Evaluation of Reproducibility of a Sporozoite Challenge Model for Plasmodium Vivax in Human Volunteers
Condition: MALARIA; FEVER; Sweating Fever; Chills; ANEMIA
MeSH terms: Malaria; Fever
Study type: Interventional
Phase: Phase 2

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): 413ABM; 414WRR; 418JAL
Intervention type(s): Other; Other; Other
Intervention description(s): +/- 3 bites of Anopheles albimanus mosquitoes infected through membrane feeding with blood of the P. vivax infected volunteers 413ABM; +/- 3 bites of Anopheles albimanus mosquitoes infected through membrane feeding with blood of the P. vivax infected volunteers 414WRR; +/- 3 bites of Anopheles albimanus mosquitoes infected through membrane feeding with blood of the P. vivax infected volunteers 418JAL
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: Malaria; Sporozoite; Challenge Model; Plasmodium vivax; Human Volunteers