Characterizing the Humoral Immune Response Against Salivary Antigens of Southeast Asian Mosquito Vectors of Malaria and Dengue With a Human Challenge Model
Phase: NA
Conditions: Malaria, Dengue
Sponsor: University of Oxford

 
 
 
 
 
 
 
Study Title: Characterizing the humoral immune response against 
salivary antigens of Southeast Asian mosquito vectors of malaria and 
dengue with a human challenge model 
 
Short title: Characterizing humoral immune response to mosquito bites 
Date and Version No: version 2.0 dated 14th June 2020 
Trial registration number: NCT04478370 
 

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Study Title: Characterizing the humoral immune response against salivary antigens of 
Southeast Asian mosquito vectors of malaria and dengue with a human challenge model 
Internal Reference Number: MAL19009 
Short title: Characterizing humoral immune response to mosquito bites  
Acronym: SG6 
OxTREC Ref: 62-19 
Date and Version No: version 2.0 dated 14th June 2020 
 
Chief Investigator: 
François Nosten 
Shoklo Malaria Research Unit (SMRU) 
68/30 Bantung Road  PO.BOX 46 Mae Sot, 63110 Tak, Thailand 
Email: Francois@tropmedres.ac 
Investigators: 
1. Victor Chaumeau 
Shoklo Malaria Research Unit (SMRU) 
68/30 Bantung Road  PO.BOX 46  Mae Sot, 63110 Tak, Thailand 
Email: victor@shoklo-unit.com 
2. Freya Fowkes 
Burnet Institute (BI) 
The Macfarlane Burnet Institute for Medical Research and Public Health 
Ltd, 85 Commercial Road, Melbourne, VIC 3004, Australia 
Email: freya.fowkes@burnet.edu.au 
3. Ellen Kearney 
Burnet Institute (BI) 
The Macfarlane Burnet Institute for Medical Research and Public Health 
Ltd, 85 Commercial Road, Melbourne, VIC 3004, Australia 
Email: ellen.kearney@burnet.edu.au 
4. Julie Simpson 
University of Melbourne 
Doherty Institute for Infection and Immunity, 207 Bouverie St, Carlton, 
VIC 3010, Australia 
Email: julieas@unimelb.edu.au 
5. David Price 
University of Melbourne 
Doherty Institute for Infection and Immunity, 207 Bouverie St, Carlton, 
VIC 3010, Australia 
Email: david.price1@unimelb.edu.au 
6. Sadudee Chotirat 

Protocol v.2.0, dated 14th June 2020 
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Malaria Vivax Research Unit, Faculty of Tropical medicine, Mahidol 
University 
Email: sadudee.chotirat@gmail.com 
7. Laurent Rénia 
The Singapore Immunology Network, Agency for Science, Technology, 
and Research 
8A Biomedical Grove, #03-15, Immunos Building, Biopolis, Singapore 
138648. 
Email: renia_laurent@immunol.a-star.edu.sg 
8. Elke Bergmann-Leitner 
The Walter Reed Army Institute of Research 
Email: elke.s.bergmann-leitner.civ@mail.mil 
Sponsor:  
Oxford University 
Funder: 
The Australian National Health and Medical Research Council and the 
Burnet Institute, the Singapore Immunology Network (Agency for 
Science, Technology, and Research, Singapore) 
 
 
 
Chief Investigator Signature:   
 
 
The investigators declare no conflict of interest. 
 
 
Confidentiality Statement 
This document contains confidential information that must not be disclosed to anyone other than the 
authorised individuals from the University of Oxford, the Investigator Team and members of the Oxford 
Tropical Research Ethics Committee (OxTREC), Ethics committee at the Faculty of Tropical medicine, 
Mahidol University, Alfred Hospital Ethics Committee and Tak Province Community Ethics Advisory Board 
(T-CAB), unless authorised to do so. 
 
 

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TABLE OF CONTENTS 
1. 
SYNOPSIS ............................................................................................................................................... 6 
2. 
ABBREVIATIONS ..................................................................................................................................... 7 
3. 
BACKGROUND AND RATIONALE ............................................................................................................ 8 
3.1. Epidemiology of malaria and dengue on the Thailand-Myanmar border ......................................... 8 
3.1.1. 
Malaria ................................................................................................................................... 8 
3.1.2. 
Dengue and other arboviruses transmitted by Aedes mosquitoes ....................................... 8 
3.2. The natural course of reactions to mosquito bites ........................................................................... 9 
3.2.1. 
Typical features ..................................................................................................................... 9 
3.2.2. 
Severe reactions .................................................................................................................... 9 
3.3. Biomarkers of human exposure to mosquito bites ......................................................................... 10 
3.3.1. 
Characteristics of the biomarkers ....................................................................................... 10 
3.3.2. 
Malaria vectors (Anopheles mosquitoes) ............................................................................ 11 
3.3.3. 
Dengue vectors (Aedes mosquitoes) ................................................................................... 11 
3.4. Study objectives .............................................................................................................................. 12 
3.5. Interest of the research ................................................................................................................... 12 
4. 
OBJECTIVES AND OUTCOME MEASURES ............................................................................................. 13 
5. 
STUDY DESIGN ..................................................................................................................................... 13 
6. 
PARTICIPANT IDENTIFICATION ............................................................................................................ 14 
6.1. Inclusion criteria .............................................................................................................................. 15 
6.2. Exclusion criteria ............................................................................................................................. 15 
7. 
STUDY PROCEDURES ........................................................................................................................... 15 
7.1. Recruitment and informed consent of study participants .............................................................. 15 
7.2. Screening visit .................................................................................................................................. 16 
7.3. Baseline visits .................................................................................................................................. 16 
7.4. Randomization ................................................................................................................................. 16 
7.5. Subsequent visits ............................................................................................................................. 17 
7.6. Blood collection ............................................................................................................................... 17 
7.7. Specimen/sample handling ............................................................................................................. 17 
7.8. Storage of Specimens/samples ....................................................................................................... 19 
7.9. Discontinuation /Withdrawal of Participants from Study ................................................................ 19 
8. 
MOSQUITO INTERVENTION ................................................................................................................. 19 
9. 
SAFETY REPORTING ............................................................................................................................. 20 

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9.1. Definition of Serious Adverse Events .............................................................................................. 20 
9.2. Reporting Procedures for Serious Adverse Events .......................................................................... 20 
9.3. Adverse event (AE) .......................................................................................................................... 21 
9.4. Safety review board ......................................................................................................................... 21 
10. STATISTICS AND ANALYSIS ................................................................................................................... 21 
10.1. 
The Number of Participants .................................................................................................... 21 
10.2. 
Analysis of Outcome Measures ............................................................................................... 21 
11. DATA MANAGEMENT .......................................................................................................................... 22 
11.1. 
Access to Data ......................................................................................................................... 22 
11.2. 
Data Handling and Record Keeping ......................................................................................... 22 
12. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES ............................................................ 22 
13. ETHICAL AND REGULATORY CONSIDERATIONS ................................................................................... 22 
13.1. 
Declaration of Helsinki............................................................................................................. 22 
13.2. 
Guidelines for Good Clinical Practice ...................................................................................... 22 
13.3. 
Approvals ................................................................................................................................. 23 
13.4. 
Participant Confidentiality ....................................................................................................... 23 
13.5. 
Risks ......................................................................................................................................... 23 
13.5.1. 
Risk of mosquito exposure .................................................................................................. 23 
13.5.2. 
Risk of blood collection ....................................................................................................... 24 
13.6. 
Benefits .................................................................................................................................... 25 
13.7. 
Compensation.......................................................................................................................... 25 
13.8. 
Reporting ................................................................................................................................. 25 
14. FINANCE AND INSURANCE .................................................................................................................. 25 
14.1. 
Funding .................................................................................................................................... 25 
14.2. 
Insurance ................................................................................................................................. 25 
15. PUBLICATION POLICY ........................................................................................................................... 25 
16. REFERENCES ........................................................................................................................................ 26 
17. APPENDIX A :Schematic diagram of study design, procedures and stages, step-by-step................... 33 
18. APPENDIX B :Study design and exposure randomization.................................................................... 34 
19. APPENDIX C: Schedule of participant’s visits including informed consent blood sampling and exposure 
to mosquito bites ........................................................................................................................................ 35 
20. APPENDIX D: Summary of blood volumes drawn from participants ................................................... 36 

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21. APPENDIX E: Classification, definition and grading of pre-specified adverse events (adapted from the 
Common Terminology for Adverse Events (CTAE) guidelines version 5.0 .................................................. 37 
22. APPENDIX F :Amendment history ....................................................................................................... 46 
 
 
 

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1. SYNOPSIS 
Study Title 
Characterizing the humoral immune response against salivary antigens of 
Southeast Asian mosquito vectors of malaria and dengue with a human 
challenge model 
Internal ref. no.  
MAL19009 
Study Design 
This study is a human challenge model with five arms corresponding to 
controlled exposure to bites of uninfected laboratory-adapted colonies of 
Anopheles minimus, An. maculatus, An. dirus, Aedes. aegypti or Ae. 
albopictus. 
Study Participants 
Healthy volunteers > 18 and ≤60 years old 
Planned Sample Size 
150 participants followed-up for 16 weeks (total of 1 venous blood sample of 
2 mL, 14 venous blood samples of 8.6 mL each, 3 venous blood samples of 
18.6 mL each, and 17 capillary blood samples of 300 uL each, yielding 178.2 
mL of venous blood and 5.1 mL of capillary blood per study participant) 
Planned Study Period 
February 2020 to December 2023 (Study duration is approximately 3 years 
after ethical approval to December 2023) 
 
Objectives 
Outcome measures 
Primary 
To identify biomarkers of exposure to 
bites of Southeast Asian mosquito 
vectors of malaria and dengue.  
1) Levels and kinetics of specific 
antibody titers against candidate 
peptides 
before 
and 
during 
repeated exposure to bites of 
laboratory-reared 
Anopheles 
minimus, An. maculatus, An. dirus, 
Aedes aegypti and Ae. albopictus 
determined 
by 
ELISA 
and 
mesoscale screening. 
2) Sequence of saliva antigens 
determined with an immuno-
proteomic 
method 
(2D 
gel 
electrophoresis of salivary gland 
protein extracts, Western blot and 
mass spectrometry). 
Secondary 
1) To characterize the relationship 
between levels of mosquito exposure 
and humoral response.to bites of 
mosquito vectors of malaria and 
dengue. 
Comparison of the antibody titers 
determined in subgroups 
corresponding to different level of 
exposure. 
 
 
2) To assess the feasibility of using 
dry blood spots made from capillary 
blood for the monitoring of exposure 
Comparison of the antibody titers 
determined in DBS and venous 
blood samples. 
EC: ethic committee; DBS: dry blood spots; ELISA: enzyme-linked immunosorbent assay. 
 

