key: cord-0993611-1zlvuari
authors: Liu, Jun; Budylowski, Patrick; Samson, Reuben; Griffin, Bryan D.; Babuadze, Giorgi; Rathod, Bhavisha; Colwill, Karen; Abioye, Jumai A.; Schwartz, Jordan A; Law, Ryan; Yip, Lily; Ahn, Sang Kyun; Chau, Serena; Naghibosadat, Maedeh; Arita, Yuko; Hu, Queenie; Yue, Feng Yun; Banerjee, Arinjay; Mossman, Karen; Mubareka, Samira; Kozak, Robert A.; Pollanen, Michael S.; Orozco, Natalia Martin; Gingras, Anne-Claude; Marcusson, Eric G.; Ostrowski, Mario A.
title: Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B
date: 2021-05-12
journal: bioRxiv
DOI: 10.1101/2021.05.11.443286
sha: cf7ee9499619beadf2177b7d91ffe52b45a93494
doc_id: 993611
cord_uid: 1zlvuari

Safe and effective vaccines are needed to end the COVID-19 pandemic caused by SARS-CoV-2. Here we report the preclinical development of a lipid nanoparticle (LNP) formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern (VOCs), including the B.1.1.7, B.1.351 and P.1 lineages. No adverse effects were induced by PTX-COVID19-B in both mice and hamsters. These preclinical results indicate that PTX-COVID19-B is safe and effective. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 1 clinical trial ongoing (ClinicalTrials.gov number: NCT04765436). One Sentence Summary PTX-COVID19-B is a SARS-CoV-2 mRNA vaccine that is highly immunogenic, safe, and effective in preventing SARS-CoV-2 infection in mice and hamsters and is currently being evaluated in human clinical trials.

of COVID-19 variants of concern (VOCs) that may escape vaccine-induced immune 77 responses (9-12), continued and concerted global efforts in SARS-CoV-2 vaccine 78 research, development, manufacturing and deployment are required to end the COVID-19 79 pandemic (13). 80 SARS-CoV-2 is an enveloped positive-sense RNA virus that uses the spike 81 protein (S) on its surface to bind the angiotensin-converting enzyme 2 (ACE2) on host 82 cells for entry to initiate replication (14-19). The S protein has two subunits: S1 and S2. 83 S1 is responsible for binding to ACE2 through its receptor-binding domain (RBD). Once 84 bound, S1 is shed from the envelope, exposing S2, which is then inserted into the host 85 cell membrane to mediate fusion of virus envelope and cell membrane to release the viral 86 genetic material into the host cells for replication. In contrast to SARS-CoV and other 87 group 2B coronaviruses, SARS-CoV-2 has a furin cleavage site between the S1 and S2 88 subunit, which promotes infection of cells expressing the transmembrane serine protease 89 2 (TMPRSS2) on their surface, e.g. human respiratory tract epithelial cells (14, (20) (21) (22) . 90

The S protein is also the main target of host generated neutralizing antibodies (nAb) that 91 can inhibit SARS-CoV-2 infection, e.g. by blocking its binding to ACE2 (23-30). Thus, 92 most of the current SARS-CoV-2 vaccines use S protein as the immunogen. 93 mRNA-based vaccines are attractive platforms for prophylactic SARS-CoV-2 94 vaccine candidates due to their unique advantages, including rapid large-scale production, 95 strong immunogenicity in both humoral and cellular immunity, and ease of adaptation to 96 tackle the emerging VOCs (31, 32). Two SARS-CoV-2 mRNA vaccines were the earliest 97 to enter phase 3 clinical trials, showing both high efficacy and safety, and were the first to 98 be approved for emergency use in humans (33, 34). Here, we report the preclinical results 99 of another SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. We found that PTX-100 COVID19-B elicited potent humoral and cellular immune responses in mice, and 101 protected both mice and hamsters from SARS-CoV-2 challenges. Based on these results, 102 PTX-COVID19-B was authorized by Health Canada to enter clinical trials with a phase 1 103 clinical trial underway (ClinicalTrials.gov number: NCT04765436). 104 length S mRNA construct, hereafter named PTX-COVID19-B, was chosen for further 149 testing and moved into the next stages of development. 150

