Determination of the Protective Efficacy of the PvCS/Montanide ISA-51 Vaccine Formulation Against Controlled Infection With Plasmodium Vivax Sporozoites
Phase: PHASE2
Conditions: Plasmodium Vivax Malaria, Plasmodium Vivax Infection, Malaria Prevention
Sponsor: Malaria Vaccine and Drug Development Center

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August 7, 2025 
Clinical Phase IIa/b P. vivax CS Chocó 
 
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THIS PROTOCOL AND ALL RELATED INFORMATION 
ARE CONFIDENTIAL AND THE PRIVATE PROPERTY OF THE 
INTERNATIONAL VACCINE CENTER - CIV 
 
 
TITLE OF THE PROTOCOL  
 
Determination of the protective efficacy of the PvCS/Montanide ISA-51 
vaccine formulation against controlled infection with Plasmodium vivax 
sporozoites 
 
 
 
 
 
 
 
 
       Protocol number:    2201-3 
 
         
 
 
 
 
 
Sponsor name:  
International Vaccine Center (CIV) 
 
 
 
REGISTERED LEGAL ADDRESS: 
CIV: km 6 vía Cali - Puerto Tejada, Cali, Colombia 
ASOCLINIC IPS:  
Calle 21A - Cra 23. Zona Minera - La Virginia, Quibdó, Chocó. 
 
 
Version: 1.0 - INVIMA 
August 7, 2025 
 
 
 
 

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Clinical Phase IIa/b P. vivax CS Chocó 
 
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SIGNATURE PAGE 
 
 
 
 
 
 
Sponsor Signature 
 
 
 
 
 
_____________________  
 
 
 
_______________________ 
Printed Name:                                                       
 
Date: 
Position: Sponsor 
 
 
 
 
 
 
 
Investigator's Signature 
 
 
________________________________________                    ____________________ 
Printed name:                                                         Date: 
Position: 
 
 
I agree to conduct this clinical trial in accordance with the design described in this 
protocol and to comply with all provisions of this protocol. 
 
 
 
 
 
 
 
The signatures recorded herein constitute acknowledgment of this protocol and its 
annexes. Furthermore, they guarantee that this clinical study will be conducted in 
accordance with the provisions of the protocol, including all confidentiality requirements 
and in compliance with legal and regulatory requirements, as well as the principles set 
forth in the GCP guidelines. 
 
 

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Clinical Phase IIa/b P. vivax CS Chocó 
 
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TABLE OF CONTENTS 
 
TITLE OF THE PROTOCOL: 
1 
REGISTERED LEGAL ADDRESS: 
1 
SIGNATURES PAGE 
2 
GLOSSARY OF ABBREVIATIONS 
7 
1. SUMMARY OF THE PROTOCOL 
10 
2. INTRODUCTION: 
15 
2.1. Epidemiology of malaria. 
15 
2.2. Development of natural immunity against P. vivax  
16 
2.3. Vaccines as tools for malaria control and elimination 
16 
2.4. Limitations to the development of vaccines against P. vivax 
18 
2.4.1. Identification and characterization of the CS protein 
20 
2.4.2. Development of the P. vivax as a candidate for an antimalarial vaccine. 21 
2.4.3. Preliminary preclinical studies in Colombia and other countries. 
21 
2.4.4. Phase I clinical trials with P. vivax  
22 
2.4.5. Infectious challenge models for P. falciparum and P. vivax. 
24 
2.4.6. Standardization of the challenge model with P. vivax sporozoites 
24 
2.5. Scientific justification 
26 
3. HYPOTHESIS 
28 
4. OBJECTIVES 
29 
4.1. General objective 
29 
4.2. Specific objectives 
29 
5. STUDY POPULATION 
29 
5.1 Steps 1 and 3. Immunization and challenge volunteers. 
29 
5.1.1 Group of healthy volunteers with no history of malaria (naïve). 
29 
5.1.2 Group of healthy volunteers with a history of malaria (semi-immune) 
31 
5.2 Step 2. Blood donor volunteers 
31 
6. STUDY DESIGN 
32 
6.1. Recruitment 
33 
6.2. Identification 
33 
6.3. Selection 
33 
6.4. Group Formation 
35 
6.5. Immunization 
35 
6.5.1. Group formation 
35 
6.5.2. Vaccine formulation 
36 
6.6. Post-immunization follow-up 
37 
6.6.1. Immediate follow-up 
37 
6.6.2. Delayed follow-up 
38 

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6.7. Step 2: Donation of infected blood for sporozoite production 
38 
6.7.1. Donor recruitment 
38 
6.7.2. Identification of the patient and blood sample 
38 
6.7.3. Blood donation 
39 
6.7.4. Laboratory testing of donated blood 
40 
6.8. Step 3: Obtaining sporozoites and infectious challenge 
42 
6.8.1. Mosquito infection 
43 
6.8.2. Preparation for challenge 
44 
6.8.3. Infectious challenge with mosquitoes infected with P. vivax 
44 
6.8.4. Post-challenge evaluation 
45 
6.8.5. Treatment for malaria 
46 
6.8.6. Follow-up after initiation of antimalarial treatment 
47 
6.8.7. Follow-up of relapses or recurrences 
48 
6.8.8 Risk and management of natural infections during the study                   48  
7. LABORATORY TESTS 
49 
7.1. Diagnosis of malaria: 
49 
7.2. Infectious screening tests: 
50 
7.3. Methodology for objective #1. Confirm the safety of the vaccine in naïve 
and previously exposed volunteers immunized with PvCS. 
50 
7.4. Methodology for objective #2. Determine the immunogenicity of PvCS in 
individuals previously exposed to malaria 
51 
7.4.1. Evaluation of B cell response 
51 
7.4.2. Evaluation of T cell response 
52 
7.5. Methodology for objective #3: Determine the protective efficacy of the 
vaccine against infectious challenge with viable P. vivax sporozoites. 
52 
7.6. Interpretation of results 
53 
8. DATA MANAGEMENT AND ANALYSIS 
53 
8.1. Record keeping 
54 
9. RISKS TO VOLUNTEERS, THE RESEARCH TEAM, AND THE 
ENVIRONMENT; PRECAUTIONS TO MINIMIZE RISK 
54 
9.1. Risks to volunteer blood donors. 
55 
9.2. Risks to volunteers associated with the malaria challenge. 
56 
9.3. Risks to volunteers associated with malaria treatment 
57 
9.4. Risk to those conducting the study 
59 
9.5. Risk and precautions associated with the environment. 
59 
10. BENEFITS 
59 
10.1. Benefits for blood donor volunteers. 
59 
10.2. Benefits for malaria challenge volunteers 
60 
11. COMPENSATION 
60 
11.1. Compensation for blood donors. 
60 
11.2. Compensation for challenge volunteers 
60 
12. CRITERIA FOR WITHDRAWAL/RETIRMENT OF VOLUNTEERS 
60 

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13. Adverse Events  
61 
 Local Systemic Adverse Events 
62 
Systemic Adverse Events Requested 
63 
Vital Signs 
64 
Serum Hematology 
66 
Urine 
67 
13.1. Serious Adverse Event (SAE) 
68 
13.2. Classification of AEs - Relationship to study activities 
69 
13.3. Reporting of SAEs 
70 
13.4. SA follow-up period 
70 
13.5. Reporting SAEs to INVIMA 
71 
14. ETHICAL CONSIDERATIONS 
71 
14.1. Approval by ethics committees and organizational plan 
71 
14.2. Affiliation of ethics committees with the US FWA 
71 
14.3. Research-related injuries 
71 
15. GOOD CLINICAL PRACTICES (GCP) AT ASOCLINIC AND GOOD 
LABORATORY PRACTICES (GLP) AT CIV 
72 
16. CONFIDENTIALITY 
73 
17. RULES FOR INTERRUPTION OF THE STUDY 
73 
18. USE OF INFORMATION AND PUBLICATIONS ARISING FROM THE 
STUDY. 
73 
19. DEVIATIONS AND MODIFICATIONS TO THE PROTOCOL. 
74 
20. WITHDRAWAL OF VOLUNTEERS FROM THE STUDY 
74 
20.1. Follow-up of volunteers who do not continue in the study 
74 
21. POTENTIAL CAUSES OF FAILURE OR FAILURE IN STUDY             74  
22. CIVIL LIABILITY POLICIES 
75 
23. TIMELINE 
75 
24. BIBLIOGRAPHY  
76

 
 
 
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GLOSSARY OF ABBREVIATIONS 
 
ABBREVIATIONS 
DEFINITIONS 
A. 
Anopheles 
Anti-HBc 
Anti-core antibodies Hepatitis B 
DNA 
Deoxyribonucleic acid 
AMA 
Artificial membrane feeding technique 
AL 
Latin America 
BUN 
Blood Urea Nitrogen (US) 
BPC/GCP 
Good Clinical Practices 
BPL/GLP 
Good Laboratory Practices 
B-HCG 
Human chorionic gonadotropin-beta subunit (US) 
C 
Terminal carboxyl 
CEI-ASOCLINIC 
ASOCLINIC Research Ethics Committee 
CI 
Informed Consent 
CIV 
International Vaccine Center 
CQ 
Chloroquine 
CS 
Circumsporozoite Protein 
EA 
Adverse Event 
SAE 
Serious Adverse Event 
EKG 
Electrocardiogram 
ELISA 
Enzyme-Linked Immunosorbent Assay (US)  
EPS 
Health Promotion Company 
ETV 
Vector-Borne Disease 
FDA 
Food and Drug Administration 
FRC/CRF 
Case Report Form 
FTA-ABS 
Fluorescent-Treponemal Antibody Absorbed (US) - Treponemal 
Antibody Absorption Test 
FWA 
Federalwide Assurance 
 
G6PD 
Glucose-6-Phosphate Dehydrogenase 
GG 
Thick Smear 

 
 
 
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HBsAg 
Hepatitis B Surface Antigen 
HTLV 
Human T-Lymphotropic Virus 
IFAT 
Immunofluorescence Antibody Test = Indirect 
Immunofluorescence Technique 
INS 
National Institute of Health (Colombia) 
INSALPA 
Pacific Health Institute 
IP/PI 
Principal Investigator 
IPS 
Health Services Provider Institution 
IRB 
Institutional Review Board = Ethics Committee 
LDH 
Lactate Dehydrogenase 
Mabs 
Monoclonal Antibodies 
    MAPs 
Multi-Antigenic Peptides  
MVDC 
Malaria Vaccine and Drug Development Center = 
International Vaccine Center – CIV) 
N 
Amino terminal 
NIH 
National Institutes of Health 
NIAID 
National Institute of Allergy and Infectious Diseases 
NMRC 
Naval Medical Research Center 
WHO 
World Health Organization 
P. 
Plasmodium 
SOP 
Standard Operating Procedure 
PQ 
Primaquine 
PSL 
Long Synthetic Peptides  
spp. 
Species 
RAS 
    Radiation-attenuated sporozoites 
SARS-CoV 2 
Severe Acute Respiratory Syndrome Coronavirus 2 (COVID-19) 
SP 
Sulfadoxine-Pyrimethamine 
SGSS 
General Social Security System for Health 
SSDC 
Departmental Health Secretariat of Choco 
SSMQ 
Municipal Health Secretariat of Quibdó 
VES 
Erythrocyte Sedimentation Rate 
HBV 
Hepatitis B virus 

 
 
 
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1. 
SUMMARY OF THE PROTOCOL 
 
Title 
To determine the protective efficacy of the 
PvCS/Montanide ISA-51 vaccine formulation against 
controlled infection with P. vivax sporozoites. 
Product Name 
PvCS.2 / M-51 
Study Objectives 
Primary Objective:  
To 
determine 
the 
protective 
efficacy 
of 
the 
PvCS/Montanide ISA-51 vaccine formulation against 
controlled infection with P. vivax sporozoites 
Specific Objectives: 
1) Confirm the safety of the vaccine in naïve volunteers 
immunized with PvCS. 
 
2) To determine the immunogenicity of PvCS in 
individuals previously exposed to malaria. 
3) Determine the protective efficacy of the vaccine 
against infectious challenge with viable P. vivax 
sporozoites in the above groups. 
Study Design 
Randomized, double-blind, controlled, Phase IIa/b study 
comparing two groups of naïve volunteers and volunteers 
previously exposed to malaria. 
Schedule 
Three intramuscular injections on days 0, 60, and 180, 
followed by infectious challenge on day 210. 
No. of Volunteers 
Step #1: volunteers (immunization) n=60; plus 4-6 
alternates. 
 
Step #2 = patients with active P. vivax infection, blood
donors to artificially infect mosquitoes: minimum 5,
maximum 15. 
 
Population 
Healthy adults of both genders: 30 volunteers with a 
history of Plasmodium vivax malaria for the semi-
immune group based on medical history and serology, 
and 30 volunteers with no history of malaria for the naïve 
group. Participants will be residents of Quibdó. 

 
 
 
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Study 
procedures 
Step 1. Selection and immunization of volunteers 
Sixty subjects who meet the inclusion criteria for each of 
the groups will be selected: a) naïve (n=30) and b) semi-
immune (n=30) from Quibdó and other municipalities in 
Chocó. The subjects in each main group (A, B) will be 
randomly distributed into two subgroups: Vaccine (A1 and 
B1, with n=20 each), Control (A2 and B2, with n=10 each). 
Subgroups A1 and B1 will be immunized with the vaccine 
(PvCS protein) and subgroups A2 and B2 with the placebo. 
The vaccine group will be immunized intramuscularly (IM) 
on days 0, 60, and 180, with the formulation, composed of 
peptides derived from the circumsporozoite protein (CS) of 
P. vivax (PvCS) at doses of 100 µg and 150 µg formulated 
in Montanide ISA-51 adjuvant (PvCS.2/M51 vaccine).  
Repeated blood samples will be taken to assess safety 
through renal, hepatic, and hematological function tests at 
0, 1, 3, 5, 7, 8, and 18 months. 
Adverse events (AEs) induced by immunization will be 
assessed during the 7 days following immunization. These 
AEs will be reported immediately to the ASOCLINIC Ltda.
research Ethics Committee (IRB). 
The immunogenicity of the vaccine will be evaluated by 
testing for specific humoral and cellular responses against 
the peptides used in immunization and against the parasite. 
Step 2. Parasite donor volunteers:  
Patients naturally infected with P. vivax (PvCS-VK210) will
be selected as parasite donors for the infection of Anopheles
mosquitoes. These donors will be identified at health posts in 
endemic areas using microscopic testing (thick smear).  
The group of parasite donor patients (n=5-15) will 
consist of individuals infected with P. vivax who will be 
asked to donate a 20 mL blood sample that will undergo 
laboratory testing to rule out co-infections detectable by 
standard blood banking. Only blood samples infected 
exclusively with P. vivax according to the PCR test will 
be used for feeding and experimental infection of 
Anopheles mosquitoes. 
 

 
 
 
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3. Infectious challenge of volunteers from step 1: To
evaluate the protective efficacy of the immunogen, 
volunteers will be challenged by bite from 3+1 mosquitoes 
infected with P. vivax sporozoites on one occasion:
approximately 1 month after the third immunization.
Starting on day 5 post-bite, medical and parasitological 
follow-up will be performed to determine the onset of 
infection. All procedures in this study will be performed 
using Good Laboratory Practices (GLP) and Good Clinical 
Practices (GCP). 
 
Protection of volunteers 
and social security      
All participants must be affiliated with the General 
Social Security System for Health (SGSS), under any 
of its schemes, and provide the corresponding 
supporting documentation. In addition, as an additional 
protective measure, the study will provide volunteers 
with a prepaid medical plan that will cover medical 
care throughout the process. Likewise, a civil liability 
policy for clinical trials will be taken out to cover any 
incident related to their participation.      

 
 
 
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Procedures to 
minimize risks 
associated with 
the challenge 
The potential risks associated with the challenge will be 
evaluated in accordance with a specific biosafety protocol, 
which includes the following aspects in more detail and 
the corresponding contingency plan:  
● 
Detailed analysis of medical history and laboratory 
tests to evaluate donor inclusion and exclusion 
criteria. 
● 
Testing of donated blood for infectious diseases to 
infect mosquitoes, in accordance with Blood Bank 
standards, to rule out the presence of other pathogens 
in the donor sample. 
● 
Restriction of personnel access to infected mosquito 
rooms to minimize the risk of accidental 
transmission of malaria to researchers and/or the 
community. 
 
● 
Close post-challenge follow-up at the time of 
diagnosis and 28 days after completion of 
antimalarial treatment (hematological, 
parasitological, and blood chemistry); in addition, 
physical examination (personalized assessment). 
● 
Antimalarial 
treatment 
will 
be 
administered 
immediately upon documentation of parasitemia ≥ 
100 p/L evaluated by thick smear. Parasitemias will 
be confirmed retrospectively by qPCR. 
● 
Immediate treatment for any individual exposed to 
accidental infection with malaria. 
 
● 
Analysis of volunteers' blood samples to detect 
antibodies against HIV, hepatitis B, and hepatitis C 
will contribute to the protection of health and 
laboratory personnel. 
● 
Standard biosafety procedures will be followed for 
the handling of blood and body fluid samples. 

 
 
 
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Data 
processing 
     Study data will be entered into an online database using REDCap 
version 5.10.1 (http://project redcap.org/). Final data processing will be 
performed using STATA®, SPSS®, or R® statistical software. 
Data will be collected using REDCap v5.10.1 and managed on a secure 
server. Data entry will be performed using electronic forms verified by 
the clinical monitor and corrected according to the SOPs. After quality 
control, the data will be analyzed using Stata, SPSS v16.0, and 
GraphPad Prism v6.0. The data will be available in the article and 
supplement; raw data may be requested from the corresponding author, 
respecting the confidentiality of the participants. 
  Study 
Duration 
30 months 
   
  Study sites 
 
Asoclinic IPS Research Center - Clinical Laboratory 
Calle 21a - Carrera 23. Zona Minera -La Virginia.  
Quibdó, Chocó 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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2. 
INTRODUCTION: 
 
Malaria continues to be a global public health problem due to high morbidity and 
mortality, mainly in countries located in tropical and subtropical areas of the planet (WHO 
2021). Despite major efforts to reduce it, in 2020 there were 241 million clinical cases and nearly 
1 million deaths reported in 85 endemic countries. In the same year in Latin America, Central 
American countries achieved reductions in incidence of more than 90%. However, in countries 
such as Brazil, Colombia, and Venezuela, which account for ~77% of the ~1.5 million cases 
reported in the region, an average reduction of 58% (to ~650,000 cases) was observed (PAHO 
2022). 
 
These large differences in the results of the fight against malaria are due, among other causes, 
to the failure of classic control measures, such as the use of insecticides and antimalarial drugs, 
and to numerous socioeconomic and political problems, which together demand their 
strengthening and the development of new control tools and strategies. 
 
2.1. Epidemiology of malaria. 
 
Malaria is a disease that currently affects ~250 million people and is responsible for 
~600,000 deaths per year, representing an enormous economic impact globally, mainly for 
populations living in developing regions in sub-Saharan African countries (Breman, 2001; Sachs 
and Malaney, 2002), but also in some regions of Asia and Latin America (LA). Epidemiological 
indicators report that infections caused by P. vivax remain widely distributed worldwide, even 
more so than infections caused by P. falciparum, representing a significant cause of morbidity 
and mortality among the 2.85 billion people living at risk of infection (Guerra, 2010). Most cases 
of P. vivax originate in Southeast Asia and the Western Pacific, accounting for ~70% of cases in 
LA and a smaller proportion (5-20%) in some African countries (Guerra, 2010; Mendis, 2001, 
WHO 2023). 
 
Most endemic areas where both P. falciparum and P. vivax are transmitted share the same 
mosquito vectors, and therefore Anopheles resistance to insecticides affects the transmission and 
control of both Plasmodium species (Rodríguez, 2009). On the other hand, although the disease 
caused by P. vivax is less lethal than that caused by P. falciparum, it is more complex due to the 
development of liver parasitic forms (hypnozoites) that remain silent (asymptomatic) and create 
a reservoir of parasites with periodic clinical reactivation of the infection (Sattabongkot 2004). 
Additionally, the hemolytic effect of Primaquine (PQ) in individuals with glucose-6-phosphate 
dehydrogenase (G6PD) deficiency is known, with the consequent risk to these patients when 
treated with conventional regimens (Galappaththy, 2013). Furthermore, in recent years, the 
limited effect of PQ in patients with certain polymorphisms of the cytochrome P450 (2D6) 
enzyme has been demonstrated, a fact that contributes to the development of relapses after 
conventional treatment (Manjurano, 2016). 
During the period 2000-2015, efforts to control malaria were intensified with a view to 
moving towards its global elimination. The Millennium Development Goals established by the 
United Nations set a target of reducing the incidence of malaria by 75% by 2015, and 

 
 
 
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contributions to global control efforts were increased by international agencies such as the 
Global Fund to Fight AIDS, Tuberculosis and Malaria, the US President's Malaria Initiative 
(PMI), and the Bill and Melinda Gates Foundation, among others (WHO, 2015). 
This led to a notable reduction in malaria worldwide, from nearly 500 million cases in 2000 to 
approximately 250 million in 2015, which, although representing a significant decrease in 
incidence, still continues to have an enormous impact on global public health. In this context, 
between 2000 and 2015, a total of 17 countries achieved the elimination of transmission in their 
territories. In addition, regions such as Central America reduced their incidence by about 90%, 
and in Colombia the reduction was around 75%. At the same time, progress was made in the 
search for vaccines that could reinforce the elimination strategy, particularly for P. falciparum, 
the species responsible for about 90% of all cases globally and 99% of cases reported in Africa. 
Progress was also made in the study of molecules with potential for the development of 
vaccines against P. vivax, a parasite with a much lower incidence but with a wider global 
distribution; this species is predominant in Asia, Oceania, and the Americas. (WHO, 2021) 
Unfortunately, due to reduced international funding and the inability of endemic countries to 
maintain control measures, primarily global access to early diagnosis and timely treatment, 
there was a resurgence in malaria transmission in the following years, and in some countries, 
such as Colombia, malaria has doubled between 2016 (~50,000 cases/year) and 2024 
(~100,000 cases/year), highlighting the importance and urgency of developing vaccines against 
P. falciparum and P. vivax, the two most prevalent species of Plasmodium.  
 
Fortunately, significant progress has been made over the last 10 years in the development of 
two vaccines aimed at preventing P. falciparum infection (WHO, 2023). Although vaccines 
will not be able to eliminate malaria on their own, their contribution to control and elimination 
strategies will be enormous; vaccines are considered the most cost-effective mechanism for 
combating communicable diseases, and the contribution of clinical immunity to reducing 
morbidity and mortality under natural conditions is incalculable (Bloland, 2002, Doolan, 2009) 
(see state of the art). 
 
2.2. Development of natural immunity against malaria caused by P. vivax 
 
In general, exposure to malaria can be fatal. As mentioned, approximately 250 million 
clinical cases are reported worldwide each year, with nearly 600,000 deaths, mainly in children 
under 5 and pregnant women (WHO 2023). However, if patients survive the first infections, 
their repeated exposure to Plasmodium produces a progressive reduction in the intensity of the 
disease and causes individuals in endemic areas to eventually develop clinical immunity, in 
which people only have mild symptoms or no clinical manifestations (asymptomatic), despite 
harboring long-term infections. These communities also develop immunity that blocks 
mosquito infection, because when the mosquito bites the patient, it ingests not only the parasite 
but also antibodies that block its transmission (Arévalo-Herrera 2011c). 
 
In this context, in highly endemic regions, children and adolescents between the ages of 10 

 
 
 
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and 15 achieve a moderate degree of immunity, which is associated with a decrease in the 
intensity of clinical manifestations, the frequency of infection (Cattani, 1986b), and the 
transmission of the infection itself. Unfortunately, this immunity disappears when individuals 
leave the endemic area (Doolan, 2009) and become susceptible again. Furthermore, this 
immunity is species-specific (Collins and Jeffery, 1999), and although there is cross-reactivity 
between parasites, this reactivity does not necessarily induce sufficiently effective natural 
cross-protection. 
 
Because P. vivax has different biological properties than P. falciparum, it is unlikely that 
one species of parasite can be effectively controlled by a heterologous vaccine, although under 
certain conditions there is cross-reactivity between components (antigens) of one species and 
another that could contribute partially to protection. Therefore, the identification of specific P. 
vivax components is required for the development of vaccines for this species and, with further 
research, the identification of antigens that would ideally allow for the development of 
multispecies vaccines. 
 
2.3. Vaccines as tools for malaria control and elimination 
 
Due to limitations in classic malaria control strategies, such as early diagnosis and timely 
treatment, vector control measures, and others, vaccines have been considered a 
complementary strategy. Over the last three decades, the scientific community has worked 
intensively to identify molecules that can be used for vaccine development as a tool to 
strengthen control and accelerate the elimination of malaria (Dhiman, 2019).  
The feasibility of malaria vaccines is based on multiple lines of evidence: 1) individuals in 
endemic areas become clinically immune through repeated exposure to malaria and can remain 
asymptomatic carriers for indefinite periods, leading an almost normal life; 2) It has been 
demonstrated that the passive transfer of specific antibodies or cells from semi-immune 
patients in endemic areas confers protection to non-immune individuals in areas without 
malaria transmission (Druilhe, 1994). 3) Human volunteers vaccinated through the bite of 
mosquitoes infected with P. falciparum and P. vivax sporozoites and irradiated develop strong 
immunity in more than 90% of those vaccinated (Clyde, 1975). 4) Similarly, inoculation with 
attenuated sporozoites, extracted from the mosquito by microscopic dissection and 
cryopreserved, induces similar protection and is being studied for industrialization (Hoffman, 
2002). 5) In the case of P. vivax, the International Vaccine Center (CIV) recently achieved 
remarkable protection in primates of the genus Aotus and in healthy adult volunteers exposed 
to vaccination with experimentally infected and irradiated mosquitoes. These mosquitoes were 
carriers of sporozoites in their salivary glands (Arévalo-Herrera, 2016). 6) After more than 
three decades of research, it has been confirmed in recent years that the Pf-RTS,S and Pf-R21 
vaccines developed in Europe by large international alliances achieve efficacy greater than 
50% and 70%, respectively, in endemic areas of Africa. This achievement recently led the 
WHO to approve their implementation in some African countries with high P. falciparum 
transmission (Penny, 2016). 6) More recently, in Colombia, the CIV found that the 
PvCS/Montanide ISA-51 formulation, which had been in development for nearly three 

 
 
 
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decades, achieved for the first time nearly 40% total protection (sterile) in naïve volunteers 
from the city of Cali and semi-immune volunteers from the municipality of Buenaventura 
(endemic area), and total protection greater than 50% and 60%, respectively, taking into 
account a notable reduction in parasitemia in those who did not produce sterile immunity 
(Arévalo-Herrera, 2022).  
 
Over the last three decades, ~30 P. falciparum antigens have been identified worldwide, 
and their immunogenicity and protective efficacy have been evaluated in animal models and 
humans (Richie and Saul, 2002). Among these antigens are those expressed in sporozoites 
(CSP, SSP2/TRAP) (Rogers, 1992), in hepatic stages (LSA1, LSA3, EXP1), in the erythrocytic 
phase (MSP-1, MSP-2, AMA-1), and in sexual stages (PfS25, PfS45/48), among others. 
Colombia, through the CIV, has contributed to the development of many of these (Arévalo-
Herrera 1998; Arévalo-Herrera 2001; Herrera 2005; Arévalo-Herrera 2010; Herrera 2011a; 
Arévalo-Herrera 2001; Arévalo-Herrera 2011a; Arévalo-Herrera 2001b; Arévalo-Herrera 
2016; Arévalo-Herrera 2022).  
 
Methodological strategies for vaccine production include, among others, synthetic 
peptides and recombinant proteins formulated in different adjuvants, as well as live 
recombinant viruses and DNA vaccines. Most of these antigens have been identified by 
screening genomic libraries with sera against whole parasites or fractions thereof, as well as 
with monoclonal antibodies (Mabs). The immunogenicity and protective efficacy of several of 
these antigens have been experimentally tested in animals and humans (Kumar, 2002), showing 
a wide range of immunogenicity and/or protection in these models (Genton and Corradin, 
2002). 
 