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2. ABBREVIATIONS 
AE 
Adverse event 
CRF 
case report form 
CTCAE 
Common Terminology Criteria for Adverse Events 
DBS 
dry blood spots 
EC 
Ethics Committee 
ELISA 
enzyme-linked immunosorbent assay 
MORU 
Mahidol-Oxford Tropical Medicine Research Unit 
OD 
Optical density 
OxTREC 
Oxford Tropical Research Ethics Committee 
PBS 
Phosphate buffered saline 
SAE 
serious adverse event 
SMRU 
Shoklo Malaria Research Unit 
T-CAB 
Tak Province Community Ethics Advisory Board 
TMB 
Tetramethylbenzidine 

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3. BACKGROUND AND RATIONALE 
According to World Health Organization, vector-borne diseases account for 17% of all infectious diseases, 
causing 700,000 deaths annually. Nearly half of the world population is at risk of contracting dengue or 
malaria. Dengue is responsible for 96 million estimated cases and 10,000 deaths every years, and the 
corresponding figure for malaria is 218 million cases and 400,000 deaths (1). The Thailand-Myanmar 
border stretches almost 2000 km from Laos to the North down to Malaysia in the South. An estimated 5 
million people live in this area, including Thai and Burmese citizens, ethnic minorities, refugees and migrant 
workers. 
3.1. Epidemiology of malaria and dengue on the Thailand-Myanmar border 
Vector-borne diseases are a major cause of morbidity and mortality for people living on the Thailand-
Myanmar border. In 2010, it was estimated that febrile episodes occurred in 5% of the pregnant women 
and that arthropod-borne (malaria, Rickettsia infections, and dengue) and zoonotic disease (leptospirosis) 
accounted for nearly half of all febrile illnesses in this population group. Malaria and dengue were 
identified as the major causes of morbidity and mortality (2). 
3.1.1. Malaria 
In this region, malaria transmission is low, seasonal and unstable (3). Plasmodium falciparum was 
eliminated from most endemic villages with widespread deployment of community-wide access to early 
diagnosis and treatment with artemisinin-based combination therapies, and mass-drug administration 
campaigns in places where submicroscopic malaria prevalence was high (4). Although the endemicity of 
vivax malaria has also declined in recent years in this region (5), it has remained much more difficult to 
tackle than falciparum malaria because of some features in the biology of Plasmodium vivax (6-8). In this 
area, the primary vectors are Anopheles minimus (Minimus Complex, Funestus Group), An. maculatus, An. 
sawadwongporni (Maculatus Group), An. dirus and An. baimaii (Dirus Complex, Leucosphyrus Group). 
Anopheles pseudowillmori (Maculatus Group), An. aconitus (Aconitus Subgroup, Funestus Group) and 
some members in the Annularis and Barbirostris Groups also play a secondary role in the transmission (9, 
10). Biting rate can be very high, thereby playing a disproportionate role in driving transmission intensity 
in this setting where Plasmodium-infection rates in mosquito populations are low (11, 12). Bed-nets and 
indoor residual spraying fail to prevent most of malaria infections (13-15) because of the ecology and 
biology of relevant anopheline species, including exophily and exophagy, zoophagy and opportunistic 
blood type selection, and activity peaks at dusk and dawn (11, 16, 17). Larval source management is 
difficult to implement because of the diverse and fragmented nature of larval habitats (18), and because 
incredibly high densities of vector larvae can be found over large areas covered with paddy fields (19). 
Several vector species pullulate in a variety of biotopes and at different time in the year, adding another 
layer of complexity to the dynamics of entomological indices (9). 
3.1.2. Dengue and other arboviruses transmitted by Aedes mosquitoes 
Dengue is a viral infection caused by four types of viruses (DENV-1, DENV-2, DENV-3, DENV-4) belonging 
to the Flaviviridae family. The spectrum of diseases severity is broad, the infection usually manifest as a 
mild fever but severe and deadly cases can occur (20, 21). The viruses are transmitted through the bite of 
infected Aedes aegypti and Ae. albopictus female mosquitoes that feed both indoors and outdoors during 
the daytime (22, 23), but the diversity and competence of other aedine mosquito species on the Thailand-
Myanmar border is not known precisely. These mosquitoes thrive in areas with standing water, including 

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puddles, water tanks, containers and old tires. Lack of reliable sanitation and regular garbage collection 
also contribute to the spread of the mosquitoes. Cases of Dengue have been increasingly reported from 
Thailand over the past two decades (2, 24-26). The most affected provinces include Chiang Rai, Chiang 
Mai, Mae Hong Son, Nakhon Pathom, Phra Nakhon Si Ayutthaya, Phetchabun, Lamphun and Phatthalung. 
Dengue is present in both urban and rural areas, with elevated risk in northeastern parts of the country. 
Peak transmission typically occurs during the rainy season, from April to December. Other arboviruses 
transmitted by Aedes mosquitoes such as Zika and Chikungunya are emerging issues in this area (22, 27-
30). Although case are rarely fatal, morbidity is high and an important proportion of the population can be 
affected at the same time during outbreaks. 
3.2. The natural course of reactions to mosquito bites 
The saliva of blood feeding arthropods is a complex mixture of biomolecules, including dozens of proteins, 
many of which have unknown function. Some of these proteins have been shown to facilitate ingestion of 
blood and pathogen transmission though tight interaction with the immune system of the host being 
bitten. The clinical and biological features of the immune reaction to mosquito saliva has been studied in 
animal and humans, both in controlled laboratory challenges and natural settings. 
3.2.1. Typical features 
Allergic reactions to mosquito bites are due to specific sensitization to the mosquito salivary proteins. 
Mosquito saliva-specific IgE and IgG antibodies as well as lymphocyte proliferation appear to be involved 
in the pathogenesis of allergic reactions to mosquito bites (31). Typical reactions to mosquito bites include 
immediate wheals 2 to 10 mm in diameter, with surrounding erythema peaking in 20 to 30 minutes 
(immediate reaction) and similarly sized pruritic papules peaking at 24 to 36 hours and diminishing in 
several days (delayed reaction). Based on clinical observations, the natural history of sensitization and 
subsequent desensitization to the saliva injected by mosquito bites has been classified into five stages (32, 
33). Individuals who have never been exposed to a specific species of mosquito, do not get a reaction from 
the initial bites (stage 1), but following subsequent bites, delayed cutaneous lesions appear (stage 2). After 
repeated bites, immediate wheals develop (stage 3). With further exposure, delayed reactions are no 
longer observed, and only immediate wheals are noted (stage 4). Individuals repeatedly exposed to 
thousands of bites from the same species of mosquito eventually lose the immediate reactions (stage 5). 
Most of the population at any given time will have some reactivity to mosquito bites: immediate reactions 
occur in 70% to 90% and delayed reactions in 55% to 65% of patients subjected to bites of common Ae sp. 
and Cx sp. mosquito species (34-36). Peng et al. further documented the correlation of skin reactions with 
specific IgE and IgG antibodies and lymphocyte proliferation response to mosquito antigens and reported 
that it took 21 weeks of repeated exposure with 100 Ae. aegypti bites per week for desensitization to 
happen (34, 37). In natural setting, desensitization occurs during infancy (38) and individuals remain at an 
intermediary stage (most often stage 3 and 4) for years before eventually progressing to the next stage 
(39). 
3.2.2. Severe reactions 
In atopic individuals, large local reactions can develop including erythematous pruritic swellings, often 
more than 3 cm, occurring in minutes to hours at the site of a bite. Delayed large local reactions may be 
papular, ecchymotic, vesicular, blistering, or bullous and persist for days or weeks (36, 40). The incidence 
of self-reported large local reactions in one study was 2.5% (8/12 cases were children less than 10 years 
old) (40). Skeeter syndrome describes patients with mosquito bite-induced large local reactions 

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accompanied by fever. This syndrome typically occurs in otherwise healthy children, and the large local 
reactions may mimic cellulitis (hot, swollen, red, and painful) but may be differentiated by their occurrence 
within hours of mosquito bites. These symptoms resolve in 3 to 10 days (41). Anaphylactic reactions to 
mosquito bites are extremely rare but have occurred in patients with underlying indolent systemic 
mastocytose (42, 43). Persons at increased risk for severe reactions include those with high exposure 
(outdoor workers) and those lacking acquired immunity (young children and immigrants) (31). In addition, 
patients with primary or acquired immunodeficiencies and those with Epstein-Barr virus (EBV) associated 
lymphoproliferative diseases are also at higher risk for severe reactions (44-46). 
3.3. Biomarkers of human exposure to mosquito bites 
Measuring exposure to mosquito bites is informative to assess the risk of disease transmission and the 
efficacy of vector-control interventions. However, direct measurement of exposure is difficult because 
diverse mosquito species pullulate in different environments at different times in the year (9). In order to 
be meaningful, estimates must also take into account the behavioural interactions between mosquito and 
human populations (47). Finally, it is difficult to scale-up entomological investigations because of the 
logistic, financial and technical constrains it implies. There is an urgent need for more efficient and 
sensitive complementary tools to improve the impact of vector control strategies and predict the risk of 
transmission of mosquito-borne diseases. The use of immunological markers of human exposure to bites 
of mosquito vectors has been proposed in this context (48, 49).  
3.3.1. Characteristics of the biomarkers 
Individuals repeatedly bitten have detectable levels of circulating antibodies directed against salivary 
antigens (50, 51). Those antibodies have been proposed as biomarkers of human exposure to mosquito 
bites (48, 52). The antibody response measured in naturally exposed individuals is highly heterogeneous 
(49, 53). This can come from varying level of exposure as some antibody titers have been shown to 
correlate with the entomological indices of exposure in specific settings (54-57), and other factors might 
also have an effect (e.g. genetic background, concomitant infection, nutritional status, history of previous 
exposure). Another important aspect of biomarker validation is the assessment of the cross-reactivity with 
the saliva proteins of other blood feeding arthropods. The immune responses elicited by mosquito bites 
have a spectrum of specificity, with some antibodies sharing reactivity with saliva from other mosquito 
species (or even phylogenetically more distant blood feeding arthropods) while some others give a species-
specific signal (53). Interestingly, transcriptomic and proteomic studies have identified groups of genus 
specific salivary proteins that are ideal candidate markers of host exposure to mosquito vectors (58-61). 
Some of those may be conserved enough to elicit a specific response specific to the mosquito genus, and 
others may exhibit enough variations in the amino-acid sequence to identify highly specific markers of a 
single mosquito species (58). Noteworthy, both genus-specific and species-specific biomarkers would be 
valuable in the context of entomological surveillance and assessment of the efficacy of vector control 
interventions. 
In order to be valid, the biomarker should fulfil four fundamental criteria: (i) to discriminate individuals 
exposed to mosquito bites from those who are not exposed, (ii) to evaluate the density and fluctuations 
of vector populations that can bite human populations, (iii) to classify individuals according to their level 
of exposure, and (iv) to be usable at a population level (48). Studies that aim at discovering biomarkers of 
exposure must be design carefully in order not to be confounded by uncontrolled factors. In this regard, 
human challenge studies with laboratory mosquitoes can achieve a very fine level of control in the 

