild 286 changes. In the SAM high dose group, a reduction in incidence and severity of inflammatory 287 infiltrates was observed (minimal severity, 4/8 animals) and epithelial erosion, luminal exudate or 288 hemorrhage in the lamina propria were absent. At Day 7, minimal or mild multifocal mixed 289 inflammatory cell infiltrates, associated with minimal hemorrhage or occasional erosion, were 290 observed in the trachea of some mock vaccinated animals (6/8). A dose-associated reduction in 291 incidence and severity of these changes was observed in vaccinated animals, with changes limited 292 to minimal inflammatory cell infiltrates in 3/8 animals from the SAM low dose groups, while all 293 SAM high dose vaccinated animals had normal tracheal morphology. At day 21 there were no 294 findings in the trachea of any animal exposed to SARS-CoV-2. 295

In summary, these data show that the SARS-CoV-2 SAM (LNP) vaccine candidate induced 296 robust and protective adaptive immunity, which reduced virus load in the upper and lower airways 297 of hamsters and protected them against weight loss and lung pathology following challenge with 298 SARS-CoV-2. No evidence of enhanced respiratory disease was found in any of the vaccinated 299 animals. 300

To further support the development of the SARS-CoV-2 SAM (LNP) vaccine, studies were 303 conducted in Sprague-Dawley rats to assess the local tolerance, potential local and/or systemic 304 toxicity, and biodistribution of the vaccine. The rat was selected for this purpose as this species is 305 considered immunologically relevant and is a routine species for the toxicity testing of 306 vaccines. 24, 25 The rat also expresses toll-like receptor 7 (TLR7), among other TLRs and RNA 307 sensors, which participate in the immune recognition of RNA. 7,26 308

The repeated-dose toxicity study was conducted in male and female rats to characterize the 309 vaccine's potential local and systemic toxicity after 3 i.m. injections (12 µg RNA/vaccination; 310 saline was used as a mock control) at two-week intervals and included a 4-week treatment-free 311 period after the 3 rd vaccination ( Figure S7A ). Overall, the SARS-CoV-2 SAM (LNP) vaccine was 312 well-tolerated. Induction of Spike-specific IgG binding antibody responses was confirmed in 313 100% of the rats at Day 32 and Day 57 (i.e., 3 and 28 days following the 3rd immunization) ( Figure  314 S8A). Injection site observations included very slight erythema after each immunization, 315 persisting until 72-hours post-vaccination, and swelling (edema) following the second and/or third 316 administration in some animals, which generally did not persist longer than 48-hours post-317 vaccination. The body weight of the animals was not affected by the consecutive administrations 318 of the SAM vaccine candidate ( Figure S8B ). 319

Slight transient increases in mean rectal temperatures in SAM vaccinated animals were 320 measured after each dose compared to the control group (i.e., mean changes up to ~2°C in males 321 and females up to 48 hours following vaccination) ( Figure 6A ). There were no SARS-CoV-2 322 SAM (LNP)-related ophthalmological findings during this study. 323

At Days 2 and 30, there were transient effects on hematology parameters relative to the 324 control group, i.e., increased white blood cells (up to 1.40-fold) and neutrophils (up to 7.97-fold), 325 and decreased lymphocyte (0.30-fold) and monocyte (0.67-fold) counts (Table S1 and (Tables S2 and S3) . 332

Increased organ weights noted on Day 32 in the iliac, inguinal and popliteal lymph nodes 333 were likely related to increased cellularity of the medullary cords (minimal to moderate), 334 characterized by increased numbers of lymphocytes, plasma cells and macrophages. This was 335 considered secondary to the expected immune stimulation of the SAM vaccine candidate within 336 the regional lymphoid tissue draining the injection site (Table S3) . After the 4-week recovery 337 period, there were no organ weight differences noted between groups. 338

Microscopic findings occurred primarily at the injection sites and draining lymph nodes. 339

