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Controlled human malaria infections by intradermal injection of cryopreserved Plasmodium falciparum sporozoites.
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PMID:  23149582     Owner:  NLM     Status:  MEDLINE    
Controlled human malaria infection with sporozoites is a standardized and powerful tool for evaluation of malaria vaccine and drug efficacy but so far only applied by exposure to bites of Plasmodium falciparum (Pf)-infected mosquitoes. We assessed in an open label Phase 1 trial, infection after intradermal injection of respectively 2,500, 10,000, or 25,000 aseptic, purified, vialed, cryopreserved Pf sporozoites (PfSPZ) in three groups (N = 6/group) of healthy Dutch volunteers. Infection was safe and parasitemia developed in 15 of 18 volunteers (84%), 5 of 6 volunteers in each group. There were no differences between groups in time until parasitemia by microscopy or quantitative polymerase chain reaction, parasite kinetics, clinical symptoms, or laboratory values. This is the first successful infection by needle and syringe with PfSPZ manufactured in compliance with regulatory standards. After further optimization, the use of such PfSPZ may facilitate and accelerate clinical development of novel malaria drugs and vaccines.
Meta Roestenberg; Else M Bijker; B Kim Lee Sim; Peter F Billingsley; Eric R James; Guido J H Bastiaens; Anne C Teirlinck; Anja Scholzen; Karina Teelen; Theo Arens; André J A M van der Ven; Anusha Gunasekera; Sumana Chakravarty; Soundarapandian Velmurugan; Cornelus C Hermsen; Robert W Sauerwein; Stephen L Hoffman
Publication Detail:
Type:  Clinical Trial, Phase I; Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-11-13
Journal Detail:
Title:  The American journal of tropical medicine and hygiene     Volume:  88     ISSN:  1476-1645     ISO Abbreviation:  Am. J. Trop. Med. Hyg.     Publication Date:  2013 Jan 
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Created Date:  2013-01-10     Completed Date:  2013-02-26     Revised Date:  2013-07-11    
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Nlm Unique ID:  0370507     Medline TA:  Am J Trop Med Hyg     Country:  United States    
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Languages:  eng     Pagination:  5-13     Citation Subset:  AIM; IM    
Radboud University Nijmegen Medical Center, Department of Medical Microbiology, Nijmegen, The Netherlands.
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MeSH Terms
Malaria, Falciparum / therapy*
Plasmodium falciparum / growth & development*
Polymerase Chain Reaction
Grant Support
R44AI058375-03/AI/NIAID NIH HHS; R44AI058375-04/AI/NIAID NIH HHS; R44AI058375-05/AI/NIAID NIH HHS; R44AI058375-05S1/AI/NIAID NIH HHS
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Am J Trop Med Hyg. 2013 Jan;88(1):3-4   [PMID:  23303797 ]

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Journal ID (nlm-ta): Am J Trop Med Hyg
Journal ID (iso-abbrev): Am. J. Trop. Med. Hyg
Journal ID (publisher-id): tpmd
ISSN: 0002-9637
ISSN: 1476-1645
Publisher: The American Society of Tropical Medicine and Hygiene
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©The American Society of Tropical Medicine and Hygiene
Received Day: 03 Month: 10 Year: 2012
Accepted Day: 19 Month: 10 Year: 2012
Print publication date: Day: 09 Month: 1 Year: 2013
pmc-release publication date: Day: 09 Month: 1 Year: 2013
Volume: 88 Issue: 1
First Page: 5 Last Page: 13
PubMed Id: 23149582
ID: 3541746
DOI: 10.4269/ajtmh.2012.12-0613

Controlled Human Malaria Infections by Intradermal Injection of Cryopreserved Plasmodium falciparum Sporozoites Alternate Title:ROESTENBERG, BIJKER AND OTHERS Title for RRH:HUMAN MALARIA BY SPOROZOITE INJECTION
Meta Roestenberg†
Else M. Bijker†
B. Kim Lee Sim
Peter F. Billingsley
Eric R. James
Guido J. H. Bastiaens
Anne C. Teirlinck
Anja Scholzen
Karina Teelen
Theo Arens
André J. A. M. van der Ven
Anusha Gunasekera
Sumana Chakravarty
Soundarapandian Velmurugan
Cornelus C. Hermsen
Robert W. Sauerwein‡
Stephen L. Hoffman*‡
Radboud University Nijmegen Medical Center, Department of Medical Microbiology, Nijmegen, The Netherlands; Sanaria Inc., Rockville, Maryland; Protein Potential LLC, Rockville, Maryland; Radboud University Nijmegen Medical Center, Department of General Internal Medicine, Nijmegen, The Netherlands
Correspondence: *Address correspondence to Stephen L. Hoffman, Sanaria Inc., 9800 Medical Center Drive, Rockville, MD 20850. E-mail:
Correspondence: †These authors contributed equally to this study.
Correspondence: ‡These authors contributed equally to this study.


Malaria caused by Plasmodium falciparum (Pf) causes approximately one million deaths and 250 million clinical cases annually.1,2 Implementation of insecticide-impregnated bed nets, residual insecticide spraying, and combinations of anti-malarial drugs, has reduced malaria-associated morbidity and mortality in many areas.1 Questions related to sustainability of this effort, however, have led to a recent delineation of requirements for new tools.3,4 A safe, long-acting anti-malarial drug and a highly effective malaria vaccine would be powerful tools for control and elimination of Pf malaria.

