key: cord-0723221-0k0ve471
authors: Boulton, Stephen; Poutou, Joanna; Martin, Nikolas T.; Azad, Taha; Singaravelu, Ragunath; Crupi, Mathieu J.F.; Jamieson, Taylor; He, Xiaohong; Marius, Ricardo; Petryk, Julia; Tanese de Souza, Christiano; Austin, Bradley; Taha, Zaid; Whelan, Jack; Khan, Sarwat T.; Pelin, Adrian; Rezaei, Reza; Surendran, Abera; Tucker, Sarah; Brown, Emily E.F.; Dave, Jaahnavi; Diallo, Jean-Simon; Auer, Rebecca; Angel, D Jonathan B.; Cameron, William; Cailhier, Jean-Francois; Lapointe, Réjean; Potts, Kyle; Mahoney, Douglas J.; Bell, John C.; Ilkow, Carolina S.
title: Single dose replicating poxvirus vector-based RBD vaccine drives robust humoral and T cell immune response against SARS-CoV-2 infection
date: 2021-10-20
journal: Mol Ther
DOI: 10.1016/j.ymthe.2021.10.008
sha: 520f6af247a922c01f835170527c771ffa4448d8
doc_id: 723221
cord_uid: 0k0ve471

The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventative responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines.

A large, sustained community outbreak of severe acute pneumonia due to a novel coronavirus was first 47 recognized with household transmission in late 2019 in Wuhan, China. Severe acute respiratory syndrome coronavirus 48 2 (SARS-CoV-2) had likely emerged from a zoonosis, and onward person-to-person transmission 1 . Variant strains 49 have emerged in and spread from the UK (alpha), South Africa (beta), the US (epsilon), Brazil (gamma) and India 50

(delta), with variable changes in infectiousness, immune evasion, and pathogenicity 51

(https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html). As of September 2021, 216 million people 52

have been infected with SARS-CoV-2 and over 4.5 million have died of COVID-19 2 . This devastating pandemic has 53 resulted in the unprecedented rapid development of several new, safe, and efficacious vaccines employing different 54 technology platforms 3 . The platforms in furthest clinical development, with regulatory emergency use authorization 55

(EUA) or full FDA-approval, include different mRNA-based vaccines 4,5 and non-replicating adenovirus-vectored 56

vaccines 6, 7 . The immunodominant target of the humoral response 8, 9 , the Spike (S) protein, emerged as the central 57

immunogen focus for SARS-CoV-2 vaccine design [4] [5] [6] [7] 10 . The S glycoprotein is a large homotrimeric complex that 58 mediates binding and entry of SARS-CoV-2 into host cells. It is divided into two subunits, S1 and S2, which control 59 binding and fusion, respectively. Within the S1 subunit is the receptor binding domain (RBD) that mediates viral 60 attachment to host cells by interacting with the host receptor, angiotensin-converting enzyme 2 (ACE2) [11] [12] [13] . Since 61 neutralization of this RBD-ACE2 interaction is capable of blocking virus infection [14] [15] [16] [17] , the RBD is a popular target 62

for drug development and vaccination strategies. 63 64

Despite the incredible progress made over the past year, there are still numerous challenges facing COVID vaccine 65 development [18] [19] [20] [21] . At this point, it remains unclear whether the currently authorized vaccines elicit long-term 66

immunogenicity. In addition, multi-dose vaccines can create financial burdens and additional challenges for 67 manufacturing and administration, which can cause delays between doses that may yet have unforeseen consequences 68 on timeliness, degree and duration of protection. Special requirements for storage and handling, especially with mRNA 69 vaccines that require -80 °C freezers, have also created challenges for delivery to remote and impoverish communities 70 that lack prerequisite equipment 22 . Altogether, this creates a demand for new vaccines with increased stability and 71

performance. 72 73

Although an estimated 190 candidates are currently undergoing preclinical development and over 90 are in early 74 clinical development 23 , few are attempting to immunize with replicating attenuated vaccinia virus vectors 24 . Previous 75 work has demonstrated that vaccinia virus (VACV) can be engineered to express heterologous antigens and can be 76 strategically attenuated to act as a safe vaccine vector 25, 26 . VACV has an excellent safety profile, in large part due to 77 its historical use as the vaccine in the worldwide campaign to eradicate smallpox 27 . In the context of vaccine 78 development against SARS-CoV, modified vaccinia Ankara (MVA), a highly attenuated VACV strain incapable of 79 productive infection 28 , expressing the Spike glycoprotein was shown to be effective in inducing a neutralizing antibody 80 response [29] [30] [31] [32] . Recent studies have explored the use of MVA as delivery vector for SARS-CoV-2 antigens 19,33-36 . 81 82

While replication-incompetent viruses such as MVA are typically favored as vaccine vectors because of their safety, 83

replicating viruses offer several additional advantages for vaccine delivery. They have increased persistence at the 84 immunization site, which locally prolongs antigen expression; they are often more effective at stimulating T cell 85 responses, and they are relatively easy to manufacture for large populations. Replicating VACV strains have been 86 used previously for vaccination campaigns against smallpox with adverse effects occurring only in a very small 87 population of individuals. Since then, many attenuations have been discovered that reduce the health risks of VACV 88 strains even further 37-42 . In our lab, we have engineered oncolytic VACV variants used to treat immunocompromised 89 cancer patients without safety concerns 37, 39, 43, 44 . 90 91

