key: cord-0796396-shrucdo8
authors: Lanini, S.; Capone, S.; Antinori, A.; Milleri, S.; Nicastri, E.; Camerini, R.; Agrati, C.; Castilletti, C.; Mori, F.; Sacchi, A.; Matusali, G.; Gagliardini, R.; Ammendola, V.; Cimini, E.; Grazioli, F.; Scorzolini, L.; Napolitano, F.; Plazzi, M. M.; Soriani, M.; De Luca, A.; Battella, S.; Sommella, A.; Contino, A. M.; Barra, F.; Gentile, M.; Raggioli, A.; Shi, Y.; Girardi, E.; Maeurer, M.; Capobianchi, M. R.; Vaia, F.; Piacentini, M.; Kroemer, G.; Vitelli, A.; Colloca, S.; Folgori, A.; Ippolito, G.
title: GRAd-COV2, a gorilla adenovirus based candidate vaccine against COVID-19, is safe and immunogenic in young and older adults
date: 2021-04-13
journal: nan
DOI: 10.1101/2021.04.10.21255202
sha: 988eb7db10b2be075294c6977ff5886ef8c5d598
doc_id: 796396
cord_uid: shrucdo8

Safe and effective vaccines against coronavirus disease 2019 (COVID-19) are urgently needed to control the ongoing pandemic. Although impressive progress has been made with several COVID-19 vaccines already approved, it is clear that those developed so far cannot meet the global vaccine demand. We have developed a COVID-19 vaccine based on a replication-defective gorilla adenovirus expressing the stabilized pre-fusion SARS-CoV-2 Spike protein, named GRAd-COV2. We aimed to assess the safety and immunogenicity of a single-dose regimen of this vaccine in healthy younger and older adults to select the appropriate dose for each age group. To this purpose, a phase 1, dose-escalation, open-label trial was conducted including 90 healthy subjects, (45 aged 18-55 years and 45 aged 65-85 years), who received a single intramuscular administration of GRAd-CoV2 at three escalating doses. Local and systemic adverse reactions were mostly mild or moderate and of short duration, and no serious AE was reported. Four weeks after vaccination, seroconversion to Spike/RBD was achieved in 43/44 young volunteers and in 45/45 older subjects. Consistently, neutralizing antibodies were detected in 42/44 younger age and 45/45 older age volunteers. In addition, GRAd-COV2 induced a robust and Th1-skewed T cell response against the S antigen in 89/90 subjects from both age groups. Overall, the safety and immunogenicity data from the phase 1 trial support further development of this vaccine.

Between August 11 and September 20, 2020, 181 potential volunteers were evaluated for 94 eligibility. Volunteers were screened in excess for speeding up enrollment and to comply with a 95 strict staggering enrollment scheme. Of the 181 subjects screened, 91 (45 in younger adults 96 cohort aged 18-55 and 46 in the older adults cohort, aged 65-85) were vaccinated. Fifty-five 97 volunteers (30 in younger age cohort and 25 in the older age cohort) were excluded as they did 98 not meet inclusion criteria and 35 (25 younger age cohort and 10 in the older age cohort) were 99 113 . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ; https://doi.org/10.1101 doi: medRxiv preprint B 1 volunteer excluded from humoral and cellular immunity analysis due SARS-CoV-2 infection 119 immediately after/before vaccination; C volunteers excluded from ELISpot analysis only, 120 because of high non-specific IFNγ secretion. D 1 volunteer excluded from immunogenicity 121 analysis and replaced, due to unspecific reactivity in anti S CLIA assay. 122 123 Vaccine safety 124 Figure 2 reports the frequency of all adverse events by type, cohort and severity. 125

