Placebo-Controlled, Randomized Trial of Ensitrelvir (S-217622) for Viral Persistence and Inflammation in People Experiencing Long COVID (PREVAIL-LC)
Phase: PHASE2
Conditions: Long COVID, Post Acute Sequelae of COVID-19, Post-Acute COVID-19
Sponsor: Timothy Henrich

 
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO 
 PLACEBO-CONTROLLED, RANDOMIZED TRIAL OF ENSITRELVIR (S-217622) FOR VIRAL 
PERSISTENCE AND INFLAMMATION IN PEOPLE EXPERIENCING LONG COVID 
(PREVAIL-LC)  
NCT06161688 
Protocol Version Date: 12/05/2024 

Protocol Number 23-38752 
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UNIVERSITY OF CALIFORNIA, SAN FRANCISCO 
 
Clinical Research Protocol 
 
PLACEBO-CONTROLLED, RANDOMIZED TRIAL OF ENSITRELVIR (S-217622) FOR VIRAL 
PERSISTENCE AND INFLAMMATION IN PEOPLE EXPERIENCING LONG COVID (PREVAIL-LC) 
 
Protocol Number: 
23-38752 
Version Date: 
2024-DEC-05 
Investigational Product: 
ENSITRELVIR (S-217622) 
IND Number: 
TBD 
Development Phase: 
Exploratory 
Sponsor: 
University of California, San Francisco 
Funding Organization: 
University of California, San Francisco 
Principal Investigator: 
Name: Timothy J. Henrich, MD, MMSc 
 
 
 
 
Approval: 
 
 
 
December 5th, 2024 
PI or Sponsor Signature (Name and Title)  
 
Date 
This confidential information about an investigational product is provided for the 
exclusive use of investigators of this product and is subject to recall at any time. 
The information in this document may not be disclosed unless federal or state law 
or regulations require such disclosure. Subject to the foregoing, this information 
may be disclosed only to those persons involved in the study who have a need to 
know, with the obligation not to further disseminate this information.  

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PROTOCOL AGREEMENT 
 
I have read the protocol specified below. In my formal capacity as Investigator, my duties include 
ensuring the safety of the study subjects enrolled under my supervision and providing University of 
California, San Francisco with complete and timely information, as outlined in the protocol. It is 
understood that all information pertaining to the study will be held strictly confidential and that this 
confidentiality requirement applies to all study staff at this site. Furthermore, on behalf of the study staff 
and myself, I agree to maintain the procedures required to carry out the study in accordance with 
accepted GCP principles and to abide by the terms of this protocol. 
 
Protocol Number:  
 
Protocol Title:  
PLACEBO-CONTROLLED, RANDOMIZED TRIAL OF ENSITRELVIR (S-217622) FOR VIRAL 
PERSISTENCE AND INFLAMMATION IN PEOPLE EXPERIENCING LONG COVID (PREVAIL-LC) 
 
Protocol Date: 2024-FEBRUARY-21 
 
 
 
December 5th, 2024 
Investigator Signature  
 
Date 
Timothy J. Henrich, MD  Professor of Medicine 
Print Name and Title 
Site # 
1 
 
Site Name 
University of California San Francisco 
Address 
 
 
 
 
 
 
 
Phone Number 
 

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TABLE OF CONTENTS 
1 
BACKGROUND 
14 
1.1 Overview of Pre-Clinical & Clinical Studies ......................................................................... 14 
2 
STUDY RATIONALE 
15 
2.1 Hypothesis .............................................................................................................................. 15 
2.2 Program Overview (LIINC Cohort) ....................................................................................... 15 
2.3 Risk/Benefit Assessment ........................................................................................................ 16 
3 
STUDY OBJECTIVES 
17 
3.1 Primary Objective ................................................................................................................... 17 
3.2 Secondary Objectives ............................................................................................................. 17 
4 
STUDY DESIGN 
17 
4.1 Study Overview ...................................................................................................................... 17 
5 
CRITERIA FOR EVALUATION 
18 
5.1 Efficacy Endpoints ................................................................................................................. 18 
5.2 Safety Evaluations .................................................................................................................. 18 
6 
SUBJECT SELECTION 
18 
6.1 Study Population .................................................................................................................... 18 
6.2 Inclusion Criteria .................................................................................................................... 18 
6.3 Exclusion Criteria ................................................................................................................... 20 
7 
CONCURRENT MEDICATIONS 
22 
7.1 Allowed Medications and Treatments .................................................................................... 22 
8 
STUDY TREATMENTS 
22 
8.1 Method of Assigning Subjects to Treatment Groups ............................................................. 22 
8.2 Blinding .................................................................................................................................. 22 
8.3 Formulation of Test and Control Products ............................................................................. 23 
8.4 Supply of Study Drug at the Site ............................................................................................ 23 
8.5 Supply of Study Drug at the Site ............................................................................................ 23 
8.6 Study Drug Accountability ..................................................................................................... 24 
8.7 Measures of Treatment Compliance ....................................................................................... 24 
9 
STUDY PROCEDURES AND GUIDELINES 
24 
9.1 Clinical Assessments .............................................................................................................. 24 
9.2 Clinical Laboratory Measurements ........................................................................................ 26 
9.3 Research Laboratory Measurements ...................................................................................... 27 
9.4 Biospecimens .......................................................................................................................... 27 
10 EVALUATIONS BY VISIT 
28 
10.1 Screening Visit (D-28) ....................................................................................................... 28 
10.2 Baseline Evaluation (D-21 to D0) ...................................................................................... 28 
10.3 Intervention (D0) ................................................................................................................ 28 
10.4 Telephone Follow-ups (Phone Follow-Up, D1, D4) .......................................................... 29 
10.5 In Person Follow-up Visits (D10, D30) ............................................................................. 29 
10.6 Telephone or In Person End-Of-Study Visit (EOS D60) ................................................... 29 

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10.7 Unscheduled (Interim) Visits ............................................................................................. 29 
10.8 Study Discontinuation Visit ............................................................................................... 29 
11 ADVERSE EXPERIENCE REPORTING AND DOCUMENTATION 
29 
11.1 Adverse Events................................................................................................................... 29 
11.2 Serious Adverse Experiences (SAE) or Serious SAR ........................................................ 31 
11.3 Liver Toxicity..................................................................................................................... 32 
11.4 Study Drug Overdose ......................................................................................................... 32 
12 DISCONTINUATION AND REPLACEMENT OF SUBJECTS 
33 
12.1 Early Discontinuation ......................................................................................................... 33 
12.3 Withdrawal of Subjects from the Study ............................................................................. 33 
12.4 Replacement of Subjects .................................................................................................... 33 
12.5 Lost to Follow Up .............................................................................................................. 34 
13 PROTOCOL VIOLATIONS 
34 
14 DATA SAFETY MONITORING 
34 
15 STATISTICAL METHODS AND CONSIDERATIONS 
35 
15.1 Data Sets Analyzed ............................................................................................................ 35 
15.2 Demographic and Baseline Characteristics ........................................................................ 35 
15.3 Analysis of Primary Endpoint ............................................................................................ 35 
15.4 Analysis of Secondary Endpoints ...................................................................................... 35 
15.5 Sample Size and Randomization ........................................................................................ 36 
16 DATA COLLECTION, RETENTION AND MONITORING 
36 
16.1 Data Collection Instruments ............................................................................................... 36 
16.2 Data Management Procedures ............................................................................................ 37 
16.3 Data Quality Control and Reporting .................................................................................. 37 
16.4 Archival of Data ................................................................................................................. 37 
16.5 Availability and Retention of Investigational Records ...................................................... 37 
16.6 Monitoring ......................................................................................................................... 37 
16.7 Subject Confidentiality ....................................................................................................... 37 
17 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS 
38 
17.1 Protocol Amendments ........................................................................................................ 38 
17.2 Institutional Review Boards and Independent Ethics Committees .................................... 38 
17.3 Informed Consent Form ..................................................................................................... 38 
17.4 Publications ........................................................................................................................ 39 
17.5 Investigator Responsibilities .............................................................................................. 39 
18 REFERENCES 
40 
 

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LIST OF ABBREVIATIONS 
AE 
Adverse Event 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
BUN 
Blood Urea Nitrogen 
CFR 
Code Of Federal Regulations 
COVID-19 
Coronavirus Disease 2019 
CRF 
Case Report Form 
CRP 
C-Reactive Protein 
DMC 
Data Monitoring Committee 
DSMB 
Data Safety Monitoring Board 
ESR 
Erythrocyte Sedimentation Rate 
FDA 
Food And Drug Administration 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act Of 1996 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
IV 
Intravenous 
LIINC 
Long-term Impact of Infection with Novel Coronavirus 
mEq 
Milliequivalent 
mITT 
Modified Intention-to-Treat 
PI 
Principal Investigator 
PK 
PRN 
Pharmacokinetic 
Pro Re Nata 
SAE 
Serious Adverse Experience 
SAP 
Statistical Analysis Plan 
SAR 
Suspected Adverse Reaction 
SARS-CoV-2 
Severe Acute Respiratory Syndrome Coronavirus 2 
SD 
Standard Deviation 
SMC 
Safety Monitoring Committee 

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PROTOCOL SYNOPSIS (TO BE COMPLETED LAST) 
TITLE 
PLACEBO-CONTROLLED, RANDOMIZED TRIAL OF ENSITRELVIR 
(S-217622) FOR VIRAL PERSISTENCE AND INFLAMMATION IN 
PEOPLE EXPERIENCING LONG COVID (PREVAIL-LC) 
 
 
SPONSOR 
University of California, San Francisco 
FUNDING 
ORGANIZATION 
University of California, San Francisco 
NUMBER OF SITES 
1 
 
 
RATIONALE 
There is growing evidence that viral antigen persistence and immune 
dysregulation are primary drivers of Long COVID (LC) symptoms. 
Recent studies have demonstrated viral persistence in a subset of 
individuals with LC, including prolonged gastrointestinal shedding, 
ongoing Spike and Nucleocapsid antigen in neuronal and astrocytic 
exosomes, and evidence of persistent viral RNA or proteins in blood 
and in deep tissues. Furthermore, we and others have recently showed 
that individuals with LC have elevated levels of inflammation, 
particularly IL-6, IL-1beta and TNF-alpha, while other groups have 
implicated excess clotting as a mechanism for end-organ disease. 
Theoretically, virus persistence may be driving inflammation and 
clotting, leading to the diffuse tissue damage and the symptoms which 
we associate with Long COVID. If this model is correct, then blocking 
virus replication will result in reduced tissue damage, either directly or 
indirectly, and resolution of symptoms. Recent anecdotal experience 
with readily available antiviral drugs (e.g., Paxlovid) in people with LC 
provide support for this concept. The finding that LC is less common in 
people who were vaccinated prior to infection is also indirect evidence 
for this model.  
Ensitrelvir (S-217622), an investigational 3CL protease inhibitor 
administered daily, has been shown to result in rapid clearance of 
SARS-CoV-2 during acute infection. The agent was well tolerated 
with few discontinuations due to adverse events and no serious 
adverse events. 
STUDY DESIGN 
Randomized, double-blind, placebo-controlled study. 
20 participants will receive 5 days of oral ensitrelvir and 20 
participants will receive 5 days of oral placebo. 
 
Following study unblinding, participants that received placebo will 
have the option of a 5 day course of ensitrelvir with repeat safety, 
clinical and laboratory measures. 
PRIMARY OBJECTIVE 
To evaluate the safety and tolerability of oral ensitrelvir in individuals 
who are experiencing Long COVID.  

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SECONDARY 
OBJECTIVES 
1. Describe the ability of ensitrelvir to affect virologic markers of 
chronic SARS-CoV-2 infection. 
2. Describe the ability of ensitrelvir to improve PASC outcomes. 
3. Describe the ability of ensitrelvir to reduce post-COVID 
inflammation. 
NUMBER OF 
SUBJECTS 
40 
SUBJECT SELECTION 
CRITERIA 
INCLUSION CRITERIA 
1. 
Age ≥18 and <70 years at Screening. 
2. 
Enrolled or willing to enroll and complete at least 1 visit in the 
UCSF Long-term Impact of Infection with Novel Coronavirus 
(LIINC) study. Any adult who has been infected with SARS-CoV-
2 or has ever received or is eligible to receive a SARS-CoV-2 
vaccination, and who is able to provide written informed consent, 
is eligible to participate in LIINC.  
3. 
History of at least one SARS-CoV-2 infection, defined as report 
of a positive nucleic acid amplification test (NAAT) and/or a 
positive SARS-CoV-2 antigen rapid diagnostic test (RDT). 
Written proof of the test will be requested but is not required as 
long as the participant attests to the positive test.  
4. 
Clinical evidence of Long COVID, as confirmed by the 
investigator’s assessment. 
a. At least two moderate symptoms or at least one 
severe symptom as assessed by the study team (see 
list) that are new or worsened since the time of a 
SARS-CoV-2 infection, not known to be attributable to 
another cause upon assessment by the PI. Symptoms 
from those listed here must be present: systemic 
symptoms (e.g., fatigue, chills, post-exertional 
malaise), neurocognitive symptoms (e.g., trouble with 
memory/concentration (“brain fog”), headache, 
dysautonomia/postural orthostatic tachycardia 
syndrome, dizziness, unsteadiness, neuropathy, sleep 
disturbance), cardiopulmonary symptoms (e.g., chest 
pain, palpitations, shortness of breath, cough, fainting 
spells), musculoskeletal symptoms (e.g., muscle 
aches, joint pain), gastrointestinal symptoms (e.g., 
nausea, diarrhea). Although other symptoms (e.g., 
skin rash, hair loss, mental health symptoms, trouble 
with smell/taste, genitourinary symptoms) will be 
recorded and tracked, at least two core symptoms 
listed above must be present. Note: the two symptoms 
can be from within the same category (for example, 
brain fog and headache).  
AND 

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b. Symptoms must have been present for at least 60 
days prior to screening. Symptoms that wax and wane 
must have been initially present at least 60 days prior 
to screening. 
AND 
c. Symptoms must be reported to be at least somewhat 
bothersome and to have an impact on quality of life 
and/or everyday functioning. 
AND 
d. At least 90 days have elapsed since the most recent 
suspected or confirmed SARS-CoV-2 infection and the 
time of screening. Note: suspected infections will be 
determined based upon assessment by the study 
investigators. 
5. 
Not currently hospitalized. 
6. 
Body mass index (BMI) 18 to 50 kilograms/meter squared 
(kg/m2), inclusive, at the time of screening. 
7. 
In otherwise stable health, as assessed by the investigator within 
28 days prior to Screening, based on medical history, physical 
assessment, laboratory findings, and vital signs. 
8. 
For Male participants, 
Participants with partners that are women of childbearing 
potential (WOCBP) are strongly advised to inform their partners 
and must agree to use effective contraception from study entry 
(defined as D0) through 14 days after the last dose of study 
intervention. Effective methods of contraception are described in 
Appendix 2. Participants with pregnant partners must agree to 
use condoms during vaginal intercourse from study entry 
(defined as D0) through 14 days after the last dose of study 
intervention administration. Participants assigned male sex at 
birth must agree to refrain from sperm donation from study entry 
through 14 days after the last dose of study intervention 
administration. 
9. 
For Female participants, 
A female participant is eligible to participate if she is not pregnant 
or breastfeeding, and the following conditions applies: 
 
Is not a WOCBP 
OR 
 
All of the following apply: 
1. 
Is a WOCBP and using a contraceptive method that is effective 
as described in Appendix 2 from –21 days from study entry 
(defined as D0), during the study intervention period, and for at 
least 14 days after the last study intervention administration. She 

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must also agree not to donate her eggs (ova, oocytes) for the 
purpose of reproduction from study entry (defined as D0), during 
the study intervention period, and for at least 14 days after the 
last study intervention administration. (Of note, participants 
taking hormonal contraception will be informed that there may be 
a possibility of decreased efficacy or contraceptive failure while 
on study drug.) 
 
