Using AS01 Adjuvant to Improve Immune Response in Older Adults Through Trained Immunity
Phase: PHASE2
Conditions: Immune System Responses and Trained Immunity After AS01 Administration, Healthy Adult
Sponsor: Singapore General Hospital

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 1 of 38 
Restricted, Sensitive (Normal) 
 
 
 
 
 
 
  
CLINICAL TRIAL PROTOCOL 
 
 
 
PROTOCOL TITLE:  
Using AS01 adjuvant to improve immune response in older adults 
through trained immunity 
 
 
 
 
 
 
 
 
 
 
PROTOCOL NUMBER:  
 
AS01-YF-01 
 
 
 
 
 
 
 
VERSION NUMBER:  
 
2.0  
 
 
 
 
 
 
 
VERSION DATE: 
 
      
15 Sep 2025   
 
 
 
 
 
 
 
 
PRINCIPAL INVESTIGATOR: 
Dr. Chan Yuen Yue Candice  
 
 
 
 
 
Senior Consultant  
 
 
 
 
 
Department of Infectious Diseases  
 
 
 
 
 
Singapore General Hospital  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 2 of 38 
Restricted, Sensitive (Normal) 
PROTOCOL SIGNATURE PAGE 
 
 
PROTOCOL TITLE: Using AS01 adjuvant to improve immune response in older adults through trained immunity
 
 
 
 
 
 
 
 
 
 
 
 
PROTOCOL NUMBER: AS01-YF-01 
 
 
 
 
 
 
 
VERSION NUMBER: 2.0  
 
 
 
 
 
 
 
VERSION DATE: 15 Sep 2025  
 
 
 
 
 
 
 
 
 
Declaration of Investigator 
 
I confirm that I have read the above-mentioned protocol and its attachments. I agree to conduct the described 
study in compliance with all stipulations of the protocol, regulations and ICH E6 Guideline for Good Clinical 
Practice (GCP).  
 
Chan Yuen Yue Candice  
___________________________________________________________________________________ 
Name of Principal Investigator: 
 
 
 
___________________________________________________________________________________ 
Signature of Principal Investigator: 
 
 
_________________________________________________________________________________ 
Date: 
 
Singapore General Hospital  
___________________________________________________________________________________ 
Name of Study Site:  
 
 
 
Note: 
- This page should be signed with wet-ink signature or digital signature. Do not paste digital image of a wet-ink 
signature.  
 
- Signed copies of this signature page should be stored in the study file and in the respective study site’s 
investigator file. 
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 3 of 38 
Restricted, Sensitive (Normal) 
Table of Contents 
1. 
BACKGROUND AND RATIONALE ...................................................................................................... 5 
1.1. 
GENERAL INTRODUCTION ................................................................................................................... 6 
1.2. 
RATIONALE AND JUSTIFICATION FOR THE STUDY ......................................................................................... 7 
1.2.1 
Rationale for the Study Purpose ........................................................................................................................... 7 
1.2.2 
Rationale for Doses Selected ............................................................................................................................. 13 
1.2.3 
Rationale for Study Population ........................................................................................................................... 14 
1.2.4 
Rationale for Study Design .................................................................................................................................. 14 
2. 
HYPOTHESIS AND OBJECTIVES ..................................................................................................... 15 
2.1. 
POTENTIAL RISKS AND BENEFITS ......................................................................................................... 16 
2.2.1 
Potential Risks ....................................................................................................................................................... 16 
2.2.2 
Potential Benefits ................................................................................................................................................. 16 
3. 
STUDY POPULATION .................................................................................................................... 16 
3.1. 
LIST THE NUMBER AND NATURE OF SUBJECTS TO BE ENROLLED. ..................................................................... 16 
3.2. 
CRITERIA FOR RECRUITMENT AND RECRUITMENT PROCESS ......................................................................... 16 
3.3. 
INCLUSION CRITERIA ....................................................................................................................... 17 
3.5 
SUBJECT REPLACEMENT ................................................................................................................... 18 
4 
STUDY DESIGN ............................................................................................................................ 18 
4.1 
CONTRACEPTION AND PREGNANCY TESTING .......................................................................................... 18 
4.2 
Screening Visits and Procedures ....................................................................................................................... 18 
4.2.1 
Study Visits and Procedures ............................................................................................................................... 19 
4.2.2 
Final Study Visit ..................................................................................................................................................... 19 
4.2.3 
Post Study Follow up and Procedures ............................................................................................................... 19 
4.3 
DISCONTINUATION/WITHDRAWAL ....................................................................................................... 19 
4.3.1 
Discontinuation Criteria ...................................................................................................................................... 19 
4.3.2 
Discontinuation Visit and Procedures ............................................................................................................... 19 
5 
TRIAL MATERIALS ......................................................................................................................... 20 
5.1 
TRIAL PRODUCT(S) ......................................................................................................................... 20 
5.2 
STORAGE AND DRUG ACCOUNTABILITY ................................................................................................. 21 
6 
TREATMENT ................................................................................................................................. 21 
6.1 
RATIONALE FOR SELECTION OF DOSE ................................................................................................... 21 
6.2 
STUDY DRUG FORMULATIONS ............................................................................................................ 21 
6.3 
STUDY DRUG ADMINISTRATION........................................................................................................... 21 
6.4 
SPECIFIC RESTRICTIONS / REQUIREMENTS ............................................................................................. 22 
6.5 
BLINDING .................................................................................................................................... 22 
6.6 
CONCOMITANT THERAPY .................................................................................................................. 22 
7 
SAFETY MEASUREMENTS .............................................................................................................. 22 
7.1 
DEFINITIONS ................................................................................................................................. 22 
7.2 
COLLECTING, RECORDING AND REPORTING OF SERIOUS ADVERSE EVENTS (SAES) TO CIRB ................................ 23 
7.3 
COLLECTING, RECORDING AND REPORTING OF SERIOUS ADVERSE EVENTS (SAES) TO THE HEALTH SCIENCES AUTHORITY (HSA)
 
23 
7.4 
SAFETY MONITORING PLAN ............................................................................................................... 23 
7.5 
COMPLAINT HANDLING .................................................................................................................... 23 
8 
DATA ANALYSIS ........................................................................................................................... 23 
8.1 
DATA QUALITY ASSURANCE ............................................................................................................... 23 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 4 of 38 
Restricted, Sensitive (Normal) 
8.2 
DATA ENTRY AND STORAGE ............................................................................................................... 23 
9 
SAMPLE SIZE AND STATISTICAL METHODS ..................................................................................... 24 
9.1 
DETERMINATION OF SAMPLE SIZE ........................................................................................................ 24 
9.2 
STATISTICAL AND ANALYTICAL PLANS ................................................................................................... 24 
10 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS ........................................................................... 27 
11 
QUALITY CONTROL AND QUALITY ASSURANCE ............................................................................... 27 
12 
ETHICAL CONSIDERATIONS .......................................................................................................... 28 
12.1 
INFORMED CONSENT ...................................................................................................................... 28 
12.2 
CONFIDENTIALITY OF DATA AND PATIENT RECORDS .................................................................................. 28 
13 
PUBLICATIONS ............................................................................................................................ 28 
14 
RETENTION OF TRIAL DOCUMENTS ................................................................................................ 29 
15 
FUNDING AND INSURANCE .......................................................................................................... 29 
16 
REFERENCES ............................................................................................................................... 29 
LIST OF ATTACHMENTS ........................................................................................................................ 36 
APPENDIX 1 – STUDY SCHEDULE ..................................................................................................................... 36 
APPENDIX 2 - BLOOD SAMPLING SUMMARY ........................................................................................................ 38 
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 5 of 38 
Restricted, Sensitive (Normal) 
1. 
BACKGROUND AND RATIONALE 
The innate immune system provides our body’s initial defence against pathogens, responding within minutes to hours of 
infection through specialized myeloid cells like monocytes, neutrophils, macrophages and dendritic cells (DCs)1. These 
myeloid cells orchestrate to mount rapid non-specific immunity and activate longer-term specific adaptive immune 
responses mediated by B and T cells. However, the robustness of the innate immune system declines with aging, leaving 
older adults more vulnerable to infections and less responsive to vaccines2. The recent COVID19 pandemic highlighted 
the vulnerabilities of older individuals to both severe disease outcomes and reduced protection to vaccines, even highly 
efficacious mRNA vaccines3,4.  
 
Various efforts have been made to enhance immunity in older adults5. Among many strategies, two adjuvanted vaccines - 
the recombinant zoster vaccine (RZV; ShingrixTM) and the respiratory syncytial virus (RSV; ArexvyTM) vaccine - 
demonstrated over 90% and 80% efficacy respectively in large phase 3 clinical trials6–9. These results prove that robust 
immune responses can be induced in older adults. Both vaccines utilise AS01, a liposome-based adjuvant containing 
MPL (a toll-like receptor 4 agonist) and QS-21 (purified plant extract)10–12. AS01 triggers early inflammatory signals and 
recruits activated monocytes and DCs to the injection site and draining lymph nodes, leading to robust antibody and T 
cell responses10–12. Beyond its adjuvant role, emerging evidence suggests AS01 possesses broader immunological 
effects. A recent study using the AS01-adjuvanted Hepatitis B vaccine in young adults showed that the vaccine induced 
trained immunity (TI) phenotype in innate immune cells via epigenetic reprogramming13. TI has been associated with 
enhanced innate immune response against unrelated pathogens, also known as ‘heterologous protection14. Supporting 
these findings, an epidemiological study during the COVID-19 pandemic found that recipients of RZV had a lower 
incidence of severe COVID-1915. Separately, our group recently revealed that the first RZV dose modulated the expression 
of genes that correlated with robust T cell response and restored the aged immune system’s ability to mount robust 
response to the second dose (preliminary data, in review). While these results are encouraging, human studies have not 
yet revealed AS01's independent effects since it has only been studied in combination with vaccine antigens. Breaking 
through this limitation, Co-I Singhal’s recent work with humanized mice demonstrated that AS01 alone could reprogram 
human hematopoietic stem cells (from the bone marrow of humanized mice) and innate immune cells at the epigenomic 
level, which is a characteristic of TI. This reprogramming manifested through alternations in gene expression across 
myeloid cells and altered chromatin accessibility at important genomic locus. Notably, AS01-alone promotes robust type 
I interferon (IFN) responses and improved antigen-presenting capability in DCs, with peak responses observed at 1-
month. When challenged with Mycobacterium tuberculosis at one-month, AS01-treated mice showed protection against 
disease, indicative of heterologous protection. (preliminary data, in review).  
 
These findings collectively reveal AS01’s potential to epigenetically reprogram innate immune cells for enhanced 
adaptive immunity. During early disease outbreaks, AS01 could serve as a rapid and broadly protective immune booster 
for older populations before pathogen-specific interventions become available, offering a novel strategy to reduce 
disease severity. Despite AS01 and TI’s promise as a new frontier in vaccinology, its effects have yet to be studied in 
humans, including the extent and durability of AS01-induced protection against pathogens. 
 
This study is the first prospective placebo-controlled, randomized trial to evaluate the molecular mechanisms and 
durability of AS01-induced TI at one and three months in healthy older adults. Serial blood samples will be collected to 
analyze changes in immune cell populations and TI induction. To evaluate heterologous protection, we will employ a virus 
challenge study using a live-attenuated yellow fever vaccine virus strain (YF17D) that causes detectable viremia safely, to 
compare viral control between AS01 and placebo groups16. If successful, our findings could enable development of novel 
vaccination strategies by stimulating TI to elicit broadly protective immunity, especially during early outbreaks when 
pathogen-specific interventions are not yet available, helping to reduce mortality and morbidity and improve vaccine 
responses in vulnerable older adults. 
 
The specific aims and hypotheses are as follows:  
Specific aim 1: Examine the host response induced by AS01 and determine the duration of these changes for up to 3 
months in healthy older adults aged 21 – 59 years old.  
This aim tests the hypothesis that the AS01 adjuvant will induce significant and persistent changes in gene expressions 
and epigenetic markers associated with innate immunity in older adults. 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 6 of 38 
Restricted, Sensitive (Normal) 
Specific aim 2: Demonstrate that innate immune reprogramming post AS01 can provide cross-protection against 
heterologous YF17D viral infection.  
This aim tests the hypothesis that AS01 recipients will have 50% reduction in detectable viremia versus placebo after 
YF17D challenge at 1 month, while also evaluating the longevity of this cross-protection at 3 months post-AS01.  
Specific aim 3: Examine the T cell and B cell responses post YF17D challenge, and its correlation with YF viremia levels in 
AS01 group versus placebo.  
This aim tests the hypothesis that the reprogramming of innate immune cells post-AS01 promotes enhanced YF viremia 
clearance without compromising humoral and cellular immune response to YF17D.  
 
1.1. 
General Introduction 
Current pandemic response limitations and aging populations:  a dual challenge  
This century has witnessed over ten transnational epidemics, underscoring the critical need for enhanced preparedness 
against novel pathogens17. With the global population rapidly aging18, the COVID-19 pandemic exposed significant 
vulnerabilities in global readiness, with older populations being particularly susceptible due to severe disease due to 
immunosenescence – the age-related decline across all arms of the immune system4. While vaccines remain essential 
tools, they face inherent limitations in addressing emerging threats: development requires substantial time, protection 
against rapidly evolving pathogens is limited, and efficacy diminishes in older populations19,20. The reduced protection of 
even highly effective COVID-19 mRNA vaccines in older adults emphasized the urgent need for more robust and 
adaptable pandemic management strategies, particularly for vulnerable populations.  
 
