A Phase 1, Dosage-Escalation Study to Assess the Safety and Immunogenicity of a COVID-19 Vaccine GRAd-COV2 in Healthy Adults and Elderly Subjects
Phase: PHASE1
Conditions: COVID-19
Sponsor: ReiThera Srl

 
 
 
 
 
 
 
 
 
ISTITUTO NAZIONALE PER LE MALATTIE INFETTIVE 
“Lazzaro Spallanzani” 
Istituto di Ricovero e Cura a Carattere Scientifico 
 
 
 
 
Full Title 
A Phase 1, Dose-Escalation Study to assess the Safety and 
Immunogenicity of a COVID-19 Vaccine GRAd-COV2 in 
Healthy Adults and Elderly Subjects 
Short Title 
GRAd-COV2 vaccine against COVID-19 
Trial ID 
RT-CoV-2 
Registration number 
2020-002835-31 
Primary study 
Vaccine(s) 
GRAd-COV2 
Version 
1.0 final 
Date 
30 July 2020 
Sponsor(s) 
ReiThera Srl 
Manufacturer 
ReiThera Srl 
Principal Investigator 
Simone Lanini 
Conducted by 
Istituto Nazionale Malattie Infettive, Lazzaro Spallanzani 
Main Co-Investigators 
Stefano Milleri, Emanuele Nicastri, Andrea Antinori 
Version log: 
Log of specific amendments by version 
Confidentiality 
statement 
Statement outlining the distribution of the document 
 
 
 

 
 
 
 
 
 
 
 
 
ISTITUTO NAZIONALE PER LE MALATTIE INFETTIVE 
“Lazzaro Spallanzani” 
Istituto di Ricovero e Cura a Carattere Scientifico 
 
SIGNATURE PAGE 
The signature below provides the necessary assurance that this trial will be conducted according to all 
stipulations of this protocol, including all statements regarding confidentiality, and according to local legal 
and regulatory requirements and applicable ICH E6 GCP guidelines. 
I agree to conduct this trial in compliance with GCP and applicable regulatory requirements. 
I agree to conduct this trial in accordance with the current protocol and will not make changes to this protocol 
without obtaining the sponsor’s approval and National Ethical committee for COVID approval, except when 
necessary to protect the safety, rights, or welfare of subjects. 
Principal Investigator Signature:
 
Signed:
Simone Lanini
Date:
30 July 2020
  
 
 
 

1 
 
 
Summary 
1. 
Synopsis ..................................................................................................................................................... 4 
2. 
Abbreviation .............................................................................................................................................. 5 
3. 
Background information and rationale ..................................................................................................... 6 
3.1. 
Coronavirus disease 2019 current situation ...................................................................................... 6 
3.2. 
The emergent SARS-CoV-2 virus and the choice of S-protein protein as vaccine antigen ................ 7 
3.3. 
Progress toward vaccination ............................................................................................................. 8 
3.4. 
TheGRAd-COV2 vaccine ..................................................................................................................... 9 
3.4.1. 
The simian adenoviral vector vaccine platform ........................................................................ 9 
3.4.2. 
The GRAd-COV2 vaccine development ................................................................................... 10 
3.5. 
Pre-clinical studies on GRAd-COV2 .................................................................................................. 11 
3.5.1. 
GRAd32 neutralizing antibodies (nAb) .................................................................................... 11 
3.5.2. 
GRAd-COV2 Immunogenicity in mice ...................................................................................... 12 
3.5.3. 
GRAd-COV2 efficacy and safety in Rhesus macaques ............................................................. 14 
4. 
Objective .................................................................................................................................................. 15 
4.1. 
General objective............................................................................................................................. 15 
4.2. 
Endpoints ......................................................................................................................................... 15 
4.2.1. 
Primary endpoints (safety) ...................................................................................................... 15 
4.2.2. 
Secondary endpoints (immunogenicity).................................................................................. 16 
4.2.3. 
Exploratory endpoints ............................................................................................................. 16 
4.2.4. 
Milestones ............................................................................................................................... 16 
4.2.5. 
Deliverables ............................................................................................................................. 17 
5. 
Study design ............................................................................................................................................ 20 
6. 
Recruitment and withdrawal ................................................................................................................... 22 
6.1. 
Recruitment strategy ....................................................................................................................... 22 
6.2. 
Adults-Cohort .................................................................................................................................. 22 
6.3. 
Elderlies-Cohort ............................................................................................................................... 22 
6.4. 
Inclusion criteria .............................................................................................................................. 22 
6.5. 
Exclusion Criteria ............................................................................................................................. 22 
6.6. 
Lost to follow-up and withdrawal ................................................................................................... 23 
6.7. 
Compliance with Dosing Regime ..................................................................................................... 24 
6.8. 
Pregnancy ........................................................................................................................................ 24 
7. 
Clinical procedures .................................................................................................................................. 24 
7.1. 
Vaccination procedure .................................................................................................................... 24 

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7.2. 
Medical visit ..................................................................................................................................... 24 
7.3. 
Blood test and other analysis for monitoring safety ....................................................................... 24 
7.4. 
Immunogenicity analysis ................................................................................................................. 27 
7.5. 
Screening ......................................................................................................................................... 27 
7.6. 
Enrolment visit (Time 0) .................................................................................................................. 27 
7.7. 
Vaccination sequence ...................................................................................................................... 27 
7.8. 
Visit (D2, W1, W2, W4, W8, W12 and W24) ................................................................................... 29 
7.9. 
Participants restrictions ................................................................................................................... 29 
8. 
Investigational vaccine production.......................................................................................................... 30 
8.1. 
Manufacturing and Presentation .................................................................................................... 30 
8.2. 
Supply and Storage .......................................................................................................................... 30 
8.3. 
Administration of Investigational Medicinal Products .................................................................... 30 
9. 
Adverse event assessment and management ......................................................................................... 30 
9.1. 
Definitions ....................................................................................................................................... 31 
9.2. 
Solicited Adverse Reactions ............................................................................................................. 31 
9.3. 
Assessment of causality ................................................................................................................... 32 
9.4. 
Guidance for assessing relationship of AE with vaccination ........................................................... 32 
9.5. 
Reporting procedure for all AE ........................................................................................................ 32 
9.6. 
Assessing severity of adverse events .............................................................................................. 33 
9.7. 
Management of participants with AE .............................................................................................. 33 
9.8. 
Halting rules and study termination due to safety issue ................................................................. 33 
10. 
Statistical plan ..................................................................................................................................... 34 
10.1. 
Sample size .................................................................................................................................. 34 
10.2. 
Statistics analysis ......................................................................................................................... 34 
11. 
Data Management ............................................................................................................................... 35 
11.1. 
Quality Control and Source Data Verification ............................................................................. 35 
11.2. 
Audit / Inspection ........................................................................................................................ 36 
11.3. 
Monitoring and Access to Source Data ....................................................................................... 36 
11.4. 
Data collection ............................................................................................................................. 36 
11.4.1. 
Data Management ................................................................................................................... 36 
11.4.2. 
Case Report Form(eCRF) .......................................................................................................... 36 
11.4.3. 
Database Release..................................................................................................................... 37 
11.5. 
Source Documents ....................................................................................................................... 37 
11.6. 
Retention of Study Documents at the Site .................................................................................. 37 
12. 
Clinical centers ..................................................................................................................................... 37 
13. 
Monitoring Committees ...................................................................................................................... 38 

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13.1. 
Data safety and monitoring board (DSMB) ................................................................................. 38 
13.2. 
Ethical committee ........................................................................................................................ 38 
14. 
Laboratory methods for immunogenicity assessment ........................................................................ 38 
14.1. 
Serology ....................................................................................................................................... 38 
14.2. 
T cell Immunology ....................................................................................................................... 39 
15. 
Ethical issues ........................................................................................................................................ 39 
15.1. 
Potential Risks for volunteers ...................................................................................................... 39 
15.1.1. 
Phlebotomy ............................................................................................................................. 40 
15.1.2. 
Vaccination: ............................................................................................................................. 40 
15.1.3. 
Vaccine induced Disease Enhancement .................................................................................. 40 
15.2. 
Known Potential Benefits ............................................................................................................ 40 
15.3. 
Declaration of Helsinki ................................................................................................................. 41 
15.4. 
Guidelines for Good Clinical Practice .......................................................................................... 41 
15.5. 
Approvals ..................................................................................................................................... 41 
15.6. 
Informed consent and participant confidentiality ...................................................................... 41 
16. 
Financing and Insurance ...................................................................................................................... 41 
16.1. 
Financing ...................................................................................................................................... 41 
16.2. 
Insurance ..................................................................................................................................... 42 
16.3. 
Participants compensation .......................................................................................................... 42 
17. 
Expected results and publication policy .............................................................................................. 42 
18. 
Reference ............................................................................................................................................. 43 
 
 
 
 

4 
 
1. Synopsis 
Trial Title 
A Phase 1, Dose-Escalation Study to assess the Safety and Immunogenicity of 
a COVID-19 Vaccine GRAd-COV2 in Healthy Adults and Elderly Subjects 
Internal ref. no.  
RT-CoV-2 
Clinical Phase  
Phase I 
Trial Centre 
Istituto per Malattie Infettive (INMI) L. Spallanzani, Via Portuense, 292, Rome; 
Coordinator Clinical Site 
Centro Ricerche Cliniche Verona srl c/o Policlinico G.B. Rossi P.le Scuro, 10 
37134 Verona 
Trial Design 
Open label study 
Trial Participants 
(i) Healthy adult volunteers (aged 18-55) including men and non-pregnant 
women  
(ii) Elderly volunteers (aged 65-85) without significant clinical conditions 
Planned Sample Size 
Arm 1: 15 Healthy younger adults volunteers (aged 18-55); 1 dose GRAd-COV2 
(5 x 1010 vp)  
Arm 2: 15 Healthy younger adults volunteers (aged 18-55); 1 dose GRAd-COV2 
(1 x 1011 vp)  
Arm 3: 15 Healthy younger adults volunteers (aged 18-55); 1 dose GRAd-COV2 
(2 x 1011 vp)  
Arm 4: 15 Healthy older adults volunteers (aged 65-85); 1 dose GRAd-COV2  
(5 x 1010 vp)  
Arm 5: 15 Healthy older adults volunteers (aged 65-85); 1 dose GRAd-COV2  
(1 x 1011 vp)  
Arm 6: 15 Healthy older adults volunteers (aged 65-85); 1 dose GRAd-COV2 (2 
x 1011 vp)  
Total = 90 volunteers 
Study duration 
12 months 
Follow up duration 
Patients will be followed up for 24 weeks after vaccination. Follow-up will 
include medical visit and test at day 2, day 7, week 2, week 4 week 8, week 12 
and week 24 
Planned Trial Period 
10 months (from screening of first participant to 6 months after final 
vaccination of last volunteer) 
 
Objectives 
Endpoints 
Primary 
 
To evaluate the safety of 
administering GRAd-COV2, 
intramuscularly in healthy 
(younger and older adults) 
volunteers  
A. Occurrence of solicited local AE signs 
and symptoms for 7 days following the 
vaccination 
B. Occurrence of solicited systemic AE 
signs and symptoms for 7 days following 
the vaccination 
C. Occurrence of unsolicited AE for 28 
days following the vaccination 
D. Change from baseline for safety 
laboratory measures at different time 
point 
E. Occurrence of severe AE during the 
whole study duration 

5 
 
Secondary 
 
To assess the cellular and 
humoral immune response to 
SARS-CoV2 elicited by the vaccine 
A. CLIA to quantify antibodies to SARS-
CoV-2 S-protein (anti-S-Ab) 
B. CLIA to quantify antibodies to SARS-
CoV-2 nucleocapsid protein (anti-N-Ab) 
C.SARS-Cov-2 micro-neutralization assay 
to quantify neutralizing antibody activity 
to the virus 
D. Ex vivo IFN-gamma ELISpot responses 
to SARS-CoV-2 S-protein 
Investigational 
Medicinal Product 
Replication-defective gorilla adenovirus (GRAd) encoding prefusion-stabilized 
SARS-CoV-2 S-protein protein, GRAd-COV2 
Formulation, Dose, 
Route of Administration 
Liquid, intramuscular needle injection 
 
2. Abbreviation 
Ad 
Adenovirus 
AE 
Adverse event 
AR  
Adverse reaction 
ChAd 
Chimpanzee Adenovirus 
CI 
Chief Investigator 
CLIA 
Chemiluminescent immunoassay 
CMV 
Cytomegalovirus 
COVID-19 
Coronavirus disease which is caused by SARS-CoV-2 
CRF 
Case Report Form 
CT 
Clinical Trials 
CTA 
Clinical Trials Authorisation 
DNA 
Deoxyribonucleic acid 
DSMB 
Data Safety Monitoring Board 
DSUR 
Development Safety Update Report 
EC 
Ethics Committee 
ECG 
Electrocardiogram 
ELISpot 
Enzyme-linked immunosorbent spot 
GCP 
Good Clinical Practice 
GMO 
Genetically Modified Organism 
GP 
General Practitioner (Medico di Medicina generale) 
GRAd 
Gorilla Adenovirus 
HLA 
Human Leukocyte Antigen 
IB 
Investigators Brochure 

6 
 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IMP 
Investigational Medicinal Product 
IFN-γ 
Interferon Gamma 
N-protein 
Nucleocapside protein 
PBMC 
Peripheral Blood Mononuclear Cell 
PIL 
Participant Information Leaflet 
RNA  
Ribonucleic acid 
S-protein 
Spike protein  
SAE 
Serious Adverse Event 
SAR 
Serious Adverse Reaction 
SARS-CoV-2 
Severe acute respiratory syndrome coronavirus 2 
SFC 
Spot-Forming Cells 
SOP 
Standard Operating Procedure 
SUSAR 
Suspected Unexpected Serious Adverse Reactions 
TMF 
Trial Master File 
vp 
Viral Particles 
WHO 
World Health Organisation 
 
3. Background information and rationale 
3.1. Coronavirus disease 2019 current situation 
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is associated with a new diverse clinical 
syndrome called Coronavirus disease 2019 (COVID-19). Common symptoms of COVID-19 include fever, 
cough, shortness of breath and dyspnea that may eventually progress towards acute respiratory distress 
syndrome and death. About 80% of patients have mild to moderate disease, 14% have severe disease and 
6% are critical (namely, they develop respiratory failure, septic shock, and/or multiple organ 
dysfunction/failure). The severity of clinical presentation is directly dependent on patients’ age. Infections 
needing hospitalization are exceptional events among children younger than 10 years while the case-fatality 
rate can exceed 10% among people older than 70 years. [1,2] 
The COVID-19 pandemic originated in Wuhan, China, in early December 2019. The World Health Organization 
declared the outbreak a Public Health Emergency of International Concern on 30 January by  on 11 March it 
has been recognized as the cause of the biggest pandemic event over the last century. [2,3] 
The first cases reported in Italy were two Chinese tourists who were identified in Rome on January 29st.[4] 
Due to the prompt intervention of Regional Infection Control network, neither of these cases did actually 
produce independent cluster. However, the epidemic sprung in the North of the country since the beginning 
of February when eleven municipalities were locked down. As for other countries, the Italian government 
implemented a series of strict collective measures for the control of local transmission. On March 11th, the 
government enforced a law to stop nearly all commercial activities except for supermarkets and pharmacies. 