To further evaluate the immunogenicity of PTX-COVID19-B, female C57BL/6 mice 152 were vaccinated twice, 3 weeks apart, with 1 or 10 µg doses of PTX-COVID19-B or, as 153 control, 10 µg of LNP formulated tdTomato mRNA. Three weeks after the boost 154 vaccination, blood and spleens were collected from the mice to measure humoral and 155 cellular immune responses. We first used an ELISA assay to measure S-specific binding 156 antibodies in the sera of the mice. As shown in Fig. 2A , both 1 and 10 µg doses of PTX-157 COVID19-B elicited very strong S-specific total IgG, IgG1, IgG2b and IgG2c responses 158 (median EC 50 titers for 1 and 10 µg PTX-COVID19-B are, respectively: 1.5×10 4 (IQR 159 8.1×10 3 -2.2×10 4 ), 1.1×10 5 (IQR 7.3×10 4 -1.5×10 5 ) for total IgG; 8.3×10 3 (IQR 3.9×10 3 160 -1.5×10 4 ), 1.7×10 4 (IQR 1.1×10 4 -2.9×10 4 ) for IgG1; 5.2×10 3 (IQR 3.0×10 3 -6.7×10 3 ), 161 5.9×10 4 (IQR 4.4×10 4 -6.3×10 4 ) for IgG2b; 2.2×10 4 (IQR 1.3×10 4 -7.6×10 4 ), 1.6×10 6 162 (IQR 1.1×10 6 -3.6×10 6 ) for IgG2c). The 10 µg dose of PTX-COVID19-B usually 163 induced higher S-specific binding antibodies than the 1 µg dose. The preponderance of 164 the Th1 antibody (IgG2b and IgG2c) over the Th2 antibody (IgG1) also indicated that 165 PTX-COVID19-B induced a Th1-biased antibody response. Very low levels of anti-S 166 antibodies were detected in the sera of control mice receiving the tdTomato mRNA. 167

We then measured nAb against SARS-CoV-2 in these C57BL/6 mouse sera. 168

Results of SARS-CoV-2 authentic virus micro-neutralization assay showed that the 10 µg 169 dose of PTX-COVID19-B elicited high nAb levels (median nAb ID 50 titer was 1259, IQR 170 652.7-1770), which was 21.0-fold higher than that of the 8 COVID-19 convalescent 171 patients ( Fig. 2B and 1C ). Low levels of nAb were induced by the 1 µg dose of PTX-172 COVID19-B, which, for the majority of mice, could only be detected by the pseudovirus 173 assay, which is more sensitive (Fig. 2B) . No detectable nAb was present in the sera of the 174 mice receiving tdTomato mRNA by either assay. 175

To further verify the ability of PTX-COVID19-B in inducing a nAb response 176 against SARS-CoV-2 virus, we vaccinated a different strain of mice, BALB/c, and 177 included both sexes in the vaccination, using the same vaccination regimen as described 178

above. Three weeks after the boost vaccination, sera were collected and nAb levels were Brazil (43) (Fig. 3A) . As shown in Fig. 3B Additionally, cytokine producing CD4 + and CD8 + T cells in splenocytes of 215 C57BL/6 mice immunized with 1 and 10 µg of vaccine were analyzed by flow cytometry 216 following overnight S peptide pool stimulation and intracellular cytokine staining (Fig.  217 4C). CD4 + T cells had increased percentages of IFN-γ, TNF-α and IL-2 producing cells, 218 and very low percentages of IL-4 and IL-5 producing cells, indicating a strong induction 219 of a Th1 response. Interestingly, CD8 + T cells showed a high number of IFN-γ producing 220 cells, which was higher in percentage than that of CD4 + T cells ( Fig. 4C and Fig. S2) . CoV-2 in the lungs of AAV6-hACE2 mice but not in control mice transduced with 239 AAV6-luciferase (Fig. S3A ). Using a real-time RT-PCR assay targeting the SARS-CoV-240 2 envelope (E) gene, we also detected a high amount of SARS-CoV-2 genomic RNA in 241 the lungs from both AAV6-hACE2 and AAV6-luciferase transduced mice, although the 242 genomic RNA copy numbers were much lower in the lungs of the AAV6-luciferase 243 transduced mice than the AAV6-hACE2 mice (Fig. S3B) . 244

