key: cord-332832-kjppd6uz authors: Ward, B. J.; Gobeil, P.; Seguin, A.; Atkins, J.; Boulay, I.; Charbonneau, P.-Y.; Couture, M.; D'Aoust, M.-A.; Dhaliwall, J.; Finkle, C.; Hager, K.; Mahmood, A.; Makarkov, A.; Cheng, M.; Pillet, S.; Schimke, P.; St-Martin, S.; Trepanier, S.; Landry, N. title: Phase 1 trial of a Candidate Recombinant Virus-Like Particle Vaccine for Covid-19 Disease Produced in Plants date: 2020-11-06 journal: nan DOI: 10.1101/2020.11.04.20226282 sha: doc_id: 332832 cord_uid: kjppd6uz Background: The stabilized prefusion form of the SARS-CoV-2 spike protein is produced by transient expression in Nicotiana benthamiana. The trimeric spike glycoproteins are displayed at the surface of self-assembling Virus-Like-Particles that mimic the shape and the size of the virus. The candidate vaccine (CoVLP) administered alone or with AS03 or CpG1018 adjuvants was evaluated in a Phase 1 trial in healthy adults. (ClinicalTrials.gov number NCT04450004) Methods: The study was a randomized, partially-blinded, prime-boost 21 days apart, dose-escalation Phase 1 study intended to assess the safety, tolerability, and immunogenicity of CoVLP at three dose levels (3.75 microgram, 7.5 microgram, and 15 microgram) unadjuvanted or adjuvanted with either CpG 1018 or AS03 in 180 SARS-CoV-2 seronegative healthy adults 18 to 55 years of age. Enrollment was staggered for dose-escalation. At each dose level, the vaccine was initially administered to a small number of subjects. Vaccination of the remaining subjects at the same dose level and the next higher vaccine dose level was administered with approval of an Independent Data Monitoring Committee (IDMC). The same procedure was followed for the second vaccine administration. Monitoring of safety signals was performed throughout the study with pre-determined pausing/stopping rules if there was clear evidence of harmful effects such as severe adverse events (AEs) related to the treatment. The primary endpoints were the safety and tolerability of the vaccine after each dose and the immunogenicity as assessed by neutralizing antibody responses assessed using a vesicular stomatitis virus (VSV) pseudovirion assay and interferon-gamma and interleukin-4 (IL-4) ELISpot assays at Days 0, 21 and 42. Secondary endpoints were anti-spike antibody responses by ELISA and neutralizing antibodies measured by live virus plaque reduction neutralization test (PRNT) assay at Days 0, 21 and 42 and immunogenicity with additional safety and immunogenicity endpoints planned for 6-months following the last vaccination. The anti-spike and neutralizing antibody responses were compared with 23 convalescent serum samples from symptomatic Covid-19 patients. We performed a primary analysis at day 42. Results: A total of 180 subjects (102 females: 78 males: average 34.3 years) were recruited to the study and interim safety and immunogenicity data up to day 42 after the first dose are reported here. There was no obvious CoVLP dose effect in safety outcomes for any of the formulations tested and all formulations were generally well-tolerated. Most solicited local and systemic AEs were mild-moderate and transient. Reactogenicity was increased in all adjuvanted formulations and was generally highest in the CoVLP+AS03 groups. Local and systemic adverse events were reported with similar frequency after the first and second doses in subjects who received either CoVLP alone or CoVLP+CpG1018 but increased in both frequency and severity after the second dose in the CoVLP+AS03 groups. CoVLP alone only elicited a weak total anti-spike IgG response at the highest dose level and little-to-no neutralization antibody response, even after the second dose. Cellular responses in the CoVLP alone groups (IFN-gamma and IL-4) were detectable after the second dose but were still only marginally above background levels. The addition of either adjuvant substantially increased both antibody and cellular responses at most CoVLP dose levels and changes were most pronounced after the second dose. However, a substantial neutralizing antibody response after the first dose was only seen in all CoVLP+AS03 groups. After the second dose, both total anti-spike IgG and neutralizing antibody titers in the CoVLP+AS03 groups were higher than those in the CoVLP+CpG1018 groups. The antibody titers achieved were either similar to (CoVLP+CpG1018) or at least 10-times higher (CoVLP+AS03) than those seen in convalescent plasma. Administration of CoVLP with either adjuvant also significantly increased the cellular responses. After 2 doses, both IFN-gamma and IL-4 responses were significantly increased in the CoVLP+CpG1018 groups. In the CoVLP+AS03 groups, significant increases in the cellular responses were observed after the first dose while