Heterologous protection by innate and trained immunity: Role of vaccines 
The immune system consists of two distinct but interconnected components: innate and adaptive immunity. The 
adaptive immune system, through B  and T cells, forms the basis of vaccinology by generating specific, long-term immune 
memory and improved responses to repeated exposures, though it requires weeks to reach optimal protection against 
new pathogens21. In contrast, the innate immune system serves as our first line of defence through its diverse 
components - myeloid cells (monocytes, macrophages), dendritic cells (DCs), innate lymphoid cells (ILCs) and 
neutrophils1. These cells provide rapid and broad-spectrum protection by recognizing conserved molecular patterns 
present across diverse microorganisms, while also being essential in activating adaptive immune responses.  
Traditionally, the innate response was considered a temporary defence mechanism. However, this dogma was changed 
by the discovery of trained immunity (TI) by Netea et al14,22,23. An increasing body of evidence suggests that a set of innate 
cells (including myeloid cells, ILCs) can display adaptive characteristics of ‘de facto’ innate immune memory. These 
memory characteristics involve a priming event through infection or vaccination whereby upon first exposure, innate 
immune cells undergo sustained metabolic and epigenetic reprogramming, such that upon re-exposure to identical or 
heterologous stimuli, they displayed heightened response and host defense14,22–24.  
This paradigm-shifting finding was first evidenced through the live-attenuated Bacillus Calmette-Guérin (BCG) vaccine, 
where vaccinated children showed unexpectedly reduced overall mortality beyond its intended protection against 
tuberculosis25,26. Further research documented BCG’s broad protective effects across age groups, including enhanced 
protection against respiratory infections in the elderly27,28, experimental malaria infection in young adults29, and 
experimental yellow fever vaccine virus infection in young males30. Live-attenuated vaccines like measles, mumps and 
rubella (MMR) have demonstrated reduced incidence of non-targeted infections and immune cell epigenetic 
reprogramming in human studies31–33, while the novel live-attenuated Bordetella pertussis vaccine showed protection 
against unrelated airway infections in mice34. However, repurposing vaccines for pandemic preparedness has limitations. 
For instance, live vaccines are contraindicated in immunocompromised individuals, and some have age restrictions or 
show reduced efficacy in older adults35,36.   
 
Adjuvant AS01 as a potential immune booster for the aging population  
 
Yet, the RZV vaccine (containing the recombinant varicella zoster virus glycoprotein E (gE) with AS01) which has 
over 90% vaccine efficacy in adults aged 50 and older with protection lasting at least 10 years6,7,37, as well as the RSV 
vaccine (containing the recombinant RSV prefusion F protein antigen and AS01) with 80% efficacy in those aged 65 and 
older8.  The AS01 adjuvant, originally developed to enhance vaccine efficacy in older adults, represents a breakthrough in 
addressing both immediate and long-term protection needs. Its combination of liposomes, MPL (a TLR-4 agonist) and QS-
21 (plant product), work synergistically to enhance both innate and adaptive immune responses10. In human studies, 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 7 of 38 
Restricted, Sensitive (Normal) 
AS01 stimulation of blood cells from both young and older adult vaccinees activates antigen-presenting cells (APCs) 
including monocytes and DCs, triggering inflammatory pathways and metabolic changes that enhance T cell response11.  
 
Recent evidence suggests AS01’s potential extends beyond its role as a vaccine adjuvant by demonstrating 
ability to induce TI changes in innate immune cells, conferring heightened protection against heterologous stimuli. 
Evidence of AS01’s broader protective effects emerged when RZV recipients demonstrated a 32% lower risk of COVID-19 
related hospitalizations over a 10-month period, suggesting potential TI induction15. A recent ex-vivo human study with 
AS01-adjuvanted hepatitis B vaccine demonstrated its ability to induce TI in innate immune cells, particularly monocytes 
and DCs, leading to reduced pro-inflammatory responses, improved response to cytokines and enhanced cell-mediated 
immunity13.  
 
Evaluating AS01 as a standalone immune booster for vulnerable older adults  
While current TI-inducing vaccines present a promising avenue for enhanced pandemic preparedness, adoption into 
clinical practice still requires deeper understanding of TI’s duration and population-specific responses. In vitro studies 
show TI effects lasting from days to weeks in myeloid cells, while epidemiological studies of BCG and MMR suggest 
protection lasting months to years15,25. Understanding the duration of TI that could be induced by AS01 in older 
populations remains crucial for determining optimal dosing intervals and establishing effective protection windows 
against emerging pathogens. To glean further insights into the immunological events and heterologous protection effects 
of AS01, our group and others have preliminary data showing AS01’s potential as a versatile immune booster. However, a 
significant knowledge gap exists – all human studies have only tested AS01 with vaccine antigens, leaving its standalone 
effects unexplored.  Building on the methods and findings from our previous studies, our CS-IRG proposal aims to 
address these critical knowledge gaps by investigating AS01-alone as an independent immune booster in older 
populations. In the short term (3 to 5 years), we aim to elucidate AS01’s mechanisms in aging immune systems and 
evaluate its broad-spectrum protection potential and duration. Our long-term vision focuses on enhancing immune 
responses in older and other vulnerable adults. By understanding the mechanistic basis of TI and heterologous 
protection, we could develop novel approaches for preventing infectious diseases, potentially transforming our ability to 
protect at-risk groups during future pandemics.  
 
1.2. 
Rationale and Justification for the Study 
Preliminary studies in humanized mice and demonstrated that AS01 alone could reprogram human hematopoietic 
stem cells (from the bone marrow of humanized mice) and innate immune cells at the epigenomic level, which is a 
characteristic of TI. When challenged with Mycobacterium tuberculosis at one-month, AS01-treated mice showed 
protection against disease, indicative of heterologous protection. (preliminary data, submitted). Separately, our group 
recently revealed that the first RZV dose (which contains the AS01 adjuvant) modulated the expression of genes that 
correlated with robust T cell response and restored the aged immune system’s ability to mount robust response to the 
second dose (preliminary data, in review).  
 
Our findings collectively reveal AS01’s potential to epigenetically reprogram innate immune cells for enhanced adaptive 
immunity. During early disease outbreaks, AS01 could serve as a rapid and broadly protective immune booster for older 
populations before pathogen-specific interventions become available, offering a novel strategy to reduce disease 
severity. Despite AS01 and TI’s promise as a new frontier in vaccinology, its stand-alone effects have yet to be studied in 
humans, including the extent and durability of AS01-induced protection against pathogens. 
 
This study is the first prospective placebo-controlled, randomized trial to evaluate the molecular mechanisms and 
durability of AS01-induced TI at one and three months in healthy older adults aged 21 –59 years old. Serial blood samples 
will be collected to analyse changes in immune cell populations and TI induction. To evaluate heterologous protection, 
we will employ a virus challenge study using a live-attenuated yellow fever vaccine virus strain (YF17D) that causes 
detectable viremia safely, to compare viral control between AS01 and placebo groups16. If successful, our findings could 
enable development of novel vaccination strategies by stimulating TI to elicit broadly protective immunity, especially 
during early outbreaks when pathogen-specific interventions are not yet available, helping to reduce mortality and 
morbidity and improve vaccine responses in vulnerable older adults.  
 
1.2.1 Rationale for the Study Purpose 
AS01 adjuvant induces TI in the bone marrow cells of humanized mice (Negishi et al., submitted)  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 8 of 38 
Restricted, Sensitive (Normal) 
To understand the long-term effects of AS01 adjuvant on the human bone marrow (BM) progenitor and myeloid cells, we 
administered AS01 intramuscularly to humanized NOG (hNOG) mice and collected BM cells at 4- and 8-week post-
inoculation (Fig 1A). 
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 9 of 38 
Restricted, Sensitive (Normal) 
 
Fig 1. AS01 induces type 1 IFN responses in diverse BM cells in humanized mice.  
(A) Schematic of the study. NOG mice were transplanted with hCD34+ HSC and after 30 days AS01 and BCG was administered 
intramuscularly and intravenously, respectively to hNOG mice. 4- and 8-week later animals were sacrificed to isolate human 
myeloid BM cells, which were processed for scRNA-seq and scATAC-seq. (B) UMAP plot of scRNA-seq showing the annotation 
and colour codes for cell types in the BM environment of hNOG mice (C) Bubble plot showing enriched pathways, based on 
upregulated genes at 4-week in each cluster for AS01 (red) and BCG (blue). The size of the bubble is Bonferroni adjusted p-
value. The intensity of the colour - fold enrichment in BCG or AS01 group. (D) Heatmap showing mean log2FC of type I IFN 
response genes in AS01 over control and BCG over control at 4 weeks post administration for each cluster in B. (E) Genomic 
views (hg38) at IFIT genes for cDC1, cDC2, monocyte and GMP. Blue shades indicate DARs in each cell type. Transcription 
factor (TF) binding track is TF binding sites derived from IRF1, STAT1 and STAT2 ChIP-seq experiments performed by the 
ENCODE project. The bottom track is cis-regulatory elements interactions predicted by Cicero co-accessibility algorithm.  
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 10 of 38 
Restricted, Sensitive (Normal) 
 
Fig 2. AS01 reprograms BM monocytes that protect against pulmonary Mycobacterium infection.  
(A) Schematic diagram of the AS01 or PBS administration (intramuscularly) in WT and CCR2-KO mice and challenge with 
either BCG (1x106 CFU) or Mtb (~200 CFU). (B) BCG load in the lungs of AS01 and PBS administered WT mice at day 7 p.i. (C) 
Flow cytometry of lung myeloid cells. Dot plots of CD64+F4/80+ macrophages are shown. (D) Median fluorescence intensity 
(MFI) of F4/80 expression. Left – overlay, right – bar graph of compiled data. (E) BCG load in the lungs of AS01 and PBS 
administered CCR2-KO mice at day 7 p.i. (F) Mtb load in the lungs of AS01 and PBS administered WT mice at day 7 and 22 p.i. 
Data is meanSEM of n=5 mice/group/timepoint, unpaired t-test. Representative of 2-3 experiments.  
In parallel, we performed intravenous BCG vaccination to analyse the effects of live BCG  vaccine on human BM 
progenitors. Of note, the effect of BCG on BM progenitors has been already reported in humans and wild type (WT) mice38–
40. From the BM of hNOG mice we flow-sorted human hCD45+hCD3-hCD19-  hCD20-hCD56- cells, which are devoid of 
mature T, B and natural killer (NK) cells, and performed single cell RNA sequencing (scRNA-seq) and single-cell 
sequencing assay for transposase-accessible chromatin (scATAC-seq) (Fig 1A). In scRNA-seq dataset we identified 12 
transcriptionally distinct clusters, which were annotated based on the known cell-type markers (Fig 1B)41,42. We next 
identified differentially expressed genes (DEGs) in BM cell subsets from control, AS01-administered and BCG-vaccinated 
hNOG. At the 4-week time point we observed profound gene expression changes in many BM cell subsets of AS01-
administered hNOG, which were dominated by genes involved in response to virus and response to type I IFN gene 
ontology (GO) terms (Fig 1C). In contrast, BCG-induced transcriptomic changes were mainly driven by genes in antigen 
processing and presentation, cytoplasmic translation and ribosome assembly (Fig 1C). Thus, our findings suggest that 
AS01-driven transcriptomic changes in BM populations are different compared to BCG-driven changes. Global scATAC-
seq data also suggested enrichment of type 1 IFN response pathway in the BM cells of AS01-administered hNOG (data 
not shown). Analysing the expression of 35 genes in this pathway showed upregulation of several interferon-stimulated 
genes (ISGs) including MX1, ISG15, OAS2, and IFI, IFIT and IFITM families in all subsets of AS01-primed BM progenitor and 
myeloid cells compared to BCG-educated BM cells (Fig 1D). Assessment of genomic loci of IFIT and IFITM genes further 
highlight the shared and specific differential accessible regions (DARs) in AS01-primed and BCG-educated cDC1, cDC2, 
monocyte and GMP subsets with some of these peaks exhibiting co-accessibility profiles (Fig 1E). Motifs of IRF1, STAT1 
and STAT2 occupy many of these differential accessible peaks, suggesting their role as master regulators of type 1 IFN 
response gene induction43. Of note, the expanded IFN response in AS01-primed BM cells at 4-week time point wanes off 
at 8-week time point (data not shown). Overall, our study indicates an epigenomic-mediated transcriptional control of 
type 1 IFN response pathway in AS01-primed BM cells.  
 
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 11 of 38 
Restricted, Sensitive (Normal) 
 
Fig 3. Fast responders achieve protective T cell levels after one dose of RZV while slow responders required two doses. 
(A) Study design. (B) Total gE-specific T cell responses to RZV measured on day 0, 14, 60, 74 using ELISpot. Slow responder 
(SR): purple (n = 11); Fast responder (FR): orange (n = 4).  
AS01 adjuvant induces TI and monocyte-mediated protection against Mycobacterium infection in mice  
Our monocyte chromatin accessibility data (not shown) indicates the induction of TI phenomenon in AS01-primed BM 
monocytes40,44. To investigate this, AS01 or PBS were administered intramuscularly to wild type mice and after 30 days 
mice were intratracheally infected with either BCG or Mycobacterium tuberculosis (Mtb) (Fig 2A). At 7- and 22-days post-
infection (p.i), the lung bacterial load and myeloid cells profile was assessed. At 7 days p.i BCG lung burden was 
significantly lower in the lungs of AS01-administered wild type mice compared to PBS-treated control (Fig 2B). The 
reduced lung bacterial load corresponded with the decreased expression of F4/80 on CD64+F4/80+ lung macrophages in 
AS01-administered mice (Fig 2C & 2D). Decreased F4/80 expression has been similarly observed in trained peritoneal 
macrophages in subcutaneously BCG-vaccinated mice45. Protection against Mtb challenge in BCG-vaccinated mice has 
been attributed to the migration of BM-derived monocytes and macrophages to the lungs40. Thus, we examined the role of 
AS01-primed BM-derived monocytes using the CCR2-KO mice model, which lacks circulatory classical Ly6chi monocytes 
(Fig 2A). The AS01-mediated protection against BCG was abolished in CCR2-KO mice (Fig 2E). This indicated the 
necessity of monocytes / macrophages for AS01 mediated protection. We next used virulent Mtb to infect AS01-primed 
WT mice and lung CFU was assessed at days 7 and 22 p.i (Fig 2A). Compared to PBS control, lungs of AS01 administered 
mice showed significantly reduced CFU (Fig 2F). The AS01-mediated protection against Mtb was abolished in CCR2-KO 
mice (not shown). Together, these data demonstrate that intramuscular administration of AS01 alone can induce training 
in BM monocytes that migrate to lung and provide protection against Mycobacterium infection. 
 
Two doses of RZV are required to induce protective levels of T cells, despite the presence of pre-existing immunity in all 
subjects (Chan et al., in review) 
Varicella zoster virus (VZV) infection initially causes chickenpox and establishes life-long latency in neurons. As immunity 
wanes with age, VZV can reactivate as herpes zoster (HZ)46. While RZV’s two-dose regimen provides over 90% efficacy in 
protection against HZ reactivation6,7, an intriguing question emerges: why are two doses necessary when here is 
widespread pre-existing immunity in older populations (>95%)?46 Elucidating these mechanisms will help develop more 
effective vaccination strategies to improve immune responses in aging populations.  
 
