Study design
The study presented here is a Phase 1 exploratory study that was conducted in four sequential single subject cohorts (ANZCTR Trial ID: ACTRN12617001502325) and performed at Q-Pharm Pty Ltd, Brisbane, Australia and the Apheresis Unit at the Royal Brisbane and Women’s Hospital (RBWH), Australia between October 2017 and May 2019. The primary objective of the study was to determine the safety of the P. vivax infection in healthy subjects following inoculation with blood-stage parasites, and the safety of apheresis for collection of P. vivax parasites from experimentally infected subjects. Secondary objectives were to assess the feasibility of apheresis as a method of harvesting, concentrating and subsequently cryopreserving P. vivax parasites from healthy subjects. Exploratory objectives were to evaluate the potential for apheresis to be used as a method for producing a P. vivax human malaria parasite bank, to evaluate the transmission of P. vivax gametocytes to mosquitoes and to collect and store plasma and peripheral blood mononuclear cells harvested using apheresis.
Specific modifications to the study protocol, such as the apheresis procedure, were required between subjects in an attempt to optimize the procedure and meet the objectives. All changes made between subjects were based on the findings from previous subjects.
Study subjects
Healthy adult males and females, aged between 18 and 55 years who met all inclusion criteria and none of the exclusion criteria were eligible for participation. Subjects were required to be malaria-naïve, Duffy blood group positive and have blood type O. Female subjects had to be Rh(D) positive. All subjects had to be available for a safety follow up period of three months. A full list of the inclusion/exclusion criteria for this study is included in the study protocol located in Additional File 1.
Study conduct
Pre-clinical component
A pre-clinical experiment was conducted prior to the clinical trial in order to confirm the feasibility of harvesting Plasmodium parasites using apheresis. The P. falciparum NF54 clone was used in these experiments [15] due to limited availability of P. vivax parasites. Plasmodium. falciparum infected red blood cells (17.6 ml; 16 ml blood with 0.1% asexual parasitaemia and 1.67 ml blood with 0.01% gametocytaemia) were added to 450 ml of fresh venous whole blood and subjected to ex vivo apheresis. Samples were collected from the 1%, 2%, 3%, 5% and 7% haematocrit (HCT) layers as determined by visualizing the colour saturation of the apheresis product. An automated haematology analyser (Sysmex XN-3000; Sysmex UK) was used retrospectively to confirm the HCT of samples collected during apheresis. Presence of parasites was assessed in each layer by 18S qPCR [16] and microscopy.
Clinical component
Following intravenous injection of P. vivax (day 0), subjects were monitored by daily telephone calls until day 4, when subjects visited the clinical unit daily until the day of apheresis. Subjects were monitored for adverse events (AEs), signs and symptoms of malaria infection, and blood was collected for 18S qPCR measurement of parasitaemia. The severity of AEs were determined by the common terminology of clinical trial adverse events (CTCAE) v. 4.03 [17].
The threshold for commencement of apheresis and treatment with artemether-lumefantrine was within 24 hours of a parasitaemia >20,000 parasites/mL, or the Malaria Clinical Score reaching >6 10], or at the Investigator’s discretion. The morning that this threshold was reached (anticipated based on previous studies to be Day 10 or 11 [9], subjects were admitted to the clinical unit (Q-Pharm) for initial safety assessments before being escorted to the Apheresis Unit at RBWH by Q-Pharm staff. The Apheresis Unit is located in the Haematology Department at RBWH where patients are subject to donor or therapeutic apheresis. At the Apheresis Unit the subjects underwent the apheresis procedure as per the Standard Operating Procedure (Additional Files 2 to 5) whilst being supervised by the apheresis specialist nurse and under the supervision of the responsible clinical haematologist (GK). The same apheresis nurse performed the apheresis procedure for all four subjects. The apheresis procedure lasted 1-4 hours. Subjects were then escorted back to the clinical unit and began treatment with artemether-lumefantrine (Riamet®, Novartis Pharmaceuticals Australia Pty Ltd). Treatment consisted of six doses of 4 tablets at 12 hourly intervals (each tablet contains 20 mg artemether and 120 mg of lumefantrine). Subjects remained confined within the clinical unit for 48-72 hours for safety monitoring. Following release from confinement, subjects attended protocol specified visits until three months post-treatment to monitor for signs of recrudescent parasitaemia and to assess late safety signals. Relapse is not a concern in the P. vivax IBSM studies as liver infection is bypassed and hypnozoites are not produced. A schematic of the study design is shown in Additional File 6 Fig. 1.