IFN-gamma and IL-4 increased further in both magnitude and number of subjects responding after the second dose. Again, the cellular responses in the CoVLP+AS03 groups were higher than those seen in the CoVLP+CpG1018 groups. Conclusion: These data demonstrate that CoVLP administered with either CpG1018 or AS03 has a safety profile similar to other candidate vaccines for SARS-CoV-2. When administered with either AS03 or CpG1018, several of the CoVLP dose levels elicited strong humoral and T cell responses after the second dose. When administered with AS03, even the 3.75 microgram CoVLP dose elicited neutralizing antibody titers that were ~10-times higher than those observed in individuals recovering from Covid-19 as well as consistent and balanced IFN-gamma and IL-4 responses. Although many CoVLP formulations were immunogenic, in the absence of established correlates of protection and given the advantages of dose-sparing in the context of the on-going pandemic, these findings suggest that CoVLP (3.75 microgram)+AS03 has a good benefit/risk ratio and support the transition of this formulation to studies in expanded populations and to efficacy evaluations A novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) 174 jumped across a species barrier in China in late 2019 1, 2 and spread rapidly around the globe 175 leading to the World Health Organization's declaration of a pandemic on March 11, 2020 3 . As 176 of October 11th, 2020, more than 37 million cases of Covid-19 have been reported with >1 177 million deaths 4 . Although many therapeutic strategies has been tried 5,6 7 , the current options 178 remain limited in both number and efficacy. Simultaneously, there has been a massive global 179 effort to develop vaccines. This effort was primed to some extent by prior experience with other 180 highly pathogenic human coronaviruses, SARS and MERS 8 Adding to the complexity of this situation is the facts that no correlate of immunity has been 190 defined for any highly pathogenic coronavirus 13, 14 and that such correlates might differ 191 between vaccines 15 . Nonetheless, a protective role for both humoral and cell-mediated 192 immunity against coronaviruses has been suggested 16, 17 . Antibody responses against the spike 193 (S) protein have the potential to protect from infection [18] [19] [20] and convalescent plasma with high 194 titers of anti-S antibody have therapeutic benefit in selected patients 7,21 . However, a substantial 195 proportion of people who develop Covid-19 fail to generate antibodies, and data from the 196 SARS-CoV-1 outbreak of 2002-2003 22 and the current pandemic suggest that antibody 197 responses can be short-lived, disappearing within months of infection in some patients 23 . In 198 contrast, T cell immunity may be critical for recovery from 25 and was shown to 199 persist for up to 11 years after SARS-CoV-1 infection 26 . T cells can provide substantial 200 protection in animal models of highly pathogenic coronavirus infection. 201 We report here the results of a Phase 1 study initiated in July 2020 evaluating the safety, 203 tolerability and immunogenicity of two doses, 21-days apart of 3.75, 7.5 or 15 µg of a virus-204 like-particle vaccine candidate for Covid-19 produced in plants (hereafter called CoVLP). This 205 allocation. On Day 0 (D0: pre-first dose), D21 (pre-second dose) and D42 (post second dose), 240 serum and peripheral blood mononuclear cells (PBMC) were processed for immune outcomes 241 as described previously 28 . 242 243 Safety 244 For details of safety monitoring, see the Protocol in Suppl. Materials. Briefly, enrollment was 245 staggered for dose-escalation with sentinel subjects at each dose level (n=6) and independent 246 data monitoring committee (IDMC) review of D3 safety data at 10% and 30% recruitment 247 before each dose acceleration. The same process was followed for the second vaccine 248 administration. Monitoring of safety signals was performed throughout the study (Suppl 249 Material: pp 2). Solicited adverse events (AEs) were assessed by the subjects as Grade 1 to 4 250 (mild, moderate, severe, or potentially life-threatening). Unsolicited AEs, and AEs leading to 251 subject withdrawal were collected up to D21 after each vaccination. All serious AEs (SAE), 252 Adverse Events of Special Interest (AESIs), and pregnancies will be collected for 6 months. 253 Potential cases of vaccine enhanced disease (VED), hypersensitivity and potential immune-254 mediated diseases (pIMDs) are being monitored throughout the study (see Suppl. Material: pp 255 7, Tables S2, S3). 