The SARS-CoV-2 SAM (LNP) vaccine candidate induced an increased incidence and severity of 340 subcutaneous and muscular inflammation and/or hemorrhage, at injection sites ( Figures 6B and  341   6C ). In the affected muscle, the predominant inflammation extended down fascial plains between 342 J o u r n a l P r e -p r o o f muscle bundles or was within intermysial regions ( Figure 6D ). Nonetheless, these findings were 343 considered a consequence of the expected immune response at the site of administration. 344

To evaluate the distribution of SARS-CoV-2 SAM (LNP) over time, a biodistribution study 345 was performed ( Figure S7B ). Male and female rats received a single i.m. administration of 6 µg 346 of the SAM vaccine candidate or saline as a mock control at Day 1 and then distribution of RNA 347 in tissues of vaccinated animals was analyzed at Days 2, 8, 15, 29, and 60 by RT-qPCR (Table 1) . 348

At Day 2, SAM RNA was detected with relatively high levels in muscle, lymph nodes, and 349 spleen, and relatively lower levels in heart, liver, gonads, lungs, gonads, and blood. RNA levels 350

progressively decreased in quantity in all tissues by Day 60, but remained detectable in lymph 351 nodes, spleen, and muscle, with at even lower quantities in the kidneys and livers of females. RNA 352 was detectable in the testes only on Day 2 and in the ovaries on Day 2 and Day 8. Additionally, 353 SARS-CoV-2 SAM was detected for longer periods in the liver of the females relative to the males. In mice, after the first and second immunization with sub-microgram doses of the SARS-373

CoV-2 SAM (LNP) candidate vaccine, robust neutralizing antibody titers were observed, which 374 were similar to and greater than the titers of a panel of SARS-CoV-2-convalescent human sera, 375 respectively. The magnitude, longevity, and quality of antibody responses induced by a vaccine 376 is determined by its ability to induce GC reactions that lead to differentiation of memory B cells 377 and long-lived plasma cells. 21 We found that the SARS-CoV-2 SAM (LNP) vaccine elicited robust 378

Spike-specific class-switched B cells and GC responses, suggesting a potential induction of high-379 affinity long-lasting B cells, which are critical for long-term protection and recovery from SARS-380

CoV-2 infection. 28 Testing at later time points after a single or a two-immunization regimen could 381 help determine the longevity of this response and the effectiveness of a possible single dose 382 regimen. 383 A SARS-CoV-2 vaccine should be effective against the emergence of SARS-CoV-2 384 variants of concern with either increased transmission or virulence. In mice, our vaccine candidate 385 elicited significant neutralizing antibody titers against SARS-CoV-2 B. 1.1.7 highlighting the dose-sparing potential of the SAM vaccine technology 10 , which will be also 419 reflected in the lower vaccine doses to be tested in clinical setting. To address pandemics, such as 420 COVID-19, where several billion doses of vaccines are needed within months, a lower vaccine 421 dose required for protection, i.e. dose-sparing, would be extremely beneficial. Interestingly, in 422 this hamster study, female and male animals responded differently to the SARS-CoV-2 SAM 423 (LNP) vaccine, and the female animals had higher neutralizing antibody titers and lower viral titers 424 at the low vaccine dose (0.03 µg RNA). As noted in several studies (for reviews, see 35 and 36 ), 425 females typically develop more robust antibody responses following vaccination than their male 426 counterparts. Dissection of the mechanisms which underlie gender differences in vaccine-induced 427 immunity is complex and has implicated hormonal, genetic, and environmental differences 428 between females and males, and the deep analyses required to study these elements exceed the 429 J o u r n a l P r e -p r o o f scope of the studies reported herein. Importantly, no evidence of enhanced respiratory disease was 430 found in any of the vaccinated hamsters, which is consistent with the Th1 cytokine signature 431 produced by resident lung cells seen in the vaccinated animals as well as the Th1 CD4+ T cell 432 response observed in mice. 433

Given the novelty and the early stage of the SAM technology, we evaluated its potential 434 toxic effects and biodistribution in rats. After two administrations, the clinical signs were limited 435 to slight local irritation at the injection site (erythema and/or edema), which did not persist for 436 more than 48 hours post vaccination. After each vaccination, there was also transient increase at 437 6-and 12-hours post-administration in mean rectal temperatures. This might be indicative of 438 innate immune responses triggered by the RNA and lipid formulation used for delivery, and/or 439 process-derived dsRNA and short aborted mRNA not completely removed from the vaccine. 37,38 440