However, the Pf-RTS-S/ASO2A) and Pf-R21-Matrix M vaccines, which are 
recombinant hybrid products containing a fragment of the P. falciparum CS protein and 
hepatitis B AgS, are the only vaccines that, after extensive phase II and III studies on the 
African continent, have been approved by the WHO for use in selected countries on that 
continent (Hill, 2011, Penny, 2016). The Pf-RTS,S/AS02A vaccine initially showed modest 
protection of approximately 30% against clinical malaria in young children, while a new 
formulation, Pf-RTS,S/AS01E, showed an increase in protective efficacy, reaching around 
53% in children aged 5 to 17 months (Bejon 2008). Meanwhile, the more recently reported 
candidate PfR21-Matrix-M achieved superior protection of 77%, making it, together with Pf-
RTS,S, one of the most advanced vaccines currently available (Samuels, 2022; Datoo, 2024). 
However, there are numerous other vaccines in preclinical and clinical trials against P. 
falciparum (Hoffman, 2015; Webster, 2021; Kublin, 2017), including other platforms, which 
more recently include mRNA vaccines (Tsoumani, 2023; Tang, 2025) and the use of 
recombinant nanoparticles (Karsk, 2017; Zhuo, 2024, Herrera, SM 2024). 
 
2.4. Limitations to the development of vaccines against P. vivax 
 
In contrast to the significant advances in the development of vaccines against P. 
falciparum, explained by its high importance and significant global investment, only a limited 

 
 
 
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number of antigens have been identified and partially characterized in P. vivax, such as the 
MSP1 protein (del Portillo, 1991), AMA1 (Thomas, 1994), MSP3, MSP4, MSP5, RBP, and 
DBP from the asexual blood stages (Barnwell and Galinski, 1995; Barnwell, 1999; Chitnis, 
2001; Galinski, 1999; Galinski., 2001; Miller, 1977), Pvs25 and Pvs28 from oocysts/oocysts, 
and PvCS and PvSSP2/TRAP antigens from the pre-erythrocytic phase (Templeton and 
Kaslow, 1997). Of this last phase of the cycle, only PvCS has been extensively analyzed in 
preclinical and clinical studies (Arévalo-Herrera, 2010). However, advances in "omic" 
technologies, mainly genomics and proteomics, are allowing for a more rapid analysis of the 
parasite. In our case, this applies to the pre-erythrocytic phase of P. vivax (Mo 2018, Herrera 
2024). On the other hand, progress has been made in other phases, such as the sexual blood 
phase of the parasite, where the proteins Pvs48/45, Pvs230, and Pvs47 expressed in 
gametocytes and in the sporogonic phase are being studied in the discovery and preclinical 
analysis phases (Malkin, 2005; Arévalo-Herrera, 2015, Molina-Cruz, 2025). Similarly, in the 
asexual erythrocytic phase, promising candidate antigens such as PvDBP, PvRBP, PvAMA-1, 
SPECT1, FALSTATIN, and CELTOS have been identified and are being analyzed in recent 
experimental studies (López-Pérez Soares 2023, and coiled-coil (Cespedes 2014). 
 
The limited progress in the study of P. vivax antigens identified as candidates for 
vaccine development can generally be explained by several factors, such as: 1) the impossibility 
of obtaining continuous cultures (in vitro) of blood forms of the parasite that in turn allow 
mosquitoes to be infected regularly in the laboratory; 2) difficulty in maintaining a constant 
and adequate production of sporozoites for infectious challenge studies; 3) the consequent 
difficulty in the experimental use of vaccination with irradiated P. vivax sporozoites, which 
would allow the generation of reagents (serum and cells) of great value for the identification 
of antigens potentially associated with protection and, therefore, candidates for the 
development of vaccines against this species (Barnwell, 1985; Arévalo-Herrera, 2010); 4) the 
existence of genetic factors that limit the development of clinical studies, such as: a) Duffy 
blood group polymorphism (Fy+/-) essential for the invasion of red blood cells by the parasite, 
b) glucose-6-phosphate dehydrogenase (G6PD) deficiency, which can affect human volunteers' 
tolerance to primaquine (PQ) treatment of the parasite, and c) the prevalence of 
hemoglobinopathies in some endemic populations.  
  Despite these difficulties, the CIV in Cali has been making progress in developing 
infrastructure and experimental models for conducting studies on vaccines against P. vivax in 
Colombia. This process includes a comprehensive approach to the parasite, ranging from 
discovery, preclinical studies, and clinical trials of proteins from the three phases of the 
parasite's cycle: 1) hepatic or pre-erythrocytic phase; 2) asexual blood phase; and 3) sexual and 
sporogonic blood phase, with a valuable component of national and international scientific 
cooperation.  
 
In this context, with financial support from national and foreign institutions and 
scientific cooperation from Latin America, Europe, and the United States, genomic and 
proteomic screening studies of the parasite have been carried out, using a collection of 
biological reagents such as sera from animal models (mice and primates) and individuals from 

 
 
 
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endemic areas on different continents, such as Latin America (Brazil, Peru, Panama, 
Guatemala, and Colombia), Africa (Mali, Burkina Faso, Senegal, Nigeria). In Colombia, the 
CIV has had access to very valuable reagents (serums and cells) from various endemic areas 
such as Nariño, Cauca, Valle, Chocó, and Córdoba, which have enabled comparative studies 
on the natural immune response to the parasite and its relationship with naturally induced 
clinical protection. On the other hand, the establishment of insectaries and the development of 
mosquito infection models and their use for controlled experimental human infection models 
has allowed access to sera and cells from volunteers experimentally vaccinated with irradiated 
sporozoites (Arévalo-Herrera, 2016a; 2016b) or naturally exposed (Molina, 2012) in the search 
for proteins critical to the parasite during the hepatic phase.  
 
Additionally, the study of the blood phase proteome in the search for protein domains, 
characterized by the expression of alpha-helix structures (coiled-coil motifs) and by the 
induction of antibodies associated with protection (Céspedes, 2014; 2017). Furthermore, the 
group has focused significant efforts on characterizing the Pvs48/45 protein expressed in P. 
vivax gametocytes/gametes, whose neutralization blocks the development of the sporogonic 
cycle in the Anopheles mosquito (Arévalo-Herrera, 2022a; 2021; 2015; 2011c; 2005). 
 
This infrastructure gives Colombia an important position as a global reference point for 
the study and development of vaccines for the different stages of the parasite, mainly but not 
exclusively against P. vivax malaria. Given the significant protective efficacy of the PfCS 
component of Pf-RTS-S and Pf-R2, which, as mentioned previously, were the first vaccines to 
be approved by the WHO for implementation in a large number of endemic countries and as 
part of regular vaccination programs after more than 30 years of study (Stoute, 1997; Hill, 
2011).  
 
The present clinical study focuses on PvCS, an orthologous protein of PfCS, 
specifically on confirming the safety and reproducibility of this vaccine candidate (Herrera, 
2011b; Herrera, 2009c; Arévalo-Herrera, 2022a). The process of immunological 
characterization and analysis of PvCS in preclinical and clinical phases is described in detail 
below.   
 
2.4.1. 
 Identification and characterization of PvCS protein 
 
The CS proteins of P. falciparum and P. vivax were identified using sera from 
individuals immunized with irradiated sporozoites (Druihle., 1998; Hoffman and Doolan, 
2000). P. vivax CS (PvCS) was identified and its gene cloned in 1985 (Arnot, 1985). Chemical 
characterization of the protein indicated that its structure is like that of the homologous protein 
in other Plasmodium species (Sinnis and Nussenzweig, 1996). PvCS is composed of 373 amino 
acids and is characterized by a central domain (90-261 aa) consisting of short repetitive units 
flanked by non-repetitive protein fragments at its amino (N) and carboxyl (C) termini. The 
flanking regions contain small, highly conserved sequences called Region I (85-89 aa) and 
Region II-plus (338-355 aa), which have been identified as parasite binding domains for the 
invasion of hepatocytes by sporozoites (Cerami, 1992; Frevert, 1993). The central domain of 

 
 
 
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the protein is composed of 19 blocks of 9 amino acids each, of which two allelic forms can be 
found in nature, the VK210 or common type (GDRADGQPA) (Arnot, 1985) and VK247 or 
variant type (ANGAGNQPG) (Tsuji and Zavala, 2001) (Figure 1 and Figure 3). Probably due 
to its repetitive nature, this region has been shown to be immunodominant and is believed to 
mask the response against the N and C flanks and their invasion domains.  
 
In addition to this dimorphism, limited polymorphism has been observed in the regions 
encoding the amino (N) and carboxyl (C) flanks of the protein. (Arnot, 1990; Gonzalez, 2001; 
Kain, 1992; Machado and Povoa, 2000; Maheswary, 1992; Mann, 1994; Qar, 1992; Rosenberg, 
1989; Wirtz, 1987). This polymorphism does not appear to have a major influence on the 
immunogenic regions (epitopes) of the protein. 
 
Over the last two decades, different research groups, including the CIV, have carried out 
extensive immunological characterization of the protein, using sera from individuals 
vaccinated with irradiated sporozoites (RAS) and from semi-immune individuals from endemic 
areas. These individuals recognize the CS protein and induce a precipitation reaction on the 
surface of live sporozoites (CSP reaction) (Cochrane, 1976), which neutralizes the invasion of 
sporozoites into hepatocytes (Nussenzweig, 1969). 
 
 
 
Figure 1. Structure of the PvCS, VK247, and VK210 variants. 
 
 
 
Using these sera, different B epitopes have been identified throughout the entire protein 
sequence (Arévalo-Herrera, 1998; Franke, 1992a), and the VK210 and VK247 sequences were 
found to be significantly recognized by sera from immune individuals from different endemic 
areas, indicating their wide distribution (Arévalo Herrera, 1998; Burkot, 1992; Cochrane, 1990; 
Franke, 1992b; Ramasamy, 1994; Wirtz, 1990). The VK210 variant contains the AGDR 
sequence, which is highly recognized by individuals from malaria-endemic communities, as 
well as by monoclonal antibodies capable of protecting Saimiri monkeys against challenge with 
infectious P. vivax sporozoites (Charoenvit, 1991). 
 
Multiple helper T epitopes have also been recognized in the context of class II Major 
Histocompatibility Complex (MHC) molecule haplotypes (Herrera, 1994; Nardin, 1991). 
Furthermore, using nona- or decapeptides containing binding motifs for class I MHC antigens, 
our group identified five peptide sequences in the CS protein of P. vivax capable of stimulating 
human CD8+ lymphocytes from HLA-A*0201 individuals (Figure 2). These peptides induced 

 
 
 
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the production of IFN-γ, a cytokine involved in protection against malaria, by mononuclear 
cells from individuals previously naturally infected with P. vivax malaria (Burkot, 1992; 
Franke, 1992b). 
 
 
 
 
Figure 2: Mapping and identification of T helper, B cell, and cytotoxic (CD8+ HLA-A2) epitopes in the CS protein 
of P. vivax 
 
 
 
2.4.2.  Development of the P. vivax CS protein as a candidate for an antimalarial 
vaccine. 
 
In 1987, the PvCS protein was initially proposed as a vaccine candidate by Dr. Ruth 
Nussenzweig's group at New York University when it was tested as a recombinant protein 
(rPVCS-1) in mice, in which it induced a strong neutralizing antibody response (Cattani, 
1986b). Later, two clinical trials were conducted in the USA using recombinant proteins, which 
failed to induce significant immune responses that would justify their development as a vaccine 
(Gunewardena, 1994; Williams, 1996); and during the following decade, no further clinical 
trials with the same protein were reported. 
 
2.4.2.1. Preliminary preclinical studies in Colombia and other countries. 
 
    
CIV has focused significant efforts on the immunological characterization of PvCS, 
with the aim of obtaining a rational vaccine design (Arévalo-Herrera, 2001). Initially, using 
epitopes identified in the protein, a series of Multi-Antigenic Peptides (MAPs) were 
constructed containing various combinations of the B and Th epitopes. Two of the seven MAPs 
designed produced strong specific antibody responses against CS and IFN-γ production in 
preclinical trials conducted in primates. However, these MAPs could not be purified in the 
quantities required for subsequent clinical trials (Herrera, 1997). Therefore, within the 
framework of cooperative studies between the CIV and the group of Dr. G. Corradin of the 
University of Lausanne (Switzerland), they decided to use the strategy of Long Synthetic 
Peptides (LSPs) with sufficient length (>70-mer) to contain multiple B, Th, and CTL epitopes. 
LSPs derived from the CS proteins of P. falciparum and P. vivax were synthesized separately 
and tested in preclinical trials in Aotus monkeys (Arévalo-Herrera, 1998). 
 

 
 
 
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Figure 3: Structure of the PvCS protein, amino (N-terminal) region, central region, and carboxyl (C-terminal) 
region with their respective amino acids. 
 
 
These studies indicated high immunogenicity and the ability of P. vivax sporozoites to 
boost this immune response (Herrera, 1997). In both trials, the animals produced high titers of 
specific antibodies capable of recognizing the native protein in antigenic preparations of the 
parasite, by immunofluorescence (IFAT), in addition to stimulating the release of IFN-γ in 
vitro, determined by the ELISpot technique. Simultaneously with these studies in Colombia, 
the Walter Reed Institute (WRAIR) group in Silver Spring (MD) in the USA developed a 
recombinant chimeric protein with sequences from different variants of PvCS. The 
recombinant vaccine formulated in Montanide ISA was highly immunogenic in mice, and the 
CS protein was recognized by sera from individuals infected with P. vivax (Yadava, 2007).  
 
 
 
 
Figure 4: Antibody response determined by ELISA, induced by the PvCS protein formulated in 
Montanide ISA-720 and ISA-51 adjuvants (a) in BALB/c mice and (b) in Aotus primates immunized 
with the same formulation. Each line represents an individual animal. 
 
 

 
 
 
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In Colombia, the CIV developed a new preclinical study of the vaccine to test its 
immunogenicity in BALB/c mice and Aotus monkeys (Céspedes, 2013; Arévalo-Herrera, 
2011a). For these studies, combinations of the three synthetic peptides corresponding to the 
amino (N), central repetitive (R), and carboxyl (C) regions of the CS protein were used, 
formulated in the adjuvants Montanide ISA-720 and Montanide ISA-51. Both formulations 
were highly immunogenic in both animal species. Mice developed a better antibody response 
against the C and R polypeptides, while the N polypeptide was more immunogenic in primates.  
 
Because Aotus lemurinus griseimembra primates develop variable infections in their prepatent 
period after infectious challenge with P. vivax sporozoites, it was not possible at that time to 
reliably test the protective efficacy of vaccination in primates. Furthermore, experimental mice 
are also not susceptible to infection with human parasites. In vitro tests were used as an 
alternative to determine the protective capacity of antibodies induced by immunization, to 
determine whether specific IgG antibodies against the PvCS protein had the ability to block the 
invasion of viable sporozoites into cultured liver cells (HepG2 cell line) (Arévalo-Herrera, 
2011b). 
 
Although during the period in which preclinical studies were conducted in mice and primates, 
it was not possible to measure protective efficacy in animal models, given the safety, 
tolerability, and immunogenicity of the PvCS + Montanide ISA 51 (PvCS.2 / M-51) 
formulation, we decided to move forward with its development in the clinical phase.  
 
However, in recent years, a murine model has been developed in which it is possible to 
determine the protective efficacy of vaccines against human malaria in mice, using murine 
(mouse) parasites such as Plasmodium berghei transfected with human parasite genes 
(Raghunandan, 2020) from the PbPf model. Subsequently, a transgenic parasite containing a 
fragment of PvCS was developed (Espinosa 2013).   
 
More recently, taking advantage of the advances made by CIV in the clinical phase and these 
developments at Johns Hopkins University in Baltimore (MD) (USA), we decided to work 
together on adapting this model to study several antigens from the pre-erythrocytic phase of P. 
vivax. This cooperation began with a modification of the transgenic parasite, and two versions 
were produced containing the PvCS gene in its two most prevalent variants in nature (PvCS 
VK210 and PvCS VK247). Using the formulation used in the phase II clinical trial in Cali 
(PvCS + Montanide ISA-51 (Arévalo-Herrera, 2022) and using the same strategy used in 
humans, in which mice are initially vaccinated with the N+C mixture and subsequently with 
N+R+C, C57Bl/6 mice were immunized. Figure 5 presents a summary of the study: panel a 
shows the general design of the trial, with an immunization phase on days 0, 30, and 60, 
followed by the infectious challenge, and finally the analysis of liver infection using 
bioluminescence. Panel b shows the antibody immune response against the central region 
determined by ELISA; panel c shows a histogram indicating the level of protection achieved. 
Immunization induced a vigorous immune response against the protein with ELISA titers > 2 
x 105 and recognition of the native PvCS protein in IFA studies. Most notably, protective 

 
 
 
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efficacy against challenge with the P. berghei/P. vivax-CS parasite was demonstrated to be 
slightly higher (68%) than that observed in humans (64%) (Herrera 2025).  
 
 
 
Figure 5: Evaluation of immunogenicity and protective efficacy of the PvCS-LSP vaccine formulated in 
Montanide ISA-51 evaluated in mice. (a) Schematic of immunization and challenge protocol. (b) Specific 
antibody response against PvCS N, R, and C peptides determined by ELISA. (c) Inhibition of liver invasion 
compared to naïve controls and Montanide ISA-51. (d) Recognition of sporozoites by antibodies from animals 
immunized with PvCS by IFA. 
 
 
This study has therefore validated not only the reproducibility of the immunogenicity 
and protective efficacy of PvCS, but also the potential usefulness of the murine model for 
predicting the future performance of vaccines for human use (see non-clinical studies in the 
Investigator's Manual). 
 
Simultaneously, in this context, studies have been conducted using the same animal 
model with nanoparticles based on self-assembled recombinant proteins (Self-Assembled 
Protein Nanoparticles - SAPN), containing the two most common natural variants, P. vivax CS 
VK210 and VK247, as well as two immunostimulants incorporated into the CpG nanoparticle 
and flagellin, meaning that the formulation does not require external adjuvants. These studies 
showed great similarity to those developed with the synthetic PvCS formulation (Figure 6) 
(Herrera, SM, 2025b submitted), indicating the consistent protective capacity of the PvCS 
protein with both synthetic and recombinant proteins, and the reproducibility of the murine 

 
 
 
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model. These latest studies have made it possible to advance more rapidly, at significantly 
lower cost and without the use of humans or experimental primates, with a new PvCS 
formulation which, although not corresponding to the studies proposed here, represents a 
promising path for this antigen and for other pre-erythrocytic candidates in the difficult field 
of vaccines against P. vivax.  
 
 
 
 
Figure 6: Evaluation of the immunogenicity and protective efficacy of the PvCS VK210-SAPN vaccine 
evaluated in mice. (a) Anti-PvCS antibody titers evaluated by ELISA in mice immunized with different 
combinations of immunostimulants (flagellin and CpG). (b) Immune response and activity induced in 
combination with immunostimulants and PvCS VK210-SAPN. 
 
 
2.4.3. Phase I clinical trials with P. vivax CS protein 
 
 
First Phase I clinical trial 
 
Based on the results of the preclinical phase, in 2000 the CIV decided to initiate Phase 
I clinical studies to determine the safety, tolerability, and immunogenicity of three (3) long 
synthetic peptides (LSPs) derived from PvCS in human volunteers residing in Cali, an area 
without malaria transmission, and subsequently conduct clinical studies aimed at 
standardizing a method for infecting healthy volunteers with viable P. vivax sporozoites, in 
preparation for the development of protective efficacy trials in subsequent Phase II trials of 
the vaccine. 
The initial studies were conducted in Cali, jointly by the CIV and the Fundación Clínica 
Valle de Lili, and were financially supported by Colciencias, the Ministry of Social 
Protection, and the US National Institutes of Health (NIH/NIAID). They were monitored by 
the WHO and are briefly described below. 
This Phase I study evaluated the safety, tolerability, and immunogenicity of PvCS LSPs 
in young (18-45 years old), healthy volunteers with no history of malaria. This trial was a 
randomized, double-blind study conducted in 69 male and female volunteers with no previous 

 
 
 
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exposure to malaria, as demonstrated by medical history and serological tests, who met all 
the inclusion criteria. The volunteers were immunized with the three LSPs corresponding to 
different regions (Amino= N; Central e Repetitive= R, Carboxyl= C) of PvCS, formulated in 
the Montanide ISA-720 adjuvant (Seppic, Inc.). The three peptides were administered in 
escalating doses of 10 µg, 30 µg, and 100 µg, which were safe and proved to be well tolerated 
and highly immunogenic. The incremental doses of the vaccine were also administered in a 
staggered manner over time; only after the first two doses of 10 µg had been administered 
was the 30 µg dose tested, and the same strategy was used for the 100 µg dose. 
Although the volunteers presented minor signs and symptoms at the injection site, none 
developed serious or severe adverse events (AEs). Table 1 describes the occurrence of AEs 
recorded after the administration of the different doses (see Annex IV Safety and Efficacy in 
the Investigator's Manual). 
 
 
 
 
Table 1: Adverse events observed after immunization with different doses of the PvCS vaccine (10, 30, and 100 
µg) compared to the control group.  
 
 
After immunization, all individuals seroconverted as demonstrated by the ELISA 
technique, although there were differences in antibody titers against the different peptides. 
These antibodies recognized the native protein in P. vivax sporozoites in IFAT tests and 
demonstrated the ability to block sporozoite invasion of liver cell lines in vitro, as assessed by 
the sporozoite invasion inhibition (ISI) test (Arévalo-Herrera, 2011b). 
 
 
 

 
 
 
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Figure 7: Specific antibodies against PvCS fragments, measured by (a) ELISA (b) IFAT (c) Inhibition 
of sporozoite invasion of human hepatoma cells (HepG2 in vitro). 
 
 
 
The CIV subsequently conducted a study of the cellular and antibody immune response in 
21 of the 69 patients in this clinical trial (Arévalo-Herrera, 2011b). The antibodies were 
predominantly of the IgG1 and IgG3 isotypes and again showed recognition of parasitic 
proteins (IFAT) and partial blocking of invasion in the ISI test. The high antibody response, its 
activity in blocking liver invasion, and the stimulation of in vitro IFN-γ production by 
peripheral blood mononuclear cells in most volunteers provided evidence and encouraged 
further studies. The first Phase I clinical trial was therefore successful and led to a new trial to 
optimize the vaccine formulation (Herrera, 2011a).  
 
 
 
 
 
Figure 8: Specific antibody response against PvCS N, R, and C peptides in volunteers immunized during Phase 
I clinical trial.  
 

 
 
 
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Second Phase I clinical trial with combined peptides. 
 
A study was designed to determine the safety, tolerability, and immunogenicity of a 
mixture of LSPs formulated in Montanide ISA-720 and Montanide ISA-51 (SEPPIC Inc.), two 
of the most potent adjuvants for use in humans (Herrera, 2011a). This second Phase I clinical 
trial was proposed to identify which of these two adjuvants generated better safety, tolerability, 
and the optimal dose for use in subsequent Phase II trials. 
The trial consisted of a randomized, double-blind, dose-escalation controlled clinical study. 
Forty healthy volunteers from the city of Cali who had not previously been exposed to malaria 
were randomized to receive three injections of the mixture of synthetic peptides N, R, and C at 
doses of 50 µg or 100 µg/dose/peptide, formulated in the Montanide adjuvants described, and 
the corresponding control group received the corresponding adjuvant as a placebo. The first 
immunization consisted of a mixture of the N+C peptides, while the second and third 
immunizations were mixtures of the three peptides N+C+R, a strategy aimed at reducing the 
immunodominance of the R region, described previously, which could affect the protective 
capacity induced by the other two fragments N and C. Once again, the vaccines were well 
tolerated and there was no association with serious or severe AEs. Table 2 describes the 
occurrence of AEs recorded after the application of the different formulations (adjuvant and 
dose) (see Annex IV Safety and Efficacy in the Investigator's Manual). 
 
 
 
 
Table 2: Adverse events related to immunization reported in healthy volunteers after administration of different 
formulations of PvCS LSP in Montanide ISA-51, Montanide ISA-720, or placebo adjuvants.  
 
The antibody response determined by ELISA again showed seroconversion, but as in the 
previous trial, the N peptide induced earlier antibodies and higher titers. The response against 
the C and R peptides appeared after the second immunization, with 97% of volunteers 
responding against these peptides. 

 
 
 
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Figure 9: Antibody response against PvCS N, R, and C peptides in volunteers from the phase IIa/b clinical trial 
in Chocó, Colombia. 
 
 
Confirmation of the safety, tolerability, and immunogenicity of these formulations 
prompted the initiation of studies aimed at establishing an infectious challenge model with 
sporozoites for use in vaccine protective efficacy trials (see 2.4.5 Infectious Challenge Models). 
 
First Phase IIa/b clinical trial 
 
Based on the success of the two Phase I trials and the establishment of the infectious 
challenge model (described below), the CIV planned and carried out, in partnership with the 
Imbanaco Medical Center (CMI) in Cali, the development of a randomized, double-blind, 
controlled clinical trial to evaluate, as a primary outcome, the safety and protective efficacy of 
the synthetic P. vivax circumsporozoite (CS) protein formulated in the Montanide ISA-51 
adjuvant in healthy young people (aged 18-45), both men and women, including a group that 
had not suffered from malaria (naïve) (phase IIa) and volunteers with previous clinical malaria, 
considered here as semi-immune (phase IIb). Participants (n=35) were randomly selected from 
a larger group (n=121) of volunteers from Cali city without local malaria transmission (naïve 
group) and from Buenaventura, an area with indigenous malaria transmission (semi-immune 
group). The 35 participants were divided into naïve (n=17) and semi-immune (n=18) groups 
and were immunized at months 0, 2, and 6 with the PvCS protein formulated in Montanide 
ISA-51 adjuvant, or with Montanide ISA-51 adjuvant alone, as a placebo.  
 

 
 
 
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Table 3: Adverse events observed in participants in phase IIa and phase IIb clinical trials after immunization with 
the PvCS protein formulated in Montanide ISA-51, compared to the control groups. 
 
 
 
After immunization, specific antibodies and the in vitro IFN-γ release response to 
stimulation of participants' mononuclear blood cells with PvCS LSPs were evaluated; all 
experimental volunteers developed specific IgG and IFN-γ production. 
 
 
 
Figure 10: IFN-γ response against PvCS N, R, and C peptides in naïve (a) and semi-immune (b) volunteers 
immunized with PvCS formulated in Montanide ISA-51. 

 
 
 
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Figure 11: Antibody response against PvCS N, R, and C peptides in naïve (a) and semi-immune (b) volunteers 
immunized with PvCS formulated in Montanide ISA-51. 
 
 
Three months after the last immunization, all participants underwent a challenge infection 
(CHMI) with P. vivax parasites (sporozoites), inoculated through mosquito bites, using the 
method developed by the CIV, described below (see 2.4.5 Challenge infection models). The 
parasites were inoculated in a controlled manner by the bite of 2-4 Anopheles mosquitoes 
previously infected by artificial feeding through a membrane.  
Within the experimental groups, there was a dramatic reduction in parasitemia, including 
sterile protection (no infection), in several volunteers in the experimental group, both in phase 
IIa (6/11) (54%, 95% CI 0.25-0.84) and phase IIb (7/11) (64%, 95% CI 0.35-0.92), however, 
no differences in parasitemia were observed between the two phase IIb subgroups. All placebo-
vaccinated controls (except one control from the semi-immune group) became infected, as 
expected.  
During the study, mild AEs occurred, like those induced by vaccination in expanded 
immunization programs (classical vaccines), as well as mild reactions in response to mosquito 
bites during the infectious challenge. Table 4 describes the occurrence of AEs recorded after 
the application of the different formulations (adjuvant and dose) (see Annex IV Safety and 
Efficacy in the Investigator's Manual). 
In conclusion, this study indicated significant protection in both naïve and semi-immune 
volunteers (Arévalo-Herrera, 2022).   

