The study is a randomized open label clinical trial to verify the reproducibility of a sporozoite challenge model for Plasmodium vivax in humans. The verification of the reproducibility of such a model will make it possible to evaluate the efficacy of candidate P. vivax vaccines in Phase 2a trials. The study is divided into two successive steps:

Step A Parasite Blood Donation: Volunteers will be recruited passively from a group of patients who present with active P. vivax infection and accept to donate infected blood. Samples of P. vivax infected blood will be collected and will be screened for infectious diseases, according to standard blood bank procedures. Colonized Anopheles albimanus mosquitoes will be fed with this blood using a Membrane Feeding Assay (MFA). Sixteen (16) days after, selected positive mosquito batches will be used for step B.

Step B Challenge: After informed consent signature, a total of 18 healthy volunteers will be randomly allocated to Groups 1, 2 and 3, of 6 volunteers each and will be challenged with the bite of 3±1, P. vivax infected mosquitoes. Each group will be exposed to a different isolated parasite. Volunteers will be closely monitored post infection, and will be treated as soon as blood infection becomes patent as ascertained by microscopic examination of thick blood smears (TBS). Comparison of data obtained in the three different groups will be used to determine reproducibility of challenge model.

Primary objective: To demonstrate that naïve human volunteers can be safely and reproducibly infected by the bite of An. albimanus mosquitoes carrying P. vivax sporozoites in their salivary glands.

Secondary objective: To determine the influence of the type of parasite isolated on reproducibility and safety of the challenge model with P. vivax in human volunteers

Hypothesis:It is possible to safely develop a reproducible P. vivax infection in human volunteers using P. vivax experimentally infected An. albimanus mosquitoes.

Study design

A randomized, open-label clinical trial was designed to standardize a P. vivax sporozoite challenge model in malaria-naïve volunteers, specifically, the relationship between the number of infectious mosquito bites and the likelihood of developing patent parasitemia detectable by Giemsa-stained thick blood smear (TBS). The study was divided into two steps. Step A was designed to produce mature P. vivax sporozoites suitable for inoculation into humans; and Step B was designed to assess the safety and reproducibility of the sporozoite challenge. For Step A, patients with infective P. vivax gametocytes detected by TBS were recruited from the outpatient clinics at the Immunology Institute (IDIV) in Cali and Buenaventura, Colombia. Patients donated 35 mL of whole blood, which was screened for co-infections that could potentially represent a threat to the health of volunteers. The blood was then artificially fed to Anopheles mosquitoes.14 For Step B, malaria-naive subjects were exposed to the bites of 3 ± 1, 6 ± 1, or 9 ± 1 infected mosquitoes to determine the prepatent period of the infection and its reproducibility.

Blood donation and blood quality assurance

The EDTA vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) were used to collect 35 mL of whole blood from each infected patient, which were divided into two aliquots: a 15 mL sample for mosquito feeding and a 20 mL sample that was transported to the blood bank of the Valle del Lili Clinic for routine screening for a panel of common infectious agents (viral, bacterial, parasitic), including confirmation of Plasmodium species ( P. vivax, P. falciparum, P. malariae) by polymerase chain reaction (PCR) (Table 2).15 The rationale behind the blood screening and direct mosquito feeding on human volunteers was based on two premises: 1) standard blood bank screening is universally accepted as a safety procedure that ensures that no pathogens are transmitted from human-to-human during a blood transfusion. Therefore, blood samples determined to be negative for pathogens other than P. vivax by standard blood bank screening should be safe enough to be transferred from human-to-human through a mosquito bite. 2) Anopheles mosquitoes have not been reported to trans mit any pathogen other than Plasmodium. Before the study, we carried out an extensive bibliographic search and consulted with U.S. and Colombian experts on virology, parasitology, and entomology about potential pathogens that might be transmitted by Anopheles and could represent a threat for the volunteers exposed to challenge. There was a consensus that as yet no other pathogen had been reported to be transmitted by Anopheles mosquitoes, including those comprised in the blood bank screening. However, mosquitoes were discarded using a biosafety procedure if after feeding blood was confirmed to have any co-infection.