To study AS01-adjuvanted RZV immune responses, we conducted a randomized trial with 30 healthy adults (50-65 years) 
with prior chickenpox exposure. Participants received either two RZV doses (n=15) at day 0 and 60, or one dose of zoster 
vaccine live (ZVL) (n=15) as control. We collected blood samples to analyse gene expression, cytokines, antibodies, and T 
cells (Fig 3A). Compared to ZVL vaccination, anti-gE IgG response to a single dose of RZV vaccination was significantly 
higher (data not shown). The second dose of RZV did not boost antibody titres, suggesting that from a humoral response 
perspective, the second RZV dose was dispensable (data not shown). However, the second dose of RZV vaccination 
significantly increased gE-specific T cell counts at day 14 after dose 2 (Fig 3B). Given that the magnitude of VZV T cell 
response is known to correlate with protection from herpes zoster47,48, and that two-doses of RZV produced 90% 
efficacy6,7, we used the 10th percentile of the gE-specific T cell counts after two doses of RZV to define a surrogate 
threshold of protection (Fig 3B). In addition, consistent with clinical findings that a single dose of RZV showed sub-clinical 
efficacy49, only four individuals (27%) achieved T cell responses exceeding the threshold associated with protection (Fig 
3B). These individuals, designated as ‘fast responders’ (FR), due to their rapid achievement protective T-cell response 
after a single dose, continued to have higher gE-specific T cell counts at day 60 post-vaccination. On the other hand, the 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 12 of 38 
Restricted, Sensitive (Normal) 
 
Fig 4. First dose of RZV resets the baseline expression of transcriptomic correlates of T cell response. (A) Venn diagram of 
the shared number of protective T cell correlates based on transcripts identified. Red denotes up-regulated genes, blue 
denotes down-regulated genes.  (B) Hierarchical clustering that had a log2FC = +/- 1.5 difference between D61 v 60 (Dose 2) and 
D1v0 (Dose 1) transcriptomic response of SR and also a FC = +/- 1.5 difference in D1v0 (Dose 1) transcriptomic response 
between FR (Orange) and SR (Purple). (C) Shared T cell correlates between protective T cell correlates for RZV in older adults (as 
in A) and T cell correlates for BNT162b2 vaccination in naïve adults. T cell correlates for BNT162b2 determined from correlating 
with ELISpot responses with D1v0 log2FC transcriptomic responses (Spearman’s pairwise correlation; r = +/- 0.3). Common 
transcript highlighted in boxes where colours indicate the direction of association. Red and blue indicates positive and negative 
correlates. *Indicate coding transcripts. (D) Averaged expression (z-score) of the T cell correlates over time for FR and SR. 
majority of other subjects, referred to as ‘slow responders’ (SR), achieved protective T cell levels only after the second 
dose. Overall, the results indicate that despite the presence of pre-existing adaptive immune response against VZV in all 
subjects, there were heterogenous T cell responses among older individuals, and a two-dose regimen was needed for 
RZV to induce sufficient T cell responses in all subjects.  
Gene expression changes associated with protective T cell levels after RZV administration 
Next, we examined characteristics associated with FR and SR. T cell response differences were independent of age, 
gender, body mass index, adverse events (AE), pre-existing antibody titers and gE-specific T cell levels at the point of 
vaccination (Data not shown). 
 
Given that demographics and baseline gE-specific T cell levels did not explain the differences in T cell response observed 
in FR and SR, we performed transcriptional profiling of whole blood from all 15 RZV recipients with the aim to identify early 
molecular signatures that could explain the differential responses between these groups. To do so, we employed a 3-step 
analysis approach. First, we compared gene expression between FR and SR after the first dose, identifying 1,145 DEGs 
(fold-change > 1.5) (data not shown). Second, since SR showed improved T cell responses after dose 2, we performed a 
similar analysis where we compared the fold-change differences between dose 2 and dose 1 in SR, revealing 615 genes 
with fold-difference >1.5 (data not shown). Finally, using Venn diagram analysis of these 2 gene sets, we identified 201 
overlapping genes that were both differentially expressed between FR and SR after dose 1 and significantly changed after 
dose 2 (Fig 4A). These 201 overlapping genes likely represent key markers of T cell responses. Indeed, unsupervised 
hierarchical clustering confirmed that these 201 genes effectively distinguished FR and SR subjects (Fig 4B).  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 13 of 38 
Restricted, Sensitive (Normal) 
As all the recruited subjects had pre-existing adaptive immune response against VZV, the 201 genes that separate FR and 
SR would also contain gene expression changes from memory responses. Thus, to identify which of these genes drove T 
cell response to vaccination independent of memory responses, we leveraged on a study where SARS-CoV-2-naïve 
individuals were given the Pfizer-BioNTech BNT162b2 vaccine50. Further Venn diagram analysis between these 201 genes 
and genes correlating with naïve T cell responses to BNT162b2 vaccine identified 27 genes (Fig 4C). The magnitude of 
fold-change of these 27 genes at day 1 post RZV vaccination also significantly predicted protective T cell levels after 
either dose of RZV with receiver operating characteristic (ROC) curve with area under the curve (AUC) = 0.98 and p < 
0.0001 (Data not shown). These genes included genes that encode for various cell functions, including the component of 
the receptor for the inflammatory cytokine IL-31 (IL31RA), endopeptidase (CAPNS2), membrane proteins (LY6G6C, MAL2), 
regulatory signalling proteins (INHBB, RGS7), and a cell cycle regulator (CEND1)51.   
 
First RZV dose induces gene expression changes that enhance T cell responses to the second dose 
The higher magnitude of log2 fold-change differences observed between FR and SRs across these 27 genes could be 
potentially explained by two distinct possibilities. The first possibility is that both FR and SR shared the same baseline 
gene expression, but FR showed greater change in expression than SR. The other possibility is that the expression of these 
genes was different between FR and SR at baseline. We thus examined the gene expression in FR and SR at day 0 and day 
1 post-RZV vaccination. The aggregate expression of the 27 genes was significantly different between FR and SR at day 0 
and day 1 post-RZV vaccination (Fig 4D). These genes were more lowly expressed in FR compared to SR before 
vaccination, which was then associated with greater increase in expression at one day post-RZV vaccination. If a lower 
expression of these 27 genes at baseline could explain T cell response to RZV, then the first RZV dose should lead to a 
lower expression of these genes before the second RZV dose. Indeed, the expression of these genes were lowered 
following the first dose of RZV in SR to levels comparable to those in FR (Fig 4D). Concomitantly, the second dose in SR 
elicited a greater increase in gene expression at day 61 (Fig 4D). Collectively, our analysis revealed 27 signature genes 
correlated with T cell responses in older adults. The first dose of RZV induced critical expression changes in these genes, 
which helped overcome heterogenicity in T-cell responses and effectively restored the aging immune system’s ability to 
mount strong T cell responses after the second dose for all subjects.  
 
Collectively, our preliminary findings reveal AS01’s potential to epigenetically reprogram innate immune cells for 
enhanced adaptive immunity. During early disease outbreaks, AS01 could serve as a rapid and broadly protective 
immune booster for older populations before pathogen-specific interventions become available, offering a novel strategy 
to reduce disease severity. Despite AS01 and TI’s promise as a new frontier in vaccinology, its effects have yet to be 
studied in humans, including the extent and durability of AS01-induced protection against pathogens. Findings from this 
study will provide unique insights into TI as a rapid and broadly protective immune booster for older populations before 
pathogen-specific interventions become available, offering a novel strategy to reduce disease severity 
 
1.2.2 Rationale for Doses Selected 
Eligible volunteers will be randomised 1:1 to receive either 1 dose of AS01 (GSK) or Placebo (NaCl 0.9%) and 
then randomised again to receive one dose of YF17D (Stamaril, Sanofi-Pasteur) on either day 30 or day 90 
according to manufacturer’s instruction.  
  
i. One dose of AS01 (0.5ml) or placebo (NaCl 0.9%, 0.5ml) on day 0 is administered intramuscularly in 
the deltoid region of the upper arm. The AS01 adjuvant component (0.5ml, which contains 50mg of 3-
O-desacyl-4’monophosphoryl lipid A and 50mg of Quillaja Saponaria Molina fraction 21) is supplied as 
a separate vial in the licensed recombinant zoster vaccine (Shingrix)52.  
 
ii. One dose of YF17D (0.5ml) on either day 30 (YF17D 1-month group) or day 90 (YF17D 3-month 
group), administered subcutaneously in the deltoid region of the upper arm53.  
 
A single dose of AS01 has been chosen based on previous studies and preliminary data demonstrating that 
one dose is sufficient to induce significant changes in immune cell populations, including epigenetic 
reprogramming13. 
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 14 of 38 
Restricted, Sensitive (Normal) 
1.2.3 Rationale for Study Population 
We will recruit 40 healthy adults 21 to 59 years of age, with a BMI ranging from >18.5 – 27.5 kg / m2. The 
rationale for recruiting people aged 21-59 is twofold: First, although the study aims to improve vaccine 
development for older adults, the primary objective is to establish the molecular mechanisms elicited by 
AS01, which can be identified regardless of participant age⁠. Second, expanding the age range increases the 
likelihood of successful recruitment, particularly when involving dengue seronegative subjects. The upper age 
limit of 60 is set because YF17D is not recommended for people above 60 years of age due to a higher risk of 
rare but potentially life-threatening AEs54. These restrictions will also ensure that our results are not 
confounded by extremes of BMI or poorly controlled diseases55,56. Subjects will be excluded if they have 
previously received yellow fever (YF) vaccines or AS01-adjuvanted vaccines, have a history of YF infection, 
dengue virus seropositivity, or known allergy to study vaccines, their components, or egg products. Dengue 
seropositivity is excluded because cross-reactive antibodies can confound yellow fever immunogenicity 
results16,57. Additional exclusion criteria that would be a risk factor for live-attenuated vaccines and blood 
taking, as established in our previous trials, will also be excluded16,58.  
(Please also refer to section on inclusion and exclusion criteria for details)  
 
1.2.4 Rationale for Study Design 
To test each of the three specific aims and corresponding hypotheses, we propose an experimental medicine 
study involving healthy older adult volunteers. We will administer AS01 and use the yellow fever live-
attenuated vaccine (YF17D) to simulate a heterologous systemic viral infection. The overview of or proposed 
study is shown (Fig 5A).   
 
The study will follow a pre-defined visit schedule and blood collection timepoints (Fig 5B, C).  Eligible 
volunteers (n = 40) will be randomised in a 1:1 ratio to receive:   
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 15 of 38 
Restricted, Sensitive (Normal) 
i) One dose of AS01 (n = 20) or NaCl 0.9% 
placebo (n = 20) on day 0: The AS01 
suspension contains 50mg of 3-O-desacyl-
4’monophosphoryl lipid A (MPL) and 50mg 
of Quillaja Saponaria Molina, fraction 21 
(QS21), licensed by GSK, available as a 
separate vial from the licensed Shingrix 
vaccine6. The placebo will consist of 0.9% 
saline solution in equivalent volume. All 
AS01/placebo administration will occur in 
morning sessions to control for circadian 
rhythm 
effects 
on 
TI 
and 
immune 
responses59. Treatment allocation will be 
determined using a computer-generated 
randomization sequence, with assignments 
sealed 
in 
opaque 
envelopes. 
These 
envelopes will be opened by the study 
coordinator only at the time of intervention 
administration. This study will employ a 
single-blinded design, where subjects will 
be unaware of AS01/placebo allocation. 
Both treatments will be administered 
intramuscularly into the deltoid muscle. A 
single dose administration strategy has 
been chosen based on previous studies and 
preliminary data demonstrating that one 
dose of AS01 is sufficient to induce significant changes in immune cell populations, including epigenetic 
reprogramming13.  
 
ii) One dose of YF17D (Stamaril, Sanofi-Pasteur) on either day 30 (YF17D 1-month group, n = 20. Fig 5B) or 
day 90 (YF17D 3-month group, n = 20. Fig. 5C): Research blood samples will be collected at pre-defined 
intervals by venipuncture to characterize host responses post-AS01/placebo for laboratory studies according 
to study schedule, as shown.  
 
2. 
HYPOTHESIS AND OBJECTIVES 
Specific aim 1: Examine the host response induced by AS01 and determine the duration of these changes for 
up to 3 months in healthy older adults aged 21 – 59 years old.  
This aim tests the hypothesis that the AS01 adjuvant will induce significant and persistent changes in gene 
expressions and epigenetic markers associated with innate immunity in older adults. 
 
Specific aim 2: Demonstrate that innate immune reprogramming post AS01 can provide cross-protection 
against heterologous YF17D viral infection.  
This aim tests the hypothesis that AS01 recipients will have 50% reduction in detectable viremia versus 
placebo after YF17D challenge at 1 month, while also evaluating the longevity of this cross-protection at 3 
months post-AS01.  
 
Specific aim 3: Examine the T cell and B cell responses post YF17D challenge, and its correlation with YF 
viremia levels in AS01 group versus placebo.  
 
Fig 5. Study Design. (A) Randomization. (B) Timeline for YF17D 1-month 
group and (C) YF17D 3-month group.  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 16 of 38 
Restricted, Sensitive (Normal) 
This aim tests the hypothesis that the reprogramming of innate immune cells post-AS01 promotes enhanced 
YF viremia clearance without compromising humoral and cellular immune response to YF17D.  
 
2.1. 
Potential Risks and Benefits  
2.2.1 Potential Risks 
Obtaining blood can cause bleeding, bruising, or swelling at the sight of needlestick, as well as 
thrombophlebitis. Infection at the site of needlestick and fainting rarely occurs. In addition, there are potential 
risks of adverse reactions from taking the vaccines.  
 
For AS01 (According to published adverse events for Shingrix): Solicited local AEs in subjects aged 50 years 
and older were pain (78.0%), redness (38.1%), and swelling (25.9%). Solicited general AEs were myalgia 
(44.7%), fatigue (44.5%), headache (37.7%), shivering (26.8%), fever (20.5%), and gastrointestinal symptoms 
(17.3%)60. 
 
