key: cord-287205-k64svq6n
authors: Pollet, Jeroen; Chen, Wen-Hsiang; Versteeg, Leroy; Keegan, Brian; Zhan, Bin; Wei, Junfei; Liu, Zhuyun; Lee, Jungsoon; Kundu, Rahki; Adhikari, Rakesh; Poveda, Cristina; Mondragon, Maria-Jose Villar; de Araujo Leao, Ana Carolina; Rivera, Joanne Altieri; Gillespie, Portia M.; Strych, Ulrich; Hotez, Peter J.; Bottazzi, Maria Elena
title: SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice
date: 2020-11-05
journal: bioRxiv
DOI: 10.1101/2020.11.04.367359
sha: 
doc_id: 287205
cord_uid: k64svq6n

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel® containing formulation and possibly in combination with other immunostimulants.

Introduction 34

The number of coronavirus disease 19 (COVID-19) cases globally is readily approaching the 50-35 million-person mark, with over 1.2 million deaths. In response to the pandemic, an international 36 enterprise to develop effective and safe vaccines is underway. There are many ways to categorize the 37 more than 100 potential COVID-19 vaccine candidates 1 , but one approach is to divide them as those 38 employing new technologies for production, but that have not yet been licensed for use, versus terms of production, scale-up, potential efficacy and safety, and delivery. 47

We have previously reported on recombinant protein-based coronavirus vaccine candidates, 48 formulated with Alhydrogel ® to prevent severe acute respiratory syndrome (SARS) 9-11 and Middle 49 East Respiratory Syndrome (MERS) 12 . In both cases, the receptor-binding domain (RBD) of the 50 SARS or MERS spike proteins was used as the target vaccine antigen. In a mouse model, the SARS-51

CoV RBD219-N1/Alhydrogel ® vaccine induced high titers of virus-neutralizing antibodies and 52 protective immunity against a mouse-adapted SARS-CoV virus challenge. It was also found to 53 minimize or prevent eosinophilic immune enhancement compared to the full spike protein 9 . 54

The RBD of SARS-CoV-2 has likewise attracted interest from several groups now entering 55 clinical trials with RBD-based vaccines 7,13-17 . Our approach was to apply the lessons learned from the 56 development of the SARS-CoV vaccine candidate and accelerate the COVID-19 vaccine induction temperature was set to 25 °C and the pH to 6.5 and, the methanol feed rate was between 1-95 15 ml/L/hr. The fermentation supernatant (FS) was filtered (0.45 m PES filter) and stored at -80 °C 96 before purification. 97

A hexahistidine-tagged SARS-CoV-2 RBD219-WT was purified from fermentation 98 supernatant (FS) by immobilized metal affinity chromatography followed by size exclusion 99 chromatography (SEC). The FS was concentrated and buffer exchanged to buffer A (20 mM Tris-100 HCl pH 7.5 and 0.5 M NaCl) using a Pellicon 2 cassette with a 10 kDa MWCO membrane 101 

To evaluate the size of RBD219-WT and RBD219-N1C1, 2 μg of these two proteins were loaded 118 onto a 4-20% tris-glycine gel under non-reduced and reduced conditions. These two proteins were 119 also treated with PNGase-F (NEB, Ipswitch, MA, USA) under the reduced condition to remove N-120 glycans and loaded on the gel to assess the impact of the glycans on the protein size. Gels were 121 stained using Coomassie Blue and analyzed using a Bio-Rad G900 densitometer with Image Alhydrogel ® formulations were centrifuged at 13,000 x g for 5 min, and the supernatant was 131 removed. The protein in the supernatant fraction and the pellet fraction were quantified using a micro 132 BCA assay (ThermoFisher, Waltham, MA, USA). 133 134