7 
 
[5] On March 21st, the quarantine was strengthened, all non-essential businesses were stopped and people’s 
movement severely restricted. [6] These collective measures adopted in the first phase of the epidemic, have 
contributed decisively to the flattening of the epidemic curve with a reduction in new cases and a consequent 
lightening of the health care burden. Although these measures made it possible to overcome the emergency 
phase, in another respect they had deleterious impact on economics and social life, which can be 
unsustainable in the medium-long term. Therefore, Italy, like many countries in Europe, has been easeing 
collective measures in favor of a progressive recovery of the economic and social life. [7] However, due to 
the still low level of immunity of the population, a rapid resumption of sustained transmission in the 
community may occur. [8] There are three main strategies for controlling the spreading of a new emergent 
pathogens during a massive event such as the current COVID-19 pandemic. 
Firstly, implementing and strengthening of a robust surveillance system that it is capable of promptly 
identifying and of properly managing incident cases and their contacts in order to intercept local clusters and 
curb the subsequent epidemic waves. [9] 
Secondly, endorsing research on new drugs with high direct antiviral activity that can be used either to 
prevent infection in high-risk individuals (pre-exposure prophylaxis), prevent infection in exposed people 
(post-exposure prophylaxis) or care infected people in order to curb their capability to transmit the infection 
and prevent clinical progression toward severe diseases. [10] 
Finally develop effective vaccine strategies for either preventing infection in general population or, at least, 
preventing severe clinical presentation diseases in frailest subjects. [11] 
Here we propose a program for rapidly scale up a new investigational vaccine based on an innovative simian 
adenoviral vector. The study design has been tailored on currents needs of Italy and it is aimed at rapidly 
demonstrating safety and immunogenicity of  a single vaccine administration at escalating doses either in 
healthy adult or elderly people. The results of the study will be eventually implemented into phase 2/3 clinical 
trial designed to meet the immediate need of those groups of people at the highest risk of severe COVID-19  
3.2. The emergent SARS-CoV-2 virus and the choice of S-protein protein as vaccine 
antigen 
SARS-CoV-2 belongs to the broad family of viruses known as coronaviruses. It is a lipid enveloped virus sizing 
approximately 100–120 nM, and the inner nucleocapsid houses the single-stranded non-segmented positive-
sense RNA genome (ssRNA+). SARS-CoV-2 is the seventh known coronavirus that can be transmitted human-
to-human, after 229E, NL63, OC43, HKU1, MERS-CoV, and the original SARS-CoV. As other SARS-like 
coronavirus, including MERS and SARS, SARS-CoV-2 is a member of the subgenus Sarbecovirus (beta-CoV 
lineage B) and has been associated with severe clinical presentation in human beings. SARS-CoV-2 genome 
is approximately 30,000 bases in length. SARS-CoV-2 is unique among known betacoronaviruses in its 
incorporation of a polybasic cleavage site, a characteristic known to increase pathogenicity and 
transmissibility in other viruses. [12] It is believed that SARS-COV-2 has zoonotic origins due to close genetic 
similarity to bat coronaviruses, suggesting it emerged from a bat-borne virus. There is no evidence yet to 
clarify the animal-human interface, including an animal species or special environmental conditions to link 
the virus natural reservoir and its introduction to human communities. However, the little genetic diversity 
among current SARS-CoV-2 strains suggest that the spillover event might have been occurred in late 2019. 
[13]  
Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (S-protein), E (envelope), 
M (membrane), and N (nucleocapsid) proteins (Figure 1); the N protein holds the RNA genome, and the S, E, 
and M proteins together create the viral envelope. Coronavirus S-protein (S) and nucleocapsid (N) envelope 
proteins are highly immunogenic and constitute important antigenic targets for the development of serologic 
assays. [14] 

8 
 
The S-protein has a pivotal central role in viral infection and pathogenesis. S-protein is, in fact, the central 
element for allowing the virus to attach to and fuse with the membrane of a host cell; specifically, its S1 
subunit catalyzes attachment, the S2 subunit fusion. [15-16] 
 
Figure 1 The SARS-CoV-2  
Recent evidence suggest that anti-S neutralizing antibodies from sera of COVID-19 convalescent patients can 
prevent cell infection in vitro by blocking viral binding to ACE2 and the eventual formation of syncytia.[17-
18]  
SARS-CoV-2 cell-adhesion and cell-entry is a complex mechanism. Functional S-protein has a dimeric 
structure including 2 subunits (S1 and S2). Cleavage of S-protein into the functional dimer is mediated by 
attachment of the S1 RBD to the ACE2 enzyme which exposes a cleavage site on S2 that is acted on by host 
cell proteases to initiate the process of cellular entry. Although SARS-CoV also uses the ACE2 receptor, SARS-
CoV2 has a distinct furin cleavage site, not found in SARS-CoV, which may explain some of the biological 
differences. In fact, removal of this motif affects cellular entry: the furin cleavage site expands the versatility 
of SARS-CoV-2 for cleavage by cellular proteases and potentially the tropism and transmissibility owing to 
the wide cellular expression of furin proteases especially in the respiratory tract. [19-20] 
The critical role of the S-protein, both in infection and pathogenesis process suggest that it is an attractive 
target for both for new drugs and vaccinations. 
3.3. Progress toward vaccination 
As of June 2020, WHO reports 148 vaccines under development for SARS-CoV-2. Of those 148 candidates, 17 
has already entered clinical testing while the remaining 131 are at different stage of preclinical testing. 
Around 10% of the candidate listed are based on “classical” vaccine approaches, such as either inactivated 
or live attenuated SARS-CoV-2 virus. Subunit protein in adjuvants or VLPs are well represented in the list, 
representing around 45% of candidates. The remaining 45% of candidate landscape is made of genetic 
vaccine platforms, with around one half based on either DNA or formulated RNA, and the remaining half 
using either replication deficient or replication competent viral vectors to deliver SARS-CoV-2 antigens. [21] 
Amongst the most advanced vaccine candidates already in clinical trials, four are based on inactivated virus, 
three are a subunit vaccine, the other ten are genetic vaccines: two based on DNA, five on RNA and three on 
non-replicating adenoviral vectors. 

9 
 
The majority of the candidate vaccines are based solely on the SARS-CoV-2 S-protein surface glycoprotein or 
the corresponding gene, either in full length form or limited to the S1 subunit or to the receptor binding 
domain (RBD), aiming at focusing the immune response on the critical epitopes to induce neutralizing 
antibodies. Additional variations include the introduction of two prolines in the S2 region, to stabilize the 
protein in the prefusion conformation [22], or the expression of the S-protein either as a soluble protein by 
deleting C terminus and transmembrane domain. 
Genetic platforms represent an innovative approach for implementing effective vaccine for emerging viral 
infections. A genetic vaccine platform based on VSV viral vector has been successfully used for developing a 
vaccine against Ebola virus. [23] Genetic vaccine platforms have several interesting advantage over 
conventional vaccines based either in whole pathogens (inactivated or live attenuated virus) or components 
(viral proteins).  
Whole pathogens vaccine are potently immunogenic, as witnessed by the fact that most of the licensed 
vaccines are based on this approach. However, they pose manufacturing issues mainly related to biosafety 
and containment. Moreover, if live-attenuated pathogens are used there is a risks of reversion to 
pathogenicity. The issue of reversion is particularly relevant when frail subjects are vaccinated or novel 
viruses whose biology is little known. Moreover, genetic vaccines can mimic natural interaction between the 
host and the pathogen much better than either inactivated or component vaccine. Indeed, in genetic vaccine 
the immunogenic antigens are produced intracellularly by host cell machinery. This results in Th1 polarized 
phenotype response. This Th1 skewed response pattern is highly desirable in vaccination against viral 
infection, such as SARS-CoV-2, as it is associated with clinical recovery and prevent potential concerns about 
vaccine-induced enhanced disease phenomena that are generally associated with Th2 response. [24-25] 
In contrast genetic vaccine platforms are endowed with intrinsic safety, since they only encode for one or 
few antigens of the target pathogen and thus a natural infection cannot be generated by the vaccine itself. 
Moreover, genetic vaccine platforms are very attractive in term of programming public health preparedness 
and response. The development of a new genetic vaccine vector is framework of response against new 
epidemics rather than the simple vaccine strategy for a unique pathogen in a special setting. In fact, the same 
genetic vector can be re-designed to carry diverse antigen sequences and thus for targeting multiple 
pathogens. This approach is pivotal in term of public health decision as the implementation of several safe 
vaccine platforms may speed up the production of different vaccine tailored on the specific needs of different 
epidemic settings.  
Adenoviral vectors are currently among the most attractive genetic vectors used for the development of 
vaccines.  
3.4. TheGRAd-COV2 vaccine 
3.4.1. The simian adenoviral vector vaccine platform 
Replication-defective adenoviruses are among the most potent vectors for induction of antibody and T cell 
responses to encoded antigens. Since their discovery, adenoviruses (Ads) showed features that made them 
an attractive platform for vaccine design: ease of genetic manipulation, broad tissue tropism, robust 
immunogenicity, and favorable safety profile. [26] In addition, they can be easily propagated in cell culture 
and are relatively inexpensive to manufacture.  
Ads vectors can infect many different cell types, including quiescent, long-lived cells and professional antigen 
presenting cells. They are not cytolitic, and their genome do not integrate in the host cell genome but remains 
episomal, conferring a safer profile compared to other viral vectors. [27] Ads genomes have been shown to 
persist at low levels in the target tissues. For example, upon intramuscular injection Ads genomes can be 
detected for months in muscle and draining lymph nodes. [28-29] These genomes have been shown to 

10 
 
maintain a transcriptionally active form for an extended period of time [30-31-32], thus constantly re-
exposing the immune system to the encoded vaccine antigen. Indeed, adenoviral vectored-vaccines have 
shown to induce long-lasting cellular immune responses with a large representation of T effector memory 
cells in the antigen-specific T cell pool, a subset that can offer immediate protection to invading pathogens. 
[33-34] 
Adenoviral vectors can be considered self-adjuvanting vaccines. Indeed, upon infection, the viral proteins 
and nucleic acids act as pathogen associated molecular patterns (PAMPs) to potently trigger many extra and 
intracellular innate immunity sensors in the host cell, such as toll-like receptors (TLR) and other pattern 
recognition receptors (PRRs). The resulting innate immune response in turn can potentiate the adaptive 
responses to the vaccine-encoded antigens, without any need for chemical/synthetic adjuvants. [35-36] A 
major drawback of Ads vectored vaccine is that their efficacy can be significantly impaired by pre-existing 
humoral anti-vector immunity, [37] with 60% and 90% anti-Ad5 seroprevalence rates in Europe and sub-
Saharan Africa, respectively [38]. The high prevalence of immunity against Ads in human population has 
endorsed the use of other related viruses, such as simian adenoviruses (SAds) that have the double advantage 
of having very low level of sero-prevalnce among human population and being associated with no human 
diseases. [39-40]Starting from 2007 and as of 2020, six chimpanzee-derived (three species C-ChAd3, PanAd3, 
ChAd155; three species E-ChAd63, ChAdOx1 and ChAdOx2) and one gorilla-derived (species C GAd20-same 
family of GRAd23) adenoviral vectors have been evaluated in many different clinical investigations as 
prophylactic and therapeutic vaccine candidates against infectious diseases or cancer. [41] Over the past 13 
years, candidate vaccines based on these seven different simian vectors have been safely administered to 
thousands of volunteers in clinical trials up to phase 2b. The safety profile described in many publications 
was invariantly favorable not only in healthy adults of various geographical origin (Europe, Africa, US), but 
also in more vulnerable populations like elderly, children and infants, and population with concomitant 
healthy conditions (intravenous drug users, HCV and HIV infected patients. Of particular relevance are the 
results of Phase I and II trials with the chimpanzee-derived adenoviral vector ChAd3 encoding Ebola virus 
glycoprotein (EBOV GP) which showed that a single dose of the vaccine induced neutralizing antibody titers 
comparable to those induced by the protective VSV Ebola vaccine, besides a better safety profile in humans. 
[42-43] 
3.4.2. The GRAd-COV2 vaccine development 
The GRAd-COV2 vaccine developer, ReiThera Srl (former Okairos Srl), has pioneered the development, 
manufacture and clinical testing of ChAd-vectored vaccines against several infectious diseases, now owned 
by GlaxoSmithKline (GSK).  
Independently from GSK, ReiThera has recently developed a novel proprietary, species C SAd vector, GRAd32, 
from a strain isolated from gorilla stools. Gorilla adenovirus vaccine vectors have been described to be 
potently immunogenic and shown to be unfrequently recognized by neutralizing antibodies in the human 
population (20). Recently, vaccine candidates based on gorilla adenovectors have been successfully assessed 
in animal models to protect against malaria and Zika. [44]  
The GRAd32 vector has been rendered replication deficient by deleting the genomic region E1, and 
production is achieved by trans complementation of the E1 gene products in Hek293 cells. To further increase 
the cloning capacity, the E3 region has been deleted from the GRAd32 genome backbone, a region also 
known to play a role in counteracting host innate immunity. To improve productivity, the E4 region has also 
been deleted and the Ad5ORF6 has been inserted to provide optimal interaction with the complementing 
Ad5 E1 region. A technological improvement in GRAd vector productivity and genetic stability was obtained 
by silencing transgene expression during production, thanks to constitutive expression of a tetracycline 
repressor by the ReiThera proprietary production cell line ReiCell35S.  