 
 
 
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Table 4: Results of the infectious challenge in naïve (phase IIa) and semi-immune (phase IIb) volunteers 
immunized with PvCS formulated in Montanide ISA-51. 
 
 
 
Figure 12: Kaplan-Meier curves of survival without parasitemia in naïve (a) and semi-immune (b) volunteers 
after infectious challenge. 

 
 
 
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To the best of our knowledge and after a systematic literature search, this study is the first 
report of a clinical trial of protective efficacy in P. vivax indicating these levels of protection. 
A previous study conducted by the Walter Reed Army Institute of Research (WRAIR) and 
reported in news reports and clinical records indicated that the vaccine evaluated did not 
achieve significant protection against parasitemia; it did show a small but significant delay in 
the time to parasitemia in a percentage of vaccinees (≈59%), and led to the conclusion that it 
was necessary to optimize candidates and models before advancing to field trials (Bennet, 
2016). 
These results prompted a clinical trial of the PvCS vaccine in a larger number of volunteers 
and in other endemic areas of the country with a higher prevalence of malaria, such as the 
Department of Chocó. For logistical reasons, the study will be conducted in Quibdó, a 
municipality with better infrastructure and health conditions than the rest of the department.   
 
2.4.4. 
Establishment of infectious challenge models for P. vivax. 
 
Although chronologically the establishment of the controlled human malaria infection 
model (CHMI) was developed prior to the phase II clinical trial, it is included here so as not to 
alter the sequence of the safety studies (phase I) and the protective efficacy study (phase II) 
reported above. Therefore, we describe below the background of experimental human infection 
with malaria.  
   
The possibility of experimentally infecting human volunteers with Plasmodium is a practice 
that has been carried out for nearly 100 years (Grassi, 1899) (Fairley, 1947), and during the 
1950s and 1960s, experimental malaria infection was routinely used as a treatment for 
neurosyphilis ("malaria therapy") (Glynn, 1995). Later, a challenge model with P. falciparum 
sporozoites was developed and used to test the protective efficacy of a vaccine against P. 
falciparum based on radiation-attenuated sporozoites (Clyde, 1975; Clyde, 1973; Egan, 1993; 
Herrington, 1991; Rieckmann, 1979). More recently, the P. falciparum challenge model was 
improved and used extensively in major research centers around the world, primarily at the 
Naval Medical Research Center (NMRC) in the United States (Rockville, MD) (Hoffman, 
2002), The Center for Clinical Vaccinology and Tropical Medicine, Oxford University 
(Walther, 2005) and the Department of Medical Microbiology at the University Medical Center 
in Nijmegen, Netherlands, and more recently at SANARIA Inc. under the direction of Dr. S. 
Hoffman (Hoffman, 2015), to determine the protective efficacy of various vaccines based 
mainly on the use of whole attenuated parasites. However, the challenge with P. vivax 
sporozoites was only used in a few patients and about 40 years ago. 
 
Given the need to develop vaccines against the hepatic forms of P. vivax (pre-erythrocytic 
stages) and, in particular, the need to test the protective efficacy of the PvCS protein, the CIV 
invested significant efforts in developing a Controlled Human Malaria Infection (CHMI) 
challenge system (Controlled Human Malaria Infection - CHMI) under controlled conditions 
of biosafety, volunteer protection, statistical significance, and reproducibility, taking into 
account the enormous logistical difficulties involved in working with this species.  

 
 
 
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2.4.5. Standardization of the challenge model with P. vivax sporozoites for the 
evaluation of protective efficacy 
 
The lack of continuous in vitro cultures of P. vivax creates the need to use parasites from 
infected patients, which is why work on this species must be carried out in endemic areas with 
greater access to patients with parasites circulating in peripheral blood. For obvious reasons, 
this work is extremely difficult in non-endemic areas and in developed countries without 
malaria transmission.  
 
Faced with the need for a method to test the protective efficacy of the described PvCS 
candidate and other parasite antigens, the CIV, together with partners such as the Pacific Health 
Institute, ASOCLINIC Ltda., and others, has worked systematically on the standardization, 
optimization, and use of infection protocols using parasites from patients infected with P. vivax 
from the endemic area of the Pacific coast of Colombia, specifically Buenaventura (Valle). 
 
Using the facilities of the Pacific Health Institute, particularly its malaria diagnostic service, 
and a colony of An. albimanus laboratory mosquitoes, preliminary studies were conducted to 
determine the susceptibility of mosquitoes to experimental infection under captive conditions, 
and optimal conditions were established to achieve reproducible infections (Hurtado, 1997). 
Subsequently, another experimental colony of Anopheles was established at the CIV 
headquarters in Cali, with the required biosafety measures.  The biosecurity of these colonies 
includes, but is not limited to, the use of enclosed spaces, restriction of access by personnel not 
belonging to the unit, use of safety suits for operators, use of aspirators and light traps, and the 
use of security locks.  
 
Under these conditions, a first clinical challenge trial in humans was conducted, in 
which 17 out of a total of 18 healthy young volunteers (aged 15-45), both men and women, 
were successfully infected. They had been screened to determine the presence of the Duffy 
group in their red blood cells (Fy+), which is essential for the parasite to invade the blood. 
Individuals (Fy-) are refractory to P. vivax infection. In addition, the normality of the glucose-
6-phosphate dehydrogenase (G6PD) protein was examined, an enzyme that protects the 
integrity of cell membranes and whose deficiency can affect patients' tolerance to primaquine 
(PQ) treatment. The volunteers were exposed to P. vivax sporozoites administered through the 
bites of batches of 2 to 10 mosquitoes. All volunteers exposed to the bites developed malaria, 
with pre-patent periods of 9-11 days, determined by thick smear (TS) and PCR, and all were 
treated immediately upon confirmation of the presence of parasites in the blood, when 
parasitemia was minimal. The volunteers were closely monitored clinically and 
parasitologically to ensure early detection of potential adverse events (AEs), which were mild 
and most frequently related to mosquito bites; these events resolved within 24 to 72 hours after 
curative treatment (Herrera, 2009a; Herrera, 2009b).  The study showed that there was no 
difference in the development of patent parasitemia in volunteers exposed to the different doses 
used, which is why it was decided that in future studies the lowest dose consisting of 3+1 bites 
from infected mosquitoes, confirmed microscopically, will be used. The minimum effective 
dose used here (2 infected mosquitoes) contrasts with the minimum dose used in P. falciparum 

 
 
 
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models in the United States and Europe, where at least 5 infected mosquitoes have been 
required to ensure infection in Controlled Human Malaria Infection (CHMI) studies 
(Roestenberg, 2013). Table 5 describes the occurrence of AEs recorded after volunteers were 
exposed to mosquito bites (see Annex IV Safety and Efficacy in the Investigator's Manual). 
 
 
 
 
 
Table 5: Local adverse events associated with P. vivax-infected mosquito bites in naïve volunteers included in 
the challenge model. The most frequent events were pruritus and erythema, both reported in 12 cases in total. All 
events were mild and transient. 
 
 
 
To ensure the reproducibility of infection with potentially different P. vivax strains, a 
second clinical challenge trial was conducted in healthy volunteers selected using the same 
parameters as in the previous study. In addition, a control group of five volunteers selected for 
being Fy- and consequently refractory to P. vivax infection (n=20) was included. They were 
randomly assigned to groups 1, 2, and 3, which were exposed to doses of 2-4 bites from 
mosquitoes infected with parasites from three different donors, with the aim of determining 
their possible variability when exposed to genetically potentially different parasites.  This time, 
infection occurred in all volunteers, with pre-patent periods like those in the first study, 
confirming the reproducibility of the challenge system (Solarte, 2011), except in Fy- 
volunteers.  The AEs were again mild and like those in the previous study, and the volunteers 
were treated as soon as the infection in peripheral blood was diagnosed by thick smear 
examination. Table 6 describes the occurrence of AEs recorded after the volunteers' exposure 
to the bite of the mosquitoes (see Annex IV Safety and Efficacy in the Investigator's Manual). 
 
 

 
 
 
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Table 6: Adverse events associated with the challenge in naïve and semi-immune volunteers. The frequency, 
severity, and duration of symptoms recorded after infection are shown, including fever, chills, headache, 
myalgia, arthralgia, asthenia, among others. In general, the events were mild to moderate, self-limiting, and 
comparable between groups, with no serious complications. 
 
 
Finally, with the aim of accelerating the clinical development of the vaccine against P. 
vivax and taking into account that, following studies in malaria-free areas such as Cali, 
vaccines should be evaluated in endemic communities with a semi-immune population due 
to their previous contact with the parasite, a third clinical challenge trial was conducted with 
experimentally induced infections in naïve and semi-immune volunteers, which aimed to 
compare the evolution of malaria infection in individuals with and without malaria 
experience. In this trial, seven healthy adults with no malaria experience and nine semi-
immune adults (total n=16) were subjected to bites from 2–4 Anopheles mosquitoes infected 
with parasitized blood from a single donor; all volunteers developed infections confirmed by 
microscopy and RT-qPCR. No significant differences were observed in the pre-patent period 
(mean 12.5 and 12.8 days for naïve and semi-immune patients, respectively). The volunteers 
developed classic signs and symptoms of malaria, while the semi-immune volunteers showed 
minor symptoms or no symptoms on the day of diagnosis. The infection induced an increase 
in specific antibody levels in both groups; this trial was safe and reproducible with mild AEs 
similar to the two previous studies (Arévalo-Herrera, 2022). Table 7 describes the occurrence 
of AEs recorded after exposure of volunteers to bites from mosquitoes (see Annex IV Safety 
and Efficacy in the Investigator's Manual). 
 
 
 

 
 
 
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Table 7: Local adverse events associated with the bite of infected mosquitoes in naïve and semi-immune 
volunteers. The main events reported were pruritus, erythema, and pain at the bite site, which were mild to 
moderate in intensity and of short duration. No serious events were observed. 
 
 
Once the remarkable safety and reproducibility of this infectious challenge model (CHMI) had 
been established, it was proposed to determine the efficacy of immunization of naïve and semi-
immune volunteers with the synthetic N+R+C peptides of PvCS (Herrera 2009, Herrera 2011) 
described above (section 2.4.6), and that of naïve, healthy volunteers with irradiated 
sporozoites described below (Arévalo-Herrera 2016a).  
As mentioned previously (section 2.3), in 1967, at the beginning of the search for a malaria 
vaccine, Clyde and colleagues conducted a study in which healthy volunteers were exposed to 
vaccination with irradiated sporozoites inoculated through mosquito bites. This study, 
conducted in the United States (Maryland, Walter Reed Army Institute of Research), 
demonstrated for the first time the extraordinary efficacy of this vaccination, which achieved 
protection greater than 90–95% for a duration of approximately 12 months (Nussenzweig, 
1967, Clyde, 1973). 
Subsequently, multiple studies with similar protocols were conducted and, as described in the 
last decade, Stephen Hoffman and his company SANARIA Inc. have worked to industrialize a 
vaccine that uses irradiated, dried, and cryopreserved sporozoites for intravenous 
administration (Hoffman et al., 2010; Epstein et al., 2011). 
With regard to P. vivax, the model of immunization with radiation-attenuated sporozoites was 
only tested in 1970 by Collins et al. in two volunteers, and then not again until 2014, when the 
CIV (Cali, Colombia) conducted the first human study with P. vivax, which evaluated the safety 
of experimental exposure to infection by mosquitoes infected with irradiated and non-irradiated 
sporozoites, thus establishing the Controlled Human Malaria Infection (CHMI) platform for 
P. vivax (Arévalo-Herrera et al., 2014, 2016). 

 
 
 
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Table 8: Systemic adverse events observed during immunizations and after CHMI in volunteers. In the group 
vaccinated with radiation-attenuated sporozoites (n=14), the most frequent symptoms were headache, fever, 
myalgia, chills, and nausea, mostly mild or moderate and without serious outcomes. In the control group (n=7), 
fewer events were reported, mainly limited to mild headache and fever after immunization or CHMI. 
 
 
2.5. State of the art 
 
The recent approval by the WHO to proceed with an implementation phase for the RTS,S and 
R21 vaccines against P. falciparum represents an important achievement for the international 
scientific community in general and, in particular, for their developers, Glaxo Smith Kline, and 
the PATH Malaria Vaccine Initiative consortium, which for nearly four decades have pursued 
the goal of developing a vaccine that is sufficiently effective, cost-effective, and accessible to 
communities affected by malaria globally. On the other hand, it is also a significant 
achievement for the University of Oxford, which has worked intensively to improve the 
protective capacity of a vaccine related to the former; the two represent the most advanced 
vaccines in development worldwide. 
 
Pf-RTS,S is a recombinant chimeric protein composed of fragments from the central and 
carboxyl-terminal regions of the P. falciparum circumsporozoite protein (Pf-CS), linked to the 
hepatitis B virus surface antigen (HBsAg), initially formulated in AS02A adjuvants and 
subsequently in AS01E. The proportions of the protein correspond to approximately 25% Pf-
CS fragments and 75% HBsAg. This vaccine was a pioneer in the development of malaria 
vaccines and paved the way for others in research. In phase II studies formulated with AS02A, 
it induced protection of close to 34%, but when replaced with the AS01E adjuvant, efficacy 
increased to 50–55% in children in endemic areas. 

 
 
 
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Pf-R21, which is very similar to Pf-RTS,S, optimized its structure by increasing the proportion 
of Pf-CS relative to HBsAg and reducing the number of redundant epitopes, as well as being 
formulated with the Matrix-M (MM) adjuvant, which has greater immunostimulatory potential. 
Both vaccines have been tested in endemic communities in several African countries (including 
Ghana, Malawi, and Kenya for Pf-RTS,S; and Burkina Faso and Mali for R21). They have now 
been approved by the WHO for pilot implementation programs in several African countries. 
On the other hand, several vaccines based on the use of live parasites attenuated by radiation 
(Radiated Attenuated Sporozoites-RAS) or genetic manipulation (Genetically Attenuated 
Sporozoites-GAP) have been progressing in studies in recent years. In contrast to Pf- or PvCS-
based vaccines, these attenuated parasites, being complete, trigger responses against many of 
the more than 5,500 Plasmodium proteins. These formulations induce more vigorous and 
comprehensive responses in terms of the number of recognized antigens, but they represent a 
challenge from an industrial standpoint.  
 
As mentioned previously, the development of vaccines against P. vivax represents a 
phenomenal challenge due to the lack of continuous in vitro cultures, the scarcity of animal 
models, the limitations imposed by the lack of insectaries with access to parasite samples, and, 
consequently, limited financial support. Nevertheless, the CIV's efforts to overcome these 
obstacles have led to the development of a very comprehensive infrastructure for the study of 
P. vivax and the development of vaccines to the level discussed above, which can be 
summarized as follows: a) exhaustive immunological analysis of the PvCS protein (Arévalo-
Herrera, 1998, 2001, 2002, 2010, 2011a, 2011b); b) antigenicity studies of the protein in 
humans naturally exposed to the parasite in endemic areas (Herrera, 2009c, 2011b, Solarte 
2011); c) preclinical trials (Herrera 1994, 1997, Arévalo-Herrera 2010, 2011a, ) and d) clinical 
phase trials (Herrera 2005, 2011a, Arévalo-Herrera 2001, 2010, 2016a, 2022). Interaction with 
international colleagues and bibliographic analysis allow us to affirm that, although two 
decades ago the Walter Reed Army Institute of Research (WRAIR) in Silver Spring (MD, 
USA) produced different formulations of the P. vivax circumsporozoite protein (PvCS) that 
reached phase IIa clinical trials, these studies (Benett 2016, Lata De 2022), and probably the 
program in general, were suspended around 2003. 
More recently, a group at the University of Oxford (UK) published a preclinical study in a 
mouse model on the immunogenicity and protective efficacy of the PvCSP-21  protein (known 
as Pv21), formulated in the Matrix-M™ adjuvant (Novavax AB, Sweden), against challenge 
with a transgenic P. berghei parasite expressing PvCS. This work demonstrated high 
immunogenicity and significant levels of protection (Salman 2017). However, to date, there 
has been no concrete progress with this candidate toward clinical development phases in 
humans. 
Given this background, the progress made by the CIV in Colombia probably represents the 
most advanced P. vivax vaccine candidate in development worldwide. This progress is due to 
the comparative advantages of the CIV and the country described above, in terms of: 1) access 

 
 
 
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to endemic communities in different latitudes (continents); 2) availability of insectaries in 
endemic and non-endemic areas; 3) access to animal facilities with rodents and non-human 
primates; 4) laboratory infrastructure with capacity for immunological, proteomic, chemical, 
and clinical studies; 5) cooperation with top-level national clinical research centers on an 
international scale; 6) national and international  scientific partnerships and funding. 
2.6. Scientific Justification  
 
Except for Africa, where 99% of malaria cases are caused by P. falciparum infections, 
most endemic areas of the world, including Colombia, experience simultaneous transmission 
of P. falciparum and P. vivax. For this reason, despite valuable advances in the development 
of vaccines against P. falciparum, the identification and production of P. vivax antigenic 
components is required both for monovalent vaccines specific to this species and for the 
possible production of multi-species vaccines that simultaneously protect against P. vivax and 
P. falciparum. 
 
The PvCS protein was recognized four decades ago, and like PfCS and several CSPs from other 
Plasmodium species, it was shown to have the same structure, immunogenic and protective 
capabilities. This protein is highly immunogenic and contains functional regions (epitopes) that 
are essential for the parasite's invasion of host cells in humans and mosquitoes. Antibodies 
against these epitopes or domains (RI, R2, Repeat) have consistently demonstrated the ability 
to block invasion of the liver cell in in vitro and in vivo studies, as well as to generate protection 
through both active immunization and passive transfer of specific antibodies ( Atcheson 2020), 
anti-mosquito infection (RII) (Sinnis 1994) and anti-repeat (Arévalo-Herrera 1998, 2011b, 
2016, Herrera 2005, 2011a).    
Additionally, numerous pieces of evidence indicate the feasibility of developing vaccines 
against malaria and represent a solid scientific justification for the development of the proposed 
clinical study.  
 
1) Immunization with radiation-attenuated sporozoites has consistently demonstrated the 
induction of sterile immunity (>90%) to several species of parasites, including P. falciparum 
and P. vivax in animals and humans (Nussenzweig 1969, Druille 1998, Hoffman 2002, 
Arévalo-Herrera 2016a). 
2) Several groups are advancing in the development of vaccines based on the use of P. falciparum 
attenuated by different methods, including genetic manipulation of the parasites, with 
promising results (Richie TL, 2023, Van der Boor SC, 2023; Vaughan AM, 2017). 
A significant number of trials conducted over the last decade have demonstrated the protective 
capacity of the RTS-S and R-21 vaccines containing P. falciparum CS in both individuals with 
no previous exposure to malaria and in endemic communities (Guinovart, 2009; Macete, 2007) 
(Hill, 2011; WHO, 2023), currently representing the most advanced vaccines approved by the 
WHO for implementation in Africa. 
3) Although, for the reasons outlined above, vaccine research in the case of P. vivax has not 
progressed as far, the CIV has successfully developed several Phase Ia clinical trials with PvCS,  
which demonstrated the safety and tolerability of the PvCS + Montanide ISA 720 and 
Montanide ISA - 51 formulations. (Herrera, 2011a; Herrera, 2005). 

 
 
 
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4) The CIV has developed a safe and reproducible infectious challenge system essential for 
studies of the protective efficacy of antimalarial vaccines. This model has been successful in 
three (3) clinical studies using parasite isolates (strains) from different donor patients, even 
with doses as low as two (2) infected mosquitoes. (Herrera, 2011b; Herrera, 2009c, Arévalo-
Herrera 2014). 
5) The immunization of healthy, naïve volunteers in Colombia with irradiation-attenuated 
parasites (PvRAS) demonstrated significant protective efficacy in Phase II clinical trials, 
comparable to that obtained in the United States with P. falciparum, and the sera of protected 
individuals preferentially recognized PvCS, establishing a correlation between anti-PvCS 
antibodies and protection (Arévalo-Herrera M, 2016a, Lopez-Perez 2024). These reagents have 
enabled the initiation of a program to discover new antigens with potential for vaccine 
development (NIAID Contract 1U01AI155363), (Herrera SM 2025). 
6) More importantly, as evidence for the present study, the formulation proposed here, PvCS + 
Montanide ISA 51, recently demonstrated high safety, tolerability, and protective efficacy 
greater than 60% (Arévalo-Herrera M, 2022).  
7) Additionally, CIV, in association with Johns Hopkins University in Baltimore (MD) (USA), 
confirmed the protective capacity of this formulation in a murine (mouse) model that induced 
a vigorous immune response against the same formulation previously used in humans and 
equivalent protective efficacy against infection with a murine parasite (P. berghei) transfected 
with the PvCS protein (Herrera SM 2025) (see non-clinical studies). 
All these studies represent solid scientific evidence supporting the feasibility and potential 
success of the study proposed in this protocol.  
 
 
3. 
HYPOTHESIS 
 
 
● The application of the N, R, and C peptides of P. vivax CS formulated in Montanide ISA-51 
adjuvant offers protection against malaria infection in naïve individuals and those previously 
exposed to malaria in malarial areas. 
 
● Immunization with the PvCS protein is safe in volunteers previously exposed to malaria in a 
natural way (semi-immune). 
 
● Immunization of volunteers naturally exposed to malaria with the PvCS protein produces a 
booster effect on the previous immune response present in these volunteers. 
 
● Vaccination with PvCS in individuals previously exposed to malaria induces a response that 
may offer sterile immunity. 
 
 
 
 
4. 
OBJECTIVES 
 

 
 
 
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4.1.General objective 
 
To determine the protective efficacy induced by the PvCS vaccine formulated with the 
Montanide ISA-51 adjuvant in naïve and previously exposed volunteers in the department of 
Chocó. 
 
 
4.2.Specific objectives 
 
1. 
Confirm the safety of the vaccine in naïve volunteers immunized with PvCS. 
2. 
To determine the immunogenicity of PvCS in individuals previously exposed to malaria. 
3. 
Determine the protective efficacy of the vaccine against infectious challenge with viable P. 
vivax sporozoites in the above groups. 
 
 
5. 
STUDY POPULATION 
 
The study will have a total population of approximately 80 young volunteers aged 18-
45, both men and women, residing in the urban area of Quibdó (Chocó), of whom 65 will be 
volunteers selected from a larger population of individuals who agree to participate in the 
immunization process and subsequently in an infectious challenge with P. vivax parasites 
administered through mosquito bites. Of these 65 volunteers, 60 will be regular participants in 
the process and the remaining 5 will be potential substitutes (or alternates) who would 
participate only if any of the regular participants are suspended or decide to withdraw early 
from the study (before the second vaccine dose). These participants must be semi-immune 
(n=30) and naïve (n=30) volunteers who may come from malaria-endemic areas of Chocó but 
reside in Quibdó and must meet all inclusion criteria and not present any exclusion criteria to 
be included in the study. The other participants will be a group of between 5 and 15 volunteer 
parasite donors in whom acute infection with P. vivax is confirmed. 
 
The study has been structured in three main steps. In step #1, the 65 participants in the 
immunization and infectious challenge and their potential substitutes or alternates will be 
selected. In step #2, volunteer parasite donors will be selected for feeding and infecting 
Anopheles mosquitoes, and in step #3, the infectious challenge will be performed on the 60 
final immunized volunteers.  
 
5.1  Steps 1 and 3. Immunization and infectious challenge volunteers. 
 
A total of 60 volunteers, semi-immune and naïve volunteers from malaria-endemic areas 
of Chocó, who are residents of the urban area of Quibdó, who meet the inclusion criteria and 
do not present any exclusion criteria, will be included in the study. 
 
 
5.1.1.1 Group of healthy volunteers with no history of malaria (naïve). 
 
Subjects must meet all the following inclusion criteria to be selected as participants in 

 
 
 
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this study: 
 
● Healthy men and women between the ages of 18 and 45. 
● Freely and voluntarily sign an informed consent form (IC), accompanied by two witnesses 
who will also sign. 
● Be a resident of the urban area of the municipality of Quibdó. 
● No history of malaria as assessed by medical history and serology.  
● For women, use an appropriate contraceptive method from the start of the study until the 
contraceptive restriction is lifted by a study physician. 
● Agree not to travel to areas considered to have higher malaria transmission in the department 
of Chocó or Colombia during the infectious challenge period (1 month). 
● Be reachable by telephone throughout the study period. 
● Hemoglobin (Hb) levels >11 g/dl. 
● Availability to participate during the study period. 
● Not participating in another clinical study. 
 
To be selected as participants in this study, subjects must NOT meet any of the following 
exclusion criteria. 
 
● Confirmed pregnancy by laboratory test, breastfeeding, plans to become pregnant from the 
time of recruitment. 
● Duffy negative phenotype (Fy-). 
● G-6-PD deficiency (d-G-6-PD). 
● Any hemoglobinopathy. 
● Personal history of allergies to medications or insect bites. 
● Having received vaccination against malaria. 
● Positive IFAT antibodies > 1:20 or ELISA greater than 1:200 for P. vivax in screening tests. 
● Living in a malaria-endemic area during the 6 months prior to the study (municipalities with 
recognized endemic areas in Chocó, the Pacific Coast, and others). 
● Clinical or laboratory evidence of systemic disease, including kidney, liver, cardiovascular, 
lung, psychiatric, or other diseases that could have a significant negative impact and alter the 
study results. 
● Evidence of active hepatitis B and C or HIV infection. 
● History of transfusion of any blood product in the six (6) months prior to the study. 
● Plans to undergo surgery from the recruitment period until the end of post-challenge follow-
ups. 
● Presence or history of autoimmune disease (lupus, rheumatoid arthritis, thyroiditis, or other). 
● Splenectomized volunteers. 
● Volunteers undergoing treatment with drugs that affect the immune system (steroids, 
immunosuppressive agents, or immunomodulators). 
● History of alcoholism or drug abuse is defined as a habit that interferes with the individual's 
normal social functioning. 
● Any condition that may interfere with the ability to provide free and voluntary Informed 

 
 
 
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Consent (IC). 
 
5.1.1.2 Healthy volunteer group with a history of malaria (semi-immune) 
 
Subjects must meet all of the following inclusion criteria to be eligible to participate in this 
study: 
 
● 
Non-pregnant, healthy men and women between the ages of 18 and 45. 
● 
Freely and voluntarily sign an IC, accompanied by two witnesses who will also sign. 
● 
Have a history of malaria infection(s) and positive serological tests for P. vivax. 
● 
Be a resident of the urban area of the municipality of Quibdó 
● 
For women, use an appropriate contraceptive method from the start until the contraceptive 
restriction is lifted by a study physician. 
● 
Agree not to travel to areas considered endemic for malaria during the infectious challenge 
period (1 month) (municipalities with recognized endemic areas in Chocó, the Pacific Coast, 
and others). 
● 
Be reachable by phone throughout the study period. 
● 
Availability to participate during the period in which the study will be conducted. 
To be selected as participants in this study, subjects must NOT meet any of the following 
criteria: 
● 
Be negative for antibodies against P. vivax by the IFAT method <1:20 or ELISA less than 
1:200 in screening tests. 
● 
All other criteria used in the case of naïve volunteers, except for a history of living in the 
endemic area during the last 6 months. 
 
To be selected as participants in this study, subjects must NOT meet any of the following 
exclusion criteria described above, except for a history of malaria determined by clinical history 
and serological tests: 
 
5.2  Step 2. Volunteers donating blood infected with P. vivax 
 
A total of 5-15 people infected with P. vivax who attend diagnostic centers in Quibdó or 
other malaria-endemic areas near Quibdó, with detected parasitemias ≥ 0.1% and who meet the 
inclusion criteria for parasite donors and do not have any exclusion criteria, will be included in 
the study. 
 
To be selected as volunteer blood donors, subjects must meet all the following inclusion 
criteria:  
 
● Non-pregnant men and women between 15 and 60 years of age 
● Have a positive diagnosis of P. vivax malaria determined by thick smear examination. 
● The patient must not have other detectable Plasmodium species in their bloodstream, 
such as P. falciparum or P. malariae. 
● Have a P. vivax parasitemia ≥ 0.1% by thick smear. 