Having confirmed that the AAV6-hACE2 mouse model was susceptible to SARS- intranasally with SARS-CoV-2. Body weight of the hamsters was measured 1 day before 279 the SARS-CoV-2 challenge and then on 1, 3, 5, 7 and 8 dpi. Oral swabs were taken from 280 the hamsters on 1, 3, 5 and 7 dpi. On 4 and 8 dpi, half (n=4) of the hamsters from each 281 group were humanly euthanized, and nasal turbinates and lungs were collected. 282

When compared to pre-challenge, the body weight of the control hamsters 283 decreased from 3 dpi to 8 dpi, while that of the PTX-COVID19-B vaccinated hamsters 284 decreased slightly on 3 dpi and then increased from 4 dpi to 8 dpi ( Lung pathology was also examined in all hamsters, using a semiquantitative 300 grading system to score the severity of the lung pathology (Fig. 6D , 6E, and Table S1 ). 301

There was a significant difference in the lung pathology of control animals (n=4) and The 2P mutation (K986P and V987P) was reported to stabilize the ectodomain of 358 the S protein in the prefusion conformation (17), which was regarded as crucial in 359 inducing nAb, and thus was adopted in some SARS-CoV-2 vaccines (32, 53, 59, 60) . vaccines using the 2P mutations in their S immunogens, suggest that the 2P mutation 368 might not be essential for induction of protective immunity (32, 53). We also designed 369 and tested another S construct, S furinmut , in which the furin cleavage site between S1 and 370 S2 subunits was removed. Removing this site was presumed to stabilize the ectodomain 371 of the S protein by keeping the S1 subunit from shedding from S, and was utilized in 372 some SARS-CoV-2 vaccines (17, 59, 60) . However, we did not find that the S furinmut 373 mRNA performed better in eliciting nAb responses in mice compared to the S mRNA. 

The objective of this study was to evaluate the immunogenicity, safety and efficacy of a 403 SARS-CoV-2 mRNA vaccine in mice and hamsters. The sample size of mice was 404 determined by power analysis assuming 60% protection efficacy. Due to the capacity 405 limit in our animal facility, a total of 16 hamsters in two groups were used in this study. 

Female C57BL/6 mice of 6-to 8-week old were vaccinated intramuscularly twice with a 470 3-week interval. In some experiments, both male and female BALB/c mice of 6-to 8-471

week old were used. Various doses of mRNA vaccines or control tdTomato mRNA in 472 50µl total volume were injected into the hind leg muscle for each immunization. Naïve 473 mice received the same volume of either DPBS or the vaccine formulation buffer. Each 474 day before vaccination, blood was collected from the mice through the saphenous vein. 475

Three weeks after boost vaccination, mice were humanly euthanized and spleen and 476 blood samples were collected. Serum was isolated from the blood by centrifugation at 477 10,000 g for 30 seconds at 4°C. 

A micro-neutralization assay was used to measure the neutralizing titers of the sera(45). 500

Briefly, VeroE6 cells cultured in DMEM-10 were seeded into 96-well plates and cultured 501 overnight. Sera were heat-inactivated at 56°C for 30 minutes. Serial dilutions of the sera 502 were mixed with 100 TCID 50 SARS-CoV-2 virus (isolate SARS-CoV-2-SB2-P3 PB 503 Clone 1, passage 3(40)) in serum free DMEM, incubated at 37°C for 1 hour, and then 504 added onto the VeroE6 cells. The cell plates were then incubated at 37°C for 1 hour, 505

shaking every 15 minutes. Inoculums were then removed and DMEM-2 (DMEM 506 supplemented with 2% FBS, 100U penicillin, 100µg streptomycin, and 2mM L-507 glutamine) was added to the cells. Cell plates were incubated at 37°C for 5 days and 508 cytopathic effect (CPE) was checked every day. 50% neutralization titer (ID 50 ) was 509 defined as the highest dilution factor of the serum that protected 50% of the cells from 510 CPE and calculated by using the 4-parameter logistic regression analysis in GraphPad 511 Prism 8. The performer of the assay was blinded to the grouping of the mice. 512

Spike-pseudotyped lentiviral assays were performed as previously described with 514 reagents kindly provided by Dr. J. D. Bloom (Department of Genome Sciences, 515