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exposure (species, dose and duration) and therefore are expected to yield invaluable information for 
biomarker validation. In order to discriminate individuals exposed to mosquito bites from those who are 
not exposed, appropriate negative controls must be used to define the positivity threshold of the assay. 
Typically, the positivity threshold of a given assay is defined as three times the standard deviation of the 
signal measured in a population of individuals who have not been exposed to the mosquito species of 
interest (49, 62, 63). Moreover, it is possible to ensure the specificity of the signal during prospective 
laboratory challenge studies by performing multiple baseline assessment of serum reactivity, by including 
a follow-up after the exposure to observe the decay of specific antibodies after the exposure ceases, and 
avoid or limit the exposure that may happen independently of the study (64). Competition ELISA can also 
be informative to assess the cross reactivity of the antibodies between several antigens (65). In order to 
evaluate the density and fluctuations of vector populations, and to classify individuals according to their 
level of exposure, validation of the biomarker must assess the kinetic and the dose-response relationship 
between the number of bites received by a given individual and the titer of specific antibodies produced 
in response to exposure (49). Field studies are usually limited to answer these questions because the 
exposure cannot be well controlled. The kinetic and dose response relationship can be better assessed by 
conducting laboratory studies with controlled exposure to insectary mosquitoes, including several doses 
and a post-exposure follow-up in the study design. Finally, in order to support the use of a given biomarker 
at the population level, assessment must be performed on a sufficient number of individual in order to 
assess inter-individual variations in the development of the immune responses against saliva antigens. 
3.3.2. Malaria vectors (Anopheles mosquitoes) 
Regarding exposure to Anopheles mosquito bites, two An. gambiae salivary proteins have been tested so 
far, namely the gSG6 protein and the antithrombin cE5. The gSG6 was the first immunogenic Anopheles 
saliva protein identified and the gSG6-P1 appeared as the best candidate peptide to assess anti-SG6 
humoral response in Africa (55). Anti-gSG6-P1 antibodies were shown to be labile (66), sensitive enough 
to detect low-level exposure (63), and to cross-react with An. gambiae, An. arabiensis and An. funestus 
(54, 67). Since, gSG6-P1 has been used in a wide variety of African transmission settings to assess malaria 
transmission dynamics (68-72) and to monitor the efficacy of vector-control interventions (66, 73-75). The 
studies conducted with gSG6-P1 outside Africa (76-79) must be interpreted cautiously because the SG6 
protein is absent from the genome of some dominant vector species or shares only a moderate identity 
with its orthologues in phylogenetically diverse Anopheles species (58). Antithrombin cE5 was reported to 
be more immunogenic, to induce long lasting antibodies and less immune tolerance than the SG6 protein 
(80). 
3.3.3. Dengue vectors (Aedes mosquitoes) 
The development of biomarker of exposure to Aedes mosquito is more recent (48). Antigenic epitopes 
were identified using sera from naturally exposed individuals and 2D gel electrophoresis of salivary gland 
extracts (81, 82). The Nterm-34kDa salivary peptide was validated as a species-specific biomarker of 
human exposure to bites of several Aedes species usable for monitoring transmission risk and vector-
control intervention in a wide variety of settings and geographic area (62, 83-89). More recently, the use 
of protein D7 was also investigated as a marker of risk of dengue infection and as a vaccine to block the 
transmission of dengue virus (90-92). 

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3.4. Study objectives 
The characteristics of the humoral response directed against mosquito saliva antigens are not known 
precisely (31). This is a major limitation for using immunological markers as an outcome in epidemiological 
trials and as an indicator for operational deployment of interventions. Recent advances in the assembly of 
the genome of some Anopheles and Aedes mosquito vector species (93-95) has facilitated the 
identification of new candidate peptides in silico, using the sequences of orthologous salivary gland 
proteins and B-cell prediction algorithms. The objective of this study is to assess the humoral immune 
response directed against candidate peptides following controlled exposure to laboratory-adapted 
colonies of An. minimus, An. maculatus and An. dirus, Ae. aegypti and Ae. albopictus. 
3.5. Interest of the research 
This research will provide essential information to identify and validate immunological markers of human 
exposure to malaria and dengue mosquito vectors in Southeast Asia. Immunological markers would be 
useful to understand transmission dynamics and predict the risk of transmission as part of a surveillance 
system, and to assess the efficacy of vector-control interventions in entomological trials or during 
operational deployment of interventions in the region. 
 
 

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4. OBJECTIVES AND OUTCOME MEASURES 
Objectives 
Outcome Measures 
Timepoint(s) of evaluation of 
this outcome measure a 
Primary Objective 
To identify biomarkers of 
exposure to bites of 
Southeast Asian 
mosquito vectors of 
malaria and dengue. 
 
1) Levels and kinetics of specific antibody 
titers against candidate peptides before 
and during repeated exposure to bites of 
laboratory-reared Anopheles minimus, 
An. maculatus, An. dirus, Aedes aegypti 
and Ae. albopictus determined by ELISA 
and mesoscale screening. 
2) 
Sequence 
of 
saliva 
antigens 
determined with an immuno-proteomic 
method (2D gel electrophoresis of 
salivary gland protein extracts, Western 
blot and mass spectrometry). 
 
1) Day 0, 7 (baseline), 14, 21, 
28, 35, 42, 49, 56 (exposure 
period), 63, 70, 77, 84, 91, 98, 
105 and 112 (post-exposure 
period). 
 
2) Day 14 (baseline), 63 and 
112 (post-exposure period). 
Secondary Objectives 
1) To characterize the 
relationship between 
levels of mosquito 
exposure and humoral 
response. 
 
Comparison of the antibody titers 
determined in subgroups corresponding 
to different level of exposure. 
 
 
Day 0, 7 (baseline), 14, 21, 28, 
35, 42, 49, 56 (exposure 
period), 63, 70, 77, 84, 91, 98, 
105 and 112 (post-exposure 
period). 
2) To assess the 
feasibility of using dry 
blood spots made from 
capillary blood for the 
monitoring of exposure 
to bites of mosquito 
vectors of malaria and 
dengue. 
Comparison of the antibody titers 
determined in DBS and venous blood 
samples. 
Day 0, 7 (baseline), 14, 21, 28, 
35, 42, 49, 56 (exposure 
period), 63, 70, 77, 84, 91, 98, 
105 and 112 (post-exposure 
period). 
a See Appendix C. 
DBS: dry blood spots; ELISA: enzyme-linked immunosorbent assay. 
5. STUDY DESIGN 
A schematic diagram detailing the study design, procedures and stages step-by-step is presented in the 
Appendix A. This study is a human challenge model with five arms corresponding to controlled exposure 
to bites of uninfected laboratory-adapted colonies of Anopheles minimus, An. maculatus, An. dirus, Aedes. 
aegypti or Ae. albopictus (see sections 8 and 13.5.1). Following screening and eligibility assessment, 150 
participants will be enrolled in the study using a block-randomized design stratified on the mosquito 
species and number of bites (35 or 305 bites in total) such as to constitute 10 groups of 15 individuals for 

Protocol v.2.0, dated 14th June 2020 
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each of the study condition (Appendix B). Participants will be in the study for 112 days (Appendix C). The 
baseline will consist of 2 visits over 2 weeks (day 0 and day 7). In the low-exposure groups, participants 
will be exposed to 5 mosquito bites at weekly intervals from day 14 to day 56 (seven challenges with 5 
mosquito bites/challenge over six weeks, yielding a total of 35 mosquito bites). In the high-exposure group, 
participants will be exposed to 5 mosquito bites on day 14 and then to 50 mosquito bites at weekly 
intervals from day 21 to day 56 (one challenge with 5 mosquito bites and six challenges with 50 mosquito 
bites/challenge over 6 weeks, yielding a total of 305 mosquito bites). The number of bites and modalities 
of the follow up used in this study were chosen based on previous entomological investigation conducted 
in this area (10, 11, 96), published reports of human challenge with mosquito bites (37, 97-99) and current 
knowledge on the characteristic of the humoral response against saliva antigens (31, 37, 52). Both 
immediate and delayed skin reactions will be recorded after day 14 and day 21 challenges, requiring 
additional visits at day 15 and day 22. Participants with hypersensitivity to mosquito bites will be 
withdrawn from the study. Both capillary and venous blood samples will be collected weekly from each 
study participant (Appendix D). Eighteen venous blood samples will be collected including one sample of 
2 mL drawn during screening visit, 14 samples of 8.6 mL during visits 2, 3, 6, 8, 9, 10, 11, 12, 14, 15, 16, 17, 
18 and 19, and 3 samples of 18.6 mL during visits 4, 13 and 20 (178.2 mL of venous blood per participant 
in total). Seventeen dry blood spot (DBS) samples will be made from of 300 uL of capillary blood collected 
during visits 2, 3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 (5.1 mL of capillary blood per 
participant in total). Candidate peptides will be identified in silico using publicly available genomes 
sequences and B-cell epitope prediction algorithms. The kinetic of antibody titers against candidate 
biomarkers will be assessed by enzyme-linked immunosorbent assay (ELISA) and mesoscale screening 
performed with serum and DBS specimens (100). Briefly, the positivity threshold of the assay will be 
determined using reference sera specimens from individuals not exposed to mosquito bites, as described 
previously (62). The ELISA signal measured in 60 reference sera obtained from unexposed individuals (30 
Thai sera obtained from the Thai Red Cross and 30 Australian sera obtained from the Burnett Intstitute) 
will be used to define the positivity threshold of the assay. The positivity threshold of the assay will be 
defined as three standard deviation (SD) above the mean optic density (ΔOD) measured for the unexposed 
control population. A test in the study population with a ΔOD above this cut-off will be considered positive 
(i.e. an immune response). The specific antibody titer of study samples will be determined by performing 
serial dilution experiments (101). Serial dilution will be made from study samples and the ΔOD will be 
determined for each dilution. The antibody titer will be defined as the highest dilution giving a positive 
signal. Results obtained with DBS and serum specimens will be compared in order to assess the feasibility 
of using DBS samples to monitor exposure to bites of mosquitoes during epidemiological studies. Serum 
specimens collected during visits 4 (baseline), 13 (early after the end of exposure) and 20 (post-exposure) 
will also be analysed with immuno-proteomic approach (2D gel electrophoresis of salivary gland protein 
extracts, Western blot and mass spectrometry) yielding additional information on the antigenic properties 
of mosquito salivary gland proteome (102). 
6. PARTICIPANT IDENTIFICATION 
Study participants are defined as healthy participants aged between 18 and 60 years old and willing to 
participate in the study. This study will take place at the Shoklo Malaria Research Unit (SMRU) research 
centre in Mae Sot. 