256 257 The primary immunological outcomes were neutralizing antibody (NAb) responses measured 259 using a VSV pseudovirion assay (Nexelis Inc, Laval, QC) and IFN and IL-4 cellular responses 260 measured by ELISpot at D0, 21 and 42. Secondary immunological outcomes were total anti-261 spike IgG responses by ELISA and NAb responses by plaque reduction neutralization test 262 (PRNT: Vismedri S.r.l., Siena, Italy) Details of these assays are provided in the Suppl. severity of disease ranged from mild-moderate (n=23) to severe/critical (n=11) (Suppl. 269 Material: Table S1 for patient characteristics: pp 6). 270 271 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi. org/10.1101 org/10. /2020 Overall, 180 healthy SATRS-COV-2 seronegative male and female subjects 18 to 55 years of 274 age were randomized in a 1:1:1:1:1:1:1:1:1 ratio into nine treatment groups. The sample size 275 made it possible to perform the initial evaluation of the vaccine immunogenicity and detect 276 major differences in rates of AEs between groups. The sample size was not large enough to 277 detect all types, including less frequent or rare, AEs. Table 1 and subject disposition up to D42 is 286 presented in Table 2 and in Suppl. Materials: Fig. S1 : pp. 19. More women (56.7%) than men 287 (43.3%) were enrolled but the female:male ratio was the same for each CoVLP dose level and 288 in each of the overall groups (unadjuvanted CoVLP, CoVLP+AS03, CoVLP+CpG1018) . Reactogenicity for all formulations was generally mild in severity ( Figure 1 ) and of short 295 duration. Both adjuvants increased the frequency of reported AEs. The frequency and severity 296 of AEs were similar after the first and second doses in the unadjuvanted CoVLP and 297 CoVLP+CpG1018 groups but increased after the second dose in subjects who received AS03-298 adjuvanted formulations. Details of solicited AEs and TEAEs by treatment group are provided 299 in Suppl. Materials: Tables S4-S9) . 300 There was no consistent impact of CoVLP dose level on safety outcomes in any group. After 302 the first dose, 74.3% of participants reported >1 solicited AE, 66.5% reporting a local reaction 303 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi. org/10.1101 org/10. /2020 and 39.7% reporting >1 systemic event. Pain at the injection site was the most common local 304 reaction (66.5%) while headache and fatigue were reported by 25.7% and 20.7% respectively. 305 The incidence of headache and fatigue were generally higher in the adjuvanted treatment 306 groups. AEs were mostly mild-moderate Grade 1-2) and only one Grade 3 report of fatigue 307 that started the evening following vaccination and resolved the same day. After the second 308 dose, 68.5% of participants reported >1 solicited AE, 62.9% reporting a local reaction and 309 47.8% reporting >1 systemic event. Pain at the injection site was again the most reported local 310 reaction (61.2%) while headache and fatigue were reported by 33.1% and 33.1% respectively. 311 Again, most symptoms were mild but there were more moderate AEs after the second dose. 312 Nine Grade 3 solicited severe AEs (fatigue, redness at injection site, swelling at injection site, 313 feeling of general discomfort or uneasiness) were reported in 6 subjects after the second dose. 314 All but one of the Grade 3 reactions were reported by subjects who receivedAS03-adjuvanted 315 formulations. One Grade 3 reaction occurred after the second dose in the CoVLP (7.5 316 g)+CpG1018 group. All Grade 3 AEs resolved in one to four days. No clinically significant 317 lab abnormalities were reported after any dose. No SAEs, AESIs or pregnancy 318 exposures have been reported at the time of writing. A small number of subjects (12/180; 6.7%) had detectable pre-existing antibodies for the spike 322 protein in one or more of the assays used. As illustrated in Figure 2 , unadjuvanted CoVLP 323 elicited no detectable antibody response after the first dose and humoral responses after even 324 the second dose were modest and inconsistent. Although a minor dose-effect for the 325 unadjuvanted CoVLP was seen on the anti-spike IgG response (ELISA) after the second 326 vaccination, the responses in the two NAb assays remained low and variable even at the highest 327 dose tested. Both adjuvants had a significant impact on antibody responses at all dose levels. 328 Although there was no convincing effect of increasing CoVLP dose for either adjuvant, there 329 was a trend towards increasing consistency of response in the CoVLP+CpG1018 groups at the 330 higher CoVLP doses (ie: a greater response and a larger proportion of subjects responding). 