 
 
 
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● Have Hb ≥9 g/dL at the time of malaria diagnosis. 
● Be able to provide informed consent freely and voluntarily. If illiterate, be able to affirm 
their decision to participate by placing their fingerprint on the informed consent form. 
Minors between the ages of 15 and 17 who wish to participate must sign the informed 
consent form, and one of their parents must sign the informed consent form, accompanied 
by two witnesses who will also sign. 
 
To be selected as volunteer blood donors, subjects may NOT meet any of the following 
criteria: 
● Have chronic or acute diseases other than P. vivax malaria. 
● Have a history of diseases or clinical conditions that, according to medical criteria, 
could significantly increase the risk associated with participation in this study. 
● Hb levels <9 g/dL at the time of recruitment. 
● Have received antimalarial treatment prior to diagnosis.  
● Have a history of diseases or clinical conditions that, according to medical criteria, could 
significantly increase the risk associated with participation in this study. 
 
6. 
STUDY DESIGN 
Diagram #3 provides an overview of the main activities of the study, which are described in 
detail below. These activities include: 1) the immunization process, 2) the infectious 
challenge, 3) clinical and laboratory analysis to determine the immunogenicity of the 
formulation administered and the laboratory parameters used to determine safety. 
Additionally, it indicates 4) the scheduled times for evaluation by the DSMB. 
 
 
Figure 13: Summary diagram of the main procedures of the study. 
 
6.1 Recruitment process 
 
Candidates will be recruited through radio messages targeting the general population and 
through graphic material (posters, flyers) targeting those who attend health facilities, with an 
emphasis on the diagnostic centers of the Vector-Borne Diseases (ETV) network of the SDS 

 
 
 
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of Chocó in Quibdó. Candidates must be residents of the urban area of Quibdó, although they 
may come from municipalities such as Tadó, Lloró, Alto Baudó, and others. All candidates for 
this study must meet the inclusion criteria and will be excluded if any of the exclusion criteria 
are present. Naïve volunteers will be those who, based on their medical history and serological 
tests (ELISA, IFAT), are determined to be negative for antibodies against P. vivax and P. 
falciparum, and individuals who have antibodies against P. vivax will be considered semi-
immune. Some volunteers may react serologically with P. falciparum, but this reactivity will 
not be considered for admission. 
 
During the call for volunteers, only individuals who are affiliated with the general social 
security health system (SGSS), in any of its regimes, can prove it with an official document 
will be considered. Eligible volunteers must sign up for an IC after being instructed by a team 
member on the purpose of the study. The person(s) providing this information will try to 
simplify the language to make it understandable to all candidates, avoiding the use of technical 
terms as much as possible. In addition, volunteers will be given a verbal test of their 
understanding of the study, particularly regarding the implications of informed consent (IC), 
the concept of blinding, confidentiality, and the risks and benefits of participating in the study, 
among others, to ensure their freedom and acceptance of the IC.  
 
If it is found that the potential volunteer donor does not understand the study, up to three 
attempts will be made to explain it to them, and if the lack of understanding persists, the subject 
will not be included. Understanding of their participation in the study will be ensured by asking 
them to repeat the explanation given. Any elements that are not fully understood through this 
test will be explained again by the evaluator. Additionally, they will be encouraged to ask 
questions if they have any doubts. All IC procedures and volunteer questionnaires will be 
documented in the participants’ table. In addition to the IC for participation in the vaccine trial, 
an IC form will be signed for HIV testing. If the individual is HIV-positive, they will be 
informed and referred for counseling and treatment. All volunteers will receive a copy of the 
IC. 
 
6.1. Identification process  
 
Volunteers will be assigned a five-character identification code: characters #1, 2, and 3 
correspond to the project initials: Vaccine CS (VCS), character #4 is the description of the 
process (1: Immunization; 2: Infectious challenge; and 3: Selection of P. vivax-infected donors 
for mosquito feeding). Characters #5-6 correspond to the Quibdó site (12). The next two 
characters,#7-8, will be the number randomly assigned to each volunteer according to the 
randomization table for each group designated by the person in charge. Example: VCS11201 
would correspond to being the first volunteer immunized in the randomly and blindly assigned 
group. 
 
 

 
 
 
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Figure 14: Structure of the identification code for volunteers in the CS vaccine study. 
 
 
6.2. Selection process 
 
The selection procedures (medical history, physical examination, and blood sampling) will 
be carried out after the volunteer signs the IC. If a volunteer has been selected and 
immunization has not begun within 12 weeks, the selection tests will be repeated. At the 
screening visit, medical history and concomitant therapy will be documented by one of the 
clinical investigators. A complete physical examination will be performed, including 
assessment of the sensory organs, cardiovascular (CV), pulmonary, neurological, 
gastrointestinal (GI), musculoskeletal, and dermatological systems. A spontaneous urine 
sample and 35 mL of blood will be collected for laboratory screening tests. When a woman is 
considered eligible for the study, she will be asked to use a hormonal method of contraception 
during the clinical trial period until the follow-up period is completed, at which time a final 
pregnancy test will be performed. 
 
A physician at the study site (Quibdó) will oversee the clinical and paraclinical evaluation of 
volunteers during recruitment and selection for the study. 
 
The following volunteer screening tests will be performed within 12 weeks prior to the first 
immunization: 
 
Hematological tests: 
Complete blood count, G-6-PD determination, Fy phenotype, hemoglobin electrophoresis, 
blood classification, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). 
 
Renal function tests: Urinalysis, creatinine, and BUN. 
 
Infectious diseases other than P. vivax: 
HIV (two rapid tests), Hepatitis B surface antigen (HBsAg), Hepatitis C virus (HCV), Human 
T-cell Lymphotropic Virus (HTLV) 1 and 2, RPR test for syphilis, and rapid test for Chagas 
disease. 

 
 
 
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Confirmatory tests: 
If the result of either (or both) rapid HIV tests is positive, a fourth-level confirmatory ELISA 
test will be performed. 
 
If the RPR is positive for syphilis (at any dilution), an FTA-ABS test will be performed. 
 
If HBsAg is positive, a Hepatitis B anti-Core antibody test (anti-HBc) will be 
performed. 
 
Liver function tests: 
ALT, AST, total bilirubin, conjugated bilirubin, PT, and PTT.  
Pregnancy test: determination of β-HCG in urine and serum. 
OTHERS: 
Blood glucose, electrocardiogram. 
 
Immunological tests: 
IFAT: for antimalarial antibodies and ANAs (antinuclear antibodies) 
 
6.3. Group formation 
 
A total of 60 people between the ages of 18 and 45 who voluntarily agree to participate in 
the study by signing an informed consent form and who meet the inclusion criteria will be 
included in the study and assigned to one of two main groups: naïve (groups A, n=30) and 
semi-immune (groups B, n=30) and will be randomly distributed into four subgroups 
corresponding to two experimental subgroups (A1 and B1) of 20 individuals per subgroup and 
two control subgroups (A2 and B2) of 10 individuals each. In the event of withdrawal of any 
volunteer, only those who withdraw from the study before the second immunization (month 
two) will be replaced; after this stage, it will not be possible to replace volunteers.   
The vaccination schedule for new volunteers (replacements) will be adjusted so that their last 
vaccine dose (3rd) matches that of the other volunteers. Volunteers will be stratified into 
subgroups as shown in Table 1: 
 
 
 
Experimental 
 
Control 
 
 
  
Naïve 
A1 (n=20) 
 
A2 (n= 10) 
 
 
 
 
Semi-immune 
B1 (n= 20) 
 
B2 (n= 10) 
 
 
 
 
Total 
40 
 
20 
 
Table 9: Group formation (Step 1) 
 

 
 
 
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6.4. Immunization process  
 
6.4.1. Group formation 
 
Groups A1 and B1 (n = 20 volunteers/group) will be immunized with the vaccine, and 
groups A2 and B2 (n = 10 each) will be immunized with placebo [See immunization schedule 
(Figure 2)]. The preparation of the assigned vaccine will be pre-coded to reduce potential 
selection biases and balance potential confounding factors, and its code translation must remain 
blind to the vaccinating investigator and clinical study staff (Herrera, 2005). To ensure double 
blinding of the study, the statistical staff will be responsible for randomizing the volunteers' 
codes and assigning them to the experimental and placebo groups. Software (OxMaR) will be 
used for randomization. 
 
Once the scientific staff has formulated the vaccine, a member of the data management group 
will assign the codes of the subjects in each group to a sealed envelope, which will be delivered 
to the clinical staff who will immunize the volunteers with knowledge of the composition of 
the groups. Neither the principal investigator nor the laboratory staff nor the statistician in 
charge of data analysis will be allowed to know this composition. Before each immunization, 
each volunteer will be evaluated by one of the research physicians. If the volunteer reports the 
presence of any AE, it will be recorded on a Case Report Form (CRF). To determine the safety 
and tolerability of the vaccine, groups A1 and B1 will receive three doses of the vaccine. The 
first dose will consist of a mixture of peptides N and C only (50 µg/peptide; total dose 100 
µg/dose) formulated in Montanide ISA 51, immunizations 2 and 3 will consist of peptides N, 
R, and C (50 µg/peptide/dose; total 150 µg/protein/dose). This peptide escalation strategy is 
aimed at reducing the volunteer's exposure to the central R fragment, considered 
immunodominant under natural conditions, which apparently competes with protective 
responses from the N and C fragments. Volunteers in groups A2 and B2 will be immunized 
with a placebo consisting of a saline emulsion in the same adjuvant. The immunization phase 
will last 6 months. 
 
6.5.2 Vaccine formulation 
 
The vaccine will be prepared directly in Quibdó by a pharmaceutical chemist (QF), as 
described in Annex 1 (Biosafety); this QF will have access to the composition of the groups. 
Using a 1 mL syringe with a 25G needle, a total of 500 µL of solution will be taken. The 
vaccine will be injected intramuscularly into the deltoid muscle opposite to the one used for 
blood sampling. A physician will oversee administering the vaccine to volunteers during this 
phase. 
 
 

 
 
 
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Figure 15: Immunization schedule. 
 
 
6.5. Post-immunization follow-up 
 
6.5.1. Immediate follow-up 
 
Volunteers will be under direct medical observation for one hour after immunization to 
detect any adverse reactions to the vaccine injection. After a one-hour observation period, a 
physical examination will be performed to confirm the volunteer's condition. If the volunteer 
is physically well, they will be discharged and may leave the vaccination site. Eight hours after 
immunization, each volunteer will receive a phone call to check on their physical condition. 
Any manifestation of AE will be reported as detailed in the AE section. 
In the event of any serious acute AE that compromises the patient's physical stability, e.g., 
severe allergic reaction (anaphylaxis), it will be treated immediately by the treating physician 
using specific equipment and medications (Epinephrine 1:1000, diphenhydramine, cimetidine, 
and methylprednisolone). If hospital care is required, the volunteer will be transported by 
available ambulance, accompanied by the study physician, to San Francisco de Asís Hospital, 
where an ICU is available if necessary.  
 
 

 
 
 
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6.5.2. Late follow-up 
 
Additionally, personal follow-ups will be conducted the day after each immunization and then 
two weeks before the next immunization. These follow-ups will include a new clinical 
evaluation and AE report. Volunteers will be provided with all the information required to 
contact members of the research team at any time (including cell phone numbers); in addition, 
they will be encouraged to ask questions if they have any concerns. In this phase of the study, 
the clinical and paraclinical follow-up of the immunized volunteers will be carried out by the 
study physician.  
 
6.6. Step 2: Donation of infected blood to produce sporozoites 
 
In this step, patients infected with P. vivax who consult the laboratories of IPS ASOCLINIC 
Ltda. or others in the public malaria diagnosis network in Quibdó and referred to ASOCLINIC 
Ltda. will be identified for parasite donation. Each selected donor will be asked to sign an 
informed consent form freely and voluntarily prior to donating parasitized blood.  
 
From each donor, 35 mL blood will be obtained by venipuncture of the arm, which will be 
distributed as follows: 5 mL will be used for infectious disease screening like that used in blood 
banks and that performed on naïve and semi-immune volunteers. The remaining 30 mL will be 
used to feed batches of adult An. albimanus mosquitoes. Infectious disease screening tests will 
be performed in the laboratory and mosquito infection will be carried out in the ASOCLINIC 
Ltda. insectarium, located in the same building (Pacific Health Institute - INSALPA) in 
Quibdó, in accordance with standard protocols. 
 
 
 6.7.1 Donor recruitment 
 
Patients will be seen by a certified bacteriologist or microscopist, who will perform the 
thick smear test and read the results. If the result is positive for P. vivax without other 
concomitant species and if the parasitemia is ≥ 0.1%, a physician assigned to the study will 
explain to the donor the methodology and objectives of the study and invite them to participate 
in the clinical trial as a parasite donor. If the patient agrees to participate in the study, they will 
be asked to sign two informed consent forms: the first for their participation as a parasite donor, 
and the second for HIV testing. 
 
6.7.2 Patient identification and collection of the parasitized blood sample 
 
Volunteer donors will be assigned an identification code with two characters preceded by 
the study code, as explained previously in section 6.2: Example: VCS11201: 01 would 
correspond to the first immunized volunteer in the randomly assigned and blinded group. Blood 
samples will have a code following the patient identification according to the time of collection 
and type of sample taken (see Table 9) as follows: Example: VCS1120156: the time was 
collection 1 at the time of challenge and 6 corresponds to a sample taken on filter paper. 

 
 
 
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Table 9: Structure of the identification code for blood samples. 
 
 
6.7.3. Blood donation and donor treatment 
 
Once the volunteer has signed the IC and been assigned an identification code, one of the 
doctors from the research group will take a medical history and perform the corresponding 
physical examination. This step will result in one of two possible situations: 
 
 The subject does not meet the inclusion criteria: they will be given medication for the 
treatment of malaria, in accordance with the national guidelines of the Colombian 
Ministry of Social Protection (MPS). In addition, the reasons why they will not continue 
in the study and therefore will not donate blood will be explained to them. Even if the 
subject is not included in the study, they will be asked to return two weeks later (Day 
15 after starting treatment) for a GG to ensure that the malaria has been cured. If the 
GG is positive on Day 15, the treatment regimen will be repeated. If CQ resistance is 
present, it will be managed as described in the treatment and follow-up section (see 
below). 
 
 
The subject meets the inclusion criteria: 35 mL of blood will then be taken by brachial 
venipuncture, of which 5 mL will be used for infectious disease screening tests similar 
to those used by blood banks, a process that will take between 3 and 5 days, and the 
other 30 mL will be used immediately to feed mosquitoes through an artificial 
membrane (AMA); The blood must be kept at 37°C between collection and mosquito 
feeding. Immediately after blood donation, volunteers will receive medication 
according to current national guidelines for the treatment of malaria recommended by 
the MSP, and the response to treatment will be confirmed as mentioned in the previous 
point. In addition, volunteers will be asked to return approximately one week later to 
receive the results of the blood bank screening tests. In the event of a positive result for 
any of the tests performed, including HIV, the volunteer will be referred to the 
appropriate health care provider according to the Social Security Health regime to 
which they are affiliated, with a copy of their results for counseling and appropriate 
medical assistance. If the volunteer has a private doctor, they will be referred to their 
doctor with a copy of all the results, and if they are not affiliated with any social security 

 
 
 
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system, they will be referred to one of the hospitals in the public health network 
affiliated with the SSDC. The case will be handled by the corresponding EPS or the 
SSDC, but the study team will follow up to ensure that the EPS provides the required 
care. 
 
Treatment and follow-up of volunteer donors according to MSP parameters: 
 
 
CQ: 600 mg (4 tablets of 150 mg) on the first day, 450 mg (three tablets) on 
the second day, and 450 mg (three tablets) on the third day. 
 
PQ: 30 mg (2 tablets of 15 mg) per day for 14 days. 
 Follow-up: they will be asked to return two weeks after starting treatment (day 
15 after the first dose) for a GG to ensure that the malaria has been cured. If 
the GG is positive on day 15, the treatment will be repeated. 
 
In case of resistance to CQ (*), the infection will be treated with the combination 
sulfadoxine/pyrimethamine (Falcidar® 25 mg pyrimethamine/500 mg sulfadoxine) 3 tablets 
single dose as an alternative treatment. 
 
If the patient has a contraindication to Falcidar® (e.g., allergy to sulfa drugs), they will receive 
amodiaquine in doses of 3 tablets (each tablet contains 200 mg, dose of 600 mg/day) for 3 days 
(Bosman, 2001) and will be asked to return a week later for a GG to confirm cure. 
 
(*) If the donor's parasite resistance to CQ is confirmed in a timely manner (7-12 days), the 
batch of mosquitoes infected with that sample will be discarded. In the exceptional case that 
the donor only reports when the batch of mosquitoes has already been used for the challenge, 
the treatment of immunized and challenged volunteers (step 3) will be modified as described 
below. 
 
6.7.4 Laboratory tests on donated blood 
 
Plasmodium PCR: 500 μL of the donor sample will be used to perform molecular PCR testing 
for P. vivax, P. falciparum, and P. malariae, to rule out the presence of mixed malaria in a 
timely manner (before the mosquitoes are used, approximately 7-12 days). 
 
The PCR technique has higher specificity and sensitivity values than any other diagnostic 
method currently available (WHO, 2000); it is therefore considered the gold standard for 
malaria diagnosis in the research setting. The test achieves sensitivities and specificities of up 
to 100% compared to other available diagnostic methods (Pöschl B., 2009). In the clinical 
setting, however, PCR is not the diagnostic method of choice, as it is a test that requires 
expensive equipment and reagents, which means it is not readily available in many clinical 
centers. Furthermore, as it is a time-consuming test to process and report, having to wait for 
the result would delay the start of treatment for the patient. This difficulty will not arise in this 
study, as thick smears will be used as the diagnostic method, and appropriate treatment will 
begin immediately if the result is positive, as confirmed by the two microscopists. 

 
 
 
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PCR testing to rule out infection by other Plasmodium species will be performed on the sample 
collected from the donor after it has been used to infect mosquitoes but before the infected 
batch is used to challenge healthy volunteers (7-12 days), so that it can be ensured that the 
sample only contains P. vivax. If PCR is positive for a Plasmodium species other than P. vivax, 
the fed mosquito batch will be discarded in accordance with biosafety regulations and will not 
be used for challenge under any circumstances. 
 
Although the possibility of false negatives for P. falciparum by PCR is very low, if a donor 
tests positive for mixed malaria not initially detected by PCR, this may be evidenced during 
post-treatment follow-up of the donor, as their symptoms would not improve with the 
antimalarial regimen initiated against P. vivax, given that this regimen (based on the use of CQ) 
is not effective against P. falciparum. At that point, a thick smear and PCR would be performed 
again, and appropriate treatment against P. falciparum would be initiated. Additionally, 
mosquitoes fed with such blood would be discarded. 
 
● Blood bank analysis: 5 mL of donor blood will be used to perform the following tests 
: Two rapid HIV tests from different manufacturers, antibodies against HTLV 1 and 2, 
hepatitis B surface antigen (HBsAg), rapid test for hepatitis C, rapid test for Chagas 
disease, and RPR for syphilis. 
 
● Confirmatory tests: If any of the rapid HIV tests are positive, fourth-generation ELISA tests 
will be performed as a confirmatory test. If HBsAg is positive, hepatitis B core antibodies will 
be tested to confirm the diagnosis. Similarly, if the RPR for syphilis is reported positive at any 
dilution, an FTA-ABS will be performed. Although it is theoretically possible for mosquitoes 
to transmit the hepatitis B virus within 72 hours after feeding on blood (Blow, 2002), no 
evidence has currently been found that any of these diseases are transmitted by Anopheles 
mosquitoes. In addition, mosquitoes have an incubation period of about 15 days, during which 
transmission of any of these diseases is unlikely. 
 
● Additional analysis: In addition to routine blood bank testing, other pathogens that could be 
inadvertently transmitted by Anopheles mosquitoes during a challenge are considered. Below 
are the diseases that have been found in Colombia that could be transmitted by Anopheles 
mosquitoes. 
 
Discussions held prior to challenge test #1 (Herrera, 2009) with experts in vector-borne 
diseases indicated that: there is no evidence of current transmission of any species of filaria in 
Colombia and that testing for these parasites is not necessary.  
Although Leishmania spp. are endemic in the country, they are not transmitted by Anopheles 
mosquitoes. Similarly, there is no evidence of the need to carry out evaluations of viruses other 
than those studied in the Blood Bank tests.  In addition, more than a decade of follow-up of 
volunteers participating in our group's previous trials (Herrera, 2009; Herrera, 2011) does not 
report any type of pathology associated with experimental infection.  Furthermore, although 

 
 
 
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the SARS-CoV-2 virus is not known to be transmitted by arthropod vectors, if the parasite 
donor is diagnosed with this virus during the mosquito infection stage (14 days), the 
corresponding batch of mosquitoes will be discarded as explained above. 
 
 
6.7. Step 3: Obtaining sporozoites and infectious challenge 
6.7.1. Infection of mosquitoes  
The mosquitoes to be used are kept in the Center's Insectarium under standardized conditions 
previously established in Cali and Quibdó, as set out in the maintenance and biosafety SOPs. 
(EN-04-mca-001 Entomology Unit Biosafety Manual). Female An. albimanus mosquitoes will 
be prepared under GLP conditions in the Quibdó insectarium. The reason for selecting only 
females is that male mosquitoes do not feed on blood and do not transmit the infection. The 
blood samples selected in step 2, with a parasitemia greater than 0.1% of P. vivax confirmed 
by PCR, correspond to P. vivax and correspond to the PvCS VK210 variants. These will be 
used to infect an equal number of batches of mosquitoes containing 10,000 mosquitoes per 
batch. The blood samples will be centrifuged at 3000 rpm for 5 min at room temperature, and 
the autologous plasma will be removed. The blood will be washed with RPMI-1640 medium 
and reconstituted to 50% hematocrit with equal volumes of a pool of non-immune AB human 
serum obtained from a blood bank. The AB serum complement will be inactivated by heating 
at 56°C for 30 min. After washing the blood, 3-4-day-old female mosquitoes that have been 
fasted overnight will be fed using an artificial membrane feeder at 37°C, as previously 
described (Hurtado, 1997). The mosquito boxes will be labeled with a feeding code and the 
date of infection. The day after feeding, females without blood will be removed from the box, 
and the fed mosquitoes will be kept under strict biosafety standards in the temperature and 
humidity conditions described in the respective Standard Operating Procedures (SOPs) for 
optimal operation of the mosquito colony. 
 
As previously mentioned, batches of mosquitoes fed with blood samples confirmed to be 
co-infected with P. falciparum will be discarded under biosafety conditions, while batches fed 
with samples containing only P. vivax will be retained. 
 
 Mosquito samples acceptable for challenge will be examined microscopically by 
abdominal dissection on days 7-8 after feeding to determine the presence of oocysts in their 
midgut and on days 14-15 to assess the sporozoite load in their salivary glands. For oocyst 
analysis, 40 mosquitoes will be dissected and their midguts stained with 2% mercurochrome 
and examined microscopically, as described by Eyles (Eyles, 1950). Oocyst infection will be 
calculated according to the equation Nx79 /N + 79, where N is the number of live mosquitoes 
on the day of mosquito dissection. The results will be expressed as the percentage of infected 
mosquitoes and the arithmetic means of the number of oocysts per dissected mosquito intestine. 
Positive mosquito batches will be kept inside the insectary's biosafety room, where they will 
remain for another seven (7) days until they are examined for the presence of sporozoites. 
 

 
 
 
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Sporozoites will be examined in a sample of 38 dissected mosquitoes/batches and examined 
microscopically on day 14. To do this, the salivary glands will be dissected (6 lobes) on a non-
slip slide in a drop of PBS (phosphate-buffered saline), crushed by applying pressure, and 
examined microscopically at 400x magnification to assess the density of sporozoites (spz) per 
pair of glands; each preparation will be classified as 1+ (1-10 spz), 2+ (11-100 spz), 3+ (101-
1000 spz), or 4+ (>1001 spz) (Chulay, 1986).  
 
Exclusion criteria for challenging mosquito batches 
 
● Mosquito batches are infected with blood from volunteers exposed to mixed malaria (Pv + Pf), 
HIV, HTVL-1-2, hepatitis B, C, Chagas, syphilis, and any other criteria determined by the 
investigator, such as the presence of therapeutic failure in the donor, which raises suspicion of 
resistance to the P. vivax strain to CQ. 
● Batches with a percentage of infected mosquitoes (with sporozoites) <50%. 
 
 
6.7.2. Preparation for the infectious challenge 
Volunteers participating in the study will be advised to contact one of the study physicians 
immediately if any of them become pregnant in the period between the selection visit and the 
challenge. One day before the challenge, the women will be asked to come to the CIV facilities 
for a blood test to detect pregnancy. If any of them test positive, they will be immediately 
excluded from the study. If this test reveals any cases of pregnancy, the volunteer will be 
replaced by one of the alternate volunteers. 
 
Two (2) days before the challenge, volunteers will be invited to visit the Entomology Unit 
at the INSALPA facilities in Quibdó. This visit will allow them to familiarize themselves with 
the place where the infection procedure will take place, which will reduce anxiety on the day 
of the challenge. At this time, 35 mL of blood will be drawn from all volunteers to establish a 
baseline for their immune system. 
 
On the day of the challenge, all participating volunteers will be evaluated by one of the 
study physicians, who will take a medical history and perform a brief evaluation, including 
vital signs. If any of the volunteers are found to have an acute illness that, in the opinion of the 
evaluator, requires the volunteer's exclusion, they will be replaced by one of the alternate 
volunteers. 
The mosquitoes from the batches selected for the challenge will be distributed in small 
7x7x7 cm "feeding cages." Prior to the challenge, the necessary cages will be prepared using 
mosquitoes from the same batch, with four (4) mosquitoes in each cage. 
 
 
 

 
 
 
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6.7.3. Infectious challenge with mosquitoes infected with P. vivax 
The 60 volunteers immunized in step 1 will be challenged on day ~210 of the study, 1 month 
after the third immunization, by exposure to the bite of 2-4 infected mosquitoes according to 
the previously established procedure. The procedure will be carried out in a safety room 
prepared for the study within the Entomology Unit. 
 
The "feeding cages" will be placed on the volunteer's forearm for 10 minutes, allowing the 
feeding window covered by a mesh to rest against the surface of the volunteer's skin. The box 
and forearm will be covered with dark cloth to create dim light, as this favors mosquito biting 
activity.  
 
After feeding, the volunteers will remain in the insectarium while technicians from the 
Entomology Unit determine the number of mosquitoes that are fed and, through dissection of 
the salivary glands and microscopic examination, determine the number of infected mosquitoes 
per cage and the sporozoite load in the salivary glands. If the bite rate (determined by the 
presence of blood in the mosquito's abdomen) and the infectivity rate (determined by the 
sporozoite load in the salivary glands) are below the minimum number of infective mosquitoes 
(n=2) (infective bites) expected for each volunteer, the volunteer will be required to be exposed 
to additional mosquitoes for feeding, until a total of 2-4 fed mosquitoes (positive for 
sporozoites in their salivary glands) are reached.  
 
Volunteers will be directly observed by one of the study physicians for one hour after the 
challenge to immediately detect any adverse reactions induced by the mosquito bites. 
Approximately eight (8) hours after the challenge, volunteers will be contacted by telephone 
to document their progress. Subsequently, a personal check-up will be carried out 24 hours 
later, and daily telephone follow-ups will continue until day 7 post-challenge. Volunteers will 
have all the necessary information to contact the researchers 24 hours a day (including a cell 
phone number) and will be encouraged to consult them if they have any questions or require 
guidance. This direct contact will continue for three weeks. The clinical and paraclinical 
follow-up of the volunteers in this phase will be carried out by the doctor in charge at each 
study site. 
 