For YF17D, adverse events include mild headaches, myalgia, low-grade fevers, or other minor symptoms for 5 
- 10 days. Local reactions include oedema, hypersensitivity, pain or mass at the injection site. Immediate 
hypersensitivity reactions, characterised by rash, urticaria, and/or asthma, anaphylaxis occur principally 
among persons with histories of allergy to eggs or other substances contained in the vaccine. Other adverse 
events include Yellow Fever Vaccine-Associated Neurotrophic Disease (YEL-AND) with incidence rate of 1 per 
100,000, Guillain-Barre-syndrome, acute disseminated encephalomyelitis (ADEM) and bulbar palsy. Yellow 
Fever Vaccine-Associated Viscerotropic Disease (YEL-AVD) has been reported (Incidence 1 per 400,000 
doses), with age > 60 being a risk factor53 and hence they will be excluded from this study. 
 
 
2.2.2 Potential Benefits 
Participants will receive a licensed yellow fever vaccine, which will help reduce the risk of developing yellow 
fever as well as associated complications should they travel to endemic countries in the future. 
 
Novel knowledge gained on the induction of trained immunity by AS01 would help improve the development of 
broadly protective vaccines in the future for this group of population. This information is currently missing, and 
we believe that addressing this knowledge gap would revolutionise vaccine development. Since older adults 
are particularly susceptible to the complications of emerging infectious diseases, the development of trained 
immunity vaccines that confer broad protection will ensure they are better protected in future outbreaks. 
 
3. 
STUDY POPULATION 
3.1. 
List the number and nature of subjects to be enrolled.  
We plan to recruit 40 adults with 21 to 59 years of age. (Please also refer to section on inclusion and exclusion 
criteria for details)  
 
3.2. 
Criteria for Recruitment and Recruitment Process 
Subjects will be recruited from SGH Clinical Trials and Research Centre (CTRC) healthy volunteer database 
and through poster advertisement. Subjects will be given a copy of the Participant Information Sheet and 
Informed Consent Form to read upon arrival. A briefing session on the study will be conducted by the 
Investigator. Thereafter, subjects will be ushered into a private room where written informed consent will be 
obtained and where they can freely ask questions about the study. Subjects will not be rushed into deciding to 
participate in the study and will be permitted to defer their decision (without any prejudice) after careful 
consideration and discussion with their family members. 
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 17 of 38 
Restricted, Sensitive (Normal) 
3.3. 
Inclusion Criteria 
Inclusion Criteria  
1) Adults aged 21 to 59 years of age at time of screening.   
2) BMI 18.5 – 27.5 kg / m2 (BMI values for Asian population according to MOH guideline NIH Consensus 
Conference)61.  
3) Satisfactory baseline medical assessment as assessed by physical examination and a stable health 
status. For subjects with underlying comorbidities, the conditions must be deemed stable by the 
investigators, and they must not have any hospitalisation relating to these conditions in the last 6 
months. 
4) Voluntarily participate, understand and sign an informed consent form approved by the Ethical Review 
Board. 
5) Subjects who are willing to comply with the requirements of the study protocol and scheduled visits. 
These requirements include completion of the subject diary, return for follow-up visits. Subjects 
should also be willing to make themselves available for the duration of the study, with access to a 
consistent means of contact. 
6) Accessible vein at the forearm for blood taking.  
7) Female subjects of non-childbearing potential due to surgical sterilisation (hysterectomy or bilateral 
oophorectomy or tubal ligation) or menopause. Post-menopausal subjects must have had at least 12 
months of natural (spontaneous) amenorrhoea.  
 
Exclusion Criteria 
1) Previous vaccination against yellow fever, dengue either with a registered product or from participation 
in a previous vaccine study.  
2) Previously received AS01-adjuvanted vaccines (e.g. Recombinant zoster vaccine, RTS,S/AS01, RSVPre-
F3-AS01), either with a registered product or from participation in a previous vaccine study.  
3) Planned administration of a AS01-adjuvanted vaccine or yellow fever vaccine other than the study 
vaccine during the study.  
4) Subjects who have been unwell in the last 7 days prior to screening. 
5) History of documented yellow fever and / or dengue infection.  
6) Dengue seropositivity upon screening.  
7) History of smoking within the last 1 year.  
8) Planned travel to yellow fever endemic countries during the study.   
9) Known allergy to AS01 and YF17D vaccine or their components (e.g. egg products). 
10) Diagnosis of diabetes HBA1c > 6.5 according to American Diabetes Association criteria62.  
11) Any medical condition that in the judgment of the investigator will make intramuscular injection unsafe 
(e.g. thrombocytopenia with platelet count < 50x10^9/L, coagulopathy, anti-coagulant therapy).  
12) Risk factor for live-attenuated vaccines, including any confirmed or suspected primary or acquired 
immunodeficiency based on history and physical examination:  
• 
History of thymus gland disease  
• 
Haematologic neoplasms including leukaemia, lymphoma, myelodysplastic syndromes  
• 
Diagnosed with cancer or treatment for cancer (except for localised basal cell carcinoma) 
within 3 years prior to screening  
• 
Post-transplant: solid organ and haematopoietic stem cell transplant 
• 
Immunocompromised due to primary or acquired (including HIV/AIDS) immunodeficiency  
• 
Other significantly immunocompromising conditions  
  
13) Administration of anti-inflammatory drugs for the past 7 days (e.g. NSAIDs, Paracetamol, aspirin).  
14) Use of metformin for the last 1 month.  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 18 of 38 
Restricted, Sensitive (Normal) 
15) Use of corticosteroids within the last 6 months prior to the first vaccine dose (defined as 
prednisolone > 10 mg / day or equivalent for > 2 weeks, or prednisolone > 40mg / day or > 1 week). 
Inhaled and topical steroids are allowed.  
16) Received biologics (such as anti-TNF inhibitors, IL-1 inhibitors, co-stimulation blockers, B-cell 
depleting therapy) for the last 12 months.  
17) Any condition (e.g. extensive psoriasis, chronic pain syndrome, severe hearing loss, cognitive 
impairment, dialysis, autoimmune disorders) that in the opinion of the investigator, would 
complicate or compromise the study or wellbeing of the subject, or prevent completion of the study.  
18) Evidence of substance abuse, or previous substance abuse. 
19) Clinically significant anaemia (Hb < 10 g/dL).  
20) Blood donation exceeding > 450 ml in the past 3 months. 
21) Participation in a study involving administration of an investigational or non-investigational compound 
within the past four months or planned participation during the duration of this study.  
22) Administration of any licensed vaccine within 30 days before the first study vaccine dose or planned to 
receive such products within 30 days after the study vaccination.  
23) Received immunoglobulin or any blood products within the 90 days preceding the first dose of study 
vaccine or planned to receive such products during the study period.  
 
3.5 Subject Replacement 
Subjects who drop out will not be replaced. 
 
4 
STUDY DESIGN  
This is a single-centre, randomised, single-blind, placebo-controlled outpatient trial in healthy older adults. 
Study will be conducted at the Clinical Trials and Research Centre (CTRC) of Singapore General Hospital.  
 
4.1 Contraception and Pregnancy Testing 
All female subjects of child-bearing potential will need to undergo a urine pregnancy test at screening and on 
the day of administration of the vaccine. Only those with a negative urine pregnancy test will be considered 
eligible for the study, provided that other eligibility criteria are fulfilled.  
 
4.2 Screening Visits and Procedures 
40 healthy volunteers 21 to 59 years of age at time of screening will be recruited through CTRC database and 
screened for eligibility (Refer to section on inclusion and exclusion criteria). Informed written consent will be 
sought from subjects who fulfil criteria for enrolment. All consented subjects will undergo screening, which 
includes medical history, physical examination, urinary pregnancy test (for female subjects of child-bearing 
potential), screening blood tests (full blood count, dengue serology, HBA1c).  
 
Randomisation and Study Arms:  
All eligible and enrolled subjects will undergo two sequential randomisations (Fig. 5A):  
i) 
First randomisation (1:1 ratio):  
- 
AS01 group (intervention group): n = 20 
- 
Placebo (NaCl 0.9%) group: n = 20 
 
ii) Second randomisation (1:1 ratio):  
- 
YF17D at 1 month (n = 20)  
- 
YF17D at 3 months (n = 20)  
 
Both randomisations will be computer-generated sequences with allocations sealed in opaque envelopes, 
opened by the study coordinator at the time of intervention administration.  
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 19 of 38 
Restricted, Sensitive (Normal) 
4.2.1 Study Visits and Procedures 
i) 
Initial intervention: Intramuscular administration of AS01 or placebo in deltoid muscle. Investigational 
bloods will be collected by venepuncture at pre-defined timepoints (Fig. 5B, C):  
a. YF17D 1-month group: Immediately before injection (day 0), day 1, day 7, day 14. 
b. YF17D 3-month group: Immediately before injection (day 0), day 1, day 7, day 14, day 21, day 
30, day 60.  
ii) Second intervention: YF17D administration at either 1 month (day 30) or 3 months (day 90). 
Investigational bloods will be collected by venepuncture at pre-defined timepoints (Fig. 5B, C):  
a. YF17D 1-month group: Immediately before YF17D (Day 30), day 32, day 33, day 34, day 35, day 
36, day 37, day 44, day 60.  
b. YF17D 3-month group: immediately before YF17D (day 90), day 92, day 93, day 94, day 95, day 
96, day 97, day 104, day 120.  
 
The comprehensive blood sampling schedule in this study is essential to fully characterise the temporal 
dynamics and duration of trained immunity induced by AS01 and effect on YF vaccine-induced viraemia and 
immune (acute, adaptive) responses. The blood volume requested represents the minimum amount needed 
to perform the full panel of assays required to address the three specific aims, as supported by published 
studies in the field of trained immunity.  
 
4.2.2 Final Study Visit 
The last study day will be on day 60 for YF17D 1-month group and D120 for YF17D 3-month group. 
 
4.2.3 Post Study Follow up and Procedures 
There is no requirement for post-study follow-up and procedures.  
 
4.3 Discontinuation/Withdrawal 
4.3.1 Discontinuation Criteria 
Subjects are free to withdraw consent and discontinue their participation at any time without prejudice to 
them or effect on their medical care. Subjects may also be withdrawn or removed, if necessary, to protect their 
health or integrity of the study. The investigator also reserves the right to withdraw participants from the study 
in event of inter-current illness, adverse events, protocol violations, administrative, or other reasons. Once a 
subject withdraws from the study, no new data will be collected for study purposes unless the data concerns 
safety or adverse events related to the study. All details available will be reported and recorded for any 
subjects that withdraws or is removed from the study.  
 
The PI may stop subject’s participation in the study at any time for one or more of the following conditions:  
• 
Failure to follow the instructions of the investigators or study staff.  
• 
The PI decides that continuing the participation could be harmful.  
• 
Pregnancy 
• 
Subject needs treatment not allowed in the study.  
• 
The study is cancelled.  
• 
Other unanticipated circumstances.  
 
4.3.2 Discontinuation Visit and Procedures 
Participants are free to withdraw consent and discontinue participation at any time without penalty and are 
not obliged to give their reason. Subjects may also be withdrawn or removed, if necessary, to protect their 
health or the integrity of the study. The investigator also reserves the right to withdraw participants from the 
study in event of inter-current illness, adverse events, protocol violations, administrative, or other reasons. 
Once a subject withdraws from the study, no new data will be collected for study purposes unless the data 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 20 of 38 
Restricted, Sensitive (Normal) 
concerns safety or adverse events related to the study. The subject will be asked to continue scheduled 
evaluations, complete an end of study evaluation, and be given appropriate care under medical supervision 
until the symptoms of any adverse events resolve or the subject’s condition becomes stable. All details 
available will be reported and recorded for any subjects that withdraws or is removed from the study.  
 
5 
TRIAL MATERIALS 
5.1 Trial Product(s) 
 
i) AS01 Adjuvant  
Content:  
Throughout this protocol, we refer to the AS01B adjuvant system as “AS01”. The GlaxoSmithKline (GSK) 
proprietary AS01B adjuvant system is composed of the plant extract Quillaja saponaria Molina, fraction 21 
(QS-21) (50 micrograms) and 3-O-desacyl-4’monophosphoryl lipid A (MPL) from Salmonella minnesota (50 
micrograms). The AS01 suspension is an opalescent, colourless to pale brownish liquid.  
 
Dosage and administration:  
The AS01 adjuvant is supplied as a component of the recombinant zoster vaccine (RZV; GSK), which normally 
contains 2 separate vials: a 0.5ml vial containing the AS01 adjuvant system and another vial containing the 
varicella zoster virus (VZV) glycoprotein E (gE) antigen. While these vials are typically reconstituted together for 
RZV administration, in this study, subjects will only receive the AS01 adjuvant vial (0.5ml) without 
reconstitution with the gE antigen. AS01 0.5ml will be given as intramuscular injection in the deltoid muscle.  
 
Contraindications:  
Do not administer Shingrix to anyone with a history of a severe allergic reaction (e.g. anaphylaxis) to any 
component of the vaccine or after a previous dose of RZV.  
 
Adverse reactions (based on RZV data, which contains reconstituted AS01 plus VZV gE antigen):  
Adverse reactions (AEs): Solicited local AEs in subjects aged 50 years and older were pain (78.0%), redness 
(38.1%), and swelling (25.9%). Solicited general AEs in subjects aged 50 years and older were myalgia (44.7%), 
fatigue (44.5%), headache (37.7%), shivering (26.8%), fever (20.5%), and gastrointestinal symptoms (17.3%).  
 
Storage:  
Adjuvant suspension component vials: Store refrigerated between 2oC to 8oC. Protect vials from light. Do not 
freeze. Discard if the adjuvant suspension has been frozen.  
 
ii) Yellow Fever Vaccine  (Stamaril)  
Content:  
Stamaril, the live-attenuated yellow fever vaccine, utilises the YF17D strain. Stamaril is supplied in the form of 
powder and solvent for suspension for injection in pre-filled syringe, Yellow fever vaccine (live). Before 
reconstitution, the powder is homogeneous, beige to orange beige, and the solvent is a clear and colourless 
solution.  
 
Dosage and administration: 
Stamaril (1 dose, 0.5ml after reconstitution) is injected by the subcutaneous route.  
 