11 
 
The GRAd-COV2 vaccine vector contains the full length S-protein (Wuhan NCBI No QHD43416) cloned into 
the E1 region. The protein contains two mutations K986P, V987P which are described to stabilize the trimer 
in the pre-fusion conformation. [45] The rationale for the choice of a pre-fusion stabilized S-protein as a 
vaccine antigen is due to the fact that the wild type trimer has a metastable conformation, and the binding 
of the RBD to hACE2 receptor likely leads to the shedding of S1 protein from S2 protein, thus promoting S2-
mediated virus-host membrane fusion and virus entry. Given the critical role of the RBD in initiating invasion 
of SARS-CoV-2 into host cells, it is obviously a vulnerable target for neutralizing antibodies. Stabilization of 
the protein in the pre-fusion conformation should avoid the dynamic of conformational states of the S-
protein and provide a long window for the immunogenic epitopes of RBD exposure to specific B cells. 
The C-terminus of the S-protein is fused to a peptide tag (HA tag) derived from the human influenza 
hemagglutinin protein, which is composed of a 9-amino acid sequence, YPYDVPDYA, that is recognized with 
high affinity by commercially available antibodies. 
The HA tag is a commonly used epitope which does not seem to affect the activity or distribution of the 
expressed protein, and is useful when protein-specific antibodies are not available, as it was the case when 
the GRAd-COV2 vector was constructed. 
3.5. Pre-clinical studies on GRAd-COV2 
3.5.1. GRAd32 neutralizing antibodies (nAb)  
The presence and levels of antibodies in human sera that are able to cross-neutralize the novel gorilla derived 
GRAd32 vector compared to neutralization profile of the common human Ad5 was assessed in a panel of 40 
human sera, using a standard neutralization assay based on infection of HEK293 cells with Ad5 and GRAd32 
vectors encoding secreted alkaline phosphatase reporter gene. [46]  
Figure 2 shows that while neutralizing antibodies to Ad5 are frequently detected at high titers, cross-
neutralizing nAb titers to GRAd32 are found and at significantly lower titers (geometric mean 443 vs 48). In 
previous clinical trials of an Ad5-based candidate vaccine for HIV, nAb titers above 200 were reported to 
negatively impact on immune response to vaccination. [47] Indeed, 67,5% of subjects in the present cohort 
had titers above 200 for Ad5, while only 15% of sera had cross-neutralizing activity on GRAd32. 
Ad5
GRAd32
10
100
1,000
10,000
nAb titer
18
200
✱✱✱✱
 
Figure 2 Seroprevalence of Ad5 and GRAd32 nAb in a cohort of 40 human sera. Data are expressed as the reciprocal of serum dilution 
able to neutralize inhibit SEAP expression by 50% compared to the SEAP expression of virus infection alone. 
This feature is likely to be relevant for the potency in humans of the GRAd-COV2 vaccine, since a substantial 
negative impact on SARS-CoV2 humoral and cellular response to vaccination in volunteers with baseline nAb 

12 
 
titers >200 was reported recently by CanSino in the phase I trial of their Ad5 based COVID-19 vaccine.[48] 
Specifically, seroconversion rate and titers of SARS-CoV-2 neutralizing antibodies and peak T cell response 
magnitude post vaccination were both clearly affected by Ad5 pre-existing immunity. 
3.5.2. GRAd-COV2 Immunogenicity in mice 
Immunogenicity and potency studies were conducted in 6 to 8-weeks old female BALB/c mice.  
Groups of 8 animals were vaccinated intramuscularly (IM) with a single administration of GRAd-COV2 at the 
dose of 109 or 108 viral particles (vp). Serum samples were taken 14 and 35 days post vaccination, and 
endpoint titers oftotal IgG or IgG2a/IgG1 ratio as a measure of Th1/Th2 polarization, were measured by ELISA 
using as a coated antigen the full length recombinant baculovirus expressed SARS-CoV-2 S-protein (Sino 
Biological). Neutralization titers in sera at week 5 post immunization were assessed by means of a plaque 
reduction assay on VERO E6 cells with SARS-CoV-2 virus. 
The antigen specific T cell response in mouse splenocytes 5 weeks post immunization was characterized in 
depth by means of different assays. Magnitude and specificity of cellular responses were measured by IFNγ 
ELISpot, using as antigenic stimulus 2 pools of overlapping 15mer peptides encompassing the SARS-Cov-2 S-
protein sequence: pool S1 and S2 (158 and 157 peptides each respectively, JPT PepMix). To explore the 
Th1/Th2 balance of the vaccine-induced response, IL4 secretion by antigen-stimulated splenocytes was also 
assessed by ELISpot. Finally, to characterize the antigen specific T cell subsets (CD8/CD4) and additional Th 
polarization profile c, splenocytes were subjected to IFNγ, IL2, IL4+IL13 and IL17 intracellular staining (ICS) 
upon antigen restimulation and FACS analysis. 
108 vp
109vp
COVID-19 pts
0
50
100
150
200
250
300
350
600
700
SARS-CoV-2 neutralizing titer
 (IC50)
108 vp
109vp
2
3
4
5
Spike endpoint IgG titer
(log10)
w2
w5
109 vp w5
0.0
0.5
1.0
1.5
2.0
IgG2a/IgG1 ratio
A
B
C
 
Figure 3: humoral response induced in BALB/c mice at two different time points after vaccination (2 and 5 weeks) with 108 and 109 vp 
of GRAd-COV2, and comparison with neutralizing response in convalescent COVID-19 patients. A) S-protein binding total IgG titrated 
on individual animals by ELISA on a recombinant S-protein. Data are expressed as endpoint titer. B) IgG1 and IgG2a titers measured 
in week 5 sera by ELISA developed in house with appropriate secondary antibodies. Data are shown as the ratio between IgG2a and 
IgG1. C) SARS-CoV-2 neutralizing antibodies in week 5 mouse sera and in a panel of 28 sera from convalescent COVID-19 patients 
detected by SARS-CoV-2 (2019-nCoV/Italy-INMI) microneutralization assay on VERO E6 cells. Data are expressed as IC50, or the 
reciprocal of serum dilution achieving 50% neutralization. Horizontal lines in all graphs represent geometric mean. 
A single GRAd-COV2 administration at both the tested doses rapidly induced S-protein binding antibodies in 
all mice 2 weeks post immunization and IgG titers increased further 5 weeks post immunization. The S-
protein specific antibody response was composed of both IgG1 and IgG2a, with IgG2a/IgG1 ratio>1 indicative 
of Th1 polarization. Importantly, GRAd-COV2 vaccination induces SARS-CoV-2 neutralizing antibodies (Nabs) 
in a dose-dependent fashion, which were titrated by means of a microneutralization assays. NAbIC50 titers 
in the serum of GRAd-COV immunized mice were in the range of those observed in a panel of sera from 
convalescent COVID-19 patients. 

13 
 
DMSO
pool S1
pool S2
DMSO
pool S1
pool S2
0
200
400
600
800
1,000
1,200
1,400
1,600
IFNγ- IL4 SFC/106 splenocytes
IL4
IFNγ
108 vp
S1
S2
S1
S2
0.01
0.1
1
10
% of CD4 or CD8 T cells
CD4-IFNγ
CD4-IL2
CD8-IFNγ
CD8-IL2
108 vp
109 vp
Spike
S1
S2
0
500
1,000
1,500
2,000
2,500
IFNγ SFC/106 splenocytes
109 vp
108 vp
A
B
C
IFNγ
IL4
0
500
1,000
1,500
2,000
2,500
IFNγ-IL4 SFC/10^6 splenocytes
108 vp
D
 
Figure 4 T cell response induced in BALB/c mice five weeks after vaccination with 108 and 109 vp of GRAd-COV2. A) IFNγ ELISpot on 
splenocytes. Data are expressed as IFN-γ Spot Forming Cells (SFC)/106 splenocytes.  Individual data points represent total S-protein 
response in each animal, obtained by summing reactivity to each of the 2 S-protein peptide pools and subtracting 2 times the DMSO 
background. Responses on S1 and S2 pools are also shown. IFNγ and IL4 ELISpot on splenocytes from mice immunized at 108 vp dose. 
Data are shown as B) response to individual S1 and S2 pools stimulation compared to mock stimulation (DMSO) or C) as the total 
response to S-protein. D) IFNγ/IL2 intracellular staining and FACS analysis on splenocytes. Data are expressed as the percentage of 
either CD8 or CD4 secreting IFNγ or IL2 in response to S-protein peptide pools stimulation, subtracted of mock stimulation (DMSO) 
background. Horizontal lines in all graphs represent geometric mean 
At both the tested doses, potent T cell responses were detected in spleens of immunized mice five weeks 
after a single GRAd-COV2 administration (Figure 4A), a hallmark of adenoviral vectors. As also indicated by 
the IgG2a/IgG1 ratio, the immune response was skewed towards Th1 , as shown by a massive IFNγ secretion 
in response to S-protein restimulation, compared to a low level of IL4 secretion, mostly S2 targeted (Figure 
4B-4C). In this mouse strain, an immunodominant CD8 epitope is located in the first S1 S-protein subunit and 
is responsible for the majority of IFNγ secretion reaching as high as 7.5% and 5% of all CD8, respectively for 
109 and 108 dose (Figure 4D). However, additional CD8 responses in S2, as well as a clear Th1 CD4 responses 
in both S1 and S2, were induced at both vaccine dose tested.  
IL2 secretion was also easily detected in response to S-protein stimulation in both CD8 and CD4 subsets. A 
small proportion (around 10-15%) of S-protein-specific IFNγ-secreting CD8 co-secreted IL2, representing 
most probably central memory cells precursors, with no antigen specific CD8 producing IL2 only. S-protein-
specific CD4 were more heterogeneous, with either IFNγ or IL2 single-producers represented and around 60-
70% of S-protein specific CD4 co-producing both cytokines (not shown). S-protein-specific Th2 and Th17 CD4 
T cells were not induced, or below the limit of detection of our ICS method. 
On the ground of previous studies with simian group C vectors encoding different transgenes, two low 
vaccine doses (107 and 106vp) were chosen to establish the so-called immunological breakpoint (Colloca et al 
2012), defined as the minimal dose capable of inducing T cell responses to the encoded transgene in 40% of 
vaccinated animals. T cell response in splenocytes and in lung infiltrating lymphocytes (at 107 vp dose only) 
were measured by IFNγ, IL4 and Il17 ELISpot 3 weeks post vaccination. 

14 
 
Spike
S1
S2
0
500
1,000
1,500
2,000
2,500
3,000
IFNγ SFC/106 splenocytes
Spleen 107 vp
Spleen 106 vp
Lung 107 vp
IFNγ
IL4
IL17
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
2,000
IFNγ-IL4-IL17 SFC/10^6 splenocytes
A
B
C
Spleen CD4
Spleen CD8
0.01
0.1
1
10
% IFNγ+ CD4 or CD8
 
Figure 5. T cell response induced in BALB/c mice three weeks after vaccination with 107 and 106 vp of GRAd-COV2. A) IFNγ ELISpot on 
splenocytes and lung infiltrating lymphocytes. Data are expressed as IFN-γ Spot Forming Cells (SFC)/106 splenocytes.  Individual data 
points represent total S-protein response in each animal, obtained by summing reactivity to each of the 2 S-protein peptide pools and 
subtracting 2 times the DMSO background. Responses on S1 and S2 pools are also shown. B) IFNγ, IL4 and IL17 ELISpot on splenocytes 
from mice immunized at 107 vp dose. Data are shown as the total response to S-protein. Horizontal lines in A and B graphs represent 
geometric mean.C) IFNγ intracellular staining and FACS analysis on splenocytes. Median+IQR is shown. Data are expressed as the 
percentage of either CD8 or CD4 secreting IFNγ in response to S-protein peptide pools stimulation.  
Clear dose dependent T cell responses were detected by IFNγ ELISpot in the spleen of all mice at both low 
doses tested (107 and 106), indicating that GRAd32 ranks amongst the most potent group C adenoviral 
vectors.  
Importantly, S-protein-specific T cells were found at high levels in the lungs of intramuscularly vaccinated 
mice three weeks post immunization (Figure 5A).  
SARS-CoV-2 specific T cell response targeted epitopes in both S1 and S2 region of the protein, with same 
recognition hierarchy seen at higher vaccine dose. Overall, the T cell response was skewed towards Th1 also 
at lower vaccine dose, and while low levels of IL4 secretion were clearly detectable, S-protein-specific Th17 
cells were not induced in immunized mice (Figure 5B). As expected, the majority of the IFNγ secreting S-
protein-specific T cells were CD8, but lower levels of CD4 Th1 responses were also clearly detectable (Figure 
5C). 
Taken together the mouse data show that GRAd-COV2 candidate vaccine is highly immunogenic and induces 
S-protein-binding as well as SARS-CoV-2 neutralizing Ab in serum, as well as potent CD8 responses to the 
encoded antigen in spleen and lungs, with CD4 responses mostly indicative of Th1 polarization. 
3.5.3. GRAd-COV2 efficacy and safety in Rhesus macaques 
An efficacy study is being conducted in collaboration with NIH/VRC in Rhesus macaques, a species shown to 
be susceptible to SARS-CoV-2 infection. NHPs are considered among the key animal models to determine 
vaccine protective efficacy and safety, in terms of assessment of vaccine-mediated enhanced disease.(49-
50-51). Indeed, upon intranasal/intratracheal SARS-COV-2 challenge the animals develop clinical symptoms 
and pneumonia, and virus shedding can be detected in nasal/throat swabs and in bronchoalveolar lavage 
(BAL). 
A vaccination and SARS-CoV-2 challenge study in Rhesus macaques is being conducted in collaboration with 
NIH/VRC, to demonstrate vaccine immunogenicity, efficacy and safety. 
The study is composed of 3 groups of 6 Rhesus macaques (macaca mulatta) each, 4-8 years old. Groups 1 and 
2 received a single dose of 1x1011vp or 2.5x1010 vp GRAd-COV2 at day 0; while control gp 3 received one 
1x1011 vp dose of a GRAd32 vector encoding a SARS-CoV-2 irrelevant antigen, as a mock vaccination.  