Self-amplifying mRNA molecules are at least 10 kb in length and present additional challenges 441 for purification compared to conventional mRNA molecules. Preliminary data in mice have shown 442 that the intracellular influx of the self-amplifying mRNA, rather than its amplification, is mainly 443 responsible for eliciting type I IFN response. 39 However, additional studies are needed to dissect 444 the contribution of incoming and amplifying mRNA to innate activation and possible vaccine 445 reactogenicity. Inflammatory responses, indicating the establishment of an innate immune 446 response, were also observed with a transient and reversible effects on hematology and chemistry 447 parameters and by the activated appearance of the draining lymph nodes, at both the microscopic 448 and macroscopic levels. Overall, these changes were similar to those seen after the administration 449 of a cationic nanoemulsion (CNE) formulated SAM vaccine as well as of conventional vaccines, 450 including adjuvanted recombinant proteins and virus like particles-based vaccines. 40-42 451 In the biodistribution study in rats, the vaccine delivered RNA was found in the muscle at 452 the injection site until 60 days post vaccination. RNA was also found in the draining lymph nodes 453 at Day 2, suggesting that it quickly traffics to the host's lymphoid organs. It remains unclear, 454 however, whether the RNA distributed to the lymph nodes or whether it entered immune cells that 455 subsequently trafficked to the lymph nodes. Similar to the previous report on a CNE formulated 456 SAM vaccine 40 , SAM RNA was detected also at systemic sites, including spleen, blood, lung and 457 liver. Although expression from a LNP delivered self-amplifying mRNA has been reported in the 458 draining lymph nodes of mice after intradermal administration 43 , it remains to be determined 459 whether the signal found in the various organs for a prolonged period of time in our study is from 460 injected material or amplified material (i.e., generation of multiple RNA copies through self-461 amplification in target cells), and whether it represents full-length RNA or truncated pieces of 462 RNA resulting from degradation. will be critical to generate clinical data from other SAM-based vaccines to fully understand the 473 potential of this technology. Each SAM vaccine candidate could have different performance in 474 humans, for example, due to difference in RNA sequences and manufacturing processes, levels of 475 purity, enrichment in full-length SAM molecules, and delivery systems. 476

The results from the ongoing GSK clinical trial will inform if the current SAM vaccine is 477 sufficiently tolerated and immunogenic, or if further optimization, including RNA engineering, 478 new delivery systems, and modulation of innate and adaptive immune responses, among others, 479 will be needed, contributing to define the true prospects of the technology. 480

The primary objective of these studies was to determine and characterize the 483 immunogenicity, protective efficacy, safety and biodistribution of a SARS-CoV-2 SAM (LNP) 484 vaccine candidate in preclinical models (mice, hamsters and rats). 

The mouse studies were carried out at GSK (Upper Providence, PA). Female BALB/c 502 mice 7-8 weeks of age (Charles River, Raleigh, NC) (N=10 mice/group; randomly distributed 503 between groups) received 2 i.m. injections 3 weeks apart in the hind leg thigh muscle with the 504 SARS-CoV-2 SAM (LNP) vaccine at three dose levels of 1.5 µg, 0.15 µg or 0.015 µg RNA or 505 saline (mock). Serum samples were collected 21 days after the first immunization (3wp1) and 14 506 days after the second immunization (2wp2) to assess IgG binding and neutralizing antibody titers, 507 as described in the supplemental methods. Spleens and draining inguinal lymph nodes were 508 collected 2wp2 to characterize Spike-specific B cell and CD4 + and CD8 + T cell responses, as 509 described in the supplemental methods. Details of the mice study design are provided in Figure  510 S2A. 511