Progress has been facilitated by the capacity to infect volunteers under controlled conditions to test new vaccines and drugs. Infection of volunteers by exposure to laboratory-reared Anopheles spp. mosquitoes transmitting Pf sporozoites (SPZ)5 was first introduced for treatment of neurosyphilis in the 1920s.6 The development of drugs such as chloroquine,7 primaquine,8 and atovaquone9 were facilitated by these controlled human malaria infections (CHMIs). The ability to culture Pf gametocytes1012 enhanced the capacity to produce infected mosquitoes for CHMI studies. Although potentially serious, Pf malaria can be radically cured at the earliest stages of blood infection when risks are virtually absent. CHMIs are restricted to a few specialized centers that can produce PfSPZ-infected mosquitoes, where more than 1,300 volunteers have been safely infected by the bites of PfSPZ-infected mosquitoes since 1986, primarily for clinical trials of drugs9 and malaria vaccines,5,1321 but also for trials of diagnostic tests,22 and studying human immune responses to Pf.23

In addition to the use of CHMIs for testing vaccines and drugs, controlled infections can also be used to immunize against malaria. For example, immunization with radiation-attenuated PfSPZ by bites of mosquitoes protects > 90% of volunteers according to the published literature,2426 and recently 100% protection against CHMI was achieved by immunization of volunteers taking a prophylactic regimen of chloroquine, with PfSPZ administered by mosquito bites.27,28

These highly protective immunization strategies could not be translated into an implementable vaccine, because they depended on inoculation of SPZ by mosquito bites. Inoculation of SPZ by injection would be a more feasible method and was performed through the early 1950s. The SPZ preparations used, however, were heavily contaminated with bacteria and mosquito material, and rates of infection with frozen and thawed SPZ were highly variable.2933 A contemporary approach to production of SPZ for infection or vaccination requires generating aseptic SPZ-infected mosquitoes, purifying SPZ from mosquito tissues, vialing, preserving, and administering the SPZ by needle and syringe. Sanaria has met these requirements to produce infectious aseptic, purified, vialed, cryopreserved PfSPZ (PfSPZ Challenge), and produced and tested the world's first vaccine composed of these sporozoites.34,35 Here, we report infection of volunteers with PfSPZ Challenge administered intradermally (ID) by needle and syringe.

Materials and Methods
Study population and study design.

This open label, Phase 1 clinical trial was performed at Radboud University Nijmegen Medical Center, the Netherlands, from October 2010 to July 2011. Volunteers 18–35 years of age were screened for eligibility by medical history, physical examination, and laboratory tests of blood, serum, and urine, including standard hematological, biochemical, and pregnancy tests, and malaria, human immunodeficiency virus (HIV), hepatitis B and hepatitis C serology. The main exclusion criteria were pregnancy; residence in a malaria-endemic area within the previous 6 months; positive Pf serology; symptoms, physical signs, or laboratory test results suggestive of systemic disorders; and history of drug or alcohol abuse interfering with normal social function. All volunteers gave written informed consent.

Eighteen healthy malaria-naive volunteers were included in this trial. Groups of six volunteers were injected intradermally (ID) with 2,500, 10,000, or 25,000 PfSPZ Challenge. The sample size of six per group had a power of 75% to show a difference between 2 of 6 volunteers infected in the 2,500 PfSPZ group and 6 of 6 volunteers infected in the 25,000 PfSPZ group. Dose escalation was done at a minimum interval of 3.5 weeks.

The trial was performed in accordance with Good Clinical Practice and an Investigational New Drug application filed with the U.S. Food and Drug Administration, and approved by the Central Committee for Research Involving Human Subjects of The Netherlands (CCMO NL31858.091.10). identifier: NCT 01086917.

Study intervention (PfSPZ Challenge).

The PfSPZ Challenge contains aseptic, purified, cryopreserved PfSPZ isolated from salivary glands of aseptically reared mosquitoes.34,35Anopheles stephensi mosquitoes were raised under aseptic conditions, and then fed on cultured Stage V gametocytes of the NF54 strain of Pf.36 Approximately 2 weeks later, mosquito salivary glands containing PfSPZ were dissected, and PfSPZ were purified, formulated, vialed (15,000 PfSPZ per vial), and cryopreserved in liquid nitrogen vapor phase at −140°C to −196°C.34,35 The PfSPZ Challenge released for clinical use met quality control specifications including sterility (USP 71 compendial assay), purity (Supplemental Figure S1), and potency (Table 1).

Potency was assessed as previously described34,35 by quantification of late liver stage parasites expressing Pf merozoite surface protein 1 (PfMSP-1)37 in cultured human hepatocytes (HC-04 cells)38 6 days after addition of PfSPZ (Table 1, Supplemental Figure S2). For this 6-day hepatocyte potency assay, 4.0 × 104 HC-04 (1F9) cells/well in triplicate were infected with 5.0 × 104 PfSPZ and incubated for 6 days with daily media change. Late liver stage parasites expressing PfMSP-1 were counted by staining the slides with an anti-PfMSP-1 mAb and fluorescently labeled secondary antibody. As previously described34,35 the membrane integrity of PfSPZ was used to assess cell viability (Table 1). For the sporozoite membrane integrity assay, propidium iodide and SYBR Green were added to 15,000 PfSPZ. PfSPZ were applied to a hemocytometer and incubated in a dark humidity chamber for 20 minutes, at which point the red PfSPZ (those with compromised membranes) and green PfSPZ (those with intact membranes) were counted under a fluorescent microscope. Those with intact membranes were considered viable, and viability is expressed as the percentage of total green PfSPZ over the total number of PfSPZ. Sporozoites was assessed before cryopreservation, for release of the lot, and to assess stability at defined time points after cryopreservation.