6.7.4. 
 Post-challenge evaluation of volunteers  
Monitoring of the pre-patent parasitemia period: Volunteers will receive instructions about 
malaria symptoms such as fever, headache, chills, myalgia, and general malaise, which may 
occur between days 7 and 23 after the challenge. However, from day one (1) to day six (6) 
post-challenge, volunteers will be monitored by telephone by study staff. According to 
previous studies, it is unlikely that parasitemia will appear before the ninth day, however, the 
study team will be available to attend to any volunteer who presents early symptoms of malaria. 
From day 7 post-challenge until day 28, volunteers will be evaluated daily by a study physician, 
monitored daily with GG, and a blood sample will be taken for subsequent PCR-TR for 
comparative purposes at the end of the study. According to our previous studies, most exposed 

 
 
 
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volunteers develop overt infection around day 10 and the rest within the following week, so it 
would be exceptional for late infections to appear after day 20. Once all volunteers test negative 
in response to treatment, the daily collection and examination of thick smears will be 
suspended.  
 
If the daily GG is negative but the volunteer has a fever (axillary temperature >38°C) and/or 
other signs/symptoms of malaria, GG and peripheral blood smears will be taken twice a day. 
If a volunteer presents symptoms compatible with malaria, but their thick smear is negative on 
three successive occasions, a PCR-TR malaria diagnostic test (Rougemont, 2004) will be 
performed immediately to clarify the diagnosis. However, the gold standard for treatment 
should be the thick smear. If any of the volunteers develop the infection and need to be 
hospitalized, they will be treated at the Ismael Roldán or San Francisco de Asís Hospital in 
Quibdó by the clinical team physician. 
 
If 28 days after the challenge the volunteers are negative for malaria, they will continue 
with their parasitological follow-up (GG) once or twice a week, depending on the logistical 
conditions of access to the volunteer, until 60 days after the challenge. During this period, 
volunteers will be monitored daily by telephone. Based on the previous clinical trial, it is 
expected that at least some of the volunteers will develop sterile immunity; however, some may 
be only partially protected and develop longer pre-patent periods or lower parasitemia. 
Individuals who become infected and have pre-patent periods or parasitemia densities like 
those of the control group, i.e., whose pre-patent periods do not differ statistically significantly 
from those of the unvaccinated controls, will be considered unprotected. If any of the volunteers 
develop malaria during this phase, the study physician will be responsible for administering 
and monitoring treatment. 
 
6.7.5. 
Treatment of volunteers infected with malaria 
Once malaria infection is confirmed, volunteers will be treated with the antimalarial 
regimen recommended by the MSP for the treatment of P. vivax, consisting of: CQ (a total of 
1,500 mg of CQ orally in divided doses: 600 mg initially, followed by 600 mg 24 hours later 
and 300 mg 48 hours after the first dose) and PQ for fourteen days (15 mg/day) administered 
with food. All antimalarial drugs will be administered with food, as they can cause stomach 
pain, nausea, and vomiting (gastritis) if taken on an empty stomach. Volunteers without 
parasitemia on day 60 will be treated from that day onwards with the same antimalarial 
regimen. PQ will be administered directly at ASOCLINIC Ltda., under daily medical 
supervision for fourteen (14) days. Because PQ can cause hemolytic anemia in individuals with 
G6PD deficiency, all volunteers accepted for the study must have a normal screening test.  
 
6.7.6. 
 Follow-up after initiation of antimalarial treatment 
Each day after the initiation of CQ treatment, a thick smear will be taken until 3 consecutive 
negative GG results are obtained. In addition, GG follow-up will be performed on days 7, 14, 

 
 
 
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and 21 after the initiation of treatment to ensure cure. If any volunteer develops a fever or any 
symptoms compatible with malaria during this period, a GG will be performed again on the 
day of the symptoms and, if positive, a new treatment will be started using the alternative 
regimen, if necessary. In two previous trials, this therapeutic regimen has been shown to be 
effective in completely controlling the infection within 1-2 days. On day 45 after starting 
antimalarial treatment, volunteers will be evaluated at ASOCLINIC Ltda. by a study physician; 
in addition, 10 mL of blood will be drawn to measure hematological, renal, and hepatic function 
and to assess for pregnancy. 
 
6.7.7. 
Follow-up for relapses or recurrences 
In Colombia, there are no documented cases of relapse with supervised high doses of PQ 
(30 mg/day/14 days). In our previous studies, no relapses were observed during a 2-year follow-
up, although on two occasions, infections were diagnosed in volunteers who visited the 
endemic area after the study, which were interpreted as reinfections. All volunteers will be 
contacted by telephone at 3-month intervals after completion of PQ treatment and confirmation 
of negative GG (see Table 10). P. vivax resistance to CQ has been documented only rarely in 
Colombia (Comer, 1968; Soto, 2001) and has not been observed with mixed treatment (CQ + 
PQ) (Soto, 2001). However, in the unexpected event that a GG-positive sample is found at any 
time during post-treatment follow-up (days 7, 14, 28 after initiation of treatment), the volunteer 
will receive alternative treatment with Fansidar® (SP) with 3 tablets in a single dose (25 mg of 
Pyrimethamine plus 500 mg of Sulfadoxine per tablet). If the patient has a contraindication to 
Fansidar® (e.g., allergy to sulfa drugs), he or she will receive Amodiaquine as described above 
and will be followed up with additional GG to confirm cure. 
 
Any relapse of P. vivax will be treated with CQ and repeat treatment with PQ (at doses identical 
to the first treatment regimen). Follow-ups will be performed on the same days as the first 
cycle, as explained above. Long-term follow-up will be performed to detect possible relapses 
due to P. vivax hypnozoites.  Although it is not possible to determine unequivocally whether a 
relapse is due to the reactivation of hypnozoites or natural reinfection, the treatment for the 
event is similar regardless of its origin. It is also not possible to distinguish whether relapses 
are due to new hypnozoites (originated by challenge) or old hypnozoites generated by natural 
infection. Once supervised treatment with PQ (2 weeks) has been completed and negative GG 
results are obtained in post-treatment controls, all volunteers will be contacted at least by 
telephone at intervals defined in Table 10. In addition, the study physician will be the clinical 
consultant for the rest of the team in all the phases and in the event of a relapse. 
 
All volunteers who have been exposed to the infectious challenge will be asked to contact 
the study physician or the people providing medical assistance to report any cases related to 
the diagnosis and treatment of malaria infection, and in case of fever. If fever, accompanied by 
chills, convulsions, or other malaria-related symptoms, occurs at any time within 1 year (12 
months) after the challenge, the physician examining the volunteer should be informed that 
they have been exposed to experimental malaria infection, and the volunteer should undergo a 

 
 
 
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GG test, peripheral blood smear, and PCR. The study procedure schedule, including volunteer 
follow-up, is outlined below. 
 
 
 
  
 
Accepted Window 
 
  
 
 
 
 Within the first month 
Weeks 1, 2, 3, and 4 
± 3 days 
 
  
 
 
 
 For two months 
Weeks 5 and 8 
± 5 days 
 
  
 
 
 
 For three months 
Weeks 10 and 12 
± 7 days 
 
  
 
 
 
 For six months 
Week 16, 20, 24 
± 10 days 
 
  
 
 
 
 During the first year 
Weeks 30, 38, 46, and 52 
± 10 days 
 
  
 
 
 
 For a year and a half 
Weeks 52, 60, 68, and 76  
± 14 days 
 
  
 
 
 
 
Table 10: Clinical and parasitological follow-up schedule for detecting relapses or recurrences and accepted 
windows. 
 
 
6.8.8 Risk and management of natural infections during the study 
Although the urban area of Quibdó is largely free of malaria transmission, given that the study 
will be conducted in proximity to areas of malaria transmission, and despite the preventive 
measures implemented to minimize the risk of natural infections, there is a possibility that some 
volunteers may acquire P. vivax or P. falciparum infection during the immunization period 
(days 0 to 180), diagnosed by thick smear. 
In the event of a natural infection, the volunteer will be treated immediately in accordance with 
the therapeutic protocol established for each species. The AE will be reported to the IRB and 
documented in detail in the CRF and in the study database. The volunteer will not be excluded 
from the primary efficacy analysis, as the study will adopt an "Intention to Treat" approach. 
This principle implies that all participants will be analyzed according to the group to which 
they were originally assigned at the time of randomization, regardless of whether they 
completed the immunization schedule, had intercurrent events (such as natural infections), or 
deviated from the protocol. This approach preserves the statistical and clinical validity of the 
results, as it more accurately reflects the efficacy of the schedule under natural conditions in 
endemic areas. 
 
Additionally, a complementary "Per-Protocol" analysis will be performed, in which 
participants who have experienced natural infections during the immunization phase will be 

 
 
 
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excluded from the efficacy analysis, with the aim of estimating biological efficacy under ideal 
compliance conditions. 
 
In all cases, volunteers who develop natural infections will remain enrolled in the study and 
will be followed according to the procedures and schedule established in the protocol. 
 
 
7. 
LABORATORY TESTS 
 
7.1. Malaria diagnosis: 
 
The IPS ASOCLINIC Ltda. laboratory, as an accredited laboratory, can perform the 
diagnosis to identify malaria infection. Each sample will be read by two independent 
microscopists. In GG tests, a total of 200 microscopic fields will be examined with immersion 
oil (x 1000) before reporting it as negative, as no parasites were found. 
 
● Thick smear. This microscopic test will be used as the standard test for making decisions 
related to infection. It will be performed in step #2 for donor group selection and in step #3 
during post-challenge and post-treatment follow-up. In addition, it will be used for the 
diagnosis of any volunteer who presents symptoms of malaria during the immunization phase 
and in extended follow-ups after definitive treatment. For testing, ~2 drops of blood will be 
collected by finger prick, GG and thin smears will be performed, which will be stained using 
the Field staining method employing POE CD-POE-001-03 recommended by the Colombian 
Ministry of Health. 
 
● PCR-TR for malaria (steps #2 and #3): This will be performed retrospectively but will not be 
used for decision-making regarding infection. For this purpose, DNA will be extracted from 
whole blood to perform PCR-RT diagnosis for Plasmodium (P. vivax, P. malariae, and P. 
falciparum) (Rougemont, 2004), with a sample of 500 μl of donor blood, using POE ML-03-
POE 007. Procedure for molecular diagnosis of malaria. This technique is characterized by 
having an analytical level sensitivity that detects up to 1 parasite/μL. Plasmo 1 and Plasmo 2 
primers and TaqMan species-specific probes for P. vivax and P. falciparum will be used for 
this analysis. For each species and assay, positive and negative controls will be used, and the 
standard curve will be constructed from plasmids for both species to establish and quantify the 
number of copies per sample. 
 
 
7.2 Methodology for objective #1.  
 
Confirm the safety of the vaccine in naïve volunteers and those previously exposed to 
malaria, immunized with PvCS. 
 
1. To determine the safety of the vaccine, the following tests will be performed: complete blood 
count, PT, PTT, ALT, AST, bilirubin, alkaline phosphatase, BUN, creatinine, partial urine test, 
pregnancy test (for women), monthly and when deemed appropriate, in order to determine 

 
 
 
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changes in any of the parameters of these tests. Any changes that occur will be reported, their 
severity will be established, and their relationship to the vaccine will be determined. If there 
are any changes that require additional clinical or paraclinical studies, these will be performed, 
as well as additional controls, beyond those already established. Follow-up will be performed 
until these parameters return to normal. 
 
 
7.3 Methodology for objective #2.  
 
Determine the immunogenicity of PvCS in individuals previously exposed to malaria 
 
 
7.2. Specific immune response assessment tests 
 
The analyses to determine the immunogenicity of the formulation in PvCS2-Montanide ISA 
51 studies will include analysis of the B immune response (IFAT and ELISA) and analysis of 
the T cell-mediated response (IFN-γ by ELISPOT). All serum samples will be stored at -20°C 
and cells will be stored in liquid nitrogen until use. Results will be archived in the laboratory 
for further analysis. 
 
7.2.1. 
Evaluation of B cell response 
 
Specific antibody titers will be measured using IFAT and ELISA techniques on day 0 (zero), 
and subsequently in months 1, 2, 3, 6, 7, and 8 using as antigens, sporozoites produced by 
artificial feeding of An. albimanus (Hurtado, 1997) and synthetic peptides derived from PvCS. 
- 
IFAT tests will be performed according to the protocol previously described (Herrera, 2005). 
Briefly, the slides will be incubated in a humid chamber in darkness for 1 hour at 37°C, with 
25 µl of serum diluted in 2% PBS-BSA, starting with a 1:20 dilution. After 3 washes in PBS, 
FITC-labeled human anti-IgG (1:100 in PBS-Evans Blue 0.05%) will be added. It will be 
washed again and mounted for reading. Antibody titers will be expressed as the last dilution 
that showed fluorescence. 
- 
Enzyme-linked immunosorbent assay/ELISA. Antibodies against the peptides included in the 
vaccine (N, R, and C) will be measured by ELISA (Herrera, 2005). Briefly, 96-well plates 
(Nunc-Immuno Plate, Maxisorp, Roskilde-Denmark) will be sensitized with 100 µl of synthetic 
peptides N, R, and C at 1 µg/mL overnight at 4°C ( ). They will then be blocked with 200 µl of 
PBS1X/Tween 0.05%, 5% milk for 2 hours at room temperature. Next, 100 µl of each of the 
serum samples diluted in PBS1X/Tween 0.05%, 2.5% milk will be added and incubated for 1 
hour at room temperature. They will then be washed 5 times with PBS 1X/Tween 0.05%. Add 
100 µl of goat anti-human IgG conjugate with alkaline phosphatase diluted 1:1000 in 
PBS1x/Tween 0.05%, milk 2.5%, incubate for 1 hour at room temperature, and wash 4 times 
with PBS 1X/Tween 0.05%. The reaction will be revealed after incubating for 30 minutes with 
100 µl per well of the para-nitrophenol phosphate substrate. The optical density of the reaction 
will be determined at 405 nm in an ELISA reader (Dynex Technologies, INC MRX Chantilly 
VA). A sample is defined as positive when the optical density (OD) of the sample is 3 times 

 
 
 
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greater than the average OD of the negative control. 
- 
 
 
7.2.2. 
Evaluation of T-cell response 
 
The cellular immune response will be evaluated by measuring the in vitro production of the 
cytokine IFN-γ by peripheral mononuclear cells (PBMC) using ELIspot techniques. Cells will 
be separated by Ficoll-Hypaque gradients from whole blood (Herrera, 2005).  
 
- ELI spot. The in vitro production of IFN-γ by total PBMCs stimulated with different antigens 
and/or synthetic peptides will be determined by ELIspot using commercial kits (MABTECH, 
Stockholm, Sweden). For IFN-γ, microplates are sensitized with 5 µg/mL of human anti-IFN-
γ 
monoclonal 
antibody 
(1-D1K 
MABTECH 
AB, 
Sweden) 
overnight 
at 
4°C. 
Subsequently,2x10⁵PBMC/well stimulated with peptides (PvCS=N, R, C) at 1 µg/mL are added, 
using PHA as a positive control and RPMI medium alone as a negative control. The plates were 
incubated at 37°C for 40 hours in a 5% CO2 atmosphere and washed with PBS/Tween-20 
0.05%. Biotinylated anti-IFN-γ monoclonal antibody (7-B6-1, MABTECH AB, Sweden) was 
added and incubated at room temperature for 2 hours. Alkaline phosphatase with streptavidin 
will then be added, and the reaction will be revealed by adding BCIP/NBT (5-bromo-2-chloro-
3-indolyl Phosphatase/Nitroblue Tetrazolium) (Sigma, St Louis, MO). Spot-forming cells 
(SFCs) will be counted using a counting system (AID ELISpot Reader System ELR07, AID 
GmbH, Germany). 
 
7.4 Methodology for objective #3:  
 
Determine the protective efficacy of the vaccine against infectious challenge with viable P. 
vivax sporozoites. 
 
To determine protective efficacy, infection will be monitored by medical examination, GG, 
peripheral blood smear, and diagnostic PCR starting on day 7 post-infection. Once the 
volunteer is diagnosed as positive for malaria, they will be treated according to the MPS 
protocol described above. The frequency (number) of individuals who become infected, the 
pre-patent period of the disease, and the severity and frequency of symptoms will be compared 
in the study groups. 
 
- Thick smear: The slides will be stained with Field's stain and read independently by two 
experienced microscopists. Parasitemia will be quantified by observing the microscopic fields 
corresponding to 300 leukocytes and the estimated leukocyte count per µL of blood. 
 
- Diagnostic PCR: This will be performed using genomic DNA obtained from the parasite from 
500 μL blood samples from study volunteers. Species-specific detection of Plasmodium will 
be performed using the previously described and validated PCR-RT technique (Rougemont, 
2004). 
 

 
 
 
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7.5 Interpretation of results 
 
B-cell response: Due to the low antibody titers induced by P. vivax under natural conditions 
(compared to P. falciparum), responses in this study will be quantified as low, medium, and 
high, bearing in mind that any positive reaction will indicate previous contact with the parasite 
and an anti-malarial immune response in volunteers (Table 11). 
 
 Technique           Low 
Medium 
High 
 
  
 
 
 
 
 IFAT 
<1:40 
>1:40 -1:320 
>1:320 
 
  
 
 
 
 
 ELISA 
1:100 - 1:500 
>1:500-1:5000 
>1:5000 
 
  
 
 
 
 
 
 
Table 11: Antibody titers against P. vivax 
 
 
T-cell response. IFN-γ production will be determined using the ELIspot technique. In this 
technique, spot-forming cells (SFCs) will be quantified and expressed as: i) the average number 
of SFCs per10⁶PMBCs, which will be considered significant if the average number of cells in 
each well with the experimental antigen is greater than the well with the control antigen (P < 
0.05, Student's t-test), ii) the net number of SFCs per well (average number of SFCs in the well 
with the experimental antigen minus the number of SFCs in the well with the control antigen), 
and iii) the number of SFCs per well (average number of SFCs in the well with the experimental 
antigen minus the number of SFCs in the well with the control antigen). 0.05, Student's t-test), 
ii) the net number of SFCs per well (average SFCs in the well with the experimental antigen 
minus the number of SFCs in the well with the control antigen) is greater than 5 SFCs per well, 
and iii) The average ratio between the SFCs in the well with the experimental antigen and the 
average SFCs in the well with the control antigen is greater than 2.0. 
 
8. 
DATA MANAGEMENT AND ANALYSIS 
 
The data obtained from the study and the medical history will be entered into a database 
designed with the REDcap program (Vanderbilt University, 2012) and stored on a server with 
password-restricted access. The data will be entered by study staff into an Electronic Case 
Report Form (ECRF), verified by the clinical monitor in accordance with standard operating 
procedures, and corrected, if necessary, by the principal investigator. The data entered in the 
ECRF will be verified by referring to the source documents and comparing them with the 
printed data from the database. The Clinical Monitor will report any inconsistencies found, to 
be reviewed and corrected by authorized personnel. After quality control, the information will 
be analyzed with Stata (TM) 9.1, and the analysis will be performed using the statistical tests 
indicated according to the type and distribution of the variables. The significance level for 
statistical tests will be α = 0.05. The statistician in charge of data analysis will not have access 
to the distribution of the groups.  

 
 
 
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Differences between groups when the variable studied is dichotomous will be analyzed using 
the Chi2test (Fisher's exact test will be used when the number of data points is less than 5). The 
comparison of continuous variables between groups will be done using Student's t-test, and the 
comparison between several groups will be done using one-way ANOVA (Scheffe's evaluation 
for post-hoc analysis). 
 
8.1. Record keeping 
 
During the study, medical records and FRCs, participants' source documents, ICs, inclusion 
questionnaires, and all information relevant to volunteers will be stored in a secure location at 
the ASOCLINIC Ltda. Center (and CIV). Electronic databases will be stored on non-rewritable 
optical media. Participant records will be transported to the CIV (Km 6 Vía Cali-Puerto Tejada, 
Corregimiento El Hormiguero, Cali, Colombia) in a portable, secure, waterproof box by 
authorized research personnel. Once these documents have been used, they will be archived 
again at the CIV. At the end of the study, all reports, consent forms, questionnaires, and other 
relevant protocol records will be archived at the CIV for a period of 20 years, after which they 
will be identified as dead files. (See Annex 2) (Data and confidentiality). 
 
9. 
RISKS TO VOLUNTEERS, THE RESEARCH GROUP, AND THE 
ENVIRONMENT; PRECAUTIONS TO MINIMIZE RISK 
 
Using the challenge system described with P. vivax sporozoites, the CIV group has 
conducted five (5) controlled human infection trials (CHMIT) involving more than 100 
participants and more than ten inoculations.  In two consecutive trials with more than 35 healthy 
naïve volunteers, the procedure was shown to be safe with doses between 2-10 infectious bites 
(Herrera, S. 2009; Herrera, S. 2011; Arévalo-Herrera M, 2016b; Arévalo-Herrera M, 2016a; 
Arévalo-Herrera, M. 2022). The infections showed pre-patent periods ranging from 9-18 days 
with an approximate average of 11 days. The duration of symptoms was similar in all 
volunteers (1.5-4.5 days) and their responses to treatment were rapid and similar in all 
volunteers. All volunteers eliminated parasitemia within the first 48 hours after starting 
treatment (Herrera, 2009; Herrera, 2010). In these studies, the pre-patent period was evaluated 
by GG and PCR from day 7. In some cases, PCR was able to detect parasitemia before GG, but 
in none of the cases did PCR detect it before 9 days post-challenge. GG was sensitive, detecting 
parasitemia levels as low as those described above (geometric mean of 46 parasites/μL). 
 
Similarly, with P. falciparum, hundreds of volunteers have been safely and reproducibly 
infected in the USA, with 97% of these volunteers developing moderate and short-lived 
symptoms (average duration, 3 days) (Hoffman, 1997). These volunteers were able to be treated 
without complications due to the early initiation of treatment, when parasitemia was still very 
low (geometric mean of 46 parasites/µL), and because the sensitivity of the parasite to the 
antimalarials used was known. 
 
Splenic rupture is a very rare event that has only been observed in patients with chronic 

 
 
 
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established natural infection (Yagmur, 2000). In one of the previous challenge studies, only 
one of the 17 volunteers presented mild splenomegaly as an AE related to the infection 
(Herrera, 2010). In the proposed study, volunteers will be thoroughly examined during the 
admission consultation, and once admitted, they will be closely monitored during the 
immunization period and even more closely and strictly after the challenge. Therefore, the 
development of splenomegaly is not expected, beyond minimal splenomegaly, which should 
respond to antimalarial treatment, which is administered immediately upon detection of the 
parasite in the bloodstream. 
 
9.1. Risks for volunteer blood donors. 
 
Potential risks associated with blood donation may include redness, itching, infection at the 
puncture site, or vasovagal symptoms such as dizziness and fainting. Parasite samples will be 
collected from donors by venipuncture under aseptic and antiseptic conditions, using new, 
sterile, disposable equipment. A study physician will be present to provide primary medical 
care, e.g., to treat possible vasovagal episodes (fainting). 
 
A short delay (15-20 minutes) in receiving the first dose of antimalarials may occur for blood 
donor volunteers; however, this risk will not affect the volunteer's recovery. Every effort will 
be made to expedite procedures so that antimalarial treatment can be started as quickly as 
possible. A complete blood count will be performed for the purpose of early detection of 
hematological abnormalities related to malaria. 
 
There is a potential risk that a positive HIV result in a volunteer may not be handled 
appropriately and may create negative effects in their personal and/or work environment. This 
risk will be mitigated by strictly adhering to confidentiality rules. Volunteers will personally 
receive a copy of the results one week (7 days) after they are performed.  , if volunteers test 
positive for any infectious disease, they will be referred to their healthcare provider in 
accordance with Law 100, Article 179 of 1993, or, failing that, to the SSDC in accordance with 
Law 1543 of 1997, Chapter II of the MSP, to provide them with counseling and medical care. 
If a volunteer already has health insurance, he or she will be referred to their private doctor 
with the test results. These results will only be given to the volunteer. 
 
9.2. Risks to volunteers associated with the malaria challenge. 
 
Risks associated with the challenge include a very low risk of anaphylaxis, the possible 
transmission of other infectious agents through mosquito bites, and the risk associated with the 
use of antimalarial drugs. As mentioned previously, the CIV has conducted 10 experiments 
with parasite inoculation through direct mosquito bites without any reported cases of 
anaphylaxis. 
 
 
Precautions to minimize the risk associated with malaria challenge: 
 
o Anaphylaxis management: Medications for anaphylaxis management, such as epinephrine 

 
 
 
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1:1000, diphenhydramine, cimetidine, and methylprednisolone, will be available at the 
challenge site and will be used by the study physician who will remain in the infection area. 
These medications will be available at the immunization and challenge site from the start of 
the study. In addition, an ambulance will be available and will be used to transport volunteers 
who need it from the Entomology Unit to the San Francisco de Asis Hospital in Quibdó, a trip 
that takes approximately 10-15 minutes. In this case, the patient will be permanently assisted 
by the study physician and the ambulance paramedical staff. As background, the vaccine has 
been administered to more than 100 volunteers who have only experienced mild AE, like those 
of classic vaccines (Expanded Program on Immunization - EPI vaccines). On the other hand, 
volunteers and the general population remain routinely exposed to mosquito bites without any 
reported cases of anaphylaxis.    
 
o Blood screening: Blood from donors infected with P. vivax will be screened for infectious 
diseases as described above. 
 
Volunteer selection and follow-up: Volunteers will be selected if they meet each of the 
inclusion criteria and none of the exclusion criteria. They will be monitored clinically and 
parasitologically, and once infection is documented, the volunteer will begin treatment 
according to the protocol described above. Early initiation of medication will minimize the risk 
of developing serious complications, which are usually uncommon in P. vivax infections. To 
ensure adequate follow-up of volunteers, each volunteer will have all the contact details of the 
research group. Under the carefully controlled conditions for conducting this study, the 
possibility of a late diagnosis is remote. During infection, some transient signs and symptoms 
may occur, such as fever, headache, myalgia, nausea, vomiting, mild anemia and/ , asthenia, 
leukopenia, and/or thrombocytopenia, which are usually mild and short-lived due to close 
clinical monitoring and early diagnosis. The only serious and direct complication of P. vivax 
infection in healthy adults is splenic rupture (Yagmur, 2000), which occurs in patients with 
long-term infections. Therefore, it is highly unlikely to occur during the study, since the 
diagnosis will be made as soon as parasitemia becomes evident (patent in peripheral blood), 
and treatment will be administered without delay. As mentioned previously, standard treatment 
results in the disappearance of parasitemia within 48 hours of initiation. However, as a 
precaution, volunteers will be informed of this risk and advised to avoid contact sports or 
strenuous activity that could result in abdominal trauma, especially during the two weeks 
following the start of treatment. 
 
o Pregnancy and P. vivax infection: Although the effects of P. vivax malaria during pregnancy 
are less severe than those caused by P. falciparum (Nosten, 1999), P. vivax infection during 
pregnancy has been associated with high maternal parasitemia (compared to parasitemia in 
non-pregnant women), maternal anemia, and low birth weight (Nosten, 1999; Singh, 1999). 
Women will be advised to use contraceptive methods for at least six months after the challenge. 
Women participating in the study will be advised to inform their physician of their participation 
in the study so that the physician is aware in the event of pregnancy. If, despite the precautions 
described any of the women experience a relapse of P. vivax while pregnant during the 12-
month follow-up period, they will be given immediate treatment, which significantly reduces 

 
 
 
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the risk of adverse outcomes for both the mother and the fetus during pregnancy. CQ is safe 
during pregnancy (McGready, 2002), as is amodiaquine as an alternative therapy. Treatment 
with PQ will be administered after pregnancy. 
 
o Relapses: Although no cases of P. vivax relapse have been documented with the administration 
of a supervised combination regimen of PQ and CQ (Baird, 2002; Hoffman, 2002), supervised 
therapy with CQ and high doses of PQ will be administered. Volunteers will be closely 
monitored after treatment. 
 