Protocol v.2.0, dated 14th June 2020 
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6.1. Inclusion criteria 
 
Healthy male or female participant judged by a responsible physician with no abnormality 
identified on a medical evaluation; 
 
Thai, Burmese or Karen ethnicity; 
 
Aged ≥ 18 to <60 years old; 
 
Living in Mae Sot city for the last 12 months; 
 
Willing to participate in the activity and able to give informed consent for participating in the 
study; 
 
Able to tolerate direct mosquito exposure. 
6.2. Exclusion criteria 
 
History of travel in a malaria endemic are (rural village) in the last 12 months, or plan to do so 
during the study; 
 
Medication or condition deemed to interfere with the outcome measure by a responsible 
physician; 
 
Moderate and severe anemia (haemoglobin concentration less than 110 g/L of blood); 
 
Hypersensitivity or anaphylaxis to mosquito bites; 
 
Pregnant women; 
 
Breastfeeding women. 
7. STUDY PROCEDURES 
7.1. Recruitment and informed consent of study participants 
The study will be spread through word of mouth in Mae Sot by the study team and then interested 
participants will contact SMRU. The persons interested in participating in the study will be invited to 
individual and group discussions with the study team during which more details of the study will be 
presented and potential participants will be given the opportunity to ask questions. The persons willing to 
take part in the study will be appointed for the screening visit. Participants’ presenting to the screening 
visit must personally and voluntarily sign and date the latest approved version of the informed consent 
form before any study specific procedures are performed. The Participant Information and Informed 
Consent Form will be presented to the participants detailing no less than: the exact nature of the study; 
what it will involve for the participant; the implications and constraints of the protocol and any risks and 
benefits involved in taking part. It will be clearly stated that the participant is free to withdraw from the 
study at any time for any reason without prejudice to future care, and with no obligation to give the reason 
for withdrawal. The participant will be allowed as much time as they wish to consider the information, and 
the opportunity to question the Investigator or other independent parties to decide whether they will 
participate in the study. The informed consent and procedures visit will take approximatively one hour. 
Written Informed Consent will be obtained by asking the participant to sign and date the informed consent 
form. If the participant is illiterate, they will be asked to give a thumb print, leaving the date field blank 
and a literate impartial witness will be asked to sign and date the consent form, confirming that the 
participant understands what they are being asked to do and have given consent. The form will also be 
signed and dated by the person who presented and obtained the Informed Consent. The person who 
obtained the consent must be suitably qualified and experienced, and authorised to do so by the Principal 

Protocol v.2.0, dated 14th June 2020 
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Investigator. A copy of the signed Informed Consent will be given to the participant. The original signed 
form will be retained at the study site. 
7.2. Screening visit  
Subjects will be screened to assess eligibility (visit 1). Informed consent will be obtained before any 
screening procedures are conducted. The following screening procedures will be carried out no longer 
than 30 days before the first baseline visit: 
• 
Personal data collection including name, age, date of birth, sex, ethnicity, address and telephone 
number; 
• 
A comprehensive physical examination including measurement of height, body weight and vital 
signs (blood pressure, respiratory rate, body temperature and heart rate) and medical history 
assessment including travel history outside Maesot town during the past 3 months; 
 
A pregnancy test if indicated (cost of the test will be covered by the study); 
 
Venous blood collection (2 mL) in order to perform complete blood count and measure 
haemoglobin concentration. 
Participants will also be informed about the importance of avoiding being bitten by mosquitoes 
independently of the study challenges during the follow-up. They will be provided with insecticide-
impregnated mosquito bed-nets and skin repellent (N,N-diethyl-meta-toluamide 20%). Participants who 
will be allocated to the Aedes groups will receive counselling about larval source management in order to 
limit exposure to mosquito bites that may happen independently of the study challenges. Sand Abate 
granules (temephos 1%) will be provided free of charge to participants who need it. 
Data collected during the screening and subsequent visits will be recorded in the Case Report Form (CRF) 
and stored in the study database. The CRF will be pseudonymized using the participant identification code, 
date of enrolment, initials, sex and date of birth. Personal data (name, telephone number and address) 
will be recorded and stored separately, allowing linkage of study data with participant’s details by the 
study team. 
7.3. Baseline visits 
One hundred and fifty healthy subjects who fulfil the eligibility criteria will be recruited to the study. 
Baseline visits will be scheduled at day 0 and day 7 (visit 2 and 3). Baseline visits will include capillary blood 
and venous blood collection (Appendix C). Randomization will be performed during visit 2.  
For baseline and subsequent visits, in case participant fails to come on the exact day a visit is scheduled, 
he/she will be allowed to come for a retake any other working day of the study team until the next 
scheduled visit. 
7.4. Randomization 
A block randomization schedule will be generated using the block.random() function of the R package 
psych version 1.8.12 using variables species (An. minimus, An. maculatus, An. dirus, Ae. aegypti and Ae. 
albopictus) and dose (35 or 305 bites in total) yielding a list of 15 blocks with 10 participants each (total of 
150 participants). Following screening and eligibility assessment, participant will be assigned a study arm 
during visit 2 through the randomization schedule. Individual, sealed and sequentially numbered 
envelopes will be provided for the study site with one envelope per participant, indicating the allocation. 

Protocol v.2.0, dated 14th June 2020 
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7.5. Subsequent visits 
From day 14 to day 56, participant will be exposed to bites of laboratory-adapted colonies of Anopheles 
minimus, An. maculatus and An. dirus, Aedes aegypti or Ae. albopictus reared at the SMRU. Appropriate 
quality control measures will be implemented such as to guarantee the absence of human pathogen in the 
mosquito batches used in this study and withdraw any participant with hypersensitivity to mosquito bites 
(see section 13.5.1. Risk of mosquito exposure). Participants will receive a total of either 35 or 305 
mosquito bites per participant in 7 challenges split over 6 weeks (see mosquito intervention section). Each 
visit will include physical examination, vital signs assessment, mosquito exposure, capillary blood and 
venous blood collection according to Appendix C. Immediate skin reactions (20-30 min after mosquito 
bites) will be recorded after each mosquito feeding assay. Delayed skin reactions (24-36 hours after 
mosquito bites) will be recorded after the day 14, 21 challenges, requiring additional visits at day 15, 22. 
After day 56, each visit will include capillary blood and venous blood collection according to Appendix C. 
The study procedure will take approximatively 1 hours for medical history assessment, physical 
examination and vital sign assessment, 15 minutes for blood sampling and 30-90 minutes for the challenge 
with mosquito bites (depending on the number of repeats needed to reach the target number of mosquito 
bites). During the exposure visits, the participants will be kept in observation for an additional hour after 
the challenge before being allowed to leave the study site. Participants will be requested not to apply any 
lotions, ointments, creams, powders, perfumes, antiperspirants, repellents, or attractants prior to each 
exposure visit. Participants will be provided palliative antipruritic medication consisting in 1% 
diphenhydramine hydrochloride cream and oral cetirizine at the dose of 10 mg per day in order to relieve 
itching from mosquito bites (103, 104). In addition, participants will be provided with a mosquito bed net 
and skin repellent in order to minimize uncontrolled natural exposure to mosquito bites that can happen 
independently of the study challenges. 
7.6. Blood collection 
Both capillary blood and venous blood will be collected weekly from each study participant over 112 days 
with an additional venous sample collected during the screening visit, yielding 17 capillary blood samples 
(5.1 mL of capillary blood in total) and 18 venous blood samples (178.2 mL of venous blood in total) per 
study participant (Appendix C). Venous blood samples will be collected by venepuncture. In addition, 
participants will undergo a finger-prick using a single usage sterile lancet, from which capillary blood will 
be collected on filter paper in order to constitute three DBS. All blood samples will be collected on site by 
SMRU study teams (trained and qualifies medics, nurses and medical doctors). The volume of blood 
collected in this study is justified based on the technical requirement to perform the assays in the different 
collaborative centres (see details below) (53, 101). 
7.7. Specimen/sample handling 
Sample pre-treatment, storage and shipment: 
Venous blood samples will be spun down to separate the serum. Three serum aliquots of 2 mL, 2 mL and 
0.1 mL each will be made during visits 2, 3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. Moreover, 
an additional 5 mL serum aliquot be made during visits 4, 13 and 20. Hence, 8.6 mL of venous blood will 
be collected from participants during visits 2, 3, 6, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18 and 19 (4.1 mL of 
serum in total), and 18.6 mL of venous blood will be collected during visits 4, 13, 20 (9.1 mL of serum in 
total). The volume of blood was calculated considering a 50% hematocrit (upper limit of the reference 
range) and an additional 0.2 mL of buffer volume of serum (0.4 mL of blood) to avoid contamination with 

Protocol v.2.0, dated 14th June 2020 
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the buffy coat when preparing the serum aliquots. Spotted capillary blood will be dried at room 
temperature. Dry blood spot specimens will be put into a zip lock bag containing silica gel. DBS and serum 
aliquots will be stored at -20°C until shipment to the collaborative centres. Coded specimen will be shipped 
to the Burnet Institute (Melbourne, Australia) for ELISA and immuno-proteomic analysis, the Singapore 
Immunology Network (Agency for Science, Technology, and Research, Singapore) for ELISA and to the 
Walter Reed Army Institute of Research (Silver Spring, United States of America) for mesoscale screening. 
No personally identifiable information will be transferred. Samples will be kept accessible only by 
authorised researchers. 
A 2-mL serum aliquot and the DBS samples collected during visits 2, 3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 
17, 18, 19 and 20 will be shipped to the Burnet Institute (Melbourne, Australia) for doing ELISA screening. 
A 2-mL serum aliquot collected during visits 2, 3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 will 
be shipped to the Singapore Immunology Network (Agency for Science, Technology, and Research, 
Singapore) for doing ELISA screening. A 0.1-mL serum aliquot collected during visits 2, 3, 4, 6, 8, 9, 10, 11, 
12, 13, 14, 15, 16, 17, 18, 19 and 20 will be shipped to the Walter Reed Army Institute of Research (Silver 
Spring, United States of America) for doing mesoscale screening. The 5-mL serum aliquots collected during 
visits 4, 13 and 20 will be shipped to the Burnet Institute (Melbourne, Australia) for being analysed with 
immuno-proteomic methods. 
Enzyme-linked immunosorbent assay procedures: 
Antibody titer against a broad panel of candidate peptides will be determined on the serum specimens. 
This panel will include, but will not be limited to, five peptides specific to each mosquito species, in addition 
to the Anopheles gambiae salivary peptide SG6-P1 (target of 10 peptides per panel). Serial dilution of the 
serum specimens will be carried out in order to determine antibody titer (the titer is defined as the last 
dilution giving a positive response; a positive response is defined as the mean + 3 standard deviation of 
naive serum used as negative control) (101). Each sample will be tested in duplicate with a reaction volume 
of 100 uL. Therefore, the volume of serum needed for each peptide is 200 uL of serum /peptide (2 mL of 
serum for 10 peptides). Dry blood spots will be eluted separately in 200 uL of PBS such as to have 3 
technical replicates and analysed using the same ELISA protocol. Samples will also undergo further 
serological analyses for antigen and antibody characterisation, including but not limited to competition 
ELISAs (to quantify the antibody/antigen interaction) (100), functional antibody assays (e.g. complement 
fixation assays) (101). 
Mesoscale assay procedures: 
The serum specimens collected will be diluted 250 folds and applied to test plates. The testing will require 
750 uL diluted sample (3 uL of neat serum) per test panel. A minimum volume of 6 uL of neat serum is 
needed in case technical issue happens and rerun is needed. It is impossible to ship such small volumes 
(evaporation/drying up) and therefore a minimum of 100 uL will be sent. 
Immuno-proteomic procedure: 
Immuno-proteomic analysis will be carried out using a procedure adapted from previously published 
studies (53, 102, 107). This analysis will be performed with the 5 mL serum aliquots collected from study 
participants during visit 4 (baseline), visit 13 (early after exposure period) and visit 20 (end of the post-
exposure period). Single serum specimens will be used in order to assess inter-individual variation of the 
antibody response against salivary gland proteome. 