In this study, we evaluate the use of a replication-competent attenuated Tiantan (TT) strain of VACV as vector for a  92 COVID-19 vaccine. With its role as the initial point of contact between SARS-CoV-2 and the host cellular receptor 93 ACE2, the RBD was selected as the primary immunogen for our vaccine [11] [12] [13] . While there are other potential 94 mechanisms to neutralize virus entry through targeting of S, direct inhibition of the RBD-ACE2 interaction is one of 95 the best characterized methods for blocking SARS-CoV-2 infection [45] [46] [47] [48] [49] . In the full S trimer, the RBD transitions 96 between two discrete conformations: an 'up' state, which is capable of interacting with ACE2 and a 'down' state in 97 which the receptor is inactive and shielded from neutralizing antibodies 20 . It is still not fully clear what controls the 98 conformation of the RBD in the S complex, but it is known that mutations in both the S1 and S2 subunits can change 99 these two conformations and influence antibody binding 6 . Thus, exposure of the RBD antigen for antibody generation 100 in full length S constructs is dependent on a conformational selection process that is controlled by other domains 101 within S. In contrast, an isolated RBD vaccine exposes epitopes that when targeted can neutralize interactions with 102 host cells. We therefore engineered a chimeric RBD construct fused to the transmembrane region of S (henceforth 103

termed CovAg) to expose essential epitopes for neutralizing antibody generation (Fig. 1A) . Membrane tethered 104 expression of RBD was used, as opposed to a secreted form, as membrane anchoring has been found to increase 105 immunogenicity of Spike-based mRNA vaccine 50 . We found that one individual dose of our strategically attenuated 106

TT-CovAg vaccine is sufficient to induce strong and long lasting RBD-targeted humoral and T cell immune responses 107

in vaccinated mice and cynomolgus macaques. The TT-based viral vector presented in this paper, TOH-VAC1, holds 108 great potential as an additional vaccine platform in the ongoing fight against COVID-19. 109 110

Characterization of poxvirus vaccine vectors 112

The RBD-based CovAg immunogen (Fig. 1A , Material and Methods) was encoded into two poxvirus strains, Tiantan 113

(TT) and modified vaccinia Ankara (MVA), to compare humoral and cellular immunities from both a replicating and 114 non-replicating viral vector, respectively. In both strains, CovAg was inserted into the B14R locus under the control 115 of a robust early/late promoter (H5R) (Fig. 1A ) 51 . For detection and purification of the virus, a GFP-firefly luciferase 116

reporter cassette under the control of a synthetic early/late promoter (O2A12) was incorporated following the CovAg 117 gene (Fig. 1A) . Recombinant TT and MVA vaccine vectors were generated through homologous recombination and 118 purified by selection of GFP positive plaques. Virus purity was confirmed via PCR analyses and Nanopore deep 119 sequencing (Fig. S1 ). 120 121

In order to compare viral kinetics of the antigen-encoding viruses with the parental wildtype (WT) strains, cells were 122 infected at an MOI of 1 and titers were quantified at 24 and 48 hours post-infection (hpi). Standard plaque assays 123 revealed that MVA CovAg replicated similarly to the WT virus in DF-1 chicken embryo fibroblast cells (Fig. 1B) . 124

Similar titers were observed for TT CovAg and TT WT in U2-OS cells (Fig. 1B) . Taken together, these results suggest 125

CovAg expression does not significantly impair replication of either MVA or TT viral vectors. 126 127

Expression of CovAg from MVA and TT vectors was assessed via immunoblotting with an HA antibody (Fig. 1C ). 128

Since RBD glycosylation is integral for maintenance of antigenic conformation and generation of neutralizing 129 antibodies against SARS-CoV-2 40,41,52,53 , glycosylation of the RBD was assayed by immunoblot following treatment 130 with glycosidase ( Fig. 1D, S2 ). The deglycosylated RBD had increased electrophoretic mobility, consistent with the 131 loss of two essential N-linked glycosylation sites at positions N331, N343 53,54 . Expression and localization of CovAg 132

was assessed by immunofluorescence ( Fig. 1E ) and flow cytometry (Fig. 1F ). Together, these data confirmed that 133

CovAg is translocated to the plasma membrane with the RBD exposed on the extracellular surface, and suggest that 134 the poxvirus vectors mediate expression of CovAg in an antigenically relevant conformation. 135 136 We investigated the short-term temperature stability of MVA and TT vaccine vectors, which has previously been 137 demonstrated to be stable long-term under a range of temperature conditions 55-57 . Both viruses were stored at -80°C, 138

4°C and room temperature for 1 week and then active vaccine particles were quantified by plaque assay. Under these 139 conditions, there was no significant drop in titer TT vectors, but replication of MVA was impaired by storage at room 140 temperature (Fig. 1G ). 141 142

Comparison of MVA and TT viral vaccine vectors 143

To assess and compare the safety profiles of our MVA and TT CovAg vaccines, BALB/c mice were injected 144 intraperitoneally (I.P.) at varying doses and their individual body weights were monitored over time, as an indicator 145 of mouse health. At the doses used for vaccination within this study, neither TT nor MVA caused any significant loss 146 average weight ( Fig. S3A-B) , and no signs indicative of pathogenic infection were observed. We further investigated 147 the safety of TT by injecting BALB/c intracranially (I.C.) at a dose of 1E6 plaque forming units (pfu), as previously 148 performed 58,59 . All mice from these injections survived with no observable signs of cognitive impairment or 149 neurotoxicity (Fig. S3C ). As a positive control, we observed that all mice injected I.C. with WT vesicular stomatitis 150 virus (VSV), a virus with well-known neurotoxicity 60-62 , died within 4 days. In addition, we did not detect any 151 significant difference in mouse body weight between intranasal (I.N.) administration of TT WT or TT CovAg (Fig.  152 S3D). 153 154