The lot of PfSPZ Challenge used in this study had been cryopreserved in liquid nitrogen vapor phase for 27 (dose of 2,500 PfSPZ) to 30 months (dose of 25,000 PfSPZ) before administration. Immediately before use, a vial of PfSPZ Challenge was thawed and diluted with phosphate buffered saline containing human serum albumin. Volunteers were injected within 30 minutes of thawing.


Three groups of six volunteers each were injected ID with PfSPZ Challenge over the deltoid muscle, one injection in each upper arm. Each injection of 50 μL contained half the total dose. After injection, volunteers were observed for at least 60 minutes. Inoculations of volunteers were spaced 60 minutes apart. In each dose group, two volunteers were inoculated 3 days before the remaining four volunteers.

Volunteers made at least one daily outpatient clinical visit beginning 5 days after inoculation of PfSPZ Challenge. All symptoms and signs (solicited and unsolicited) were recorded and graded by the attending physician as follows: mild (easily tolerated), moderate (interferes with normal activity), or severe (prevents normal activity); fever was recorded as grade 1 (> 37.5–38.0°C), grade 2 (> 38.0–39.0°C), or grade 3 (> 39.0°C). Hematological and biochemical parameters were monitored daily. Because of a previous cardiac-related serious adverse event (SAE) following CHMI with Pf infection,39 markers of cardiac damage and coagulation were assessed. Troponin, lactate dehydrogenase (LDH), platelets, and D-dimer were assessed daily during the period when blood stage parasitemia was expected, and for 3 days after initiating curative treatment with atovaquone/proguanil. If D-dimer or LDH were abnormal, blood samples were tested for fragmentocytes and von Willebrand cleaving protease activity, as markers for vascular endothelial cell activation.40 Final follow-up visits were on Days 35 and 140 after infection.

As soon as parasites were detected by microscopic examination of blood smears, volunteers were treated with atovaquone/proguanil (1,000/400 mg) administered orally once daily for 3 days. Complete cure was confirmed in all volunteers by two consecutive parasite-negative blood slides after treatment, at least 4 days apart. Volunteers who did not develop parasitemia by Day 21 after challenge were presumptively treated with the same regimen.


The primary outcome was occurrence of Pf parasitemia detected by microscopic examination of blood smears. Sampling was done twice daily on Days 5 and 6 post-inoculation, thrice daily on Days 7–11, twice daily on Days 12–15, once daily on Days 16–21, and for 2 days after initiation of treatment. To make thick blood smears, 15 μL of EDTA-anti-coagulated blood was spread on each well of a 3-well glass slide (CEL-LINE Diagnostic Microscope Slides, 30-12A-black-CE24, Braunschweig, Germany). After drying, wells were stained with Giemsa for 45 minutes, and examined at 1,000 × magnification to assess 0.5 μL of blood. The smear was scored as positive if two unambiguous parasites were found. Thus, volunteers could be diagnosed with as few as 4 parasites/μL of blood. The pre-patent period was defined as the period between inoculation of PfSPZ Challenge and appearance of first positive blood smear.

Retrospectively, parasitemias were determined by real-time quantitative polymerase chain reaction (qPCR), performed on all samples collected after challenge, as previously described.41 The sensitivity of qPCR was 20 parasites/mL of blood.

Statistical analysis.

Data analysis was performed using SPSS software version 16.0. The qPCR results were assessed by analysis of variance (ANOVA) on log-transformed data.

Parasitemia after injection of PfSPZ Challenge.

Thirty-six healthy, malaria-naive volunteers were screened and 18 were included. All volunteers completed follow-up (Supplemental Figure S3). After ID injection of PfSPZ Challenge, 15 of the 18 volunteers developed a positive blood smear for Pf, five of six volunteers from each group (Table 2). The slide-negative volunteers in each group were presumptively treated with atovaquone/proguanil at 21 days post-infection.

Blood slides were first positive 11 to 14.3 days after administration of PfSPZ Challenge. The geometric mean (GM) pre-patent period was similar for all groups, i.e., 13.0, 12.7, and 13.0 days for the groups receiving 2,500, 10,000, and 25,000 PfSPZ Challenge, respectively (ANOVA P = 0.92). The GM parasite densities by microscopy at the time of diagnosis were 12.4, 11.2, and 23.4 parasites/μL blood (ANOVA P = 0.69 on log-transformed data) (Table 2).

Quantitative PCRs were first positive 9.0 to 12.0 days after challenge (Table 2). Volunteers in the 2,500, 10,000, and 25,000 PfSPZ Challenge groups had similar GM times to first detection of parasites by qPCR of 10.6, 10.3, and 9.9 days (ANOVA P = 0.486) at a GM parasite density of 0.07, 0.2, and 0.2 parasites/μL blood (ANOVA P = 0.24), respectively. The GM parasite densities by PCR at the time of thick smear diagnosis were 35, 5, and 132 parasites/μL (ANOVA P = 0.23). qPCR was negative throughout the 21-day follow-up for the three slide-negative volunteers. Parasite growth was cyclical, and was similar in all dose groups (Figure 1), and the parasite replication rate in the bloodstream was comparable to that seen after CHMI by exposure to the bite of PfSPZ-infected mosquitoes, ∼11.5-fold every 48 hours.42


Local reactogenicity was not observed after ID administration of PfSPZ in any of the volunteers. All volunteers, including the three volunteers who did not develop parasitemia, reported solicited adverse events (AEs) considered possibly, probably, or definitely related to the trial procedures (clinical malaria) (Table 3). Headache was the most frequently reported AE, and occurred in all volunteers including the three who did not develop parasitemia. There were no significant differences among the groups in solicited AEs, which were most frequently reported between Days 12 and 18 post-injection. The percentage of volunteers with related grade 3 AEs was comparable to historical data from subjects subjected to CHMIs by mosquito-bites (44% versus 49%, respectively).42 The total number of solicited and unsolicited AEs reported over time is shown in Figure 2. There were few AEs before Day 7; PfSPZ Challenge inoculations were well tolerated.