Protocol v.2.0, dated 14th June 2020 
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Two-dimensional gel electrophoresis will be performed with protein extracts made from the dissected 
salivary glands of 200 female adult mosquito specimens. In order to obtain a sufficient resolution, protein 
separation will be done on an 11 cm × 20 cm gel format. The gel proteins will be then transferred to a 
membrane. The membrane will be incubated with 50 mL of serum diluted 10 folds (5mL of neat serum) in 
order to assess the reactivity of serum IgG by Western blot (53). Reactive spots of interest will be detected 
by comparing the staining patterns from baseline, end of exposure and post-exposure samples. Spots of 
interest will be excised from the 2D gel and used to perform in gel digestion with trypsin. The protein 
digest will be analysed with liquid chromatography-mass spectrometry. Immunogenic proteins will be 
identified by searching the mass spectra with Mascot (Matrix Science Ltd.). 
7.8. Storage of Specimens/samples 
Left over specimens will be stored no longer than 10 years using codes assigned by the investigators or 
their designee(s). Access to research samples will be limited using either a locked room or a locked freezer. 
Only investigators or their designee(s) will have access to the samples. 
In the future, investigators from this protocol and other investigators besides those listed in this protocol 
may wish to study these samples. A proposal of the planned research will be submitted to the IRB for their 
consideration. 
7.9. Discontinuation/Withdrawal of Participants from Study 
Each participant has the right to withdraw from the study at any time.  
In addition, the Investigator may discontinue a participant from the study at any time if the Investigator 
considers it necessary for any reason including: 
 
Ineligibility (either arising during the study or retrospectively having been overlooked at screening) 
 
Significant protocol deviation 
 
Significant non-compliance with study requirements 
 
Loss to follow up 
 
Hypersensitivity to mosquito bites or anaphylaxis 
The data collected until the point of withdrawal will be included in the study and this is stated in the 
participant information sheet unless participant refuse to do so. The participant will be replaced with 
another person and the replacement will be assigned to the same study arm. 
7.9. 
Definition of End of Study 
The end of the study is defined as the last day of sample collection from the last participant. 
8. MOSQUITO INTERVENTION 
From day 14 to day 56, participant will be exposed to bites of laboratory-adapted colonies of An. minimus, 
An. maculatus and An. dirus, Ae. aegypti or Ae. albopictus reared at the SMRU. Appropriate quality control 
measures will be implemented such as to guarantee the absence of human pathogen in the mosquito 
batches used in this study and withdraw any participant with hypersensitivity to mosquito bites (see 
section 13.5.1. Risk of mosquito exposure). In the low-exposure groups, participants will be exposed to 5 
mosquito bites at weekly intervals from day 14 to day 56 (seven challenges with 5 mosquito 
bites/challenge over six weeks, yielding a total of 35 mosquito bites). In the high-exposure group, 

Protocol v.2.0, dated 14th June 2020 
Page 20 of 46 
participants will be exposed to 5 mosquito bites on day 14 and then to 50 mosquito bites at weekly 
intervals from day 21 to day 56 (one challenge with 5 mosquito bites and six challenges with 50 mosquito 
bites/challenge over 6 weeks, yielding a total of 305 mosquito bites). 
For the low-dose challenge, five 5 to 7-day-old nulliparous female imagoes (i.e. that have never blood fed 
before) will be introduced individual into 50 mL plastic tube covered with netting material. For high-dose 
challenge, 47 mosquitoes will be split into four plastic cups of 10 cm in diameter covered with netting 
material (three cups with 12 mosquitoes and one cup with 11 mosquitoes), and 3 mosquitoes will be 
introduced individually into 50 mL plastic tubes. At first, three mosquito bites from individual 50 mL tubes 
will be administered on participant’s arm. Then, the remaining mosquito containers will be placed on 
participant’s left arm, calf, thigh or back skin, and the lights will be dimmed to encourage mosquito blood 
feeding. Bite site will be chosen such as potential skin reactions do not overlap. Mosquito bites 
administered on participant’s arm will be used to assess immediate and delayed skin reactions as described 
previously (37). Immediate and delayed skin reactions will be recorded respectively 20-30 min and 24-36 
hours after the day 14 and day 21 challenges, requiring additional visits at day 15 and 22. 
Mosquitoes will be given the opportunity to feed on participant’s skin for 30 min. The number of bites 
actually received by participants will be assessed by counting the number of engorged mosquitoes at the 
end of the 30-minute exposure time. If not all mosquitoes would have successfully engorged, participant 
will be exposed to additional mosquitoes using the same procedure in order to reach the target number 
of bites (participant may be exposed to mosquito bites up to three times at each visit). Partially engorged 
mosquitoes will not be discarded and we be counted as fully engorged specimens. In case the challenge 
will be carried out in the morning, mosquitoes will be starved overnight. In case the challenge will be 
carried out in the afternoon, mosquitoes will be staved since 9 am. Mosquitoes used to challenge each 
study participant will be immediately killed at the end of the challenge using 70% ethanol. The mosquito 
exposure will be performed by study staff under supervision of a medical doctor. The challenge with 
mosquito bites will take 30 minutes and may be repeated twice in case not all mosquito engorge during 
the first challenge. The participants will be kept in observation for an additional hour before being allowed 
to leave the study site. During that time, participants will be provided with a snack and refreshment. 
 
9. SAFETY REPORTING 
9.1. Definition of Serious Adverse Events 
A serious adverse event (SAE) is any untoward medical occurrence that: 
 
results in death; 
 
is life-threatening; 
 
requires inpatient hospitalisation or prolongation of existing hospitalisation; 
 
results in persistent or significant disability/incapacity. 
The term "life-threatening" in the definition of "serious" refers to an event in which the participant was at 
risk of death at the time of the event; it does not refer to an event which hypothetically might have caused 
death if it were more severe. 
9.2. Reporting Procedures for Serious Adverse Events 
A SAE occurring to a participant should be reported to ethic committee at the Faculty of Tropical Medicine, 
Mahidol University and to the Safety Review Board within 5 working days of investigator’s awareness.  

Protocol v.2.0, dated 14th June 2020 
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9.3. Adverse event (AE) 
At each visit, a CRF and an AE form will be completed. AEs will be documented according to standard 
definitions and procedures following the Common Terminology Criteria for Adverse Events version 5.0 
guidelines (108). Pre-specified AEs include blood and lymphatic system disorders (leucocytosis and 
eosinophilia), general disorders and administration site conditions (chills, fatigue, fever, challenge site 
reaction, malaise, pain, challenge site lymphadenopathy), immune system disorders (allergic reaction, 
anaphylaxis), infections and infestations (papulopustular rash, rash pustular, sepsis, skin infection), injury, 
poisoning and procedural complications (bruising, venous injury), skin and subcutaneous tissue disorders 
(bullous dermatitis, eczema, erythema multiforme, pain of skin, pruritus, rash acneiform, rash maculo-
papular, skin induration, skin ulceration, urticaria, skin atrophy, skin hyperpigmentation, skin 
hypopigmentation). Definition and detailed grading procedure of pre-specified adverse events are given 
in the Appendix E. Any unexpected AEs will be recorded according to the same guidelines. 
9.4. Safety review board 
All data from the study participant will be submitted to a safety review board after days 15 and 22 in order 
to review the adverse events (AEs) and decide whether the participant should be withdrawn from the 
study. Reactions that will exclude the participant include immediate or delayed local reactions greater 
than 30 mm diameter, any ecchymotic, vesicular, blistering or bullous manifestation, Skeeter syndrome or 
any systemic symptom (e.g. generalized urticaria, angioedema or anaphylaxis). 
10. STATISTICS AND ANALYSIS 
10.1. 
The Number of Participants 
There is no preliminary data to calculate a priori a required sample size. The groups are 15 participants (15 
in the low-exposure and 15 in the high-exposure groups), and a comparison between these groups in 
expected. Because there is such variation in individual’s antibody responses, and the specificity and 
immunogenicity of the antigens of interest is not known at this stage, it is important to ensure sure that 
any measured responses are not due to chance. Therefore, 15 participants per study condition was 
deemed appropriate for this study. 
10.2. Analysis of Outcome Measures 
Antibody titers will be analysed as (i) a continuous outcome measure and (ii) used to define a binary 
responder status. Briefly, the positivity threshold of the assay will be determined using reference sera 
specimens from individuals not exposed to mosquito bites, as described previously (62). The ELISA signal 
measured in 60 reference sera obtained from unexposed individuals (30 Thai sera obtained from the Thai 
Red Cross and 30 Australian sera obtained from the Burnett Intstitute) will be used to define the positivity 
threshold of the assay. The positivity threshold of the assay will be defined as three standard deviation 
(SD) above the mean optic density (ΔOD) measured for the unexposed control population. A test in the 
study population with a ΔOD above this cut-off will be considered positive (i.e. an immune response). The 
specific antibody titer of study samples will be determined by performing serial dilution experiments. Serial 
dilution will be made from study samples and the ΔOD will be determined for each dilution. The antibody 
titer will be defined as the highest dilution giving a positive signal. The kinetics and longevity of antibody 
titers will be assessed using generalised linear mixed-effects linear modelling (family = binomial, link = logit 
for binary variables) showing changes in antibody titre over time, adjusting for variables of interest 

Protocol v.2.0, dated 14th June 2020 
Page 22 of 46 
(including age (continuous), sex (binary) and biting exposure group (binary)) and with the inclusion of 
random intercepts for individual volunteers. Interaction terms between variables and time will be 
investigated to determine whether kinetics and longevity vary according to variables of interest. Estimates 
from these equations will be used to calculate the half-life of Ig-responses to gSG6-P1 overall and according 
to variables of interest. Results obtained with DBS and serum specimens will be compared in order to 
assess the feasibility of using DBS samples to monitor exposure to bites of mosquitoes during 
epidemiological studies. 
11. DATA MANAGEMENT 
11.1. Access to Data 
Direct access will be granted to authorised representatives from sponsor, the Burnet Institute, the 
Singapore Immunology Network, the Walter Reed Army Institute of Research, the Shoklo Malaria Research 
Unit/Mahidol Oxford Research Unit, and any host institution, ethics committee and regulatory authorities 
for monitoring, audits and/or inspections of the study to ensure compliance with regulations. 
11.2. 
Data Handling and Record Keeping 
All data collected specifically for the study will be recorded on paper forms, and entered to a study 
database. The database will be built in MACRO and managed by MORU data management team. 
Laboratory samples will be labelled using unique participant identifiers and date, which will be used to 
merge laboratory results with other study data. Participant identifiable information such as participant’s 
names and telephone numbers will be stored separately from other study data and will only be accessible 
by authorised members of the research team. 
Non-identifiable data will be shared with researchers via secure data transfer platforms for analysis. 
Analysis will be conducted on anonymized datasets. In accordance with regulations, study staff will retain 
all study records on site for at least 5 years after study closure. Data will be stored indefinitely in secure 
MORU servers and on secure Burnet Institute servers for a minimum of 7 years. 
Volunteer’s data and results from blood analyses stored in our database may be shared with other 
researchers to use in the future. However, the other researchers will not be given any information that 
could identify the subject. 
12. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
The study will be conducted in accordance with relevant regulations and standard operating procedures. 
13. ETHICAL AND REGULATORY CONSIDERATIONS 
13.1. Declaration of Helsinki 
The Investigator will ensure that this study is conducted in accordance with the principles of the 
declaration of Helsinki version 2013. 
13.2. 
Guidelines for Good Clinical Practice 
The Investigator will ensure that this study is conducted in accordance with relevant regulations and with 
Good Clinical Practice. 