15 
 
Vaccine dosages correspond to either the intended clinical trial intermediate dose or to a lower dose for 
comparison with previously published data with a SARS-Cov-2 vaccine based on a different simian adenoviral 
vector [34]. 
Seven weeks post vaccination the animals will be challenged intranasally/intratracheally (1ml and 3ml 
volumes respectively) with ~106 PFU of SARS-CoV-2 USA/WA1/2020 strain. 
Immunogenicity of the vaccine will be assessed before and after challenge in serum, PBMC and BAL cells. 
Infected animals will be followed for clinical, radiological (chest X-ray) and virological endpoints such as viral 
load by subgenomic PCR analysis and detection of infectious virus in nasal swabs and BALs, until sacrifice. At 
necropsy, viral load will be assessed in upper and lower respiratory tract samples, and lung pathology will be 
assessed by immunohistochemistry.  
Study results will be available while the phase I trial is ongoing. 
4. Objective  
4.1. General objective 
This is an accelerated vaccine development program to be implemented during a massive global epidemic 
due to a newly emergent viral pathogen, SARS-CoV-2. The aim of the program is to prove first in human safety 
and immunogenicity of the new vaccine GRAd-COV2 considering both adult healthy population and special 
groups at risk such as the elderlies. Our approach includes a phase 1A/1B dose escalation trial with six 
sequential arms in two cohorts. 
Adult-Cohort. Will enroll 45 healthy adults aged 18-55 to produce evidence on vaccine safety and 
immunogenicity at different vaccine doses in the general population (first in human safety). 
Elderly-Cohort. Will enroll 45 people with no major comorbidities aged 65-85 to produce evidence on vaccine 
safety and immunogenicity at different vaccine doses in a special population. 
Our goals are to: 
A. Provide preliminary information of vaccine safety and immunogenicity in healthy adults within 4 
months since trial approval (Adults-Cohort) 
B. Confirm vaccine safety and immunogenicity in a special population (such as older adults) within 6 
months (Elderly Cohort) 
C. Implement an eventual phase 2/3 trial tailored on current Italian epidemic situation at the beginning 
of 2021. 
D. Provide full data about safety and immunogenicity at 6 months after vaccination within one year 
since the approval of the program. 
Main endpoints and expected results for either Adults-Cohort or Elderlies-Cohort are reported in figure 6 and 
figure 7, respectively. 
4.2. Endpoints 
4.2.1. Primary endpoints (safety)  
The primary endpoint of the study is to assess safety in term of occurrence of solicited and unsolicited 
adverse events (AE). Occurrence of AE will be monitored by preplanned medical visits and blood tests (active 
surveillance) and by recording participant’s reported AE (passive surveillance). This includes: 
A. Occurrence of solicited local AE signs and symptoms for 7 days following the vaccination 
B. Occurrence of solicited systemic AE signs and symptoms for 7 days following the vaccination 

16 
 
C. Occurrence of unsolicited AE for 28 days following the vaccination 
D. Change from baseline for safety laboratory measures at different 2 days, 7 days, 2 weeks, 4 week, 8 
weeks, 12 weeks and 24 weeks 
E. Occurrence of serious AE (SAE) during the whole study duration 
Volunteers will undergo clinical follow up for AE for 24 weeks following completion of the vaccination 
regimen. SAEs will be collected throughout the study. 
4.2.2. Secondary endpoints (immunogenicity)  
The secondary endpoint for the study is the assessment of cellular and humoral immune response elicited by 
the vaccine at different time weeks after the administration of three different doses of vaccine, namely 
5x1010, 1x1011 and 2x1011 viral particles, either in adults and elderlies . This include: 
A. CLIA to quantify antibodies to SARS-CoV-2 S-protein (anti-S-Ab) at week 2, 4, 8, 12 and 24 in adults 
who receive either 5x1010, 1x1011 and 2x1011 viral particles; 
B. CLIA to quantify antibodies to SARS-CoV-2 S-protein (anti-S-Ab) at week 2, 4, 8, 12 and 24 in elderlies 
who receive either 5x1010, 1x1011 and 2x1011 viral particles; 
C. CLIA to quantify antibodies to SARS-CoV-2 N-protein(anti-N-Ab) at week 2, 4, 8, 12 and 24 in adults 
who receive either 5x1010, 1x1011 and 2x1011 viral particles; 
D. CLIA to quantify antibodies to SARS-CoV-2 N-protein(anti-N-Ab) at week 2, 4, 8, 12 and 24 in elderlies 
who receive either 5x1010, 1x1011 and 2x1011 viral particles; 
E. SARS-Cov-2 micro-neutralization assay to quantify neutralizing antibody activity to the virus at week 
4 and 24 in adults who receive either 5x1010, 1x1011 and 2x1011 viral particles; 
F. SARS-Cov-2 micro-neutralization assay to quantify neutralizing antibody activity to the virus at week 
4 and 24 in elderlies who receive either 5x1010, 1x1011 and 2x1011 viral particles; 
G. Ex vivo IFNγ ELISpot responses to SARS-CoV-2 S-protein at week 2, 4, 8, 12 and 24 in adults who 
receive either 5x1010, 1x1011 and 2x1011 viral particles 
H. Ex vivo IFNγ ELISpot responses to SARS-CoV-2 S-protein at week 2, 4, 8, 12 and 24 in elderlies who 
receive either 5x1010, 1x1011 and 2x1011 viral particles 
4.2.3. Exploratory endpoints 
The exploratory objectives of this study are to provide preliminary evidence on kinetics of immune response 
for 3 doses (namely 5x1010, 1x1011 and 2x1011 viral particles) of GRAd-COV2 either in healthy adults or in 
elderlies. Kinetics will be described as the variation of humoral and cellular immune response at time of 
enrollment and eventually at week 2, 4, 8, 12 and 24 after vaccination in each arm.  Several T cells assay will 
be performed to identify T cell subset mainly involved in the response to vaccination and to evaluate their 
polyfunctionality and phenotypic profile. Level of Neutralizing antibodies (nAb) to GRAd32 will be measured 
in serum at time of enrollment, 4 and 24 weeks after vaccination to evaluate any potential impact on 
vaccination efficiency. A correlation between humoral and cellular immune response will be evaluated. 
Within-the-cohort comparisons will be carried out to assess difference in immune response according to 
different doses in the same population. Across-the-cohort comparison will be carried out to assess whether 
elderlies and adults have different pattern of immune response after vaccination at different end points.  
4.2.4. Milestones 
We expect to start enrollment within 4 weeks after final study approval. Eventual milestones include: 
A. DSMB authorizes ARM1 group B and ARM2 Group A 
B. DSMB authorizes ARM2 group B and ARM3 Group A 
C. DSMB authorizes ARM3 group B  
D. DSMB revises 4-week safety of whole Arm-1. DSMB authorize enrollment in ARM4 Group A. 

17 
 
E. DSMB authorizes ARM4 Group B and ARM5 Group A 
F. DSMB authorize ARM5 Group B and ARM6 Group A 
G. DSMB authorize ARM6 Group B 
4.2.5. Deliverables 
1. Within five months after the study approval a report on safety and immunogenicity at 4 weeks for 
Adult-Cohort will be developed; this report will include: 
a. an analysis of safety issue in the three adults-arms  
b. pivotal consideration used for authorizing enrollment in the Elderlies-Cohort 
c. a preliminary analysis of immunogenicity in the three adults-arms aimed at identifying the 
most convenient doses to be used in the eventual phase 2/3 trial. 
2. Within seven months after the study approval a report on safety and immunogenicity at 4 weeks for 
Elderly-Cohort will be developed; this report will include pivotal consideration on  
a. an analysis of safety issue in the three elderlies-arms  
b. a preliminary analysis of immunogenicity in the three elderlies-arms aimed at identifying the 
most convenient doses to be used in the eventual phase 2/3 trial. 
c. comparison of safety and immunogenicity between adults and elderlies (across the cohort 
comparison) to define the most convenient doses to be used either in general population or 
special groups at risk. 
3. Within one year since study approval final report will be produced including: 
a. Final analysis on safety and immunogenicity at 24 weeks after vaccination in Adults-Cohort 
b. Final analysis on safety and immunogenicity at 24 weeks after vaccination in Elderlies-Cohort 

18 
 
 
 Figure 6 Estimated time (week) for enrollment and follow-up for Adults-Cohort. Figures within cells represent time (in weeks) after vaccination Grey bars represent timing of authorization for 
enrollment. Blue cells represent time of participants’ enrollment. Emerald green cells represent time for safety assessment. Golden cells represent time of sampling carried out for immunogenicity 
assay on frozen specimens Red cells represent time of sampling carried out for immunogenicity assay on fresh samples. Orange cells represent milestones (letters represent the specific milestone). 
Olive green cells represent deliverables (numbers represent the specific deliverable). 
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A
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3
Milestonne
D
1
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Deliverables
Time (week)
Adults-Cohort
ARM-1       
low dose
Group A N=3
Group B N=12 
ARM-2 
intermediate 
dose
Group A N=3
Group B N=12 
ARM-3       
high dose
Group A N=3
Group B N=12 
Cohort
ARM
Item

19 
 
 
 
Figure 7 Estimated time (week) for enrollment and follow-up for Elderlies-Cohort. Figures within cells represent time (in weeks) after vaccination Grey bars represent timing of authorization for 
enrollment. Blue cells represent time of participants’ enrollment. Emerald green cells represent time for safety assessment. Golden cells represent time of sampling carried out for immunogenicity 
assay on frozen specimens Red cells represent time of sampling carried out for immunogenicity assay on fresh samples. Orange cells represent milestones (letters represent the specific milestone). 
Olive green cells represent deliverables (numbers represent the specific deliverable). 
 
 
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Group B N=12 
ARM-5 
intermediate 
dose
Group A N=3
Group B N=12 
ARM-6       
high dose
Group A N=3
Group B N=12 
Milestonne
D
Deliverables
1
2

20 
 
5. Study design  
This is a first-in-human, open-label, dose escalation, phase 1A/1B clinical trial to assess the safety and 
immunogenicity of the candidate GRAd-COV2 vaccine in healthy volunteers aged 18-55 years and elderly 
volunteers aged 65-85 years. The vaccine will be administered intramuscularly once in time.  
We aim to enroll 90 patients in two cohorts with three arms each (i.e. 15 participants per arm in six arms). 
Each of the study arm will assess a unique dose of the candidate GRAd-COV2 in a particular population, either 
adults or elderlies.  
• 
Adults-Cohort (Phase 1A): 
o Arm-1 will investigate safety and immunogenicity of a dose of 5x1010 viral particles in adults.  
o Arm-2 will investigate safety and immunogenicity of a dose of 1x1011 viral particles in adults. 
o Arm-3 will investigate safety and immunogenicity of a dose of 2x1011 viral particles in adults. 
• 
Elderlies-Cohort (Phase 1B) 
o Arm-4 will investigate safety and immunogenicity of a dose of 5x1010 viral particles in elderlies. 
o Arm-5 will investigate safety and immunogenicity of a dose of 1x1011 viral particles in elderlies. 
o Arm-6 will investigate safety and immunogenicity of a dose of 2x1011 viral particles in elderlies. 
To minimize the risk of severe adverse event in frail subjects enrollment of Elderlies-cohort will start after 
that 4-week safety results in Adults-Cohort will be available. Moreover, as it is a first in human trial, we have 
scheduled volunteers to be enrolled according to a strict frame of authorization rules. In particular, each arm 
includes two groups (A and B). Group As are made of 3 volunteers each to be enrolled throughout a week. 
The occurrence of SAE within 48h since the injection will be reported to data safety monitory board (DSMB) 
and the enrolment suspended until a direct effect of the vaccination is excluded. Group Bs are made of 12 
participant each to be enrolled over a week. DSMB provide binding advice on sequential enrollment across 
groups and arms; this is:  
• 
enrolment of Arm-1 group B and Arm-2 group A is bound to a positive decision on 7-day safety of 
Arm-1 group A; 
• 
enrolment of Arm-2 group B and Arm-3 group-A is bound to a positive decision on 7-day safety of 
Arm-2 group A; 
• 
enrolment of Arm-3 group B is bound to a positive decision on 7-day safety of Arm-3 group A; 
• 
enrolment of Arm-4 group A is bound to a positive decision on 4-week safety Arm-1; 
• 
enrolment of Arm-4 group B and Arm-5 group A is bound to a positive decision on 7-day safety of 
Arm-4 group A; 
• 
enrolment of Arm-5 group B and Arm-6 group-A is bound to a positive decision on 7-day safety of 
Arm-5 group A; 
• 
enrolment of Arm-6 group B is bound to a positive decision on 7-day safety of Arm-5 group A. 
Figure 8 reports the flow-charts for authorization and enrollment. 