331 Although both adjuvants elicited modest IgG titers after the first dose, only the groups that 332 received COVLP+AS03 formulations mounted significant NAb responses at D21 (36/60; 60%) 333 and across all dose levels (ie: overall GMT of 33.3 in the pseudovirus assay). Both adjuvants 334 induced more robust responses after the second dose with the large majority of subjects at all 335 dose levels mounting a ≥4-fold rise in total IgG (117/118; 99.1%) and in both NAb assays 336 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi. org/10.1101 org/10. /2020 93 .8% in the pseudovirion assay and 106/116; 91.3% in the PRNT). At all dose 337 levels, both anti-spike IgG and pseudovirion NAb titers at D42 were 10-to 50-fold higher in 338 subjects who had received AS03-adjuvanted formulations compared to those who had received 339 CpG1018-adjuvanted formulations. For example, after the second CoVLP(3.75 g) dose, the 340 GMTs in the PRNT were 7.2 for CoVLP alone, 56.6 for CoVLP+CpG1018 and 811.3 for 341 CoVLP+AS03. Overall, the levels of NAb induced in the groups that received two doses of 342 CoVLP with an adjuvant were either similar to (CoVLP+CpG1018) or substantially greater 343 (CoVLP+AS03) than those seen in subjects 3-4 weeks after recovering from natural Covid-19 344 infection. Details of serologic response results by treatment group are presented in Suppl. 345 Materials: Tables S10-S12. 346 347 There was a strong correlation between the VSV pseudovirion neutralization assay and the 349 PRNT for responses in all groups: r =0.85 (p < 0.0001) after the first ( Figure 3A ) and r = 0.68 350 (p < 0.0001) second vaccinations ( Figure 3B ). 351 352 Immunogenicity: T Cell Response 353 As illustrated in Figure 4 , the IFN and IL-4 responses in PBMC (ELISpot) elicited by CoVLP 354 ± adjuvants were more variable than the antibody responses. A substantial minority of subjects 355 in all groups had what appeared to be pre-existing IFN responses to the S protein peptide pool 356 that were, in some subjects, substantial (ie: >200 spots) 33 . Although low level 'background' IL-357 4 activity was seen in a small number of subjects, these responses were close to the limit of 358 detection of the assay used (generally <10 spots). Unlike antibody responses, CoVLP alone was 359 able to induce a substantial IFN response and, to a lesser extent, IL-4 response in many 360 subjects after the second doses at all dose levels but that was most consistently seen at the 361 highest dose (CoVLP 15g). Both adjuvants generally increased IFN and IL-4 responses 362 above background levels after the first dose that were further and substantially increased in 363 magnitude and consistency by the second dose. Compared to unadjuvanted CoVLP, IFN 364 responses were slightly higher and IL-4 responses were slightly lower in the CpG1018-365 adjuvanted groups but most of these differences did not reach statistical significance. Once 366 again, the IFN and IL-4 responses to the CoVLP+AS03 formulations at all dose levels were 10 367 to 50-fold higher than those seen in the equivalent CoVLP+CpG1018 groups. For example, at 368 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10. 1101 In the current study, the adjuvants performed as expected with enhancement of humoral 400 and cellular responses to the S protein and in promoting responses at lower CoVLP 401 doses. In both dose-sparing and enhancing immune responses, AS03 appeared to be 402 more effective than CpG1018. For example, a substantial minority of the subjects who or CpG1018-adjuvanted groups. In the AS)3-adjuvanted groups, the IL4 response was 423 10-fold lower than the IFN responses after the first dose but rose to near equivalence 424 with the second dose suggesting a balanced Th1-and Th2-type response. It is noteworthy that a substantial proportion of the subjects in this study appeared to 427 have pre-existing IFN responses to the S protein peptide pool used for PBMC 428 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint restimulation. Such cross-reactive T cell memory, possibly due to prior exposure to 429 common human coronaviruses has been seen in 40-60% of adults and may provide 430 some protection against highly pathogenic strains 33 . Prior exposure to circulating 431 coronaviruses may also explain the small number of subjects in this study (6.7%) who 432 were 'seronegative' at screening based upon a commercial N-based ELISA but who 433 were sero-positive at D0 in our assays targeting the S protein. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint Like any early-phase clinical trial, this study has several limitations beyond the obvious 459 concern regarding small group size when testing multiple dose levels and formulations 460 (n=20/group). For comparisons between formulations however, the small group risk 461 was mitigated by the fact that results across the three CoVLP dose levels were highly 462 consistent (n=60 for CoVLP alone or with each adjuvant). Another obvious concern is 463 the relatively limited range of immune response parameters available at the time of (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint 21 TABLES (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint Subjects were monitored for solicited local (panel A) and systemic (panel B) AEs from the time of vaccination through 7 days after vaccine administration. There was no grade 4 (life-threatening) events. Participants who reported 0 events make up the remainder of the 100% calculation (not shown). If any of the solicited AEs persisted beyond Day 7 after each vaccination (when applicable), it was recorded as unsolicited AEs. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint Serum antibodies of subjects vaccinated with 3.75, 7.5, or 15 µg CoVLP with or without AS03 or CpG1018 adjuvant, were measured to spike protein by ELISA (panel A) or by neutralization of pseudovirus (panel B), or live virus (panel C). Convalescent sera from recovered COVID-19 infected mildly, moderately, or severely ill patients were analyzed by anti-spike ELISA (n=35), and sera from mildly or moderately ill patients and one severely ill patient were analyzed by pseudovirion neutralization assay (n=21); results are shown in the right panels. All results are presented as reciprocal mid-point titers. Bars indicate geometric means. Error bars indicate 95% confidence intervals. Significant differences between days 0 and 21, or 0 and 42, for each vaccine regimen are indicated by # (#p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; unpaired T-test of log-transformed values, GraphPad Prism, v8.1.1). Significant differences between unadjuvanted and adjuvanted regimens for days 21 and 42 are indicated by * (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; 2-way ANOVA of log-transformed values, GraphPad Prism, v8.1.1). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint Neutralizing serum antibodies of subjects vaccinated with 3.75, 7.5, or 15 µg CoVLP with or without AS03 or CpG1018 adjuvant, were measured by neutralization of pseudovirion (x-axis) or live virus (y-axis) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint twenty-one days after the first vaccination (panel A) or twenty-one days afters the second vaccination (panel B). Results are presented as reciprocal titers. R are Pearson correlation coefficients (GraphPad Prism, v8.1.1). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint Frequencies of antigen-specific T-cells producing interferon-gamma (Panel A) and interleukin-4 (Panel B) cellular immune responses at baseline (day 0) and 21 days after one immunization (day 21) or two immunizations (day 42) with 3.75, 7.5 or 15 µg doses of CoVLP with or without adjuvants (CpG1018 and AS03) after restimulation ex vivo with recombinant spike peptide pool. Bars indicate medians and error bars indicate 95% CI. Significant differences between days 0 and 21 or between day 0 and 42 for each vaccine regimen are indicated by # ( # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001; Unpaired T-test. Comparisons for each test to day 0 data are to the same pre-vaccination data set that include all subjects. The figure illustrates matched subject data. GraphPad Prism, v8.1.1). Significant difference between adjuvanted vaccine and unadjuvanted vaccine regiments at day 21 and 42 are indicated by * (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Kruskal-Wallis, GraphPad Prism, v8.1.1). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint Safety monitoring of safety signals were and will be performed throughout the study. Stopping rules or conditions for stopping this clinical trial would occur if there was clear evidence of harm or harmful effects such as SAEs related to the treatment (study vaccine). A SAE which was thought to be unrelated to the study vaccine would not warrant stopping the trial. The following event(s) may result in a halt to the study, for further review and assessment of the event(s): • Any death occurring during the study; • Any vaccine-related SAE during the study; • Any life-threatening (Grade 4) vaccine-related AE during the study; • If 10 % or more of subjects in a single treatment group, experience the same or similar listed event(s) that cannot be clearly attributed to another cause: o a severe (Grade 3 or higher) vaccine-related AE during the study; o a severe (Grade 3 or higher) vaccine-related vital sign(s) abnormality; o a severe (Grade 3 or higher) vaccine-related clinical laboratory abnormality. In the case that a pre-defined safety signal is met in any treatment group, subsequent dosing will result in at least a transient halt in the study to permit a complete evaluation of the reported event(s) and to consult an IDMC. A decision as to whether the study can progress as planned must be made and documented in the event of any safety signal. If a stopping rule has been met once all subjects have been vaccinated in the study, the IDMC will be notified by a Note To File (NTF) for their information purposes. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint Evaluation of SARS-CoV-2 Humoral Response SARS-CoV-2 Spike Protein ELISA Briefly, SARS-Cov2 Spike protein (SARS-Cov2/Wuhan/2019, Immune Technology Corp.) was coated onto a flat-bottom 96-well microplate at a concentration of 1ug/mL in sodium carbonate 50mM (overnight; 4°C). Following washing steps (PBS-Tween), plates were blocked using Blotto 5% (Rockland Inc.) in PBS (1-2 hours; 37°C). Following washing steps, serially diluted sera (starting dilution 1/100, 4-fold dilutions, 8 dilutions, in PBS-Tween-Blotto) were added to the wells, in duplicates, and incubated at 37°C for 1 hour. Plates were washed and incubated with secondary antibody (anti-Human IgG (H+L) antibody, Peroxidase-labeled, Seracare), diluted at 1/20 000 in PBS-Tween-Blotto and incubated at 37°C for 1 hour. Plates were washed and incubated with peroxidase substrate (SureBlue TMB, Seracare) for 20 minutes at room temperature. Reaction was stopped using hydrochloric acid and absorbance was read at 450nm, within 2 hours (Variaskan Flash microplate reader, Thermo Scientific). Optical density (OD) results for the serial dilutions were used to generate a 4-parameter logistic regression (4PL). The titer was defined as the reciprocal dilution of the sample for which the OD is equal to a fixed cut-point value. Samples below cut-point were attributed a value of 50 (half the minimum required dilution). Neutralizing antibody analysis was performed using a cell-based pseudotyped virus neutralisation assay (Nexelis, Quebec, Canada). Pseudotyped virus particles were first generated using a genetically modified Vesicular Stomatitis Virus backbone from which the glycoprotein G was removed and luciferase reporter introduced (rVSVΔG-luciferase, Kerafast) to allow quantification using relative luminescence units with SARS-Cov-2 Spike glycoprotein (NXL137-1 in POG2 containing 2019-nCOV Wuhan-Hu-1; Genebank: MN908947) from which the last nineteen amino acids of the cytoplasmic tail were removed (rVSVΔG-Luc-Spike ΔCT). Serial dilutions (starting dilution of 1/10; 2-fold; 8 dilutions, in complete growth media) of the heatinactivated human sera (56°C; 30min) were prepared in a 96-well plate, in duplicates. The SARS-Cov-2 pseudovirus (in complete growth media) was added to the sera dilutions to reach a target concentration equivalent to approximately 150,000 RLU/well and mixture was incubated at 37°C with 5% CO2 supplementation for 1 hour. Serum-pseudovirus complexes were then transferred onto plates previously seeded overnight with Vero E6 cells (ATCC CRL-1586), expressing ACE-2 receptor, and incubated at All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint 37°C with 5% CO2 supplementation for 20-24 hours. Once incubation was completed, cells were lysed and samples equilibrated using ONE-Glo EX luciferase assay system (Promega), incubated for 3 minutes at room temperature, and luminescence level read using a luminescence plate reader (i3x plate reader, Molecular Devices). The resulting RLU was inversely proportional to the level of neutralizing antibodies present in the serum. For each sample, the neutralizing titer was established as the reciprocal dilution corresponding to the 50% neutralization (NT50), when compared to the pseudoparticle control. The NT50 was interpolated from a linear regression using the two dilutions flanking the 50% neutralisation. Samples below cut-off were attributed a value of 5 (half the minimum required dilution). Neutralizing antibody analysis was also performed using a cell-based cytopathic effect (CPE) assay (VisMederi, Sienna, Italy). Sera sample were first heat inactivated (56°C; 30min) and then serially diluted (starting dilution of 1/10, 2-fold; 8 dilutions, in complete growth media). Wild-type SARS-Co-2 virus (2019 nCOV ITALY/INMI1, provided by EVAg; Genebank: MT066156) was then added at final concentration of 25 TCID50/mL (in complete growth media), and plates were incubated for 1 hour at 37°C with 5% CO2 supplementation. At the end of the incubation, the mixture was transferred onto duplicate 96-well microtiter plates pre-seeded overnight with Vero E6 cells (ATCC CRL-1586), expressing ACE-2 receptor. Plates were then incubated for 3 days at 37°C with 5% CO2 supplementation. Cytopathic effect (CPE) was then quantified using an inverted optical microscope. The microneutralization titer (MNt) was defined as the reciprocal of the highest sample dilution that protects from CPE at least 50% of the cells. If no neutralization was observed, samples were attributed a titer value of 5 (half