A WOCBP must have a negative urine pregnancy test 
within 24 hours before the first dose of study 
intervention. If a urine pregnancy test cannot be 
confirmed as negative (e.g., an ambiguous result), a 
serum pregnancy test must be negative. 
10. Willingness and ability to comply with the study protocol. This 
includes reliable transportation and sufficient time to attend all 
visits. 
11. Written informed consent (and assent when applicable) obtained 
from subject or subject’s legal representative and ability for 
subject to comply with the requirements of the study. 
EXCLUSION CRITERIA 
1. 
Previously received COVID-19 convalescent plasma treatment or 
a SARS-CoV-2-specific mAb within 60 days prior to planned Day 
0 or plan to receive such treatment before exiting the study. 
2. 
Previously received a SARS-CoV-2 antiviral within 60 days prior 
to planned Day 0 or plan to receive such treatment before exiting 
the study. 
3. 
Plans to receive any investigational or approved vaccine or 
booster for SARS-CoV-2 within 60 days prior to planned Day 0 or 
before Day 30 following planned Day 0. 
4. 
Active cardiovascular disease, defined as known: 
a. Myocardial infarction within 90 days of screening 
OR 
b. Coronary artery bypass procedure within 90 days of 
screening 
OR 
c. Current heart failure with reduced ejection fraction 
(<45%)  
OR 
d. Current pulmonary arterial hypertension. 
5. 
Known stroke within 3 months prior to planned Day 0. 
6. 
Known active bacterial, fungal, viral, or other infection besides 
SARS-CoV-2 requiring treatment within the 28 days prior to 
planned Day 0 and meeting criteria for systemic involvement 
upon review by the PI. Note: Mild or limited infections such as 

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uncomplicated urinary tract or yeast infections, sexually 
transmitted infections, and mild dermatophyte infections may be 
reviewed with the study PI and/or Safety Monitoring Committee 
chair, but are not exclusionary. 
7. 
Major surgery within 6 months prior to planned Day 0 or planned 
major surgery during the first 60 days following planned Day 0. 
8. 
History of unplanned hospitalization for >24 hours within 28 days 
prior to Screening. 
9. 
Active Hepatitis B (Hep B) infection (defined as Hep B surface 
antigen (sAg) positive). Note: A known positive Hep B core 
antibody (cAb) in the absence of positive sAg is not considered 
exclusionary. 
10. Active Hepatitis C (Hep C) infection (defined as Hep C Ab 
positive or indeterminate with detectable Hep C RNA). Note: 
Those with cured Hep C (Ab positive or indeterminate but 
negative Hep C RNA) will remain eligible. 
11. Laboratory abnormalities at Screening, specifically including: 
a. ANC <1500 per mm3 
b. Platelet count <100,000 per mm3 
c. AST/ALT > 1.5x ULN 
d. CrCl < 30 mL/min 
e. Hemoglobin <9 g/dL 
12. Known HIV infection.  
13. End stage kidney disease requiring dialysis. 
14. Severe hepatic impairment (Child-Pugh Class C). 
15. Moderate or severe immunocompromise, according to the 
current NIH COVID-19 Treatment Guidelines as of March 6, 
2023. The detailed list is in Appendix 2, and includes the 
following: (a) receiving active treatment for solid tumor or 
hematologic malignancy, including use of systemic 
chemotherapy for treatment of cancer within the year prior to 
screening, (b) prior solid-organ transplant with active 
immunosuppressive therapy, (c) CAR-T cell therapy or 
hematopoietic cell transplant, on immunosuppressive therapy or 
transplant within the prior 2 years, (d) primary immunodeficiency 
syndromes, advanced or untreated HIV infection (any HIV 
infection is exclusionary), (f) on active high-dose corticosteroids 
(i.e., >= 20mg prednisone or equivalent daily per day for >= 2 
weeks) at the time of screening.  
16. Known prior diagnosis of myalgic encephalomyelitis/chronic 
fatigue syndrome (ME/CFS), preceding and not related to SARS-
CoV-2 infection and not worsened since SARS-CoV-2 infection. 
17. Known prior diagnosis of dysautonomia, preceding and not 
related to SARS-CoV-2 infection and not worsened since SARS-
CoV-2 infection. 

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18. Pregnant, breastfeeding, or unwilling to practice birth control 
abide by the contraception requirements outlined in the inclusion 
criteria. 
19. Participation in a clinical trial with receipt of an investigational 
product within 28 days or 5 half-lives (whichever is longer) prior 
to planned Day 0. The following exceptions will apply: 
a. If the investigational product is a SARS-CoV-2-specific 
monoclonal antibody, convalescent plasma, or 
equivalent (e.g., IVIG), refer to Criterion #1 (60 days 
must have elapsed prior to planned D0).  
b. If the investigational product is a PET imaging tracer 
that is not expected to have significant biological 
activity, has a biological half-life less than 24 hours 
and radiological half-life less than 6 hours; more than 
14 days must have elapsed between receipt of the 
tracer and planned Day 0. 
20. Current alcohol or illicit drug use as determined by the 
investigator to preclude participation. 
21. Presence of a condition or abnormality that in the opinion of the 
Investigator would compromise the safety of the patient or the 
quality of the data. 
22. Study site personnel directly affiliated with the study or family of 
directly involved personnel. 
23. Known allergy to any components used in the formulation of the 
intervention. 
24. Current use of or anticipated need for any medications prohibited 
with study intervention, as described in Appendix 3.  
25. Participants who have used any of the following drugs within 14 
days prior to enrollment: 
 
Strong cytochrome P450 (CYP) 3A inducer 
2. 
Products containing St. John’s wort  
TEST PRODUCT, 
DOSE, AND ROUTE OF 
ADMINISTRATION 
Ensitrelvir Fumaric Acid (S-217622) given orally 375 mg on day 1 
followed by 125 mg daily for 4 additional days 
CONTROL PRODUCT, 
DOSE AND ROUTE OF 
ADMINISTRATION 
Placebo, given orally daily for 5 days 

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DURATION OF 
SUBJECT 
PARTICIPATION AND 
DURATION OF STUDY 
Screening: up to 28 days 
Treatment: 5 days 
Follow-up: 60 days 
 
For optional cross-over from placebo to study drug arm following 
main study unblinding: 
Treatment: 5 days 
Follow-up: 60 days 
CONCOMMITANT 
MEDICATIONS 
All concomitant medication and concurrent therapies taken within 28 
days prior to Screening will be documented. Dose, route, unit 
frequency of administration, and indication for administration and 
actual or estimated dates of medication will be captured. 
Details regarding all SARS-CoV-2 vaccinations and/or boosters, as 
well as COVID-specific therapies, will be collected. 
EFFICACY 
EVALUATIONS 
• 
The change in patient-reported outcomes (PROs) PROMIS-29 
score from Baseline to D30.  
PRIMARY ENDPOINT 
• 
Safety and tolerability. 
SECONDARY 
ENDPOINTS 
• 
Change in other assessments (EuroQoL Quality of Life, 
neurocognitive assessment, DASI, 6MWT performance) between 
Baseline and at D15, D30, and D60. 
 
OTHER EVALUATIONS 
• 
Percentage of participants with no detection of SARS-CoV-2 
plasma remnants (i.e., viral detection by reverse transcriptase-
polymerase chain reaction ((RT-PCR) and Spike protein 
fragments) compared to baseline and at D15 and D30 post 
administration. 
• 
Proportion with reduction in inflammatory markers (e.g., 
interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha) at 
Baseline and at D15 and D30. 
SAFETY 
EVALUATIONS 
Number of participants with adverse events (AE)s (Treatment 
Emergent Adverse Events (TEAE)s, Serious Adverse Events (SAE)s, 
and Adverse Events of Special Interest (AESI)). The primary outcome 
will be the number of individuals in each group experiencing a severe 
(Grade 3 or greater) AE thought to be possibly, probably, or definitely 
related to study treatment throughout the study duration. 
PLANNED INTERIM 
ANALYSES  
Approximately 3 months after enrollment of the first participant and 
then every 3 months thereafter, the Independent Medical Monitor 
(IMM) will review accrual (including screening and enrollment), AE 
summaries, including all reported Grade ≥3 AEs, retention of 
participants including off-study rates. 
Serious adverse events will be monitored by the IMM on an ongoing 
basis throughout the study. 

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STATISTICS 
Primary Analysis Plan 
Eligible patients who are randomized into the study and receive at 
least one dose of the study drug during initial blinded, placebo-
controlled phase will comprise the modified intent to treat (mITT) 
population and will be the primary analysis population for all 
analyses.  
Exploratory analyses will include mITT population with addition of 
participants original assigned to placebo who opt to undergo 
ensitrelvir therapy following unblinding in the optional cross-over 
stage. 
Safety and tolerability data will be summarized by treatment group.  
Adverse event rates will be coded by body system. Adverse events 
will be tabulated by treatment group and will include the number of 
patients for whom the event occurred, the rate of occurrence, and the 
severity and relationship to study drug. Adverse events between 
groups will be summarized by proportions with associated 95% 
Clopper-Pearson confidence intervals and will be compared using a 
two-sided 0.05 level Fisher exact test.  
Rationale for Number 
of Subjects 
This is an exploratory study, and the primary results will be 
descriptive. We will use ensitrelvir as a probe to better understand LC 
biology and determine whether a signal is present to warrant further 
study. 
For PROMIS-29, assuming a standard deviation (SD) of the within-
person difference of 10, then the planned sample size will have 80% 
power to detect a difference of 11.2 on a two-sided 0.05 level test.  
For biomarkers, such as IL-6, we will be able to detect a difference 
from a mean value of 2.5 pg/mL to 1.1 times the within-person 
standard deviation with 80% power. 

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1 
BACKGROUND 
Coronavirus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Coronavirus type 2 
(SARS-CoV-2), a ribonucleic acid (RNA) virus which emerged in Wuhan, China, in December 2019 and 
spread across the world at an unprecedented pace through human-to-human transmission. The spike 
protein of the 2019 novel coronavirus is a surface protein which binds to angiotensin-converting 
enzyme-2 (ACE-2) on human cells. The S1 subunit catalyzes attachment to ACE-2 and the S2 subunit 
allows fusion with cell membranes and subsequent entry into the cell. As a result, the spike protein is a 
relevant target for drug development. Moreover, antibodies (Abs) against the spike protein are able to 
neutralize the virus, prevent infection, and reduce the severity of disease.  
The COVID-19 pandemic has resulted in a growing population of individuals recovering from SARS-
CoV-2 infection. Approximately one in five American adults who have had COVID-19 still have 
symptoms of Long COVID, a type of post-acute sequelae of SARS-CoV-2 infection (PASC).1 Some 
aspects of this recovery may be unique to COVID-19, but many appear to be similar to recovery from 
other viral illnesses, critical illness, and/or sepsis.2 LC is comprised of a broad range of symptoms that 
develop during or after COVID-19, continue for ≥2 months (i.e., three months from the onset of illness), 
have an impact on the patient's life, and are not explained by an alternative diagnosis. Consensus 
around the clinical definition of LC has progressed significantly, and as of October 1, 2021, there has 
been an International Classification of Diseases, Tenth Revision, Clinical Modification, (ICD-10), for 
unspecified post-COVID conditions (U09.9). The World Health Organization (WHO) has also created a 
global COVID-19 clinical platform CRF for clinicians and patients to collect and report information.3 The 
United States (US) Department of Health and Human Services (HHS) and the Department of Justice 
(DoJ) released a guidance statement on LC as a disability under the Americans with Disabilities Act, 
the Rehabilitation Act of 1973, and the Patient Protection and Affordable Care Act. 
Persistent viral infection with viral reservoirs after the initial acute illness is one potential pathogenic 
mechanism for Long COVID.4 Theoretically, virus persistence may be driving inflammation and clotting, 
leading to the diffuse tissue damage and the symptoms which we associate with Long COVID. If this 
model is correct, then blocking virus replication will result in reduced tissue damage, either directly or 
indirectly, and resolution of symptoms. Recent anecdotal experience with readily available antiviral drugs 
(e.g., Paxlovid) in people with LC provide support for this concept.5 The finding that LC is less common 
in people who were vaccinated prior to infection is also indirect evidence for this model. Therefore, this 
trial will study the safety and efficacy of S-217622, an investigational 3CL protease inhibitor to treat 
individuals with Long COVID in an adult population. 
1.1 
Overview of Pre-Clinical & Clinical Studies 
Ensitrelvir fumaric acid (S-217622; hereafter, ensitrelvir) is a novel oral SARS-CoV-2 3C-like protease 
inhibitor which has shown to have antiviral efficacy against many SARS-CoV-2 variants, including 
Omicron subvariants.6-9 A phase 1 study demonstrated that one-daily oral doing was well tolerated with 
favorable pharmacokinetics (c). In phase 2a study, ensitrelvir led to a reduction in SARS-CoV-2 RNA 
levels and viral titers compared to placebo in acutely infected individuals.10 Subsequent data from the 
phase 2b part of a phase 2/3 study in 341 participants the change from baseline in SARS-CoV-2 titers 
on day 4 as significantly greater in both of the oral Ensitrelvir dosing arms [125 mg (375 mg on day 1) 
or 250 mg (750 mg on day 1) once daily for 5 days] compared with placebo (-0.41 log10 TCID50) and a 
faster time-weighted average change from baseline to 120 hours in some acute and respiratory 
symptoms.11 Adverse events were generally mild and included headache, diarrhea and a transient 
increase in HDL (which was more pronounced in the higher dosage group)11. In a post-hoc analysis, 
post hoc analysis, compared with placebo, ensitrelvir demonstrated a reduced time to resolution of 5 
symptoms in patients with mild-to-moderate COVID-19.8  

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2 
STUDY RATIONALE 
Early in the pandemic, the common assumption was that SARS-CoV-2 would prove to be a transient 
infection, as is the case with coronaviruses in general. This assumption was challenged by early reports 
that viral nucleic acid and proteins could be detected in the gut mucosa months after infection.12 Case 
reports emerged indicating that immunocompromised individuals including those with advanced 
malignancy, Human immunodeficiency virus (HIV)/Acquired Immunodeficiency Syndrome (AIDS), or on 
immunosuppression for autoimmune conditions, can harbor active replicating virus for many months.13-
16 More recently, similar observations have been made in immunocompetent people.16-21 Autopsy 
studies of people post-COVID dying from related or unrelated reasons also began to report presence of 
viral nucleic acid or protein in various tissues months after the infection was apparently cleared.20 
Another provocative study demonstrated SARS-CoV-2 in neonatal stool following remote maternal 
COVID-19.22 Taken together these studies provide growing support for SARS-CoV-2 persistence. 
SARS-CoV-2 can infect several cell types and in theory any infected cell is at risk of harboring 
persistent virus. The precise localization of SARS-CoV-2 persistence is unknown, but it is widely 
assumed that this occurs in tissues. This may or may not include immune-privileged sites as has been 
observed with other ribonucleic acid (RNA) viruses (e.g. Ebola).23 Because tissue studies are generally 
impractical due to invasiveness of these procedures, there are now efforts to develop less-invasive 
measures to assess this. Some studies identified detectable SARS-CoV-2 RNA in plasma and stool 
during the early post-acute phase.24 One recent study found that a large proportion of individuals with 
LC had at least intermittently detectable circulating antigen in the plasma for up to a year post-
infection.25 However, it remains unclear whether antigen can also persist in asymptomatic individuals, 
whether what is being detected represents remnants of a long extinguished infection or ongoing virus 
production from a long-lived reservoir or ongoing replication. 
2.1 
Hypothesis 
We hypothesize that tissue viral persistence contributes to LC pathophysiology, and that administration 
of oral ensitrelvir to individuals experiencing LC will result in symptom reduction as well as improvement 
in biomarkers of inflammation in comparison to those receiving placebo.  
2.2 
Program Overview (LIINC Cohort) 
This study will occur within the Long-term Impact of Infection with Novel Coronavirus (LIINC) cohort,26 
an observational study of post-COVID conditions that has been ongoing at UCSF since April 2020. 
Since that time, LIINC has been characterizing the natural history and biology of LC. The cohort has 
recruited over 700 individuals, of whom over 350 report mild-to-severe LC symptoms. The retention 
rate at 1 year is 89%. Individuals are evaluated at scheduled visits and biological specimens (plasma, 
serum, peripheral blood mononuclear cells (PBMCs)) are collected and stored. A subset of the cohort is 
referred for additional studies including advanced cardiopulmonary (echocardiogram, cardiopulmonary 
exercise testing, cardiac magnetic resonance imaging (MRI), pulmonary function tests, and tilt table 
tests) neurologic (neuropsychiatric testing, lumbar puncture), imaging (fluorodeoxyglucose-positron 
emission tomography/computed tomography (FDG-PET/CT), and tissue assessment (gut biopsy, lymph 
node fine needle aspiration). To date, the LIINC team has conducted over 3000 participant-encounters 
and have enrolled over 200 individuals into the more intensive protocols. LIINC has also described the 
relationship between LC and adaptive immune responses,27 inflammation,28-30 autoimmunity,31,32 
microbial translocation,33 neurocognitive changes,34,35 and cardiopulmonary physiology.29,36 The LIINC 
team has for decades conducted investigator-initiated clinical trials as part of the associated HIV 
program (SCOPE) and has the capacity to implement the study described in this protocol using 
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Potential participants will be required to enroll in LIINC, and the trial participants will be recruited 
directly from LIINC if they have had LC subsequent to an acute COVID episode  
2.3 
Risk/Benefit Assessment  
The potential benefits to participants and society are likely to outweigh the minimal risks from 
participation in the study. 
2.3.1 Potential Risks 
Confidentiality. Participation in research may involve loss of privacy. Participants’ records will be 
handled as confidentially as possible. All research records will be coded with a four-digit study 
identification (ID) code. Only the study investigators and their staff will have access to study records 
and test results. Study charts will be kept in a locked file cabinet in a locked office. Electronic data will 
be protected with a password and kept on a secure network. All collaborators will receive specimens 
only identified by the four-digit study ID. No individual identities will be used in any reports or 
publications resulting from this study. 
Phlebotomy. Drawing blood from a vein may cause some discomfort, bleeding, or bruising where the 
needle enters the skin, and rarely, fainting or infection may occur. No more than 500 mL (2 cups) of 
blood will be drawn over any two-month period. This is within Red Cross guidelines. Risks of blood 
collection include anemia (low blood counts). Symptoms of anemia include tiredness, weakness, and 
dizziness. Participants will be checked for signs and symptoms of anemia at each visit, and 
CBC/diff will be checked as outlined in the schedule of events. If the investigator feels that a participant 
is at significant risk for anemia, the amount of blood collected will be reduced. 
Nasal or oral swab. These collections may cause temporary discomfort. 
Viral testing. Testing for viruses such as SARS-CoV-2 and viral hepatitis and testing may be performed 
as part of this study. Being tested for these infections may cause anxiety, regardless of the test results. 
Receiving a positive test result may cause a lot of anxiety. If the test result is negative, there is still the 
possibility that a participant could be infected and test positive at some time in the future. In addition, 
there is always the rare possibility that the test results could be wrong. Newly positive test results for 
SARS-CoV-2, HIV, hepatitis B, hepatitis C, and Tuberculosis (TB) infections are required to be reported 
to the Department of Public Health. 
Drug side effects. No serious adverse events were observed in the published phase 2b data of 
ensitrelvir.11 Possible side effects may include: 
1) Headache, diarrhea or back pain. These appear to be mild with resolution following 
completion of drug. 
2) Allergic reaction, including rash, may occur after any novel antiviral medication. 
3) Transient increases in blood HDL and triglycerides while on antiviral therapy. 
3) Participants may experience return or change in Long COVID symptoms following cessation 
of study drug as has been anecdotally reported with other direct antiviral use in Long COVID. 
5) Unanticipated AEs. These are not all the possible side effects.  
2.3.2 Protection Against Risk 
Confidentiality. Participants’ records will be handled as confidentially as possible. Participants will be 
assigned a unique four-digit study ID code that will appear on all specimens and in our database. All 
biologic specimens and clinical data obtained from this study will be linked to this code and not to 
personal identifying information (e.g., name, social security number, medical record number). A key 