Routine daily laboratory tests showed no clinically significant abnormalities before initiation of anti-malarial treatment. Three or 4 days after receiving the first dose of atovaquone/proguanil, four volunteers had thrombocyte levels in the range 78–95 × 109/L, which was below the lower limit of normal (120 × 109/L). Leukocyte counts decreased after initiation of treatment in all thick smear positive volunteers (minimum 2.89 × 109/L compared with 5.46 × 109/L at baseline). In 13 volunteers, D-dimers were > 500 ng/mL, the upper limit of normal (ULN), at 1 or 2 days after initiation of anti-malarial treatment (range of peaks: 540–10,200 ng/mL). D-dimer increases most likely reflect non-specific inflammatory responses to parasite-derived material released after initiation of treatment. In all volunteers, D-dimer concentrations normalized without complications. One volunteer had abnormal liver function tests at Day 2 post atovaquone/proguanil initiation. Maximum values were 526 U/L ASAT (ULN 40 U/L), 745 U/L ALAT (ULN 45 U/L), 777 U/L LDH (ULN 450 U/L), and 74 U/L γGT (ULN 50 U/L). Bilirubin and alkaline phosphatase were normal. Abnormal values had returned to baseline levels at Day 100 after infection.

One SAE occurred in a volunteer who reported chest pain 1 day after the first dose of atovaquone/proguanil. Based on medical history, the chest pain was initially considered possibly consistent with angina pectoris. Pain resolved within 1 hour without treatment. The volunteer was admitted to the cardiac care unit for monitoring for 6.5 hours. The first electrocardiogram (ECG) had a negative T-wave in V2, which was absent at the time of study initiation. All subsequent ECGs, beginning 2.5 hours after the first ECG, were comparable to baseline, with a negative T in V1 only. Troponin T levels were normal at the time of chest pain, 6 and 17 hours later, daily for the next 3 days and at trial Days 28 and 35. As per protocol, the trial was put on hold, and the event was reported to the Safety Monitoring Committee (SMC) and regulatory authorities. The SMC concurred with the principal investigator's attribution of the chest pain as “possibly related” to participation in the trial. The SMC concluded that although the cause of chest pain was not clear, the clinical data suggested that the SAE was not a serious cardiac event, and recommended resumption of the trial within 3 days of the event. The regulatory authorities concurred.


We report for the first time that healthy, malaria-naive volunteers can be infected with P. falciparum malaria by injection of aseptic, purified, cryopreserved PfSPZ manufactured in compliance with regulatory standards. Five of six volunteers became infected when 2,500, 10,000, or 25,000 PfSPZ were inoculated ID. The AEs were comparable with those in mosquito bite challenge trials.17,19,42,43 Virtually all related AEs were attributed to malaria, not to the inoculations with PfSPZ Challenge.

The capacity to infect volunteers with PfSPZ Challenge is dependent on the efficiency of administration and the infectiousness/fitness of the cryopreserved PfSPZ. It can be expressed by the success rate of infection in the exposed individuals and/or the pre-patent period, i.e., the time from inoculation until first detected parasitemia. Since 1986 CHMIs have been performed by exposing volunteers to bites of laboratory-reared mosquitoes infected by feeding on Pf gametocyte-infected erythrocytes grown in culture.12 Essentially, all volunteers challenged by bites of five PfSPZ-infected mosquitoes develop Pf parasitemia.5,12,17,19 When numbers are reduced to one or two mosquitoes, success rates drop to 50% or less.4345 The ID inoculation of the lowest dose of 2,500 cryopreserved PfSPZ Challenge, which resulted in infection of 5 of 6 volunteers in the current study, was thus at least as effective in achieving blood stage infection as the bites of 1–2 infected mosquitoes.

In regard to the pre-patent period the results were not straightforward. The pre-patent period in the 2,500 PfSPZ group was longer than was observed after 1–2 bites of PfSPZ (NF54)-infected mosquitoes at RUNMC43 but shorter than after 1–2 bites of PfSPZ (3D7)-infected mosquitoes at the Naval Medical Research Center.44 The longer pre-patent period in our study compared with the pre-patent period after exposure to NF54-infected mosquitoes may have been caused by fewer developing liver stage schizonts after inoculation than after exposure to the bites of 1–2 PfSPZ-infected mosquitoes. Alternatively, replication in the liver stage could have been of lower magnitude or slower with the aseptic, purified, cryopreserved PfSPZ as compared with the fresh PfSPZ delivered by the mosquito bite. Finally, the findings may just reflect expected biologic variability, because the study with 1–2 3D7 infected mosquitoes showed a longer pre-patent period than after PfSPZ Challenge.44