Mosquito infection

Anopheles albimanus mosquitoes Buenaventura strain, have been reared under laboratory conditions at IDIV in Cali for about 10 years. This mosquito colony has been successfully used to regularly produce sporozoites14,16 and for studies on malaria transmission-blocking immunity.17 Here, mosquitoes were fed within 2-3 hours after the P. vivax-infected blood (gametocytemia ≥ 0.1%) was collected in Buenaventura. The 15 mL blood was centrifuged at 500 × g for 5 minutes at room temperature, plasma was removed, and cells were washed once with RPMI 1640 medium (Gibco Cell Culture Systems, Invitrogen, Grand Island, NY ). Parasitized erythrocytes were reconstituted to 50% hematocrit with pooled human AB non-immune, complement-inactivated serum obtained from the Red Cross blood bank and were used to infect lots of 4,000 female mosquitoes (3-4 days old) using a water-jacketed membrane apparatus at 37°C, described previously.14 Infected mosquitoes were maintained under strict biosafety conditions at 27 ± 1°C and relative humidity of 82% and a sugar solution supplemented with 0.05% para-aminobenzoic-acid was provided.18 Samples of fed mosquitoes were dissected and microscopically examined 7-8 days after the blood meal to determine the presence of oocysts in the midgut, and on Days 14 and 15 to assess the presence of sporozoites in salivary glands.19 Mosquito infections were graded as 1+ (1-10 spz), 2+ (11-100 spz), 3+ (101-1000 spz), and 4+ (> 1001 spz).7

Sporozoite challenge

The proposed infective biting dose was to be 3, 6, and 9 mosquitoes, respectively. However, a range of plus or minus one bite per dose was allowed. Therefore, screen-meshed boxes (7 × 7 × 7 cm) were filled with 4, 7, and 10 mosquitoes, to have a better chance of achieving the targeted mosquito dose in a single biting round. Participants who did not complete the minimal targeted dose were subjected to a second round of biting. Eighteen malaria-naïve volunteers were randomly assigned to one of three groups (N = 6) and were exposed by group, from the fewest (3 bites) to the greatest (9 bites) number of bites. Sporozoite challenge was carried out under strict adherence to experimental protocol in a secure room in the entomology unit at the IDIV. Volunteers were asked not to use any topical chemicals (e.g., soap, deodorant, perfume) that could affect mosquito feeding. Mosquitoes were allowed to bite the flexor side of the forearm for a 10 to 15-minute period, previously determined to be sufficient for full An. albimanus engorgement. After biting, all mosquitoes were dissected to confirm the presence of blood meal and sporozoites. Mosquitoes were considered positive if any number of sporozoites (> 1) was detected microscopically. Study participants were observed for 1 hour at the entomology unit, followed up by phone 8 hours later, and examined again 24 hours thereafter to assess the response to challenge.

Malaria diagnosis and patient follow-up

From Day 7 post-challenge onward, volunteers had daily follow-up visits during which symptoms and signs of malaria were assessed and blood was collected for TBS and Plasmodium PCR (the latter performed retrospectively).

The TBS were performed using 50 μL of whole blood collected by finger prick that were spread over a rectangular area of approximately 1.5 sq cm and were stained with freshly prepared 10% Giemsa stain.20 A total of 300 microscopic fields were examined before a slide was considered negative. When TBS were found positive, parasitemia was quantified by estimating the number of parasites counted in presence of 300 leukocytes and calculating absolute parasitemia according the leukocyte counts per μL. Smears were read by experienced microscopists and a random sample of TBS was subjected to quality control by a microscopist from the malaria control program.