This study used an iterative adaptive design approach where subject safety and outcome data were analysed after each subject and modifications made to improve the chances of meeting the exploratory objectives in the subsequent subject. A summary of the changes instituted is shown in Table 1.
CMNC; continuous mononuclear cell collection, HCT; haematocrit. Protocols for subjects 1 to 4 and all experiments can be found in Additional Files 7 to 10. *Biological duplicates involved repeat 18S qPCR testing from two separate blood samples from each HCT layer collected using apheresis.
^When a HCT range is included the sample was taken from multiple HCT layers e.g. 5-7% = 5%, 6% and 7% HCT. +Originally aimed to sample 8% HCT layer but actual sample consisted of 11% HCT. #During cohort 4 a red cell depletion was carried out, producing an intermediate bag sample, followed by a second apheresis procedure on the red cell depletion product. The second apheresis procedure involved sampling of ~ 100 ml of the lowest HCT layers of the sample (final bag) followed by ~ 100mls of the subsequent lowest HCT layers (spare bag) and then the remainder ~ 100mls (waste bag).
Malaria challenge agent
The P. vivax human malaria parasite (HMP) bank HMP013 was derived from blood group O rhesus positive blood donated from a returned traveller from India who presented with clinical manifestations of malaria [9]. The inoculum was prepared as previously described [18].
Measurement of parasitaemia by qPCR
Parasitaemia was quantified using 18S qPCR targeting the highly conserved Plasmodium 18S ribosomal RNA gene [16, 19]. Quantitative reverse transcriptase PCR (qRT-PCR) assays were used to quantify gametocyte levels with assays targeting the P. falciparum pfs25 (female) and pfMGET (male) gametocyte mRNA transcripts [20] and P. vivax pvs25 (female) gametocyte mRNA transcripts [21].
Flow cytometry
Flow cytometry was performed to characterize cell populations present in samples collected during the apheresis process. A combination of stains and antibodies were used to identify cells containing DNA/RNA (SYBR Green I), white blood cells (WBCs) (CD45 antibody) and/or reticulocytes (CD71 antibody). Samples from subject 1 were stained with SYBR Green I (Molecular Probes) ; samples from subjects 2 and 3 were stained with SYBR Green I and CD45-PacificBlue; and samples from subject 4 were stained with SYBR Green I, CD45-Pacific Blue and/or CD71-APC. Samples were kept on ice or at 4-8ºC until analysed by flow cytometry.
SYBR Green I staining
A volume of 2.5 μl or 1 x 106 cells from each sample was stained with 30-50 μl of SYBR Green I at 10x for 30 minutes in the dark. After incubation, 200 μl of FACS buffer (2% fetal bovine serum in phosphate buffered saline) was added.
Antibody staining
Approximately 1 x 106 cells were stained with 5-10 μg/ml of CD45-Pacific Blue or 2.5 μl of CD71 stock solution for 30 minutes at 4-8ºC in the dark. Cells were washed twice with PBS by centrifugation at 1455 xg for 4 minutes, at 4ºC. After the last wash 200 μl of FACS buffer was added to the cells.
Double staining with SYBR Green I and CD45-Pacific Blue: A volume of 30 μl of SYBR Green I at 10x was added to pelleted cells that were previously stained with CD45-Pacific Blue (as mentioned above) for 30 minutes at 4-8ºC, in the dark. After incubation a volume of 200 μl of FACS buffer was added.