Contraindications:  
• 
Hypersensitivity to the active substance or to any of the excipients listed in Section 6.1 or to eggs or 
chicken proteins.  
• 
Severe hypersensitivity reactions (e.g., anaphylaxis) after a previous dose of any yellow fever vaccine.  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 21 of 38 
Restricted, Sensitive (Normal) 
• 
Immunosuppression, whether congenital, or acquired. This includes individuals receiving 
immunosuppressive therapies such as treatment with high-dose systemic corticosteroids (e.g. daily 
dose of 20 mg or 2 mg/kg body weight of prednisone or equivalent for 2 weeks or more or daily dose of 
40 mg or more of prednisone for more than one week), any other medicinal products including 
biologicals with known immunosuppressive properties, radiotherapy, cytotoxic drugs or any other 
condition which may result in immunocompromised status.  
• 
History of thymus dysfunction (including myasthenia gravis, thymoma).  
• 
Thymectomy (for any reason).  
• 
Symptomatic HIV infection.  
• 
Asymptomatic HIV infection when accompanied by evidence of impaired immune function   
• 
Moderate or severe febrile illness or acute illness.  
 
Adverse reactions:  
The most frequently reported reactions (between 12 – 18% of subjects) were headache, asthenia, injection site 
pain and myalgia. Adverse reactions usually occurred within the first three days following vaccination except 
pyrexia, which occurred between day 4 and day 14 and usually lasted for not more than 3 days. Both local and 
systemic reactions were usually of mild intensity; however at least one severe injection site reaction was 
reported in 0.8% of subjects and at least one severe systemic reaction was reported in 1.4% of subjects in 
general population. Other adverse reactions (> 1 in 100 to < 1 in 10) include rash, nausea, and arthralgia. Very 
rarely (< 1 in 10,000), YEL-AND and YEL-AVD have been reported following vaccination with sequelae or with 
fatal outcomes and risk appears to be higher in those aged over 60 years of age.  
 
Storage:  
Store in a refrigerator (2°C - 8°C). Do not freeze. Keep the vial of powder and the syringe of solvent in the outer 
carton in order to protect from light.  
 
5.2 Storage and Drug Accountability 
Upon receipt of the vaccine product, the delegated research staff at CTRC will be responsible for taking an 
inventory of the investigational products. A record of this inventory must be kept and dispensation of vaccines 
must be documented on drug inventory forms. The record must be available for inspection by the medical 
monitor, IRB or regulatory authorities.  
 
6 
TREATMENT 
 
6.1 Rationale for Selection of Dose 
Yellow fever vaccine containing the YF17D strain (Stamaril) has been licensed and approved for use. The 
above dose and method of administration are chosen based on vaccine manufacturer’s recommendations.  
 
AS01 adjuvant has been licensed and approved for use together with the recombinant zoster vaccine 
(Shingrix). The above dose and method of administration are chosen based on vaccine manufacturer’s 
recommendations, except VZV gE antigen will not be included in this study.  
 
6.2 Study Drug Formulations 
Live-attenuated yellow fever vaccine (Stamaril) and recombinant zoster vaccine (Shingrix) have been licensed 
and approved for use in Singapore.   
 
6.3 Study Drug Administration 
 
i) 
AS01 adjuvant or placebo 
0.5ml AS01 or placebo (NaCl 0.9%) will be given as intramuscular injection in the deltoid muscle.  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 22 of 38 
Restricted, Sensitive (Normal) 
 
ii) YF17D (Stamaril) vaccine:  
Stamaril (1 dose, 0.5ml after reconstitution) is injected by the subcutaneous route.  
 
6.4 Specific Restrictions / Requirements  
Subjects will be educated to refrain from self-administration of other prescription-only or over-the-counter 
oral or topical anti-inflammatory drugs, if possible, at any time throughout the study period. Subject should 
inform the CTRC or study team members if they are not feeling well for assessment before seeing General 
Practitioner. If the subject has seen a General Practitioner and was prescribed with drugs, they should be 
advised to inform study team members.  
 
6.5 Blinding 
Blinding:  
- 
First intervention (AS01/placebo): single blinded – subjects are blinded to treatment allocation. 
Subjects will not be told their assignments unless unblinding is required to protect participant safety.  
 
- 
Second intervention (YF17D timing): Open-label – no blinding.  
 
Planned Unblinding:  
- 
The investigators will receive unblinded data for analysis.  
 
Emergency Unblinding:  
- 
Emergency unblinding may occur in case of serious adverse events (SAEs) or medical emergencies 
where knowledge of the treatment is essential for subject care.  
- 
The PI or designated physician may request emergency unblinding.  
- 
All instances of emergency unblinding must be documented with:  
o Date and time of unblinding  
o Reason for unblinding 
o Name of person requesting unblinding  
o Name of person performing unblinding  
 
6.6 Concomitant therapy 
All vaccinations, medications (prescription and over the counter), vitamin and mineral supplements, and / or 
herbs taken by the participant should be documented. 
 
 
7 
SAFETY MEASUREMENTS 
 
7.1 Definitions 
An adverse event (AE) is any untoward medical occurrence in a trial participant administered a therapeutic 
products (TP)/ cell, tissue and gene therapy products (CTGTP)/ medicinal products (MP) and which does not 
necessarily have a causal relationship with this treatment. 
 
An AE can therefore be any unfavourable and unintended sign (including an abnormal laboratory finding, for 
example), symptom, or disease temporally associated with the use of a TP/CTGTP/MP, whether or not 
considered related to the TP/CTGTP/MP. 
 
A serious adverse event (SAE) is any untoward medical occurrence that at any dose: 
a. results in death 
b. is life-threatening 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 23 of 38 
Restricted, Sensitive (Normal) 
c. requires inpatient hospitalisation or prolongation of existing hospitalisation 
d. results in persistent or significant disability/incapacity, or 
e. is a congenital anomaly/birth defect 
 
7.2 Collecting, Recording and Reporting of Serious Adverse Events (SAEs) to CIRB  
The reporting requirements will be in accordance to the reporting requirements published on CIRB website at 
the time when the event took place. 
 
Only related SAEs (definitely/ probably/ possibly) will be reported to CIRB. Related means there is a 
reasonable possibility that the event may have been caused by participation in the clinical trial.   
 
The investigator is responsible for informing CIRB after first knowledge that the case qualifies for reporting. 
Follow-up information will be actively sought and submitted as it becomes available.  
 
Related AEs will not be reported to CIRB.  However, the investigator is responsible to keep record of such AEs 
cases at the Study Site File. 
 
7.3 Collecting, Recording and Reporting of Serious Adverse Events (SAEs) to the Health Sciences 
Authority (HSA) 
The reporting requirements will be based on the reporting requirements published on HSA website at the time 
when the event took place. 
 
7.4 Safety Monitoring Plan 
The study may be evaluated by government inspectors/regulatory authorities who must be allowed access to 
electronic case report forms (e-CRFs), source documents, and other study files. The inspectors will review 
CRFs and compare them with source documents to verify accurate and complete collection of data and 
confirm that the study is being conducted according to the protocol, ICH-Good Clinical Practices (ICH-GCP) 
and all applicable regulations.  
 
7.5 Complaint Handling  
Subjects can complain to the Principal Investigator or Centralised Institutional Review Board (CIRB). The 
contact numbers are listed in the Participant Information and Informed Consent Form. 
 
8 
DATA ANALYSIS 
 
8.1 Data Quality Assurance 
The PI and Co-Is will periodically review the study for data and safety monitoring. Internal quality checks will 
be performed by two CRC who are study team members. The data entered by one CRC will be checked by 
another using the source documents. The study may also be evaluated by government inspectors / regulatory 
authorities who must be allowed to access to e-CRFs, source documents, and other study files. The 
inspectors will review CRFs and compare them with source documents to confirm accurate and complete 
collection of data. The inspectors will also ensure that the study is being conducted according to the protocol, 
ICH-Good Clinical Practices and all applicable regulations.  
 
8.2 Data Entry and Storage 
Demographic and clinical data will be collected using computer notebooks. The data will be kept confidential 
in password-protected computers accessible only by delegated research staff. Subject identifiers will be kept 
in a separate file in another office, and every effort will be made to protect the privacy of study participants. 
Data analysis will be conducted only on de-identified data. An electronic data capture system will be used.  
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 24 of 38 
Restricted, Sensitive (Normal) 
 
Table 1: Markers for 
immunophenotyping  
9 
SAMPLE SIZE AND STATISTICAL METHODS 
 
9.1 Determination of Sample Size 
Based on power calculation, we determined that 16 subjects per group (total 32) would 
provide 80% power to detect a true difference in gene expression of at least 2 standard 
deviations between vaccine groups, with a false discovery rate of < 10%. This sample 
size is also sufficient for achieving reproducibility in gene set analysis63. To account for 
potential dropouts during the study period, we will enrol 40 subjects (20 per group). 
 
9.2 Statistical and Analytical Plans 
Specific Aim 1: Examine the host response induced by AS01 and determine the 
duration of these changes for up to 3 months in healthy older adults aged 21 – 59 
years old.   
Sample collection, laboratory investigations and safety considerations  
Samples (PBMCs, serum, plasma) will be stored at – 80oC and analysed collectively. 
Subjects will undergo clinical review during visits, including medical history, vaccination 
history, drug history and physical examination. They will be instructed to observe for 
adjuvant-related AEs such as injection site reactions, fever, myalgia, fatigue and 
headache throughout the study period. Participants will maintain a diary to record any 
AEs and report to CTRC should they require medical attention.  
 
Early AS01-induced effects on immune cell populations will be analysed through flow cytometry, focusing 
particularly on myeloid cells (monocytes, DCs, plasmacytoid dendritic cells (pDCs), NK cells, and innate 
lymphoid cells (ILCs) due to their crucial role in innate immune response and TI. Flow cytometric analysis for 
immunophenotyping of blood leukocytes will be carried out using a 26-marker panel (Table 1), with PBMCs 
being thawed and stained according to our established protocols64. After staining, cells will be fixed in 2% 
paraformaldehyde before acquisition on BD Symphony cytometer. Flow cytometry data will be analyzed with 
FlowJoTM version 10.3 (Tree Star, Inc., USA). 
 
Comprehensive analysis of gene expression profiles in individual immune cell types will be conducted using 
scRNA-seq on PBMCs, followed by comprehensive functional and pathway analyses over baselines (day 0) 
and post-vaccination timepoints (Fig. 5B, C). In brief, scRNA-seq library preparation and sequencing will be 
performed on thawed PBMCs resuspended in PBS + 1% BSA for processing by the Chromium Next GEM Single 
Cell 5' Kit (10x Genomics) v2. The Chromium Controller will be used to target droplet encapsulation of 5,000 
cells, and gene expression libraries will be prepared following the manufacturer’s protocol. The libraries will 
be quantified on the Agilent Bioanalyzer and sequenced at 2×151 cycles on Illumina NovaSeq 6000 at a 
targeted sequencing depth of 50,000 reads/cell. Impact of AS01 on epigenome will be investigated by genome-
wide scATAC-seq. Processing and analytical pipelines for the single cell studies is already established in Co-I 
Singhal’s group65 (Nigeshi et al., submitted). scATAC-seq library preparation and sequencing will be performed 
on nuclei from thawed PBMCs and generated following the low cell input nuclei isolation option of the 10x 
Genomics Demonstrated Protocol CG000169, using 1x Lysis Buffer and 3 min lysis time. Nuclei will be 
processed by the Chromium Single Cell ATAC Kit (10x Genomics) v2 following the manufacturer’s protocol, 
targeting the capture of 5,000-6,000 nuclei or fewer if the sample was limiting. After quantification on the 
Agilent Bioanalyzer, 2-3 ATAC-seq libraries will be pooled for sequencing per lane of Illumina HiSeq 4000.  
 
Plasma cytokines and chemokines will be measured at pre-vaccination baseline (Day 0) and post-vaccination 
timepoints (Fig. 5B, C)  using the Olink Target 48 Cytokine panel as previously performed and published by our 
group66. Olink’s proximity extension assay technology permits quantitative detection of 45 protein biomarkers 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 25 of 38 
Restricted, Sensitive (Normal) 
simultaneously, including cytokines and chemokines to depict the immune response following infection or 
vaccination. This analysis will complement our flow cytometry and sequencing data by providing insights into 
the early systemic immune environment that regulate cellular responses. The Olink analysis will be conducted 
at the Viral Research and Experimental Medicine Centre @ SingHealth Duke-NUS (ViREMiCS). 
 
Analysis of scRNA-seq data 
Raw FASTQ files data will be aligned to human reference genome GRCh38 by Cell Ranger (version 7.1.0, 10x 
Genomics)67. The output files will then be loaded into R package Seurat (version 4.4.0)68. Quality control (QC) 
will be performed for each library and low-quality cells will be removed. Cells containing less than 10% of 
mitochondrial reads will be retained in the analysis. After dimensional reduction using Principal Component 
Analysis (PCA) followed by Uniform Manifold Approximation and Projection (UMAP), unsupervised graph-
based clustering (resolution=0.5) will be performed using FindClusters function of Seurat package. Major cell 
types will be annotated according to expression of canonical cell type markers, which will be further refined in 
downstream sub-clustering of each cell group. After identification of different cell types, DEGs will be 
evaluated for similar cell type across groups. FindMarkers function of Seurat package will be used with options 
logfc.threshold=0 and min.pct=0. Genes with adjusted p-values less than 0.05 were considered as DEGs. The 
enriched pathways in DEGs were identified using R package clusterProfiler (version 4.2.0)69. The R package 
pheatmap (v1.0.12) will be used to create heatmaps70. The scRNA-seq data will be utilized to quantitatively 
infer cell-cell communication networks, for this R package CellChat71 (version 1.6.1) will be used. 
 
Analysis of scATAC-seq data  
FASTQ files containing raw scATAC-seq reads will be mapped to human reference genome CRCh38 using Cell 
Ranger ATAC (version 2.0.0)72. The output files will be loaded into R package Signac (version 1.10.0)68 for 
downstream processing. Nuclei with <200 genes detected, and high percentage mitochondria genes will be 
filtered out. Genes will be annotated to ATAC-seq peaks by Annotation function and Ensembl based 
annotation database EnsDb.Hsapiens.v86 (version 2.99.0)73. scATAC-seq data will be pre-processed and 
undergo dimensional reduction independently before calculating the weighted nearest neighbor (WNN)graph 
and cluster identification. This will be followed by performing differential accessible (DA) peak analysis. Peaks 
with adjusted p-value less than 0.05 will be considered as DA peaks. Pathway enrichment will be performed 
on the closet genes annotated to DA peaks using clusterProfiler package. Enriched transcription factor (TF) 
motifs will be determined using Signac 74 and chromVAR 75 to calculate per-cell accessibility scores for known 
motifs. 
 