To determine which vector (MVA or TT) is more effective at stimulating immune responses against the CovAg 155 immunogen, BALB/c mice were vaccinated with each virus via I.P., I.N., or subcutaneous (S.C.) injections. Antibody 156 responses were measured by ELISA against recombinant RBD and the antibody titer was calculated by fitting of 157 dilution curves ( Fig. 2A -B, Materials and Methods). For I.N. and I.P. administration routes, TT CovAg was 158 significantly more effective than MVA CovAg at stimulating humoral responses against the RBD. On average, the 159 antibody titer for TT CovAg was 3-4 orders of magnitude greater than those of MVA CovAg. In contrast, the antibody 160 titers did not significantly differ between vaccines administered S.C, but the antibody titers induced by TT CovAg 161 S.C. administration were also 3-4 orders of magnitude lower than I.P. and I.N. administration (Fig. 2B ). 162 163

MVA and TT vectors were further compared for their ability to stimulate RBD-specific T cell responses via IFNγ 164 ELISPOT assays. Both the MVA CovAg and TT CovAg vaccines induced T cell responses against RBD epitopes by 165 both I.P. and I.N. administration routes ( Fig. 2C-D) . In addition, the T cell responses were more robust for both vectors 166 when administered I.N, and TT had stronger responses compared to MVA. Interestingly, before commercially 167 available peptide pools were available, we identified an immunodominant RBD epitope for BALB/c mice, which had 168 elevated responses for TT CovAg, similar to the commercial S1 peptide pool, as compared to DMSO controls (Fig.  169 S4A-C, Table S1 ). The responses against this single peptide were weaker for MVA CovAg than TT CovAg ( Fig. S4A -170 C). Lastly, we examined T cell responses against the VACV backbone using two well-established T cell epitopes 63 . 171

Both MVA and TT had similar responses to these peptides and we did not observe any difference between I.P. and 172 I.N. administration routes (Fig. 2E ). 173 174

Overall, the comparative analyses of antibody and T cell responses for TT CovAg and MVA CovAg showed that TT 175 was a more suitable vaccine platform. Not only did TT CovAg generate antibody titers that were 3-4 orders of 176 magnitude greater than those generated by MVA (Fig. 2B ) , but ELISPOT results were on average 1.5-2.5 times higher 177 with TT CovAg (Fig. 2D ). Of note, this was accomplished with a TT dose that was 10-fold less than MVA, and no 178 toxicity was observed. Given that TT outperformed MVA through these initial in vivo experiments and given the 179 relative challenge of manufacturing MVA compared to TT, we decided to focus on the TT CovAg platform for further 180 investigation into its suitability as a COVID-19 vaccine. 181 182

TT is a potent single-dose vaccine that offers longstanding protection 183

To determine whether our TT platform could be further enhanced, we designed a homologous prime/boost strategy 184 for our vaccine. BALB/c mice were initially inoculated I.P. and I.N. and ELISAs were performed every week to 185 evaluate RBD antibody responses. Upon detection of a slight decrease in antibody titer (around day 35), mice were 186 boosted I.N. with a second dose of the same vaccine. At day 49, the prime/boost responses were compared for each 187 route of administration, and we observed no significant increase in antibody titers after the boost dose (Fig. 3A) . We 188 continued measuring antibody titers for more than six months after the initial immunization, and we showed sustained 189 total antibody titers for both prime only and prime:boost vaccination regimes ( Fig. 3B -C). We also quantified the 190 neutralizing capacity of antibodies produced with and without the boosting dose using a replication-competent VSV 191 pseudotyped with the SARS-CoV-2 spike. This VSV-based surrogate system has been previously demonstrated to 192 resemble both SARS-CoV-2 entry and neutralizing antibody sensitivity 64 . Consistent with the ELISA data, antibodies 193 generated over a six-month period from a single dose TT CovAg vaccination resulted in robust neutralization without 194 drop in titer overtime ( Fig. 3D-F ). In addition, there was no noticeable difference in the neutralizing response of mice 195 receiving either one or two doses of the vaccine. Lastly, we showed high antibody titer in mice that were immunized 196 through I.M. administration of TT CovAg, but not TT WT control (Fig. S4D ).

Development of attenuated TT vaccine with improved safety 199

Given the compelling immunological responses observed in mice vaccinated with TT CovAg, we evaluated its 200 potential as a vaccine candidate in non-human primates. Since mice are more resistant to VACV compared to primates, 201

there was concern that TT might pose additional safety risks at the administered dose. To address that potential 202 concern, we further attenuated the VACV backbone by inserting mCherry into the A56R gene of the TT CovAg 203 vaccine to disrupt its function (TOH-VAC1). A56R was selected based on prior studies that showed its deletion 204 significantly attenuated the lethality of the virus in vivo 58 . CovAg expression was unaffected by the A56R deletion 205 ( An interesting feature of TT vector with A56R deletion is its enhanced syncytium formation capacity (Fig. S5C ). To 214 determine whether the attenuated toxicity or syncytia formation could impact the immune response against the CovAg 215 antigen, BALB/c mice were immunized and tested for neutralizing antibodies and T cell responses ( Fig. S5D -E). The 216

titer of neutralizing antibodies and the T cell response against RBD peptides were not significantly impacted by the 217 A56R deletion. Therefore, the novel TOH-VAC1 construct offers a vaccine platform safer than the parental TT vector 218 and without any loss in immunogenicity. 219 220