The hamster study was carried out at Colorado State University (Fort Collins, CO). Male 513 and female Golden Syrian hamsters (Mesocricetus auratus) approximately 8 weeks of age 514 (Envigo, Indianapolis, IN) (N=27 animals/group, 15 females and 12 males) received 2 i.m. 515 injections, 3 weeks apart in the hind leg thigh muscle with the SARS-CoV-2 SAM (LNP) vaccine 516 at a dose of 3 µg or 0.03 µg or with saline (mock). A non-treated group of hamsters (N=8, 4 517 females and 4 males) was also included in the study to provide a baseline for the histopathologic 518 analysis. Serum samples were collected 21 days after the first immunization (3wp1) and 21 days 519 after the second immunization (3wp2) to assess neutralizing antibody titers. Three weeks after the 520 second immunization, hamsters were anesthetized (ketamine-xylazine) and challenged under 521 BSL3 containment by intranasal instillation of 10 4 plaque forming units (PFU) of the USA-522 WA1/2020 isolate of SARS-CoV-2 virus (product NR-52281, BEI resources) propagated in Vero 523 C1008 cells (Vero E6) as previously described. 47 524 Animals were monitored for body weights and temperatures (thermal microchips) once 525 daily starting at one day prior to viral challenge and continuing until the day of scheduled 526 euthanasia. Oropharyngeal swabbing for viral load assessment in the upper respiratory tract was 527 performed at on Days 1, 2, 3, and 7 post challenge. Samples were collected in viral transport 528 medium, frozen at -80 o C, and assayed for virus by plaque assay on Vero cells. 529

Scheduled euthanasia of 8 hamsters per group (4 males and 4 females) was performed on 530

Days 3 (acute), 7 (subacute), and 21 (resolving) post-challenge. At Day 3, nasal turbinates and 531 two lobes of lung (cranial and caudal lobes of the right lung) were harvested for virus titration by 532 plaque assay on Vero cells. At Days 3, 7, and 21, a selected panel of tissues was harvested for 533 macroscopic observation and histopathology. Details of the hamster study design are provided in 534 Figure S5A , while methods for plaque assay, cytokine profiling and histopathology are described 535 in the supplemental methods. 536

Male and female Sprague-Dawley rats (Rattus norvegicus) were obtained from Charles 540 River Laboratories (Laval, Quebec, Canada) and were acclimated for at least 12 days to be 10 541 weeks old at the initiation of dosing (Day 1). The male rats weighed 310-388 g and the female 542 rats weighed 205-271 g on Day 1. The repeated-dose toxicity and biodistribution studies were 543 carried out at the Charles River Laboratories (Laval, Quebec, Canada). Description of welfare 544 principles and non-clinical guidelines 48,49 are described in the supplemental methods. 545

The dosing schedule (3 doses) was intended to cover the number of injections planned in 547 human clinical trial subjects +1 additional dose (i.e., n + 1 dosing). During the acclimation period, 548 15 male and 15 female rats were allocated to 2 groups by a randomizing stratification system based 549 on body weights. On Days 1, 15 and 29, the animals received one i.m. injection of the SARS-550

CoV-2 SAM (LNP) vaccine (100 μL, corresponding to 12 μg RNA) into the posterior part of the 551 thigh muscle. Animals in the control group received saline in the same conditions as in the treated 552 groups. The study design is depicted in Figure S7A . Detailed methods for clinical examinations, 553 serology, hematology, coagulation and blood biochemistry investigations as well as for necropsy, 554 tissue processing and histopathological examination are provided in the supplemental information. 555

To evaluate the biodistribution of the SARS-CoV-2 SAM (LNP) vaccine, one i.m. injection 557 (100 μL, corresponding to 6 μg RNA) was performed into the posterior part of the thigh muscle of 558 the right hind limb of male and female Sprague-Dawley rats. The biodistribution was evaluated 559 in tissues by reverse transcription quantitative polymerase chain reaction (RT-qPCR) on extracted 560 RNA, targeting the non-structural region of the SAM construct, over a 59-day observation period 561 ( Figure S7B ). Rats in the treatment group were randomly allocated into 5 subgroups (1 562 group/timepoint) of 10 animals (5 animals/sex/group). Treated animals were necropsied at Days 563 2, 8, 15, 29 and 60. In the control group, 5 males and 5 females received an injection of saline, 564 and 1 animal/sex was sacrificed at the same time points as in the treated groups. 565