Triple staining with SYBR Green I, CD45-Pacific Blue and CD71-APC: Approximately 1 x 106 cells were stained with 10 μg/ml of CD45-Pacific Blue, 2.5 μl of CD71 stock solution and 30 μl of SYBR Green I at 10x. Samples were incubated for 30 minutes in the fridge (4-8ºC) in the dark. Cells were washed twice with PBS by centrifugation at 1455 xg for 4 minutes, at 4ºC. After incubation a volume of 200 μl of FACS buffer was added.
Flow cytometry analysis
Samples from subjects 1, 2 and 3 were acquired on a FACS CANTO II (BD Biosciences), using the 488 nm and 405 nm lasers. SYBR Green I positive cells were detected using a 530/30 nm band-pass filter and CD45-Pacific Blue positive cells were detected using a 450/50 nm band-pass filter. Samples from subject 4 were acquired on a LSR FORTESSA (BD Biosciences), using the 488 nm, 640 nm and 405 nm lasers. SYBR Green I positive cells were detected using a 530/30 nm filter, CD45-Pacific Blue positive cells were detected using a 450/50 nm filter and CD71-APC positive cells were detected using a 670/14 nm filter. Flow cytometry data was analysed using FlowJo® software (version 10.8, Tree Star Inc, Oregon, USA).
Microscopy
Thick and thin smears were stained with Giemsa and examined under a 100× oil immersion objective by level 1 or 2 WHO certified malaria microscopists. Apheresis samples were expected to have a significantly different composition in terms of proportions of RBCs and WBCs when compared to whole blood (e.g. RBCs make up 1% and approximately 46% of 1% HCT and whole blood samples respectively). As such, standard parasitaemia measures by microscopy were not feasible. It was decided that the expert microscopists would estimate parasitaemia based on sample composition.
Mosquito feeding assays
Transmissibility of pre-apheresis samples and post-apheresis samples to An. stephensi was evaluated using membrane feeding assays (MFA) [9, 22]. For enriched MFA, gametocytes present in 80 mL of whole blood (pre-apheresis) were enriched in 70% Percoll gradient. For direct MFA (DMFA), 650µL of pellet from whole blood (pre-apheresis) or from each apheresis sample was reconstituted to 50% haematocrit with malaria naïve AB+ serum. Infection in midguts was assessed by qPCR [23] 8 days after the feeding assays. For logistic reasons, enriched MFA was not carried out in subject 4. Following consideration of gametocyte levels measured by qRT-PCR targeting pvs25, DMFA was not carried out in subject 2.
Apheresis procedures
Apheresis was carried out using a Spectra Optia v11.3 apheresis system (Terumo BCT, Inc Tokyo Japan) as detailed in the Additional File 11.
The continuous mononuclear cell collection procedure was used to sample from the targeted HCT layers from the blood of subjects 1 to 3. The targeted HCT layers that were sampled from these subjects ranged from the platelet rich layer through to the red cell rich layer.
A double stage procedure was used to collect the targeted HCT layers from subject 4. In the first stage, a red cell depletion procedure was used to collect approximately 500 ml of packed red blood cells from the subject. Targeted HCT layers were then collected from the red cell concentrate using the polymorphonuclear (PMN) collection procedure. The starting product (red cell concentrate) for the second stage of this procedure had a significantly higher HCT than the whole blood of subjects 1 to 3 and sampling focussed on the higher HCT layers. This was the rationale for using a PMN collection in subject 4 rather than the CMNC collection used in subjects 1 to 3.
Statistical analysis
Continuous data was summarized using descriptive statistics (mean and standard deviation, or median and interquartile range). Categorical data was presented using N and %. Descriptive statistics were produced using Microsoft Excel® (version 1903). GraphPad® Prism was used for the construction of all figures.