For the ACE-2 binding study, the Alhydrogel ® -RBD vaccine formulations were blocked overnight 136 with 0.1% BSA. After hACE-2-Fc (LakePharma, San Carlos, CA, USA) was added, the samples 137 were incubated for 2 hours at RT. After incubation, the Alhydrogel ® was spun down at 13,000 x g for washed once with 300 L PBST using a Biotek 405TS plate washer and diluted mouse serum 156 samples were added to the plate in duplicate, 100 L/well. As negative controls, pooled naïve mouse 157 serum (1:200 diluted) and blanks (0.1% BSA PBST) were added as well. Plates were incubated for 2 158 hours at room temperature, before being were washed four times with PBST. Subsequently, 1:6,000 159 diluted goat anti-mouse IgG HRP antibody (100 L/well) was added in 0.1% BSA in PBST. Plates 160 were incubated 1 hour at room temperature, before washing five times with PBST, followed by the 161 addition of 100 L/well TMB substrate. Plates were incubated for 15 min at room temperature while 162 protected from light. After incubation, the reaction was stopped by adding 100 L/well 1 M HCl. The absorbance at a wavelength of 450 nm was measured using a BioTek Epoch 2 spectrophotometer. 164

Duplicate values of raw data from the OD450 were averaged. The titer cutoff value was calculated 165 using the following formula: Titer cutoff = 3 x average of negative control + 3 x standard deviation 166 of the negative control. For each sample, the titer was determined as the lowest dilution of each 167 mouse sample with an average OD450 value above the titer cutoff. When a serum sample did not 168

show any signal at all and a titer could not be calculated, an arbitrary baseline titer value of 67 was 169 assigned to that sample (baseline). sample/ RLU of negative control) x 100. Serum from vaccinated mice was also characterized by the 186 IC50-value, defined as the serum dilution at which the virus infection was reduced to 50% compared with the negative control (virus + cells). When a serum sample did not neutralize 50% of the virus 188 when added at a 1:10 dilution, the IC50 titer could not be calculated and an arbitrary baseline titer 189 value of 10 was assigned to that sample (baseline). As a control, human convalescent sera for SARS- For the re-stimulation assays, splenocyte suspensions were diluted to 8x10 6 live cells/mL in a 206 2-mL deep-well dilution plate and 125 L of each sample was seeded in two 96-well tissue culture 207 treated culture plates. Splenocytes were re-stimulated with 10 g/mL RBD219-WT, 20 ng/mL PMA 208 + 1 g/mL Ionomycin or just media (unstimulated). For the flow cytometry plate, the PMA/I was not added until the next day. 125 L (2x concentration) of each stimulant was mixed with the 125 L 210 splenocytes suspension in the designated wells. After all the wells were prepared, the plates were 211 incubated at 37 °C 5% CO2. One plate was used for the cytokine release assay, while the other plate 212 was used for flow cytometry. For flow cytometry, another plate was prepared with splenocytes, 213 which would be later used as fluorescence minus onecontrols (FMOs). 214

After 48 hours in the incubator, splenocytes were briefly mixed by pipetting. Then plates were 216 centrifuged for 5 min at 400 x g at RT. Without disturbing the pellet 50 L supernatant was 217 transferred to two skirted PCR plates and frozen at -20 °C until use. 218

For the in vitro cytokine release assay, splenocytes were seeded in a 96-well culture plate at 219 1x10 6 live cells in 250 µL cRPMI. Splenocytes were then (re-)stimulated with either 10 µg/mL 220 RBD219-WT protein, 10 µg/mL RBD219-N1C1 protein, PMA/Iomycin (positive control), or nothing 221 (negative control) for 48 hours at 37 °C 5% CO2. After incubation, 96-well plates were centrifuged to 222 pellet the splenocytes down and supernatant was transferred to a new 96-well plate. The supernatant 223 was stored at -20°C until assayed. A Milliplex Mouse Th17 Luminex kit (MD MilliPore) with 224 analytes IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12(p70), IL-13, IL-17A, IL-23, IFN-γ, and TNF-α was 225 used to quantify the cytokines secreted in the supernatant by the re-stimulated splenocytes. An 226 adjusted protocol based on the manufacturers' recommendations was used with adjustments to use 227 less sample and kit materials 23 . The readout was performed using a MagPix Luminex instrument. 228

Raw data was analyzed using Bio-Plex Manager software, and further analysis was done with Excel 229 and Prism.