 
9.3. Risks to volunteers associated with malaria treatment 
 
Potential side effects from the use of antimalarial drugs include nausea, vomiting, diarrhea, 
abdominal pain, vertigo, headache, sleep disturbances, blurred vision, pruritus, tinnitus, and 
photosensitivity. The following adverse reactions have been reported by the FDA in connection 
with the use of these drugs:  
● Chloroquine phosphate (CQ): Gastrointestinal reactions (vomiting, nausea, diarrhea, 
cramps), mild transient headache, auditory effects such as sensorineural hearing loss, tinnitus, 
and decreased hearing acuity in those with a history of hearing loss. Visual effects, 
dermatological reactions (pruritus and alopecia), and cardiovascular reactions (hypotension or 
changes in the EKG) may also occur. The use of CQ is contraindicated in people with psoriasis 
or other types of dermatological conditions. 
 
● Primaquine (PQ): The most common side effects are related to gastrointestinal disorders such 
as nausea and abdominal discomfort, especially if the medication is administered on an empty 
stomach. PQ will be administered with food in this study. PQ has been reported to cause 
leukopenia, and mild methemoglobinemia occurs in most individuals. The concomitant use of 
substances that predispose to this side effect, such as sulfonamides, will be avoided. PQ is not 
recommended for pregnant women. The administration of a 30 mg daily dose of PQ for more 
than one year in healthy adults has been shown to be well tolerated if it is accompanied by food 
intake. There are no significant effects related to renal or hepatic damage, as evidenced by 
serum creatinine, BUN, AST, ALT, LDH, alkaline phosphatase, and the methemoglobinemia 
that occurs is reversible and asymptomatic (Fryauff, 1995). 
 
● Falcidar® (Sulfadoxine-Pyrimethamine): Toxic manifestations are rare and usually 
attributable to the sulfadoxine component. Severe skin reactions (such as erythema multiforme, 
Steven-Johnson syndrome, and toxic epidermal necrosis) have been reported in individuals 
using a weekly regimen as prophylaxis. The safety of the combination during pregnancy has 
not been established, but the drug has been used to treat many pregnant women. 
 
● Amodiaquine: Adverse reactions to amodiaquine are generally like those of CQ, the most 
common being nausea, vomiting, abdominal pain, diarrhea, and pruritus; a less common effect 
is bradycardia. There is evidence that pruritus is less common with amodiaquine than with CQ. 
 

 
 
 
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● Treatment precautions: Volunteers will receive supervised treatment, allowing for close 
monitoring to observe side effects. Any AEs that arise will be documented, as will any potential 
associations with the treatment, which will be given a causality score. In the previous trial 
(Arévalo-Herrera, M., 2022), the AEs most frequently associated with treatment were 
gastrointestinal in origin (nausea, dizziness, and epigastric pain). The symptoms reported by 
volunteers did not significantly affect their daily activities. 
 
9.4. Risk to those conducting the study 
 
There is a low risk of accidents with the needles used in procedures with volunteers for 
researchers responsible for taking and processing blood samples.  
 
o   Precautions for staff: To reduce the risk, all workers who meet blood or blood products will 
strictly follow standard precautions. In addition, volunteers with HIV, hepatitis B, or hepatitis 
C infection and their biological products (blood) will be excluded from the study. 
 
9.5. Risk and precautions associated with the environment. 
 
The risk of accidental transmission of malaria to anyone in the community is negligible; 
infected mosquitoes will only be found in a secure, restricted area of the insectarium and will 
not be removed from this location at any time. Infections in volunteers will be treated promptly, 
before gametocytes can develop (this is generally 10 days after the first appearance of parasites 
in the blood). Volunteers will only be allowed to stay in the Quibdó area, which, despite being 
located within an endemic zone, is considered to have natural malaria transmission occurring 
specifically in localized and recognized areas of the city. If any member of the group is 
accidentally bitten by an infected mosquito or develops symptoms of malaria, they will be 
immediately evaluated with GG to confirm the presence of infection. If the result is positive, 
treatment will be given with standard doses of CQ and PQ under supervised therapy. 
 
 
10.BENEFITS 
 
10.1.Benefits for volunteer blood donors 
 
There will be no direct benefits for volunteers participating in this study. However, volunteers 
will receive indirect benefits such as blood testing for infectious diseases. In the event of a 
positive result for any infectious disease other than malaria, including HIV, the volunteer will 
receive a copy of their results and will be referred to the appropriate provider under their social 
security health plan for counseling and medical assistance. The trial team will follow up to 
ensure that the institution responsible for their social security takes the necessary steps to 
address the volunteer's needs in this regard. 
 
10.2. Benefits for malaria challenge volunteers 
 
There are no financial benefits for volunteers participating as subjects in the vaccination and 

 
 
 
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infectious challenge phases or for those participating as parasite donors (infected blood) in this 
study. However, all volunteers may receive some indirect benefits, such as comprehensive 
laboratory screening that includes the study of infectious diseases, as well as hematological and 
blood chemistry tests. If, during the selection phase, a positive result for any infectious disease, 
including HIV, is found, the volunteer will be referred with a copy of their results to the 
appropriate health care provider according to the insurance plan to which they are affiliated, 
for counseling and medical assistance in accordance with Law 100, Article 179 of 1993, or to 
the SSDC in accordance with Law 1543 of 1997, Chapter II of the MSP. 
 
 
11.COMPENSATION 
 
11.1.Compensation for blood donors. 
 
As mentioned above and established by the MSP, no monetary compensation will be given to 
volunteers for their participation in the study. However, they will not incur any financial 
expenses because of participating in this study, for which reason the study will cover 
(compensate) the costs of transportation, food, and work absence caused by their attendance at 
study activities. In addition, the study will cover any additional expenses not covered in the 
protocol that are incurred because of participation in the study. A complete medical evaluation 
will be performed, and donors would receive counseling if any pathological conditions 
associated or not associated with malaria are found. Volunteers will be provided with a snack 
after blood donation. 
 
11.2. Compensation for volunteers participating in the immunization and infectious 
challenge phases  
 
There will be no remuneration for volunteers participating in this study during the 
immunization and infectious challenge phases. However, volunteers will receive indirect 
benefits such as screening for infectious diseases and other laboratory tests, including screening 
for infectious diseases, hematological tests, blood chemistry tests, among others. Volunteers 
will not incur any financial expenses because of participating in this study. Therefore, 
transportation costs will be reimbursed, and refreshments on the day of the challenge and 
follow-up days will be covered by the study. Additionally, each time a volunteer is called in 
for a study-related procedure, they will receive the equivalent of one day's minimum wage as 
compensation for the time dedicated to the study and any possible loss of wages due to their 
absence from work. 
 
 
12.CRITERIA FOR WITHDRAWAL/RETIRMENT OF VOLUNTEERS 
 
Volunteers may withdraw freely at any time during the study. If a volunteer withdraws from 
the study, they will be treated at the time of withdrawal using the protocols described. If a 
volunteer is excluded from the study for any reason, a final evaluation (physical and 
paraclinical examination) will be performed. The reason for the withdrawal of any volunteer 

 
 
 
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will be reported in an FRC and accompanied by supporting information.  
On the other hand, regardless of their withdrawal from the study, they will be provided with 
timely treatment and medical care in the event of malaria and/or related complications. 
 
 
13.ADVERSE EVENTS 
 
An AE is any sign, inconvenience, harm, dysfunction, adverse reaction to a medication, or any 
other undesirable outcome that occurs in any of the volunteers participating in the study, 
including those that have already been defined as expected risks. Each of these events will be 
reported in an AE and will be given a degree of severity and causality related to the study 
activities (e.g., blood donation or malaria challenge). Non-serious AE will be reported to 
INVIMA in the Investigator's Manual, annually or as required. 
 
The intensity of the AE recorded in the CRF corresponds to the highest degree reached during 
an episode. For example, if a person has a fever, the intensity is graded according to the highest 
temperature recorded. 
 
AEs will be divided into two groups: solicited and unsolicited. Solicited AEs: will be asked 
about by clinical trial staff to volunteers at all contacts and will be recorded in an FRC at the 
periods determined as described below. 
 
 
Local AEs occurring in the region of the body where volunteers were exposed to 
experimental mosquitoes. These will be verified from the time of the challenge until 7 
days after the challenge. 
 
 
All local AEs will be recorded in the CRFs, and their severity will be classified 
according to the following table, adapted from the document "Guidance for Industry – 
Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in 
Preventive Vaccine Clinical Trials (FDA, 2007)". 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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Local Systemic Adverse Events 
 
Local 
Reaction 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Does not 
interfere 
with 
activity 
Repeated use of 
NSAIDs >24 
hours or interferes 
with activity 
Any use of opioid 
analgesics or that 
interferes with daily 
activity 
Emergency care for >12 
hours or requiring 
hospitalization 
Sensitivity 
Mild 
discomfort 
to touch 
Discomfort with 
movement 
Significant discomfort 
at rest 
Emergency care for > 12
hours or requiring 
hospitalization 
Erythema 
2.5-5 cm 
5.1-10 cm 
> 10 cm 
Necrosis or exfoliative 
dermatitis 
Induration 
2.5-5 cm 
and does 
not 
interfere 
with 
activity 
5.1-10 cm or 
interferes with 
activity 
> 10 cm or interferes 
with daily activity 
Necrosis 
 
 
 Systemic AEs will be monitored from the time of challenge until 7 days after completion 
of antimalarial treatment. AEs may be due to the body's reaction to the challenge or to 
the administration of antimalarial drugs. Events occurring from the time of challenge 
until the diagnosis of malaria will be attributed to P. vivax infection. Events occurring 
from the time of administration of antimalarial treatment until 7 days after completion 
of treatment will be attributed to the drug. A margin of 7 days after treatment is given 
because subtherapeutic levels of the drug circulate during this time. 
 
 All systemic AEs reported will be recorded in the corresponding CRF according to the 
following table, adapted from the document "Guidance for Industry – Toxicity Grading 
Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine 
Clinical Trials (FDA, 2007)". 
 
 
 
 
 
 
 
 
 
 

 
 
 
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Systemic Adverse Events Reported 
 
Systemic 
Reaction 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Adverse 
clinical 
event or 
disease 
Does not 
interfere 
with 
activity 
Interferes with 
activity, but does 
not require 
medical 
intervention 
Medical 
Interferes with 
daily activities 
and requires 
medical 
attention 
Emergency care for > 12 
hours or requiring 
hospitalization 
Nausea 
Does not 
interfere with 
activity 
Interferes with 
activity 
Interferes with 
daily activity 
Emergency care for > 12 
hours or requiring 
hospitalization 
 
 
 
 
 
 
 
Vomiting 
1-2 
episodes 
> 2 episodes 
Requires LEV 
outpatient 
Emergency care for > 
12 hours or requiring 
hospitalization 
Diarrhea 
2-3 
loose stools 
4-5 loose stools 
6 or more 
liquid stools or 
requiring 
outpatient 
treatment 
Emergency care for > 
12 hours or requiring 
hospitalization 
Headache 
Does not 
interfere 
with activity 
Repeated use of 
NSAIDs >24 hours 
or interfering with 
activity 
Any use of opioid 
analgesics or that 
interferes with 
daily activity 
Emergency care for >12 
hours or requiring 
hospitalization 
Fatigue 
Does not 
interfere 
with activity 
Interferes with 
activity 
Significant 
interferes with 
daily activity 
Emergency care for > 12 
hours or requiring 
hospitalization 
Myalgia 
Does not 
interfere with 
activity 
Interferes with 
activity 
Significant 
interferes with 
daily activities 
Emergency care for > 12 
hours or requiring 
hospitalization 

 
 
 
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Vital signs 
 
Vital signs 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Fever °C 
38-38.4 
38.5-38.9 
39-40 
> 40 
Tachycardia bpm 
101-115 
116-130 
> 130 
Emergency care for > 12 
hours or requiring 
hospitalization 
Bradycardia l/m 
50-54 
45-49 
< 45 
Emergency care for > 12 
hours or requiring 
Hospitalization 
Hypertension 
(systolic) mm Hg 
141-150 
151-155 
> 155 
Emergency care for > 12 
hours or requiring 
hospitalization 
Hypertension 
(diastolic) mm Hg 
91-95 
96-100 
> 100 
Emergency care for > 12 
hours or requiring 
hospitalization 
Hypotension 
(systolic) mm Hg 
85-89 
80-84 
< 80 
Emergency care for > 12 
hours or requiring 
hospitalization 
Respiratory rate r/m 
17-20 
21-25 
> 25 
Intubation 

 
 
 
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Serum 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Glucose 
Hypoglycemia 
65-69 
55-64 
45-54 
> 45 
Random 
glucose 
Hyperglyce
mia 
110-125 
126-200 
> 200 
Requires insulin or 
hyperosmolar coma 
BUN mg/dL 
23-26 
27-31 
> 31 
Requires dialysis 
Creatinine 
1 
 
1.8-2.0 
2.1-2.5 
Requires dialysis 
ALT, AST 
increase in factor 
1.1-2.5 x 
ULN 
2.6-5.0 x 
ULN 
5.1-10 x ULN 
> 10 x ULN 
Bilirubin - 
accompanied by 
alteration in 
AST/ALT 
increase in factor 
1.1-1.25 
x ULN 
1.26-1.5 x 
ULN 
1.51-1.75 x ULN 
&gt; 1.75 x ULN 
Bilirubin - no 
change in 
AST/ALT 
increase in factor 
1.1-1.5 x 
ULN 
1.6-2.0 x 
ULN 
2.0-3.0 x ULN 
> 3.0 x ULN 

 
 
 
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Serum Hematology 
Note: The lower cutoff point for neutrophils is set below the reference range due to the association between benign 
ethnic neutropenia and populations of African descent. 
 
Urine 
 
Urine 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Proteinuria 
Traces 
1+ 
2+ 
Hospitalization or dialysis 
Glucosuria 
Traces 
1+ 
2+ 
Hospitalization or 
hyperglycemia 
Hematuria 
(microscopic) – red 
blood cells/field 
1-10 
11-50 
>50 or 
macroscopic 
    Hospitalization   
or red blood cell 
transfusion 
 
 
 
Hematology 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Hb Women-g/dL 
11-12 
9.5-10.9 
8.0-9.4 
> 8 
Hb Men - g/dL 
12.5-13.5 
10.5-12.4 
8.5-10.4 
> 8.5 
Leukocytosis - 
cells/mm3 
10,800-15,000 
15,001-20,000 
20,001-25,000 
> 25,000 
Leukopenia - 
cells/mm3 
2,500-3,500 
1,500-2,499 
1,000-1,499 
> 1,000 
Lymphopenia - 
cells/mm3 
750-1000 
500-749 
250-499 
> 205 
Neutropenia - 
cells/mm3 
1,000-1,499 
500-999 
499-300 
> 300 
Eosinophils - 
cells/mm3 
650-1500 
1501-5000 
>5000 
Hypereosinophilia 
Thrombocytopenia 
cells/mm3 
125,000- 
140,000 
100,000-124,000 
25,000-99,000 
> 25,000 
PT - increase in 
factor 
1.0-1.1 x ULN 
1.11-1.20 x 
ULN 
1.21-1.25 x ULN 
> 1.25 x ULN 
PTT - increase in 
factor 
1.0-1.2 x ULN 1.21-1.4 x ULN 
1.41-1.5 x ULN 
> 1.5 x ULN 

 
 
 
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Unrequested AEs: These correspond to all AEs reported by volunteers that were not included 
among the requested AEs. All these events will be recorded in an FRC from the time of the 
challenge until 7 days after the end of antimalarial treatment. After this time, only events related 
to treatment or indicating suspected malaria will be recorded in the corresponding medical 
record. In addition, volunteers who visit a malaria-endemic area will undergo routine GG 
diagnosis, taking a blood sample on filter paper to confirm the diagnosis by PCR. This sample 
will help determine whether it is a new malaria infection or a relapse due to the clinical trial. 
 
To grade the severity of unreported AEs, the values assigned for symptoms, signs, and laboratory 
results in the Common Toxicity Criteria will be applied. The classification of clinical AEs will 
be made according to the clinical judgment of the evaluating physician and the principal 
investigator based on the categories specified in the document "Guidance for Industry - Toxicity 
Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine 
Clinical Trials (FDA, 2007). If there are any abnormalities that require additional clinical or 
paraclinical studies, these will be performed in addition to the established controls. Follow-up 
will continue until these parameters return to normal.  
 
The severity of symptoms will be assigned by the physician after evaluating the volunteer and in 
accordance with the definitions described below: 
 
● 
Grade 1 = Mild 
● 
Grade 2 = Moderate 
● 
Grade 3 = Severe 
● 
Grade 4 = Potentially life-threatening 
 
Mild: A transient, self-limiting event with minor symptoms that do not interfere with the individual's 
normal activities (e.g., the volunteer is able to work or study ) and does not require any medical 
intervention. Example: parasite inoculation site (challenge): pain and erythema; malaria: myalgia. 
 
Moderate: Events requiring minimal medical intervention to improve the volunteer's condition. In these 
cases, once the intervention has been performed, the individual is expected to be able to carry out routine 
activities; some degree of functional limitation may occur. Example: Bite site: Itching and/or pain 
sufficient to limit movement; malaria: Fever that improves with nonsteroidal anti-inflammatory drugs. 
 
Severe: Symptoms that require treatment and prevent the individual from effectively carrying out their 
daily activities. Volunteers with severe AE who are generally unable to work but can be safely managed 
as outpatients. Example: malaria: flu-like reaction or fever resulting in prostration. 
 
Potentially life-threatening: Any event that results in emergency care for more than 12 hours or requires 
hospitalization. Example: bronchospasm requiring parenteral medication in the emergency room or 
seizures evaluated in the emergency room but not resulting in hospitalization. 
 
 
 

 
 
 
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13.1.1.  Serious adverse events (SAE) 
 
SAEs will be reported according to the classification in the Document of the Americas. 
 
According to the following: 
 
1. 
Results in death. 
2. 
Threatens life, requires hospitalization of the patient, or prolongation of existing 
hospitalization. 
3. 
Results in persistent or significant disability/invalidity or is a congenital 
anomaly/birth defect. 
 
13.1.2.  Classification of AEs: Relationship to study activities 
 
According to GCP standards, AEs may occur during any interaction with the volunteer, including 
screening and selection, as well as during study processes or subsequent follow-ups. Each AE will be 
classified as definitely related, probably unrelated, possibly related, or unrelated to study activities (e.g., 
blood sampling, challenge, or antimalarial treatment). This classification will be made according to the 
medical judgment of the principal investigator and in coordination with other physicians evaluating AEs. 
Any AE that occurs during the study will be reported, whether it is considered to be related to the 
infectious bites under study. These definitions include intercurrent illnesses, injuries, and exacerbations 
of preexisting conditions. 
 
Degrees of causality: 
 
4. 
Unrelated: The event has no temporal relationship to participation in the research and is definitely 
related to another etiology. 
 
5. 
Probably unrelated: The timing and nature of the event are not temporally related to the 
intervention performed in the research. 
 
6. 
Possibly related: The timing and nature of the AE may be the result of participation in the research, 
but another explanation may be more likely. 
 
7. 
Probably related: The timing and nature of the AE suggests that it is related to participation in the 
study (e.g., arm erythema following mosquito exposure). A different potential etiology is apparent but 
less likely. 
 
8. 
Definitely related: Those AEs that have a temporal relationship to the study intervention and 
cannot be attributed to another etiology. 
 
The occurrence of AEs will be classified as expected or unexpected. Severe or serious AEs are not 
expected to occur in this study. 
 
13.1.3.  AE reporting 
 

 
 
 
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Each AE reported by volunteers, whether related to study procedures or not, will be recorded in an CRF, 
in accordance with GCP standards. 
 
Serious AEs or those that compromise the lives of volunteers and are classified as possibly or probably 
related to participation in the study will be reported by the investigator immediately to the CIV (sponsor), the 
Research Ethics Committee, and the clinical monitor by telephone, all SAEs within 24 hours of becoming aware of 
the event, maintaining the confidentiality of the information. Detailed SAE follow-up reports will be prepared, 
informing them on the status of the subject participating until the outcome of the SAE. The IRB will report the SAE 
to INVIMA within seven (7) business days of the first report made by the investigator. They must also prepare a 
follow-up report on the serious adverse event, which must be submitted to INVIMA within fifteen (15) days of the 
investigator's initial report. The SAE outcome report must also be sent in the format defined by the institution in 
accordance with Resolution No. 2011020764 (June 2011).   
 
This report must include the following points: 
 
● 
Date/time of SA reporting. 
● 
Volunteers code. 
● 
Date of birth, gender, and ethnicity of volunteer. 
● 
Name of the principal investigator. 
● 
Step of the study in which the AEF occurred. 
● 
Procedures performed on the volunteer during the study and their corresponding dates. 
● 
Date/time of SAE occurrence. 
● 
Complete description of the AEE. 
● 
    Signs or symptoms of the AEFI and their causality. 
● 
Interventions performed on the volunteer after the AEE, including medications used with their  
 
doses, route of administration, and the date of the first and last doses. 
● 
    Date/time of resolution of the AEE or death. 
● 
   Consequences for the volunteer's health and continued participation in the trial. 
● 
   Assessment and categorization of the AEFI in relation to study activities. 
● 
   Specific recommendations to ensure the safety of volunteers, which may result in changes to the          
               protocol. 
 
The written report will be reviewed with the local clinical safety monitor and then sent to the chairs 
of the ethics committees within the first 3 business days after the AE is reported. All AEs and interventions 
will be recorded in the CRF for each volunteer and included in the reports submitted to the ethics 
committees. 
 
13.2. 
AE follow-up period 
 
All AEs will be followed up until the outcome is classified into one of the following options: 
 
1. Fatal 
2. Unresolved 
3. Resolved 
4. Resolved with sequelae 

 
 
 
Page 79 from 98 
 
5. In resolution 
6. Unknown 
Pregnancies that occur in the period between the infectious challenge and the seventh day after 
completion of antimalarial treatment will be followed by the clinical trial group until their conclusion. 
 
 
 
13.3.  Reporting of AEs to INVIMA 
The CIV, in its capacity as sponsor, will report the SAE to INVIMA's Subdirectorate of Medicines and 
Biological Products within seven (7) business days of the first report made by the investigator to the CIV and 
the IRB. SAE reports shall be made through INVIMA's official Vigiflow platform or through its authorized 
interfaces such as eReporting industria or Vigiflow eForms, in accordance with the provisions of current 
regulations and external circulars 3000-0526-2021 and 3000-0471-2021. In addition, CIV will submit an EAS 
follow-up report to INVIMA within fifteen (15) business days of the initial event report and the event outcome 
report.   
 
In order to ensure the protection of participants, regulatory compliance, and the integrity of the study, a detailed 
procedure has been established to guide the detection, documentation, reporting, analysis, and follow-up of all 
SAEs and AEFI that occur during the clinical trial of the vaccine candidate (see Annex 3) (EAS and AEFI 
Management Plan) and Annex 4 (DSMB). 
 
14. 
ETHICAL CONSIDERATIONS 
 
14.1. 
 Approval by ethics committees and organizational plan 
 
The protocol will be submitted for review to the CIV Human Ethics Committees (CEICIV) in 
their capacity as sponsor and subsequently to the CEI of the ASOCLINIC Ltda. Clinical Research Center, 
in its capacity as a GCP-certified Research Center. This protocol contains the IC forms that must be signed 
by the participants (Informed Consents - IC), which include the conditions regarding the nature and 
scientific integrity of the research and information on the guarantees provided for volunteers participating 
in the study. During the study, the PI will be responsible for reporting all events that may affect the safety 
of individuals and the continuation of the clinical trial. Recruitment activities may not begin until the local 
Ethics Committees have issued their approval, and the protocol has been endorsed by INVIMA, 
Colombia's regulatory agency. 
 
14.2. Affiliation of ethics committees with the US FWA 
 
The IPS ASOCLINIC Ltda. research ethics committee is GCP certified by INVIMA (Resolution 
No. 2025013111 of April 4, 2025), and CECIV is registered with the US Federal Wide Assurance (FWA) 
for the protection of human subjects of the US Department of Health and Human Services (DHHS), Office 
for Human Research Protections (OHRP), under the guidelines of regulation 45CFR46.103. (CECIV: 
FWA00016072). The activities of these institutions with human subjects and all activities of the Ethics 
Committee will be conducted in accordance with the provisions of the Declaration of Helsinki (as adopted 
in 1996 or 2000). 
 

 
 
 
Page 80 from 98 
 
14.3. Research-related injuries 
 
Once volunteers are included in steps #1 and #3 of the study, they will be enrolled in the Prepaid 
Medicine service and a life insurance policy. These services will be provided at no cost to them for the 
duration of the study. 
 
If the volunteer belongs to the subsidized regime, they will be transferred to the contributory regime and 
enrolled in the Prepaid Medical Service; if they already belong to the contributory regime, the study will 
assume the volunteer's payments and enroll them in the Prepaid Medical Service as well. However, if the 
volunteer is a beneficiary of the "Social Subsidy Beneficiary Identification System - SISBEN," they may 
decide to remain in it so as not to lose the various benefits and subsidies, waiving the benefit of affiliation 
to the contributory regime and Prepaid Medicine, which are provided for their participation in the study. 
In this case, they will only receive the benefit of life insurance. 
 
Participants who are injured because of their cooperation in the study will receive medical care at no cost 
to them at a level III complexity health institution. They will not receive any other compensation for the 
injury. 
 
This situation does not suppress the volunteer's right to seek legal assistance to which they are entitled. 
 
 
15. GOOD PRACTICES CLINICAL (GCP) and GOOD LABORATORY PRACTICES 
(GLP) AT CIV AND ASOCLINIC LTDA - IPS 
 
ASOCLINIC Ltda., in Quibdó will be the sites for conducting post-challenge follow-up visits 
with volunteers. IPS ASOCLINIC Ltda. was created in 2022 and authorized as an IPS in 2023. Asoclinic 
was certified in April 2025 as a GCP institution through INVIMA resolution 2025013111. 
 
Starting in 2000, in partnership with CIV and with the advice of the WHO (World Health Organization), 
specifically the TDR program (WHO-Tropical Diseases Research and Training Program) and under the 
direction of Dr. Myriam Arévalo-Herrera (PhD), it began implementing Good Laboratory Practices (GLP) 
in its scientific projects and later established GLP training programs for health professionals in Colombia. 
Over the last 10 years, the CIV, in partnership with ASOCLINIC Ltda., has conducted a total of seven (7) 
clinical trials related to this vaccine (Arévalo-Herrera, 2022; 2016a; Herrera, 2005; 2009b; 2011a; 2011b; 
2011c). During the same period, members of both institutions participated in GLP and GCP workshops 
organized and sponsored by the US government through the National Institute of Allergy and Infectious 
Diseases (NIAID). In the present clinical study, quality controls related to study materials will be ensured 
by clinical study monitors who will be directly linked to the CIV. 
 
16. 
CONFIDENTIALITY 
 
All information collected from volunteers will be stored in strict confidence. Each person 
participating in the selection process will be assigned a 5-digit identification code. Although the names of 
participants will be available in the inclusion form, as noted above, this information will be filed under 
lock and key. All volunteer data will be entered into the electronic database in the REDcap program. The 

 
 
 
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list of volunteer names and codes will have a username and password that only people authorized by the 
PI will have access to. If any abnormalities are found in the results of the volunteers' tests, they will be 
contacted as soon as possible to personally deliver the laboratory reports and relevant medical 
recommendations. The records may be examined by monitors, auditors, and/or regulatory authorities. All 
individual reviews of the records are subject to strict confidentiality rules. 
 
As mentioned above, to ensure confidentiality and quality of the procedures for collecting, 
storing, processing, transmitting, and archiving clinical trial data, in accordance with national and 
international regulations, we have developed a Study Confidentiality and Data Management Plan (see 
Annex 2). National regulations include [Res 8430 of 1993; Res. 2378 of 2008; Law 1581 of 2012 and 
Decree 1377 of 2013] and international regulations [ICH E6(R3) – BPC (sec. 2.11 and 2.12) on 
confidentiality and data protection; Helsinki Declaration (principle 24: protection of privacy and personal 
information); CIOMS Guide to the Protection of Confidentiality and Secondary Use of Data; European 
Data Protection Regulation (GDPR, where applicable].  
 