University of Washington, Seattle, WA) and with minor modifications for mouse 516 samples(74). Briefly, Spike-pseudotyped lentivirus particles (both wild-type Wuhan-Hu-517 1 and tested VOCs) were generated and used at ~1:25 virus stock dilution (a virus 518 dilution resulting in >1000 relative luciferase units (RLU) over control). For the 519 neutralization assay, diluted mouse sera (1:40 from stock sera) were serially diluted (from 520 2.5 to 4-fold dilutions over 7 dilutions to encompass a complete neutralization curve per 521 sample) and incubated with diluted pseudovirus at a 1:1 ratio for 1 hour at 37°C before 522 being transferred to plated HEK293T-ACE2/TMPRSS2 cells and incubated for an 523 additional 48 hours at 37°C and 5% CO 2 . After 48 hours, cells were lysed, and Bright-524

Glo luciferase reagent (Promega, Madison, WI) was added for 2 minutes before reading 525 with a PerkinElmer Envision instrument (PerkinElmer, Waltham, MA). 50% 526 neutralization titer (ID 50 ) were calculated with nonlinear regression (log[inhibitor] versus 527 normalized response -variable slope) using GraphPad Prism 8. The assay was performed 528 in the same manner for all VOCs tested. The performer of the assay was blinded to the 529 grouping of the mice. 530 ELISPOT assay: To perform IFN-γ ELISPOT for C57BL/6 mice, ELISPOT plates 531 (Sigma-Aldrich) were coated with rat anti-mouse IFN-γ antibody (BD Bioscience) 532 overnight. Plates were washed and blocked with RPMI-10 medium (RPMI-1640 533 supplemented with 10% FBS, 100U penicillin, 100µg streptomycin, and 2mM L-534 glutamine. All were purchased from Wisent Bioproducts) for 2 hours. Splenocytes were 535 added into the plates, and stimulated with a SARS-CoV-2 S peptide pool (15-mer 536 peptides with 11 amino acids overlap covering the full-length S, total 315 peptides, JPT 537

Peptide Technologies GmbH, Berlin, Germany) at 1 µg/ml/peptide. The same volume of 538 40% DMSO (Sigma-Aldrich), the solution to dissolve the peptide pool, was used as the 539 negative control. PMA/Ionomycin (Sigma-Aldrich) was used as the positive control. 540

After overnight incubation, the cells were washed away, and biotinylated anti-mouse 541 IFN-γ (BD) was added, and the plates were incubated for 2 hours. After washing with 542 PBS/0.01% Tween 20, Streptavidin-HRP enzyme conjugate (Thermo Fisher Scientific) 543 was added into the plates, which was incubated for 1 hour. After washing with 544 PBS/0.01% Tween 20, TMB ELISPOT substrate (Mabtech, Cincinnati, OH) was added 545 into the plates, and the spots were developed and read with an ImmunoSpot® Analyzer 546 were used for RNA extraction while 500 µl of homogenates were used for quantification 607 of SARS-CoV-2. For oral swabs, anesthetized animals were swabbed (9-11 seconds 608 swabbing) and swabs were then introduced into 1ml DMEM-2. All oral swab samples 609 were frozen until further processing. 500 µl of each oral swab sample was used for 610 quantification of SARS-CoV-2. 611

VeroE6 cells cultured in DMEM-10 were seeded into 96-well plates and incubated 613 overnight at 37°C. On the following day culture medium was removed and tissue 614 samples 10-fold serially diluted in DMEM supplemented with 1% FBS were added onto 615 the cells. The plates were then incubated at 37°C for 1hour. After incubation lung 616 homogenates were replaced with 100µl/well DMEM-2, and the cells were incubated at 617 37°C for 5 days. Cytopathic effect (CPE) was checked on day 3 and day 5. TCID 50 was 618 defined as the highest dilution factor of the inoculum that yielded 50% of the cells with 619 CPE and determined by using the Spearman-Karber TCID 50 method. 620

Real-time RT-PCR to quantify the genomic copies of SARS-CoV-2 in tissue 622 homogenates was done according to the published protocol(40 GenScript, Piscataway, NJ) was also run at the same time for conversion of Ct value to 632 genomic copies, by using the Rotor-Gene Q software (QIAGEN). 633