A single intramuscular administration of GRAd-COV2 was well tolerated at all doses and in both 126 cohorts. Overall, we observed 328 adverse events of any grade; of those, 255 were considered to 127 be related with vaccination. In the younger age cohort we observed 143 solicited AEs (118 mild 128 and 25 moderate) and 8 unsolicited AEs (4 mild and 4 moderate). In the older age cohort (we 129 observed 104 solicited AEs (79 mild, 13 moderate and 2 severe) and 10 unsolicited AEs (5 mild, 130 3 moderate and 2 severe). Twenty-four volunteers (26%; 7 younger and 17 older) reported no 131 AE, 44 volunteers (48%; 23 younger and 21 older) reported at least one mild AE and 21 132 volunteers (48%; 15 younger and 6 older) reported at least one moderate AE and 2 (2%; both 133 older age volunteers) reported at least one severe adverse event. No serious AE was reported, 134 and no pre-specified trial-halting rules were met. Nineteen volunteers (15 younger and 4 older) 135 received antipyretics to control AEs. One older age volunteer received inhalator steroids for 136 cough started the day after the vaccination that resolved in 9 days. 137

Most AEs occurred in the first 48 hours after vaccination and were short-lived (median time to 138 resolution 24 hours, IQR less than 1 day-2 days). 139

Overall, no clinically significant blood count changes were observed. A transient reduction of 140 neutrophils with concomitant increase in monocytes was detected at day 2 in the majority of 141 volunteers in all age and vaccine dose groups that mostly reverted at pre-dose level by week 1 142 ( Fig S1) . Other hematological parameters were mostly unaffected (Fig. S1 , lymphocytes and 143 platelets shown for reference), and no vaccine-related trends were noted for biochemistry 144 parameters. 145 . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. The severity of AE is reported according to intensity scale in the protocol. (A) Younger adults 149 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 13, 2021. Antibody response to GRAd-COV2 vaccination was primarily monitored by a clinically 154 validated CLIA, revealing similar kinetics of anti-S IgG induction in all study groups (Fig. 3A  155 and table S1). Seroconversion to S occurred at week 2, when anti-S IgG became detectable in 23 156 (52%) out of 44 of the younger volunteers and in 15 (33%) out of 45 of the older age subjects. At 157

week 4 after vaccination, high levels of anti-S IgG were measurable in 40 (91.0%) out of the 44 158 analyzed younger and in 42 (93.3%) out of the 45 older age volunteers. In the younger age 159 cohort, three volunteers (1 in ID and 2 in HD arm) showed a weak increase of anti-S-IgG (10.8 160 AU/mL, 11.5 AU/mL and 12.8, respectively), that are below the diagnostic cutoff applied in the 161 clinical practice (15.0 AU/mL). Only one volunteer in the LD arm showed anti-S-protein IgG 162 below the assay limit of detection (LOD; <3.8 AU/mL). Also in the older age cohort, three 163 volunteers (1 in LD, 1 in ID, 1 in HD arm) showed a weak increase of anti-S-IgG (5.0 AU/mL, 164 8.5 AU/mL and 9.7 respectively) that were below the diagnostic cutoff. 165

Antibodies measured by CLIA did not show a dose response relationship in the younger adult 166 cohort (IgG levels at W4 were 59.5, 60.6 and 61.8 AU/mL for LD, ID and HD respectively, 167 IgG (including both antibodies against the whole Spike protein and specific anti-RBD) at 4 177 weeks after vaccination (Fig 3B-C and table S1) . 178 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Median titers to S and RBD showed a dose-response trend both in the younger and older age 179 cohorts. We also observed that median level of IgG either against S or RBD were higher in 180 younger than in older volunteers, although the difference between the two age cohorts were not 181 significant ( Fig. 3B -C and table S1). 182