Surface staining and intracellular cytokine staining followed by flow cytometry was performed to 232 measure the amount of activated (CD44=) CD4+ and CD8+ T cells producing IFN-, IL-2, TNF-,  233 and IL-4 upon re-stimulation with S2RBD219 WT. 234

Five hours before the 24-hour re-stimulation incubation, Brefeldin A was added to block 235 cytokines from secretion. PMA/I was also added to designated wells as a positive control. After the 236 incubation, splenocytes were stained for the relevant markers. A viability dye and an Fc Block were 237 also used to remove dead cells in the analysis and to minimize non-specific staining, respectively. 

Results 251

Here we report on the expression of a modified, recombinant RBD of the SARS-CoV-2 spike protein 252 using the yeast (P. pastoris) expression system. The candidate antigen selection, modifications, and 253 production processes were based on eight years of process development, manufacture, and preclinical 254 prior experience with a SARS-CoV recombinant protein-based receptor-binding domain (RBD) 9-11 . 255

The RBDs of the SARS-CoV-2 and SARS-CoV share significant amino acid sequence similarity 256 (>75% identity, >80% homology) and both use the human angiotensin-converting enzyme 2 (ACE2) 257 receptor for cell entry 25,26 . Process development using the same procedures and strategies used for the 258 production, scale-up, and manufacture of the SARS-CoV recombinant protein allowed for a rapid 259 acceleration in the development of a scalable and reproducible production process for the 260 SARS-CoV-2 RBD219-N1C1 protein, suitable for its technological transfer to a manufacturer. 261

We found that the modifications used to minimize yeast-derived hyperglycosylation and 262 optimize the yield, purity, and stability of the SARS-CoV RBD219-N1 protein were also relevant to 263 the SARS-CoV-2 RBD expression and production process. The modified SARS-CoV-2 antigen, 264 RBD219-N1C1, when formulated on Alhydrogel ® , was shown to induce virus-neutralizing antibodies 265 in mice, equivalent to those levels elicited by the wild-type (RBD219-WT) recombinant protein 266 counterpart. 267

The wild-type SARS-CoV-2 RBD amino acid sequence comprises residues 331-549 of the spike (S) 269 protein (GenBank: QHD43416.1) of the Wuhan-Hu-1 isolate (GenBank: MN908947.3) (Figure 1) . 270

In the RBD-219-WT construct, the gene fragment was expressed in P. pastoris. After fermentation at 271 the 5 L scale, the hexahistidine-tagged protein was purified by immobilized metal affinity 272 chromatography, followed by size-exclusion chromatography. We observed glycosylation and 273 aggregation during these initial expression and purification studies, and therefore, similar to our previous strategy 10 , we generated a modified construct, the RBD219-N1C1, by deleting the N331 275 residue and mutating the C538 residue to alanine. The additional mutation of C538 to A538 was done 276 because we observed that in the wild-type sequence nine cysteine residues likely would form four 277 disulfide bonds. Therefore, the C538 residue was likely available for intermolecular cross-linking, 278 leading to aggregation. As a result, in the RBD219-N1C1 construct, and based on the modifications, 279

the Pichia-derived hyperglycosylation, as well as aggregation via intermolecular disulfide bridging, 280 were greatly reduced. We note that the deleted and mutated residues are structurally far from the 281 immunogenic epitopes and specifically the receptor-binding motif (RBM) of the RBD (Figure 1) 

When mixing 25 µg of either RBD219-WT or RBD219-N1C1 proteins to 500 µg of Alhydrogel ® , we 300 observed that >98% of the proteins bind to Alhydrogel ® after 15 min of incubation. Only when the 301 Alhydrogel ® was reduced to less than 100 µg (Alhydrogel ® /RBD219 ratio <4), the Alhydrogel ® 302 surface was saturated, and protein started to be detected in the supernatant (Figure 2A) . It is known 303 that unbound protein may impact the immunogenicity of the vaccine formulation, therefore we 304 proceeded to only evaluate formulations with Alhydrogel ® /RBD219 ratios higher than 4. 305 Figure 2B shows that hACE-2-Fc, a recombinant version of the human receptor used by the 306 virus to enter the host cells, can bind with the RBD proteins that are adsorbed on the surface of the 307 Alhydrogel ® . This demonstrates that bound RBD proteins are structurally and possibly functionally 308 active and that after adsorption the protein does not undergo any significant conformational changes 309 that could result in the loss of possible key epitopes around the receptor-binding motif (RBM). 310