Investigation of TOH-VAC1 efficacy in cynomolgus macaques 221

Cynomolgus macaques were immunized I.M. with TOH-VAC1 at a dose of 1E7 pfu. With exception of some 222 macaques developing minor swelling or lesions at the injection site, which eventually made a full recovery (Fig. 4A ), 223

there were no adverse side effects from the TOH-VAC1 vaccine. Average body weight and temperature remained 224 unchanged ( Fig. S7A-B ). In addition, virus shedding was not observed in either macaque saliva or urine after 225 immunization, as indicated (Fig. 4B, S7C and Tables S3-S5 ). Antibody responses were measured by ELISA for 43 226 days following vaccination and from day 15 onwards, we observed a robust and consistent response (Fig. 4C ). 227

Similarly, the neutralization response against spike-pseudotyped VSV was consistent over the 43 days with values 228 similar to patients that had previously tested positive for COVID-19 (Wuhan strain) (Fig. 4D) . RBD specific T cell 229 responses were also measured from the PBMCs of macaques immunized with TOH-VAC1 (Fig. 4E 

To address potential concerns about the increase in resistance to spike-based vaccines observed for SARS-CoV-2 238 variants 65-67 , we developed a new biosensor assay (Fig. 4F, S8 ), based on our previous work 12,52 , to test the binding of 239 antibodies produced from TOH-VAC1 vaccination against common RBD variants that have emerged around the 240 world. Using this assay, we tested the antibodies produced in macaques against the WT (Wuhan sequence) and three 241 RBD variants, including the N501Y mutation found in the Alpha variant and the E484K mutation found in the Beta 242

and Gamma variants. Both of these variants can reduce virus sensitivity to immune sera from vaccinated individuals 243 and when combined together, resistance to pre-existing antibodies is even further increased 65-67 . However, with our 244 biosensor assay we found that sera from macaques immunized with TOH-VAC1 interacted with both single and 245 double mutants (Fig. 4F ). 246 247

As In our study, the comparative analysis of humoral and cellular immune responses between TT and MVA demonstrated 260 that TT is far more effective at stimulating neutralizing antibody production and alloreactive T cell responses against 261 the SARS-CoV-2 RBD (Fig. 2) . Antibody levels were 3-4 orders of magnitude lower for MVA immunized mice 262 compared to TT and T cell responses were 2-fold lower. The use of an attenuated non-replicating vector has several 263 advantages from a safety standpoint, but its inability to replicate in vivo also decreases the quantity and duration of 264 immunogen expression, which consequently leads to reduced immunity. With the replication-competent TT backbone, 265

our vaccine was capable of stimulating robust humoral and cellular immunity with only a single dose, whereas MVA-266 based SARS-CoV-2 vaccines require two doses to achieve strong immunity 18, 19, 36, 77, 78 . Antibody titers from our 267 vaccine were also similar to those reported for the mRNA and Adenovirus vector-based vaccines currently in 268 use 4,5,74,79 . 269 270 In mice, immunization with a second dose of TT CovAg did not further improve the levels of RBD-specific 271 neutralizing antibodies. In macaques, a single dose of TOH-VAC1 was sufficient to generate neutralizing antibodies 272 titers that are similar to those from former symptomatic COVID-19 patients. As for safety concerns with the use of a 273 replicating vector, we have shown in this study that TT is sufficiently safe for immunization of both mice and 274 macaques. However, if any concerns arise, it is possible to further attenuate TT using deletions like our A56R 275 knockout 58,59,80 . In contrast, it is much more challenging to reverse engineer MVA to become more immunogenic or 276 more persistent for prolonging immunogen expression. 277 278

Based on our results it is clear there are many inherent advantages to using a replicating poxvirus vector for a SARS-279

CoV-2 vaccine. It produces stronger immune responses with only a single injection at a 10-fold lower dose than its 280 non-replicating MVA counterpart. Biomanufacturing of MVA is also more challenging as it requires growth in 281 specialized cells such as primary chicken embryonic fibroblasts 35 , as opposed to TT which can be produced using 282 many common immortalized cell lines and at very high yields. Therefore, it will be faster to produce more doses and 283

improve global accessibility to vaccination with our TT vaccine than with a similar MVA-based vaccine platform. 284

Since many nations are also facing challenges obtaining enough vaccines to provide two doses in a timely manner, 285 this single-dose TT vaccine could improve global accessibility to vaccination. This will be essential for getting 286 COVID-19 rates under control, especially with new, highly infectious variants. 287 288

Anti-vector immunity can pose a challenge to many viral-vector vaccines. However, for VACV, pre-existing 289 immunity fades over time, and can be evaded through the use of mucosal routes of administration [81] [82] [83] . There is also 290 existing evidence that VACV-based vaccines suffer little to no impairment in protective efficacy when administered 291

to individuals that have received prior vaccination against smallpox 81,84-86 . These have been met with successful 292

boosting of the immune response, and in at least one case providing increased viral control in the previously vaccinated 293 population 87 . There is also an additional advantage in providing a vaccine that provides immunity against both SARS-294

CoV-2 and smallpox. Although smallpox has been eradicated on the world stage, there are still potential concerns that 295 it could be used for bioterrorism 88 . 296 297

A key factor for a vaccine to provide long-lasting immunity against SARS-CoV-2 and protection against novel SARS-298

CoV-2 variants will be the generation of T cell responses against highly conserved immunodominant antigens. Despite 299 being a key target for neutralizing antibodies, the RBD, which comprises the main component of the CovAg 300 immunogen used in this study, is not considered an immunodominant antigen 21,89,90 . However, our vaccine stimulates 301 robust T cell immunity against the RBD, which is likely attributed to using a replicating viral vector for delivery. 302