None of the study volunteers showed seroconversion to N during the 4 weeks following 183 vaccination, suggesting that no SARS-CoV-2 infection occurred. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. triangles) and high dose (HD-2x10 11 vp-upside down triangles). Blue and red color shades 210 identify younger and older age cohorts, respectively. Horizontal black lines are set at median 211 across all panels. HCS: human convalescent sera (diamonds), obtained from either previously 212 hospitalized (hosp-dark grey) or from non-hospitalized (non-hosp-light grey) COVID-19 213 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Notably, 80% of evaluable subjects across the two age cohorts showed a response above 1000 234 SFC/million PBMC. Only one volunteer in arm1 did not show a detectable anti-S T cell response 235 but still had measurable level of antibodies to S and RBD (ELISA titer of 1494 and 721 236 respectively). 237 Figure 4 shows that an incremental level of T cell response was observed in younger (median 238 response 1162, 2857 and 2272 SFC/per million PBMC in the LD, ID and HD arms, respectively; 239 P=0.154) and older (median response 1917, 2262 and 3142 SFC in the LD, ID and HD arms, 240 respectively, P=0.206) volunteers. However, neither trend was associated to a significant dose-241 response within the cohorts. Moreover, we did not find significant differences between younger 242 . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ;  and older study arms receiving the same vaccine dose (P was 0.116, 0984 and 0.152 for LD, ID 243 and HD, respectively). 244 T cell response induced by vaccination was directed to multiple epitopes, as most of the 245 volunteers in both cohorts had a detectable response against all four S peptide pools analyzed 246 ( Fig. S2A and B) . Moreover, we found that all regions of the S protein had a similar degree of 247 immunogenicity in both age cohorts (Fig. 4B) . 248

S-specific T cell response was generally higher in GRAd-COV2 vaccinated subjects than in 249 SARS-CoV-2 convalescent controls who were sampled 1-2 months after symptoms onset. 250 Intracellular staining for cytokine production (ICS) and FACS analysis revealed that vaccine-251 induced responses involved both S protein specific CD4 and CD8 T lymphocytes in younger and 252 older volunteers ( Fig. 4C -D and E-F), with a slightly higher S-specific CD4 than CD8 T cell 253 responses. Importantly, among GRAd-COV2 vaccine induced S-specific CD4, IFNγ production 254 was more prominent than IL4 and IL17 in both age cohorts, indicating that the vaccine induced a 255 predominantly T helper 1 (Th1) response (tables below Fig. 4C and 4E report comparisons). 256

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The copyright holder for this preprint this version posted April 13, 2021. ; 

GRAd neutralization was not set as a screening or a randomization parameter, but was anyway 278 assessed as an exploratory endpoint at baseline and 4 weeks after vaccination. Out of 90 279 volunteers, we measured neutralization titers >200, a level above which an impact on vaccine 280 immunogenicity has been reported, in only 10 volunteers (11%). 281

As expected, the vaccination with GRAd-COV2 induced or boosted anti-vector immunity in 282 most volunteers (Fig. 5A) , with no significant differences attributable to vaccine dose level or 283 age. 284

To assess the impact of anti-GRAd immunity, pooled immunogenicity data at peak post 285 vaccination (T, binding and neutralizing Ab responses) were stratified according to their baseline 286 GRAd neutralizing titers (Fig. 5B-G) . A trend for reduced S binding antibody response to the 287 vaccine antigen was noted in volunteers with GRAd nAb >200 at baseline, with IgG titers 288 measured by ELISA significantly lower (P=0.018 on full length S and P=0.0145 on RBD). 289 . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ; https://doi.org/10.1101/2021.04.10.21255202 doi: medRxiv preprint Similar trend was noted for T cell response and SARS-CoV-2 neutralizing Ab. Conversely, 290 vaccine-induced immune responses in individuals with GRAd nAb titers in the 18-200 range (i.e. 291 low but detectable) at baseline were undistinguishable or similar to those of volunteers with no 292 anti-vector immunity. 293

294 . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ; https://doi.org/10.1101/2021.04.10.21255202 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. Here we report the first-in-human data on the safety and immunogenicity of a single 330 administration of GRAd-COV2 given at different doses in healthy younger adults aged 18-55 331 and in healthy older adults aged 65-85 years. GRAd-COV2 is a COVID-19 candidate vaccine 332 . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ; https://doi.org/10.1101/2021.04.10.21255202 doi: medRxiv preprint based on a replication-defective gorilla adenovirus vector. Our analysis provides evidence that 333 GRAd-COV2 is well tolerated in both younger and older age cohorts at all three doses assessed. 334