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which will link the four-digit code to the personal information will be maintained on a secure server only 
accessible to designated study staff and maintained by the PI. Lab personnel and database 
programmers will have access only to the coded number and the participant’s date of birth; no other 
personal identification information will be available to them. Collaborators will receive specimens only 
identified by the four-digit study ID. No individual identities will be used in any reports or publications 
resulting from this study. 
Phlebotomy. Relevant personnel are trained and certified phlebotomists. We will stay within Red Cross 
Guidelines of less than 500 mL every two months. Participants will be checked for anemia at each 
study visit. If the investigator feels that an individual is at significant risk for anemia, the amount of blood 
collected will be reduced. If the study participant’s hemoglobin falls below 9 grams/deciliter (g/dl) or 
HCT falls below 27%, we will draw 5 mL of blood to check safety labs such as Hgb and HCT. Other 
than the blood required to check safety labs, the participant will not have more blood drawn until the 
Hgb rises above 9 g/dl or the HCT rises above 27%. 
2.3.3 Benefits to Participants 
There is currently no accepted treatment for LC. Participants should not anticipate a personal benefit 
from participation in the study. However, it is possible that if the therapeutic intervention has the 
hypothesized effects, participants could experience relief from LC symptoms. Furthermore, it is possible 
that participants might benefit in general from engagement in research. 
2.3.4 Benefits to Society 
The identification of a treatment for LC symptoms could benefit the likely millions of individuals suffering 
from this condition. 
3 
STUDY OBJECTIVES 
3.1 
Primary Objective 
The primary objective of the study is to evaluate the safety and tolerability of oral ensitrelvir in 
individuals who are experiencing Long COVID.  
3.2 
Secondary Objectives 
Secondary objectives include the following: 
• 
Describe the ability of ensitrelvir to affect virologic markers of chronic SARS-CoV-2 infection. 
• 
Describe the ability of ensitrelvir to improve PASC outcomes. 
• 
Describe the ability of ensitrelvir to reduce post-COVID inflammation. 
4 
STUDY DESIGN 
4.1 
Study Overview 
This is an exploratory, 1:1 randomized, double-blind, placebo-controlled study with optional post-
blinding placebo to treatment to assess the safety and efficacy of ensitrelvir to treat LC.  
The study will enroll approximately 40 participants who meet the WHO LC criteria (defined above in 
protocol synopsis). Participants will be enrolled at a single center and randomized 1:1 to receive 
ensitrelvir fumaric acid (S-217622) given orally 375 mg on day 1 followed by 125 mg daily for 4 
additional days or placebo. Randomization will be stratified by duration of infection (<6 months and ≥6 

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months) at Day 0. Evaluations will take place at baseline and at timepoints up to 60 days after initiation 
of study drug. 
Screening data will be reviewed to determine subject eligibility. Subjects who meet all inclusion criteria 
and none of the exclusion criteria will be entered into the study.  
The following treatment regimens will be used: 
• 
Ensitrelvir Fumaric Acid (S-217622) given orally 375 mg on day 1 followed by 125 mg daily for 4 
additional days  
• 
Placebo 
The total duration of subject participation will be approximately 2 months.  
5 
CRITERIA FOR EVALUATION 
5.1 
Efficacy Endpoints 
● The change in patient-reported outcomes (PROs) PROMIS-29 score from Baseline to D30.  
● Change PROMIS-29 score in those with symptoms at Baseline and at D10, and D30. 
● Change in EuroQoL Quality of Life between Baseline and at D10, D30, and D60. 
● Change in other assessments (neurocognitive assessment and 6MWT performance) between 
Baseline and at D10. 
● Change in other assessments (DASI, Dysautonomia assessment) between Baseline and at 
D10, and D30. 
● Percentage of participants with no detection of SARS-CoV-2 plasma remnants (i.e., viral 
detection by reverse transcriptase-polymerase chain reaction ((RT-PCR) and Spike protein 
fragments) compared to baseline at D10 and D30 post ensitrelvir administration. 
● Proportion with reduction in inflammatory markers (e.g., interleukin-6 (IL-6), tumor necrosis 
factor alpha (TNF-alpha) at Baseline and at D10 and D30. 
5.2 
Safety Evaluations 
● Number of participants with adverse events (AE)s (Treatment Emergent Adverse Events 
(TEAE)s, Serious Adverse Events (SAE)s, and Adverse Events of Special Interest (AESI)). The 
primary outcome will be the number of individuals in each group experiencing a severe (Grade 3 
or greater) AE thought to be possibly, probably, or definitely related to study treatment 
throughout the study duration. 
6 
SUBJECT SELECTION 
6.1 
Study Population 
Subjects with a history of SARS-CoV-2 infection who meet the case definition for Long COVID, and 
who meet the inclusion and exclusion criteria will be eligible for participation in this study.  
6.2 
Inclusion Criteria 
1. 
Age ≥18 and <70 years at Screening. 
2. 
Enrolled or willing to enroll and complete at least 1 visit in the UCSF Long-term Impact of Infection 
with Novel Coronavirus (LIINC) study. Any adult who has been infected with SARS-CoV-2 or has 
ever received or is eligible to receive a SARS-CoV-2 vaccination, and who is able to provide 
written informed consent, is eligible to participate in LIINC.  

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3. 
History of at least one SARS-CoV-2 infection, defined as report of a positive nucleic acid 
amplification test (NAAT) and/or a positive SARS-CoV-2 antigen rapid diagnostic test (RDT). 
Written proof of the test will be requested but is not required as long as the participant attests to 
the positive test.  
4. 
Clinical evidence of Long COVID, as confirmed by the investigator’s assessment. 
a. At least two moderate symptoms or at least one severe symptom as assessed by the 
study team (see list) that are new or worsened since the time of a SARS-CoV-2 
infection, not known to be attributable to another cause upon assessment by the PI. At 
least two symptoms from those listed here must be present: systemic symptoms (e.g., 
fatigue, chills, post-exertional malaise), neurocognitive symptoms (e.g., trouble with 
memory/concentration (“brain fog”), headache, dysautonomia/postural orthostatic 
tachycardia syndrome, dizziness, unsteadiness, neuropathy, sleep disturbance), 
cardiopulmonary symptoms (e.g., chest pain, palpitations, shortness of breath, cough, 
fainting spells), musculoskeletal symptoms (e.g., muscle aches, joint pain), 
gastrointestinal symptoms (e.g., nausea, diarrhea). Although other symptoms (e.g., skin 
rash, hair loss, mental health symptoms, trouble with smell/taste, genitourinary 
symptoms) will be recorded and tracked, at least two core symptoms listed above must 
be present. Note: the two symptoms can be from within the same category (for example, 
brain fog and headache).  
AND 
b. Symptoms must have been present for at least 60 days prior to screening. Symptoms 
that wax and wane must have been initially present at least 60 days prior to screening. 
AND 
c. Symptoms must be reported to be at least somewhat bothersome and to have an impact 
on quality of life and/or everyday functioning. 
AND 
d. At least 90 days have elapsed since the most recent suspected or confirmed SARS-
CoV-2 infection and the time of screening. Note: suspected infections will be determined 
based upon assessment by the study investigators. 
5. 
Not currently hospitalized. 
3. 
Body mass index (BMI) 18 to 50 kilograms/meter squared (kg/m2), inclusive, at the time of 
screening. 
6. 
In otherwise stable health, as assessed by the investigator within 28 days prior to Screening, 
based on medical history, physical assessment, laboratory findings, and vital signs. 
7. 
For Male participants, 
Participants with partners that are women of childbearing potential (WOCBP) are strongly advised 
to inform their partners and must agree to use effective contraception from study entry (defined as 
D0) through 14 days after the last dose of study intervention. Effective methods of contraception 
are described in Appendix 2. Participants with pregnant partners must agree to use condoms 
during vaginal intercourse from study entry (defined as D0) through 14 days after the last dose of 
study intervention administration. Participants assigned male sex at birth must agree to refrain from 
sperm donation from study entry through 14 days after the last dose of study intervention 
administration. 
8. 
For Female participants, 

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A female participant is eligible to participate if she is not pregnant or breastfeeding, and the 
following conditions applies: 
 
Is not a WOCBP 
OR 
 
All of the following apply: 
4. 
Is a WOCBP and using a contraceptive method that is effective as described in Appendix 2 from    
–21 days from study entry (defined as D0), during the study intervention period, and for at least 14 
days after the last study intervention administration. She must also agree not to donate her eggs 
(ova, oocytes) for the purpose of reproduction from study entry (defined as D0), during the study 
intervention period, and for at least 14 days after the last study intervention administration. (Of 
note, participants taking hormonal contraception will be informed that there may be a possibility of 
decreased efficacy or contraceptive failure while on study drug.) 
 
A WOCBP must have a negative urine pregnancy test within 24 hours before the first dose 
of study intervention. If a urine pregnancy test cannot be confirmed as negative (e.g., an 
ambiguous result), a serum pregnancy test must be negative. 
9. 
Willingness and ability to comply with the study protocol. This includes reliable transportation and 
sufficient time to attend all visits. 
10. Written informed consent (and assent when applicable) obtained from subject or subject’s legal 
representative and ability for subject to comply with the requirements of the study. 
6.3 
Exclusion Criteria 
5. 
Previously received COVID-19 convalescent plasma treatment or a SARS-CoV-2-specific mAb 
within 60 days prior to planned Day 0 or plan to receive such treatment before exiting the study. 
1. 
Previously received a SARS-CoV-2 antiviral within 60 days prior to planned Day 0 or plan to 
receive such treatment before exiting the study. 
2. 
Plans to receive any investigational or approved vaccine or booster for SARS-CoV-2 within 60 
days prior to planned Day 0 or before Day 30 following planned Day 0. 
3. 
Active cardiovascular disease, defined as known: 
a. Myocardial infarction within 90 days of screening 
OR 
b. Coronary artery bypass procedure within 90 days of screening 
OR 
c. Current heart failure with reduced ejection fraction (<45%)  
OR 
d. Current pulmonary arterial hypertension. 
4. 
Known stroke within 3 months prior to planned Day 0. 
5. 
Known active bacterial, fungal, viral, or other infection besides SARS-CoV-2 requiring treatment 
within the 28 days prior to planned Day 0 and meeting criteria for systemic involvement upon 
review by the PI. Note: Mild or limited infections such as uncomplicated urinary tract or yeast 
infections, sexually transmitted infections, and mild dermatophyte infections may be reviewed with 
the study PI and/or Safety Monitoring Committee chair, but are not exclusionary. 

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6. 
Major surgery within 6 months prior to planned Day 0 or planned major surgery during the first 60 
days following planned Day 0. 
7. 
History of unplanned hospitalization for >24 hours within 28 days prior to Screening. 
8. 
Active Hepatitis B (Hep B) infection (defined as Hep B surface antigen (sAg) positive). Note: A 
known positive Hep B core antibody (cAb) in the absence of positive sAg is not considered 
exclusionary. 
9. 
Active Hepatitis C (Hep C) infection (defined as Hep C Ab positive or indeterminate with detectable 
Hep C RNA). Note: Those with cured Hep C (Ab positive or indeterminate but negative Hep C 
RNA) will remain eligible. 
10. Laboratory abnormalities at Screening, specifically including: 
a. ANC <1500 per mm3 
b. Platelet count <100,000 per mm3 
c. AST/ALT > 1.5x ULN 
d. CrCl < 30 mL/min 
e. Hemoglobin <9 g/dL 
11. Known HIV infection.  
12. End stage kidney disease requiring dialysis. 
13. Severe hepatic impairment (Child-Pugh Class C). 
14. Moderate or severe immunocompromise, according to the current NIH COVID-19 Treatment 
Guidelines as of March 6, 2023. The detailed list is in Appendix 2, and includes the following: (a) 
receiving active treatment for solid tumor or hematologic malignancy, including use of systemic 
chemotherapy for treatment of cancer within the year prior to screening, (b) prior solid-organ 
transplant with active immunosuppressive therapy, (c) CAR-T cell therapy or hematopoietic cell 
transplant, on immunosuppressive therapy or transplant within the prior 2 years, (d) primary 
immunodeficiency syndromes, advanced or untreated HIV infection (any HIV infection is 
exclusionary), (f) on active high-dose corticosteroids (i.e., >= 20mg prednisone or equivalent daily 
per day for >= 2 weeks) at the time of screening.  
15. Known prior diagnosis of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), 
preceding and not related to SARS-CoV-2 infection and not worsened since SARS-CoV-2 
infection. 
16. Known prior diagnosis of dysautonomia, preceding and not related to SARS-CoV-2 infection and 
not worsened since SARS-CoV-2 infection. 
17. Pregnant, breastfeeding, or unwilling to practice birth control abide by the contraception 
requirements outlined in the inclusion criteria. 
18. Participation in a clinical trial with receipt of an investigational product within 28 days or 5 half-lives 
(whichever is longer) prior to planned Day 0. The following exceptions will apply: 
a. If the investigational product is a SARS-CoV-2-specific monoclonal antibody, 
convalescent plasma, or equivalent (e.g., IVIG), refer to Criterion #1 (60 days must have 
elapsed prior to planned D0).  
b. If the investigational product is a PET imaging tracer that is not expected to have 
significant biological activity, has a biological half-life less than 24 hours and radiological 
half-life less than 6 hours; more than 14 days must have elapsed between receipt of the 
tracer and planned Day 0. 
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20. Presence of a condition or abnormality that in the opinion of the Investigator would compromise the 
safety of the patient or the quality of the data. 
21. Study site personnel directly affiliated with the study or family of directly involved personnel. 
22. Known allergy to any components used in the formulation of the intervention. 
23. Current use of or anticipated need for any medications prohibited with study intervention, as 
described in Appendix 3.  
24. Participants who have used any of the following drugs within 14 days prior to enrollment: 
 
Strong cytochrome P450 (CYP) 3A inducer 
 
Products containing St. John’s wort  
7 
CONCURRENT MEDICATIONS 
All subjects should be maintained on the same medications throughout the entire study period, as 
medically feasible, with no introduction of new chronic therapies unless deemed necessary by a 
medical provider with the exception of exclusionary medications as listed above. Initiation of over-the-
counter supplements will be discouraged.  
7.1 
Allowed Medications and Treatments 
Participation in the study will not interfere with the participant’s standard of care. Routine or standard of 
care vaccinations (such as influenza, pneumococcus, etc.) are allowed and will be documented in the 
CRFs; wherever possible these will be spaced greater than or equal to two weeks from study visits. 
8 
STUDY TREATMENTS 
8.1 
Method of Assigning Subjects to Treatment Groups 
The 40 participants will be randomized, 20 to ensitrelvir and 20 to placebo. The randomization will be 
stratified by duration of COVID-19 related symptoms (< 6 months vs. ≥ 6 months). Within the strata, 
randomization will use permuted blocks of 3 and 6 enrollees with equal likelihood.   Following study 
unblinding, participants in the placebo arm will be given the option to receive ensitrelvir with similar 
safety and efficacy evaluations as in the primary study population. 
8.2 
Blinding 
Treatment assignments will be blinded to the investigator, subjects, and all clinical and research staff 
for the entire study, except for designated pharmacy staff or delegate who will remain unblinded to 
prepare the study drug or placebo.  
Unblinding can occur at any time for a medical emergency or any other significant medical event and 
when a treatment decision is contingent on knowing the subject’s treatment assignment. When 
possible, the investigator or delegate should discuss with Shionogi prior to unblinding. The date and 
reason for unblinding must be recorded and Shionogi will be notified.  
The Independent Medical Monitor (IMM) may review any unblinded clinical analysis during the study 
and provide the recommendations to the Investigator. Subject level unblinded data will not be shared 
with the Investigator until the end of the trial.  