Statistical analysis 
The raw data will be analyzed by GraphPad Prism (version 9.5.1). Generally, unpaired t-test will be applied to 
compare means between two groups of data after passing normality check using Shapiro-Wilk test and 
confirming equal variances using F test. If more than two groups are involved, Kruskal-Wallis test will be used 
with Dunn’s multiple comparisons test. 
 
Specific aim 2: Demonstrate that innate immune reprogramming post AS01 can provide cross-protection 
against heterologous YF17D viral infection 
Recent evidence suggests that AS01’s ability to induce TI could provide broad protection against heterologous 
infections through epigenetic and metabolic reprogramming of innate immune cells. However, the duration 
and extent of the AS01-alone mediated cross-protection in older populations remain undefined. This aim 
seeks to establish the protective capacity and temporal dynamics of AS01-alone induced immune 
enhancement.  
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 26 of 38 
Restricted, Sensitive (Normal) 
Study design 
To test this hypothesis, study participants from Specific Aim 1 who received either AS01 (n = 20) or placebo (n 
= 20) will be randomized in a 1:1 ratio to receive the live-attenuated yellow fever vaccine YF17D (Stamaril, 
Sanofi-Pasteur) at either 1 month (YF17D 1-month group) or 3 months (YF17D 3-month group) (Fig 5B, C). 
YF17D serves as an ideal experimental viral challenge model due to its well-characterized safety profile and 
reliable induction of measurable viremia, as demonstrated in our previous studies16,76–78. 
 
To assess the impact of AS01 on YF17D RNAemia, baseline blood samples will be collected on the day before 
YF17D administration (day 30 for the YF17D 1-month group, or day 90 for the YF17D 3-month group). This will 
be followed by daily collection on days 2 – 7 post-YF17D vaccination for measuring YF17D RNAemia. This 
sampling schedule follows our previously established protocols and aligns with known YF17D RNAemia 
dynamics and typical detection window for viral RNAemia58,77. Quantification of viral RNAemia in blood will be 
determined by viral RNA extraction followed by quantitative RT-PCR directed against the NS5 gene of YF17D. 
An in vitro transcribed RNA of the YF17D NS5 gene will be used to construct a standard curve to quantify viral 
RNAemia77. Viremia area under the curve (AUC) will be calculated as previously described77.  
 
Furthermore, we will characterize the immunological mechanisms underlying the protective effects of AS01 
through comprehensive analysis of the host response. To understand the mechanisms of protection, YF17D 
RNAemia will be correlated with immune cell profiles and host responses induced 7 days post-YF17D 
challenge. Host response parameters to be evaluated include flow cytometry of immune cell populations, 
bulk RNA expression profiles, epigenetic changes and cytokine profiles. Notably, the sampling points selected 
for correlation analysis aligns with previous work from our group and others which demonstrates peak gene 
expression changes at these time points77. 
 
To perform bulk RNASeq, total RNA from PAXgene tubes will be extracted using the manufacturer’s protocol 
(Qiagen) and quality-assessed via Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). cDNA libraries 
will be prepared using SMARTSeq v2 protocol with 500 pg total RNA, followed by indexed PE sequencing (2x51 
cycles, Illumina Hiseq 2000)64. Reads will be mapped to the human genome using STAR, counted with 
featureCounts (Subread package)79, and analyzed for differential expression using DESeq2 (R, version 3.3.3, p 
< 0.05)80. DEGs will undergo pathway enrichment analysis.  
 
Specific aim 3: Examine the T and B cell responses post YF17D challenge, and its correlation with YF 
viremia levels in AS01 group versus placebo.  
In the context of emerging pandemic threats, enhancing vaccine response is critical for establishing both 
individual protection and herd immunity to prevent sustained community transmission. This is particularly 
critical for older populations who face dual challenges: increased vulnerability to severe infections and 
reduced vaccine responsiveness due to immunosenescence.  
 
Our preliminary data (Chan et al., submitted) demonstrates that a single dose of RZV can effectively modulate 
baseline expression of genes that correlate with T cell responses, leading to enhanced protective T cell 
responses following administration of the second RZV dose. Previous studies on BCG-induced TI in human 
monocytes have shown sustained immunological changes, particularly in T cell responses against ex-vivo 
experimental heterologous infections, suggesting important implications for improving overall vaccine 
responses81. Importantly, T cell immunity has been demonstrated to play an important role in protection 
against symptomatic infection and viral clearance for SARS-CoV282,83 and herpes zoster47.  
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 27 of 38 
Restricted, Sensitive (Normal) 
However, established studies from our group have shown that YF17D RNAemia duration positively correlates 
with both humoral and cellular immunity16,84. This raises a critical question: could the hypothesized reduction 
in vial RNAemia duration through AS01-induced innate immune reprogramming potentially impact the 
development of T cell immunity against YF17D? 
 
For specific aim 3, we will investigate whether innate immune cell reprogramming post-AS01 administration 
can achieve enhanced YF17D RNAemia clearance while maintaining robust cellular immune responses to 
YF17D. This investigation will examine T cell responses following YF17D challenge and the correlation with 
YF17D RNAemia levels. The findings will provide crucial insights into the relationship between TI, viral 
RNAemia clearance, and the development of protective T cell responses in vivo.  
 
Sample collection and laboratory investigations (Fig. 5B, C)   
To quantify YF17D-specific T cells, peripheral blood samples will be collected at specific timepoints: days 30, 
44, 90 for the YF17D 1-month group, and days 90, 104, and 120 for the 3-month group. PBMCs will be isolated 
and stored at – 80oC until further analysis as previously described85. YF17D-specific T cell response will be 
measured using YF17D specific IFN ELISPOT assays. In brief, PBMCs will be stimulated with YF17D peptide 
pools that span the structural (capsid, pre-membrane, envelope) and non-structural (NS1 to NS5) proteins. 
PBMCs will be stimulated with YF17D peptide pools inside IFN-coated ELISpot plates, followed by incubation 
with biotinylated anti-human IFN antibodies and treatment with streptavidin-alkaline phosphatase. Signal 
development will utilize BCIP/NBT phosphatase substrate. To quantify positive peptide-specific response, 
mean spots of the unstimulated wells are subtracted from the peptide-stimulated wells, and the results 
expressed as spot-forming units (SFU) per 106 PBMCs. Concomitant measurement of neutralizing antibodies 
induced by YF17D challenge will also be measured by plaque reduction neutralization test (PRNT), as 
previously described using sera obtained on day 0 and 1-month after YF17D administration16. Differences in 
YF17D-specific T cell response and YF17D neutralizing antibody titers between participants challenged with 
YF17D 1 month and 3 months post-AS01 administration will be performed using unpaired t-tests. Correlation 
analysis between viral RNAemia duration and T and B cell responses will be performed by nonparametric 
Spearman’s correlation. Statistical significance is defined by p-value less than 0.05. To better understand the 
immunomodulatory role of AS01 on T and B cell responses against YF17D, we will also conduct 
comprehensive analyses integrating immune cell profiles, gene expression and epigenetic data between AS01 
and placebo groups. This systematic evaluation will enable correlation of immunological parameters with 
adaptive immune responses, providing valuable insights into AS01’s immunomodulatory effects and 
advancing our understanding of its immunomodulatory effects in older adults. 
 
10 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
The investigator(s)/institution(s) will permit study-related monitoring, audits and/or IRB review and regulatory 
inspection(s), providing direct access to source data/document. 
 
11 
QUALITY CONTROL AND QUALITY ASSURANCE 
The PI and Co-Is will conduct periodic reviews of the study for data and safety monitoring, with the frequency 
of these reviews to be determined by the committee and regulatory bodies based on their established policies. 
Internal quality control measures will be implemented through two CRCs who are members of the study team. 
To ensure data accuracy, a dual-verification system will be employed where data entered by one CRC will be 
independently verified by the second CRC using source documents. 
 
The study will be subject to evaluation by government inspectors and regulatory authorities, who will be 
granted full access to e-CRFs, source documents, and other study files. These inspectors will conduct 
thorough reviews of the CRFs, comparing them against source documents to verify accurate and complete 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 28 of 38 
Restricted, Sensitive (Normal) 
data collection. Additionally, they will assess study conduct to ensure compliance with the protocol, 
International Conference on Harmonisation-Good Clinical Practices (ICH-GCP), and all applicable 
regulations. The frequency and scope of these regulatory inspections will be determined by the respective 
regulatory bodies according to their standard procedures and policies. 
 
12 
ETHICAL CONSIDERATIONS 
This study will be conducted in accordance with the ethical principles that have their origin in the Declaration 
of Helsinki and that are consistent with the Good Clinical Practice and the applicable regulatory requirements. 
 
This final Clinical Trial Protocol, including the final version of the Participant Information and Consent Form, 
must be approved in writing by the Centralised Institutional Review Board (CIRB), prior to enrolment of any 
patient into the study. 
 
The principal investigator is responsible for informing the CIRB and HSA of any amendments to the protocol or 
other study-related documents, as per local requirement. 
 
12.1 
Informed Consent 
The informed consent process will begin with potential subjects being identified through the CTRC healthy 
volunteer database, followed by initial contact from CTRC to assess interest and availability. Eligible 
candidates, determined through preliminary telephone screening against basic inclusion and exclusion 
criteria, will be invited for a physical screening visit at CTRC. Prior to any study procedures, blood sample 
collection, randomisation, or interventions, written informed consent will be obtained by the study PI or CO-I 
from subjects who meet all inclusion criteria. For participants who are not fluent in English, qualified 
interpreters or translators will assist in the informed consent process to ensure comprehensive understanding 
of study procedures and participant rights. 
 
In accordance with Good Clinical Practice (GCP) guidelines and the ethical principles outlined in the 
Declaration of Helsinki, the investigators will maintain strict compliance throughout the consent process. Two 
separate consent forms will be utilized: one for study participation and another for future use of specimens, 
ensuring clear differentiation between immediate study involvement and potential future research 
applications. This dual consent approach allows participants to make informed decisions about both their 
current participation and the future use of their biological samples. 
 
12.2 
Confidentiality of Data and Patient Records 
All information collected for this study will be kept confidential. The subject records, to the extent of the 
applicable laws and regulations, will not be made public available. Only investigators and delegated study 
team members will have access to the confidential information. Singapore General Hospital, Regulatory 
Agencies, Institutional Review Board and Ministry of Healthy may be granted direct access to the original 
medical records to check study procedure and data, without making any of the information public. Hardcopy 
research data will be kept under lock and key in a secured office and can only be accessed by the study team. 
In publications, identifiable information will not be used.  
 
13 
PUBLICATIONS 
In the event of any publications pertaining to this study, identifiable information will not be used. PI, CO-I and 
collaborators will oversee all publications that arise from this study. The publication process encompasses 
manuscript preparation, reviews and revisions, with authorship granted to substantial contributors. 
Publications will follow the journal’s review timelines. All publications shall acknowledge trial participants, the 
funding support provided by Grantor and where appropriate, the scientific and other contributions of other 
Institutions and Research Personnel in accordance with established norms. 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 29 of 38 
Restricted, Sensitive (Normal) 
14 
RETENTION OF TRIAL DOCUMENTS 
Records for all participants, including CRFs, all source documentation (containing evidence to study eligibility, 
history and physical findings, laboratory data, results of consultations, etc.) as well as IRB records and other 
regulatory documentation should be retained by the PI in a secure storage facility. The records should be 
accessible for inspection and copying by authorized authorities. They will be kept for 15 years then 
subsequently destroyed.  
 
15 
FUNDING AND INSURANCE 
This grant is funded by National Medical Research Council (NMRC) under the CS-IRG scheme. The study is 
covered under National Clinical Trial Insurance, with the documentation and coverage details to be verified 
and updated as appropriate through the Research Innovation Office, SGH.   
 