We saw no statistical differences between the binding of hACE-2-Fc to RBD219-WT (red, 311 Figure 2B ) or RBD219-N1C1 (green, Figure 2B ) proteins, based on an unpaired t-test (P=0.670). 312

Likewise, we saw no relation between the amount of Alhydrogel ® to which the RBD was bound and 313 the interaction with hACE-2-Fc, indicating that the surface density of the RBD proteins on the 314 Alhydrogel ® plays no role in the presentation of ACE binding sites. Alhydrogel ® , produced a lower IgG response, albeit slightly higher than the negative control that had 338 been immunized with 500 g Alhydrogel ® alone (Figure 3B, Supplemental Table 1) . Importantly, 339 based on a Mann-Whitney test, we determined that there was no statistical difference between the 340 groups vaccinated with the modified and the wild-type version of the RBD protein (p=0.3497). The 341 average neutralizing antibody titers observed on day 35 (IC50 range: 5.0x10 3 to 9.4x10 3 , 342

Supplemental (Figure 3C) . 345

On day 43, 22 days after receiving the boost vaccination, half of the mice in each group 346 (N=4), those with the highest IgG titers, were sacrificed to determine the total IgG, the IgG subtypes, 347 and the neutralizing antibody titers. As we observed on day 35, all animals that had received the 348 vaccine produced strong antibody titers, with the groups receiving >200 g Alhydrogel ® eliciting a 349 higher titer than those that received only 100 g of Alhydrogel ® , albeit no statistical significance was 350 detected (Figures 3B) . For all animals, as typical for vaccine formulations containing aluminum, the 351 IgG2a:IgG1 titer ratio was <0.1 (Supplemental Figure 3) . In the pseudovirus neutralization assay 352 for the day 43 samples (Figure 3C) , all vaccines containing >200 g Alhydrogel ® elicited IC50 titers 353 that, on average, were several-fold higher than on day 35 (IC50 range: 1.1x10 4 to 1.2x10 5 , 354

Supplemental Table 2 ). There again was no difference between the RBD219-WT and RBD-N1C1 355 vaccines.

On day 57, all remaining animals were sacrificed. In contrast to the animals studied on days 357 35 and 43, these animals had received a second boost vaccination. A robust immune response in all 358 vaccinated mice, including those immunized with the protein adsorbed to 100 g Alhydrogel ® 359 achieved high average IgG titers. The total IgG titers in the mice sacrificed on day 57, had increased 360 after the third vaccination, compared to the titers seen on day 35. Likewise, we observed a 361 corresponding increase in the average IC50 values (IC50 range: 3.8x10 2 to 1.1x10 4 , Supplemental 362 Table 2 ) for all animals, including those immunized with the protein adsorbed to 100 g 363 Alhydrogel ® . Interestingly, for this time point, the cohort receiving 25 g RBD219-N1C1 with 500 364

g Alhydrogel ® appeared to show higher neutralizing antibody titers than the corresponding 365 For all samples, we employed Flow Cytometry to quantify intracellular cytokines in CD4+ and CD8 + 379 cells after restimulation ( Figure 4A) . On day 43, high percentages of CD4 + -IL-4 and, to a slightly 380 lesser extent CD4 + -TNF producing cells were detected. Conversely, as expected for an 381 Alhydrogel ® -adjuvanted vaccine, low levels of IL-2 producing CD4 + cells were seen. In a cytokine 382 release assay, strong IFN-, IL-6, and IL-10 secretion was observed independent of whether the 383 animals had received two or three immunizations, whereas low amounts of secreted Th1-typical 384 cytokines such as IL-2 or IL-12 were seen ( Figure 4B ). 

Cytokine concentrations of non-stimulated controls were subtracted from re-stimulated samples.