Replicating viral vectors such as TT can often stimulate more effective cellular immunity relative to non-replicating 303 vectors because they achieve higher and longer expression of the target immunogen and act as stronger adjuvants for 304

the immune system 22,89-91 . TOH-VAC1 is therefore a promising backbone for the next generation of vaccination 305

strategies implementing immunodominant T cell antigens. 306 307

The large coding capacity of VACV could permit encoding of multiple SARS-CoV-2 antigens, which could serve to 308 both increase T cell immunity against the virus and reduce the chance of novel variants arising that are resistant to the 309 vaccine. The S protein, which has been the target of available vaccines 4,5,74 , is prone to mutations that have resulted 310 in increased infectivity and/or vaccine resistance 65 . Currently, the four SARS-CoV-2 variants of concern and four 311

variants of interest that have been classified by the WHO all contain one or more mutations within the S protein. In 312 light of this, COVID-19 vaccine development has begun to incorporate other targets such as the nucleocapsid (N), 313 membrane (M) and envelope (E) proteins 92 . The N protein is a particularly promising target since it is highly 314 conserved in sequence and structure among coronaviruses, has lower rates of mutations compared to S and is highly 315 immunogenic 92 , driving T cell responses. In addition, a multi-antigen SARS-CoV-2 vaccine may be able to replace 316 S-based vaccines and alleviate potential concerns about vaccine induced thrombotic thrombocytopenia 93-95 . 317 318

As the emergence of new SARS-CoV-2 variants continues to increase it is necessary to assess the risk of vaccine 319 resistance 65 . Here, we developed a novel biosensor assay using a split luciferase system, that measures antibody 320

interactions with RBD mutants. The assay can be rapidly performed on samples from vaccinated or infected 321 individuals to reveal whether their existing antibodies are capable of binding mutant RBD and conferring some 322 protection against emerging SARS-CoV-2 variants. Using this assay, we demonstrated that the antibodies generated 323 from our TOH-VAC1 vaccine recognize four RBD variants. 324 325

Here, we report the development of a replicating VACV-based vaccine, TOH-VAC1, that expresses the RBD of 326 SARS-CoV-2. An RBD-based MVA vaccine has been generated previously by Liu et al., but antibody responses for 327 it were much weaker than its full length S counterparts 19 . This was likely due to the removal of key T cell epitopes 328 elsewhere in S that were capable of aiding humoral immunity. However, the study also found that the RBD-vaccine 329 was more effective than full length S constructs in generating neutralizing antibodies when used as a booster 5 . In our 330 case, we may have been able to overcome the weaker humoral and T cell responses from an RBD antigen with the use 331 of TT, a replicating poxvirus vector. This does open possibilities for heterologous prime-boost strategies with other 332 viral and subunit vaccines incorporating RBD or full-length S as an antigen. In many places, there are currently long 333 waits between doses for approved vaccines due to shortages and issues with manufacturing. TOH-VAC1 provides a 334 J o u r n a l P r e -p r o o f potential solution as a single, low-dose of replicating viral vaccine that is easy to manufacture and store. Clinical 335 testing will be important to determine the efficacy of this vaccine in humans, but TOH-VAC1 promises to be a valuable 336 tool in the fight against COVID-19. 337 338

Cell Lines and Viruses 341

All cell lines were purchased from the American Type Culture Collections (Manassas, VA All experimental absorbance readings were normalized relative to the blank and the positive control (monoclonal RBD 374 antibody at 1 µg/ml) and fit using a quadratic binding polynomial assuming 1:1 binding. The fitting was performed 375 using a Monte Carlo simulation with the non-linear curve fitting tool in QtGrace. The reciprocal antibody titer (LDF) 376 was determined by interpolating the dilution factor that intersected with a minimum detection threshold defined by 377

10x the standard deviation of the responses from mice vaccinated with Tiantan WT, or to a fixed value of 0.025 378 (whichever was larger). 379 380

ELISPOTs 381 BALB/c mice were inoculated either intranasally or intraperitoneally with 1E7 pfu MVA or 1E6 pfu Tiantan vaccines. 382

Seven days after injection, mice were sacrificed, and spleens were harvested for IFN-γ ELISPOT assays. Splenocytes 383 J o u r n a l P r e -p r o o f were isolated and incubated at a density of 2E5 cells/well on murine IFN-γ Single-Color ELISPOT plates 384 (ImmunoSpot) with either 1 µM of PepTivator SARS-CoV-2 Prot S1 pool (Miltenyi Biotec) or 10 µM of the 385 CYGVSPTKL peptide (CanPeptide). The CYG peptide was identified via a primary screen of previously discovered 386 SARS-CoV-1 T cell epitopes as a potent SARS-CoV-2 T cell epitope for BALB/c mice (Summary of peptides is found 387

in Table S2 ). In addition, DMSO was utilized as a negative control, while peptides from VacV F2/E3 63 388 (SPGAAGYDL/ VGPSNSPTF; Genscript) were used as positive controls for VACV, respectively. Splenocytes were 389 stimulated with peptides for 20 h and then the ELISPOT was performed according to the manufacturer's protocol. 390

Plates were imaged and spots were counted using an ImmunoSpot Analyzer. 391 392

Pseudovirus neutralization assay 393

Vero E6 cells were seeded in 96-well plates such that there were 40, 000 cells per well at the time of infection. Serum 394 was first diluted in a separate 96-well at a 1:10 dilution in serum free DMEM and a serial 1 in 2 dilution series was 395

performed. VSV pseudotyped with the SARS-CoV-2 spike glycoprotein 64 and co-encoded with eGFP was then added 396

to the serum in an equal volume of serum free DMEM for a final dilution of 2000 pfu per well and incubated for 1h 397

at 37 o C. After 1h, media on the cell was replaced with 60uL of the virus/serum and incubated for 1h at 37 o C. Wells 398

were then topped up with carboxymethylcellulose (CMC) in DMEM (supplemented with 10% FBS) for a final 399 concentration of 3% CMC and incubated 24 h at 34 o C. GFP foci were imaged and counted using a Cellomics 400

ArrayScan VTI HCS Reader.