At their respective scheduled termination (Days 2, 8, 15, 29 and 60) , animals were 566 anesthetized with isoflurane and blood and tissues (brain, lung liver, spleen, kidneys, heart, gonads, 567 lymph nodes (popliteal, inguinal and iliac) and muscle at the injection site) were collected, 568 preserved, and RNA was extracted and analyzed by RT-qPCR as previously described. 40 Detailed 569 methods are described in the supplemental materials. 570

Mixed model repeated measurements with group, day and interaction between group and day as 572 fixed effects with appropriate variance and co-variance structure was fitted on log10 transformed 573 for Spike-specific IgG binding antibody titers, pVNT50 and VNT50 for the mouse data, and for 574 pVNT50 and oropharyngeal swab viral titers for each sex for the hamster data. An analysis of 575 variance (ANOVA) was fitted on log10 transformed data with group as fixed factors with 576 appropriate variance assumption for pVNT50 and VNT50, antigen specific B cell and T cell 577 responses in mice and for nasal and lung tissue virus titer (nasal turbinates, cranial lung, caudal 578 lung for each sex) in hamsters. For hamsters' body weight and body temperature, an analysis of 579 variance (ANOVA) was performed with group as fixed factors on separated day for each sex. All 580 data shown were derived from one experiment, except for B cells data where data from two 581 individual experiments were combined. For the comparison of mean body weight, organ weights 582 and relative organ weights, mean body temperature, food consumption, hematology, coagulation, 583 clinical chemistry mean values or Draize-based scoring parameters in the rat study, statistical 584 methods and criteria were used following those described in Stokes et al, 2020. 40 indicate distribution of the Tc0/Tc1/Tc2/Tc17 cytotoxic cells within the total CD8+ and the 851 Th0/Th1/Th2/Th17 T helper cells within the CD4 + Spike-specific T cells (Mean ± SEM). Boolean 852 combinations for Spike-specific CD8 + and CD4 + T cell cytokines from splenocytes of individual 853 immunized mice were background subtracted using Pestle software, and pies were generated using 854 SPICE software (C, D). Each pie slice represents a proportion of the total Spike-specific CD8 + or 855 CD4 + T cell responses for a unique sub-population comprising of CD107a, IFN-γ, IL-2 or TNF-α, 856 as indicated in the legend. Bars under p value asterisks indicate significantly different groups 857 comparisons with *, p<0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. 858 

Pandemic Preparedness: Developing Vaccines and Therapeutic Antibodies For COVID-633 19

Safety and 638 Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

FDA Approves First COVID-19 Vaccine

Rapidly produced 644 SAM((R)) vaccine against H7N9 influenza is immunogenic in mice

Nonviral delivery of self-648 amplifying RNA vaccines

Induction of 652 an IFN-Mediated Antiviral Response by a Self-Amplifying RNA Vaccine: Implications 653 for Vaccine Design

Self-Amplifying RNA 656 Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much 657 Lower Doses

A Nanostructured Lipid 660 Carrier for Delivery of a Replicating Viral RNA Provides Single, Low-Dose Protection 661 against Zika

A single dose of self-664 transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective 665 adaptive immunity in mice

Low-dose single-shot COVID-19 mRNA vaccines lie ahead

A new coronavirus associated with human 670 respiratory disease in China

Key residues of the receptor binding motif in the spike protein of 673 SARS-CoV-2 that interact with ACE2 and neutralizing antibodies

Structural basis for the 676 recognition of SARS-CoV-2 by full-length human ACE2

Mechanisms of SARS-CoV-2 679 entry into cells

Cryo-EM structure of the 2019-nCoV spike in the 682 prefusion conformation

Immunization with SARS coronavirus vaccines 685 leads to pulmonary immunopathology on challenge with the SARS virus

A Double-689 Inactivated Severe Acute Respiratory Syndrome Coronavirus Vaccine Provides 690

Incomplete Protection in Mice and Induces Increased Eosinophilic Proinflammatory 691 Pulmonary Response upon Challenge

A high-throughput neutralizing antibody assay for COVID-695 19 diagnosis and vaccine evaluation