Discussion 396

Here we report on a yeast-expressed SARS-CoV-2 RBD219-N1C1 protein and its potential as a 397 vaccine candidate antigen for preventing COVID-19. Building on extensive prior experience 398 developing vaccines against SARS-CoV and MERS-CoV 10-12 , we initially selected and compared the 399 SARS-CoV-2 RBD219-WT and the SARS-CoV-2 RBD219-N1C1 proteins for their potential to 400 induce high titers of virus-neutralizing antibodies, T-cell responses, and protective immunity. 401

Previously we observed that the SARS-CoV RBD219-N1 antigen, formulation with 402 Alhydrogel ® elicited high levels of neutralizing antibodies without evidence of eosinophilic immune 403 enhancement. That RBD-based vaccine was even superior to the full-length spike protein in inducing 404 specific antibodies and fully protected mice from SARS-CoV infection while preventing eosinophilic 405 pulmonary infiltrates in the lungs upon challenge 9 . 406

In this work, using the SARS-CoV-2 RBD219 protein analog, we observed that, just like in 407 the case of the SARS-CoV RBD antigen, the deletion of the N-terminal asparagine residue reduced 408 hyperglycosylation, thus allowing for easier purification of the antigen obtained from the yeast 409 expression system. Moreover, mutagenesis of a free cysteine residue further improved protein 410 production through the reduction of aggregation. Based on the predicted structure of the RBD, no 411 impact on the functionality of the RBD219-N1C1 antigen was expected, and using an ACE-2 in vitro 412 binding assay we indeed showed similarity to the RBD219-WT antigen. In addition, we showed that, 413 in mice, the modified RBD219-N1C1 antigen triggered an equivalent immune response to the 414 RBD219-WT protein when both proteins were adjuvanted with Alhydrogel ® . 415 Similar to our previous findings with the SARS-CoV RBD antigen 9 , we show the RBD219-416 N1C1 protein when formulated with Alhydrogel ® elicits a robust neutralizing antibody response with 417 IC50 values up to 4.3x10 5 in mice, as well as an expected T-cell immunological profile. Some of the titers of virus-neutralizing antibodies exceed the titer, 2.4x10 4 , measured in-house with human 419 convalescent serum research reagent for SARS-CoV-2 (NIBSC 20/130, National Institute for Biological 420 Standards and Control, UK). 421

In a mouse virus challenge model for the SARS CoV RBD recombinant protein vaccine, we 422 found that Alhydrogel ® formulations induced high levels of protective immunity but did not 423 stimulate eosinophilic immune enhancement, suggesting that Alhydrogel ® may even reduce immune The selection of the P. pastoris expression platform for the production of the RBD antigen 442 was motivated by the intent to develop a low-cost production process that could easily be transferred 443 to manufacturers in LMICs. Currently, there are several types of COVID-19 vaccine candidates in 444 advanced clinical trials 6,40-45 . The focus of some of the initiatives behind these vaccines is to provide 445 vaccines for the developed world that might struggle to be successful without advanced 446 infrastructure. Being able to match the existing experience in LMICs with the production of other 447 biologics in yeast increases the probability of successful technology transfer 20 . For example, 448 currently, the recombinant hepatitis B vaccine is produced in yeast by several members of the 449 Development Country Vaccine Manufacturers Network (DCVMN), and we foresee that, given the 450 existing infrastructure and expertise, those facilities could be repurposed to produce a yeast-produced 451 COVID-19 vaccine 46 . Recently, the research cell bank and production process for the RBD219-N1C1 452 antigen was technologically transferred to a vaccine manufacturer in India and produced under cGMP 453 conditions with the intent to enter into clinical development. In addition, preclinical studies using the 454 RBD219-WT and RBD219-N1C1 antigens are ongoing to further optimize and evaluate other novel 455 formulations, including a challenge study in a non-human primate model. 456

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The authors declare that Baylor College of Medicine recently licensed the RBD219-N1C1 482 technology to an Indian manufacturer for further development. The research conducted in this paper 483 was performed in the absence of any commercial or financial relationships that could be construed as 484 a potential conflict of interest. 485