21 
 
 
Figure 8 Study design and enrollment flow charts according to DSMB binding decisions  

22 
 
6. Recruitment and withdrawal  
6.1. Recruitment strategy 
Participants may be recruited by use of an advertisement +/- registration form formally approved by the 
ethics committee. All participants will sign and date the informed consent form (ICF) before any study-related 
procedures can be performed. Doctors enrolling the patients are responsible for obtaining the ICF before 
enrolling patients (i.e. before making screening test)  
The volunteer’s GP (Medico di Medicina Generale) will be informed and relevant clinical information 
registered if it is possible. 
6.2. Adults-Cohort 
Healthy people aged 18–55 years and full details of the eligibility criteria are described in section 6.5. 
6.3. Elderlies-Cohort 
Healthy people aged 65–85 years and full details of the eligibility criteria are described below 
6.4. Inclusion criteria 
A subject must meet all of the following criteria to be eligible to participate in this study: 
1. Provides written informed consent prior to initiation of any study procedures. 
2. Be able to understand and agrees to comply with planned study procedures and be available for all study 
visits. 
3. Agrees to the collection of venous blood per protocol. 
4. Confirms to have not donated blood three months before the study 
5. Agrees to refrain from blood during the study and until the three months after the end of the study. 
6. Body Mass Index 18-29 kg/m2, inclusive, at screening. 
7. Premenopausal women must agree to use one acceptable primary form of contraception. 
8. Premenopausal women must have a negative urine pregnancy test the day of vaccination and are 
routinely using - and willing to use up to six months from vaccine administration - an effective method 
of birth control resulting in a low failure rate (i.e., hormonal contraception, condoms in combination with 
a spermicidal cream, male partner sterilization-vasectomy or total sexual abstinence). 
9. Oral temperature ≤37.0 degrees Celsius the day of the administration of the vaccine 
10. Pulse no greater than 100 beats per minute. 
11. Systolic blood pressure (BP) is 85 to 139 mmHg, inclusive the day of vaccination.  
12. Should not show laboratory values outside the normal range which may have clinical significance even in 
absence of specific signs or symptoms. 
6.5. Exclusion Criteria 
A subject who meets any of the following criteria will be excluded from participation in this study: 
1. Positive serology for anti-HIV-Ab 
2. Positive HbBsAg 
3. Positive anti-HCV-Ab 
4. Positive for SARS-CoV-2 (either anti-S-Ab or anti-N-Ab) 
5. Acute illness, as determined by the site PI or appropriate sub-investigator, the day of vaccination. 
6. Breastfeeding women 
7. Autoimmune and hyper-inflammatory condition 
8. History of atopy (or any IgE associated condition) who had required treatment over the last 6 months; 
9. History of hypersensitivity or severe allergic reaction (e.g., anaphylaxis, generalized urticaria, 
angioedema, other significant reaction) to any previous licensed or unlicensed vaccines;  

23 
 
10. Assumption of any immunomodulatory medication over the last 4 months (including, but not limited to, 
systemic corticosteroids, allergy injections, immunoglobulin, interferon, immunomodulators, cytotoxic 
drugs, or other similar or toxic drugs). The use of low dose topical, ophthalmic, inhaled, and intranasal 
steroid preparations will be permitted. 
11. Presence of self-reported or medically documented significant medical condition 
12. Presence of self-reported or medically documented significant psychiatric condition 
13. Significant cardiovascular disease needing therapy or history of myocarditis or pericarditis or heart 
surgery. Patients in treatment with Sartans or ACE-Inhibitors and good response to therapy may be 
included.  
14. Neurological or neurodevelopmental conditions (e.g., history of migraines in the past 5 years, epilepsy, 
stroke, seizures in the last 3 years, encephalopathy, focal neurologic deficits, Guillain-Barré syndrome, 
encephalomyelitis or transverse myelitis). 
15. Ongoing malignancy or recent diagnosis of malignancy in the last five years excluding basal cell and 
squamous cell carcinoma of the skin, which are allowed. 
16. Primary or secondary immunodeficiency of any cause. 
17. Participated in another investigational study involving vaccination of biologic compounds in the last 12 
months.  
18. Currently enrolled in or plans to participate in another clinical trial with an investigational agent that will 
be received during the study-reporting period. 
19. Administration of immunoglobulins and/or any blood or blood products within the 4 months before the 
first vaccine administration or at any time during the study. 
20. Has any significant disorder of coagulation. 
21. Has any chronic liver disease, including fatty liver. 
22. Has a history of alcohol abuse or other recreational drug use within 6 months before the first vaccine 
administration. 
23. Has any abnormality or permanent body art (e.g., tattoo) that would interfere with the ability to observe 
local reactions at the injection site (deltoid region). 
24. Received or plans to receive additional vaccination within 4 weeks before or after each vaccination. 
25. Has been reported as a case (confirmed or probable) of COVID-19 form the regional health system 
26. Has any clinical conditions that, in the opinion of the site PI or appropriate sub-investigator, precludes 
study participation, this includes any acute, subacute, intermittent or chronic medical disease or 
condition that would place the subject at an unacceptable risk of injury, render the subject unable to 
meet the requirements of the protocol, or may interfere with the evaluation of responses or the subject's 
successful completion of this trial 
6.6. Lost to follow-up and withdrawal 
In accordance with the principles of the current revision of the Declaration of Helsinki and any other 
applicable regulations, a participant has the right to withdraw from the study at any time and for any reason, 
and is not obliged to give his or her reasons for doing so. The Investigator may withdraw a participant at any 
time in the interests of the participant’s health and well-being. The reason for withdrawal will be recorded in 
the CRF (if available). If withdrawal is due to an AE, appropriate follow-up visits or medical care will be 
arranged, with the agreement of the participant, until the AE has resolved.  
Investigator may decide to replace a participant withdrawn or lost to follow-up if that is possible within the 
specified enrollment timeframe. 
If a participant withdraws from the study, blood samples collected before their withdrawal from the trial will 
be used/stored unless the participant specifically requests otherwise. In all cases of participant withdrawal, 
except those of complete consent withdrawal, long-term safety data collection, including some procedures 
such as safety bloods, will continue as appropriate if subjects have received vaccine. 

24 
 
6.7. Compliance with Dosing Regime 
All doses in this vaccine study will be administered under the supervision of a medical investigators by 
registered healthcare workers and recorded in the CRF. The study medication will be at no time in the 
possession of the participantand compliance will, therefore, not be an issue. 
6.8. Pregnancy 
Should a participantbecome pregnant during the trial, she will be followed up as other volunteers and in 
addition will be followed until pregnancy outcome. We will not routinely perform venopuncture in a pregnant 
volunteer. 
7. Clinical procedures 
All participants will receive the GRAd-COV2 vaccine and undergo follow-up at specific endpoint for a total of 
24 weeks. All follow-up visit will include a medical visit and a blood sampling. Moreover, all participants will 
receive a telephone number to be contacted for receiving assistance should any medical condition occur, 
potentially associated with the vaccination. Additional visits or procedures may be performed at the 
discretion of the investigators based on participants needs. 
7.1. Vaccination procedure 
Vaccines will be administered intramuscularly in the deltoid of non-dominant arm according to different 
doses: 
A. Dose 5x1010 vp (arm 1 and arm 4) 
B. Dose 1x1011 vp (arm 2 and arm 5) 
C. Dose 2x1011 vp (arm 3 and arm 6)  
The injection site will be covered with a sterile dressing and the participants will stay in observation per 4 
hours, in case of immediate adverse events. Investigator will decide whether the participant can leave or 
need additional time in hospital monitoring. 
All participants will be vaccinated with the same procedure and according to the same schedule of clinical 
record. A standard electronic case report forms (e-CRF) will be produced and filled in. 
7.2. Medical visit 
Medical visit will be carried out at the endpoints indicated in the schedule of procedures and will always 
include a check of medical history, physical examination, weight and height (at screening only), pulse, blood 
pressure, after 10 min rest in sitting position, and body temperature. 
7.3. Blood test and other analysis for monitoring safety 
Biological testing for monitoring safety will be carried out at the endpoints indicated in the schedule of 
procedures at the laboratory of the clinical centers that enroll volunteers and will include: 
A. Full Blood Count 
B. Urine analysis 
C. Biochemistry: Sodium, Potassium, , , Albumin, Liver Function Tests (ALT, ALP, Bilirubin total and 
fractionated), Renal function (Creatinine and BUN), lactate dehydrogenases (LDH), alkaline 
phosphatases (ALP) 
D. Diagnostic serology: HBsAg, HCV antibodies, HIV antibodies (specific consent will be gained prior to 
testing blood for these blood-borne viruses) 
E. Coagulation: aPTT (ratio), PT (ratio) and INR. 

25 
 
Urine will be tested for pH, Glucose Protein, Haemoglobin, Leukocyte esterase, Ketones, Bilirubin, 
Urobilinogen and Nitrites at screening. For female participants only, urine will be tested for beta-human 
chorionic gonadotrophin (β-HCG) at screening.  
Participants presenting with respiratory symptoms consistent with COVID-19 infection will undergo to 
molecular test for SARS-CoV-2 and admitted to hospital in isolation room until a negative is available. 
Additional safety blood tests may be performed if clinically relevant at the discretion of the medically 
qualified investigators. 

26 
 
 
 
 
Item 
Time of the visit since vaccination 
Type of 
intervention 
Scr 
T0 
D2 
W1 
W2 
W4 
W8 
W12 W24 
Window (days) 
≤ 21 
0 
0 
±1 
±1 
±2 
±4 
±7 
±14 
  
Informed consent 
x 
  
  
  
  
  
  
  
  
Procedures 
Eligibility 
x 
  
  
  
  
  
  
  
  
Vaccination 
  
x 
  
  
  
  
  
  
  
AE reporting 
  
x 
x 
x 
x 
x 
x 
x 
x 
Diary card 
  
A 
  
  
  
B 
  
  
  
Medical history 
x 
 
 
 
 
 
 
 
 
Medical visit 
Physical examination 
x 
x 
x 
x 
x 
x 
x 
x 
x 
TC 
x 
x 
x 
x 
x 
x 
x 
x 
x 
SpO2 % 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Pulse 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Blood pressure 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Urine analysis 
x 
  
  
  
  
  
  
  
  
Urine 
Urine B-HCG 
x 
x 
  
  
  
  
  
  
  
Blood counts 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Blood test 
Biochemistry 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Coagulation 
x 
  
  
  
  
  
  
  
  
HIV-Ab 
x 
  
  
  
  
  
  
  
  
HBsAg and HCV 
x 
  
  
  
  
  
  
  
  
HLA Typing 
  
  
  
x 
  
  
  
  
  
SARS-CoV-2 Serology * 
x 
  
  
  
  
  
  
  
  
immuno 
CLIA anti-S (IgG) 
  
x 
  
x 
x 
x 
x 
x 
x 
CLIA anti-N (IgG) 
  
x 
  
x 
x 
x 
x 
x 
x 
Micro neutralization 
  
  
  
  
  
x 
  
  
x 
Elispot SARS-CoV-2 
  
x 
  
  
x 
x 
x 
x 
x 
Total blood volume (mL) 
36 
60 
10 
27 
60 
60 
60 
60 
60 
433 

27 
 
Figure 9 Timing of participant follow-up after vaccination. Scr=screening to be done no more than 21 days before vaccination; T0= 
day of the vaccination; D2= day 2 after vaccination; W1= one week after vaccination; W2= two weeks after vaccination W4= four 
weeks after vaccination; W8= eight weeks after vaccination; W12= twelve weeks after vaccination; W24= twenty-four weeks after 
vaccination. A) Diary given to participants; B) Diary received form participants.   
* This is any serological assay to detect anti-SARS-CoV-2 total IgG which is validated for clinical use in Italy (i.e. validated for diagnosis 
of SARS-CoV-2 infection) 
  
7.4. Immunogenicity analysis 
Tests for assessing immunogenicity will be carried out at INMI Lazzaro Spallanzani laboratory at the endpoints 
indicated in the schedule of procedures. Management of samples and methodology of testing are reported 
in section 6. Testing for immunogenicity includes:  
A. CLIA to quantify antibodies to SARS-CoV-2 S-protein (anti-S-Ab) 
B. CLIA to quantify antibodies to SARS-CoV-2 N-protein(anti-N-Ab) 
C. SARS-Cov-2 micro-neutralization assay to quantify neutralizing antibody activity to the virus 
D. Ex vivo IFN-gamma ELISpot responses to SARS-CoV-2 S-protein 
Other exploratory immunological assays including analysis polyfunctional T cell analysis and other antibody 
assays will be performed at the discretion of the investigators.  
7.5. Screening 
A month before starting the enrolment a set of subjects will undergo medical screening including a medical 
visit (pulse, blood pressure, temperature, medical history, physical examination, ECG) blood testing and urine 
analysis. Testing for screening is reported in Figure 9. Biochemical test valid for enrollment should be carry 
out no more than 21 days before vaccination. 
7.6. Enrolment visit (Time 0) 
Before vaccination, the eligibility of each participant will be reviewed. Investigator will check pulse, blood 
pressure, temperature, medical history, physical examination and carry out a blood sampling. Testing for 
time 0 is reported figure 9. 
7.7. Vaccination sequence 
For safety reasons enrolment of first in human vaccine administration will be carried out on a weekly 
sequence in the six different arms. Each arm will be divided into 2 groups. Group-As are made of 3 volunteers 
each and group-Bs are made of 12 volunteers each.  
A the beginning of the study clinical center will smoothly screen eligible subjects.  
At enrollment on day 1 the first participant (arm 1 group-A) will be vaccinated ahead of any other participants 
and the profile of adverse events will be reviewed 48 hours after vaccination. Provided there are no safety 
concerns, as assessed by the local medical investigators, another two participants will be vaccinated at the 
same dose after at least 48 hours has elapsed following the first participant. Thus within one week, three 
participants will be vaccinated. 
The following week the three participants vaccinated will undergo clinical screening for AE including a 
medical visit and a laboratory tests up to 7 days after vaccination. DSMB will analyze data on safety for arm 
1 group-A and provide binding advice for the eventual enrolment. Eventual enrollment will include two blocks 
of participants this is: 
A. Arm 1 group-B (12 participants) 
B. Arm 2 group-A (3 participants) 