Indeed at 4 week of follow-up most of the observed adverse events were short lived and mild or 335 moderate in intensity. Only 2 volunteers reported at least one severe adverse event and none 336 reported serious adverse events. Solicited adverse events were less common in older age 337 volunteers, similarly to what described for other COVID-19 vaccines (5-7). In general, the 338 adverse event profile did not differ from those reported in published work for other vector-based 339 vaccines (7-12) while they were milder than those reported for mRNA formulated in 340 liponanoparticles (LNP) and for adjuvanted vaccines (2, 13, 14) . -1 (8, 9) , Ad-26 (7) and Ad5-vectored 12) , 360

suggesting that the measured lower nAb titers could result from different assay sensitivities. 361 . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ; https://doi.org/10.1101/2021.04.10.21255202 doi: medRxiv preprint

There is a lack of knowledge on reliable immune correlates that would predict the efficacy of 362 . However, preliminary evidence from a non-human primate model 363 (21), clinical observation of the natural infection (22) as well as evidence of protective efficacy 364 after first dose for several authorized vaccines (5, 17, 23) suggested that even low titer of 365 neutralizing titers may be protective. The single administration of GRAd-COV2 elicits robust 366 and Th1-skewed immune response, similar to that induced by a single dose of other vectored 367 COVID-19 vaccines such as ChAdOx1 nCoV-19 (8, 9), Ad5 (11, 12) and Ad26 (7), and by two 368 doses of mRNA-1273 (13, 15). Indeed, a single administration of GRAd-COV2 elicits, within 2 369 weeks, a Th1 immune response that is broadly directed to multiple S protein epitopes. 370 Importantly, a robust T cell response, such as that elicited by GRAd-COV-2, likely determines 371 the duration of anti-COVID-19 immunity (24, 25) and prevents unintended immune reactions 372 such as the vaccine-associated enhanced respiratory disease (VAERD) (26, 27) . Moreover, S 373 protein-specific T cell responses directly correlated with binding and neutralizing antibodies, 374

suggesting that GRAd-COV2 vaccine is able to shape a well-balanced and coordinated cellular 375 and humoral specific immune response, as this naturally occurs in mild 29) . 376

New SARS-CoV-2 variants are becoming predominant through a process of evolutionary 377 selection that is not well understood. Among others, a variant of concern is the B.1.351 strain 378 which is less effectively neutralized by convalescent plasma from COVID-19 patients and by 379 sera from those vaccinated with several vaccines in development (30-32). The decrement in 380 neutralization can be more than 10-fold with convalescent plasma and averages 5-to 6-fold less 381 with sera from vaccinated individuals. The interim data from a randomized placebo-controlled 382 vaccine study, of rAd26 from Janssen in the US, Brazil, and South Africa showed efficacy 383 against COVID-19 at 72%, 66%, and 57%, respectively (https://www.jnj.com/johnson-johnson-384 announcessingle-shot-janssen-covid-19-vaccine-candidatemet-primary-endpoints-in-interim-385 analysis-of-itsphase-3-ensemble-trial). Viral sequence data from infected patients showed that 386 the B.1.351 strain was responsible for the majority of infections in South Africa. Lower vaccine 387 efficacy in the South Africa cohort could be related to antigenic variation or to geographic or 388 population differences. Interestingly, despite the reduced efficacy, the single dose of rAd26 389 vaccine was 85% effective overall in preventing severe COVID-19, and protection was similar in 390 all regions. Given the important reduction of neutralizing activity on the B.1.351 variant, it is 391 . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 tempting to speculate that the vaccine-induced T cell responses might be predominant in 392 protecting from severe disease. 393

As for other candidate vaccines using viral vectors, pre-existing immune responses against the 394 vector can compromise the induction of an immune response against the target antigen (33, 34) 395 and indeed this was shown also for a candidate COVID-19 vaccine based on the common human 396

Ad5 serotype (11). In our study, high levels of neutralizing antibodies against GRAd-COV2 397 were associated with a reduced median T cell response and antibody levels against S protein. This study has some limitations due to the low number of volunteers per arm (N=15), and to the 414 lack of subject randomization amongst study arms on the basis of GRAd neutralizing titer at 415 baseline. Both these aspects could have had an impact in the poor vaccine dose effect observed. 416