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8.3 
Formulation of Test and Control Products 
Ensitrelvir and placebo will be provided by the manufacturer who will be responsible for ensuring that 
ensitrelvir and placebo are manufactured in accordance with applicable current Good Manufacturing 
Practice (GMP) regulations and requirements.  
8.3.1 Packaging and Labeling 
The study drugs will be labeled according to the requirements of local law and legislation. The study 
drugs will be dispensed according to GCP by the clinical site’s pharmacy in accordance with the site’s 
standard operating procedures (SOPs).  
8.4 
Supply of Study Drug at the Site 
The manufacturer (or designee) will ship ensitrelvir and placebo to the investigational site’s clinical 
research pharmacy. The initial ensitrelvir and placebo shipment will be shipped after site activation (i.e., 
all required regulatory documentation has been received by Shionogi and a contract has been 
executed). Subsequent ensitrelvir and/or placebo shipments will be made after site request for 
resupply. 
8.4.1 Dosage/Dosage Regimen 
Ensitrelvir is administered orally over 5 days. On the first day, 3 125 mg pills (total 375 mg) will be taken 
orally followed by 4 additional days of one 125 mg pill per day. Placebo is also administered orally, with 
three pills being taken on the first day followed by 1 pill daily for 4 additional days. Details regarding the 
preparation and administration of the study drugs are provided in the pharmacy manual. 
There is no adjustment for weight, age, meals, or other factors.  
8.4.2 Dispensing 
Participants will receive the full 5-day course of treatment at the time of the D0 visit. Participants will 
receive a treatment log to document the time that each dose was taken. Compliance will be assessed 
during the D1 and D4 phone follow-up and participants will be asked to return their treatment log at the 
in-person follow-up visit.  
8.4.3 Administration Instructions 
The date and time of each dose will be reported to the study team by the participants and to be started 
on the day designated by the study team. 
8.5 
Supply of Study Drug at the Site 
The investigator will delegate responsibility for drug receipt, storage, accountability, and disposition to 
the clinical site’s research pharmacist. The clinical site’s pharmacist will maintain an inventory record of 
the study drugs received, stored (in a secure restricted area), and dispensed. Study drugs and placebo 
will be provided to study participants only. 
8.5.1 Storage 
Ensitrelvir pills and placebo will be shipped from the manufacturer or manufacturer resources to the 
clinical site’s pharmacy. The study medication will be stored at room temperature.  

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8.6 
Study Drug Accountability 
An accurate and current accounting of the dispensing for each subject will be maintained on an ongoing 
basis by the research pharmacist. The number of study drug dispensed will be recorded on the 
Investigational Drug Accountability Record. The study monitor will verify these documents throughout 
the course of the study. 
8.7 
Measures of Treatment Compliance 
Since this study involves a short course of oral antiviral pills, participants will be asked to keep a written 
log of the timing of each oral dose to be provided to the study team once they complete the treatment 
or placebo course. 
9 
STUDY PROCEDURES AND GUIDELINES 
A Schedule of Events representing the required testing procedures to be performed for the duration of 
the study is diagrammed in Appendix 1. The Schedule of Events will serve as the final resource for 
guiding study activities. 
Prior to conducting any study-related activities, written informed consent and the Health Insurance 
Portability and Accountability Act (HIPAA) authorization must be signed and dated by the subject or 
subject’s legal representative. If appropriate, assent must also be obtained prior to conducting any 
study-related activities. 
9.1 
Clinical Assessments 
9.1.1 Concomitant Medications 
All concomitant medication and concurrent therapies taken within 28 days prior to Screening will be 
documented. Dose, route, unit frequency of administration, and indication for administration and actual 
or estimated dates of medication will be captured. 
Details regarding all SARS-CoV-2 vaccinations and/or boosters, as well as COVID-specific therapies, 
will be collected. 
9.1.2 Demographics  
Demographic information (date of birth, gender, race) will be recorded at Screening. 
9.1.3 Medical History  
Relevant medical history, including history of current disease, other pertinent respiratory history, and 
information regarding underlying diseases will be recorded at Screening. 
9.1.4 Physical Examination 
A complete physical examination at Screening will be performed by a qualified study clinician 
(physician, nurse, or physician assistant). Qualified staff (MD, DO, NP, RN, or PA) may also complete 
the abbreviated (targeted) physical exam at all other visits. New abnormal physical exam findings must 
be documented and will be followed by a physician or other qualified staff at the next scheduled visit. 
9.1.5 Vital Signs and Oximetry 
Weight, body temperature, blood pressure, pulse, respirations, and oximetry on room air will be 
performed after resting for 5 minutes or more as indicated in the Schedule of Events (SOE). Vital signs 
in the lying, seated, and standing positions may also be performed. 

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9.1.6 Questionnaire-based Measurements 
Long COVID Symptom Assessment. Signs and symptoms related to Long COVID will be reviewed at 
all visits, using forms such as the LIINC CRFs. At D0 and all subsequent visits, all grades of signs and 
symptoms that are newly developed or changed since the previous visit must be recorded. Duration 
(start and stop dates), severity/grade, outcome, treatment, and relation to study drug will be recorded 
on the case report forms. All clinical events and new diagnoses or changes in diagnoses should be 
recorded.  
Other Symptom Assessment (AE Review). At D0 and all subsequent visits, all grades of signs and 
symptoms that are newly developed or changed since the previous visit will be recorded. Symptoms not 
previously experienced as part of Long COVID, or which have worsened beyond the degree previously 
experienced by the participant, will be considered to represent adverse events and recorded as such. 
For these symptoms, duration (start and stop dates), severity/grade, outcome, treatment, and relation 
to study drug will be recorded on the case report forms. All clinical events and new diagnoses or 
changes in diagnoses should be recorded.  
Quality of Life. Quality of life (QoL) will be obtained using the EQ-5D-5L scale which is part of the 
LIINC CRFs. This will include the 100-point visual analogue scale (VAS). 
PROMIS-29. The PROMIS-29 form will be completed as outlined in the schedule of events. This scale 
measures self-reported health, using a collection of short forms assessing fatigue, physical function, 
anxiety, depression, pain, sleep disturbance, and ability to participate in social roles and activities. The 
questionnaire uses a computer interface over approximately 5 minutes. It is available in English and 
Spanish. This instrument has been used for ME/CFS and Long COVID. 
Patient Global Impression of Change (PGIC). The self-report measure Patient Global Impression of 
Change (PGIC) reflects a patient’s belief about the efficacy of their treatment. We will use a modified 
PGIC scale which has been used to study pain syndromes and has been employed in other Long 
COVID clinical trials. It is a common data element developed by the National Institutes of Mental 
Health. 
Everyday Cognition Form (ECOG). The ECOG is performed in the parent LIINC study and asks 
participants to self-rate issues with cognition. It is a patient-reported measure for neurocognitive 
symptoms.  
Neurocognitive Assessment. A neurocognitive assessment such as the NIH Toolbox or similar 
assessment will be administered as outlined in the schedule of events. Neurocognitive assessments 
can assess motor, emotional, sensory, and cognitive function. It is available in English and Spanish and 
is administered using an iPad or computer.  
Duke Activity Status Index. The Duke Activity Status Index is a patient-reported estimate of functional 
capacity, maximal oxygen consumption (VO2 max) and maximum metabolic equivalent of tasks 
(METs). The DASI questionnaire produces a score between 0 and 58.2 points, which is linearly 
correlated with a patient’s VO2 max and METs, as measured from cardiopulmonary exercise testing 
(CPET). It inquires about a person’s ability to perform self-care, walk, climb stairs, run, do house and 
yard work, engage in sexual intercourse, and perform moderate recreational activities. 
Dysautonomia Assessment (e.g., COMPASS-31 or similar Scale). The Composite Autonomic 
Symptom Score (COMPASS)-31 is an assessment of autonomic dysfunction. This abbreviated 
questionnaire provides a quantitative measure of autonomic symptoms that otherwise might not be 
adequately recorded using the above instruments. It includes measures of orthostatic intolerance, 
vasomotor and secretomotor symptoms, GI and urinary symptoms, syncope, and genitourinary 
symptoms. We will utilize this questionnaire or a similar instrument to determine the impact of treatment 
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DSQ-PEM (Short Form). The DePaul Symptom Questionnaire (DSQ) post-exertional malaise (PEM) 
questionnaire will be used to assess post-exertional malaise at baseline, and again at the primary 
endpoint as specified in the schedule of events.  
WHO-DAS (Short Form). The World Health Organization Disability Assessment Schedule 2.0 
questionnaire asks about difficulties due to health conditions. Health conditions include diseases or 
illnesses, other health problems that may be short or long lasting, injuries, mental or emotional 
problems, and problems with alcohol or drugs. We will utilize this questionnaire at baseline, and again 
at the primary endpoint as specified in the schedule of events.  
6 Minute Walk Test. A 6MWT will be performed by the research team. 
9.1.7 Adverse Events 
Information regarding occurrence of adverse events will be captured throughout the study. Duration 
(start and stop dates), severity/grade outcome, treatment and relation to study drug will be recorded on 
the case report form (CRF). 
This is a study of Long COVID symptoms. As a result, symptoms experienced prior to receipt of the 
study intervention will be documented, and a symptom will only be considered to represent an AE if its 
quality or severity differs from or exceeds that which was previously experienced by the participant. 
9.1.8 Participants with Positive COVID Tests During the Study 
Due to the ongoing pandemic, it is anticipated that some individuals may test positive for COVID-19 
during the course of the study. If an individual exhibits acute COVID-19 symptoms during the study, we 
will arrange for or navigate to testing. If an individual tests positive for SARS-CoV-2 during the course 
of the study, we will leverage existing testing and treatment programs in our hospital system and/or 
geographic region to navigate the participant to care. In addition to navigation for clinical care, all 
participants who test positive during the study will be referred to a home-based research collection 
protocol for more frequent sampling.  
9.2 
Clinical Laboratory Measurements 
9.2.1 Hematology 
Blood will be obtained and sent to each site’s clinical hematology lab for a complete blood count 
(hemoglobin, hematocrit, red blood cell count, white blood cell count, white blood cell differential, and 
platelet count), and serum C-reactive protein (CRP) determinations for assessment of systemic 
evidence for infection and/or inflammation. These measurements will be performed as outlined in the 
Schedule of Events. 
9.2.2 Blood Chemistry Profile 
Blood will be obtained and sent to the site’s clinical chemistry lab for determination of serum sodium, 
potassium, chloride, bicarbonate, random glucose, BUN, creatinine, aspartate aminotransferase 
(AST/SGOT), alanine aminotransferase (ALT/SGPT), alkaline phosphatase, total bilirubin, direct 
bilirubin, and albumin. These measurements will be performed as outlined in the Schedule of Events. 
9.2.3 Pregnancy Testing 
A urine or serum pregnancy test will be obtained from female subjects who are of childbearing potential 
prior to their participation in the study. These measurements will be performed as outlined in the 
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9.2.4 Hepatitis B Testing 
Participants will undergo Hep B surface antigen testing at Screening, if they have not had such 
screening within the preceding 6 months. Individuals with active Hep B will be excluded. This is defined 
as detectable Hep B surface antigen. Individuals without detectable Hep B surface antigen will remain 
eligible, regardless of their vaccination or antibody status (e.g., a positive Hep B cAb or negative Hep B 
sAb is not exclusionary). 
9.2.5 Hepatitis C Testing 
Participants will undergo Hep C antibody testing at Screening, if they have not had such screening 
within the preceding 6 months. Individuals with active Hep C, defined as positive or indeterminate 
antibody with the presence of Hep C RNA, will be excluded. Positive or indeterminate Hep C Ab in the 
absence of Hep C RNA is not exclusionary. 
9.2.6 SARS-CoV-2 PCR or Antigen Testing 
Swabs of the anterior nares will be performed to assess for SARS-CoV-2 shedding as outlined in the 
Schedule of Events. 
9.3 
Research Laboratory Measurements 
9.3.1 Plasma Measures of Viral Persistence 
Plasma levels of SARS-CoV-2 antigens (Spike, nucleocapsid, etc.) may be performed in collaboration 
with research laboratories to evaluate the effect of antiviral therapy on measures of viral persistence. 
9.3.2 Immunophenotyping 
Single- or multi-plex platforms will be used to measure targeted biomarkers previously shown to be 
elevated in Long COVID, which may include interleukin-6, tumor necrosis factor-alpha, monocyte 
chemoattractant protein-1, and interferon-gamma induced protein 10. In addition, we may use high-
throughput platforms such as Olink proteomics to conduct within-person and/or between-group 
comparisons in order to identify pathways that are altered with treatment, or that differ between those 
with and without improvement during the study period. Additional measures of cellular and humoral 
immunity may also be performed. 
9.3.3 SARS-CoV-2 Genotyping 
Genotyping may be performed on any samples collected during the study in which SARS-CoV-2 virus 
is recovered. Participants with a positive test at the time of screening are not eligible for the study. 
Participants with suspected acute SARS-CoV-2 infection or who report rebound symptoms at the time 
of an in-person follow-up visit will undergo a SARS-CoV-2 test and if positive the sample will be banked 
for genotyping analyses.  
9.4 
Biospecimens 
Within the UCSF LIINC infrastructure, initial sample processing is performed on peripheral blood units 
which are then transported by a staff member or courier to a biorepository, which include the Molecular 
and Cellular Core (formerly Core Immunology Laboratory) and the UCSF Specimen Processing and 
Banking Subcore (formerly AIDS Specimen Bank). All samples are tracked through laboratory 
information systems and managed with sample storage inventories, monthly reports, and institutionally 
managed data storage and back-up systems. All biospecimens will eventually be stored long-term via 
the UCSF Specimen Processing and Banking Subcore (formerly AIDS Specimen Bank), where the 
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ultra-low temperature freezers (including liquid nitrogen) with back-up power systems, in which 
temperature is monitored by a programmable scanning alarm system wired to the university’s 
telephone system. After completion of the primary analyses, samples may be stored indefinitely via the 
LIINC biorepository for use on other COVID-related pathogenesis studies (as addressed in the consent 
form), at the discretion of the PI.  
10 EVALUATIONS BY VISIT 
The details of the visit schedule are outlined below. The Schedule of Events will be the final guide for 
what events are to occur at each specific study visit. 
10.1 Screening Visit (D-28) 
Following the informed consent discussion and signed informed consent form, the Screening 
Assessment will be performed. The inclusion and exclusion criteria will be reviewed with the participant. 
Screening labs, medical history, and the details of concomitant medications will be obtained. Details of 
the participant’s COVID-19 history (including COVID-19 treatments) will be reviewed and/or confirmed. 
The Screening Assessment will include a physical assessment by a study clinician (physician, nurse, or 
physician assistant). Other assessments as outlined in the Schedule of Events will be performed. 
Laboratory tests as outlined in the Schedule of Events will be performed. Laboratory tests or other 
assessments performed for a clinical indication (not exclusively to determine study eligibility) may be 
used for screening values even if the studies were performed before informed consent was obtained. 
An individual who recently had laboratory tests performed as part of routine clinical care need not 
repeat these tests if they are within the window for the test (e.g., within 6 months for hepatitis testing). 
For example, someone with recent Hepatitis B or C or HIV testing may not need to have the test 
repeated, as indicated in the Schedule of Events. However, these measurements may be repeated at 
the discretion of the PI.  
Screening evaluations should be completed within 28 days of planned D0. If more than 28 days lapse, 
screening procedures may be repeated to re-confirm eligibility prior to D0. At a minimum, this will 
include the metabolic panel, complete blood count, and pregnancy testing. Laboratory tests that are not 
expected to change (such as coagulation studies or viral hepatitis status) are not required to be 
repeated but may be at the discretion of the PI. 
Rescreening: A participant who does not meet all eligibility criteria at the time of Screening may be 
given the opportunity to rescreen for the study at a later date.  
10.2 Baseline Evaluation (D-21 to D0) 
An additional visit will be performed following Screening, at least 7 days after the screening visit and 
prior to D0. The target date for this visit is within 7 days prior to the scheduled date of study product 
administration, although up to 21 days is acceptable. The purpose of this visit is to make additional 
assessments prior to the intervention, to establish baseline symptomatology and measurements. This 
will include a symptom assessment and various clinical assessments as outlined in the Schedule of 
Events. Certain real-time laboratory tests will be obtained to establish a baseline.  
Note, after biospecimen collection has occurred, the participant may immediately move on to the 
intervention visit (medications can be dispensed after the baseline evaluations are complete).  
10.3 Intervention (D0)  
Participants who screen into the study and complete baseline measurements will be randomized to 
receive either ensitrelvir or placebo. D0 refers to the day that the participants start taking oral study 
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Note: During the D0 encounter or at the reminder call prior to that visit, a team member will confirm that 
there have been no major changes to the participant’s health since the baseline visit that would affect 
eligibility. If there have been major changes, this will be discussed with a study clinician prior to product 
administration to confirm whether or not it is acceptable to proceed. If the Baseline and Entry visit are 
on the same day (i.e., combined), blood will be collected only once. 
Note: For persons of childbearing potential, a negative pregnancy test result must be obtained prior to 
dispensing the study product. 
10.4 Telephone Follow-ups (Phone Follow-Up, D1, D4)  
The participant will be contacted by telephone to check on the overall response to the first and last 
dose of medication. The Phone FU D1 visit will focus on determining whether there have been any 
adverse events from the study product. The Phase FU on D4 will focus on determining whether there 
have been any adverse events from the study product, as well as whether there has been any short-
term efficacy. 
10.5 In Person Follow-up Visits (D10, D30) 
In-person follow-up visits will occur as outlined in the Schedule of Events. At each visit, vital signs will 
be collected, and the participant will undergo a targeted physical assessment as needed. They will 
complete clinical assessments as outlined in the Schedule of Events. Laboratory tests will be ordered 
and biospecimens collected and stored for later testing as per the Schedule of Events.  
10.6 Telephone or In Person End-Of-Study Visit (EOS D60) 
The participant will be contacted by telephone or by in person interview with study staff to undergo 
clinical questionnaire assessments as outlined in the Schedule of Events. 
10.7 Unscheduled (Interim) Visits 
An unscheduled (or interim) visit is defined as any visit not pre-specified in the study protocol. An 
unscheduled visit may be scheduled in order to assess a participant in person, repeat a laboratory test, 
or to collect additional biospecimens at the discretion of the PI.  
10.8 Study Discontinuation Visit 
If a subject withdraws or discontinues from the study, they should complete a Study Discontinuation 
Visit. If D0 has occurred, this visit should at a minimum include AE assessment and documentation of 
the reason for discontinuation or withdrawal. Whenever possible, the study events for the Study 
Discontinuation Visit should be as follows: 
• 
Prior to D0: No further assessment needed. 
• 
Between D0 and D10: Complete D10 assessments. 
• 
After D10: Complete D30 assessments (even if D30 has elapsed). 
11 ADVERSE EXPERIENCE REPORTING AND DOCUMENTATION 
11.1 Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a clinical investigation of a patient 
administered a pharmaceutical product and that does not necessarily have a causal relationship with 
the treatment. An AE is therefore any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom or disease temporally associated with the administration of an 
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of a type not identified in nature, severity, or frequency in the current Investigator’s Brochure or of 
greater severity or frequency than expected based on the information in the Investigator’s Brochure. 
Suspected Adverse Reaction (SAR) – any AE for which there is a reasonable possibility that the drug 
caused it. It implies a lesser degree of certainty about causality than adverse reaction (any AE caused 
by a drug). 
An unexpected AE or unexpected SAR is one of a type not identified in nature, severity, or frequency in 
the current Investigator’s Brochure or of greater severity or frequency than expected based on the 
information in the Investigator’s Brochure. 
The Investigator will probe, via discussion with the subject, for the occurrence of AEs during each 
subject visit and record the information in the site’s source documents. Adverse events will be recorded 
in the patient CRF. Adverse events will be described by duration (start and stop dates and times), 
severity, outcome, treatment and relation to study drug, or if unrelated, the cause. 
11.1.1 AE Severity 
The National Institutes of Health Division of AIDS (DAIDS) Table for Grading the Severity of Adult and 
Pediatric Adverse Events, Corrected Version 2.1 (July 2017) will be used to assess and grade AE 
severity, including laboratory abnormalities judged to be clinically significant. The modified criteria can 
be found in the study manual. If the experience is not covered in the modified criteria, the guidelines 
shown in Table 1 below should be used to grade severity. It should be pointed out that the term 
“severe” is a measure of intensity and that a severe AE is not necessarily serious. 
Table 1. AE Severity Grading 
Severity (Toxicity Grade) 
Description 
Mild (1) 
Transient or mild discomfort; no limitation in activity; no medical 
intervention or therapy required. The subject may be aware of 
the sign or symptom but tolerates it reasonably well. 
Moderate (2) 
Mild to moderate limitation in activity, no or minimal medical 
intervention/therapy required. 
Severe (3) 
Marked limitation in activity, medical intervention/therapy 
required, hospitalizations possible. 
Life-threatening (4) 
The subject is at risk of death due to the adverse experience as 
it occurred. This does not refer to an experience that 
hypothetically might have caused death if it were more severe. 
11.1.2 AE Relationship to Study Drug 
The relationship of an AE to the study drug should be assessed using the following the guidelines in 
Table 2 below. 
Table 2. AE Relationship to Study Drug 
Relationship 
to Drug 
Comment 