17. 
RULES FOR INTERRUPTION OF THE STUDY 
 
This study will have a total duration of 6 months from the moment the volunteers are included. 
However, from that moment on, there will be a 12-month post-study follow-up to evaluate the immune 
response and the rate of reinfection.  
 
 The clinical monitor of the study and the PI will review all serious AEs in accordance with GCP 
standards. The occurrence of an AE possibly related to the study procedures, as determined by the PI and 
the clinical monitor, will result in the suspension of the study. All reviewing institutions and Ethics 
Committees will be informed of the development of these serious AEs by the PI and the monitor. The 
medical records will be reviewed by the clinical monitor and sent to the chairs of the committees within a 
period of no more than three business days. The ethics committees will review the AEs and decide whether 
the study can continue. The CIV, in its capacity as sponsor, will also review the reports and the 
recommendations of the ethics committees and make the final decision regarding the continuation of the 
study. The PI will be informed of the final decision by the IBC. 
 
18.   DEVIATIONS AND MODIFICATIONS TO THE PROTOCOL. 
 
Any inadvertent non-compliance with any part of this protocol will be reported as a deviation 
from the protocol and will be reported to each of the monitors, as well as to the Ethics Committees. Any 
modification to the protocol will be reported and submitted for consideration to the Ethics Committees of 
each of the participating institutions. 
 
19.  WITHDRAWAL OF VOLUNTEERS FROM THE STUDY 
 
Volunteers participating in any stage of the clinical trial may withdraw from the study at any 
time. A memorandum will be written during the study to document the departure or withdrawal of 
volunteers. There will be an FRC for reporting on the withdrawal of volunteers. Withdrawn volunteers 
retain the right to receive the results obtained during their participation in the study (see Annex 5).  

 
 
 
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19.1. 
Follow-up of volunteers who do not continue in the study 
 
If a volunteer is excluded from the study for any reason after the challenge but before infection 
is detected, they will be treated immediately according to the protocol, and every effort will be made to 
follow up on the volunteer appropriately. If the volunteer presents clinical manifestations secondary to 
malaria, every effort will be made to provide appropriate treatment and perform the relevant follow-up 
evaluations for up to 1 year after the challenge. The reason for the withdrawal of any volunteer will be 
recorded in the CRF and accompanied by a memorandum supporting such information. 
 
20. 
POTENTIAL CAUSES OF STUDY FAILURE 
 
Based on national and international standards, we have established three levels of potential 
failures for this study: logistical, clinical, immunological, and parasitological.      
 
From a logistical point of view, failure could occur due to a) failure to recruit a sample size that 
allows for statistical analysis, e.g., recruitment of less than 60% of the proposed number of participants. 
A reduction of up to 40% is considered acceptable, as it would allow for vaccinated groups of 12 
volunteers and 6 volunteers in the control groups. b)  Abandonment or withdrawal from the study due to 
these limits, e.g., withdrawal of >40% of volunteers (8) in the vaccine group and (4) in the control group. 
c) Loss of data or poor data quality. d) Suspension of the study for safety reasons due to public order 
disturbances.  
 
Clinical failure, a) presence of P. falciparum cases after challenge. b)  development of 
complicated malaria due to P. vivax. Highly unlikely, due to close monitoring of volunteers after challenge 
and immediate treatment at minimal parasitemia.  c) suspension of the study due to the presence of AES 
in one or more study volunteers, uncertain factor. 
 
Failure for immunological reasons such as lack or reduction of vaccine immunogenicity, or 
reduction of the response to a significant level compared to previous studies.  
 
Failure due to parasitological reasons, protective efficacy less than 50%, with the presence of 
parasitemia in >50% of vaccinees, with pre-patent parasitemia periods like or shorter than those of the 
controls.  
 
21.CIVIL LIABILITY POLICIES 
 
The study will be covered by a clinical trial insurance policy. The insurance covers 
claims made for damages arising from the clinical trial covered, provided that such claims are 
made during the period of the study.  
 
22. STUDY MONITORING  
To ensure that the CT is conducted in accordance with the protocol approved by INVIMA, in 
accordance with Good Clinical Practices (GCP), national regulations (Resolution 8430/93 and Resolution 

 
 
 
Page 83 from 98 
 
2378/08) and international guidelines (ICH E6(R3), E8(R1), E9, E19). To protect the rights, safety, and 
well-being of participants and the research team, and to ensure the scientific validity and reliability of the 
data, the responsibilities of the clinical monitor are described here.  
The monitor will be responsible for monitoring activities related to the different stages of the study.  
Before the start of the study, they must: 
● Verify that ASOCLINIC Ltda. complies with the infrastructure, personnel, and regulations defined 
for the study. 
● Review essential documents related to ethical aspects, contracts, resumes (CVs), and GCP/GCP. 
● Evaluate the team's knowledge and familiarity with protocol, safety, and procedures. 
During implementation  
● Supervise recruitment and informed consent: verification of eligibility and signing of consent 
forms. 
● Clinical and laboratory procedures: compliance with the protocol. 
● Control collection, storage, and shipment of biological samples. 
● Data recording in CRF/eCRF: accuracy, consistency (CRF/eCRF vs. source), and traceability. 
● Handling of the investigational product: storage, dispensing, and returns. 
● Control vaccine administration and investigational product management. 
● Safety report: identification, recording, and reporting of AEs/SAEs/AEFI (Adverse Events 
Following Immunization) 
● Essential file: verify data quality, proper preservation of documents and records. 
During monitoring visits 
● Review compliance with the visit schedule. 
● Ensure consistency in malaria diagnoses (thick blood smear microscopy, PCR, external quality 
control). 
● Reconcile vaccine and reagent inventories. 
At the end of the study  
● Confirm that all documentation and databases are complete and corrected. 
● Reconcile return or destruction of surplus product. 
● Prepare final monitoring report and ensure regulatory filing. 
To ensure the quality and accuracy of monitoring, a detailed plan has been established called the Clinical 
Trial Monitoring Plan – Phase II PvCS2/M51), which sets out in more detail the specific activities and 
responsibilities established to guarantee the quality and safety of the clinical trial. Its implementation will 
strengthen the credibility of the data generated, as well as the protection of participants, volunteers, and 
the research team, ensuring compliance with national and international regulations (see Annex 6).  
23. USE OF INFORMATION AND PUBLICATIONS ARISING FROM THE 

 
 
 
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STUDY. 
 
The results of this study are confidential and will be published only after authorization by the PI and 
the sponsoring institution. It is anticipated that the results of this protocol will be presented to the scientific 
community through oral presentations at scientific meetings and events and in written publications in 
specialized international journals. Researchers who are not named from the outset in this protocol will be 
required to submit to the Researcher Assurance Agreement. The participation of researchers in 
publications resulting from this study will be determined in accordance with international principles of 
scientific authorship. 
 
     
24. 
TIMELINE 
 
 
 
 
 
 

 
 
 
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23. 
 BIBLIOGRAPHY 
 
Alonso P.L., Sacarlal J., Aponte J.J., Leach A., Macete E., Milman J., Mandomando I., Spiessens B., 
Guinovart C., Espasa M., Bassat Q., Aide P., Ofori-Anyinam O., Navia M.M., Corachan S., Ceuppens 
M., Dubois M.C., Demoitie M.A., Dubovsky F., Menendez C., Tornieporth N., Ballou W.R., Thompson 
R., Cohen J. (2004) Efficacy of the RTS,S/AS02A vaccine against Plasmodium falciparum infection and 
disease in young African children: randomised controlled trial. Lancet 364:1411-20. DOI: 
S0140673604172231 [pii]10.1016/S0140-6736(04)17223-1. 
 
Arévalo-Herrera M., Herrera S. (2001) Plasmodium vivax malaria vaccine development. Mol Immunol 
38:443-55. DOI: S0161589001000803 [pii]. 
 
Arévalo-Herrera M., Roggero M.A., Gonzalez J.M., Vergara J., Corradin G., Lopez J.A., Herrera S. (1998) 
Mapping and comparison of the B-cell epitopes recognized on the Plasmodium vivax circumsporozoite 
protein by immune Colombians and immunized Aotus monkeys. Ann Trop Med Parasitol 92:539-51. 
 
Arévalo-Herrera M., Vera O., Castellanos A., Cespedes N., Soto L., Corradin G., Herrera 
S. (2011a) Preclinical vaccine study of Plasmodium vivax circumsporozoite protein derived-synthetic 
polypeptides formulated in montanide ISA 720 and montanide ISA 51 adjuvants. Am J Trop Med Hyg 
84:21-7. DOI: 84/2_Suppl/21 [pii] 10.4269/ajtmh.2011.10-0110. 
 
Arévalo-Herrera M., Soto L., Perlaza B.L., Cespedes N., Vera O., Lenis A.M., Bonelo A., Corradin G., 
Herrera S. (2011b) Antibody-mediated and cellular immune responses induced in naïve volunteers by 
vaccination with long synthetic peptides derived from the Plasmodium vivax circumsporozoite protein. 
Am J Trop Med Hyg 84:35-42. DOI: 84/2_Suppl/35 [pii]10.4269/ajtmh.2011.09-0507. 
 
Arévalo-Herrera M., Chitnis C., Herrera S. (2010) Current status of Plasmodium vivax 
vaccine. Hum Vaccin 6:124-132. 
 
Arévalo-Herrera M, Valencia AZ, Vergara J. et al., (2002). Identification of HLA-A2 restricted CD8(+) T-
lymphocyte responses to Plasmodium vivax circumsporozoite protein in individuals naturally exposed 
to malaria. Parasite Immunol. 24(3):161-9. doi: 10.1046/j.1365 3024.2002.00449.x. PMID: 12078650. 
 
Arévalo-Herrera M, Vásquez-Jiménez JM, Lopez-Perez M, Vallejo AF, Amado-Garavito AB, Céspedes N, 
Castellanos A, Molina K, Trejos J, Oñate J, Epstein JE, Richie TL, Herrera S (2016a). Protective 
Efficacy of Plasmodium vivax Radiation-Attenuated Sporozoites in Colombian Volunteers: A 
Randomized 
Controlled 
Trial. 
PLoS 
Negl 
Trop 
Dis. 
19;10(10):e0005070. 
doi: 
10.1371/journal.pntd.0005070. PMID: 2776014

 
 
 
Page 86 from 98 
 
Arévalo-Herrera M, Lopez-Perez M, Dotsey E, Jain A, Rubiano K, Felgner PL, Davies DH, Herrera S. 
(2016b). Antibody Profiling in Naïve and Semi-immune Individuals Experimentally Challenged with 
Plasmodium 
vivax 
Sporozoites. 
PLoS 
Negl 
Trop 
Dis. 
Mar 
25;10(3):e0004563. 
doi: 
10.1371/journal.pntd.0004563. eCollection 2016 Mar. PMID: 27014875. 
 
Arévalo-Herrera M., Forero-Peña D.A., Rubiano K., Gómez-Hincapie J., Martínez N.L., Lopez-Perez M., 
Castellanos A., Céspedes N., Palacios R., Oñate J.M., Herrera S. Plasmodium vivax sporozoite 
challenge in malaria-naive and semi-immune Colombian volunteers. (2014). PLoS One.  doi: 
10.1371/journal.pone.0099754. eCollection 2014. PMID: 24963662 
 
Arévalo-Herrera, M. et al. (2022) Randomized clinical trial to assess the protective efficacy of a 
Plasmodium vivax CS synthetic vaccine. Nat Commun 13, 1603. 10.1038/s41467-022-29226-3. 
 
Arévalo-Herrera M, Miura K, Solano E, Sebastián Ramírez J, Long CA, Corradin G, Herrera S. (2022b). 
Immunogenicity of full-length P. vivax rPvs48/45 protein formulations in BALB/c mice. Vaccine. Jan 
3;40(1):133-140. doi: 10.1016/j.vaccine.2021.11.036. Epub 2021 Nov 19. PMID: 34802791 
 
Arévalo-Herrera M, Miura K, Cespedes N, Echeverry C, Solano E, Castellanos A, Ramírez JS, Miranda 
A, Kajava AV, Long C, Corradin G, Herrera S. (2021). Immunoreactivity of Sera From Low to 
Moderate Malaria-Endemic Areas Against Plasmodium vivax rPvs48/45 Proteins Produced in 
Escherichia coli and Chinese Hamster Ovary Systems. Front Immunol. Jun 24;12:634738. doi: 
10.3389/fimmu.2021.634738. eCollection 2021. PMID: 34248932. 
 
Arévalo-Herrera M, Vallejo AF, Rubiano K, Solarte Y, Marín C, Castellanos A, Céspedes N, Herrera S. 
(2015). Recombinant Pvs48/45 antigen expressed in E. coli generates antibodies that block malaria 
transmission in Anopheles albimanus mosquitoes. PLoS One. Mar 16;10(3):e0119335. doi: 
10.1371/journal.pone.0119335. eCollection 2015. PMID: 25775466. 
 
Arévalo-Herrera M, Solarte Y, Rocha L, Álvarez D, Beier JC, Herrera S. (2011c). Characterization of 
Plasmodium vivax transmission-blocking activity in low to moderate malaria transmission settings of 
the Colombian Pacific coast. Am J Trop Med Hyg. Feb;84(2 Suppl):71-7. doi: 10.4269/ajtmh.2011.10-
0085. PMID: 21292881. 
 
Arévalo-Herrera M, Solarte Y, Yasnot MF, Castellanos A, Rincón A, Saul A, Mu J, Long C, Miller L, 
Herrera S. (2005) Induction of transmission-blocking immunity in Aotus monkeys by vaccination with 
Plasmodium vivax clinical grade PVS25 recombinant protein. Am J Trop Med Hyg. Nov;73(5 
Suppl):32-7. doi: 10.4269/ajtmh.2005.73.32.PMID: 16291764. 
 
Arévalo-Herrera, M., Gaitán, X., Larmat-Delgado, M., Caicedo, M. A., Herrera, S. M., Henao-Giraldo, 
J., ... &amp; Herrera, S. (2022). Randomized clinical trial to assess the protective efficacy of a 
Plasmodium vivax CS synthetic vaccine. Nature communications, 13(1), 1603. 
 
Arévalo-Herrera M, Miura K, Solano E, Sebastian Ramirez J, Long CA, Corradin G, et al. (2022). 
Immunogenicity of full-length P. vivax rPvs48/45 protein formulations in BALB/c mice. Vaccine. doi: 

 
 
 
Page 87 from 98 
 
10.1016/j.vaccine.2021.11.036. 
PubMed 
PMID: 
34802791; 
PubMed 
Central 
PMCID: 
PMCPMC9109962. 
 
Arnot D.E., Stewart M.J., Barnwell J.W. (1990) Antigenic diversity in Thai Plasmodium vivax 
circumsporozoite proteins. Mol Biochem Parasitol 43:147-9. DOI: 0166- 6851(90)90140-H.  
 
Arnot D.E., Barnwell J.W., Tam J.P., Nussenzweig V., Nussenzweig R.S., Enea V. (1985) 
Circumsporozoite protein of Plasmodium vivax: gene cloning and characterization of the 
immunodominant epitope. Science 230:815-8. 
 
Barnwell J.W., Galinski M.R. (1995) Plasmodium vivax: a glimpse into the unique and shared biology of 
the merozoite. Ann Trop Med Parasitol 89:113-20. 
 
Barnwell J.W., Galinski M.R., DeSimone S.G., Perler F., Ingravallo P. (1999). Plasmodium vivax, P. 
cynomolgi, and P. knowlesi: identification of homologue proteins associated with the surface of 
merozoites. Exp Parasitol 91:238-49. DOI: S0014- 4894(98)94372-7 [pii] 10.1006/expr.1998.4372. 
Bejon Ph., Lusingu J., Olotu A., Leach A., Lievens M., Vekemans J., Mshamu S., Lang T., Gould J., et 
al. (2008). Efficacy of RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. N Engl 
J Med.  doi: 10.1056/NEJMoa0807381. PMID: 19064627. PMCID: PMC2655100 
Bennett JW, Yadava A, Tosh D, Sattabongkot J, Komisar J, Ware LA, McCarthy WF, Cowden JJ, Regules 
J, Spring MD, et al. (2016) Phase 1/2a Trial of Plasmodium vivax Malaria Vaccine Candidate 
VMP001/AS01B in Malaria-Naive Adults: Safety, Immunogenicity, and Efficacy. PLoS Negl Trop 
Dis . doi: 10.1371/journal.pntd.0004423. .PMID: 26919472 
Bloland P., Williams HA. (2002).  Malaria Control During Mass Population Movements and Natural 
Disasters  Roundtable on the Demography of Forced Migration, National Research Council, Program 
on Forced Migration and Health at the Mailman School of Public Health of Columbia University. 
ISBN: 
0-309-50362-0, 
184 
pages. 
https://www.ncbi.nlm.nih.gov/books/NBK221163/pdf/Bookshelf_NBK221163.pdf 
 
Bojang K.A., Milligan P.J., Pinder M., Vigneron L., Alloueche A., Kester K.E., BallouW.R., Conway 
D.J., Reece W.H., Gothard P., Yamuah L., Delchambre M., Voss G., Greenwood B.M., Hill A., 
McAdam K.P., Tornieporth N., Cohen J.D., Doherty T. (2001) Efficacy of RTS,S/AS02 malaria 
vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a 
randomised trial. Lancet 358:1927-34. DOI: S0140- 6736(01)06957-4 [pii] 10.1016/S0140-
6736(01)06957-4. 
 
Breman J.G., Egan A., Keusch G.T. (2001) The intolerable burden of malaria: a new look at the numbers. 
Am J Trop Med Hyg 64:iv-vii. 
 
Burkot T.R., Wirtz R.A., Paru R., Garner P., Alpers M.P. (1992) The population dynamics in mosquitoes 
and humans of two Plasmodium vivax polymorphs distinguished by different circumsporozoite protein 
repeat regions. Am J Trop Med Hyg 47:778-786. 

 
 
 
Page 88 from 98 
 
Céspedes N., Arévalo-Herrera M., Felger I., Reed S., Kajava A.V., Corradin G., Herrera S. Antigenicity 
and immunogenicity of a novel chimeric peptide antigen based on the P. vivax circumsporozoite protein 
(2013).doi: 10.1016/j.vaccine.2013.05.082. PMID: 23954378. 
 
Céspedes N, Habel C, Lopez-Perez M, Castellanos A, Kajava AV, Servis C, Felger I, Moret R, Arévalo-
Herrera M, Corradin G, Herrera S. (2014). Plasmodium vivax antigen discovery based on alpha-helical 
coiled coil protein motif. PLoS One. Jun 24;9(6):e100440. doi: 10.1371/journal.pone.0100440. 
eCollection 2014. PMID: 24959747. 
 
Céspedes N, Li Wai Suen CSN, Koepfli C, França CT, Felger I, Nebie I, Arévalo-Herrera M, Mueller I, 
Corradin G, Herrera S. (2017). Natural immune response to Plasmodium vivax alpha-helical coiled 
coil protein motifs and its association with the risk of P. vivax malaria. PLoS One. Jun 
26;12(6):e0179863. doi: 10.1371/journal.pone.0179863. eCollection 2017. PMID: 28651021. 
 
Céspedes, N., Vallejo, A., Arévalo-Herrera, M., & Herrera, S. (2013). Malaria vaccines: high-throughput 
tools for antigen discovery with potential for their development. Colombia Médica, 44(2), 121-128. 
 
Cattani J.A., Tulloch J.L., Vrbova H., Jolley D., Gibson F.D., Moir J.S., Heywood P.F., Alpers M.P., 
Stevenson A., Clancy R. (1986a) The epidemiology of malaria in a population surrounding Madang, 
Papua New Guinea. Am J Trop Med Hyg 35:3-15. 
 
Cattani J.A., Tulloch J.L., Vrbova H., Jolley D., Gibson F.D., Moir J.S., Heywood P.F., Alpers M.P., 
Stevenson A., Clancy R. (1986b) The epidemiology of malaria in a population surrounding Madang, 
Papua New Guinea. Am J Trop Med Hyg 35:3-15. 
 
Cerami C., Frevert U., Sinnis P., Takacs B., Clavijo P., Santos M.J., Nussenzweig V. (1992) The 
basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite 
protein of Plasmodium falciparum sporozoites. Cell 70:1021- 33. DOI: 0092-8674(92)90251-7. 
 
Clyde D.F. (1975) Immunization of man against falciparum and vivax malaria by use of attenuated 
sporozoites. Am J Trop Med Hyg 24:397-401. 
 
Clyde D.F., McCarthy V.C., Miller R.M., Hornick R.B. (1973) Specificity of protection of man 
immunized against sporozoite-induced falciparum malaria. Am J Med Sci 266:398-403. 
 
Cochrane A.H., Aikawa M., Jeng M., Nussenzweig R.S. (1976) Antibody-induced ultrastructural changes 
of malarial sporozoites. Journal of Immunology 116:859-67. 
 
Cochrane A.H., Nardin E.H., de Arruda M., Maracic M., Clavijo P., Collins W.E., Nussenzweig R.S. 
(1990) Widespread reactivity of human sera with a variant repeat of the circumsporozoite protein of 
Plasmodium vivax. Am J Trop Med Hyg 43:446-51. 
 
Collins W.E., Jeffery G.M. (1999) A retrospective examination of sporozoite- and trophozoite-induced 
infections with Plasmodium falciparum in patients previously infected with heterologous species of 
Plasmodium: effect on development of parasitologic and clinical immunity. Am J Trop Med Hyg 

 
 
 
Page 89 from 98 
 
61:36-43. 
 
Collins W.E., Sullivan J.S., Morris C.L., Galland G.G., Richardson B.B. (1996) Observations on the 
biological nature of Plasmodium vivax sporozoites. J Parasitol 82:216-9. 
 
Comer R.D., Young M.D., Porter J.A., Jr., Gauld J.R., Merritt W. (1968) Chloroquine resistance in 
Plasmodium falciparum malaria on the Pacific coast of Colombia. Am J Trop Med Hyg 17:795-9. 
 
Charoenvit Y., Collins W.E., Jones T.R., Millet P., Yuan L., Campbell G.H., Beaudoin R.L., Broderson 
J.R., Hoffman S.L. (1991) Inability of malaria vaccine to induce antibodies to a protective epitope 
within its sequence. Science 251:668-71. 
 
Chitnis C.E. (2001) Molecular insights into receptors used by malaria parasites for erythrocyte invasion. 
Curr Opin Hematol 8:85-91. 
 
Chulay J.D., Schneider I., Cosgriff T.M., Hoffman S.L., Ballou W.R., Quakyi I.A., Carter R., Trosper 
J.H., Hockmeyer W.T. (1986) Malaria transmitted to humans by mosquitoes infected from cultured 
Plasmodium falciparum. Am J Trop Med Hyg 35:66-8. 
 
Datoo, M. M., Dicko, A., Tinto, H., Ouédraogo, J. B., Hamaluba, M., Olotu, A., ... &amp; Hill, A. V. A 
Phase III Randomized Controlled Trial Evaluating the Malaria Vaccine Candidate R21/Matrix-M™ 
in African Children. 
 
Del Portillo H.A., Longacre S., Khouri E., David P.H. (1991) Primary structure of the merozoite surface 
antigen 1 of Plasmodium vivax reveals sequences conserved between different Plasmodium species. 
Proc Natl Acad Sci U S A 88:4030-4. 
 
Dhiman, S. (2019) Are malaria elimination efforts on the right track? An analysis of gains achieved and 
challenges ahead. Infect Dis Poverty 8, 14.10.1186/s40249-019-0524x. 
 
Doolan DL, Dobaño C, Baird JK. (2009). Acquired immunity to malaria.Clin Microbiol Rev. 22(1):13-
36, doi: 10.1128/CMR.00025-08. PMID: 19136431. 
 
Druihle P., Reina L., Fidock D.A. (1998) Immunity to liver stages, in I. Sherman (Ed.), Malaria: Parasite 
biology, pathogenesis and protection Washington DC. pp. 513-543. 
 
Druilhe P, Pérignon JL (1994) Mechanisms of defense against P. falciparum asexual blood stages in 
humans. Immunol Lett. 41(2-3):115-20. doi: 10.1016/0165- 2478(94)90118-x. PMID: 8002025. 
 
Egan J.E., Hoffman S.L., Haynes J.D., Sadoff J.C., Schneider I., Grau G.E., Hollingdale M.R., Ballou 
W.R., Gordon D.M. (1993) Humoral immune responses in volunteers immunized with irradiated 
Plasmodium falciparum sporozoites. Am J Trop Med Hyg 49:166-73. 
Espinosa, D.A., Yadava, A., Angov, E., Maurizio, P.L., Ockenhouse, Ch.F., Zavala, F. Development of a 
chimeric Plasmodium berghei strain expressing the repeat region of the P. vivax circumsporozoite 

 
 
 
Page 90 from 98 
 
protein for in vivo evaluation of vaccine efficacy. (2013) Infect Immun. doi: 10.1128/IAI.00461-13. 
PMID: 23716612. PMCID: PMC3719583. DOI: 10.1128/IAI.00461-13 
 
Eyles D.E. (1950) A stain for malarial oocysts in temporary preparations. J Parasitol 36:501. 
 
Fairley N.H. (1947) Sidelights on malaria in man obtained by sub-inoculation experiments. Trans R Soc 
Trop Med Hyg 40:621-76. 
 
Franke E.D., Lucas C.M., San Roman E., Wirtz R.A. (1992a) Prevalence of antibody to the variant repeat 
of the circumsporozoite protein of Plasmodium vivax in Peru. Am J Trop Med Hyg 46:708-10. 
 
Franke E.D., Lucas C.M., Chauca G., Wirtz R.A., Hinostroza S. (1992b) Antibody response to the 
circumsporozoite protein of Plasmodium vivax in naturally infected humans. Am J Trop Med Hyg 
46:320-6. 
 
Frevert U., Sinnis P., Cerami C., Shreffler W., Takacs B., Nussenzweig V. (1993) Malaria 
circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane 
of hepatocytes. J. Exp. Med. 177:1287-1298. 
Galappaththy, G.N. et al. (2013) Primaquine for preventing relapse in people with Plasmodium vivax 
malaria treated with chloroquine. The Cochrane database of systematic reviews, CD004389. 
10.1002/14651858.CD004389.pub3 
 
Galinski M.R., Corredor-Medina C., Povoa M., Crosby J., Ingravallo P., Barnwell J.W. (1999) 
Plasmodium vivax merozoite surface protein-3 contains coiled-coil motifs in an alanine-rich central 
domain. Mol Biochem Parasitol 101:131-47. DOI: S0166- 6851(99)00063-8. 
 
Galinski M.R., Ingravallo P., Corredor-Medina C., Al-Khedery B., Povoa M., Barnwell J.W. (2001) 
Plasmodium vivax merozoite surface proteins-3beta and-3gamma share structural similarities with P. 
vivax merozoite surface protein-3alpha and define a new gene family. Mol Biochem Parasitol 115:41-
53. DOI: S0166-6851(01)00267-5 [pii]. 
 
Genton B., Corradin G. (2002) Malaria vaccines: from the laboratory to the field. Curr Drug Targets 
Immune Endocr Metabol Disord 2:255-67. 
 
Glynn J.R., Collins W.E., Jeffery G.M., Bradley D.J. (1995) Infecting dose and severity of falciparum 
malaria. Trans R Soc Trop Med Hyg 89:281-3. 
 
Gonzalez J.M., Hurtado S., Arévalo-Herrera M., Herrera S. (2001) Variants of the Plasmodium vivax 
circumsporozoite protein (VK210 and VK247) in Colombian isolates. Mem Inst Oswaldo Cruz 96:709- 
12. DOI: S0074-02762001000500023. 
 
Grassi B.A., Bignami, Bastianelli G. (1899) Further research on the cycle of human malaria parasites in 
the body of the mosquito. Atti Reale Accad. Lincei 5:8-21. 
 