However, the explored dose range (4-fold between low and high dose) was limited, and no major 417 dose effect was expected. Nevertheless, for phase 2/3 single-dose regimen we selected the 2x10 11 418 vp (high) dose, due to higher and more consistent immunogenicity especially in the elderly 419 cohort. As for the two-dose regimen, the intermediate dose of 1x10 11 vp was selected which 420 represents the best compromise between tolerability and immunogenicity. Finally, data beyond 421 . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ; https://doi.org/10.1101/2021.04.10.21255202 doi: medRxiv preprint w4 post vaccination on persistence of humoral and cellular responses will be part of a future 422 report once the study follow up will be completed. 423

This study is a phase 1, dose-escalation, open label clinical trial designed to determine the safety 427 and immunogenicity of GRAd-COV2. The study included two age cohorts, of either younger 428 The dose-escalation staggered enrollment included 3 sentinel volunteers in the LD arm followed 444 by enrollment of the full LD arm. ID and HD sentinel volunteers were enrolled 7 days after that 445 safety data of LD or ID arm were available, respectively. All the enrollment, stages were 446 supervised by an independent data safety monitoring board. Volunteers recorded local and 447 systemic reactions on a diary card for 28 days. The severity and relatedness with vaccination or 448 adverse events were assessed by the medical team in each center. 449

. CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 As comparator for immunogenicity analysis we used three independent sets of anonymized 451 specimens (sera and PBMC) from COVID-19 patients either hospitalized or recovering from 452 mild symptomatic disease, collected 20 to 60 days after symptom onset. The Research reagent 453 for anti-SARS-CoV-2 Ab (NIBSC code 20/130), a human plasma from a donor recovered from 454 COVID-19, was included as a positive control. 

Immunoassay 467

Two commercial Ig assays were used according to manufacturer's protocols: 1) DiaSorin 468 LIAISON® SARS-CoV-2 S1/S2 IgG test on LIAISON® XL analyzers, a chemiluminescence 469 immunoassays (CLIA) detecting anti-S1/S2 IgG (IgG antibody concentrations CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 NUNC Maxisorp plates (ThermoFisher Scientific) at optimized concentration (5 μ g/ml for R121, 480 2,5 μ g/ml for RBD) in PBS overnight at 4°C. The following day, plates were washed with PBS 481 0,05% Tween-20 (PBS-T), then blocked with PBS-T+3% non-fat dry milk for 1,5h at 25°C in 482

shaking. After wash, serum dilution curves (six 3-fold serial dilution of human sera from 1:100 483 to 1:24,300) prepared in PBS-T+1% non-fat dried milk were plated and incubated for 2h at 25°C containing 2% Formaldehyde was added to each well. After 30 min, the fixing solution was 506 removed by washing with tap water and cell viability measured by photometer at 595 nm 507 (Synergy™ HTX Multi-Mode Microplate Reader, Biotek). The highest serum dilution inhibiting 508 at least 90% of the CPE was indicated as the neutralization titre and expressed as the reciprocal 509 . CC-BY-ND 4.0 International license It is made available under a 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 April 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 of serum dilution (MNA 90 ). Serum from the National Institute for Biological Standards and 510

Control, UK (NIBSC) with known neutralization titer was used as reference in MNA (Research 511 reagent for anti-SARS-CoV-2 Ab NIBSC code 20/130). 512

As an exploratory endpoint, the SARS-CoV-2 PRNT 50 titers of vaccinated volunteers' sera was 514 determined by means of a plaque reduction neutralization test (PRNT) developed and run at 515 Viroclinics Biosciences. Briefly, a standard number of SARS-CoV-2 (Bav/Pat1/2020 strain) 516 infectious units were incubated with eight two-fold serial dilutions of heat inactivated sera, 517 starting from 1:8 and up to 1:1024. After 1h pre-incubation, the virus/serum mixtures were 518 inoculated on VeroE6 cells for 1 hour, than washed, replaced with infection medium and the 519 cells were left overnight. After 16-24h the cells were formalin-fixed, permeabilized with ethanol, 520 and incubated with primary anti SARS-CoV-2 Nucleocapsid monoclonal antibody followed by a 521 secondary anti-mouse IgG peroxidase conjugate and TrueBlue substrate, which forms a blue 522 precipitate on positive cells. Images of all wells were acquired by an ImmunoSpot analyzer 523 (Cellular Technology Limited-CTL), equipped with software capable to accurately count the 524 virus positive cells. The 50% neutralization titers were calculated according to a method 525 described earlier (35). The NIBSC standard 20/130 was tested as reference in the same run than 526 the RT-COV-2 trial serum samples, with resulting PRNT 50 of 697 and 934 for younger and older 527 adults assay runs, respectively. 528