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Definitely 
Previously known toxicity of agent; or an event that follows a reasonable 
temporal sequence from administration of the drug; that follows a known or 
expected response pattern to the suspected drug; that is confirmed by 
stopping or reducing the dosage of the drug; and that is not explained by any 
other reasonable hypothesis. 
Probably 
An event that follows a reasonable temporal sequence from administration of 
the drug; that follows a known or expected response pattern to the suspected 
drug; that is confirmed by stopping or reducing the dosage of the drug; and 
that is unlikely to be explained by the known characteristics of the subject’s 
clinical state or by other interventions. 
Possibly 
An event that follows a reasonable temporal sequence from administration of 
the drug; that follows a known or expected response pattern to that 
suspected drug; but that could readily have been produced by a number of 
other factors. 
Unrelated 
An event that can be determined with certainty to have no relationship to the 
study drug. 
11.2 Serious Adverse Experiences (SAE) or Serious SAR 
An SAE is defined as any AE occurring at any dose that results in any of the following outcomes: 
• 
death 
• 
a life-threatening adverse experience 
• 
inpatient hospitalization or prolongation of existing hospitalization 
• 
a persistent or significant disability/incapacity 
• 
a congenital anomaly/birth defect  
Other important medical events may also be considered an SAE when, based on appropriate medical 
judgment, they jeopardize the subject or require intervention to prevent one of the outcomes listed.  
A life-threatening AE or life-threatening SAR is an occurrence that places the patient or subject at 
immediate risk of death. It does NOT include an AE or SAR that, had it occurred in a more severe form, 
might have caused death. 
SUSARS are fatal or life threatening serious unexpected SARs. The team must notify the FDA as soon 
as possible, but no later than 7 calendar days after the initial receipt of information.  Serious non-fatal or 
non-life-threatening SARs will be reported to Shionogi as soon as possible, but no later than 15 
calendar days after initial receipt of information. 
11.2.1 Serious Adverse Experience Reporting 
Study sites will document all SAEs that occur (whether or not related to study drug) per UCSF CHR 
Guidelines. The collection period for all SAEs will begin after informed consent is obtained and end 
after procedures for the final study visit have been completed. 
In accordance with the standard operating procedures and policies of the local Institutional Review 
Board (IRB)/Independent Ethics Committee (IEC), the site investigator will report SAEs to the IRB/IEC.  
Shionogi, the manufacturer of the IP, shall be notified within 24 hours of study team investigator 
awareness of an SAE. In addition, an AE master list shall be sent to Shionogi Safety monthly. The 
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monitoring. This will include liver toxicity monitoring, symptom assessment, and any other monitoring 
deemed indicated by the study investigator.  
12 DISCONTINUATION AND REPLACEMENT OF SUBJECTS 
12.1 Early Discontinuation 
A subject may be discontinued from study treatment at any time if the subject or the investigator feels 
that it is not in the subject’s best interest to continue. The following is a list of possible reasons for study 
treatment discontinuation:  
• 
Subject withdrawal of consent 
• 
Subject is not compliant with study procedures 
• 
Adverse event that in the opinion of the investigator would be in the best interest of the subject 
to discontinue study treatment 
• 
Protocol violation requiring discontinuation of study treatment 
• 
Lost to follow-up 
• 
Manufacturer request for early termination of study 
If a subject is withdrawn from treatment due to an adverse event, the subject will be followed and 
treated by the Investigator until the abnormal parameter or symptom has resolved or stabilized.  
All subjects who discontinue study treatment should come in for an early discontinuation visit (as 
outlined above) as soon as possible and then should be encouraged to complete all remaining 
scheduled visits and procedures. 
All subjects are free to withdraw from participation at any time, for any reason, specified or unspecified, 
and without prejudice. 
Reasonable attempts will be made by the investigator to provide a reason for subject withdrawals. The 
reason for the subject’s withdrawal from the study will be specified in the subject’s source documents 
Refer to Section 10 for study discontinuation procedures. 
12.2 Withdrawal of Subjects from the Study 
A subject may be withdrawn from the study at any time if the subject or the investigator feels that it is 
not in the subject’s best interest to continue.  
All subjects are free to withdraw from participation at any time, for any reason, specified or unspecified, 
and without prejudice. 
Reasonable attempts will be made by the investigator to provide a reason for subject withdrawals. The 
reason for the subject’s withdrawal from the study will be specified in the subject’s source documents. 
As noted above, subjects who discontinue study treatment early (i.e., they withdraw prior to D60) 
should have an early discontinuation visit. Refer to Section 10 for early termination procedures.  
12.3 Replacement of Subjects 
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12.4 Lost to Follow Up 
A subject will be considered LTFU if s/he repeatedly fails to return for scheduled visits and is unable to 
be contacted by the study site. The following actions must be taken if a subject fails to return to the 
clinic for a required study visit: 
• 
The site must attempt to contact the subject and reschedule the missed visit as soon as possible, 
counsel the subject on the importance of maintaining the assigned visit schedule and ascertain 
whether the subject wishes to and/or should continue in the study. 
• 
Before a subject is deemed LTFU, the investigator or designee must make every effort to regain 
contact with the subject (at a minimum, 2 telephone calls and, if necessary, a certified letter to the 
subject’s last known mailing address or local equivalent methods). These contact attempts should 
be documented in the subject’s source documents. 
Should the subject continue to be unreachable, s/he will be considered to have withdrawn from the 
study 
13 PROTOCOL VIOLATIONS 
A protocol violation occurs when the subject or investigator fails to adhere to significant protocol 
requirements affecting the inclusion, exclusion, subject safety, and primary endpoint criteria. Protocol 
violations for this study include, but are not limited to, the following: 
• 
Failure to meet inclusion/exclusion criteria 
• 
Use of a prohibited concomitant medication 
Failure to comply with Good Clinical Practice (GCP) guidelines will also result in a protocol violation. 
The Investigator will determine if a protocol violation results in withdrawal of a subject. 
When a protocol violation occurs, it will be discussed with the investigator and a Protocol Violation 
Form detailing the violation will be generated. This form will be signed by the Investigator or delegate. A 
copy of the form will be filed in the site’s regulatory binder and in the Investigator’s files. 
14 DATA SAFETY MONITORING 
Safety Monitoring Committee (SMC): We will develop an independent SMC prior to the initiation of 
the study.  We expect that the committee will be composed of at least 3 independent individuals from 
the scientific community. These individuals will be selected based on their expertise in either the clinical 
management of SARS-CoV-2 infection and/or their expertise with other viral infections and clinical 
studies. The committee will be chaired by a senior investigator who has experience in the regulatory 
aspects of clinical studies.   
Approximately 4 months after enrollment of the first participant and then every 4 months thereafter, the 
SMC will review accrual (including screening and enrollment), AE summaries, including all reported 
Grade ≥3 AEs, retention of participants including off-study rates. 
In addition to the regularly scheduled reviews, a safety review will be conducted by the SMC for any of 
the following criteria: 
• 
A participant develops a Grade 3 or 4 AE that is definitely, probably, or possibly related to the 
study product, as determined by the investigator.  
• 
A participant develops an SAE that is deemed possibly, probably, or definitely related to the 
study product, as determined by the investigator. 
If the above criteria are met, the PI will request a review by the SMC, to be held within 3 business days 
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In addition, enrollment into the study will be paused (defined as newly scheduled screenings or new 
dispensing of study product) until the SMC review has taken place and a determination has been made 
that enrollment can resume. In addition, administration of study products will be paused until a course 
of action is recommended by the SMC. Previously scheduled screening, baseline, and follow-up visits 
can continue during this period of review as these are not expected to affect the safety of participants 
who have not yet received the study IP or who have already received the study IP. However, new study 
IP will not be dispensed while the study is paused. 
The SMC will recommend, based on the results of the review, whether the study can proceed as 
planned, proceed with modifications, or should be discontinued.  
The SMC will review progress towards pre-specified benchmarks of enrollment and retention of 
participants, completion of study procedures, and collection of viable samples. If progress towards any 
benchmark is not adequate, as determined by the SMC, the SMC will recommend protocol modification 
if necessary. 
15 STATISTICAL METHODS AND CONSIDERATIONS 
Prior to the analysis of the final study data, a detailed Statistical Analysis Plan (SAP) will be written 
describing all analyses that will be performed. The SAP will contain any modifications to the analysis 
plan described below.  
15.1 Data Sets Analyzed 
Eligible patients who are randomized into the study and receive at least one dose of the study drug 
during the randomized stage of this study will comprise the modified intent to treat (mITT) population 
and will be the primary analysis population for all analyses.  
Per protocol analysis will be based on description of the results per study group excluding anyone who 
has received a non-study COVID antiviral therapy during the trial period. 
Exploratory analyses will include mITT population with addition of participants original assigned to 
placebo who opt to undergo ensitrelvir therapy following unblinding in the optional cross-over stage. As 
this is exploratory, results of the primary and secondary endpoints from the initial randomized cohort 
may be reported prior to completion of the cross-over option. 
15.2 Demographic and Baseline Characteristics 
The following demographic variables at screening will be summarized by treatment group.  
15.3 Analysis of Primary Endpoint 
Safety and tolerability data will be summarized by treatment group.  
Adverse event rates will be coded by body system. Adverse events will be tabulated by treatment group 
and will include the number of patients for whom the event occurred, the rate of occurrence, and the 
severity and relationship to study drug. Adverse events between groups will be summarized by 
proportions with associated 95% Clopper-Pearson confidence intervals and will be compared using a 
two-sided 0.05 level Fisher exact test.  
15.4 Analysis of Secondary Endpoints 
The key secondary outcome will be the change in the PROMIS-29 scores from baseline to D10 follow 
up. This will be analyzed from the mITT population using a linear regression model for follow-up 
PROMIS-29 with terms for the baseline value, randomized treatment and randomization strata (<26 v. 

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>= 26 weeks of symptoms) – an analysis of covariance model (ANCOVA) and tested using a two-sided 
0.05 level test. 
Continuous secondary events (e.g., IL-6) will be compared using the ANCOVA approach. Antigen 
detection will be summarized by proportion with 95% Clopper-Pearson confidence intervals and will be 
compared using a two-sided 0.05 level Fisher exact test 
 
Primary and secondary endpoint analyses will be performed on the mITT of the pre-unblinded 
randomized portion of the study. Exploratory analyses will be performed including the roll-over 
participants from placebo to treatment group.  Primary and secondary endpoint analyses may proceed 
prior to completion of this optional roll-over study stage. 
15.5 Sample Size and Randomization 
This is an exploratory study, and the primary results will be descriptive. We will use ensitrelvir as a 
probe to better understand LC biology and determine whether a signal is present to warrant further 
study. 
The study will enroll 40 participants who meet the WHO LC criteria. Participants will be enrolled at a 
single center and randomized 1:1 to receive ensitrelvir or placebo. Randomization will be stratified by 
duration of infection (<6 months and ≥6 months) at Day 0.  
For PROMIS-29, assuming a standard deviation (SD) of the within-person difference of 10, then the 
planned sample size will have 80% power to detect a difference of 11.2 on a two-sided 0.05 level test.  
For biomarkers, such as IL-6, we will be able to detect a difference from a mean value of 2.5 pg/mL to 
1.1 times the within-person standard deviation with 80% power.  
We assume that 25% of participants will have positive antigen at entry, we will have 62% power to 
detect a reduction to < 1% with positive antigen on the treatment arm.  
16 DATA COLLECTION, RETENTION AND MONITORING 
16.1 Data Collection Instruments 
The Investigator will prepare and maintain adequate and accurate source documents designed to record 
all observations and other pertinent data for each subject treated with the study drug.  
Study personnel at each site will enter data from source documents corresponding to a subject’s visit 
into the protocol-specific electronic Case Report Form (eCRF) or paper CRF when the information 
corresponding to that visit is available. Subjects will not be identified by name in the study database or 
on any study documents to be collected by the Investigator (or designee) but will be identified by their 
unique PID. 
For eCRFs: If a correction is required for an eCRF, the time and date stamps track the person entering 
or updating eCRF data and creates an electronic audit trail. For paper CRFs: If a correction is made on 
a CRF, the study staff member will line through the incorrect data, write in the correct data and initial 
and date the change. 
The Investigator is responsible for all information collected on subjects enrolled in this study. All data 
collected during the course of this study must be reviewed and verified for completeness and accuracy 
by the Investigator.  