The asexual erythrocytic stage parasites in our study replicated ∼11.5-fold every ∼48 hours. Thus, with a 10-fold increase in PfSPZ, the theoretical time until parasitemia by microscopic examination (pre-patent period) should have been 2 days less in the 25,000 PfSPZ group as compared with the 2,500 PfSPZ group. However, this was not the case as pre-patent periods of 13.0 and 13.0 days by microscopy and 10.59 and 9.95 days by qPCR were obtained in the 2,500 PfSPZ and 25,000 PfSPZ groups, respectively. Thus, increasing the dose of PfSPZ Challenge 10-fold from 2,500 PfSPZ to 25,000 PfSPZ administered ID did not increase the percentage of infected volunteers or reduce the pre-patent period. Apparently, increasing the dose administered in two 50 μL injections did not result in higher numbers of PfSPZ getting from the skin to the circulation, invading and maturing in hepatocytes, eventually resulting in merozoites that invaded and multiplied in erythrocytes. Understanding this lack of dose response will be important for optimization of administration of PfSPZ Challenge. A possible explanation for this lack of dose response may be trapping of PfSPZ at the inoculation site. The use of five mosquitoes that probe in multiple sites must result in distribution of PfSPZ in the dermis and subcutaneous tissue in at least five different sites, and probably considerably more. Therefore, increase in the number of inoculation sites and injection of much smaller volumes (< 0.5 μL) may result in better infections. Such strategies may also be useful for improving the efficiency of administration of the irradiated PfSPZ in the PfSPZ Vaccine. Although not as profound, there was a lack of a linear dose response in the first trial of the PfSPZ Vaccine in which irradiated PfSPZ were administered in 120 μL ID or SC.35

To determine the minimal numbers of PfSPZ required to achieving 100% infection rates, and a pre-patent period similar to five PfSPZ-infected mosquitoes, it would be most useful to assess intravenous (IV) administration of PfSPZ Challenge. Data from studies in mice show that administration of purified cryopreserved Plasmodium yoelii (Py) SPZ required ∼23 times more PySPZ administered ID than IV to achieve 80% infection rates (ID80) (Table 4). Similar differences in liver load in vivo between IV and ID routes of administration were demonstrated using luciferase-labeled, bioluminescent fresh Plasmodium berghei (Pb) SPZ (Nganou-Makamdop and others, Parasite Immunol, published online ahead of print, doi:10.1111/j.1365-3024.2012.12000.x). Thus, we will conduct studies to investigate the minimal IV-dose and to optimize non-IV administration by modifying the route of administration (e.g., ID, subcutaneous, intramuscular), inoculation volume, numbers of inoculations, and sites of injection.

Next to route of administration, our manufacturing/cryopreservation process may also be responsible for reduced infectivity. In vitro assays of potency and viability estimate a maximum difference of 25–30% between fresh and cryopreserved PfSPZ Challenge (Table 1). Rodent model in vivo data, however, suggest that a ∼7-fold loss in infectivity caused by cryopreservation is more likely (Table 5). Therefore, we will continue to concentrate our efforts on improvement of infectivity of PfSPZ Challenge. Interestingly, once the merozoites are released from the liver into the bloodstream they are as fit as non-cryopreserved, mosquito-administered parasites, as their replication rates are similar.

Successful development and application of PfSPZ Challenge will increase the global capacity to conduct CHMIs, including in Africa where a CHMI consortium has been established with representative institutes from seven countries. This expansion of clinical sites conducting CHMIs will facilitate the clinical development of malaria vaccine candidates and anti-malarial drugs.3,46 Another advantage of CHMI by PfSPZ Challenge may be a better-defined number of injected PfSPZ compared with the numbers administered by mosquito bites. This may decrease the large inter-individual variation in the estimated number of infected hepatocytes.47 Furthermore, using needle administration of defined quantities of PfSPZ Challenge from the same lot, will allow for comparisons of parallel and sequential clinical trials at multiple sites, including malaria-endemic areas. Finally, needle and syringe administration of cryopreserved PfSPZ is critical for potential development of whole PfSPZ vaccines where parasite development is arrested by radiation, anti-malarial drugs, or genetic modification.

In summary, we show that aseptic, purified, vialed, cryopreserved PfSPZ (PfSPZ Challenge) are infectious to humans for at least 2.5 years after cryopreservation. These data provide the rationale and foundation for a clinical trials program aimed at establishing a dose and route of PfSPZ that consistently achieves 100% infection rates. This will allow for the global expansion of sites that can conduct CHMIs for assessment of malaria vaccines and new drugs, and the potential to develop whole parasite vaccines based on cryopreserved PfSPZ.

Supplementary Material Supplemental Figures.


FN1Financial support: This work was supported by the Top Institute Pharma (TI Pharma) (grant T4-102). A. C. T. is supported by the European Malaria Vaccine Development Association. K. N. M. is supported by a Mozaiek grant from the Netherlands Organization for Scientific Research. The development and manufacturing of PfSPZ Challenge was also supported by Small Business Innovation Research (SBIR) (grants R44AI058375-03, 04, 05, 05S1) from the National Institute of Allergy and Infectious Diseases at the National Institute of Health (NIAID/NIH) and through Agreement 07984 from the PATH Malaria Vaccine Initiative.

FN2Disclosure: Sanaria Inc. manufactured PfSPZ Challenge, and Protein Potential LLC is affiliated with Sanaria. Thus, all authors associated with Sanaria or Protein Potential have potential conflicts of interest. There are no other conflicts of interest.

FN3Authors' addresses: Meta Roestenberg, Else M. Bijker, Guido J. H. Bastiaens, Anne C. Teirlinck, Dunja Zimmerman, Karina Teelen, Theo Arens, Pieter Beckers, Annemieke Jansens, Anja Scholzen, Krystelle Nganou-Makamdop, Jorien Wiersma, Cornelus C. Hermsen, and Robert W. Sauerwein, Radboud University Nijmegen Medical Center, Department of Medical Microbiology, Nijmegen, The Netherlands, E-mails:,,,,,,,,,,,,, and Adrian J. F. Luty, Institut de Recherche pour le Développement, UMR 216 Mére et enfant face aux Infections Tropicales, Paris, France, and Faculté de Pharmacie, Université Paris Descartes, Sorbonne Paris Cité, France, E-mail: André J. A. M. van der Ven, Radboud University Nijmegen Medical Center, Department of General Internal Medicine, Nijmegen, The Netherlands, E-mail: B. Kim Lee Sim and Richard E. Stafford, Sanaria Inc., Rockville, MD, and Protein Potential LLC, Rockville, MD, E-mails: and Minglin Li, Protein Potential LLC, Rockville, MD, E-mail: Peter F. Billingsley, Eric R. James, Anusha Gunasekera, Adam Richman, Sumana Chakravarty, Soundarapandian Velmurugan, Tao Li, Anita Manoj, Adam J. Ruben, Abraham G. Eappen, and Stephen L. Hoffman, Sanaria Inc., Rockville, MD, E-mails:,,,,,,,,,, and