Neutralizing antibody (nAb) titers in human sera were assayed as previously described (36). 530

Briefly, 8×10 4 HEK293 cells per well were seeded in 96 well plates the day before the assay. 531

GRAd vector encoding for the reporter gene secreted alkaline phosphatase (SEAP) at a pre-532 optimized multiplicity of infection (MOI) was preincubated for 1h at 37°C alone or with serial 533 dilutions of control or test serum samples and then added to the 80-90% confluent HEK293 cells. 534

After incubation for 1h at 37°C, the serum/infection mix was removed and replaced with 10% 535 FBS in DMEM. SEAP expression was measured 24 hours later in cell supernatant by means of 536 the chemiluminescent substrate from the Phospha-Light™ kit (Applied Biosystems). 537

Neutralization titers were defined as the dilution at which a 50% reduction of SEAP activity 538 from serum sample was observed relative to SEAP activity from virus alone. 539 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Peripheral venous blood (40 ml) was collected in 7 ml lithium-heparin Vacutest blood collection 541 tubes (Kima). Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood 542 by density gradient centrifugation (Histopaque 1077, Sigma Aldrich). After separation, PBMC 543 were suspended in RPMI 1640 (Sigma Aldrich) supplemented with 10% heat-inactivated highly 544 defined fetal bovine serum (FBS) (HyClone), 2 mmol/L L-glutamine, 10 mmol/L HEPES buffer 545 (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, Sigma Aldrich), and 100 U/ml penicillin, 546 and 100 µg/mL streptomycin (Gibco), hereafter termed R10. PBMC count and viability were 547 performed by using Guava Muse (Luminex). A set of 316 15mer peptides overlapping by 11 548 amino acids (synthetized by Elabscience Biotech Inc, and distributed by TEMA RICERCA), 549 designed to cover the full-length S protein, was arranged into 4 pools (S1a, S1b-including RBD 550 domain, S2a and S2b). PBMC (2x10 6 /ml) were stimulated with the four Spike peptides pools for 551 18 hours (3μg/ml each peptide final concentration). For flow-cytometry experiments, brefeldin A 552 (10ug/ml, Sigma Aldrich) was added to block endoplasmic reticulum and Golgi apparatus. 553

The frequency of IFNγ-producing T cells was assessed by enzyme-linked immunosorbent spot-555 forming cell assay (ELISpot) after specific stimulation. PBMCs were resuspended in R10, 556 stimulated with peptides pools, as described above, and plated at 2 × 10 5 cells/well in ELISpot 557 plates (Human IFN-γ ELISpot plus kit; Mabtech). PBMCs were incubated for 18-20 hours with 558 5% of CO 2 . At the end of incubation, the ELISpot assay was developed according to 559 manufacturer's instructions. Spontaneous cytokine production (background) was assessed by 560 incubating PBMC with DMSO, the peptides diluent (Sigma,). Results are expressed as spot 561 forming cells (SFC)/10 6 PBMCs in stimulating cultures after subtracting spontaneous 562 background. A result was considered positive if matching two criteria: i) higher than >48 563 SFC/10 6 PBMC and ii) higher than 3 times Background. Data from three volunteers were 564 excluded from all ELISpot analyses since their spontaneous IFNγ secretion in DMSO wells 565 (yLD-101027, 450 SFC; yID-101034, 465 SFC; oID-101072, 233 SFC on DMSO) was above 566 the mean+2standard deviation of the study population (mean=42 SFC, SD=72.27, 567 mean+2SD=186 SFC/million PBMC), leading to unreliable quantitative analysis. 568

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The copyright holder for this preprint this version posted April 13, 2021. the immunological plot (SideScatter (SSC)/CD45), the CD45+ cells were gated, followed by 582 gating CD3+ lymphocytes. Among the CD3+ cells, CD4+ or CD8+ cells were selected and the 583 percentage CD4 or CD8 cells producing cytokines were evaluated. Spontaneous cytokines 584 production (DMSO stimulation) was subtracted. 585