Protocol v.2.0, dated 14th June 2020 
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13.3. 
Approvals 
The protocol, informed consent form, and participant information sheet and consent form will be 
submitted to the Oxford Tropical Research Ethics Committee (OXTREC), the Ethics Committee of the 
Faculty of Tropical Medicine, Mahidol University and the Alfred Hospital Ethics Committee for written 
approval. Furthermore, the protocol, informed consent form, and participant information sheet will be 
reviewed by the Tak Community Advisory Board, a community-based committee assembling members of 
the communities in which the study will be performed. 
The Investigator will submit and, where necessary, obtain approval from the above parties for all 
amendments to the original approved documents. 
13.4. 
Participant Confidentiality 
The study staff will ensure that the participants’ anonymity is maintained. The participants will be 
identified only by a participant ID number on all study documents and any electronic database. All 
documents will be stored securely and only accessible by study staff and authorised personnel. The study 
will comply with the Data Protection Act, which requires data to be anonymised as soon as it is practical 
to do so. 
13.5. 
Risks 
13.5.1. Risk of mosquito exposure 
The main risk associated with mosquito feeding experiments are allergic reactions, skin infections at the 
site of the bites and accidental transmission of vector-borne disease causative agents. 
Allergic reactions to mosquito bites: 
Most of the population at any given time will have some reactivity to mosquito bites. Typical reactions to 
mosquito bites include immediate wheals 2 to 10 mm in diameter, with surrounding erythema peaking in 
20 to 30 minutes (immediate reaction) and similarly sized pruritic papules peaking at 24 to 36 hours and 
diminishing in several days (delayed reaction). Immediate reactions occur in 70% to 90% and delayed 
reactions in 55% to 65% of patients subjected to bites of common Ae sp. and Cx sp. mosquito species (34-
36). 
In atopic individuals, large local reactions can develop including erythematous pruritic swellings, often 
more than 3 cm, occurring in minutes to hours at the site of a bite. Delayed large local reactions may be 
papular, ecchymotic, vesicular, blistering, or bullous and persist for days or weeks (36, 40). The incidence 
of self-reported large local reactions in one study was 2.5% (8/12 cases were children less than 10 years 
old) (40). Skeeter syndrome describes patients with mosquito bite-induced large local reactions 
accompanied by fever. This syndrome typically occurs in otherwise healthy children, and the large local 
reactions may mimic cellulitis (hot, swollen, red, and painful) but may be differentiated by their occurrence 
within hours of mosquito bites. These symptoms resolve in 3 to 10 days (41). Anaphylactic reactions to 
mosquito bites are extremely rare but have occurred in patients with underlying indolent systemic 
mastocytose (42, 43). Persons at increased risk for severe reactions include those with high exposure 
(outdoor workers) and those lacking acquired immunity (young children and immigrants) (31). In addition, 
patients with primary or acquired immunodeficiencies and those with Epstein-Barr virus (EBV) associated 
lymphoproliferative diseases are also at higher risk for severe reactions (44-46). 

Protocol v.2.0, dated 14th June 2020 
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A few previous studies reported the safety of human challenges performed with large numbers of 
(infected) malaria mosquito bites and no serious adverse event was observed (97-99). Immediate skin 
reactions (20-30 min after mosquito bites) will be recorded after each mosquito feeding assay. Delayed 
skin reactions (24-36 hours after mosquito bites) will be recorded after the day 14, 21, requiring additional 
visits at day 15, 22. All data from study participant will be submitted to a safety review board after day 15, 
22 for reviewing potential adverse events (AEs) and eventually request withdrawal of the participants from 
the study. Reactions that will exclude the participant include immediate or delayed local reactions greater 
than 30 mm diameter, any ecchymotic, vesicular, blistering or bullous manifestation, Skeeter syndrome or 
any systemic symptom (e.g. generalized urticaria, angioedema or anaphylaxis). In addition, all mosquito 
feeding experiments will be conducted under the supervision of a medical doctor and resuscitation 
material will be available at the site of the study. In case SAE happen and cannot be managed on site, the 
participants will be referred to Mae Sot general hospital (located at 5 min from the study site). 
Skin infection at the challenge site: 
Secondary development of a skin infection may develop at the site of the challenge, especially if severe 
allergic skin reactions develop (36). Participants with severe reaction to mosquito bites will not be enrolled 
or will be withdrawn from the study, therefore limiting the risk of skin infection. In addition, the skin will 
be disinfected with 70% alcohol before the challenge, and appropriate diagnosis and treatment of eventual 
infection will be ensured during participant follow-up. 
Accidental transmission of vector-borne disease causative agents: 
The mosquito strains used in this study were colonized decades ago, and reared under laboratory 
conditions since. All feeds will be performed with nulliparous females (i.e., that never blood fed) so it is 
not possible for these mosquitoes to be infected with pathogen transmitted only “horizontally” (i.e. from 
one host to another) such as Plasmodium malaria parasites and lymphatic filaria. For pathogens that can 
be “vertically” transmitted in the mosquito (i.e. transmitted from the female mosquito to its eggs), such 
as dengue, Zika and Chikungunya viruses, there is a very small risk that nulliparous females inherit the 
pathogen from the previous generation, if the colony is accidentally fed on blood containing viable viral 
particles during routine maintenance procedures. This is very unlikely because colonies are maintained on 
human blood procured from authorized blood banks that screen donors for relevant infections including 
dengue, Zika and Chikungunya viruses. In addition, Aedes mosquito colonies will be screened for dengue, 
Zika and Chikungunya before the study. All mosquitoes used for challenging participants will be 
immediately killed and disposed at the end of the experiment. 
All participants will be followed up and observed from the visit 1 until the visit 20 which corresponds to a 
total period of 112 to 142 days (according to the timing of the screening visit), including 56 days after the 
last challenge, in order to make sure that they do not get malaria or dengue fever from being bitten by 
mosquitoes during study procedure. 
13.5.2. Risk of blood collection 
Blood sampling is associated with a low risk of bruising, bleeding, swelling, fainting. Infection is very rare 
but may occur. Blood sampling and mosquito feeds will be made by trained and qualified SMRU staff using 
single-usage sterile materials. 

Protocol v.2.0, dated 14th June 2020 
Page 25 of 46 
13.6. 
Benefits 
There is no individual benefit in participating to this study for participants. This study will benefit to the 
population exposed to mosquito-borne diseases transmitted by An. minimus, An. maculatus, An. dirus, Ae. 
aegypti and/or Ae. albopictus as a whole, by providing tools for improving entomological surveillance in 
the context of malaria elimination in the Greater Mekong Subregion. 
13.7. 
Compensation 
Study participants will be compensated for loss of work and transportation costs. We have determined 
that 350 THB (USD 11) per visit is locally appropriate. As the study consists of 20 visits the maximum 
compensation per participant is 7000 THB (USD 220).  
13.8. 
Reporting 
The chief investigator shall submit an Annual Progress Report to OxTREC and local EC on the anniversary 
of the date of approval of the study. In addition, the CI shall submit an End of Study Report to OxTREC and 
local EC. 
14. FINANCE AND INSURANCE 
14.1. Funding 
This project is funded by The Australian National Health and Medical Research Council, the Burnet Institute 
(Melbourne, Australia) and the Singapore Immunology Network (Agency for Science, Technology, and 
Research, Singapore) via Oxford University and the Mahidol Oxford Tropical Medicine Research Unit 
(MORU). 
14.2. Insurance 
The project is covered under the Oxford University sponsorship. 
The University has a specialist insurance policy in place which would operate in the event of any participant 
suffering harm as a result of their involvement in the research (Newline Underwriting Management Ltd, at 
Lloyd’s of London). 
15. PUBLICATION POLICY 
All publications will abide by the International Committee of Medical Journal Editors (ICMJE) 
recommendations of the role of authors and contributors. 
 

Protocol v.2.0, dated 14th June 2020 
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94. 
Chen XG, Jiang X, Gu J, Xu M, Wu Y, Deng Y, et al. Genome sequence of the Asian Tiger mosquito, 
Aedes albopictus, reveals insights into its biology, genetics, and evolution. Proc Natl Acad Sci U S A. 
2015;112(44):E5907-15. 
95. 
Matthews BJ, Dudchenko O, Kingan SB, Koren S, Antoshechkin I, Crawford JE, et al. Improved 
reference genome of Aedes aegypti informs arbovirus vector control. Nature. 2018;563(7732):501-7. 
96. 
Somboon P, Aramrattana A, Lines J, Webber R. Entomological and epidemiological investigations 
of malaria transmission in relation to population movements in forest areas of north-west Thailand. 
Southeast Asian J Trop Med Public Health. 1998;29(1):3-9. 
97. 
Egan JE, Hoffman SL, Haynes JD, Sadoff JC, Schneider I, Grau GE, et al. Humoral immune responses 
in volunteers immunized with irradiated Plasmodium falciparum sporozoites. Am J Trop Med Hyg. 
1993;49(2):166-73. 
98. 
Hoffman SL, Goh LM, Luke TC, Schneider I, Le TP, Doolan DL, et al. Protection of humans against 
malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J Infect Dis. 
2002;185(8):1155-64. 
99. 
Clyde DF, Most H, McCarthy VC, Vanderberg JP. Immunization of man against sporozite-induced 
falciparum malaria. Am J Med Sci. 1973;266(3):169-77. 
100. 
Charnaud SC, McGready R, Herten-Crabb A, Powell R, Guy A, Langer C, et al. Maternal-foetal 
transfer of Plasmodium falciparum and Plasmodium vivax antibodies in a low transmission setting. Sci Rep. 
2016;6:20859. 
101. 
Miura K, Orcutt AC, Muratova OV, Miller LH, Saul A, Long CA. Development and characterization 
of a standardized ELISA including a reference serum on each plate to detect antibodies induced by 
experimental malaria vaccines. Vaccine. 2008;26(2):193-200. 