Participants were treated with anti-malarial drugs as soon as parasites were detected by TBS. Treatment consisted of chloroquine (1,500 mg chloroquine base provided orally in divided doses: 600 mg initially followed by 450 mg given 24 and 48 hours later) and primaquine (two 15 mg doses given once per day for 14 days). Volunteers were provided with intravenous fluids, analgesics, and antiemetics as needed. Signs and symptoms consistent with malaria were assessed by clinical examination and were graded 1-5 according to their severity following the National Cancer Institute (NCI) criteria for adverse events (CTCAE ) as follows: Grade 1 = Low, Grade 2 = Moderate, Grade 3 = Severe, Grade 4 = Serious, and Grade 5 = Death. Pruritus was graded from 1 to 3 (1 = mild or localized; 2 = intense or widespread; and 3 = intense or widespread and interfering with activities of daily living). Because there is no common terminology AE agreement, for edema and erythema following mosquito bites, an adaptation from the CTCAE v 3 for "injection site/extravasation changes" was used as follows: Grade 1 = local pain/edema and erythema; Grade 2 = pain or swelling with inflammation or phlebitis; Grade 3 = ulceration or necrosis. Pain was scored as Grade 1 = mild not interfering with function; Grade 2 = moderate pain (pain or analgesics interfering with function, but not interfering with activities of daily living); Grade 3 = severe pain (pain or analgesics severely interfering with activities of daily living).

Clinical laboratory tests

Clinical laboratory screening in -clouding an electrocardiogram confirmed the health status of volunteers 1 month before challenge and after the study was completed (Table 1). Any abnormality found in the electrocardiogram was considered as an exclusion criterion. Tests for hemoglobin, white blood cell count, platelet count, and total bilirubin were performed again on Days 11 and 53 post-challenge.

Statistical methods

Sample size (N = 6 per group) was based on the minimum number of individuals that would be allowed to observe the occurrence of rarer events (e.g., events that occur in approximately 5% of individuals) with reasonable probability based on a binomial assumption. The overall sample size of 18 would be sufficient to detect even rare events. A titration of mosquito bites (biting doses) was established; beginning with that arbitrarily considered the minimum number required causing an infection. Prepatent periods and duration of symptoms were expressed as geometric means. Differences among the groups were estimated by the Kruskal-Wallis test, and were considered statistically significant at P values less than 0.05.

Inclusion Criteria:

• Healthy, adult male or non-pregnant females (18-45 years of age).
• Capacity to sign a free informed consent form of participation along with two witnesses.
• Use of adequate contraceptive method from the initiation of the study until three months after sporozoite challenge.
• No plans to travel to malaria endemic areas during the course of the study (a year).
• No plans to travel outside the study area from the 7th day until 31st day after the challenge.
• Reachable by phone during the whole study period.
• Able to participate during the whole study period.

Exclusion Criteria:

• Are less than 18 and over 45 years of age.
• Are female who is pregnant at serum positive B-HCG screening, planning to become pregnant or who is nursing.
• Have a Duffy negative phenotype.
• Have a G-6-PD deficiency or any other hemoglobinopathy.
• Current or past infection with any species of malaria as demonstrated by a positive TBS on screening or history of a documented positive blood smear. P. vivax IFAT of 1
• Have a known history of allergy to antimalarial drugs or immediate type hypersensitivity reactions to mosquito bites.
• Clinical or laboratory evidence of significant systemic disease, including hepatic, renal, cardiac, immunologic or hematological disease, HIV positive or have any other known immunodeficiency (including receiving immunosuppressive therapy or a history of splenectomy); infected with hepatitis B or C virus; have a history of autoimmune disease (including inflammatory bowel disease, hemolytic anemia, autoimmune hepatitis, rheumatoid arthritis, lupus, etc.) or connective tissue disease or have any other serious underlying medical condition.
• Clinically significant laboratory abnormalities as determined by the Investigator(s).
• Plan to have surgery between enrollment and the end of the challenge follow-up.
• Previous history of alcoholism or drugs use which interfere with social activities of the volunteer
• Have any other conditions that are determined by at least two concurring investigators that may interfere with the capacity to provide free and willing informed consent.

3 months

Volunteers were directly observed by a study physician for one hour post-challenge, they were phone called approximately eight hours after challenge to control her/his evolution and then were followed by by the physician at day 1 after challenge.