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16.2 Data Management Procedures 
The data will be entered into a validated database (REDCap). The Data Management group will be 
responsible for data processing, in accordance with procedural documentation.  
All procedures for the handling and analysis of data will be conducted using good computing practices 
meeting FDA guidelines for the handling and analysis of data for clinical trials. 
16.3 Data Quality Control and Reporting 
After data have been entered into the study database, a system of computerized data validation checks 
will be implemented and applied to the database on a regular basis. Query reports (Data Clarification 
Requests) pertaining to data omissions and discrepancies will be forwarded to the study team for 
resolution. The study database will be updated in accordance with the resolved queries.  
16.4 Archival of Data 
The database is safeguarded against unauthorized access by established security procedures; 
appropriate backup copies of the database and related software files will be maintained. Databases are 
backed up by the database administrator in conjunction with any updates or changes to the database.  
At critical junctures of the protocol (e.g., production of interim reports and final reports), data is cleaned 
per established procedures. 
16.5 Availability and Retention of Investigational Records 
The Investigator must make study data accessible to the monitor, IRB/IEC, and Regulatory Agency 
(e.g., FDA) inspectors upon request. A file for each subject must be maintained that includes the signed 
Informed Consent, HIPAA Authorization and Assent Form and copies of all source documentation 
related to that subject. The Investigator must ensure the reliability and availability of source documents 
from which the information on the CRF was derived. 
All study documents (patient files, signed informed consent forms, copies of CRFs, Study File 
Notebook, etc.) must be kept secured for a period of two years following marketing of the 
investigational product or for two years after centers have been notified that the IND has been 
discontinued. There may be other circumstances for which the Investigator is required to maintain study 
records and, therefore, the Investigator should be contacted prior to removing study records for any 
reason. 
As outlined above, after completion of the primary analyses, samples may be stored indefinitely via the 
LIINC biorepository for use on other COVID-related pathogenesis studies (as addressed in the consent 
form), at the discretion of the PI. 
16.6 Monitoring 
Monitoring visits will be conducted by representatives of the Investigator according to the U.S. CFR 
Title 21 Parts 50, 56, and 312 and ICH Guidelines for GCP (E6). By signing this protocol, the 
Investigator grants permission to the safety medical committee (SMC), and appropriate regulatory 
authorities to conduct on-site monitoring and/or auditing of all appropriate study documentation. 
16.7 Subject Confidentiality 
In order to maintain subject confidentiality, only a subject PID will identify all study subjects on CRFs 
and other documentation. Additional subject confidentiality issues (if applicable) are covered in the 
Clinical Study Agreement. 

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17 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS 
The study will be conducted according to the Declaration of Helsinki, Protection of Human Volunteers 
(21 CFR 50), Institutional Review Boards (21 CFR 56), and Obligations of Clinical Investigators (21 
CFR 312). 
To maintain confidentiality, all laboratory specimens, evaluation forms, reports and other records will be 
identified by a coded number and initials only. All study records will be kept in a locked file cabinet and 
code sheets linking a patient’s name to a patient identification number will be stored separately in 
another locked file cabinet. Clinical information will not be released without written permission of the 
subject, except as necessary for monitoring by the FDA. The Investigator must also comply with all 
applicable privacy regulations (e.g., Health Insurance Portability and Accountability Act of 1996, EU 
Data Protection Directive 95/46/EC). 
17.1 Protocol Amendments 
Any amendment to the protocol will be written by the Investigator or their designee. Protocol 
amendments cannot be implemented without prior written IRB/IEC approval except as necessary to 
eliminate immediate safety hazards to patients. A protocol amendment intended to eliminate an 
apparent immediate hazard to patients may be implemented immediately, provided the IRBs are 
notified within five working days. Protocol amendments will be sent to Shionogi in addition to the 
IRB/IEC and FDA as required. 
17.2 Institutional Review Boards and Independent Ethics Committees 
The protocol and consent form will be reviewed and approved by the IRB/IEC of each participating 
center prior to study initiation. Serious adverse experiences regardless of causality will be reported to 
the IRB/IEC in accordance with the standard operating procedures and policies of the IRB/IEC, and the 
Investigator will keep the IRB/IEC informed as to the progress of the study. The Investigator will obtain 
assurance of IRB/IEC compliance with regulations. 
Any documents that the IRB/IEC may need to fulfill its responsibilities (such as protocol, protocol 
amendments, Investigator’s Brochure, consent forms, information concerning patient recruitment, 
payment or compensation procedures, or other pertinent information) will be submitted to the IRB/IEC. 
The IRB/IECs written unconditional approval of the study protocol and the informed consent form will be 
in the possession of the Investigator before the study is initiated. The IRB/IECs approval statement will 
be transmitted by the Investigator to Shionogi. This approval must refer to the study by exact protocol 
title and number and should identify the documents reviewed and the date of review. 
Protocol and/or informed consent modifications or changes may not be initiated without prior written 
IRB/IEC approval except when necessary to eliminate immediate hazards to the patients or when the 
change(s) involves only logistical or administrative aspects of the study. Such modifications will be 
submitted to the IRB/IEC and written verification that the modification was submitted and subsequently 
approved should be obtained.  
The CanRB/CanEC must be cannformed of re”cansio’s to other documents originally submitted for 
review; serious and/or unexpected adverse experiences occurring during the study in accordance with 
the standard operating procedures and policies of the IRB; new information that may affect adversely 
the safety of the patients of the conduct of the study; an annual update and/or request for re-approval; 
and when the study has been completed. 
17.3 Informed Consent Form  
Informed consent will be obtained in accordance with the Declaration of Helsinki, ICH GCP, US Code of 
Federal Regulations for Protection of Human Subjects (21 CFR 50.25[a,b], CFR 50.27, and CFR Part 

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56, Subpart A), the Health Insurance Portability and Accountability Act (HIPAA, if applicable), and local 
regulations. 
The Investigator will prepare the informed consent form and HIPAA authorization for submission to the 
IRB/IEC. The consent form generated by the Investigator must be approved by the IRB/IEC. The 
written consent document will embody the elements of informed consent as described in the 
International Conference on Harmonisation and will also comply with local regulations. The Investigator 
will store an IRB/IEC-approved copy of the Informed Consent Form for the study file. 
A properly executed, written, cannformed consent will be obtained from each subject prior to entering 
the subject into the trial. Information should be given in both oral and written form and subjects (or their 
legal representatives) must be given ample opportunity to inquire about details of the study. If 
appropriate and required by the local IRB/IEC, assent from the subject will also be obtained. If a subject 
is unable to sign the informed consent form (ICF) and the HIPAA authorization, a legal representative 
may sign for the subject.  A copy of the signed consent form will be offered to the subject or legal 
representative of the subject and the original will be maintained with the subject’s records. 
17.4 Publications  
The preparation and submittal for publication of manuscripts containing the study results shall be in 
accordance with a process determined by mutual written agreement among the study Investigator and 
participating institutions. The publication or presentation of any study results shall comply with all 
applicable privacy laws, including, but not limited to, the Health Insurance Portability and Accountability 
Act of 1996.  
17.5 Investigator Responsibilities 
By signing the Agreement of Investigator form, the Investigator agrees to: 
1. 
Conduct the study in accordance with the protocol and only make changes after notifying Shionogi, 
except when to protect the safety, rights or welfare of subjects. 
25. Personally conduct or supervise the study (or investigation). 
26. Ensure that the requirements relating to obtaining informed consent and IRB review and approval 
meet federal guidelines, as stated in § 21 CFR, parts 50 and 56. 
27. Report to Shionogi or designee any AEs that occur in the course of the study, in accordance with 
§21 CFR 312.64. 
28. Ensure that all associates, colleagues and employees assisting in the conduct of the study are 
informed about their obligations in meeting the above commitments. 
29. Maintain adequate and accurate records in accordance with §21 CFR 312.62 and to make those 
records available for inspection with Shionogi (or designee). 
30. Ensure that an IRB that complies with the requirements of §21 CFR part 56 will be responsible for 
initial and continuing review and approval of the clinical study. 
31. Promptly report to the IRB and Shionogi (or designee) all changes in the research activity and all 
unanticipated problems involving risks to subjects or others (to include amendments and IND 
safety reports). 
32. Seek IRB approval before any changes are made in the research study, except when necessary to 
eliminate hazards to the patients/subjects. 
33. Comply with all other requirements regarding the obligations of clinical investigators and all other 
pertinent requirements listed in § 21 CFR part 312. 

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18 REFERENCES 
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Nalbandian A, Sehgal K, Gupta A, et al. Post-acute COVID-19 syndrome. Nat Med 2021. DOI: 
10.1038/s41591-021-01283-z. 
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Global Burden of Disease Long CC, Wulf Hanson S, Abbafati C, et al. Estimated Global 
Proportions of Individuals With Persistent Fatigue, Cognitive, and Respiratory Symptom 
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Proal AD, VanElzakker MB, Aleman S, et al. SARS-CoV-2 reservoir in post-acute sequelae of 
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Peluso MJ, Anglin K, Durstenfeld MS, et al. Effect of Oral Nirmatrelvir on Long COVID 
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Kuroda T, Nobori H, Fukao K, et al. Efficacy comparison of 3CL protease inhibitors ensitrelvir 
and nirmatrelvir against SARS-CoV-2 in vitro and in vivo. J Antimicrob Chemother 2023. DOI: 
10.1093/jac/dkad027. 
7. 
Noske GD, de Souza Silva E, de Godoy MO, et al. Structural basis of nirmatrelvir and ensitrelvir 
activity against naturally occurring polymorphisms of the SARS-CoV-2 Main Protease. J Biol 
Chem 2023;299(3):103004. DOI: 10.1016/j.jbc.2023.103004. 
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10.1097/MD.0000000000033024. 
9. 
Kawashima S, Matsui Y, Adachi T, et al. Ensitrelvir is effective against SARS-CoV-2 3CL 
protease mutants circulating globally. Biochem Biophys Res Commun 2023;645:132-136. DOI: 
10.1016/j.bbrc.2023.01.040. 
10. 
Mukae H, Yotsuyanagi H, Ohmagari N, et al. A Randomized Phase 2/3 Study of Ensitrelvir, a 
Novel Oral SARS-CoV-2 3C-Like Protease Inhibitor, in Japanese Patients with Mild-to-
Moderate COVID-19 or Asymptomatic SARS-CoV-2 Infection: Results of the Phase 2a Part. 
Antimicrob Agents Chemother 2022;66(10):e0069722. DOI: 10.1128/aac.00697-22. 
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Mukae H, Yotsuyanagi H, Ohmagari N, et al. Efficacy and safety of ensitrelvir in patients with 
mild-to-moderate COVID-19: the phase 2b part of a randomized, placebo-controlled, phase 2/3 
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Gaebler C, Wang Z, Lorenzi JCC, et al. Evolution of antibody immunity to SARS-CoV-2. 
Nature 2021. DOI: 10.1038/s41586-021-03207-w. 
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Aydillo T, Gonzalez-Reiche AS, Aslam S, et al. Shedding of Viable SARS-CoV-2 after 
Immunosuppressive Therapy for Cancer. N Engl J Med 2020;383(26):2586-2588. DOI: 
10.1056/NEJMc2031670. 
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Choi B, Choudhary MC, Regan J, et al. Persistence and Evolution of SARS-CoV-2 in an 
Immunocompromised Host. N Engl J Med 2020;383(23):2291-2293. DOI: 
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15. 
Zollner A, Koch R, Jukic A, et al. Postacute COVID-19 is Characterized by Gut Viral Antigen 
Persistence in Inflammatory Bowel Diseases. Gastroenterology 2022;163(2):495-506.e8. DOI: 
10.1053/j.gastro.2022.04.037. 
16. 
Natarajan A, Zlitni S, Brooks EF, et al. Gastrointestinal symptoms and fecal shedding of SARS-
CoV-2 RNA suggest prolonged gastrointestinal infection. Med (N Y) 2022. DOI: 
10.1016/j.medj.2022.04.001. 
17. 
Xu Q, Milanez-Almeida P, Martins AJ, et al. Adaptive immune responses to SARS-CoV-2 
persist in the pharyngeal lymphoid tissue of children. Nat Immunol 2023;24(1):186-199. DOI: 
10.1038/s41590-022-01367-z. 
18. 
Cheung CCL, Goh D, Lim X, et al. Residual SARS-CoV-2 viral antigens detected in GI and 
hepatic tissues from five recovered patients with COVID-19. Gut 2022;71(1):226-229. DOI: 
10.1136/gutjnl-2021-324280. 
19. 
Goh D, Lim JCT, Fernaíndez SB, et al. Case report: Persistence of residual antigen and RNA of 
the SARS-CoV-2 virus in tissues of two patients with long COVID. Front Immunol 
2022;13:939989. DOI: 10.3389/fimmu.2022.939989. 
20. 
Stein SR, Ramelli SC, Grazioli A, et al. SARS-CoV-2 infection and persistence in the human 
body and brain at autopsy. Nature 2022;612(7941):758-763. DOI: 10.1038/s41586-022-05542-y. 
21. 
Peluso MJ, Ryder D, Flavell R, et al. Multimodal Molecular Imaging Reveals Tissue-Based T 
Cell Activation and Viral RNA Persistence for Up to 2 Years Following COVID-19. medRxiv 
2023. DOI: 10.1101/2023.07.27.23293177. 
22. 
Jin JC, Ananthanarayanan A, Brown JA, et al. SARS CoV-2 detected in neonatal stool remote 
from maternal COVID-19 during pregnancy. Pediatr Res 2022:1-8. DOI: 10.1038/s41390-022-
02266-7. 
23. 
Racine T, Kobinger GP. Viral pathogenesis: Unlocking Ebola persistence. Nat Microbiol 
2017;2:17124. DOI: 10.1038/nmicrobiol.2017.124. 
24. 
Tejerina F, Catalan P, Rodriguez-Grande C, et al. Post-COVID-19 syndrome. SARS-CoV-2 
RNA detection in plasma, stool, and urine in patients with persistent symptoms after COVID-19. 
BMC Infect Dis 2022;22(1):211. DOI: 10.1186/s12879-022-07153-4. 
25. 
Swank Z, Senussi Y, Manickas-Hill Z, Yu XG, Li JZ, Alter G, Walt DR. Persistent circulating 
SARS-CoV-2 spike is associated with post-acute COVID-19 sequelae. Clin Infect Dis 2022. 
DOI: 10.1093/cid/ciac722. 
26. 
Peluso MJ, Kelly JD, Lu S, et al. Persistence, magnitude, and patterns of postacute symptoms 
and quality of life following onset of SARS-CoV-2 infection: cohort description and approaches 
for measurement.  Open forum infectious diseases2022:ofab640. 
27. 
Peluso MJ, Deitchman AN, Torres L, et al. Long-term SARS-CoV-2-specific immune and 
inflammatory responses in individuals recovering from COVID-19 with and without post-acute 
symptoms. Cell Rep 2021:109518. DOI: 10.1016/j.celrep.2021.109518. 
28. 
Peluso MJ, Lu S, Tang AF, et al. Markers of Immune Activation and Inflammation in 
Individuals With Postacute Sequelae of Severe Acute Respiratory Syndrome Coronavirus 2 
Infection. J Infect Dis 2021. DOI: 10.1093/infdis/jiab490. 
29. 
Durstenfeld MS, Peluso MJ, Kelly JD, et al. Role of antibodies, inflammatory markers, and 
echocardiographic findings in postacute cardiopulmonary symptoms after SARS-CoV-2 
infection. JCI Insight 2022;7(10). DOI: 10.1172/jci.insight.157053. 

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30. 
Peluso MJ, Sans HM, Forman CA, et al. Plasma Markers of Neurologic Injury and Inflammation 
in People With Self-Reported Neurologic Postacute Sequelae of SARS-CoV-2 Infection. Neurol 
Neuroimmunol Neuroinflamm 2022;9(5). DOI: 10.1212/NXI.0000000000200003. 
31. 
Peluso MJ, Thomas IJ, Munter SE, Deeks SG, Henrich TJ. Lack of Antinuclear Antibodies in 
Convalescent Coronavirus Disease 2019 Patients With Persistent Symptoms. Clin Infect Dis 
2022;74(11):2083-2084. DOI: 10.1093/cid/ciab890. 
32. 
Bodansky A, Wang CY, Saxena A, et al. Autoantigen profiling reveals a shared post-COVID 
signature in fully recovered and long COVID patients. JCI Insight 2023;8(11). DOI: 
10.1172/jci.insight.169515. 
33. 
Giron LB, Peluso MJ, Ding J, et al. Markers of fungal translocation are elevated during post-
acute sequelae of SARS-CoV-2 and induce NF-κB signaling. JCI Insight 2022;7(18). DOI: 
10.1172/jci.insight.164813. 
34. 
Hellmuth J, Barnett TA, Asken BM, et al. Persistent COVID-19-associated neurocognitive 
symptoms in non-hospitalized patients. J Neurovirol 2021;27(1):191-195. DOI: 10.1007/s13365-
021-00954-4. 
35. 
Apple AC, Oddi A, Peluso MJ, et al. Risk factors and abnormal cerebrospinal fluid associate 
with cognitive symptoms after mild COVID-19. Ann Clin Transl Neurol 2022;9(2):221-226. 
DOI: 10.1002/can3.51498. 
36. 
Durstenfeld MS, Sun K, Tahir P, et al. Use of Cardiopulmonary Exercise Testing to Evaluate 
Long COVID-19 Symptoms in Adults: A Systematic Review and Meta-analysis. JAMA Netw 
Open 2022;5(10):e2236057. DOI: 10.1001/jamanetworkopen.2022.36057. 

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1  For participants who received placebo and are opting to participate in study drug rollover, the repeat screening and baseline visits may be combined at the 
investigators discretion but the repeat baseline visit must be repeated as with the initial schedule of events. 
2 The baseline visit and intervention visit may be combined to minimize participant burden.  
3 Assessments at the D1 Phone FU visit will focus on adverse events since taking the medication. Assessments at the D4 Phone FU visit will focus on the 
preceding 2 days (rather than the preceding month) to assess AEs and the potential impact of treatment.  
4 For persons of childbearing potential, a negative pregnancy test must be confirmed prior to the participant being dispensed the medication vs placebo. 
5 If the intervention visit is on the same day as the baseline visit, blood is collected for biospecimen storage will only be collected once. 
 