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102. 
Cantillo JF, Puerta L, Puchalska P, Lafosse-Marin S, Subiza JL, Fernandez-Caldas E. Allergenome 
characterization of the mosquito Aedes aegypti. Allergy. 2017;72(10):1499-509. 
103. 
Karppinen A, Kautiainen H, Petman L, Burri P, Reunala T. Comparison of cetirizine, ebastine and 
loratadine in the treatment of immediate mosquito-bite allergy. Allergy. 2002;57(6):534-7. 
104. 
Reunala T, Brummer-Korvenkontio H, Lappalainen P, Rasanen L, Palosuo T. Immunology and 
treatment of mosquito bites. Clin Exp Allergy. 1990;20 Suppl 4:19-24. 
105. 
Crowther JR. Basic Principles of ELISA. In: Crowther JR, editor. ELISA: Theory and Practice. Totowa, 
NJ: Humana Press; 1995. p. 35-61. 
106. 
Wouters D, Wiessenberg HD, Hart M, Bruins P, Voskuyl A, Daha MR, et al. Complexes between C1q 
and C3 or C4: novel and specific markers for classical complement pathway activation. Journal of 
Immunological Methods. 2005;298(1-2):35-45. 
107. 
Jariyapan N, Roytrakul S, Paemanee A, Junkum A, Saeung A, Thongsahuan S, et al. Proteomic 
analysis of salivary glands of female Anopheles barbirostris species A2 (Diptera: Culicidae) by two-
dimensional gel electrophoresis and mass spectrometry. Parasitol Res. 2012;111(3):1239-49. 
108. 
National Cancer Institute. Common terminology criteria for adverse events guidelines version 5.0 
2017 [Available from: https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
 

Protocol v.2.0, dated 14th June 2020 
 
Page 33 of 46 
17. APPENDIX A: Schematic diagram of study design, procedures and stages, step-by-step 
 
 

Protocol v.2.0, dated 14th June 2020 
 
Page 34 of 46 
18. APPENDIX B: Study design and exposure randomization 
 
Low-dose: total of 35 bites split into 7 direct feeding assays over 6 weeks (5 bites/visit); 
High-dose: total of 305 bites split into 7 direct feeding assays over 6 weeks (5 bites for the screening of hypersensitivity to 
mosquito bites during visit 4, then 50 bites/visit).

Protocol v.2.0, dated 14th June 2020 
Page 35 of 46 
19.  APPENDIX C: Schedule of participant’s visits including informed consent blood sampling and 
exposure to mosquito bites 
a In case participant fail to come on the exact day a visit is scheduled, he/she will be allowed to come for a retake any other 
working day of the study team until the next scheduled visit; 
b 5 bites for the screening of hypersensitivity to mosquito bites during visit 4, then 5 or 50 bites/visit according to the 
randomisation schedule (total of 35 and 305 bites/participant in low- and high-exposure groups respectively, split into 7 direct 
feeding assays); 
c before exposure to mosquito bites. 
d 2 mL during the screening visit, 8.6 mL during visits 2, 3, 6, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18 and 19 and 18.6 mL during visits 4, 
13 and 20.
Visit 
Study 
period 
Day a 
Informed 
consent 
obtained 
Medical 
History 
PE 
and 
VS  
Randomization 
Mosquito 
exposure b 
DBS 
Collection 
c 
VP 
collection 
c,d 
1 
screening 
30 days or 
less before 
enrolment 
Yes 
Yes 
Yes 
No 
No 
No 
Yes 
2 
baseline 
0 
No 
No 
No 
Yes 
No 
Yes 
Yes 
3 
7 
No 
No 
No 
No 
No 
Yes 
Yes 
4 
exposure 
14 
No 
No 
Yes 
No 
Yes 
Yes c 
Yes c 
5 
15 
No 
No 
Yes 
No 
No 
No 
No 
6 
21 
No 
No 
Yes 
No 
Yes 
Yes 
Yes 
7 
22 
No 
No 
Yes 
No 
No 
No 
No 
8 
28 
No 
No 
Yes 
No 
Yes 
Yes 
Yes 
9 
35 
No 
No 
Yes 
No 
Yes 
Yes 
Yes 
10 
42 
No 
No 
Yes 
No 
Yes 
Yes 
Yes 
11 
49 
No 
No 
Yes 
No 
Yes 
Yes 
Yes 
12 
56 
No 
No 
Yes 
No 
Yes 
Yes 
Yes 
13 
post-
exposure 
63 
No 
No 
No 
No 
No 
Yes 
Yes 
14 
70 
No 
No 
No 
No 
No 
Yes 
Yes 
15 
77 
No 
No 
No 
No 
No 
Yes 
Yes 
16 
84 
No 
No 
No 
No 
No 
Yes 
Yes 
17 
91 
No 
No 
No 
No 
No 
Yes 
Yes 
18 
98 
No 
No 
No 
No 
No 
Yes 
Yes 
19 
105 
No 
No 
No 
No 
No 
Yes 
Yes 
20 
112 
No 
No 
No 
No 
No 
Yes 
Yes 

Protocol v.2.0, dated 14th June 2020 
Page 36 of 46 
20. APPENDIX D: Summary of blood volumes drawn from participants 
Period 
Visit 
no. 
Day 
Venous 
blood 
(ml) 
Total 
volume 
of serum 
(ml) 
Volume of 
serum 
sent to BI 
(ml) a 
Volume of 
serum sent 
to A*STAR 
(ml) b 
Volume of 
serum sent 
to WRAIR 
(ml) c 
Capillary 
blood 
(ml) 
Screening 
1 
30 days or 
less before 
enrolment 
2 
NA 
NA 
NA 
NA 
0 
Baseline 
2 
0 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
3 
7 
8.6 
4.1 
2 
2 
0.1 
0.3 
Exposure 
4 
14 
18.6 
9.1 
7 
2 
0.1 
0.3 
 
5 
15 
0 
NA 
NA 
NA 
NA 
0 
 
6 
21 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
7 
22 
0 
NA 
NA 
NA 
NA 
0 
 
8 
28 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
9 
35 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
10 
42 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
11 
49 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
12 
56 
8.6 
4.1 
2 
2 
0.1 
0.3 
Post-
exposure 
13 
63 
18.6 
9.1 
7 
2 
0.1 
0.3 
 
14 
70 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
15 
77 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
16 
84 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
17 
91 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
18 
98 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
19 
105 
8.6 
4.1 
2 
2 
0.1 
0.3 
 
20 
112 
18.6 
9.1 
7 
2 
0.1 
0.3 
 
 
 
 
 
 
 
 
 
Total 
 
 
178.2 
 
 
 
 
5.1 
 
 
Grand Total for Blood collection = 183.3 mL 
a Samples sent to the Burnet Institute (BI) will be used for ELISA screening and immuno-proteomic analysis; 
b Samples sent to the Singapore Immunology Network, Agency for Science, Technology, and Research, 
Singapore (A*STAR) will be used for ELISA screening; 
c Samples sent to the Walter Reed Army Institute of Research (WRAIR) will be used for mesoscale 
screening. 
Abbreviations: NA, not applicable.

Protocol v.2.0, dated 14th June 2020 
Page 37 of 46 
21. APPENDIX E: Classification, definition and grading of pre-specified adverse events (adapted from the Common Terminology for Adverse 
Events (CTAE) guidelines version 5.0 
 
SOC 
CTCAE Term 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
Blood and lymphatic 
system disorders 
Leucocytosis 
- 
- 
>100,000/mm3 
Clinical 
manifestations 
of 
leucostasis; 
urgent 
intervention 
indicated 
Death 
Definition: A disorder characterized by laboratory test results that indicate an increased number of white blood cells in the blood. 
Eosinophilia 
>ULN 
and 
>Baseline 
- 
Steroids initiated 
- 
- 
Definition: A disorder characterized by a sensation of marked discomfort in a lymph node.      
General 
disorders 
and administration 
site conditions 
Chills 
Mild sensation of 
cold; 
shivering; 
chattering of teeth  
Moderate tremor 
of the entire body; 
narcotics indicated 
Severe or prolonged, 
not responsive to 
narcotics 
- 
- 
Definition: A disorder characterized by a sensation of cold that often marks a physiologic response to sweating after a fever.   
Fatigue 
Fatigue relieved by 
rest 
Fatigue 
not 
relieved by rest; 
limiting 
instrumental ADL 
Fatigue not relieved 
by rest, limiting self-
care ADL 
- 
- 
Definition: A disorder characterized by a state of generalized weakness with a pronounced inability to summon sufficient energy to 
accomplish daily activities.    
Fever 
38.0 -39.0 degrees 
C (100.4 -102.2 
degrees F) 
>39.0 -40.0 degrees 
C 
(102.3 
-104.0 
degrees F) 
>40.0 
degrees 
C 
(>104.0 degrees F) 
for <=24 hrs 
>40.0 
degrees 
C 
(>104.0 degrees F) 
for >24 hrs 
Death 

Protocol v.2.0, dated 14th June 2020 
Page 38 of 46 
SOC 
CTCAE Term 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
 
 
 
 
Definition: A disorder characterized by elevation of the body's temperature above the upper limit of normal. 
Challenge 
site 
reaction 
Tenderness 
with 
or 
without 
associated 
symptoms 
(e.g., 
warmth, 
erythema, itching) 
Pain; 
lipodystrophy; 
edema; phlebitis 
Ulceration 
or 
necrosis; 
severe 
tissue 
damage; 
operative 
intervention 
indicated 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Definition: A disorder characterized by an intense adverse reaction (usually immunologic) developing at the site of an injection. 
Malaise 
Uneasiness or lack 
of well being 
Uneasiness or lack 
of 
wellbeing 
limiting 
instrumental ADL 
Uneasiness or lack of 
wellbeing 
limiting 
self-care ADL 
- 
- 
Definition: A disorder characterized by a feeling of general discomfort or uneasiness, an out-of-sorts feeling. 
Pain 
Mild pain 
Moderate 
pain; 
limiting 
instrumental ADL 
Severe pain; limiting 
self-care ADL 
- 
- 
Definition: A disorder characterized by the sensation of marked discomfort, distress or agony.       
Challenge 
site 
lymphadenopathy 
Local lymph node 
enlargement 
Localized 
ulceration; 
generalized lymph 
node enlargement 
- 
- 
- 
Definition: A disorder characterized by lymph node enlargement after vaccination.       
Immune 
system 
disorder 
Allergic reaction 
Systemic 
intervention 
not 
indicated 
Oral 
intervention 
indicated 
Bronchospasm; 
hospitalization 
indicated for clinical 
sequelae; 
intravenous 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 

Protocol v.2.0, dated 14th June 2020 
Page 39 of 46 
SOC 
CTCAE Term 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
intervention 
indicated 
Definition: A disorder characterized by an adverse local or general response from exposure to an allergen. 
Anaphylaxis 
- 
- 
Symptomatic 
bronchospasm, with 
or without urticaria; 
parenteral 
intervention 
indicated; 
allergy-
related 
edema/angioedema; 
hypotension 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Definition: A disorder characterized by an acute inflammatory reaction resulting from the release of histamine and histamine-like 
substances from mast cells, causing a hypersensitivity immune response. Clinically, it presents with breathing difficulty, dizziness, 
hypotension, cyanosis and loss of consciousness and may lead to death. 
Infections 
and 
infestations 
Papulopustular rash Papules 
and/or 
pustules covering 
<10% BSA, which 
may or may not be 
associated 
with 
symptoms 
of 
pruritus 
or 
tenderness 
Papules 
and/or 
pustules 
covering 
10-30% BSA, which 
may or may not be 
associated 
with 
symptoms 
of 
pruritus 
or 
tenderness; 
associated 
with 
psychosocial 
impact; 
limiting 
instrumental ADL; 
papules 
and/or 
pustules covering > 
30% BSA with or 
Papules 
and/or 
pustules 
covering 
>30% 
BSA 
with 
moderate or severe 
symptoms; 
limiting 
self-care 
ADL; 
IV 
antibiotics indicated 
Life-threatening 
consequences 
Death 