Cynomolgus macaque study 403

Our study with 11 non-naïve cynomolgus macaques was performed at the Institute national de la recherche scientifique 404 (Laval, Québec). The macaques were previously used in an unrelated study involving inoculation with oncolytic 405

Maraba virus but had no prior exposure to any SARS-CoV-2 antigens. Pre-study data (body weight, detail clinical 406 exam, complete blood count, and liver function) were used for randomization. Each single-dose immunization (IM) 407

with TOH-VAC1 at 1E7 pfu was followed by observation for 4 h post-administration. Weekly body weight 408 measurements and detail clinical observations were recorded on bleed-day or the day before administration. Daily 409 cage-side observations were performed (e.g. animal behavior, food consumption, estrous cycle bleeding, feces 410 appearance). Images of the injection sites were acquired after vaccination and at the end of the study to monitor for 411 potential pox lesions. CBC and liver function analyses were conducted (sub-contracted by the CNBE), as well as 412 ELISA (serum) and Nabs (serum), and blood draw every 7 days. Blood draw (9-10 mL) for ELISpot day 1, pre-boost, 413

post boost and end of study (ELISA, Nabs and serum analysis performed by Dr. Lapointe's laboratory). Urine samples 414 and saliva throat-swab (in PBS) samples were collected and stored at -80°C on day -1 pre-vaccination (baseline), then 415 24 h, 48 h, and 1 week post vaccination to detect potential virus shedding. 416 417

Viral Shedding Luciferase Assay 418 U-2 OS cells were seeded at 1E4 cells/well in a 96-well white-bottom plate. The following day, 100uL of urine or 419 saliva samples or media alone was added to each well with 100uL of media supplemented with antibiotics to prevent 420 contamination. After 72 h, additional cells were infected with TOH-VAC1, which expresses Firefly luciferase, at 421 indicated pfu/ml (positive controls). At 6hpi, Renilla substrate was added to each well and readout was performed by 422

BioTEK plate reader. 423 424

Screening PCR for viral shedding of TOH-Vac1 425

To determine if macaques vaccinated with TOH-Vac1 shed viral genomes a PCR amplifying short sequences of two 426 different genomic loci was performed on throat swaps taken on d1 and d8 post vaccination. The genomic loci amplified 427 are B8R and D10R. Successful detection of parts of the viral genome results in the amplification of a 556 bp for B8R 428 and 548 bp for D10R, respectively (Tables S3-S5) . Throat swaps were boiled at 98°C for 3 minutes to inactivated 429 virus and release viral genomes. 2 µl of each sample were used per PCR reaction. As a positive control, U2-OS cells 430

were infected at an MOI of 0.1 with vaccinia Tiantan WT. The cell culture supernatant was collected 48 hpi, diluted 431 1/10 with sterile PBS and boiled at 98°C for 3 minutes. 2 µl of the positive control were used per PCR reaction. The 432 same amount of water was used as a negative control. After PCR, the complete reaction volume was analyzed on a 433

1.2% (w/v) agarose gel, stained with RedSafe™ Nucleic Acid Staining Solution (20,000x). 0.5 µg Thermo 434

Scientific™ GeneRuler 1 kb Plus DNA Ladder were used as reference. 435 436

Seroconversion Biosensor Assay 437

Recently we have developed several biosensors for detection of SARS-CoV-2 seroconversion 12,52 . Here, we updated 438 our biosensor by fusing nanoluciferase to the C-terminus of the RBD via a flexible linker. An IgK signal peptide was 439

inserted at the beginning of the RBD sequence to secrete the fusion protein from cells. All RBD mutations were 440 generated by site-directed mutagenesis. The construct was cloned into pcDNA3.1 and transfected into HEK293T cells. 441

After 2 days, the supernatant was collected and filtered to use for the seroconversion assay. Luciferase activity was 442 measured from the supernatants to adjust the relative concentrations of RBD used for the seroconversion assay. For 443 the seroconversion assay against different variants of SARS-CoV-2, 20 ul of magnetic beads conjugated with protein 444 G were combined with 50 ul of nanoluciferase-conjugated RBD and 5 ul of serum from vaccinated macaques. After 445 15 min incubation on a shaker at 25°C, the beads were collected using a magnet and washed with PBS three times to 446 remove excess nanoluciferase. The next step was followed by adding 50ul of diluted nanoluciferase substrate to it. A 447

Synergy microplate reader (BioTek, Winooski, VT, USA) was used to measure luminescence. 448 449