The following individuals from RUNMC and Sanaria contributed significantly to this project and are considered co-authors: Dunja Zimmerman, Pieter Beckers, Annemieke Jansens, Adrian J. F. Luty, Krystelle Nganou-Makamdop, Jorien Wiersma, Adam Richman, Tao Li, Anita Manoj, Adam J. Ruben, Abraham G. Eappen, Minglin Li, and Richard E. Stafford. We thank the trial volunteers, the staff from the Clinical Research Centre Nijmegen, and the staff from the RUNMC Pharmacy who made this study possible; Wendy Arts, Nanny Huiberts, Chantal Siebes, Marlou Kooreman, Paul Daemen, and Ella Driessen for reading many thick smears; and Gheorghe Pop for his cardiac monitoring of the trial volunteers. We thank Bas van Haren for his support with the ADAMTS13 measurements and Jody van den Ouweland for the performance of the Troponin T measurements. We thank the members of the Safety Monitoring Committee, Barney Graham, David Diemert, and Alexander Rennings for their participation and for their guidance and safety recommendations throughout the trial. We thank the entire Sanaria Manufacturing Team including Gametocyte Production: Yonas Abebe, Asha Patil, Yeab Getachew, Mark Loyevsky, Bingbing Deng; Mosquito Production: Steve Matheny, Yingda Wen, Keith Nelson, James Overby, Virak Pich, Thomas Mitchell; Dissection: Richard Fan; Purification: Lixin Gao, Ray Xu; Formulation and Cryopreservation: Aderonke Awe; Quality Assurance and Quality Control: Irina Belyakova, Mary King; Assays: Chinnamma Chakiath; Rana Chattopadhyay, Charles Anderson, Solomon Conteh, Gwynne Roth; Regulatory and Clinical Affairs: Tooba Murshedkar. We are grateful for Sanaria's Operations and Legal teams, especially Robert Thompson, Alexander Hoffman, and David Dolberg. We thank the entire Protein Potential Team including, Adriana Ahumada, Maria Socorro Orozco, Bing Jiang, Emily Chen. We also thank MSource (Belgium, now CromSource, Italy) for clinical monitoring.