28 
 
Participants in arm 2 group-A will be scheduled for vaccination similarly to those vaccinated in arm 1 group-
A, 48 hours apart from each other, provided there are no safety concerns, as assessed by the local medical 
investigators. Participants in arm 1 group-B will be smoothly vaccinated throughout the week. 
The participants vaccinated in arm 2 group-A and arm-1 group-B undergo clinical screening for AE including 
a medical visit and a laboratory tests up to 7 days after vaccination. DSMB will analyze data on safety for arm 
2 group-A and provide binding advice for the eventual enrolment. Eventual enrollment will include two blocks 
of participants this is: 
A. Arm 2 group-B (12 participants) 
B. Arm 3 group-A (3 participants) 
Participants in arm 3 group-As will be scheduled for vaccination similarly to those vaccinated other group-As, 
48 hours apart from each other, provided there are no safety concerns, as assessed by the local medical 
investigators. Participants in arm 2 group-B will be smoothly vaccinated throughout a week. 
The participants vaccinated in arm 3 group-A and arm 2 group-B will undergo clinical screening for AE 
including a medical visit and a laboratory tests up to 7 days after vaccination. DSMB will analyze data on 
safety for arm 2 group-A and provide binding advice for the eventual enrolment. Eventual enrollment will 
include one block of participants this is: 
A. Arm 3 group-B (12 participants) 
Participants in arm 3 group-B will be smoothly vaccinated throughout the week. 
The participants vaccinated in arm 3 group-b will undergo clinical screening for AE including a medical visit 
and a laboratory tests up to 7 days after vaccination. DSMB will analyze data and provide a general advice on 
vaccine safety at the conclusion of enrollment of the Adult-Cohort. 
When data on 4-week safety for the whole arm 1 (15 participants) will be available. DSMB will provide binding 
advice for the eventual enrolment in the Elderlies-Cohort. The decision will be done only if no less than full 
data of 12 out of 15 participants will be available.  
The enrollment the Elderlies-Cohort will follow the same scheme of the enrollment of Adults-Cohort. 
The first participant of the Elderly-Cohort (arm 4 group-A) will be vaccinated ahead of all other elderlies 
participants. Provided there are no safety concerns, as assessed by the local medical investigators, another 
two participants will be vaccinated at the same dose after at least 48 hours has elapsed following the first 
participant. By the end of a week, three participants will be vaccinated. 
All the participants vaccinated in ARM 4 group-A will undergo clinical screening for AE including a medical 
visit and a laboratory tests up to 7 days after vaccination. DSMB will analyze data on safety for arm 4 group-
A and provide binding advice for the eventual enrolment. Eventual enrollment will include two blocks of 
participants this is: 
A. Arm 4 group-B (12 participants) 
B. Arm 5 group-A (3 participants) 
Participants in arm 5 group-A will be scheduled for vaccination similarly to those vaccinated group-As, 48 
hours apart from each other, provided there are no safety concerns, as assessed by the local medical 
investigators. Participants in arm 4 group B will be smoothly vaccinated throughout the week. 
The participants vaccinated in arm 5 group-A and those vaccinated in arm 4 group-B will undergo clinical 
screening for AE including a medical visit and a laboratory tests up to 7 days after vaccination. DSMB will 

29 
 
analyze data on safety for arm 4 group-A and provide binding advice for the eventual enrolment. Eventual 
enrollment will include two block of participants this is: 
A. Arm 5 group-B (12 participants) 
B. Arm 6 group-A (3 participants) 
Participants in arm 6 group-A will be scheduled for vaccination similarly to those vaccinated group-As, 48 
hours apart from each other, provided there are no safety concerns, as assessed by the local medical 
investigators. Participants in arm 5 group B will be smoothly vaccinated throughout the week. 
The participants vaccinated in arm 5 group-A and those vaccinated in arm 5 group-B will undergo clinical 
screening for AE including a medical visit and a laboratory tests up to 7 days after vaccination. DSMB will 
analyze data on safety for arm 5 group-A and provide binding advice for the eventual enrolment. Eventual 
enrollment will include one block of participants this is: 
A. Arm 6 group-B (12 participants) 
Participants in arm 3 group-B will be smoothly vaccinated throughout the week.  
The participants vaccinated in arm 6 group-B clinical screening for AE including a medical visit and a 
laboratory tests up to 7 days after vaccination. No more participants will be vaccinated. 
7.8. Visit (D2, W1, W2, W4, W8, W12 and W24) 
Follow-up visits will take place 48h after the vaccination (D2) and eventually at week 1 (W1), 2 (W2), 4 (W4), 
8 (W8), 12 (W12), 24 (W24) and 48 (W48) after vaccination. Participants will be assessed for local and 
systemic AE, interim history, physical examination, review and blood tests at these endpoints. Blood will also 
be taken for exploratory immunology purposes. 
If a participant experience adverse events (laboratory or clinical), which the investigator (physician) 
determine necessary for further close observation, the participant may be admitted to the clinical facility of 
the enrolling center. Moreover, investigator can decide to perform additional medical examination if needed. 
Participants who experience symptoms that are associated with COVID-19 including fever ≥ 37.5 °C and/or 
respiratory symptoms will be treated according to local guideline for suspect cases of COVD-19 until an 
infection with SARS-CoV-2 is not excluded by a molecular test. A test for detection of SARS-CoV-2 on 
respiratory sample will be carried out on all participants with fever ≥ 37.5 °C persisting for more than 48 
hours at any time after vaccination. Participants with confirmed COVID-19 will be admitted to hospital until 
clearance of the infection.   
Schedule of attendance is reported in Figure 9. 
7.9. Participants restrictions 
Subjects will be asked to adhere to the following study restrictions along their study participation, which are 
also listed and explained in the ICF: 
A. not to do strenuous physical exercise during 2 days before each visit 
B. To be in fasting condition (for at least 8 h) at each visit 
C. To avoid alcohol during the 24 hours before each visit 

30 
 
8. Investigational vaccine production 
8.1. Manufacturing and Presentation 
The GRAd-COV2 vaccine consists of the replication-deficient simian adenovirus vector GRAd32, containing a 
stabilized surface glycoprotein (S-protein protein) antigen of the SARS-CoV2 expressed from the strong CMV 
IE promoter. The following vaccinations will be given in this study: 
1. GRAd-COV2 5 x 1010 vp 
2. GRAd-COV2 1 x 1011 vp 
3. GRAd-COV2 2 x 1011 vp 
GRAd-COV2 is manufactured in A195 Formulation Buffer at a concentration of 2 x 1011 vp/mL. The drug 
product is filled into 3mL glass vials (supplied by Nuova Ompi Stevanato Group) with a 13 mm grey 
chlorobutyl rubber stopper and a 13 mm aluminium seal (supplied by West Pharmaceutical Services). The 
filled vials are supplied sterile. The containers and closures are tested for compliance with defined 
specifications. The vials are made from Ph Eur borosilicate Type 1 glass. 
8.2. Supply and Storage 
GRAd-COV2 has been formulated and vialed under Good Manufacturing Practice conditions at the ReiThera 
Srl Facility, Roma, IT. At ReiThera Srl the vaccine will be certified and labelled for the trial by a Qualified 
Person (QP) before transfer to the clinical site. 
The vaccine is stored at nominal -80°C in a locked freezer, at the clinical site. All movements of the study 
vaccines will be documented in accordance with existing standard operating procedure (SOP). Vaccine 
accountability, storage, shipment and handling will be in accordance with relevant SOPs and forms. 
8.3. Administration of Investigational Medicinal Products 
The dosages of GRAd-COV2 to be used in this study are: 5x1010vp (low dose), 1x1011vp (intermediate dose) 
and 2x1011vp (high dose). See the SOP for detailed information on the preparation, labeling, storage, and 
administration of the GRAd-COV2 investigational vaccine. For administration of the high dose, 1ml of GRAd-
COV2 will be injected without dilution. For intermediate and low dose, the vaccine will be diluted in sterile 
saline solution to reach a final 1ml injection volume. Vaccine dilution will be performed by the site pharmacist 
on the same day of vaccine administration. 
On vaccination day, GRAd-COV2 will be allowed to thaw to room temperature and will be administered within 
1 hour of removal from the freezer. The vaccine will be administered intramuscularly into the deltoid of the 
non-dominant arm (preferably). Qualified medical personnel will wear gloves.  All volunteers will be observed 
in the unit for 4 hours (±10 minutes) after vaccination. During administration of the investigational products, 
Advanced Life Support drugs and resuscitation equipment will be immediately available for the management 
of anaphylaxis. Vaccination will be performed and the IMPs handled according to the relevant SOPs.  
The study will be performed in accordance with Genetically Modified Organisms (Contained Use) Regulations 
(2001/18/CE). In order to minimize dissemination of the recombinant vectored vaccine virus into the 
environment, inoculation sites will be covered with a dressing after immunization. This should absorb any 
virus that may leak out through the needle track. The dressing will be removed from the injection site after 
30 minutes (+15/- 5 minutes) and will be disposed as GMO waste by autoclaving.  
9. Adverse event assessment and management 
Safety will be assessed by the frequency, incidence and nature of adverse events and severe adverse events 
arising during the study. 

31 
 
9.1. Definitions 
Adverse event (AE). (AE) is any untoward medical occurrence in a participant, which may occur during or 
after administration of the vaccine and does not necessarily have a causal relationship with the intervention. 
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), 
symptom or disease temporally associated with the study intervention, whether or not considered related 
to the study intervention. 
Adverse reaction (AR). An AR is any untoward or unintended response to the vaccine. This means that a 
causal relationship between the vaccine and an AE is at least a reasonable possibility, i.e., the relationship 
cannot be ruled out. All cases judged by the reporting medical Investigator as having a reasonable suspected 
causal relationship to the vaccine (i.e. possibly, probably or definitely related to the vaccine) will qualify as 
adverse reactions. 
Unexpected adverse reaction (UAR). An UAR is any untoward or unintended response to the vaccine, the 
nature and severity of which is not consistent with the information about the medicinal product in question 
set out in the investigator brochure (IB) or summary of product characteristics. 
Serious adverse event (SAE). A SAE is an AE that results in any of the following outcomes, whether or not 
considered related to the study intervention. 
A. Death 
B. Life-threatening event (i.e., the participant was, in the view of the Investigator, at immediate risk of 
death from the event that occurred).  
C. Persistent or significant disability or incapacity (i.e., substantial disruption of one’s ability to carry out 
normal life functions). 
D. Hospitalization, regardless of length of stay, even if it is a precautionary measure for continued 
observation.  
E. Significant medical intervention including surgery or administration of drugs (examples of such 
medical events include allergic reaction requiring intensive treatment in an emergency room or clinic, 
blood dyscrasias, or convulsions regardless eventual need of hospitalization) 
F. A grade ≥3 abnormalities on safety blood test according to Common Terminology Criteria for Adverse 
Events (CTCAE v.4 and MedDRA 12.1). 
Serious adverse reaction (SAR). A SAE (expected or unexpected) that, is in the opinion of the reporting 
Investigator, believed to be possibly, probably or definitely due to the vaccine. 
Suspect unexpected serious adverse reaction (SUSAR). A severe adverse reaction, the nature and severity 
of which is not consistent with the information about the medicinal product in question set out in the 
investigator brochure (IB) or Summary of Product Characteristics. 
9.2. Solicited Adverse Reactions 
Solicited Adverse Reactions are those (foreseeable) AEs following vaccination with GRAd-COV2, and include 
injection site pain, erythema, warmth, swelling, pruritus, myalgia, arthralgia, headache, fatigue, fever, 
feverishness, malaise and nausea. 
Solicited Adverse Reactions are grade as following: 
A. None: Symptom does not experience 
B. Mild: Short-lived or mild symptoms; medication may be required. No limitation to usual activity 
C. Moderate: Mild to moderate limitation in usual activity. Medication may be required but 
hospitalization is not required. 
D. Severe: Considerable limitation in activity. Invasive intervention or hospitalization is required.  

32 
 
We do not expect life threatening or fatal solicited adverse reactions in this study. 
9.3. Assessment of causality 
For every AE, an assessment of the relationship of the event to the administration of the vaccine will be 
undertaken by the doctors who have enrolled the participants. An intervention-related AE refers to an AE for 
which there is a probable or definite relationship to administration of a vaccine. An interpretation of the 
causal relationship of the intervention to the AE in question will be made, based on the type of event; the 
relationship of the event to the time of vaccine administration; and the known biology of the vaccine. 
Alternative causes of the AE, such as the natural history of pre-existing medical conditions, concomitant 
therapy, other risk factors and the temporal relationship of the event to vaccination will be considered and 
investigated. 
Causality assessment will take place during planned safety reviews by DSMB.  
9.4. Guidance for assessing relationship of AE with vaccination 
A. No Relationship. No temporal relationship to vaccine and alternative etiology (clinical state, 
environmental or other interventions); and it does not follow known pattern of response to the 
vaccine. 
B. Unlikely. Unlikely temporal relationship to study product and alternative etiology likely (clinical state, 
environmental or other interventions) and it does not follow known typical or plausible pattern of 
response to study product. 
C. Possible. Reasonable temporal relationship to study product; or event not readily produced by 
clinical state, environmental or other interventions; or similar pattern of response to that seen with 
other vaccines 
D. Probable. Reasonable temporal relationship to study product; and event not readily produced by 
clinical state, environment, or other interventions or known pattern of response seen with other 
vaccines 
E. Definite. Reasonable temporal relationship to study product; and event not readily produced by 
clinical state, environment, or other interventions; and known pattern of response seen with other 
vaccines. 
9.5. Reporting procedure for all AE  
All local and systemic AEs occurring in the 28 days following each vaccination observed by the Investigator 
or reported by the participants, whether or not attributed to study medication, will be recorded. Recording 
and reporting of all AEs will be detailed in SOP. All AEs that result in a participant withdrawal from the study 
will be followed up until a satisfactory resolution occurs or until a non-study related causality is assigned (if 
the participant consents to this). SAEs will be collected throughout the entire trial period (i.e. until W24). 
SAEs will be reported on the SAE forms by doctors who enrolled participants to the principal investigator and 
DSMB. SAE will be documented accurately and notified within 48 hours to the DSMB. The DSMB will also 
review severity and causality analysis within 2 days and will provide advice on study continuation. AEs will be 
reported to DSMB within 7 days. In special circumstance the chair of DSMB may decide to involve the ethical 
committee to analyze AE reports in order to define how to continue the study. 
SAR and SUSAR that occur within week 10 of the study will determine a temporal suspension of the 
enrollment pending the decision of DSMB. The suspension is decided by the local investigator and 
immediately communicated to principal investigator and DSMB. When a SAR or a SUSAR occurs among 
participants enrolled in a clinical center the investigator of the clinical center will immediately contact the 
principal investigator who will eventually contact other enrolling centers. 