SARS-CoV-2 Delta 698 (B.1.617.2) Variant: A Unique T478K Mutation in Receptor Binding Motif (RBM

From vaccines to 701 memory and back

Syrian hamsters 704 as a small animal model for SARS-CoV-2 infection and countermeasure development

Simulation of the Clinical and 708 Pathological Manifestations of Coronavirus Disease

Syrian Hamster Model: Implications for Disease Pathogenesis and Transmissibility. Clin 710 Infect Dis

Note for guidance on preclinical pharmacological and toxicological 712 testing of vaccines

WHO guidelines on nonclinical evaluation of vaccines

Characterization of equine and other vertebrate TLR3, 720 TLR7, and TLR8 genes

Efficacy and 723 Safety of the mRNA-1273 SARS-CoV-2 Vaccine

SARS-CoV-2 mRNA 727 vaccines induce persistent human germinal centre responses

mRNA-1273 731 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 732 variants

Virus-Specific Memory CD8 T Cells Provide Substantial Protection from 735

Lethal Severe Acute Respiratory Syndrome Coronavirus Infection

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19

Alphavirus-derived replicon RNA vaccine induces SARS-CoV-2 neutralizing antibody 745 and T cell responses in mice and nonhuman primates

Self-amplifying RNA 749 SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody 750 titers in mice

Attenuated 753 activation of pulmonary immune cells in mRNA

CoV-2 infection

Sex differences in immune responses

Sex 758 differences in vaccine-induced humoral immunity

Generating the optimal 761 mRNA for therapy: HPLC purification eliminates immune activation and improves 762 translation of nucleoside-modified, protein-encoding mRNA

Corticosteroids and cellulose purification improve, respectively, the in vivo translation 767 and vaccination efficacy of sa-mRNAs

Immunogenicity and Protection Efficacy of a Naked Self-Replicating mRNA-Based Zika 772 Virus Vaccine. Vaccines (Basel)

Nonclinical safety 775 assessment of repeated administration and biodistribution of a novel rabies self-776 amplifying mRNA vaccine in rats

Non-clinical 779 safety assessment of single and repeated intramuscular administration of a human 780 papillomavirus-16/18 vaccine in rabbits and rats

Non-clinical safety assessment of single and repeated administration of gE/AS01 784 zoster vaccine in rabbits

Expression Kinetics and Innate 787 Immune Response after Electroporation and LNP-Mediated Delivery of a Self-788 Amplifying mRNA in the Skin

Shattock RJ 794 Safety and Immunogenicity of a Self-Amplifying RNA Vaccine Against COVID-19: 795 COVAC1, a Phase I, Dose-Ranging Trial

A scalable, extrusion-free method for efficient liposomal encapsulation of 799 plasmid DNA

Delivery of 802 self-amplifying mRNA vaccines by cationic lipid nanoparticles: The impact of cationic 803 lipid selection

A 806 Whole Virion Vaccine for COVID-19 Produced via a Novel Inactivation Method and 807 Preliminary Demonstration of Efficacy in an Animal Challenge Model. Vaccines (Basel) 808 9

Considerations for Plasmid DNA Vaccines for Infectious Disease 813 Indications

Considerations-for-Plasmid-DNA-Vaccines-for-Infectious-Disease-816 Indications

Statistical analysis: *, p<0

001 with t-Test, except those 886 labelled "w", which was analyzed using Wilcoxon's test. The injection site sections, which include 887 skin, dermis and underlying skeletal muscle, from saline (B) or SAM (C) vaccinated animals at 888

Day 33 (3 days post third vaccination) were stained with H&E, and representative 889 photomicrographs are represented (scale bar, 1000 μm). (D) 5x magnification of the boxed area 890 in Figure 6C showing inflammation after SAM vaccination (scale bars

caudal right (E) lung lobes of 4 female and 4 male hamsters from each group at 3 dpi. Bars 863 represent GMT + 95% CI. Asterisks above bars indicate statistically significant difference in viral 864 titers between mock and vaccine groups (****, p<0.0001; ***, p<0.001). Horizontal dotted line 865 denotes limit of detection. 866