1.. World Health Organization, Global Malaria ProgrammeYear: 2010World Malaria Report 2010: World Health Organization
2.. Murray CJ,Rosenfeld LC,Lim SS,Andrews KG,Foreman KJ,Haring D,Fullman N,Naghavi M,Lozano R,Lopez AD. Year: 2012Global malaria mortality between 1980 and 2010: a systematic analysisLancet37941343122305225
3.. Alonso PL,Brown G,Arevalo-Herrera M,Binka F,Chitnis C,Collins F,Doumbo OK,Greenwood B,Hall BF,Levine MM,Mendis K,Newman RD,Plowe CV,Rodriguez MH,Sinden R,Slutsker L,Tanner M. Year: 2011A research agenda to underpin malaria eradicationPLoS Med8e100040621311579
4.. Najera JA,Gonzalez-Silva M,Alonso PL. Year: 2011Some lessons for the future from the Global Malaria Eradication Programme (1955–1969)PLoS Med8e100041221311585
5.. Sauerwein RW,Roestenberg M,Moorthy VS. Year: 2011Experimental human challenge infections can accelerate clinical malaria vaccine developmentNat Rev Immunol11576421179119
6.. Covell G,Nicol WD. Year: 1951Clinical, chemotherapeutic and immunological studies on induced malariaBr Med Bull8515514944815
7.. Coggeshall LT,Craige B. Year: 1949Old and new plasmodicidesBoyd MFMalariology: A Comprehensive Survey of All Aspects of This Group of Diseases from a Global StandpointPhiladelphia, PAW. B. Saunders10711114
8.. Alving AS,Arnold J,Hockwald RS,Clayman CB,Dern RJ,Beutler E,Flanagan CL. Year: 1955Potentiation of the curative action of primaquine in vivax malaria by quinine and chloroquineJ Lab Clin Med4630130613242948
9.. Shapiro TA,Ranasinha CD,Kumar N,Barditch-Crovo P. Year: 1999Prophylactic activity of atovaquone against Plasmodium falciparum in humansAm J Trop Med Hyg6083183610344660
10.. Ifediba T,Vanderberg JP. Year: 1981Complete in vitro maturation of Plasmodium falciparum gametocytesNature2943643667031476
11.. Campbell CC,Collins WE,Nguyen Dinh P,Barber A,Broderson JR. Year: 1982Plasmodium falciparum gametocytes from culture in vitro develop to sporozoites that are infectious to primatesScience217104810507051285
12.. Chulay JD,Schneider I,Cosgriff TM,Hoffman SL,Ballou WR,Quakyi IA,Carter R,Trosper JH,Hockmeyer WT. Year: 1986Malaria transmitted to humans by mosquitoes infected from cultured Plasmodium falciparumAm J Trop Med Hyg3566683511753
13.. Ballou WR,Hoffman SL,Sherwood JA,Hollingdale MR,Neva FA,Hockmeyer WT,Gordon DM,Schneider I,Wirtz RA,Young JF,Wasserman GF,Reeve P,Diggs CL,Chulay JD. Year: 1987Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccineLancet1127712812884410
14.. Herrington DA,Clyde DF,Losonsky G,Cortesia M,Murphy JR,Davis J,Baqar S,Felix AM,Heimer EP,Gillessen D,Nardin E,Nussenzweig RS,Nussenzweig V,Hollingdale MR,Levine MM. Year: 1987Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoitesNature3282572592439920
15.. Egan JE,Hoffman SL,Haynes JD,Sadoff JC,Schneider I,Grau GE,Hollingdale MR,Ballou WR,Gordon DM. Year: 1993Humoral immune responses in volunteers immunized with irradiated Plasmodium falciparum sporozoitesAm J Trop Med Hyg491661738357078
16.. Hoffman SL,Edelman R,Bryan JP,Schneider I,Davis J,Sedegah M,Gordon D,Church P,Gross M,Silverman C,Hollingdale M,Clyde D,Sztein M,Losonsky G,Paparello S,Jones TR. Year: 1994Safety, immunogenicity, and efficacy of a malaria sporozoite vaccine administered with monophosphoryl lipid A, cell wall skeleton of mycobacteria, and squalane as adjuvantAm J Trop Med Hyg516036127985753
17.. Church LW,Le TP,Bryan JP,Gordon DM,Edelman R,Fries L,Davis JR,Herrington DA,Clyde DF,Shmuklarsky MJ,Schneider I,McGovern TW,Chulay JD,Ballou WR,Hoffman SL. Year: 1997Clinical manifestations of Plasmodium falciparum malaria experimentally induced by mosquito challengeJ Infect Dis1759159209086149
18.. Stoute JA,Slaoui M,Heppner DG,Momin P,Kester KE,Desmons P,Wellde BT,Garcon N,Krzych U,Marchand M. Year: 1997A preliminary evaluation of a recombinant circumsporozoite protein vaccine against Plasmodium falciparum malariaN Engl J Med33686918988885
19.. Epstein JE,Rao S,Williams F,Freilich D,Luke T,Sedegah M,de la Vega P,Sacci J,Richie TL,Hoffman SL. Year: 2007Safety and clinical outcome of experimental challenge of human volunteers with Plasmodium falciparum-infected mosquitoes: an updateJ Infect Dis19614515417538895
20.. Thompson FM,Porter DW,Okitsu SL,Westerfeld N,Vogel D,Todryk S,Poulton I,Correa S,Hutchings C,Berthoud T,Dunachie S,Andrews L,Williams JL,Sinden R,Gilbert SC,Pluschke G,Zurbriggen R,Hill AV. Year: 2008Evidence of blood stage efficacy with a virosomal malaria vaccine in a phase IIa clinical trialPLoS ONE3e149318231580
21.. Kester KE,Cummings JF,Ofori-Anyinam O,Ockenhouse CF,Krzych U,Moris P,Schwenk R,Nielsen RA,Debebe Z,Pinelis E,Juompan L,Williams J,Dowler M,Stewart J,Wirtz RA,Dubois MC,Lievens M,Cohen J,Ballou R,Heppner DG Jr. RTS,S Vaccine Evaluation GroupYear: 2009Randomized, double-blind, phase 2a trial of falciparum malaria vaccines RTS,S/AS01B and RTS,S/AS02A in malaria-naive adults: safety, efficacy, and immunologic associates of protectionJ Infect Dis20033734619569965
22.. Beadle C,Long GW,Weiss WR,McElroy PD,Maret SM,Oloo AJ,Hoffman SL. Year: 1994Diagnosis of malaria by detection of Plasmodium falciparum HRP-2 antigen with a rapid dipstick antigen-capture assayLancet3435645687906328
23.. McCall MB,Netea MG,Hermsen CC,Jansen T,Jacobs L,Golenbock D,van der Ven AJ,Sauerwein RW. Year: 2007Plasmodium falciparum infection causes proinflammatory priming of human TLR responsesJ Immunol17916217117579034
24.. Clyde DF,Most H,McCarthy VC,Vanderberg JP. Year: 1973Immunization of man against sporozoite-induced falciparum malariaAm J Med Sci2661691774583408
25.. Rieckmann KH,Carson PE,Beaudoin RL,Cassells JS,Sell KW. Year: 1974Sporozoite induced immunity in man against an Ethiopian strain of Plasmodium falciparumTrans R Soc Trop Med Hyg682582594608063