The formalin-fixed lung tissue was processed for paraffin embedding, microtomy and 635 then stained with hematoxylin and eosin. The blocks were examined at 3 separate levels 636

(3 separate slides). Histological sections were examined blind to vaccination status. 637

Semiquantitative grading of lung was conducted according to Table S1 . 638

One-way ANOVA (Kruskal-Wallis test) followed by Dunn's multiple comparison, two-640 way ANOVA followed by Sidak's multiple comparison, two-tailed paired t test, or two-641 tailed unpaired t test (Mann-Whitney) were used for comparison between groups, as 642 indicated in the figure legends. Spearman correlation test was used for correlation 643 analysis. Logistic regression was used for determining nAb ID 50 threshold titer that 644 would confer 95% predicted probability of protection from productive SARS-CoV-2 645 infection in mice. All statistical analysis was performed by using GraphPad Prism 8. 646

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) 865 Author contributions: 866 Conceptualization

RBD: SARS-CoV-2 RBD (receptor binding domain) mRNA (amino acids 319-541). tdTomato: control mRNA encoding tdTomato. S1: S1 subunit of SARS-CoV-2 Spike (amino acids 1-685). S2: S2 subunit of SARS-CoV-2 Spike (amino acids 686-1273). (B) Mice vaccination regimen. Six-to 8-week old mice were vaccinated twice with a 3-week interval. One day before each vaccination, peripheral blood was collected from the mice. Three weeks after the second vaccination

Three weeks after the second vaccination, blood was collected to test neutralization of SARS-CoV-2 authentic virus or pseudovirus by the sera. For comparison, convalescent sera from 8 SARS-CoV-2 infected human subjects (HCS in the graph) were also tested for neutralization of SARS-CoV-2 authentic virus. Each symbol represents one mouse or person. Samples that did not neutralize viruses at the lowest dilution (1:20 for real virus, 1:40 for pseudovirus) are designated a 50% neutralization titer of 1. For each group, the long horizontal line indicates the median and the short lines below and above the median indicate the 25% and 75% percentile

Three weeks after the second vaccination, blood was collected to detect serum neutralization of SARS-CoV-2 authentic virus or pseudovirus by the mouse sera. Shown are 50% neutralization titers (ID 50 ). N=10 per group except n=9 for the female 20 µg dosed PTX-COVID19-B group in the pseudovirus assay. In (B) and (C) samples that did not neutralize viruses at the lowest dilution (1:20 for real virus, 1:40 for pseudovirus) are designated a 50% neutralization titer of 1. Each symbol represents one mouse. For each group, the long horizontal line indicates the median and the short lines below and above the median indicate the 25% and 75% percentile. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 as determined by one way ANOVA (Kruskal-Wallis test) followed by Dunn's multiple comparison test

C57BL/6 mice immune sera shown in Fig. 2 A and 2B were used in the neutralization. Shown in (B) are 50% neutralization titers across all pseudoviruses

The numbers above the brackets in (C) are the ratios of the median 50% neutralization titers against the VOCs to the titers against Wuhan-Hu-1 isolate (blue: 10 µg PTX-COVID19-B group, red: 1 µg PTX-COVID19-B group). Each symbol represents one mouse

One week after the second vaccination, mice were intranasally transduced with AAV6-hACE2. Nine days after the transduction, mice were intranasally challenged with SARS-CoV-2. One day before each vaccination, blood was collected from the mice. Four days after SARS-CoV2 challenge, mice were humanly euthanized and blood and lungs were collected from the mice. (B) Amount of infectious SARS-CoV-2 virus and (C) SARS-CoV-2 RNA in the lungs of the mice. Shown in (B) are TCID 50 /100mg lung tissue (n=10 per group) and (C) RNA copies/mg lung tissue (n=10 per group). (D) Neutralization of SARS-CoV-2 authentic virus by the mouse sera collected 4 days after SARS-CoV-2 challenge. Shown are 50% neutralization titers (ID 50 , n=10 per group). Samples that did not neutralize viruses at the lowest dilution (1:20) are designated a 50% neutralization titer of 1. Each symbol represents one mouse

Six-to 10-week old male Syrian hamsters (n=8) were vaccinated with a 20 µg dose of PTX-COVID19-B or formulation buffer twice with a 3-week interval