33 
 
9.6. Assessing severity of adverse events 
The severity of the adverse events will be assessed according to the scale reported below. In particular AE 
will be judged according to inspection of injection area, medical visit and review of all biochemical and 
hematological tests carried out at each endpoint. AEs autonomously reported by participants outside medical 
visit will be immediately evaluated by investigators on a telephone interview. Investigators may decide to 
admit participant to hospital for further investigation if needed.  
9.7. Management of participants with AE 
The clinical center that enroll participants will provide care to any participant with any AE as appropriate for 
the clinical conditions.  
Each enrolling center will have a medical investigator that will ensure real-time safety oversight. The local 
medical investigator will review AEs deemed possibly, probably or definitely related to study interventions. 
The local investigator will immediately register AE and will notify them to principal investigator according to 
SOP. Moreover the local investigator can decide to place the study on hold if deemed necessary following a 
study intervention-related as severe or unexpected event such as: SAE, UAR, SAR and SUSAR. The decision to 
put the study on hold is immediately notified to principal investigator and to DSMB. 
Table 1 Adverse event severity scale 
Adverse events 
Severity 
None 
Mild 
Moderate 
Severe 
Erythema  
no 
3-50 mm 
51-100 mm 
>100 mm 
Swelling 
no 
3-20 mm 
21-50 mm 
> 50 mm 
Ulceration 
-  
 - 
-  
any 
Fever 
36.0-37.4 
37.5-38.0 
38.1-39.0 
 >39.0 
hypothermia 
35.9-35.1 
 - 
<35 
Tachycardia (bpm) 
55-100 
101-115 
116-130 
>130 
bradycardia (bpm) 
50-54 
40-49 
≤40 
Systolic hypertension 
85-140 
141-159 
160-179 
>179 
Systolic hypertension 
85-89 
80-84 
<80 
Diastolic hypertension 
<90 
91-99 
100-109 
>109 
Respiratory Rate low 
12-20 
 - 
9-11 
<9 
Respiratory Rate high 
 - 
21-24 
>24 
SpO2 at FiO2 21% 
>97 
94-96 
92-93 
<92 
Consciousness 
Alert 
 - 
-  
any 
Biochemistry 
MedDra and CTAE. Grade 1-2=mild; Grade 3/4= severe/life threatening 
Blood counts 
 
9.8. Halting rules and study termination due to safety issue  
Safety halting rules have been developed considering the fact that this is a first-in-human dose escalation 
study. Solicited adverse events are those listed as in section 9.2, occurring within the first 7 days after 
vaccination (day of vaccination and six subsequent days). Unsolicited adverse events are AE other than the 
solicited AEs occurring within the first 7 days, or any AEs occurring after the first 7 days after vaccination.  
Enrolment of participants in one arm will be upheld if: 
A. Solicited local adverse events (site of injection): 2 or more vaccinations in a group are followed by 
the same severe solicited local adverse event beginning within 2 days after vaccination (day of 

34 
 
vaccination and one subsequent day) and persisting at severe for >48 hrs and these are deemed 
possibly, probably or definitely related to study interventions.  
B. Solicited systemic adverse events: If 2 or more vaccinations in a group are followed by the same 
severe solicited systemic adverse event beginning within 2 days after vaccination (day of vaccination 
and one subsequent day) and these are deemed possibly, probably or definitely related to study 
interventions. 
C. Unsolicited adverse events: If 2 or more vaccinations in a group are followed by the same serious 
unsolicited adverse event (including the same laboratory adverse event) and relation with 
vaccination cannot be immediately excluded.  
D. One participant with serious adverse event considered possibly, probably or definitely related to 
vaccination occurs 
E. Life-threatening condition when relation with study intervention cannot be immediately excluded.  
F. Death when relation with study intervention cannot be immediately excluded. 
If a halting rule has been met, we will inform the regulatory authority. Moreover, before restarting 
enrollment the DSMB should exclude that AE has no immediate causal relation with the vaccine or that 
benefit of vaccination outweigh risk of further AE occurrence. In these latter case Ethical committee should 
be involved in the decision. Halting rules apply to one dose and the next higher one, so that if halting rule is 
met for the highest dose, only this arm is upheld. If halting is met for intermediate dose, two arms are hold. 
If halting rule is met for the lowest dose all the study is stopped pending on the investigation of causal 
association between AE and vaccination (DSMB advice). 
The DSMB will always consider: 
A. The relationship of the AE or SAE to the vaccine. 
B. The relationship of the AE or SAE to the vaccine dose, or other possible causes of the event. 
C. If appropriate, additional screening or laboratory testing for other participants to identify those who 
may develop similar symptoms and alterations to the current Participant Information Sheet are 
discussed. 
D. New, relevant safety information from ongoing research programs on the various components of the 
vaccine. 
The day of vaccination participants should have no evidence of clinical condition that may resemble AE. 
10. Statistical plan 
10.1. Sample size 
This is a descriptive phase I first in human trial to assess safety and immunogenicity of three different 
vaccine doses. Overall, the trial will include 90 participants and no formal sample size calculation is carried 
out.  
10.2. Statistics analysis 
This is a descriptive safety study, where participants will be vaccinated with different doses of GRAd-COV2 
against SARS-CoV-2. Forty-five participants per age cohort (for a total of 90 subjects) will be vaccinated in 
total. This sample size should allow an estimation to be made of the frequency and magnitude of outcome 
measures, rather than aiming to obtain formal statistical significance for differences between groups.  
Safety data will be presented according to frequency, severity and duration of adverse events. The primary 
analysis for immunogenicity will be to assess the difference in magnitude of SARS-CoV-2 specific T-cell and 
antibody responses between the groups. We will assess vaccine immunogenicity by comparing the change in 
these immunological parameters from baseline to different endpoints. At the end of the study, we expect to 

35 
 
6 measures per participant for T-cell response and antibody level. We will explore the possibility to describe 
kinetics of these parameters through non-parametric test for comparison of: 
A. within-the-arm medians at different times (i.e. baseline vs. either week-2, week-4, week-8, week-12 and 
week 24) between groups at different follow-up time); 
B. within the Adults-Cohort medians at specific time (i.e. Arm-1 vs. either Arm-2 or Arm-3 at baseline, week-
2, week-4, week-8, week-12 or week-24). 
C. within the Elderlies-Cohort medians at specific time (i.e. Arm-4 vs. either Arm-5 or Arm-6 at baseline, 
week-2, week-4, week-8, week-12 or week-24). 
D. Across the cohort medians at specific time (i.e. Arm-1 vs. Arm-4; Arm-2 vs Arm-5; Arm-3 vs. Arm-6 at 
baseline, week-2, week-4, week-8, week-12 or week-24)  
Explorative kinetics model based on regression techniques for correlated measures will be carried out if 
normal distribution of data is proven. 
11. Data Management 
11.1. Quality Control and Source Data Verification 
All subject data relating to the study will be recorded on electronic case report form (eCRF). The local 
investigator (physician) is responsible for verifying that data entries are accurate and correct by physically or 
electronically signing the eCRF. The Investigator shall assure the accuracy, completeness, legibility, and 
timeliness of the data reported on the eCRFs and in all required reports. 
The Investigator must maintain accurate documentation (source data) that supports the information entered 
in the eCRF. The Investigator must permit study-related monitoring, audits, Ethical committee review, and 
regulatory agency inspections and provide direct access to source data documents.  
Study monitors will perform ongoing source data review of data to confirm that data entered into the eCRF 
by authorized site personnel are accurate, complete, and verifiable from source documents; that the safety 
and rights of subjects are being protected and that the study is being conducted in accordance with the 
currently approved protocol and any other study agreements, 21 CFR, ICH-GCP, and all applicable regulatory 
requirements. 
Records and documents, including signed ICF, pertaining to the conduct of this study must be retained by the 
Investigator for 15 years after study completion unless local regulations or institutional policies require a 
longer retention period. No records may be destroyed during the retention period without the written 
approval of the Sponsor. No records may be transferred to another location or party without written 
notification to the Sponsor. 
All data generated by the site personnel will be captured electronically at each study center using eCRF. Once 
the eCRF clinical data have been submitted to the central server at the independent data center, corrections 
to the data fields will be captured in an audit trail. The reason for change, the name of the person who 
performed the change, together with the time and date will be logged to provide an audit trail. 
If additional corrections are needed, the responsible monitor or data manager will raise a query in the eCRF. 
The appropriate staff at the study site will answer queries sent to the Investigator. The name of the staff 
member responding to the query, and time and date stamp will be captured to provide an audit trail. Once 
all source data verification is complete and all queries are closed, the eCRF page will be ‘frozen’ to prevent 
further changes. 
The specific procedures to be used for data entry and query resolution using the eCRF will be provided to 
study sites in a training manual. In addition, site personnel will receive training on the eCRF. 

36 
 
11.2. Audit / Inspection 
The Agenzia Italiana del Farmaco the National Ethical committee for COVID and the Sponsor’s Clinical Quality 
Assurance Group may complete audit / inspections any time to assess compliance with the protocol and the 
principles of Good Clinical Practice and all other relevant regulations.  
Clinical centers and principal investigator must permit study-related monitoring, audits, Ethical committee 
review and regulatory agency inspections and provide direct access to source data documents; they will be 
carried out giving due consideration to data protection and medical confidentiality. The Investigator always 
assures the Sponsor and designated representatives of the necessary support. 
11.3. Monitoring and Access to Source Data 
During study conduct, monitoring will be performed by a Contract Research Organization (CRO). The CRO will 
conduct periodic monitoring, via telephone contacts and on-site visits (where applicable), to ensure that the 
protocol and guidelines are being followed. 
During the on-site visits the monitors may review source documents to confirm that the data recorded on 
the eCRFs are accurate, with a focus on ICF and other relevant study data. 
It is important that the physician(s) and their relevant personnel are available during the monitoring visits or 
during the phone contacts. 
Checking of the eCRF entries for completeness and clarity, and cross-checking with source documents, will 
be required to monitor the progress of the study.  
11.4. Data collection 
Subjects will be identified by a subject number, assigned at the screening visit. 
The data will then be processed, evaluated, and stored in pseudonymised form in accordance with applicable 
data protection regulations. 
11.4.1. Data Management 
The Clinical Research Organization (CRO) contracted for this study will be responsible for the study data 
management, which will process the data in accordance with procedures approved by the Sponsor, including 
data validation, resolution of errors, and quality control of defined immunogenicity analysis and safety 
variables. 
11.4.2. Case Report Form(eCRF) 
The eCRFs will be used to capture all the data related to the study. The eCRFs use third party software (Rave®) 
to capture data via an on-line system on a computer.  
Data related to the study will be recorded electronically in a central database over encrypted lines, and all 
entries and modifications to the data will be logged in an audit trail. Access to the system will only be granted 
after appropriate and documented training.  
The data will be entered in the system from the source data according to the description and timelines 
specified in the eCRF Completion Guidelines. 
Subjects/patients that fail screening will be noted as a screen failure in the eCRF.  Screen failures are 
participants who consent to participate in the clinical study but are not subsequently entered in the study. A 
minimal set of screen failure information is required to ensure transparent reporting of screen failure 
participants to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and 

37 
 
to respond to queries from regulatory authorities. Minimal information includes demography, screen failure 
details, eligibility criteria, and any SAEs.   
Electronic signatures will be used where signatures are required on pages and/or visits. Automated data 
entry checks will be implemented where appropriate; other data will be reviewed and evaluated for accuracy 
by the CRA.  
All entries, corrections, and changes must be made by the investigator or a designee. 
11.4.3. Database Release 
The database will be declared to be complete and accurate by mutual agreement between the CRO and the 
Sponsor. Once the database release has been approved by both parties, the database will be released in 
accordance with the CRO’s procedure, as agreed by the Sponsor. 
If a site closes before the study has been completed, the investigator will continue to have read-only access 
to the eCRF until the study has been completed. At database release, all user access to the eCRF will be 
changed to read-only. Renewed access to the eCRF will be given if corrections or updates to the released 
Rave® database are required. At the end of the study, the site(s) will be provided with all data related to the 
site (including eCRF data, queries, and the audit trail) using a secure electronic medium; the secure storage 
of these data at the site is the responsibility of the investigator. When confirmation of receipt of the data has 
been received from all site(s), all user access to the eCRF will be revoked. If, for some reason, the data are 
not readable for the full retention period 15 years or in accordance with national requirements, whichever is 
longer), the investigator may be requested that the data be re-sent. 
11.5. Source Documents 
Source documents provide evidence for the existence of the subject and substantiate the integrity of the 
data collected. Source documents are filed at the Investigator’s site. 
Data entered in the eCRF that are transcribed from source documents must be consistent with the source 
documents or the discrepancies must be explained. The Investigator may need to request previous medical 
records or transfer records, depending on the study. Also, current medical records must be available. 
11.6. Retention of Study Documents at the Site 
The investigator must keep the investigator’s set of documents in the Investigator Trial Master File (TMF) for 
at least 15 years after the Clinical Study Report has been approved or in accordance with national 
requirements, whichever is longer. The Sponsor will remind the investigator in writing of this obligation when 
the Clinical Study Report Synopsis is distributed to the site. Documents that belong to a hospital, institution, 
or private practice must be kept for the maximum period permitted by the institution.  
If off-site storage is used, a study-specific binder will remain at the site after the other study specific 
documents have been shipped for off-site storage. This binder is considered part of the TMF and must be 
kept in a secure place by the site for the required period. The binder must contain, at a minimum, the 
following documents: a copy of the Investigator TMF Index, a certified copy of the Subject Identification Code 
List, and a Retrieval Form. Under no circumstances may the investigator dispose of any study documents 
before having obtained written consent from the Sponsor. When the required storage period has expired, 
the documents may be destroyed in accordance with regulations. If it becomes necessary for the Sponsor or 
a regulatory authority to review any documentation related to this study, the investigator must permit access 
to such documentation 
12. Clinical centers 
GRAd-COV2 will be tested in a FIH Phase I multi-center clinical trial. 