 
 

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• APPENDIX 2. CONTRACEPTION GUIDANCE 
 
CONTRACEPTIVESa ALLOWED DURING THE STUDY INCLUDE: 
Highly Effective Methodsb That Have Low User Dependency Failure rate of < 1% per year when used consistently and correctly 
• 
Implantable progestogen-only hormone contraception associated with inhibition of ovulationc 
PLUS an additional barrier method 
• 
Intrauterine device (IUD)  
• 
Intrauterine hormone-releasing system (IUS)c 
• 
Bilateral tubal occlusion 
• 
Vasectomized partner (Vasectomized partner is a highly effective contraceptive method provided that the partner is the sole sexual partner of the woman of childbearing potential and 
the absence of sperm has been confirmed. If not, an additional highly effective method of contraception should be used. Spermatogenesis cycle is approximately 90 days.) 
• 
Combined (estrogen- and progestogen-containing) hormonal contraceptionc associated with inhibition of ovulation (eg, oral, intravaginal, transdermal, injectable) 
PLUS an additional barrier method  
• 
Progestogen-only hormone contraceptivec associated with inhibition of ovulation: oral, injectable PLUS an additional barrier method  
Highly Effective Methodsb That Are User Dependent Failure rate of < 1% per year when used consistently and correctly 
• 
Sexual abstinence (Sexual abstinence is considered a highly effective method only if defined as refraining from heterosexual intercourse during the entire period of risk 
associated with the study intervention. The reliability of sexual abstinence needs to be evaluated in relation to the duration of the study and the preferred and usual 
lifestyle of the participant.)  
a 
Contraceptive use by men or women should be consistent with regulations regarding the methods of contraception for those participating in clinical studies. 
B 
Failure rate of < 1% per year when used consistently and correctly. Typical use failure rates differ from those when used consistently and correctly. 
C 
S-217622 may lead to an increase or decrease of hormonal contraception levels. Therefore, hormonal contraception must not be used alone and must be combined with 
other barrier method. 
Note: Periodic abstinence (calendar, symptothermal, postovulation methods), withdrawal (coitus interruptus), spermicides only, and lactational amenorrhoea method are not 
acceptable methods of contraception. Male condom and female condom should not be used together (due to risk of failure with friction). 
 
 

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• APPENDIX 3. PROHIBITED MEDICATION 
 
Use of the following therapies will be prohibited from the time of informed consent 
through 28 days after the first dose of study intervention: 
 Sensitive CYP3A substrates with narrow therapeutic index. S-217622 is considered to be a strong inhibitor of CYP3A4. 
(Note: remdesivir and combined oral contraceptives are permitted). Examples of sensitive CYP3A substrates with narrow 
therapeutic index include the following medications: alfentanil, cyclosporine, sirolimus, tacrolimus and terfenadine 
 
Use of the following therapies is prohibited from the time of informed consent through 10 days after the last dose of study 
intervention. However, even if a prohibited concomitant therapy is used, the participant should continue the study procedures 
according to the scheduled of event (Appendix 1) to the extent possible. 
 Hydroxychloroquine, and ivermectin are prohibited due to DDIs with S-217622. Hydroxychloroquine and ivermectin are 
both CYP3A substrates and thus may also be substantially impacted by S-217622 with an increase in drug levels.  
 Paxlovid (nirmatrelvir/ritonavir): Potential clinically significant interaction is likely expected when administered with S-
217622. Coadministration of itraconazole, strong CYP3A4 inhibitor (200 mg once daily 8 doses), and nirmatrelvir/ritonavir 
(300/100 mg twice daily 5 doses) increased nirmatrelvir AUC and Cmax by 39% and 19%, respectively.  
 Corticosteroids administered via any route (including intranasal, inhaled, oral, intra-articular but excluding topical). However, 
prednisolone is permitted based on a lack of significant interaction in a clinical DDI study. Intranasal or inhaled fluticasone is 
permitted because the DDI potential by CYP3A inhibitor would be low. Furthermore, intranasal or inhaled beclomethasone is 
allowed based on demonstration of the lack of a significant interaction with darunavir/ritonavir, and dexamethasone can be 
administered the day after the last dose of study intervention administration based on the results of clinical DDI study with 
dexamethasone.  
 CYP3A substrates except for sensitive CYP3A substrates with narrow therapeutic index. S-217622 is considered to be a 
strong inhibitor of CYP3A4. (Note: remdesivir and combined oral contraceptives are permitted). Examples of prohibited 
CYP3A substrates include the following medications: alprazolam, anamorelin, aprepitant, atorvastatin, avanafil, azelnidipine, 
blonanserin, budesonide, buspirone, chloroquine, colchicine, conivaptan, darifenacin, darunavir, dasatinib, 
dihydroergotamine, dronedarone, ebastine, eletriptan, eliglustat, eplerenone, ergometrine, ergotamine, everolimus, felodipine, 
finerenone, ibrutinib, indinavir, ivabradine, lomitapide, lovastatin, lurasidone, maraviroc, methylergometrine, midazolam, 
naloxegol, nisoldipine, pimozide, quetiapine, quinidine, rilpivirine, riociguat, rivaroxaban, saquinavir, sildenafil, simvastatin, 
suvorexant, tadalafil, ticagrelor, tipranavir, triazolam, tolvaptan, vardenafil, and venetoclax. For additional CYP3A 

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substrates, see the Liverpool DDI checker (https://www.covid19-druginteractions.org/checker). 
 
Use of the following therapies is prohibited from the time of informed consent through the day after the last study intervention. 
 Strong CYP3A inducers. Examples include apalutamide, carbamezpine, enzalutamide, mitotane, phenytoin, phosphenytoin, 
rifampin and St. John’s wort. For additional strong CYP3A inducers, see the Liverpool DDI checker (https://www.covid19-
druginteractions.org/checker). 
 Methotrexate as organic anion transporter 3 (OAT3) substrate with a narrow therapeutic index.  
 Digoxin and dabigatran as P-gp substrates with a narrow therapeutic index.  
 High dose of rosuvastatin (20 to 40 mg, as it is considered BCRP substrate and S-217622 may influence the treatment by 
rosuvastatin when high dose of rosuvastatin is administered). 
 
 
 

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APPENDIX 5. PROTOCOL AMENDMENTS 
Date 
Version 
Section 
Changes 
2023-JUN-22 
1.1 
N/A 
N/A 
2023-AUG-15 
1.2 
Exclusion 
criteria 
Exclusion criteria was updated to allow experimental PET imaging within 14 days of 
Day 0 rather than 28 days for all other interventional studies. 
2023-AUG-15 
1.2 
Safety 
Monitoring 
The independent medical monitor has been replaced by a safety monitoring 
committee.  The monitoring schedule has not been changed. 
2023-NOV-27 
1.4 
All 
Corrected references (previously two separate reference lists now consolidated into 
one) 
2023-NOV-27 
1.4 
6.2 
1 – Removed mention of sex/gender from inclusion criteria – all adults regardless of 
sexual or gender identity are eligible and the unnecessary detail created confusion 
3 – Simplified item 3 which was previously redundant 
4 – Clarified at least two symptoms or at least one severe symptom. Added that at 
least 90 days have elapsed since the most recent SARS-CoV-2 infection 
7 – Corrected criterion 7 to indicate “In otherwise stable health prior to Screen” rather 
than “D0” since eligibility for the protocol must be determined prior to D0. 
8 – Clarified that study entry is defined as D0 
9 – Added additional detail about the timing requirements of contraception to make 
internally consistent. 
2023-NOV-27 
1.4 
6.3 
2 – Clarified that that no SARS-CoV-2 antiviral should have been administered within 
60 days prior to D0 (not just 60 days prior to D0) 
4 – Clarified cardiovascular disease within 90 days should be excluded (not ever) – 
this is consistent with other UCSF-based Long COVID study protocols; also clarified 

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that those with current heart failure or pulmonary hypertension are excluded but those 
with resolved heart failure are eligible. 
11 – Indicated that these laboratory tests occur at screening. Corrected hemoglobin 
units. 
12 
– Added ESRD (FDA recommendation) 
14 – Added severe hepatic impairment (FDA recommendation) 
15 – Clarified that any HIV infection is exclusionary. Added clarification about steroid 
therapy. 
16, 17 – specified that those with a history of ME/CFS or dysautonomia that is not 
worsened since SARS-CoV-2 infection would be ineligible 
19 – revised to create consistency with Exclusion Criterion #1 for monoclonal 
antibodies or equivalent products, and to provide additional detail about timing since 
investigational PET tracers 
Adjusted multiple other criteria to anchor on planned D0, since eligibility needs to be 
determined prior to D0. 
2023-NOV-27 
1.4 
9.1.4 
Clarified that a physician, nurse, or PA may complete the physical assessments. 
2023-NOV-27 
1.4 
9.1.5 
Indicated that vital signs in various positions may be obtained. 
2023-NOV-27 
1.4 
9.1.6 
Indicated details about Long COVID assessments versus Other Symptom 
Assessment (AE Review) 
Added detail about PGIC 
Added details of ECOG 
Clarified that a neurocognitive assessment other than NIH Toolbox might be used. 
Removed Post-COVID function scale, which has proven to be insensitive in other 
studies by our group and burdensome to participants 
Clarified that a dysautonomia assessment other than COMPASS-31 may be 
performed. 

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Added detail about DSQ-PEM. 
Added detail about WHO-DAS. 
2023-NOV-27 
1.4 
8.4.2 
Added detail about medication dispensing and logging. 
2023-NOV-27 
1.4 
10.1 
Referred to the Schedule of Events for screening procedures. 
Added note about rescreening. 
2023-NOV-27 
1.4 
10.2 
Added note that the D0 visit may be combined with the baseline visit.  
Added additional details about visit window. 
2023-NOV-27 
1.4 
10.3 
Added additional safety check prior to D0 to ensure no new changes to health have 
occurred since screen. 
Added detail that negative pregnancy test must be confirmed prior to dispensing 
study drug. 
2023-NOV-27 
1.4 
9.3.3 
Added additional detail about SARS-CoV-2 genotyping 
2023-NOV-27 
1.4 
10.4 
Added additional phone follow-up. 
2023-NOV-27 
1.4 
10.5 
Combined D7 and D15 visits into a single D10 visit to (1) minimize participant burden 
with one fewer in person visit (2) improve the consistency in timing of measurement 
for the primary outcomes of the study. 
2023-NOV-27 
1.4 
10.8 
Corrected discontinuation visits to sync with the new SOE. 
2023-NOV-27 
1.4 
12.3 
Added details about liver toxicity monitoring and overdose monitoring 
2023-NOV-27 
1.4 
14 
Edited SMC meeting frequency to every 4 months to sync with our other Long COVID 

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clinical trials. 
Indicated that the study would be paused for grade 3 or 4 AEs or SAEs in any 
participant (FDA recommendation) 
Clarified rules around study pause to indicate that scheduled visits at which study 
product is not dispensed (screening, baseline, and follow up visits) may still occur but 
no new study product may be dispensed. 
2023-NOV-27 
1.4 
SOE 
Amended schedule of events to reflect protocol changes (extra phone visit, one fewer 
in person visit) 
Added PGIC scale to be consistent with other Long COVID trials 
Added ECOG (LIINC Form) 
Removed COMPASS-31 due to licensing issues; will now use the orthostatic 
hypotension questionnaire which does not have licensing issues 
Added DSQ-PEM, an assessment of post-exertional malaise 
Added WHO-DAS as a disability scale, at recommendation of patient groups 
Removed mention of NIH Toolbox; will use CNS-Vital Signs like other UCSF-based 
Long COVID trials 
Corrected safety labs from baseline to intervention visit; this was already indicated in 
the “real-time laboratory tests” below and does not represent a change  
Removed ESR, fibrinogen, and D-dimer as real-time laboratory tests. These have not 
been useful in other Long COVID studies and may be performed post-hoc. 
Added note that baseline and intervention visit may be combined to minimize 
participant burden. 
2024-FEB-21 
1.5 
2.3.1 
Updated evaluation of anemia monitoring to match with SOE.  
2024-FEB-21 
1.5 
5.1 
1 – Corrected the endpoint visit for Promis-29 to match with SOE 
2 – Removed mention of NIH Toolbox; will use CNS-Vital Signs like other UCSF-
based Long COVID trials 

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3 – Corrected the endpoint visits for neurocognitive assessment, DASI, dysautonomia 
assessment and 6MWT to match with SOE 
4 – Corrected Day 15 to Day 10 
2024-FEB-21 
1.5 
12.3 
Corrected end of study visit from Day 360 to Day 60 
2024-FEB-26 
1.5 
10.3 
Removed language requiring first dose of study intervention to be taken at home 
2024-OCT-15 
1.6 
Inclusion 
Criteria 
Updated effective contraception methods requirement to begin 21 days prior to the 
start of study intervention 
2024-OCT-15 
1.6 
9.2.1 
Removed mention of ESR to match SOE update from version 1.4 
2024-NOV-05 
1.6 
Appendix 4 
Added Open Label Extension Schedule of Events for after unblinding rollover dosing 
2024-NOV-05 
1.6 
Appendix 5 
Appendix 4 in version 1.5 “Tracked Changes” has been relabeled Appendix 5  
2024-NOV-05 
1.6 
Entire 
Protocol 
Updated Version Date to match most current date of changes, corrected formatting 
on table of contents 
 
 
 

UNIVERSITY OF CALIFORNIA, SAN FRANCISCO 
 PLACEBO-CONTROLLED, RANDOMIZED TRIAL OF ENSITRELVIR (S-217622) FOR VIRAL 
PERSISTENCE AND INFLAMMATION IN PEOPLE EXPERIENCING LONG COVID 
(PREVAIL-LC)  
NCT06161688 
Statistical Analysis Plan Finalization Date: 02/21/2025 

Statistical Analysis Plan – PREVAIL-LC  
   
Version #:1.0 Version Date: Feb 2025 1 of 12   
Statistical Analysis Plan (SAP)  
  
Title: Placebo-Controlled, Randomized Trial of Ensitrelvir (S-217622) for Viral 
Persistence and Inflammation in People Experiencing Long COVID (PREVAIL-LC)   
  
Primary Investigator: Timothy Henrich, MD  
  
Co-Investigator: Michael Peluso, MD  
  
Biostatistician: David V. Glidden, PhD  
  
Version: 1.0  
  
Version Date: February 2025  
  
Primary Investigator: Timothy Henrich, MD 
 
Date:  2/21/2025 
Co-Investigator: Michael Peluso, MD  
 
Date:  2/20/2025 
Lead Biostatistician: David V. Glidden, PhD  
Date:  2/21/2025 
  
   
  
1. Introduction  
The objective of this statistical analysis plan (SAP) is to describe the general analytic 
strategy and the statistical methods that will be used to analyze the data for 
“PlaceboControlled, Randomized Trial of Ensitrelvir (S-217622) for Viral Persistence 
and Inflammation in People Experiencing Long COVID (PREVAIL-LC).   
  
2. Study Design  
2.1. Randomization and Follow-Up  
  

Statistical Analysis Plan – PREVAIL-LC  
   
Version #:1.0 Version Date: Feb 2025 2 of 12   
This is an exploratory, 1:1 randomized, double-blind, placebo-controlled study to assess 
the safety and efficacy of Ensitrelvir (S-217622) to treat Long COVID (LC).  The study 
will enroll approximately 40 participants who meet the WHO LC criteria.  
  
Participants will be enrolled at a single center and randomized 1:1 to receive 5 days of 
Ensitrelvir or placebo. Evaluations will take place at baseline and at timepoints up to 2 
months post-administration.   
  
Screening data will be reviewed to determine subject eligibility. Subjects who meet all 
inclusion criteria and none of the exclusion criteria (see study protocol) will be entered 
into the study.   
  
The following treatment regimens will be used:  
  
Route  
Planned 
Dose  
Dose  
Oral  
Ensitrelvir  
375 mg on day 1, followed by 125 mg daily for 4 additional 
days  
Placebo  
NA  
  
The total duration of subject participation will be approximately 3 months. In-person 
study visits will occur at screening, baseline, intervention, and days 10 and 30 (D10 and 
D30, respectively) following administration. Additional interviews will be conducted by 
phone on days 1, 4, and 60 (D1, D4 and D60).   
  
2.2. Study Endpoints   
  
Efficacy Endpoints   
● Change in PROMIS-29 Physical and Mental health score in those with symptoms at 
Baseline and at D10 and D30.   
● Change in EuroQoL Quality of Life between Baseline and at D10, D30, and D60.   
● Change in other assessments (neurocognitive assessment (i.e. ECog, CNS-VS) and 
6MWT performance) between Baseline and at D10.   
● Change in other assessments (DASI, dysautonomia assessment) between Baseline 
and at D10 and D30.   
● Percentage of participants with no detection of SARS-CoV-2 plasma remnants (i.e., 
viral detection by reverse transcriptase-polymerase chain reaction (RT-PCR) and 
Spike protein fragments) compared to baseline at D10 and D30 post-ensitrelvir 

Statistical Analysis Plan – PREVAIL-LC  
   
Version #:1.0 Version Date: Feb 2025 3 of 12   
administration.  ● Proportion with reduction in inflammatory markers (e.g. CRP) at 
Baseline and at D10 and D30.   
  
2.3. Safety Endpoints   
The National Institutes of Health Division of AIDS (DAIDS) Table for Grading the 
Severity of Adult and Pediatric Adverse Events, Corrected Version 2.1 (July 2017) will 
be used to assess and grade AE severity, including laboratory abnormalities judged to 
be clinically significant.  
  