the minimum required dilution). Interferon-γ ELISpot Cell-mediated immune response was evaluated using an Interferon-γ ELISpot assay (Human IFN-γ ELISpot assay, Cellular Technology Limited (CTL), USA). Cryopreserved peripheral blood mononuclear cells (PBMC) were rapidly thawed and allowed to rest between 2 to 3 hours at 37°C with 5% CO2 supplementation, in CTL-Test media supplemented with 1% Glutamine and 1% Penicillin/Streptomycin. Cells were enumerated and dispensed at 0.5x10 6 cells per well, in duplicates, onto PVDF filter plates precoated with an IFN-γ specific capture antibody. Cells were stimulated using a pool of peptides (15-mer peptides) overlapping the full sequence of SARS-Cov-2 spike protein (USA-CA2/2020, Genbank: MN994468.1) at a concentration of 2.19 µg/mL, for 18-24 hours, at 37°C with 5% CO2 supplementation. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi. org/10.1101 org/10. /2020 After washes (PBS-Tween), biotinylated anti-interferon-γ detection antibody was added to the plates and incubated for 2 hours at room temperature after which, following another round of washes, a streptavidinalkaline phosphatase conjugate was added and incubated for 30 minutes at room temperature. After washing steps, substrate solution was added, incubated at room temperature for 15 minutes, after which plate was rinsed and left to air-dry pending spot enumeration, using an ELISpot reader (ImmunoSpot S6 Universal Analyzer, Cellular Technology Limited). Mean of peptide pool stimulation duplicates was calculated and normalized using the mean of the negative control replicates (control media) and multiplied by a factor of 2 to express cells counts per million cells. Cell-mediated immune response was evaluated using an IL-4 ELISpot assay (Human IL-4 ELISpot assay, Cellular Technology Limited (CTL), Cleveland, OH, USA). Cryopreserved peripheral blood mononuclear cells (PBMC) were rapidly thawed and allowed to rest between 2 to 3 hours at 37°C with 5% CO2 supplementation, in CTL-Test media supplemented with 1% Glutamine and 1% Penicillin/Streptomycin. Cells were enumerated and dispensed at 0.5x10 6 cells per well, in duplicates, onto PVDF filter plates precoated with an IL-4 specific capture antibody. Cells were stimulated using a pool of peptides (15-mer peptides) overlapping the full sequence of SARS-Cov-2 spike protein (USA-CA2/2020, Genbank: MN994468.1) at a concentration of 2.19 µg/mL, for 32-48 hours, at 37°C with 5% CO2 supplementation. After washes (PBS-Tween), biotinylated anti-IL-4 detection antibody was added to the plates and incubated for 2 hours at room temperature after which, following another round of washes, a streptavidinalkaline phosphatase conjugate was added and incubated for 30 minutes at room temperature. After washing steps, substrate solution was added, incubated at room temperature for 15 minutes, after which plate was rinsed and left to air-dry pending spot enumeration, using an ELISpot reader (ImmunoSpot S6 Universal Analyzer, Cellular Technology Limited). Mean of peptide pool stimulation duplicates was calculated and normalized using the mean of the negative control replicates (control media) and multiplied by a factor of 2 to express cells counts per million cells. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi. org/10.1101 org/10. /2020 Sera from Covid-19 convalescent patients were collected from a total of 34 individuals with confirmed disease diagnosis. Time between the onset of the symptoms and sample collection varied between 27 and 105 days. Three samples were collected by Solomon Park (Burien, WA, USA), 20 by Sanguine BioSciences (Sherman Oaks, CA, USA) and 11 from McGill University Health Centre. Disease severity were ranked as mild (Covid-19 symptoms without shortness of breath), moderate (shortness of breath reported), and severe (hospitalized). These samples were analysed in parallel of clinical study samples, using the same assay as described above. Samples from severely ill were analyzed by serum IgG ELISA only. Demographic characteristics are provided in Supplementary Table 1 . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint AESI for CoVLP Vaccine -Vaccine Enhanced Disease (VED) Safety signal of VED after exposure to the Coronavirus-Like Particle COVID-19 Vaccine was closely monitored and assessed by retrieving data for this AESI as follows: AEs within the system organ class (SOC): immune system disorders and high level group term (HLGT): lower respiratory tract disorders (excluding obstruction and infection), cardiac disorders, signs and symptoms not elsewhere classified (NEC), vascular disorders, heart failures NEC, arteriosclerosis, stenosis, vascular insufficiency and necrosis, cardiac arrhythmias, myocardial disorders, and vascular hemorrhagic disorders. High level term (HLT): renal failure and impairment and preferred term (PT): pericarditis, coagulopathy, deep vein thrombosis, pulmonary embolism, cerebrovascular accidents, peripheral ischemia, liver injury, Guillain-Barre syndrome, anosmia, ageusia, encephalitis, chilblains, vasculitis, erythema multiforme (based on standardized MedDRA ® classification) 1,2 that require inpatient hospitalization (≥ 24 hours) and have laboratory confirmed SARS-Cov-2 infection will be monitored for assessment of any potential case of VED. All reported events were also monitored for hypersensitivity reactions after exposure to the Coronavirus-Like Particle COVID-19 Vaccine. In eight clinical studies conducted to date with the Quadrivalent VLP Influenza Vaccine (QVLP) produced using similar plant-based technology, all reported events were monitored for a possible hypersensitivity component (events were searched using both narrow and broad standardized MedDRA ® queries). Based on these data, there was a single case of possible early anaphylactic reaction associated with use of QVLP in humans. A small number of subjects had potential hypersensitivity reactions judged to be related to vaccine administration (no more than 0.3 % of subjects in any given QVLP treatment group experienced one of these events) and the events were distributed fairly evenly among treatment groups, including the placebo and the active comparator groups. However, since severe reactions are considered to be an important potential risk (based on the theoretical risk that using plants for the production of biotherapeutics may induce hypersensitivity), Medicago required that appropriate medical treatment and supervision were available to manage any possible anaphylactic reactions in this study. To collect data on these events, Medicago closely monitored and assessed allergic reactions assessed by the site Investigators as related to the Investigational product as AESIs. AESI for Adjuvant -Potential Immune-Mediated Diseases (pIMD) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint Potential immune-mediated diseases are a subset of AEs that include autoimmune diseases and other inflammatory and/or neurologic disorders of interest which may or may not have an autoimmune aetiology. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint • Autoimmune hemolytic anemia. • Autoimmune thrombocytopenia. • Antiphospholipid syndrome. • Pernicious anemia. • Autoimmune aplastic anemia. • Autoimmune neutropenia. • Autoimmune pancytopenia. • Autoimmune glomerulonephritis including: -IgA nephropathy. -Glomerulonephritis rapidly progressive. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https: //doi.org/10.1101 //doi.org/10. /2020 - All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 Supplementary (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 Supplementary Redness at injection site Grade 1 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20226282 doi: medRxiv preprint Redness at injection site (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. Redness at injection site (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 6, 2020. Subjects with at least one unsolicited AE 8 9 8 Eye Clinical Characteristics of Coronavirus Disease 2019 in 485 China Immunogenicity and safety of a recombinant 487 adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a 488 randomised, double-blind, placebo-controlled, phase 2 trial Repurposing 496 Therapeutics for Potential Treatment of SARS-CoV-2: A Review Drug treatment options for the 2019-new coronavirus (2019-nCoV) SARS and MERS: recent insights 503 into emerging coronaviruses World Health Organization, Draft landscape of COVID-19 candidate vaccines The COVID-19 vaccine development 508 landscape Vaccines for Novel Coronavirus Disease 2019 (COVID-19) SARS vaccines: where are we? What are the roles of antibodies versus a durable, high quality T-cell 515 response in protective immunity against SARS-CoV-2? Approaches and Challenges in SARS-CoV-2 Vaccine 517 Development Sariol A, Perlman S. Lessons for COVID-19 Immunity from Other Coronavirus Supplementary Figure 1 Consort Flow Diagram *One subject did not receive the second vaccination following Grade 3 adverse event but accepted to have blood collection for immunogenicity GMT: 14989 (1617, 138966) Severe: N=11 GMT GMFR = Geometric mean Fold Rise SCR (%) (95%CI) 2 (10.0%) (1.2, 31.7) 12 (63.2%) (38.4, 83.7) 18 (100%) (81.5, 100) 4 (20.0%) (5.7 GMFR = Geometric mean Fold Rise GMFR = Geometric mean Fold Rise Safety platform for emergency vaccines (SPEAC). D2.3 Priority list of adverse events of special interest: COVID-19 Safety platform for emergency vaccines (SPEAC). D2.3 Priority list of adverse events of special interest: COVID-19