38 
 
Enrolled Clinical Sites are: 
A. Istituto per Malattie Infettive (INMI) L. Spallanzani, Via Portuense, 292, Rome; Coordinator Clinical 
Site 
B. Centro Ricerche Cliniche Verona srl c/o Policlinico G.B. Rossi P.le Scuro, 10 37134 Verona 
The study will be conducted in accordance with the current approved protocol, ICH GCP, relevant regulations 
and standard operating procedures. Approved and relevant SOPs will be used at all clinical and laboratory 
sites. 
13. Monitoring Committees  
13.1. Data safety monitory board (DSMB) 
DSMB will include 4 voting members and 2 technical supporting members.  
The chair of the board and the 3 other voting members will be independent experts with no connection either 
with sponsor or clinical centers participating to the study. To participate to the board each member should 
disclose any potential conflict of interest according to Italian legislation.  
The decisions of DSMB are provided according to the time framework reported in milestone (section 4.2.2). 
These decisions are binding so that enrolment of each eventual group of participants cannot be carried out 
without the advice of DMSB. The board has the power to withhold the enrollment and terminate the study.  
In case the enrollment is withhold because a halting rule is met, the DSMB will provide binding advice on how 
and when the study enrollment can be restarted, or the study should be terminated.  
Principal investigator may seek the advice of DSMB in case local investigator report the occurrence of special 
situation that do not immediately meet criteria for halting rules. 
The DSMB will meet once a week throughout the enrollment of participants and once a month after the end 
of the enrollment. 
DSMB may seek the advice of ethical committee if in case local investigator or principal investigator report 
the occurrence of special situation that do not immediately meet criteria for halting rules. 
Significant variation to the study design that are proposed after the approval of the study should be approved 
by DSMB and by ethical committee. 
13.2. Ethical committee 
The Law Decree 17 March 2020 n. 18, in art. 17, indicates the ethics committee of the National Institute for 
Infectious Diseases Lazzaro Spallanzani (IRCCS) as the only national ethics committee for the evaluation of 
clinical trials on COVID-19.  In this capacity, the committee will express the authorization for this study.  
Local ethical committees of participating center will provide local authorization if needed according to 
Regional legislation. 
14. Laboratory methods for immunogenicity assessment 
14.1. Serology 
Specific IgG against S-protein and N-protein will be tested by using commercial CE-IVD chemiluminescence 
immunoassays. In particular, the anti-S IgG will recognize specifically the S1 and S2 domains of the SARS-CoV-
2 virus S-protein protein (LIAISON®SARS-CoV-2 from DiaSorin®); and the anti-N specific IgG will detect 
recombinant N-protein from SARS-CoV-2 (Abbott Architect SARS-CoV-2 IgG assay, Abbott Diagnostics).  

39 
 
SARS-CoV-2 neutralizing antibodies will be detected by microneutralization assay under BSL3 conditions at 
INMI laboratories. Briefly, sera will be heat-inactivated, diluted 1:10 in serum-free medium, and titrated in 
duplicate in two-fold dilutions. Equal volumes of 100 TCID50/well SARS-CoV-2 (2019-nCoV/Italy-INMI) and 
serum dilutions will be mixed and incubated at 37°C for 30 minutes. Virus-serum mixtures will be added to 
sub-confluent Vero E6 cells and incubated at 37 °C and 5% CO2 for two days. Plates will be then stained with 
Gram’s crystal violet + 5% formaldehyde 40% m/v, washed and read at 595nm. Neutralizing antibody titers 
will be calculated as the last serum dilution presenting the 50% of virus growth inhibition with respect to the 
control virus. 
GRAd32 nAb titers will be determined at ReiThera laboratories by means of a standard neutralization assay 
based on infection of HEK293 cells with GRAd32 vectors encoding secreted alkaline phosphatase (SEAP)  
reporter gene. [46] Briefly, SEAP-expressing GRAd32 pre-incubated with serial dilutions of heat-inactivated 
serum from trial volunteers, is added to semi-confluent HEK293 cells. SEAP activity in the cell supernatant is 
measured after 24 hours using a chemiluminescent substrate (CSPD). The neutralisation titre is defined as 
the reciprocal of sera dilution required to inhibit SEAP expression by 50% compared to the SEAP expression 
of virus infection alone.  
14.2. T cell Immunology 
To test the ability of vaccine to induce a T cell response, we will quantify and characterize SARS-CoV-2 specific 
T cells before and after vaccination by ELISpot and multiparametric flow cytometry. Peripheral Blood 
Mononuclear Cells (PBMC) will be isolated by density gradient centrifugation, counted and resuspended in 
culture medium supplemented with 10% heat inactivated FBS.  
The first immunogenicity analysis will be performed after 2 weeks from vaccination (T2) using fresh isolated 
PBMC. Specifically, fresh PBMC isolated from peripheral blood of vaccinated subjects at T2 will be stimulated 
for 18-24 hours with SARS-CoV-2 peptides mix (designed on the S-protein protein) and the IFN- production 
by single cells will be evaluated  by ELISpot assay. Results will be expressed as Spot Forming Cells (SFC) per 
million of PBMC. 
Moreover, the kinetic of T cell induction after vaccination will be evaluated on frozen PBMC using different 
T cell assays (ELISpot assay, intracellular staining/flow cytometry and Tetramer staining) with the aim to 
deeply characterize the vaccine-induced T cell response overtime. Specifically, the ELISpot assay and the 
intracellular staining/flow cytometry will be performed after in vitro stimulation with S-protein-specific 
peptides and allow defining the functionality/polyfunctionality (IFN-γ, TNF-α, IL-17, IL-2, IL-10, CD107a) of 
antigen specific T cell clones. The results of the intracellular staining will be expressed as frequency of specific 
T cells able to exert one (single functional), two (double-functional), three (triple-functional) or four 
(quadruple-functional) different functions. Moreover, the Tetramer staining and flow cytometry will allow an 
ex vivo deep characterization of S-protein-specific T cells to define their phenotype (activated, exhausted, 
effector, memory). 
15. Ethical issues 
15.1. Potential Risks for volunteers 
The potential risk to participants is considered as low. The potential risks are those associated with 
phlebotomy and vaccination. In general, recombinant adenoviral vectors are safe. Similar vaccines encoding 
different antigens have been given to several thousand volunteers (including children) with a good safety 
profile. 

40 
 
15.1.1. Phlebotomy 
The maximum volume of blood drawn over the study period (approximately 465 mL) should not compromise 
these otherwise healthy volunteers. There may be minor bruising, local tenderness or pre-syncopal 
symptoms associated with venopuncture, which will not be documented as AEs if they occur. 
15.1.2. Vaccination: 
GRAd-COV2 has not been used in humans before and therefore will be initially administered at the lower 
dose of 5 x 1010 vp before progressing to the higher doses of 1x1011 and 2x1011 in the arms 2,3 ,5 and 6. 
Potential expected risks from vaccination include local effects such as pain, redness, warmth, swelling, 
tenderness or itching. Systemic reactions that could potentially occur following immunization with a 
recombinant adenovirus vaccine include a flu-like illness with feverishness, fatigue, malaise, arthralgia, 
myalgia and headache. 
As with any vaccine, Guillain-Barré syndrome or immune-mediated reactions that can lead to organ damage 
may occur, but this should be extremely rare. Serious allergic reactions including anaphylaxis could also occur 
and for this reason volunteers will be vaccinated in a clinical area where Advanced Life Support trained 
physicians, equipment and drugs are immediately available for the management of any serious adverse 
reactions (SAR). 
15.1.3. Vaccine induced Disease Enhancement  
A potential risk exists that the immunity elicited by an investigational candidate vaccine for COVID-19 may 
provoke an enhancement of the disease upon infection with SARS-CoV-2. This phenomenon has been 
described only in animal models when testing vaccines against other coronaviruses, and it was shown to be 
due to vaccine-induced “suboptimal” immune responses, such as poorly neutralizing antibodies and Th2 
skewed immune response promoting inflammatory and allergic immune environment upon pathogen 
encounter.  
The vaccine-mediated disease enhancement has not been observed so far in animal models of SARS-CoV-2 
infection or vaccination. In addition, the GRAd-COV2 vaccine has been designed to minimize this risk: first, 
the choice of a prefusion-stabilized SARS-Cov-2 S-protein protein is meant to maximize the induction of 
properly neutralizing antibodies, and second, the adenoviral vectored vaccine is a potent inducers of Th1 
skewed immune responses that are known to prevent or blunt inflammatory and allergic immune responses. 
Nevertheless, as recommended by the International Coalition of Medicines Regulatory Authorities (ICMRA) 
in a meeting held on March 18 2020 (Global regulatory workshop on COVID-19 vaccine development, 
http://www.icmra.info/drupal/news/March2020/summary), this potential risk has been clearly stated in the 
ICF.  
During the study follow up, as an additional safety measure, extra-visits are foreseen in case any subject 
develops febrile or respiratory symptoms. If the investigator will consider these symptoms compatible with 
COVID-19, the subject will be assessed for the presence of SARS-CoV-2 infection in respiratory tract samples. 
This measure is meant to ensure that potential exposure to SARS-CoV-2 in vaccinated subjects will be 
properly monitored and scored to assess the occurrence of any vaccine-mediated disease enhancement. 
15.2.  Known Potential Benefits 
Volunteers will not benefit directly from participation in this study. However, it is hoped that the information 
gained from this study will contribute to the development of a safe and effective SARS-CoV-2 vaccine regime. 
The only benefits for participants would be information about their general health status. 

41 
 
15.3. Declaration of Helsinki 
The Investigators will ensure that this study is conducted according to the principles of the current revision 
of the Declaration of Helsinki. 
15.4. Guidelines for Good Clinical Practice 
The Investigators will ensure that this study is conducted in full conformity with the Good Clinical Practice 
(GCP). 
15.5. Approvals 
The protocol, ICF, participant information sheet and any proposed advertising material will be submitted to 
an appropriate Ethics Committee and Italian regulatory authorities (ISS and AIFA). 
The Investigator will submit and, where necessary, obtain approval from the above parties for all substantial 
amendments to the original approved documents. 
No substantial amendments to this protocol will be made without consultation with, and agreement of, the 
Sponsor and Co-sponsor. Any substantial amendments to the trial that appear necessary during the course 
of the trial must be discussed by the Investigator and Sponsor concurrently. If agreement is reached 
concerning the need for an amendment, it will be produced in writing by the Principal Investigator and will 
be made a formal part of the protocol following ethical and regulatory approval. 
15.6. Informed consent and participant confidentiality 
All participants will sign and date the informed consent form before any study specific procedures are 
performed. At the screening visit, the participantwill be fully informed of all aspects of the trial, the potential 
risks and the time to be spent for attending follow-up visit and the restriction to personal activities. The 
following general principles will be emphasized: 
A. Participation in the study is entirely voluntary 
B. Refusal to participate involves no penalty or loss of medical benefits 
C. The participant may withdraw from the study at any time 
D. The participants free to ask questions at any time to allow him or her to understand the purpose of 
the study and the procedures involved 
E. The study involves research of an investigational vaccine 
F. The study involves genetic analysis for characterization of HLA type 
G. There is no direct benefit from participating 
H. The volunteer’s GP will be contacted to corroborate their medical history 
I. 
The volunteer’s blood samples taken as part of the study will be stored indefinitely and samples may 
be sent outside of the Italy and Europe to laboratories in collaboration with the INMI Lazzaro 
Spallanzani. These will be anonymized. 
J. The aims of the study and all tests to be carried out will be explained.  
The participant will be given the opportunity to ask other questions about the trial, and will then have time 
to consider whether or not to participate. If they do decide to participate, they will sign and date two copies 
of the consent form (paper document), one for them to take away and keep, and one to be stored at clinical 
center. 
16. Financing and Insurance 
16.1. Financing 
The clinical trial is funded by ReiThera SRL and IRCCS INMI Spallanzani. IRCCS INMI Spallanzani owns the 
property of the IMP lot #RL20-0024. 

42 
 
16.2. Insurance 
Indemnity and/or compensation for harm arising specifically from an accidental injury and occurring as a 
consequence of the research participant’s participation in the trial for which ReiThera is the research Sponsor 
will be covered by a no-fault compensation policy with HDI Global SE – DPT. SPECIAL LINES. 
16.3. Participants compensation 
Participants will be compensated for their time and for the inconvenience caused by procedures.  
17. Expected results and publication policy 
Publication of the results of the trial will be agreed between ReiThera SRL and IRCCS INMI Spallanzani. If one 
of the parties is interested in proceeding with a scientific publication that covers this clinical trial as a whole 
or in part, it must give written communication, also electronically, to the other party involved. In order to 
ensure that any patent protection opportunities or other interests, including national ones, are not affected. 
Any impediments to publication must also be communicated electronically within 30 days of receipt of the 
communication of publication of the interested party. If a party deems it inappropriate to publish, it will still 
try to provide written changes and / or additions to the text that could allow its publication, albeit in a 
reduced version. All publications covering this clinical trial as a whole or in part must report the affiliation of 
all the parties involved and the recognition of the authors involved. The Authors’ list will be defined on the 
basis of the principles contained in the document Uniform Requirements for Manuscripts submitted by 
Biomedical Journals. 
 

43 
 
18. Reference 
 
 
1 European Centre for Disease Prevention and Control. Outbreak of novel coronavirus disease 2019 (COVID-
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