16 
REFERENCES 
1.  
Iwasaki A, Medzhitov R. Control of adaptive immunity by the innate immune system. Nat Immunol. 
2015 Apr;16(4):343–53.  
2.  
Shaw AC, Goldstein DR, Montgomery RR. Age-dependent dysregulation of innate immunity. Nat Rev 
Immunol. 2013 Dec;13(12):875–87.  
3.  
Jo N, Hidaka Y, Kikuchi O, Fukahori M, Sawada T, Aoki M, et al. Impaired CD4+ T cell response in 
older adults is associated with reduced immunogenicity and reactogenicity of mRNA COVID-19 
vaccination. Nat Aging. 2023 Jan;3(1):82–92.  
4.  
Esmaeili ED, Azizi H, Sarbazi E, Khodamoradi F. The global case fatality rate due to COVID-19 in 
hospitalized elderly patients by sex, year, gross domestic product, and continent: A systematic review, 
meta-analysis, and meta-regression. New Microbes New Infect. 2023 Jan 4;51:101079.  
5.  
Hou Y, Chen M, Bian Y, Hu Y, Chuan J, Zhong L, et al. Insights into vaccines for elderly individuals: 
from the impacts of immunosenescence to delivery strategies. npj Vaccines. 2024 Apr 10;9(1):77.  
6.  
Cunningham AL, Lal H, Kovac M, Chlibek R, Hwang SJ, Díez-Domingo J, et al. Efficacy of the 
Herpes Zoster Subunit Vaccine in Adults 70 Years of Age or Older. N Engl J Med. 2016 Sep 
15;375(11):1019–32.  
7.  
Lal H, Cunningham AL, Godeaux O, Chlibek R, Diez-Domingo J, Hwang SJ, et al. Efficacy of an 
adjuvanted herpes zoster subunit vaccine in older adults. N Engl J Med. 2015 May 28;372(22):2087–
96.  
8.  
Efficacy and Safety of a Bivalent RSV Prefusion F Vaccine in Older Adults | New England Journal of 
Medicine [Internet]. [cited 2024 Sep 15]. Available from: 
https://www.nejm.org/doi/full/10.1056/NEJMoa2213836 
9.  
Cunningham AL, Heineman TC, Lal H, Godeaux O, Chlibek R, Hwang SJ, et al. Immune responses to 
a recombinant glycoprotein E herpes zoster vaccine in adults aged 50 years or older. The Journal of 
infectious diseases. 2018;217(11):1750–60.  
10.  Coccia M, Collignon C, Hervé C, Chalon A, Welsby I, Detienne S, et al. Cellular and molecular 
synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine 
immunogenicity. npj Vaccines. 2017 Sep 8;2(1):1–14.  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 30 of 38 
Restricted, Sensitive (Normal) 
11.  Smith CL, Richardson B, Rubsamen M, Cameron MJ, Cameron CM, Canaday DH. Adjuvant AS01 
activates human monocytes for costimulation and systemic inflammation. Vaccine. 2024 
Jan;42(2):229–38.  
12.  Didierlaurent AM, Collignon C, Bourguignon P, Wouters S, Fierens K, Fochesato M, et al. 
Enhancement of Adaptive Immunity by the Human Vaccine Adjuvant AS01 Depends on Activated 
Dendritic Cells. The Journal of Immunology. 2014 Aug 15;193(4):1920–30.  
13.  Bechtold V, Smolen KK, Burny W, De Angelis SP, Delandre S, Essaghir A, et al. Functional and 
epigenetic changes in monocytes from adults immunized with an AS01-adjuvanted vaccine. Sci Transl 
Med. 2024 Jul 31;16(758):eadl3381.  
14.  Netea MG, Joosten LA, Latz E, Mills KH, Natoli G, Stunnenberg HG, et al. Trained immunity: a 
program of innate immune memory in health and disease. Science (New York, NY). 2016 Apr 
21;352(6284):aaf1098.  
15.  Bruxvoort KJ, Ackerson B, Sy LS, Bhavsar A, Tseng HF, Florea A, et al. Recombinant Adjuvanted 
Zoster Vaccine and Reduced Risk of Coronavirus Disease 2019 Diagnosis and Hospitalization in 
Older Adults. J Infect Dis. 2022 Jun 1;225(11):1915–22.  
16.  Chan KR, Wang X, Saron WAA, Gan ES, Tan HC, Mok DZL, et al. Cross-reactive antibodies enhance 
live attenuated virus infection for increased immunogenicity. Nat Microbiol. 2016 Sep 19;1(12):1–10.  
17.  Baker RE, Mahmud AS, Miller IF, Rajeev M, Rasambainarivo F, Rice BL, et al. Infectious disease in 
an era of global change. Nat Rev Microbiol. 2022 Apr;20(4):193–205.  
18.  Kanasi E, Ayilavarapu S, Jones J. The aging population: demographics and the biology of aging. 
Periodontol 2000. 2016 Oct;72(1):13–8.  
19.  Palacios-Pedrero MÁ, Jansen JM, Blume C, Stanislawski N, Jonczyk R, Molle A, et al. Signs of 
immunosenescence correlate with poor outcome of mRNA COVID-19 vaccination in older adults. Nat 
Aging. 2022 Oct;2(10):896–905.  
20.  Chumakov K, Avidan MS, Benn CS, Bertozzi SM, Blatt L, Chang AY, et al. Old vaccines for new 
infections: Exploiting innate immunity to control COVID-19 and prevent future pandemics. Proc Natl 
Acad Sci USA. 2021 May 25;118(21):e2101718118.  
21.  Charles A Janeway J, Travers P, Walport M, Shlomchik MJ. The course of the adaptive response to 
infection. In: Immunobiology: The Immune System in Health and Disease 5th edition [Internet]. 
Garland Science; 2001 [cited 2024 Nov 15]. Available from: 
https://www.ncbi.nlm.nih.gov/books/NBK27125/ 
22.  Netea MG, Domínguez-Andrés J, Barreiro LB, Chavakis T, Divangahi M, Fuchs E, et al. Defining 
trained immunity and its role in health and disease. Nat Rev Immunol. 2020 Jun;20(6):375–88.  
23.  Netea MG, Latz E, Mills KHG, O’Neill LAJ. Innate immune memory: a paradigm shift in 
understanding host defense. Nat Immunol. 2015 Jul;16(7):675–9.  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 31 of 38 
Restricted, Sensitive (Normal) 
24.  Baydemir I, Dulfer EA, Netea MG, Domínguez-Andrés J. Trained immunity-inducing vaccines: 
Harnessing innate memory for vaccine design and delivery. Clinical Immunology. 2024 Apr 
1;261:109930.  
25.  Biering-Sørensen S, Aaby P, Lund N, Monteiro I, Jensen KJ, Eriksen HB, et al. Early BCG-Denmark 
and Neonatal Mortality Among Infants Weighing <2500 g: A Randomized Controlled Trial. Clin 
Infect Dis. 2017 Oct 1;65(7):1183–90.  
26.  Nieminen H, Lahdenkari M, Syrjänen RK, Nohynek H, Ruokokoski E, Palmu AA. Lower incidence of 
hospital-treated infections in infants under 3 months of age vaccinated with BCG. Vaccine. 2022 Oct 
6;40(42):6048–54.  
27.  Giamarellos-Bourboulis EJ, Tsilika M, Moorlag S, Antonakos N, Kotsaki A, Domínguez-Andrés J, et 
al. Activate: Randomized Clinical Trial of BCG Vaccination against Infection in the Elderly. Cell. 
2020 Oct 15;183(2):315-323.e9.  
28.  Wardhana  null, Datau EA, Sultana A, Mandang VVV, Jim E. The efficacy of Bacillus Calmette-
Guerin vaccinations for the prevention of acute upper respiratory tract infection in the elderly. Acta 
Med Indones. 2011 Jul;43(3):185–90.  
29.  Walk J, de Bree LCJ, Graumans W, Stoter R, van Gemert GJ, van de Vegte-Bolmer M, et al. 
Outcomes of controlled human malaria infection after BCG vaccination. Nat Commun. 2019 Feb 
20;10(1):874.  
30.  Arts RJW, Moorlag SJCFM, Novakovic B, Li Y, Wang SY, Oosting M, et al. BCG Vaccination 
Protects against Experimental Viral Infection in Humans through the Induction of Cytokines 
Associated with Trained Immunity. Cell Host Microbe. 2018 Jan 10;23(1):89-100.e5.  
31.  Byberg S, Thysen SM, Rodrigues A, Martins C, Cabral C, Careme M, et al. A general measles 
vaccination campaign in urban Guinea-Bissau: Comparing child mortality among participants and non-
participants. Vaccine. 2017 Jan 3;35(1):33–9.  
32.  Sørup S, Jensen AKG, Aaby P, Benn CS. Revaccination With Measles-Mumps-Rubella Vaccine and 
Infectious Disease Morbidity: A Danish Register-based Cohort Study. Clin Infect Dis. 2019 Jan 
7;68(2):282–90.  
33.  Röring RJ, Debisarun PA, Botey-Bataller J, Suen TK, Bulut Ö, Kilic G, et al. MMR vaccination 
induces trained immunity via functional and metabolic reprogramming of γδ T cells. The Journal of 
Clinical Investigation. 2024 Jan 30;134(7):e170848.  
34.  Cauchi S, Locht C. Non-specific Effects of Live Attenuated Pertussis Vaccine Against Heterologous 
Infectious and Inflammatory Diseases. Front Immunol. 2018 Dec 7;9:2872.  
35.  Avery RK. Immunizations in adult immunocompromised patients: Which to use and which to avoid. 
Cleveland Clinic Journal of Medicine. 2001 Apr 1;68(4):337–48.  
36.  Murmu N, Sarkar M, Dey S, Manna R, Roy S, Mondal T, et al. Efficacy and limitations of repurposed 
drugs and vaccines for COVID-19. Journal of Medicine, Surgery, and Public Health. 2024 Apr 
1;2:100041.  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 32 of 38 
Restricted, Sensitive (Normal) 
37.  Boutry C, Hastie A, Diez-Domingo J, Tinoco JC, Yu CJ, Andrews C, et al. The Adjuvanted 
Recombinant Zoster Vaccine Confers Long-Term Protection Against Herpes Zoster: Interim Results of 
an Extension Study of the Pivotal Phase 3 Clinical Trials ZOE-50 and ZOE-70. Clin Infect Dis. 2022 
Apr 28;74(8):1459–67.  
38.  Sun SJ, Aguirre-Gamboa R, de Bree LCJ, Sanz J, Dumaine A, Joosten LAB, et al. BCG vaccination 
impacts the epigenetic landscape of progenitor cells in human bone marrow. bioRxiv. 2023 Nov 
28;2023.11.28.569076.  
39.  Cirovic B, de Bree LCJ, Groh L, Blok BA, Chan J, van der Velden WJFM, et al. BCG Vaccination in 
Humans Elicits Trained Immunity via the Hematopoietic Progenitor Compartment. Cell Host Microbe. 
2020 Aug 12;28(2):322-334.e5.  
40.  Kaufmann E, Sanz J, Dunn JL, Khan N, Mendonça LE, Pacis A, et al. BCG Educates Hematopoietic 
Stem Cells to Generate Protective Innate Immunity against Tuberculosis. Cell. 2018 Jan 11;172(1–
2):176-190.e19.  
41.  Zhang X, Song B, Carlino MJ, Li G, Ferchen K, Chen M, et al. An immunophenotype-coupled 
transcriptomic atlas of human hematopoietic progenitors. Nat Immunol. 2024 Apr;25(4):703–15.  
42.  Triana S, Vonficht D, Jopp-Saile L, Raffel S, Lutz R, Leonce D, et al. Single-cell proteo-genomic 
reference maps of the hematopoietic system enable the purification and massive profiling of precisely 
defined cell states. Nat Immunol. 2021 Dec;22(12):1577–89.  
43.  Lukhele S, Boukhaled GM, Brooks DG. Type I interferon signaling, regulation and gene stimulation in 
chronic virus infection. Semin Immunol. 2019 Jun;43:101277.  
44.  Kleinnijenhuis J, Quintin J, Preijers F, Joosten LAB, Ifrim DC, Saeed S, et al. Bacille Calmette-Guérin 
induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of 
monocytes. Proceedings of the National Academy of Sciences. 2012 Oct 23;109(43):17537–42.  
45.  Jeyanathan M, Vaseghi-Shanjani M, Afkhami S, Grondin JA, Kang A, D’Agostino MR, et al. 
Parenteral BCG vaccine induces lung-resident memory macrophages and trained immunity via the gut-
lung axis. Nat Immunol. 2022 Dec;23(12):1687–702.  
46.  Gershon AA, Breuer J, Cohen JI, Cohrs RJ, Gershon MD, Gilden D, et al. Varicella zoster virus 
infection. Nat Rev Dis Primers. 2015 Jul 2;1(1):1–18.  
47.  Laing KJ, Ouwendijk WJD, Koelle DM, Verjans GMGM. Immunobiology of Varicella-Zoster Virus 
Infection. The Journal of Infectious Diseases. 2018 Sep 22;218(suppl_2):S68–74.  
48.  Laing KJ, Ford ES, Johnson MJ, Levin MJ, Koelle DM, Weinberg A. Recruitment of naive CD4+ T 
cells by the recombinant zoster vaccine correlates with persistent immunity. J Clin Invest [Internet]. 
2023 Dec 1 [cited 2024 Sep 25];133(23). Available from: https://www.jci.org/articles/view/172634 
49.  Izurieta HS, Wu X, Forshee R, Lu Y, Sung HM, Agger PE, et al. Recombinant Zoster Vaccine 
(Shingrix): Real-World Effectiveness in the First 2 Years Post-Licensure. Clinical Infectious Diseases. 
2021 Sep 15;73(6):941–8.  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 33 of 38 
Restricted, Sensitive (Normal) 
50.  Syenina A, Gan ES, Toh JZN, Alwis R de, Lin LZ, Tham CYL, et al. Adverse effects following anti–
COVID-19 vaccination with mRNA-based BNT162b2 are alleviated by altering the route of 
administration and correlate with baseline enrichment of T and NK cell genes. PLOS Biology. 2022 
May 31;20(5):e3001643.  
51.  UniProt: the Universal Protein Knowledgebase in 2023 | Nucleic Acids Research | Oxford Academic 
[Internet]. [cited 2024 Oct 24]. Available from: 
https://academic.oup.com/nar/article/51/D1/D523/6835362?login=false 
52.  Shingrix - Herpes Zoster (HZ, or Shingles) vaccine (non-live recombinant, AS01b adjuvanted) 
[Internet]. GSK; 2021. Available from: 
https://gskpro.com/content/dam/global/hcpportal/en_SG/products/PDF/shingrix/pdf/shingrix-pi-
gdsv06si-approved-13dec2021.pdf 
53.  Stamaril - Powder and solvent for suspension for injection, yellow fever vaccine (live) [Internet]. 
Sanofi-Pasteur; 2022. Available from: 
https://www.sanofi.com/assets/countries/singapore/docs/Stamaril-SG-0222.pdf 
54.  Yellow fever vaccine: stronger precautions in people with weakened immunity and in those aged 60 
years or older [Internet]. GOV.UK. [cited 2024 Dec 20]. Available from: https://www.gov.uk/drug-
safety-update/yellow-fever-vaccine-stronger-precautions-in-people-with-weakened-immunity-and-in-
those-aged-60-years-or-older 
55.  Sharma NK, Das SK, Mondal AK, Hackney OG, Chu WS, Kern PA, et al. Endoplasmic Reticulum 
Stress Markers Are Associated with Obesity in Nondiabetic Subjects. The Journal of Clinical 
Endocrinology and Metabolism. 2008 Aug 26;93(11):4532.  
56.  Sheridan PA, Paich HA, Handy J, Karlsson EA, Hudgens MG, Sammon AB, et al. Obesity is 
associated with impaired immune response to influenza vaccination in humans. Int J Obes (Lond). 
2012 Aug;36(8):1072–7.  
57.  Saucha CVV, Maia M de L de S, Sousa ESS, Oliveira PMN de, Xavier JR, Castro T da M de, et al. 
Immunogenicity of a single dose of the 17DD yellow fever vaccine in a cohort of adults and children 
in a non-endemic area, and its association with dengue and Zika seropositivity. PLOS Neglected 
Tropical Diseases. 2025 Apr 9;19(4):e0012993.  
58.  Kalimuddin S, Chan YFZ, Sessions OM, Chan KR, Ong EZ, Low JG, et al. An experimental medicine 
decipher of a minimum correlate of cellular immunity: Study protocol for a double-blind randomized 
controlled trial. Front Immunol [Internet]. 2023 Mar 10 [cited 2024 Dec 17];14. Available from: 
https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1135979/full 
59.  de Bree LCJ, Mourits VP, Koeken VA, Moorlag SJ, Janssen R, Folkman L, et al. Circadian rhythm 
influences induction of trained immunity by BCG vaccination. J Clin Invest. 2020 Oct 
1;130(10):5603–17.  
60.  Safety Profile & Adverse Reactions | SHINGRIX (Zoster Vaccine Recombinant, Adjuvanted) 
[Internet]. [cited 2025 May 23]. Available from: https://ShingrixHCP.com/efficacy-safety/adverse-
reactions/ 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 34 of 38 
Restricted, Sensitive (Normal) 
61.  BMI Calculator - Singapore Heart Foundation [Internet]. https://www.myheart.org.sg/. [cited 2025 Jun 
4]. Available from: https://www.myheart.org.sg/tools-resources/bmi-calculator/ 
62.  Clinical presentation, diagnosis, and initial evaluation of diabetes mellitus in adults - UpToDate 
[Internet]. [cited 2025 Jul 9]. Available from: https://www.uptodate.com/contents/clinical-
presentation-diagnosis-and-initial-evaluation-of-diabetes-mellitus-in-adults#H7 
63.  Maleki F, Ovens K, McQuillan I, Kusalik AJ. Size matters: how sample size affects the reproducibility 
and specificity of gene set analysis. Human Genomics. 2019 Oct 22;13(1):42.  
64.  Böhme J, Martinez N, Li S, Lee A, Marzuki M, Tizazu AM, et al. Metformin enhances anti-
mycobacterial responses by educating CD8+ T-cell immunometabolic circuits. Nat Commun. 2020 
Oct 16;11(1):5225.  
65.  Lin QXX, Rajagopalan D, Gamage AM, Tan LM, Venkatesh PN, Chan WOY, et al. Longitudinal 
single cell atlas identifies complex temporal relationship between type I interferon response and 
COVID-19 severity. Nat Commun. 2024 Jan 18;15(1):567.  
66.  Ong EZ, Koh CW, Tng DJ, Ooi JS, Yee JX, Chew VS, et al. RNase2 is a possible trigger of acute-on-
chronic inflammation leading to mRNA vaccine-associated cardiac complication. Med (New York, 
N.y). 2023 Apr 26;4(6):353.  
67.  Zheng GXY, Terry JM, Belgrader P, Ryvkin P, Bent ZW, Wilson R, et al. Massively parallel digital 
transcriptional profiling of single cells. Nat Commun. 2017 Jan 16;8(1):14049.  
68.  Hao Y, Hao S, Andersen-Nissen E, Mauck WM, Zheng S, Butler A, et al. Integrated analysis of 
multimodal single-cell data. Cell. 2021 Jun 24;184(13):3573-3587.e29.  
69.  Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, et al. clusterProfiler 4.0: A universal enrichment tool for 
interpreting omics data. Innovation (Camb). 2021 Aug 28;2(3):100141.  
70.  Raivo Kolde. pheatmap: Pretty Heatmaps [Internet]. 2010 [cited 2025 Jan 16]. p. 1.0.12. Available 
from: https://CRAN.R-project.org/package=pheatmap 
71.  Jin S, Guerrero-Juarez CF, Zhang L, Chang I, Ramos R, Kuan CH, et al. Inference and analysis of cell-
cell communication using CellChat. Nat Commun. 2021 Feb 17;12(1):1088.  
72.  Satpathy AT, Granja JM, Yost KE, Qi Y, Meschi F, McDermott GP, et al. Massively parallel single-
cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion. Nat 
Biotechnol. 2019 Aug;37(8):925–36.  
73.  EnsDb.Hsapiens.v86 [Internet]. Bioconductor. [cited 2025 Jan 16]. Available from: 
http://bioconductor.org/packages/EnsDb.Hsapiens.v86/ 
74.  Stuart T, Srivastava A, Madad S, Lareau CA, Satija R. Single-cell chromatin state analysis with 
Signac. Nat Methods. 2021 Nov;18(11):1333–41.  
75.  Schep AN, Wu B, Buenrostro JD, Greenleaf WJ. chromVAR: inferring transcription-factor-associated 
accessibility from single-cell epigenomic data. Nat Methods. 2017 Oct;14(10):975–8.  