Statistical analyses 450

All graphs and statistical analyses were performed using GraphPad Prism v9. Means of more than two groups were 451 compared by one-way ANOVA using Tukey correction for multiple comparisons. Two-way ANOVA with Sidak 452 correction for multiple comparisons was used to compare the means of more than two groups split on two independent 453 variables. Dunnet's correction for multiple comparisons was used when comparing the mean of more than two groups 454 against a single baseline or control group. Alpha levels for all statistical tests were set at a threshold of 0.05. Normal 1. Zhou, P., Yang, X.-L., Wang, X.-G., Hu, B., Zhang, L., Zhang, W., Si, H.-R., Zhu, Y., Li, B., Huang, C.-L., 479 et al. (2020) Thomas, S.J., Kitchin, N., Absalon, J., Gurtman, A., Lockhart, S., Perez, J.L., Pérez Marc, G., 485 Moreira, E.D., Zerbini, C., et al. (2020) . Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. 486 Engl. J. Med. 383, 2603 -2615 5. Baden, L.R., El Sahly, H.M., Essink, B., Kotloff, K., Frey, S., Novak, R., Diemert, D., Spector, S.A., 488 Rouphael, N., Creech, C.B., et al. (2021) . Henderson, R., Edwards, R.J., Mansouri, K., Janowska, K., Stalls, V., Gobeil, S.M.C., Kopp, M., Li, D., Parks, 534 R., Hsu, A.L., et al. (2020 

Deletion of Apoptosis Inhibitor F1L in Vaccinia Virus Increases Safety 591 and Oncolysis for Cancer Therapy

Glycans on the SARS-CoV-2 Spike Control the Receptor Binding Domain Conformation

Multimerization-And glycosylation-dependent receptor 597 binding of SARS-CoV-2 spike proteins

ORF6 and ORF61 Expressing MVA 599 Vaccines Impair Early but Not Late Latency in Murine Gammaherpesvirus MHV-68 Infection. Front. 600 Immunol. 10. 601 43. Breitbach

Oncolytic Vaccinia Virus Disrupts Tumor-Associated Vasculature 603 in Humans

The emerging therapeutic potential of 605 the oncolytic immunotherapeutic Pexa-Vec (JX-594)

Key 607 residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and 608 neutralizing antibodies

Neutralizing Aptamers Block S/RBD-ACE2 Interactions and Prevent Host Cell Infection

Chemie -Int

Neutralizing antibodies targeting SARS-CoV-2 613 spike protein

Mutations in the SARS-CoV-2 spike RBD are 615 responsible for stronger ACE2 binding and poor anti-SARS-CoV mAbs cross-neutralization

Induce Highly Potent Neutralizing Antibodies

Conformation-Dependent Epitopes that Protein Contains Multiple Respiratory Syndrome Coronavirus Spike 618 Receptor-Binding Domain of Severe Acute

SARS-CoV-2 mRNA vaccine design enabled by 621 prototype pathogen preparedness

The Complete Genomic Sequence of the 623 Modified Vaccinia Ankara Strain: Comparison with Other Orthopoxviruses

Nanoluciferase complementation-based bioreporter reveals the 626 importance of N-linked glycosylation of SARS-CoV-2 S for viral entry

Site-specific glycan analysis 630 of the SARS-CoV-2 spike

Freeze-Drying Formulations 632 Increased the Adenovirus and Poxvirus Vaccine Storage Times and Antigen Stabilities

Stability of undiluted and diluted 634 vaccinia-virus vaccine

Long-lasting stability of vaccinia 636 virus (orthopoxvirus) in food and environmental samples

The vaccinia virus A56 protein: a multifunctional transmembrane 638 glycoprotein that anchors two secreted viral proteins

Molecular attenuation of vaccinia virus: mutant generation and animal characterization

Neurovirulence properties of recombinant vesicular stomatitis virus vectors 644 in non-human primates

Attenuation of 646 vesicular stomatitis virus infection of brain using antiviral drugs and an adeno-associated virus-interferon 647 vector

Altered CD8 + T Cell Immunodominance after Vaccinia Virus Infection and the Naive 652 Repertoire in Inbred and F 1 Mice

Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-655 SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2

Will SARS-CoV-2 variants of concern affect the promise of vaccines?

Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited 660 antibodies

Escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma 663 Network for Genomic Surveillance in South Africa*, COMMIT-KZN Team

Recombinant 665 vaccinia vector-based vaccine (Tiantan) boosting a novel HBV subunit vaccine induced more robust and 666 lasting immunity in rhesus macaques

HIV-1 668 vaccines based on replication-competent Tiantan vaccinia protected Chinese rhesus macaques from simian 669 HIV infection

Frequent and durable anti-hiv envelope viv2 igg responses induced by hiv-1 672 dna priming and hiv-mva boosting in healthy tanzanian volunteers

The Brighton Collaboration standardized template for collection 675 of key information for risk/benefit assessment of a Modified Vaccinia Ankara (MVA) vaccine platform. 676 Vaccine 39

IL-12 DNA Displays Efficient Adjuvant Effects Improving 679 Immunogenicity of Ag85A in DNA Prime

Safety and immunogenicity of a modified vaccinia virus Ankara vector vaccine 682 candidate for Middle East respiratory syndrome: an open-label, phase 1 trial

Safety and efficacy of an 685 rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine: an interim analysis of a 686 randomised controlled phase 3 trial in Russia

Phase 3 Efficacy Trial of Modified Vaccinia Ankara as a Vaccine against 689 Smallpox

Protective efficacy 691 of monovalent and trivalent recombinant MVA-based vaccines against three encephalitic alphaviruses

Development of a multi-antigenic SARS-CoV-2 vaccine candidate 695 using a synthetic poxvirus platform

A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune 699 pathology, and dysfunction in the lungs

ChAdOx1 nCoV-19 vaccine prevents 702 SARS-CoV-2 pneumonia in rhesus macaques

Development of a Safe and Effective Vaccinia 704

Virus Oncolytic Vector WR-Δ4 with a Set of Gene Deletions on Several Viral Pathways

Humoral Immunity to Primary Smallpox 707 Vaccination: Impact of Childhood versus Adult Immunization on Vaccinia Vector Vaccine Development in 708 Military Populations