Criteria for relatedness of adverse events is given Sec 11.1.2 of the study protocol. 
Criteria for SAE are given in Sec 11.2 of the protocol.  
  
● Safety endpoints include number of participants with adverse events (AE)s 
(Treatment Emergent Adverse Events (TEAE)s, Serious Adverse Events (SAE)s, 
and Adverse Events of Special Interest (AESI). The primary outcome will be the 
number of individuals in each group experiencing a severe (Grade 3 or greater) 
AE thought to be possibly, probably, or definitely related to study treatment 
throughout the study duration.   
  
2.4. Sample Size and Study Power   
  
This is an exploratory study, and the primary results are meant to be descriptive. We 
use Ensitrelvir as a probe to better understand LC biology and determine whether a 
signal is present to warrant further study.  
The study will enroll 40 participants who meet the WHO LC criteria. Participants will be 
enrolled at a single center and randomized 1:1 to receive Ensitrelvir or placebo.   
For PROMIS-29 Physical Health Score, assuming a standard deviation (SD) of the 
within-person difference of 10, then the planned sample size has 80% power to detect a 
difference of 11.2 on a two-sided 0.05 level test.   
For biomarkers, such as CRP, we can detect a difference from a mean value of 2.5 
pg/mL to 1.1 times the within-person standard deviation with 80% power.  
We assume that 25% of participants will have positive antigen at entry, we will have 
62% power to detect a reduction to < 1% with positive antigen on the treatment arm.  
3. Analysis Principles   
  
The following principles apply for the comparisons of the participants randomized to 
Ensitrelvir (S-217622) (“active group”) versus the participants randomized to placebo 
(“control group”).  

Statistical Analysis Plan – PREVAIL-LC  
   
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3.1. Analysis populations  
  
Comparisons for safety and efficacy outcomes will be by modified intention-to- treat 
(mITT). The modified intention-to-treat analysis is restricted to participants who 
received at least one dose of blinded Ensitrelvir/placebo; participants who did not 
receive any Ensitrelvir//placebo are excluded from the mITT population.  
  
Sensitivity analyses by intention-to-treat (ITT) will be carried out for the primary efficacy 
outcome and key secondary outcomes. The ITT population is all participants who were 
randomized during the study and assigns them to the group to which they were 
randomized.  
  
The per protocol population (PP) is restricted to participants who received any of the 
blinded Ensitrelvir/Placebo. It excludes participants who received a non-study 
COVIDantiviral therapy during the trial period (prior to Day 30).   
  
The safety population will be identical to the mITT population.  
3.2. Descriptive statistics  
  
Descriptive statistics will be reported overall and by randomization group. For 
categorical outcomes, the number and percent in each category will be reported; 
percentages will be of non-missing values, if data are not complete. Continuous 
variables will be summarized by median (interquartile range [IQR]) and/or mean (SD). 
Continuous variables may be categorized (e.g., age may be broken into categories to 
investigate the distribution across age groups).  
  
3.3. Binary outcomes  
  
For binary outcomes, probabilities will be compared between the investigational agent 
and its control group using Fisher’s exact tests or logistic regression. Proportions will be 
accompanied by Agresti Coull 95% confidence intervals1. Relative risks will be 
calculated with 2-sided 95% confidence intervals.  Risk difference (RD) with 2-sided 
95% confidence intervals (CI) will also be compared between the groups. If the numbers 
in the denominators are small, our confidence intervals will use the pseudo-observation 
approach of Agresti and Caffo.2  
  
For longitudinally measured binary outcomes, the treatment effect, in relative risks, 
through follow-up will be estimated with 95% confidence intervals using generalized 

Statistical Analysis Plan – PREVAIL-LC  
   
Version #:1.0 Version Date: Feb 2025 5 of 12   
estimating equations (GEE) with a Poisson family and a log link function; the treatment 
effect is estimated via the interaction between the indicator for randomization group and 
the binary indicator for follow-up (versus baseline) visits. Here, we may also employ the 
pseudo-observation approach to calculating the confidence intervals if the outcomes are 
rare. 2 The treatment effect, in risk differences, with 95% confidence intervals will be 
estimated by margining the models and calculating 95% confidence intervals by the 
chain rule.  
  
3.4. Ordered categorical outcomes  
  
Ordinal variables will be compared between randomization groups using 
WilcoxonMann-Whitney.  We may summarize effects quantifying the treatment effects 
using the proportional odds model with the summary OR of being in a better category in 
the  group compared to the placebo group, adjusted for baseline category, will be 
estimated with a 2-sided 95% CI  The validity of the proportional odds assumption will 
be assessed by testing the log ORs (for the treatment effect) across the dichotomized 
cumulative ordered categories in the corresponding ordered logistic regression model 
(partial proportional odds model, test for “unequal slopes”)3. For longitudinal measured 
ordinal variables, the treatment effect through follow-up will be estimated with 95% 
confidence intervals using generalized estimating equations (GEE) with a logistic link 
function; the treatment effect is estimated via the interaction between the indicator for 
randomization group and the binary indicator for follow-up (versus baseline) visits.  
3.5. Continuous outcomes   
  
Continuous outcomes will be compared between randomization groups using ANCOVA 
models for comparing means, if the ANCOVA model assumptions hold. If the 
distributions of the continuous outcomes are skewed, outcomes may be transformed or 
compared between randomization groups using rank-based methods, such as the 
Wilcoxon test, or quantile (median) regression. For example, biomarker levels often 
require log-transformation to meet model assumptions for ANCOVA analyses.  
  
Comparisons between randomization groups for a continuous outcome will be adjusted 
for baseline values of the outcome, for the purpose of variance reduction, unless there 
are concerns over model stability with such an adjustment.   
  
To estimate the treatment effect for longitudinally measured continuous outcomes, 
including those measured at baseline and just once post-baseline. We will use linear 
mixed effects models (in case of Gaussian responses) with an indicator for 
randomization group, visit and visit by group interaction. The model will include an 
unstructured covariance structure. Models will be adjusted for the baseline values of the 

Statistical Analysis Plan – PREVAIL-LC  
   
Version #:1.0 Version Date: Feb 2025 7 of 12   
3.9. Subgroup analyses  
    
We will perform subgroups analyses by participant sex and antigen detection at 
baseline. A subgroup effect will be tested by a two-sided 0.05 level test of interaction.  
  
4. Efficacy Analyses   
  
The primary efficacy analyses will be conducted in the mITT population. Analysis in the 
per protocol population will be considered a sensitivity analysis.  
  
4.1. Primary Efficacy Outcome   
  
The primary efficacy outcome is the change in PROMIS-29 Physical Health score from 
Baseline to D10. The baseline value for the analysis will be the mean of screening and 
baseline values. This will be analyzed from the mITT population using a linear 
regression model for follow-up PROMIS-29 Physical Health score with terms for the 
baseline value, randomized treatment – an analysis of covariance model (ANCOVA).   
  
The significance of the treatment comparison will be given by a two-sided 0.05 level test 
for the randomized treatment term.   
The mean PROMIS-29 Physical Health score to D10 can be calculated by margining the 
ANCOVA model above.4  
Note, these estimates will differ from both taking the mean PROMIS-29 at the baseline 
and D10 timepoints. However, the ANCOVA estimate is more efficient.  
4.2. Secondary Efficacy Analyses   
  
Secondary efficacy outcome will be analyzed from the mITT population. There will be no 
formal adjustment for multiple testing in analyzing the secondary endpoints. Secondary 
outcomes are listed in 4.2.1, 4.2.2 and 4.2.3.  
  
4.2.1 Questionnaires  
  
● Change in PROMIS-29 Physical Health summary score from Baseline to D30.  
○ The primary estimand for this outcome is the difference in mean 
PROMIS29 Physical Health summary score from baseline. The baseline 
value for the analysis will be the mean of screening and baseline values.  
○ This continuous outcome will be analyzed as described in Section 3.5, i.e.  
using a mixed effect model for repeated measurements.  

Statistical Analysis Plan – PREVAIL-LC  
   
Version #:1.0 Version Date: Feb 2025 8 of 12   
  
● Change in PROMIS-29 Mental Health summary score from Baseline to D10.  
○ The primary estimand for this outcome is the difference in mean 
PROMIS29 Mental Health summary score from baseline.  
○ This continuous outcome will be analyzed as described in Section 3.5, i.e.  
using a mixed effect model for repeated measurements.  
  
● Change in PROMIS-29 Mental Health summary score from Baseline to D30.  
○ The primary estimand for this outcome is the difference in mean 
PROMIS29 Mental Health summary score from baseline.  
○ This continuous outcome will be analyzed as described in Section 3.5, i.e.  
using a mixed effect model for repeated measurements.  
  
• LIINC Questionnaire – Quality of Life by Visual Analogue Scale (VAS) from 
Baseline to D10.  
o The primary estimand for this outcome is the difference in mean VAS 
score from baseline. The baseline value for the analysis will be the mean 
of screening and baseline values. o This continuous outcome will be 
analyzed as described in Section 3.5, i.e.  
using a mixed effect model for repeated measurements.  
  
• Quality of Life Using the 5-Item EuroQol EQ-5D-5L index value score from 
Baseline to D10. o The primary estimand for this outcome is the difference in 
mean the EQ5D-5L index value from Baseline to D10. The baseline value for the 
analysis will be the mean of screening and baseline values.  
o This continuous outcome will be analyzed as described in Section 3.5, i.e.  
using a mixed effect model for repeated measurements.  
  
• Change in Duke Activity Status Index (DASI) from Baseline to D10.  
o The primary estimand for this outcome is the difference in mean DASI 
total score from baseline. The baseline value for the analysis will be the 
mean of screening and baseline values. o This continuous outcome will be 
analyzed as described in Section 3.5, i.e.  
using a mixed effect model for repeated measurements.  
  
• Change in the World Health Organization Disability Assessment Schedule 2.0 
(WHO-DAS 2.0) questionnaire from Baseline to D10.  

Statistical Analysis Plan – PREVAIL-LC  
   
Version #:1.0 Version Date: Feb 2025 9 of 12   
o The primary estimand for this outcome is the difference in mean 
WHODAS 2.0 score from baseline.   
o This continuous outcome will be analyzed as described in Section 3.5, i.e.  
using a mixed effect model for repeated measurements.  
  
• Patient Global Impression of Change (PGIC) at D10.  
o The primary estimand for this outcome is the relative odds of better 
change on the PGIC at D10 between the arms.  o This ordinal outcome 
will be analyzed as described in Section 3.4.   
  
• Change in the Everyday Cognition Form (ECog-41) instrument from Baseline to 
D10.  
o The primary estimand for this outcome is the difference in mean ECog-41 
score from baseline. o This continuous outcome will be analyzed as 
described in Section 3.5, i.e.  
using a mixed effect model for repeated measurements.  
  
• Change in Orthostatic Intolerance Questionnaire (OHQ) composite score from 
Baseline to D10. o The primary estimand for this outcome is the difference in 
mean OHQ composite score from baseline.   
o This continuous outcome will be analyzed as described in Section 3.5, i.e.  
using a mixed effect model for repeated measurements.  
  
4.2.2 Performance Measures  
  
• Change in 6 Minute Walking Test (6MWT) from Baseline to D10.  
o The primary estimand for this outcome is the difference in mean 6MWT 
change from baseline. o This continuous outcome will be analyzed as 
described in Section 3.5 , i.e.  
using a mixed effect model for repeated measurements.  
  
• Change in Active Stand Test Result from Baseline to D10.  
o The primary estimand for this outcome is the relative proportion with 
postural orthostatic tachycardia syndrome by the stand test. o This binary 
outcome will be analyzed as described in Section 3.3, i.e.  
using a GEE model.  
  
• Change in Neurocognition Index (NCI) standard score from the CNS-VS from 
Baseline to D10.  

St ati sti c al A n al y si s Pl a n – P R E V AI L -L C  
   
V er si o n #: 1. 0 V er si o n D at e: F e b 2 0 2 5 1 0  of 1 2   
o  T h e pri m ar y e sti m a n d f or t hi s o ut c o m e i s t h e diff er e n c e i n m e a n c h a n g e i n 
t h e N CI st a n d ar d s c or e fr o m t h e C N S-V S fr o m b a s eli n e.  
o  T hi s c o nti n u o u s o ut c o m e will b e a n al y z e d a s d e s cri b e d i n S e cti o n 3. 5  , i. e.  
u si n g a mi x e d eff e ct m o d el f or r e p e at e d m e a s ur e m e nt s.  
  
4. 3.  E x pl or at or y Effi c a c y A n al y s e s   
  
4. 3. 1  Q u e sti o n n air e s  
  
● C h a n g e i n t h e LII N C Q u e sti o n n air e – S y m pt o m A s s e s s m e nt b et w e e n B a s eli n e 
a n d D 4, D 1 0, D 3 0 a n d D 6 0.  
○ T h e pri m ar y e sti m a n d f or t h e s e c o nti n u o u s o ut c o m e s ar e t h e diff er e n c e 
fr o m b a s eli n e f or e a c h r e s p e cti v e s c or e.   
○ T h e s e c o nti n u o u s o ut c o m e s will b e a n al y z e d a s d e s cri b e d i n S e cti o n 3. 5 ,  
i. e. u si n g a mi x e d eff e ct m o d el f or r e p e at e d m e a s ur e m e nt s.   
  
● C h a n g e i n D A SI, O H Q, W H O D A S ( 2. 0) fr o m B a s eli n e t o D 3 0.   
○ T h e pri m ar y e sti m a n d f or t h e s e c o nti n u o u s o ut c o m e s ar e t h e diff er e n c e 
fr o m b a s eli n e f or e a c h o ut c o m e.  
○ T h e s e c o nti n u o u s o ut c o m e s will b e a n al y z e d a s d e s cri b e d i n S e cti o n 3. 5 ,  
i. e. u si n g a mi x e d eff e ct m o d el f or r e p e at e d m e a s ur e m e nt s.  
  
● C h a n g e i n D e P a ul S y m pt o m Q u e sti o n n air e ( D S Q) p o st -e x erti o n al m al ai s e ( P E M) 
fr o m B a s eli n e D 4, D 1 0, D 3 0.  
○ T h e pri m ar y e sti m a n d f or t hi s o ut c o m e i s t h e r el ati v e pr o p orti o n wit h P E M. 
○ T hi s bi n ar y o ut c o m e will b e a n al y z e d a s d e s cri b e d i n S e cti o n 3. 3 , i. e.  
u si n g a G E E m o d el.  
  
 P ati e nt Gl o b al I m pr e s si o n of C h a n g e ( P GI C) at D 4, D 3 0, D 6 0.  
o  T h e pri m ar y e sti m a n d f or t hi s o ut c o m e i s t h e r el ati v e o d d s of b ett er 
c h a n g e o n t h e P GI C b et w e e n t h e ar m s.  o  T hi s or di n al o ut c o m e will 
b e a n al y z e d a s d e s cri b e d i n S e cti o n 3. 4 .  
  
4. 3. 2  P erf or m a n c e M e a s ur e s   
  
 C h a n g e i n A cti v e St a n d Te st r e s ult fr o m B a s eli n e t o D 3 0.  

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o The primary estimand for this outcome is the relative proportion with 
postural orthostatic tachycardia syndrome by the stand test. o This 
binary outcome will be analyzed as described in Section 3.3, i.e.  
using a GEE model.  
  
  
4.3.3 Laboratory Measures  
  
● Change from Baseline to D10, D30 in C-reactive protein (CRP)  
○ The primary estimand for these outcomes is the difference in mean change 
from baseline.  
○ These continuous outcomes will be analyzed as described in Section 3.5 ,  
i.e. using a mixed effect model for repeated measurements.  
  
● Percentage of participants with no detection of SARS-CoV-2 plasma remnants 
(i.e., viral detection by reverse transcriptase-polymerase chain reaction (RT-
PCR) and Spike protein fragments) compared to Baseline through D10 and D30.   
  
5. Safety Analysis   
  
The primary outcome will be the number of individuals in each group experiencing a 
severe (Grade 3 or greater) Adverse Event (AE) attributed to be possibly, probably, or 
definitely related to study treatment will be compared between the arms. The number 
and proportion of participants treatment-emergent adverse events (TEA), serious 
adverse events (SEA) and Adverse Events of Special Interest (AESI) will also be 
summarized across treatment groups in the mITT population. Differences will be tested 
by a 0.05 level two-sided Fisher’s exact test. Agresti and Coull confidence intervals will 
be calculated for proportions.1  
  
  
 

Statistical Analysis Plan – PREVAIL-LC  
   
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REFERENCES  
1. 
Agresti A, Coull BA. Approximate is Better than “Exact” for Interval Estimation of 
Binomial Proportions. The American Statistician. 1998 May;52(2):119–126.  
2. 
Agresti A, Caffo B. Simple and Effective Confidence Intervals for Proportions and 
Differences of Proportions Result from Adding Two Successes and Two Failures. 
The American Statistician. 2000 Nov;54(4):280–288.  
3. 
Harrell F. Assessing the Proportional Odds Assumption and Its Impact [Internet].  
Statistical Rethinking. [cited 2027 Jun 26]. Available from:  
https://hbiostat.org/ordinal/impactpo.pdf  
4. 
Easy peasy STATA-like marginal effects with R [Internet]. [cited 2024 Jun 26]. 
Available from: https://www.brodrigues.co/blog/2017-10-26-margins_r/