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 35 of 38 
Restricted, Sensitive (Normal) 
76.  Chan CY, Chan KR, Chua CJ, Nur Hazirah S, Ghosh S, Ooi EE, et al. Early molecular correlates of 
adverse events following yellow fever vaccination. JCI Insight. 2017 Oct 5;2(19):e96031, 96031.  
77.  Mok DZ, Tng DJ, Yee JX, Chew VS, Tham CY, Ooi JS, et al. Electron transport chain capacity 
expands yellow fever vaccine immunogenicity. EMBO Mol Med. 2024 Jun;16(6):1310–23.  
78.  Chan KR, Gan ES, Chan CYY, Liang C, Low JZH, Zhang SLX, et al. Metabolic perturbations and 
cellular stress underpin susceptibility to symptomatic live-attenuated yellow fever infection. Nat Med. 
2019 Aug;25(8):1218–24.  
79.  The Subread package [Internet]. [cited 2025 Jan 19]. Available from: https://subread.sourceforge.net/ 
80.  Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data 
with DESeq2. Genome Biology. 2014 Dec 5;15(12):550.  
81.  Bollampalli VP, Yamashiro LH, Feng X, Bierschenk D, Gao Y, Blom H, et al. BCG Skin Infection 
Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to 
Draining Lymph Node During CD4+ T-Cell Priming. PLOS Pathogens. 2015 Oct 6;11(10):e1005206.  
82.  Kalimuddin S, Tham CYL, Qui M, de Alwis R, Sim JXY, Lim JME, et al. Early T cell and binding 
antibody responses are associated with COVID-19 RNA vaccine efficacy onset. Med. 2021 Jun 
11;2(6):682-688.e4.  
83.  Tan AT, Linster M, Tan CW, Le Bert N, Chia WN, Kunasegaran K, et al. Early induction of functional 
SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 
patients. Cell Rep. 2021 Feb 9;34(6):108728.  
84.  Yau C, Gan ES, Kwek SS, Tan HC, Ong EZ, Hamis NZ, et al. Live vaccine infection burden elicits 
adaptive humoral and cellular immunity required to prevent Zika virus infection. EBioMedicine. 2020 
Nov 1;61:103028.  
85.  Mok DZL, Chan KR. The Effects of Pre-Existing Antibodies on Live-Attenuated Viral Vaccines. 
Viruses. 2020 May 8;12(5):520.  
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 36 of 38 
Restricted, Sensitive (Normal) 
List of Attachments 
Appendix 1 – Study Schedule 
 
YF17D 1-month group  
 
Screening D-
14 to D-1 
D0h 
D1 
D7 
D14 
D30i 
(3 
day) 
D32 
(3 
day) 
D33 
(3 
day) 
D34 
(3 
day) 
D35 
(3 
day) 
D36 
(3 
day) 
D37 
(3 
day) 
D44 
(5 day) 
D60 
(5 day) 
Informed consent  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility check  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history & 
demographic a 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical 
examination b 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signs c 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
FBCd, Dengue 
serology, HBA1c 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Pregnancy 
Teste  
X 
Xf 
 
 
 
Xg 
 
 
 
 
 
 
 
 
Research bloods 
 
Xf 
X 
X 
X 
Xg 
X 
X 
X 
X 
X 
X 
X 
X 
Randomisation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
AS01 Adjuvant 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
YF17D (Stamaril) 
vaccine 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
Adverse event 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
medication check 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
a Demographic includes DOB, gender, ethnicity, height, weight, and BMI 
b Full physical examination will be performed at Screening. On Day 0, 1, 7, 14, 30, 32, 33, 34, 35, 36, 37, 44 and 60 visits, 
brief PE will be performed by investigator as needed.  
c Vital signs include temperature (oral or tympanic), blood pressure, pulse rate, and respiratory rate. 
d Full blood count (FBC) includes haemoglobin, RBC, WBC, haematocrit, platelet count, neutrophil, lymphocyte, 
monocyte, eosinophil, basophil.  
e For female subjects of child-bearing potential only.   
f Samples to be collected before AS01 / placebo (NaCl 0.9%) administration on Day 0.  
g Samples to be collected before YF17D administration on day 30.  
h AS01 or placebo (NaCl 0.9%) intramuscular injection to be given.  
i YF17D subcutaneous injection to be given.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 37 of 38 
Restricted, Sensitive (Normal) 
YF17D 3-month group  
 
Screening 
D-14 to 
D-1 
D0h 
D1 
D7 
D14 
D21 
D30 
(3 
day) 
D60 
(3 
day) 
D90i 
(3 
day) 
D92 
(3 
day) 
D93 
(3 
day) 
D94 
(3 
day) 
D95 
(3 
day) 
D96 
(3 
day) 
D97 
(3 
day) 
D104 
(5 
day) 
D120 
(5 
Informed 
consent  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility check  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
& demographic a 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical 
examination b 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signsc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
FBCd, Dengue 
serology, HBA1c 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Pregnancy 
Teste  
X 
Xf 
 
 
 
 
 
 
Xg 
 
 
 
 
 
 
 
 
Research blood 
 
Xf 
X 
X 
X 
X 
X 
X 
Xg 
X 
X 
X 
X 
X 
X 
X 
X 
Randomisation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AS01 Adjuvant 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
YF17D 
(Stamaril) 
vaccine 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
Adverse event 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
medication 
check 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
a Demographic includes DOB, gender, ethnicity, height, weight, and BMI 
b Full physical examination will be performed at Screening. On Day 0, 1, 7, 14, 21, 30, 60, 90, 92, 93, 94, 95, 96, 97, 104 , 
and 120 visits, brief PE will be performed by investigator as needed.  
c Vital signs include temperature (oral or tympanic), blood pressure, pulse rate, and respiratory rate. 
d Full blood count (FBC) includes haemoglobin, RBC, WBC, haematocrit, platelet count, neutrophil, lymphocyte, 
monocyte, eosinophil, basophil.  
e For female subjects of child-bearing potential only.   
f Samples to be collected before AS01 / placebo (NaCl 0.9%) administration on Day 0.  
g Samples to be collected before YF17D administration on day 90.  
h AS01 or placebo (NaCl 0.9%) intramuscular injection to be given.  
i YF17D subcutaneous injection to be given.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Version <No. 2.0>, Dated <15/09/2025> 
Page 38 of 38 
Restricted, Sensitive (Normal) 
 
Appendix 2 - Blood Sampling Summary 
 
YF17D 1-month group  
 
Collection 
Tube 
Volume 
Screen 
D0# 
D1 
D7 
D14 
D30# 
D32 
D33 
D34 
 
D35 
D36 
D37 
D44 
D60 
 
FBC, HBA1c 
EDTA tube 
(clinical) 
3ml 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dengue 
serology 
(Panbio) 
Plain tube 
(research) 
3.5ml 
(BD 
367983) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
YF PRNT  
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
Gene 
expression  
(Bulk RNAseq) 
Tempus 
tube  
3ml 
 
X 
X 
X 
X 
X 
 
 
X 
 
 
X 
X 
X 
Gene 
expression 
(scRNASeq) 
10ml EDTA 
tube 
20ml 
 
X 
X 
X 
X 
X 
 
 
X 
 
 
x 
X 
X 
Epigenetic 
studies 
(scATACseq) 
 
X 
X 
X 
X 
X 
 
 
X 
 
 
X 
X 
X 
Flow cytometry 
 
X 
X 
X 
X 
X 
 
 
X 
 
 
X 
X 
X 
YF17D T cells 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X 
Cytokines 
(Olink) 
3ml EDTA 
tube 
(research) 
3ml 
 
X 
X 
X 
X 
X 
 
 
X 
 
 
X 
X 
X 
YF17D 
RNAemia 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
 
Total volume each visit  
6.5ml 
26ml 
26ml 
26ml 
26ml 
29.5ml 
3ml 
3ml 
26ml 
3ml 
3ml 
26ml 
26ml 
29.5ml 
Total volume 259.5 ml  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
#Day 0, Day 30 bloods are collected pre-vaccination 
YF17D 3-month group  
 
Collection 
Tube 
Volume 
Screen 
D0# 
D1 
D7 
D14 
D21 
D30 
D60 
D90# 
D92 
D93 
D94 
 
D95 
D96 
D97 
D104 
D120 
 
FBC, HBA1c 
EDTA tube 
(clinical) 
3ml 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dengue 
serology  
(Panbio) 
Plain tube 
(research) 
3.5ml 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
YF PRNT  
 
 
 
 
 
 
 
 
X  
 
 
 
 
 
 
 
X 
Gene 
expression  
(Bulk RNAseq) 
Tempus 
tube  
3ml 
  
X 
X 
X 
X 
X 
X 
X 
X 
 
 
X 
 
 
X 
X 
X 
Gene 
expression 
(scRNASeq) 
10ml EDTA 
tube 
 
20ml 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
X 
 
 
X 
X 
X 
Epigenetic 
studies 
(scATACseq) 
 
X 
X 
X 
X 
X 
X 
X 
X  
 
 
X 
 
 
X 
X 
X 
Flow cytometry 
  
X 
X 
X 
X 
X 
X 
X 
X  
 
 
X 
 
 
X 
X 
X 
YF17D T cells 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X  
Cytokines 
(Olink) 
3ml EDTA 
tube 
(research) 
3ml 
  
X 
X 
X  
X  
X  
X 
X 
X  
 
 
X 
 
 
X 
X 
X 
YF17D 
RNAemia 
 
 
 
 
 
 
 
 
X 
X  
X  
X  
X  
X  
X  
 
 
Total volume each visit  
6.5ml 
26ml 
26ml 
26ml 
26ml 
26ml 
26ml 
26ml 
29.5ml 
3ml 
3ml 
26ml 
3ml 
3ml 
26ml 
26ml 
29.5ml 
Total volume 337.5 ml  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
#Day 0, Day 90 bloods are collected pre-vaccination