Mucosal vaccination overcomes the barrier 710 to recombinant vaccinia immunization caused by preexisting poxvirus immunity

A Novel Replication-Competent Vaccinia Vector MVTT Is Superior to MVA for Inducing High 714 Levels of Neutralizing Antibody via Mucosal Vaccination

Effects of pre-existing 717 orthopoxvirus-specific immunity on the performance of Modified Vaccinia virus Ankara-based influenza 718 vaccines

Attenuated Modified Vaccinia Virus 720

Ankara Can Be Used as an Immunizing Agent under Conditions of Preexisting Immunity to the Vector

Phase 1 safety and immunogenicity testing of DNA and recombinant 724 modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles

Preexisting Vaccinia Virus Immunity Decreases

Specific Cellular Immunity but Does Not Diminish Humoral Immunity and Efficacy of a DNA/MVA Vaccine

Smallpox as a Weapon for Bioterrorism

SARS-CoV-2 genome-wide T cell epitope mapping reveals immunodominance and 733 substantial CD8+ T cell activation in COVID-19 patients

Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed 736 humans. Science (80-. )

Replicating and non-replicating viral vectors for vaccine development

The Nucleocapsid Protein of SARS-CoV-2: a Target for 740 Vaccine Development

SARS-CoV-2 binds platelet ACE2 to enhance thrombosis in COVID-19

SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: 746 implications for microclot formation in COVID-19

Genome Scale Patterns of Recombination between Coinfecting Vaccinia 750 Viruses

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Figure 1: Design and validation of CovAg vaccine. A) Schematic of CovAg (spanning residues

Dashed 763 lines reflect 10-fold increase or decrease points. C) Immunoblot of CovAg expressed from U2OS cells infected MVA 764 and TT as probed with HA antibody. D) Immunoblot of CovAg from infected U2OS cells after undergoing treatment 765 with glycosidase to illustrate RBD glycosylation (uncropped western can be found in Fig. S2). E) Immunofluorescence 766 of MVA/TT CovAg constructs with α-HA or α-RBD

Quantification of RBD expressed on the surface 768 of live cells infected with MVA-or TT-CovAg, by flow cytometry (n=3, mean ±SD). G) Temperature stability of 769 MVA and TT backbones as probed by plaque assay after storage at -80°C, 4°C or room temperature (RT) for 7 days 770 (n=3, log-transformed titer means ± SD; two-way ANOVA with Sidak's correction for multiple comparisons

Figure 2: Comparison of MVA and TT CovAg vaccines. A) Normalized ELISA absorbance vs. serum dilution factors 774 for mice vaccinated I.P. with MVA CovAg (black) or TT CovAg (pink)

B) Endpoint titer from RBD ELISAs for MVA and TT vaccines inoculated via different administration routes (n=5, 776 mean ±SEM; two-way ANOVA with Sidak's correction for multiple comparisons; alpha threshold = 0.05). N/D 777 indicates values were not detectable. C) Representative wells from IFNγ ELISPOT experiment for MVA and TT

Spot-forming units (SFU) of MVA and TT vaccines stimulated with spike S1 peptide pool. E) Spot-forming units 779 (SFU) of MVA and TT vaccines stimulated with Vaccinia E3 and F2 peptides to examine T cell responses against 780 Vaccinia backbone (n=5, mean ± SEM; two-way ANOVA with Sidak's correction for multiple comparisons

Analyses of humoral responses induced by TT CovAg vaccine in BALB/c mice. A) Endpoint titer of TT 784

CovAg vaccinated mice with and without boosts for various routes of administration. Data was acquired at day 49, 14 785 days after the boost injection date (n=5, mean ±SEM; two-way ANOVA with Sidak's correction for multiple 786 comparisons; alpha threshold = 0.05). B-C) Antibody endpoint titers over 195 days for IN (B) or I

D) VSV-S neutralization IC50 values for samples 788 described in panel A. E-F) VSV-S neutralization IC50 values over 195 days for prime only and prime/boost 789 vaccinations given either I.P. (C) or IN (D) (n=5, Log2 mean ±SEM; *p-value < 0.05 relative to D14; two-way 790 ANOVA with Dunnet's correction for multiple comparison

A) Images of injection sites for TOH-793 VAC1 after 7 and 100 days. B) Measurement of viral shedding in macaque urine and saliva. U2OS cells were mixed 794 with either macaque urine, saliva or pure TOH-VAC1 (virus control) and virus presence was assayed based on 795 firefly luciferase activity. Baseline (BL) samples were sera sample taken from macaques before vaccination

Endpoint antibody titer for RBD specific antibodies from macaques vaccinated I.M. with TOH-VAC1. Baseline 797 values were subtracted from all other timepoints (n=11, mean ±SEM

01 relative to D8; one-way ANOVA 798 with Dunnet's correction for multiple comparison, alpha threshold = 0.05). D) VSV-S neutralization assay results 799 from macaque samples post-immunization (vaccinated I.M. with TOH-VAC1), and results from sera of SARS-CoV-800 2 (Wuhan strain) positive patients. No observable neutralization was observed from sera prior to vaccination 801 (macaques n=11, mean ±SEM

PBMCs stimulated with S1 peptide pool. Each curve depicts results from a single macaque immunized with TOH-803

VAC1. F) Seroconversion assay for the RBD of SARS-CoV-2 and three of its variants tested against macaque sera 804

days-post immunization (n=11, mean shown

0001 relative to baseline (BL); two-way 805 ANOVA with Sidak's correction for multiple comparisons, alpha threshold = 0.05)