Protocol v.2.0, dated 14th June 2020 
Page 40 of 46 
SOC 
CTCAE Term 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
without 
mild 
symptoms 
Definition: A disorder characterized by an eruption consisting of papules (a small, raised pimple) and pustules (a small pus filled 
blister), typically appearing in face, scalp, and upper chest and back. Unlike acne, this rash does not present with whiteheads or 
blackheads, and can be symptomatic, with itchy or tender lesions. 
Rash pustular 
- 
Localized; 
local 
intervention 
indicated 
(e.g., 
topical 
antibiotic, 
antifungal, 
or 
antiviral) 
IV 
antibiotic, 
antifungal, 
or 
antiviral intervention 
indicated; 
invasive 
intervention 
indicated 
- 
- 
Definition: A disorder characterized by a circumscribed and elevated skin lesion filled with pus.   
Sepsis 
 
 
Blood 
culture 
positive with signs or 
symptoms; 
treatment indicated 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Definition: A disorder characterized by the presence of pathogenic microorganisms in the blood stream that cause a rapidly 
progressing systemic reaction that may lead to shock. 
Skin infection 
Localized, 
local 
intervention 
indicated 
Oral 
intervention 
indicated 
(e.g., 
antibiotic, 
antifungal, 
or 
antiviral) 
IV 
antibiotic, 
antifungal, 
or 
antiviral intervention 
indicated; 
invasive 
intervention 
indicated 
Life-threatening 
consequences; 
urgent intervention 
indicated 
Death 
Definition: A disorder characterized by an infectious process involving the skin such as cellulitis. 
Injury, 
poisoning 
and 
procedural 
complications 
Bruising 
Localized or in a 
dependent area 
Generalized 
- 
- 
- 
Definition: A finding of injury of the soft tissues or bone characterized by leakage of blood into surrounding tissues. 

Protocol v.2.0, dated 14th June 2020 
Page 41 of 46 
SOC 
CTCAE Term 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
Venous injury 
Asymptomatic 
diagnostic finding; 
intervention 
not 
indicated 
Symptomatic (e.g., 
claudication); 
repair or revision 
not indicated 
Severe 
symptoms; 
limiting 
self-care 
ADL; 
repair 
or 
revision indicated 
Life-threatening 
consequences; 
evidence 
of 
end 
organ 
damage; 
urgent 
operative 
intervention 
indicated 
Death 
Definition: A finding of damage to a vein.   
Skin 
and 
subcutaneous tissue 
disorders 
Bullous dermatitis 
Asymptomatic; 
blisters 
covering 
<10% BSA 
Blisters covering 10 
-30% BSA; painful 
blisters; 
limiting 
instrumental ADL 
Blisters 
covering 
>30% BSA; limiting 
self-care ADL 
Blisters 
covering 
>30% 
BSA; 
associated with fluid 
or 
electrolyte 
abnormalities; 
ICU 
care or burn unit 
indicated 
Death 
Definition: A disorder characterized by inflammation of the skin characterized by the presence of bullae which are filled with fluid.    
Eczema 
Asymptomatic or 
mild 
symptoms; 
additional medical 
intervention over 
baseline 
not 
indicated 
Moderate; topical 
or oral intervention 
indicated; 
additional medical 
intervention 
over 
baseline indicated 
Severe or medically 
significant but not 
immediately 
life-
threatening; 
IV 
intervention 
indicated 
- 
- 
Definition: A disorder characterized by skin which becomes itchy, red, inflamed, crusty, thick, scaly, and/or forms blisters.    
Erythema 
multiforme 
Target 
lesions 
covering 
<10% 
BSA 
and 
not 
associated 
with 
skin tenderness 
Target 
lesions 
covering 10 -30% 
BSA and associated 
with 
skin 
tenderness 
Target 
lesions 
covering >30% BSA 
and associated with 
oral 
or 
genital 
erosions 
Target 
lesions 
covering >30% BSA; 
associated with fluid 
or 
electrolyte 
abnormalities; 
ICU 
care or burn unit 
indicated 
Death 

Protocol v.2.0, dated 14th June 2020 
Page 42 of 46 
SOC 
CTCAE Term 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
Definition: A disorder characterized by target lesions (a pink-red ring around a pale center). 
Pain of skin 
Mild pain 
Moderate 
pain; 
limiting 
instrumental ADL 
Severe pain; limiting 
self-care ADL 
- 
- 
Definition: A disorder characterized by a sensation of marked discomfort in the skin.    
Pruritus 
Mild or localized; 
topical 
intervention 
indicated 
Widespread 
and 
intermittent; 
skin 
changes 
from 
scratching 
(e.g., 
edema, papulation, 
excoriations, 
lichenification, 
oozing/crusts); oral 
intervention 
indicated; limiting 
instrumental ADL 
Widespread 
and 
constant; 
limiting 
self-care 
ADL 
or 
sleep; 
systemic 
corticosteroid 
or 
immunosuppressive 
therapy indicated 
- 
- 
Definition: A disorder characterized by an intense itching sensation. 
Rash acneiform 
Papules 
and/or 
pustules covering 
<10% BSA, which 
may or may not be 
associated 
with 
symptoms 
of 
pruritus 
or 
tenderness 
Papules 
and/or 
pustules 
covering 
10 - 30% BSA, 
which may or may 
not be associated 
with symptoms of 
pruritus 
or 
tenderness; 
associated 
with 
psychosocial 
impact; 
limiting 
instrumental ADL; 
papules 
and/or 
Papules 
and/or 
pustules 
covering 
>30% 
BSA 
with 
moderate or severe 
symptoms; 
limiting 
self-care 
ADL; 
associated with local 
superinfection with 
oral 
antibiotics 
indicated 
Life-threatening 
consequences; 
papules 
and/or 
pustules 
covering 
any % BSA, which 
may or may not be 
associated 
with 
symptoms 
of 
pruritus 
or 
tenderness and are 
associated 
with 
extensive 
superinfection with 
Death 

Protocol v.2.0, dated 14th June 2020 
Page 43 of 46 
SOC 
CTCAE Term 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
pustules covering > 
30% BSA with or 
without 
mild 
symptoms 
IV 
antibiotics 
indicated 
Definition: A disorder characterized by an eruption of papules and pustules, typically appearing in face, scalp, upper chest and back.   
Rash 
maculo-
papular 
Macules/papules 
covering 
<10% 
BSA 
with 
or 
without symptoms 
(e.g., 
pruritus, 
burning, tightness) 
Macules/papules 
covering 10 -30% 
BSA 
with 
or 
without symptoms 
(e.g., 
pruritus, 
burning, tightness); 
limiting 
instrumental ADL; 
rash 
covering 
> 
30% BSA with or 
without 
mild 
symptoms 
Macules/papules 
covering >30% BSA 
with moderate or 
severe 
symptoms; 
limiting self-care ADL 
- 
- 
Definition: A disorder characterized by the presence of macules (flat) and papules (elevated). Also known as morbilliform rash, it is 
one of the most common cutaneous adverse events, frequently affecting the upper trunk, spreading centripetally and associated 
with pruritis. 
Skin induration 
Mild 
induration, 
able to move skin 
parallel to plane 
(sliding) 
and 
perpendicular 
to 
skin (pinching up) 
Moderate 
induration, able to 
slide skin, unable to 
pinch skin; limiting 
instrumental ADL 
Severe 
induration; 
unable to slide or 
pinch skin; limiting 
joint 
or 
orifice 
movement 
(e.g., 
mouth, 
anus); 
limiting self-care ADL 
Generalized; 
associated with signs 
or 
symptoms 
of 
impaired breathing 
or feeding 
Death 
Definition: A disorder characterized by an area of hardness in the skin. 
Skin ulceration 
Combined area of 
ulcers 
<1 
cm; 
Combined area of 
ulcers 1 -2 cm; 
Combined area of 
ulcers >2 cm; full-
Any size ulcer with 
extensive 
Death 

Protocol v.2.0, dated 14th June 2020 
Page 44 of 46 
SOC 
CTCAE Term 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
nonblanchable 
erythema of intact 
skin 
with 
associated 
warmth or edema 
partial 
thickness 
skin loss involving 
skin 
or 
subcutaneous fat 
thickness skin loss 
involving damage to 
or 
necrosis 
of 
subcutaneous tissue 
that 
may 
extend 
down to fascia 
destruction, 
tissue 
necrosis, or damage 
to muscle, bone, or 
supporting 
structures with or 
without 
full 
thickness skin loss 
Definition: A disorder characterized by a circumscribed, erosive lesion on the skin.  
Urticaria 
Urticarial 
lesions 
covering 
<10% 
BSA; 
topical 
intervention 
indicated 
Urticarial 
lesions 
covering 10 -30% 
BSA; 
oral 
intervention 
indicated 
Urticarial 
lesions 
covering >30% BSA; 
IV 
intervention 
indicated 
- 
- 
Definition: A disorder characterized by an itchy skin eruption characterized by wheals with pale interiors and well-defined red 
margins. 
Skin atrophy 
Covering 
<10% 
BSA; 
associated 
with 
telangiectasias or 
changes in skin 
color 
Covering 10 -30% 
BSA; 
associated 
with 
striae 
or 
adnexal 
structure 
loss 
Covering >30% BSA; 
associated 
with 
ulceration 
- 
- 
Definition: A disorder characterized by the degeneration and thinning of the epidermis and dermis.    
Skin 
hyperpigmentation 
Hyperpigmentatio
n covering <10% 
BSA; 
no 
psychosocial 
impact 
Hyperpigmentation 
covering >10% BSA; 
associated 
psychosocial 
impact 
- 
- 
- 
Definition: A disorder characterized by darkening of the skin due to excessive melanin deposition. 

Protocol v.2.0, dated 14th June 2020 
Page 45 of 46 
SOC 
CTCAE Term 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
Skin 
hypopigmentation 
Hypopigmentation 
or depigmentation 
covering 
<10% 
BSA; 
no 
psychosocial 
impact 
Hypopigmentation 
or depigmentation 
covering >10% BSA; 
associated 
psychosocial 
impact 
- 
- 
- 
Definition: A disorder characterized by loss of skin pigment (e.g., vitiligo). 
ADL: activities of daily living; BSA: body surface area; CTCAE: Common Terminology Criteria for Adverse Events; ICU: intensive care unit; IV: intra-venous; SOC: 
system organ class; ULN: upper limit of the normal.

Protocol v.2.0, dated 14th June 2020 
Page 46 of 46 
22. APPENDIX F: Amendment history 
 
Amendment 
No. 
Protocol 
Version 
No. 
Date 
issued 
Author(s) of changes 
Details of Changes made 
 
 
 
 
 
 
List details of all protocol amendments here whenever a new version of the protocol is produced.  This is 
not necessary prior to initial Ethics Committee submission.