Guerra C.A., Howes R.E., Patil A.P., Gething P.W., Van Boeckel T.P., Temperley W.H., Kabaria C.W., 

 
 
 
Page 91 from 98 
 
Tatem A.J., Manh B.H., Elyazar I.R., Baird J.K., Snow R.W., Hay S.I. (2010) The international limits 
and population at risk of Plasmodium vivax transmission in 2009. PLoS Negl Trop Dis 4:e774. DOI: 
10.1371/journal.pntd.0000774. 
 
Guinovart C., Aponte J.J., Sacarlal J., Aide P., Leach A., Bassat Q., Macete E., Dobano C., Lievens M., 
Loucq C., Ballou W.R., Cohen J., Alonso P.L. (2009) Insights into long-lasting protection induced by 
RTS, S/AS02A malaria vaccine: further results from a phase IIb trial in Mozambican children. PLoS 
One 4:e5165. DOI: 10.1371/journal.pone.0005165. 
 
Gunewardena D.M., Carter R., Mendis K.N. (1994) Patterns of acquired anti-malarial immunity in Sri 
Lanka. Mem Inst Oswaldo Cruz 89:63-5. 
 
Gysin J., Moisson P., Pereira da Silva L., Druilhe P. (1996) Antibodies from immune African donors with 
a protective effect in Plasmodium falciparum human infection are also able to control asexual blood 
forms of the parasite in Saimiri monkeys. Res Immunol 147:397-401. DOI: 0923249496820487 [pii]. 
 
Herrera S., Escobar P., de Plata C., Avila GI., Corradin G., Herrera MA. (1992) Human recognition of T 
cell epitopes on the Plasmodium vivax circumsporozoite protein. J Immunol1992 Jun 
15;148(12):3986-90 PMID: 1376347 
 
Herrera M.A., de Plata C., Gonzalez J.M., Corradin G., Herrera S. (1994) Immunogenicity of multiple 
antigen peptides containing Plasmodium vivax CS epitopes in BALB/c mice. Mem Inst Oswaldo Cruz 
89:71-6. 
 
Herrera S., De Plata C., Gonzalez M., Perlaza B.L., Bettens F., Corradin G., Arévalo Herrera M. (1997) 
Antigenicity and immunogenicity of multiple antigen peptides (MAP) containing P. vivax CS epitopes 
in Aotus monkeys. Parasite Immunol 19:161-70. 
 
Herrera S., Fernandez O.L., Vera O., Cardenas W., Ramirez O., Palacios R., Chen Mok M., Corradin G., 
Arévalo-Herrera M. (2011a) Phase I safety and immunogenicity trial of Plasmodium vivax CS derived 
long synthetic peptides adjuvanted with montanide ISA 720 or montanide ISA 51. Am J Trop Med 
Hyg 84:12-20. DOI: 84/2_Suppl/12 [pii] 10.4269/ajtmh.2011.09-0516. 
 
Herrera S., Solarte Y., Jordan-Villegas A., Echavarria J.F., Rocha L., Palacios R., Ramirez O., Velez J.D., 
Epstein J.E., Richie T.L., Arévalo-Herrera M. (2011b) Consistent safety and infectivity in sporozoite 
challenge model of Plasmodium vivax in malaria-naïve human volunteers. Am J Trop Med Hyg 84:4- 
11. DOI: 84/2_Suppl/4 [pii] 10.4269/ajtmh.2011.09-0498. 
 
Herrera S., Fernandez O., Manzano M.R., Murrain B., Vergara J., Blanco P., Palacios R., Velez J.D., 
Epstein J.E., Chen-Mok M., Reed Z.H., Arévalo-Herrera M. (2009c) Successful sporozoite challenge 
model in human volunteers with Plasmodium vivax strain derived from human donors. Am J Trop Med 
Hyg 81:740-6. DOI: 81/5/740 [pii] 10.4269/ajtmh.2009.09-0194. 
 
Herrera S., Bonelo A., Perlaza B.L., Fernandez O.L., Victoria L., Lenis A.M., Soto L., Hurtado H., Acuna 
L.M., Velez J.D., Palacios R., Chen-Mok M., Corradin G., Arévalo Herrera M. (2005) Safety and e -

 
 
 
Page 92 from 98 
 
elicitation of humoral and cellular responses in Colombian malaria-naïve volunteers by a Plasmodium 
vivax circumsporozoite protein-derived synthetic vaccine. Am J Trop Med Hyg 73:3-9. DOI: 
73/5_suppl/3. 
 
Herrera, S., Fernández, O., Manzano, M.R., Murrain, B., Vergara, J., Blanco, P., Palacios, R., Vélez, J.D., 
Epstein, J., Chen-Mok, M., Reed, Z., Arévalo-Herrera, M. 2009. Case report: Successful sporozoite 
challenge model in human volunteers with Plasmodium vivax strain derived from human donors. Am. 
J. Trop. Med. Hyg, 81(5):740-746. doi: 10.4269/ajtmh.2009.09-0194. PMID: 19861603 
 
Herrera, S., Solarte, Y., Jordán-Villegas, A., Echavarría, J.F., Rocha, L., Palacios, R., Ramírez, O., Vélez, 
J.D., Epstein, J.E., Richie, T.L. and Arévalo-Herrera, M. 2011. Consistent safety and infectivity in 
sporozoite challenge model of Plasmodium vivax in malaria-naïve human volunteers. Am J. Trop. 
Med. Hyg, 84(2):4-11 doi: 10.4269/ajtmh.2011.09-0498. PMID: 21292872 
 
Herrera, S., De Plata C., González M., Perlaza BL., Bettens F., Corradin G., Arévalo-Herrera M. (1997). 
Antigenicity and immunogenicity of multiple antigen peptides (MAP) containing P. vivax CS epitopes 
in Aotus monkeys. Parasite Immunol., 19(4): p. 161-70. doi: 10.1046/j.1365-3024.1997.d01-193.x. 
PMID: 9149283  
 
Herrera, SM; Flores Y; Corradin G; Arevalo-Herrera M; Herrera S; Zavala F, (2025; Submitted). 
Validation of the human protective efficacy of a Plasmodium vivax CS synthetic vaccine candidate in 
a rodent model 
 
Herrington D.A., Nardin E.H., Losonsky G., Bathurst I.C., Barr P.J., Hollingdale M.R., Edelman R., 
Levine M.M. (1991) Safety and immunogenicity of a recombinant sporozoite malaria vaccine against 
Plasmodium vivax. Am J Trop Med Hyg 45:695-701. 
 
Hill AV. (2011) Vaccines against malaria. Philos Trans R Soc Lond B Biol Sci. 366(1579):2806–2814. 
doi: 10.1098/rstb.2011.009 
 
Hoffman S.L., Doolan D.L. (2000) Malaria vaccines-targeting infected hepatocytes. Nat Med 6:1218- 9. 
DOI: 10.1038/81315. 
 
Hoffman S.L., Goh L.M., Luke T.C., Schneider I., Le T.P., Doolan D.L., Sacci J., de la Vega P., Dowler 
M., Paul C., Gordon D.M., Stoute J.A., Church L.W., Sedegah M., Heppner D.G., Ballou W.R., Richie 
T.L. (2002) Protection of humans against malaria by immunization with radiation-attenuated 
Plasmodium falciparum sporozoites. J Infect Dis 185:1155-64. DOI: JID010922 [pii] 10.1086/339409. 
 
Hoffman, S. L., Vekemans, J., Richie, T. L., &amp; Duffy, P. E. (2015). The march toward malaria 
vaccines. Vaccine, 33, D13-D23. 
 
Hurtado S., Salas M.L., Romero J.F., Zapata J.C., Ortiz H., Arévalo-Herrera M., Herrera 
S. (1997) Regular production of infective sporozoites of Plasmodium falciparum and P. vivax in 
laboratory-bred Anopheles albimanus. Ann Trop Med Parasitol 91:49-60. 

 
 
 
Page 93 from 98 
 
 
Kain K.C., Brown A.E., Webster H.K., Wirtz R.A., Keystone J.S., Rodriguez M.H., Kinahan J., Rowland 
M., Lanar D.E. (1992) Circumsporozoite genotyping of global isolates of Plasmodium vivax from dried 
blood specimens. J Clin Microbiol 30:1863-6. 
Karch Ch.P., Doll T.,   Paulillo S.M.,  Nebie I., Lanar D.E., Corradin G., Peter Burkhard. The use of a P. 
falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to 
prevent malaria. J Nanobiotechnology. PMID: 28877692. doi: 10.1186/s12951-017-0295-0. 
 
Kublin, J. G., Mikolajczak, S. A., Sack, B. K., Fishbaugher, M. E., Seilie, A., Shelton, L., ... & Kappe, S. 
H. (2017). Complete attenuation of genetically engineered Plasmodium falciparum sporozoites in 
human subjects. Science translational medicine, 9(371), eaad9099. 
 
Kumar S., Epstein J.E., Richie T.L., Nkrumah F.K., Soisson L., Carucci D.J., Hoffman S.L. (2002) A 
multilateral effort to develop DNA vaccines against falciparum malaria. Trends Parasitol 18:129-35. 
DOI: S1471492201022073 [pii]. 
 
Lata De S., Ntumngia F.B., Nicholas J., Adams J.H. (2022). Expert Rev Vaccines Progress towards the 
development of a P. vivax vaccine. PMC7994195  NIHMSID: NIHMS1667293  PMID: 33481638 
doi: 10.1080/14760584.2021.1880898 
 
 
Lopez-Perez, M., Jain, A., Davies, D. H., Vásquez-Jiménez, J. M., Herrera, S. M., Oñate, J., ... &amp; 
Arévalo-Herrera, M. (2023). Human sterile immunity to Plasmodium vivax malaria correlates with 
specific antibody response elicited by immunization with radiation-attenuated sporozoites. 
 
Lopez-Perez M, Jain A, Davies DH, Vasquez-Jimenez JM, Herrera SM, Onate J, Felgner PL, Herrera S, 
Arevalo-Herrera M: Profiling the antibody response of humans protected by immunization with 
Plasmodium vivax radiation-attenuated sporozoites. Sci Rep 2024, 14:2790. 
 
Malkin, E. M. et al. Phase 1 vaccine trial of Pvs25H: a transmission blocking vaccine for Plasmodium 
vivax malaria. Vaccine 23, 3131–3138, doi:10.1016/j.vaccine.2004.12.019 (2005). 
 
Manjurano, A. et al. (2015) African glucose-6-phosphate dehydrogenase alleles associated with protection 
from severe malaria in heterozygous females in Tanzania. PLoS Genetics 11, e1004960 
 
Macete E., Aponte J.J., Guinovart C., Sacarlal J., Ofori-Anyinam O., Mandomando I., Espasa M., 
Bevilacqua C., Leach A., Dubois M.C., Heppner D.G., Tello L., Milman J., Cohen J., Dubovsky F., 
Tornieporth N., Thompson R., Alonso P.L. (2007) Safety and immunogenicity of the RTS, S/AS02A 
candidate malaria vaccine in children aged 1-4 in Mozambique. Trop Med Int Health 12:37-46. DOI: 
TMI1754 [pii] 
10.1111/j.1365-3156.2006.01754.x. 
 
Machado R.L., Povoa M.M. (2000) Distribution of Plasmodium vivax variants (VK210, VK247 and P. 
vivax-like) in three endemic areas of the Amazon region of Brazil and their correlation with 
chloroquine treatment. Trans R Soc Trop Med Hyg 94:377-81 

 
 
 
Page 94 from 98 
 
 
Maheswary N.P., Perpanich B., Rosenberg R. (1992) Presence of antibody to a heterologous 
circumsporozoite protein of Plasmodium vivax (VK247) in southeastern Bangladesh. Trans R Soc Trop 
Med Hyg 86:28. 
 
Mann V.H., Huang T., Cheng Q., Saul A. (1994) Sequence variation in the circumsporozoite protein gene 
of Plasmodium vivax appears to be regionally biased. Mol Biochem Parasitol 68:45-52. DOI: 0166-
6851(94)00148-0 [pii]. 
 
Mendis K., Sina B.J., Marchesini P., Carter R. (2001) The neglected burden of 
Plasmodium vivax malaria. Am J Trop Med Hyg 64:97-106. 
 
Miller L.H., McAuliffe F.M., Mason S.J. (1977) Erythrocyte receptors for malaria merozoites. Am J Trop 
Med Hyg 26:204-8. 
 
Mo, AX, McGugan. Understanding the Liver-Stage Biology of Malaria Parasites: Insights to Enable and 
Accelerate the Development of a Highly Efficacious Vaccine (2018). Am J Trop Med Hyg. 
Oct;99(4):827-832. doi:10.4269/ajtmh.17-0895. PMID: 30141395. PMCID: PMC6159572 DOI: 
10.4269/ajtmh.17-0895 
 
Molina DM, Finney OC, Arévalo-Herrera M, Herrera S, Felgner PL, Gardner MJ, Liang X, Wang R. 
(2012) Plasmodium vivax pre-erythrocytic-stage antigen discovery: exploiting naturally acquired 
humoral responses. Am J Trop Med Hyg. Sep;87(3):460-9. doi: 10.4269/ajtmh.2012.12-0222. Epub 
2012 Jul 23. PMID: 22826492. 
Molina-Cruz A, Gonzalez-Ceron L, Dwivedi A, Torres TZB, Raytselis N, Young M, Kamath N, McNinch 
C, Su X, Ford A, Ferreira MU, Arévalo-Herrera M, Herrera S, Lo E, Silva JC, Barillas-Mury C.  (2025). 
Worldwide genetic diversity of Plasmodium vivax Pv47 is consistent with natural selection by 
anopheline mosquitoes. Nat Commun. doi: 10.1038/s41467-025-62680-3. PMID: 40783499.  
Nardin E., Clavijo P., Mons B., van Belkum A., Ponnudurai T., Nussenzweig R.S. (1991) T cell epitopes 
of the circumsporozoite protein of Plasmodium vivax. Recognition by lymphocytes of a sporozoite-
immunized chimpanzee. J Immunol 146:1674-8. 
 
Nussenzweig R., Vanderberg J., Most H. (1969) Protective immunity produced by the injection of x-
irradiated sporozoites of Plasmodium berghei. IV. Dose response, specificity and humoral immunity. 
Mil Med 134:1176-82. 
 
 Nussenzweig RS, Vanderberg J, Most H, Orton C Protective immunity produced by the injection of x-
irradiated sporozoites of plasmodium berghei. (1967). Nature. doi: 10.1038/216160a0. PMID: 6057225  
 
PAHO (2022). Malaria. In Salud, O.P.d.l., ed. https://www.paho.org/es/temas/malaria 
 
Penny MA, Verity R, Bever CA, et al. (2016). Public health impact and cost-effectiveness of the RTS, 
S/AS01 malaria vaccine: a systematic comparison of predictions from four mathematical models. 
Lancet; 387:367–375. doi: 10.1016/S0140-6736(15)00725-4 

 
 
 
Page 95 from 98 
 
Qari S.H., Goldman I.F., Povoa M.M., di Santi S., Alpers M.P., Lal A.A. (1992) Polymorphism in the 
circumsporozoite protein of the human malaria parasite Plasmodium vivax. Mol. Biochem. Parasitol. 
55:105-114 
 
Ramasamy R., Nagendran K., Ramasamy M.S. (1994) Antibodies to epitopes on merozoite and sporozoite 
surface antigens as serologic markers of malaria transmission: studies at a site in the dry zone of Sri 
Lanka. Am J Trop Med Hyg 50:537- 47. 
 
Richie T.L., Saul A. (2002) Progress and challenges for malaria vaccines. Nature 415:694-701. DOI: 
10.1038/415694a415694a [pii]. 
 
Richie TL, Church LWP, Murshedkar T, Billingsley PF, James ER, …, Hoffman SL. Expert Rev 
Vaccines. 2023 Jan-Dec;22(1):964-1007. Sporozoite immunization: innovative translational science to 
support the fight against malaria.  doi: 10.1080/14760584.2023.2245890. Epub 2023 Aug 11.  
 
Rieckmann K.H., Beaudoin R.L., Cassells J.S., Sell K.W. (1979) Use of attenuated sporozoites in 
immunizing human volunteers against falciparum malaria. Bull World Health Organ 57:261-5. 
 
Rodriguez M.H., Betanzos-Reyes A.F., Hernandez-Avila J.E., Mendez-Galvan J.F., Danis-Lozano R., 
Altamirano-Jimenez A. (2009) The participation of secondary clinical episodes in the epidemiology of 
vivax malaria during pre-and post-implementation of focal control in the state of Oaxaca, Mexico. Am 
J Trop Med Hyg 80:889-95. DOI: 80/6/889 [pii]. 
 
Rogers W.O., Malik A., Mellouk S., Nakamura K., Rogers M.D., Szarfman A., Gordon D.M., Nussler 
A.K., Aikawa M., Hoffman S.L. (1992) Characterization of Plasmodium falciparum sporozoite surface 
protein 2. Proc Natl Acad Sci U S A 89:9176-80. 
 
Rosenberg R., Wirtz R.A., Lanar D.E., Sattabongkot J., Hall T., Waters A.P., Prasittisuk 
C. (1989) Circumsporozoite protein heterogeneity in the human malaria parasite. 
Plasmodium vivax. Science 245:973-6. 
Roestenberg M, Bijker EM, Sim BKL, et al. Controlled human malaria infections by intradermal 
injection of cryopreserved Plasmodium falciparum sporozoites. (2013). Am J Trop Med Hyg. 
doi:10.4269/ajtmh.2012.12-0613. 
Rougemont M., Van Saanen M., Sahli R., Hinrikson H.P., Bille J., Jaton K. (2004) Detection of four 
Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-
time PCR assays. J Clin Microbiol 42:5636-43. DOI: 42/12/5636 [pii] 10.1128/JCM.42.12.5636-
5643.2004. 
Raghunandan R, Mayer BT, Flores-Garcia Y, Gerber MW, Gottardo R, Jhun H, Herrera SM, Perez-Ramos 
DW, Locke E, King CR, Zavala F. (2020). Characterization of two in vivo challenge models to measure 
functional activity of monoclonal antibodies to Plasmodium falciparum circumsporozoite protein. 
Malar J. doi: 10.1186/s12936-020-03181-0. PMID: 32183833. 
 

 
 
 
Page 96 from 98 
 
Sachs J., Malaney P. (2002) The economic and social burden of malaria. Nature 415:680- 5. DOI: 
10.1038/415680a 415680a [pii]. 
 
Samuels, A. M., Ansong, D., Kariuki, S. K., Adjei, S., Bollaerts, A., Ockenhouse, C., ... & Ivinson, K. 
(2022). Efficacy of RTS, S/AS01E malaria vaccine administered according to different full, fractional, 
and delayed third or early fourth dose regimens in children aged 5–17 months in Ghana and Kenya: an 
open-label, phase 2b, randomized controlled trial. The Lancet Infectious Diseases, 22(9), 1329-1342. 
 
Stanisic DI, McCarthy JS, Good MF. Controlled Human Malaria Infection: Applications, Advances, and 
Challenges. Infect Immun. 2017 Dec 19;86(1):e00479-17. doi: 10.1128/IAI.00479-17. PMID: 
28923897; PMCID: PMC5736798. 
 
Sattabongkot J., Tsuboi T., Zollner G.E., Sirichaisinthop J., Cui L. (2004) Plasmodium vivax transmission: 
chances for control? Trends Parasitol 20:192-8. DOI: 10.1016/j.pt.2004.02.001 S1471492204000327 
[pii]. 
 
Sinnis P, Clavijo P, Fenyö D, Chait BT, Cerami C, Nussenzweig V.J Structural and functional properties 
of 
region 
II-plus 
of the 
malaria 
circumsporozoite 
protein. 
(1994). 
Exp 
Med. 
doi: 
10.1084/jem.180.1.297.PMID: 8006589  
 
Sinnis P., Nussenzweig V. (1996) Preventing sporozoite invasion of hepatocytes, in: S.L. Hoffman (Ed.), 
Malaria vaccine development. A multi-immune response approach, ASM Press, Washington, D.C. pp. 
15-34. 
 
Soares da Veiga G.T., Ribeiro Moriggi M., Vettorazzi J.F., Müller-Santos M., Albrecht L. Plasmodium 
vivax vaccine: What is the best way to go? (2023). Front Immunol. PMID: 36726991 PMCID: 
PMC9885200. DOI: 10.3389/fimmu.2022.910236. 
 
Solarte Y., Manzano M.R., Rocha L., Hurtado H., James M.A., Arévalo-Herrera M., Herrera S. (2011) 
Plasmodium vivax sporozoite production in Anopheles albimanu 
 
Mosquitoes for vaccine clinical trials. The American journal of tropical medicine and hygiene 84:28-
34. 
 
Soto J., Toledo J., Gutierrez P., Luzz M., Llinas N., Cedeno N., Dunne M., Berman J. (2001) Plasmodium 
vivax clinically resistant to chloroquine in Colombia. Am J Trop Med Hyg 65:90-3. 
 
Stoute J.A., Slaoui M., Heppner D.G., Momin P., Kester K.E., Desmons P., Wellde B.T., Garcon N., 
Krzych U., Marchand M. (1997) A preliminary evaluation of a recombinant circumsporozoite protein 
vaccine against Plasmodium falciparum malaria. RTS, S Malaria Vaccine Evaluation Group. N Engl J 
Med 336:86-91. DOI: 10.1056/NEJM199701093360202. 
 
Stoute J.A., Heppner D.G., Jr., Mason C.J., Siangla J., Opollo M.O., Kester K.E., Vigneron L., Voss G., 
Walter M.J., Tornieporth N., Cohen J.D., Ballou W.R. (2006) Phase One safety and immunogenicity 

 
 
 
Page 97 from 98 
 
trial of malaria vaccine RTS, S/AS02A in adults in a hyperendemic region of western Kenya. Am J 
Trop Med Hyg 75:166-70. DOI: 75/1/166. 
 
Stoute J.A., Kester K.E., Krzych U., Wellde B.T., Hall T., White K., Glenn G., Ockenhouse C.F., Garcon 
N., Schwenk R., Lanar D.E., Sun P., Momin P., Wirtz R.A., Golenda C., Slaoui M., Wortmann G., 
Holland C., Dowler M., Cohen J., Ballou W.R. (1998) Long-term efficacy and immune responses 
following immunization with the RTS, S malaria vaccine. J Infect Dis 178:1139-44. 
Tang J., Amin M.A., Campian J.L. Past, Present, and Future of Viral Vector Vaccine Platforms: A 
Comprehensive Review. (2025). Vaccines (Basel). doi: 10.3390/vaccines 13050524. PMID: 40432133 
Templeton T.J., Kaslow D.C. (1997) Cloning and cross-species comparison of the thrombospondin-
related anonymous protein (TRAP) gene from Plasmodium knowlesi, Plasmodium vivax and 
Plasmodium gallinaceum. Mol Biochem Parasitol 84:13- 24. DOI: S0166-6851(96)02775-2 [pii]. 
 
Thomas A.W., Trape J.F., Rogier C., Goncalves A., Rosario V.E., Narum D.L. (1994) High prevalence 
of natural antibodies against Plasmodium falciparum 83-kilodalton apical membrane antigen 
(PF83/AMA-1) as detected by capture-enzyme-linked immunosorbent assay using full-length 
baculovirus recombinant PF83/AMA-1. Am J Trop Med Hyg 51:730-40. 
 
Tsoumani M.E., Voyiatzaki C., Efstathiou A. Malaria Vaccines: From the Past towards the mRNA 
Vaccine Era. (2023) Vaccines (Basel). doi: 10.3390/ vaccines 11091452. PMID: 37766129  
 
Tsuji M., Zavala F. (2001) Peptide-based subunit vaccines against pre-erythrocytic stages of malaria 
parasites. Mol Immunol 38:433-42. DOI: S0161589001000797 [pii]. 
 
Vallejo AF, García J, Amado-Garavito AB, Arévalo-Herrera M, Herrera S. (2016) Plasmodium vivax 
gametocyte infectivity in sub-microscopic infections. Malar J. Jan 28;15:48. Doi: 10.1186/s12936-016-
1104-1. PMID: 26822406 
Van der Boor SC, Alkema M, van Gemert GJ, Teelen K, van de Vegte-Bolmer M, Walk J, van Crevel R, 
de Mast Q, Ockenhouse CF, Sauerwein RW, McCall MBB. BMC Med. 2023 Apr 7;21(1):137. Whole 
sporozoite immunization with Plasmodium falciparum strain NF135 in a randomized trial. doi: 
10.1186/s12916-023-02788-9 
 
Vaughan 
AM, 
Kappe 
SHI. 
Expert 
Rev 
Vaccines. 
2017 
Aug;16(8):765-767.  
Genetically attenuated malaria parasites as vaccines. doi: 10.1080/14760584.2017.1341835. Epub 
2017 Jun 16. 
 
Walther M., Dunachie S., Keating S., Vuola J.M., Berthoud T., Schmidt A., Maier C., Andrews L., 
Andersen R.F., Gilbert S., Poulton I., Webster D., Dubovsky F., Tierney E., Sarpotdar P., Correa S., 
Huntcooke A., Butcher G., Williams J., Sinden R.E., Thornton G.B., Hill A.V. (2005) Safety, 
immunogenicity and efficacy of a pre-erythrocytic malaria candidate vaccine, ICC-1132 formulated in 
Seppic 
ISA 
720—vaccine 
23:857-64. 
DOI: 
S0264-410X(04)00642-5 
[pii] 
10.1016/j.vaccine.2004.08.020. 
 

 
 
 
Page 98 from 98 
 
Webster, R., Sekuloski, S., Odedra, A., Woolley, S., Jennings, H., Amante, F., ... &amp; McCarthy, J. S. 
(2021). Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine. 
BMC medicine, 19(1), 1-13. 
 
WHO. (2010) World Malaria Report 2010, World Health Organization, Washington. 
 
WHO. (2021) World Malaria Report 2021: regional data and trends. 
https://www.who.int/es/publications/m/item/WHO-UCN-GMP-2021.09 
 
WHO, (2023) WHO recommends R21/Matrix-M vaccine for malaria prevention in updated advice on 
immunization. https://www.who.int/news/item/02-10-2023-who-recommends-r21-matrix-m-vaccine-
for-malaria-prevention-in-updated-advice-on-immunization 
 
Williams T.N., Maitland K., Bennett S., Ganczakowski M., Peto T.E., Newbold C.I., Bowden D.K., 
Weatherall D.J., Clegg J.B. (1996) High incidence of malaria in alpha thalassaemic children. Nature 
383:522-5. DOI: 10.1038/383522a0. 
 
Wirtz R.A., Burkot T.R., Graves P.M., Andre R.G. (1987) Field evaluation of enzyme-linked 
immunosorbent assays for Plasmodium falciparum and Plasmodium vivax sporozoites in mosquitoes 
(Diptera: Culicidae) from Papua New Guinea. J Med Entomol 24:433-7. 
 
Wirtz R.A., Rosenberg R., Sattabongkot J., Webster H.K. (1990) Prevalence of antibody to heterologous 
circumsporozoite protein of Plasmodium vivax in Thailand. Lancet 336:593-5. DOI: 0140-
6736(90)93393-4 [pii]. 
 
Wombou Toukam CM, Solano P, Bengaly Z, Jamonneau V, Bucheton B. (2011) Experimental evaluation 
of xenodiagnosis to detect trypanosomes at low parasitaemia levels in infected hosts. Parasite 
18(4):295-302 
 
Yadava A., Sattabongkot J., Washington M.A., Ware L.A., Majam V., Zheng H., Kumar S., Ockenhouse 
C.F. (2007) A novel chimeric Plasmodium vivax circumsporozoite protein induces biologically 
functional antibodies that recognize both VK210 and VK247 sporozoites. Infect Immun 75:1177-85. 
DOI: IAI.01667-06 [pii] 10.1128/IAI.01667-06. 
 
Zhuo Y., Zeng H., Su C., Lv Q., Cheng., Lei L. Tailoring biomaterials for vaccine delivery. (2024). J 
Nanobiotechnology. doi: 10.1186/s12951-024-02758-0. PMID: 39135073