26.. Hoffman SL,Goh LM,Luke TC,Schneider I,Le TP,Doolan DL,Sacci J,de la Vega P,Dowler M,Paul C,Gordon DM,Stoute JA,Church LW,Sedegah M,Heppner DG,Ballou WR,Richie TL. Year: 2002Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoitesJ Infect Dis1851155116411930326
27.. Roestenberg M,McCall M,Hopman J,Wiersma J,Luty AJ,van Gemert GJ,van de Vegte-Bolmer M,van Schaijk B,Teelen K,Arens T,Spaarman L,de Mast Q,Roeffen W,Snounou G,Renia L,van der Ven A,Hermsen CC,Sauerwein R. Year: 2009Protection against a malaria challenge by sporozoite inoculationN Engl J Med36146847719641203
28.. Roestenberg M,Teirlinck AC,McCall MB,Teelen K,Makamdop KN,Wiersma J,Arens T,Beckers P,van Gemert G,van de Vegte-Bolmer M,van der Ven AJ,Luty AJ,Hermsen CC,Sauerwein RW. Year: 2011Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up studyLancet3771770177621514658
29.. James SP,Shute PG. Year: 1926Report on the First Results of Laboratory Work on Malaria in EnglandGenevaPublications of the League of Nations, Health Organization130
30.. Maynes B. Year: 1933The injection of mosquito sporozoites in malaria therapyPublic Health Rep48909913
31.. Mayne B,Young M. Year: 1941The technique of induced malaria as used in the South Carolina State HospitalJ Vener Dis Inf22271276
32.. Jeffery GM,Rendtorff RC. Year: 1955Preservation of viable human malaria sporozoites by low-temperature freezingExp Parasitol444545413262073
33.. Glynn JR,Bradley DJ. Year: 1995Inoculum size, incubation period and severity of malaria. Analysis of data from malaria therapy recordsParasitology1107197845714
34.. Hoffman SL,Billingsley PF,James E,Richman A,Loyevsky M,Li T,Chakravarty S,Gunasekera A,Li M,Stafford R,Ahumada A,Epstein JE,Sedegah M,Reyes S,Richie TL,Lyke KE,Edelman R,Laurens M,Plowe CV,Sim BK. Year: 2010Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malariaHum Vaccin69710619946222
35.. Epstein JE,Tewari K,Lyke KE,Sim BK,Billingsley PF,Laurens MB,Gunasekera A,Chakravarty S,James ER,Sedegah M,Richman A,Velmurugan S,Reyes S,Li M,Tucker K,Ahumada A,Ruben AJ,Li T,Stafford R,Eappen AG,Tamminga C,Bennett JW,Ockenhouse CF,Murphy JR,Komisar J,Thomas N,Loyevsky M,Birkett A,Plowe CV,Loucq C,Edelman R,Richie TL,Seder RA,Hoffman SL. Year: 2011Live attenuated malaria vaccine designed to protect through hepatic CD8+ T cell immunityScience33447548021903775
36.. Ponnudurai T,Lensen AH,Van Gemert GJ,Bensink MP,Bolmer M,Meuwissen JH. Year: 1989Infectivity of cultured Plasmodium falciparum gametocytes to mosquitoesParasitology981651732668861
37.. Holder AA. Year: 2009The carboxy-terminus of merozoite surface protein 1: structure, specific antibodies and immunity to malariaParasitology1361445145619627632
38.. Sattabongkot J,Yimamnuaychoke N,Leelaudomlipi S,Rasameesoraj M,Jenwithisuk R,Coleman RE,Udomsangpetch R,Cui L,Brewer TG. Year: 2006Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivaxAm J Trop Med Hyg7470871516687667
39.. Nieman AE,de Mast Q,Roestenberg M,Wiersma J,Pop G,Stalenhoef A,Druilhe P,Sauerwein R,van der Ven A. Year: 2009Cardiac complication after experimental human malaria infection: a case reportMalar J827719958549
40.. de Mast Q,Groot E,Lenting PJ,de Groot PG,McCall M,Sauerwein RW,Fijnheer R,van der Ven A. Year: 2007Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium falciparum malariaJ Infect Dis19662262817624850
41.. Hermsen CC,Telgt DS,Linders EH,van de Locht LA,Eling WM,Mensink EJ,Sauerwein RW. Year: 2001Detection of Plasmodium falciparum malaria parasites in vivo by real-time quantitative PCRMol Biochem Parasitol11824725111738714
42.. Roestenberg M,O'Hara GA,Duncan CJ,Epstein JE,Edwards NJ,Scholzen A,van der Ven AJ,Hermsen CC,Hill AV,Sauerwein RW. Year: 2012Comparison of clinical and parasitological data from controlled human malaria infection trialsPLoS ONE7e3843422701640
43.. Verhage DF,Telgt DS,Bousema JT,Hermsen CC,van Gemert GJ,van der Meer JW,Sauerwein RW. Year: 2005Clinical outcome of experimental human malaria induced by Plasmodium falciparum-infected mosquitoesNeth J Med63525815768480
44.. Rickman LS,Jones TR,Long GW,Paparello S,Schneider I,Paul CF,Beaudoin RL,Hoffman SL. Year: 1990Plasmodium falciparum-infected Anopheles stephensi inconsistently transmit malaria to humansAm J Trop Med Hyg434414452240371
45.. Fries LF,Gordon DM,Schneider I,Beier JC,Long GW,Gross M,Que JU,Cryz SJ,Sadoff JC. Year: 1992Safety, immunogenicity, and efficacy of a Plasmodium falciparum vaccine comprising a circumsporozoite protein repeat region peptide conjugated to Pseudomonas aeruginosa toxin AInfect Immun60183418391563771
46.. WHOYear: 2011Malaria Vaccine Rainbow Tables Available at. Accessed November 15, 2011.
47.. Hermsen CC,De Vlas SJ,van Gemert GJ,Telgt DS,Verhage DF,Sauerwein RW. Year: 2004Testing vaccines in human experimental malaria: statistical analysis of parasitemia measured by a quantitative real-time polymerase chain reactionAm J Trop Med Hyg7119620115306710


[Figure ID: F1]
Figure 1. 

Parasite density as measured by quantitative polymerase chain reaction (qPCR) in the 2,500 (A), 10,000 (B), and 25,000 (C) PfSPZ Challenge dose groups. Panels A, B, and C show geometric mean parasite density of positive volunteers per group with confidence intervals (N = 5 for all groups) from day of inoculation through last day of positivity after initiation of treatment. Panel D shows an overlay of geometric mean parasite densities of positive volunteers in each group.

[Figure ID: F2]
Figure 2. 

Number of possibly, probably, or definitely related solicited and unsolicited adverse events reported over time in the 2,500 (black dashed), 10,000 (red dotted), and 25,000 PfSPZ Challenge dose (green straight) groups.

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