This is a descriptive phase I first in human trial to assess safety and immunogenicity of three 587 different vaccine doses and no formal sample size calculation is carried out. Categorical 588 variables including occurrence of adverse events (AE) and detectable levels of binding and 589 neutralizing antibody against SARS-CoV-2 and GRAd were reported as proportions. Continuous 590 variables including results of CLIA anti-S IgG, MNA 90 titers, PNRT 50 titers and ELISpot values 591

for IFNγ secretion were reported as median and interquartile range. Comparison of medians 592 across arms and the impact of pre-existing immunity against GRAd were evaluated by two-tailed 593

Kruskal-Wallis one-way variance analysis. Association between categorical variable was carried 594 out by Fisher exact test. Correlations between immunogenicity assays were assessed by non-595 parametric Spearman's rank tests. 596 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 13, 2021. ; https://doi.org/10.1101/2021.04.10.21255202 doi: medRxiv preprint Fig. S2 . Breadth of GRAd-COV-2 induced T cell response. 599 Table S1 . Median and Interquartile range of main immunological measures. 600

RT-CoV-2 study Protocol 601 NUCLEIC ACID-AND AMINO ACID-SEQUENCES, VECTORS CONTAINING SAME, 841 AND USES THEREOF". All remaining authors declare that they have no competing interests. 842

All data are available in the main text or the supplementary materials. This trial is registered with 844 ClinicaTrials.gov, NCT04528641. 845 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine

m, 699 Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults

Safety and 705 Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates

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

What 715 defines an efficacious COVID-19 vaccine? A review of the challenges assessing the 716 clinical efficacy of vaccines against SARS-CoV-2

Immunological surrogate endpoints of COVID-2019 718 vaccines: the evidence we have versus the evidence we need

Looking beyond COVID-19 vaccine phase 3 trials

Correlates of protection against SARS-CoV-2 in rhesus macaques

Neutralizing Antibodies Correlate with

Protection from SARS-CoV-2 in Humans during a Fishery Vessel Outbreak with a High 731 Attack Rate

Single-dose administration and the influence of 745 the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 746 (AZD1222) vaccine: a pooled analysis of four randomised trials

COVID-19-neutralizing antibodies predict disease severity and 752 survival

Covid-19 vaccines: delivering 754 protective immunity

Rapid COVID-19 vaccine development

Prospects for a safe COVID-19 vaccine

T cell responses in patients with COVID-19

Sinai 766 Immunology Review, Immunology of COVID-19: Current State of the Science

Increased Resistance of SARS-CoV-2 Variants 771 B.1.351 and B.1.1.7 to Antibody Neutralization

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 776 donor plasma

mRNA-1273 vaccine induces neutralizing antibodies against spike mutants 780 from global SARS-CoV-2 variants. bioRxiv

Step Study Protocol, 784 Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a 785 double-blind, randomised, placebo-controlled, test-of-concept trial

Seroprevalence of neutralizing 788 antibodies to human adenoviruses type-5 and type-26 and chimpanzee adenovirus type-789 68 in healthy Chinese adults

Development of an 791 improved microneutralization assay for respiratory syncytial virus by automated plaque 792 counting using imaging analysis

Quantitative adenovirus neutralization assays based 796 on the secreted alkaline phosphatase reporter gene: application in epidemiologic studies 797 and in the design of adenovector vaccines

Alessandra Vergori (INMI), Alessandra D'Abramo 801 (INMI), Antonella Petrecchia (INMI)

Rita Casetti (INMI), Veronica Bordoni (INMI), Stefania Notari (INMI)

Silvia Meschi (INMI), Daniele Lapa (INMI), Licia Bordi (INMI)

Alessandra Grillo (INMI), Erminia Masone 805 (INMI), Elisa Marchioni (INMI), Dorian Bardhi (INMI), Ottavia Porzio

Francesca Cocca (Ospedale Pediatrico Bambino GesuSilvia Murachelli (INMI)