The use of P. falciparum sporozoite-infected A. stephensi mosquitoes reared aseptically under cGMPs is safe, efficiently and reliably transmits malaria, and is associated with a very precise pre-patent period. This first trial was a proof-of-principle study, establishing that these mosquitoes are viable and capable of transmitting malaria to volunteers at least as efficiently as mosquitoes reared under conventional conditions. Moreover, a precise prepatent period of 11 days was demonstrated in this study along with a determination that the quantity of mosquito bites required to confer malaria in 100% of the volunteers is at most, 3 bites, and may be one bite.
The traditional malaria challenge technique without the benefit of asepticity is safe and predictably transmits malaria 
. The aseptic malaria challenge model appears similarly safe and well tolerated and advances the science of malaria challenge as well as the protection of human subjects. Symptom severity was generally mild to moderate with only five volunteers suffering a severe symptom (i.e. fever >102.2°F) defined by criteria consistent with FDA guidelines. The symptoms associated with malaria peaked 2–3 days after diagnosis coinciding with clearance of parasites, and thus probably represent an inflammatory response to the clearing infection. The solicited symptoms noted in this trial were generally mild and even less in frequency than previously reported () 
. Similarly, laboratory abnormalities were transient, and mild to moderate in grade with the exception of a single case of severe thrombocytopenia (84,000/mm3
); a level that is not associated with an increased risk of bleeding. Leukopenia was noted in 41% of participants with malaria and occurred a mean of 2.1 days (1–4 days) after onset of detectable parasitemia by blood smear, and lasted between 1 and 8 days 
. The severity of laboratory abnormalities did not appear to correlate with the bite quantity or sporozoite load within the biting mosquitoes. None of the symptoms or laboratory abnormalities met World Health Organization criteria for severe malaria 
The traditional challenge methodology relies on the bite of five mosquitoes to reliably transmit malaria to 100% of volunteers. Utilizing mosquitoes raised aseptically under cGMPs, all participants in the 3 and 5 bite groups and 5 of 6 volunteers in the 1 bite group developed malaria. One volunteer withdrew from the study on day 13 and was treated with chloroquine before developing parasitemia. Excluding this volunteer, whose ultimate infection status is unknown, 100% infectivity was achieved with a single bite. The number of mosquitoes required to achieve a successful blood feed varied per individual. In instances where a higher number of mosquitoes were required to achieve a successful blood feed, it is possible that the total number of mosquitoes to which the volunteers were exposed was underestimated as mosquitoes can inoculate sporozoites as they probe for blood, even if they did not take a blood meal. However, of the volunteers randomized to the one bite arm, two were exposed to the bite of only one mosquito and the rest were exposed to only two or three mosquitoes (total of 10 mosquitoes for infecting 5 volunteers, ) before achieving a successful blood feed indicating that ≤3 mosquitoes are required to achieve malaria infection using this technique. It should be noted that all other studies have used the same criteria for a successful blood feed. Thus, regardless of probing without feeding, our data are distinct from the other studies, despite use of similar methodology.
Of the 17 participants who were infected, 14 (82%) developed a positive blood smear on Day 11 (range 9–12) after challenge. This compares favorably with previous published results where 18/47 (38%) participants developed malaria on Day 11 (range 9–14, mean 10.52 days) but the pre-patent period was more variable. Increased sporozoite inoculation and liver burden could result in reduced prepatent periods 
or prolonged parasitemias 
. Conversely, the physical characteristics of the mosquito salivary duct may limit the number of sporozoites that can be inoculated during probing and feeding 
. In the P. yoelli
model, sporozoite injection has proven to be highly variable ranging from 0–1,297 per bite (mean 123) and is only weakly correlated to sporozoite gland quantity 
. Therefore, increased numbers of sporozoites per mosquito may not translate into increased sporozoites inoculated. Complicating interpretation, the traditional method of determining sporozoite loads is imprecise, relying upon qualitative estimation of total sporozoites on salivary glands squashes, with gland scores graded from 0 to 4+ 
. In virtually all previous studies, mosquitoes with a gland score of ≥2+ (11–100 sporozoites) were considered infectious 
. Utilizing a more precise counting technique, 16,753 sporozoites per mosquito (range 1,000–57,500) were present in the challenge mosquitoes. Our data indicate that mosquitoes raised aseptically in compliance with cGMPs can successfully transmit malaria to 100% of participants through the bite of 3, and likely 1, rather than 5 mosquito bites, and suggest they may lead to a more precise pre-patent period. The mosquitoes used in this study had more sporozoites than those used in most other studies. Thus, it is possible that the infectivity of 1 and 3 infected mosquitoes may have been due to an increased sporozoite inoculum when compared to traditionally-raised mosquitoes, but experimental data in P. yoelii 
do not support this interpretation.
The use of PCR to achieve diagnosis earlier in malaria challenge studies is attractive, but it also carries risks. A false negative or false positive result in a small challenge trial of a malaria vaccine could profoundly alter study results. There were no false positive results in our preliminary use of this technique. While the ability to detect low-level parasitemia days before blood smear detection could, theoretically, avert symptoms associated with malaria, we did not find a correlation between PCR and symptom onset (data not shown). Moreover, the severity of symptoms increased once therapy was initiated and peaked after 48–72 hours when parasites were no longer detectable. Performance of the PCR assay could be increased to every 8–12 hours to enhance detection, but PCR is, currently, more labor-intensive procedure than is the reading of blood smears, and would require round-the-clock staffing in a challenge setting. Further study of diagnostic PCR in the context of volunteer challenges is required to fully document the pre-test specificity of the assay.
The development of a metabolically active, non-replicating (radiation attenuated) P. falciparum
sporozoite vaccine is based on the successful immunization of volunteers by the bite of irradiated, non-aseptic mosquitoes 
. It has been hypothesized that the contaminants, including bacterial and fungal material, accompanying these mosquitoes may provide some adjuvant effects that enhance sporozoite-induced immunity. Our study does not give any indication of the immunogenicity of the sporozoites administered by the bite of aseptically-reared mosquitoes, but establishing that the sporozoites produced under aseptic conditions remain fully virulent and capable of eliciting malaria is nonetheless important data for the effort to develop a metabolically active, non-replicating whole sporozoite P. falciparum
vaccine, which is produced in aseptic A. stephensi
mosquitoes using the same methodology.
Now that the aseptic malaria challenge model has been established with the NF54 strain of P. falciparum
, which has historically been utilized in malaria challenge studies, we plan to develop challenges with additional P. falciparum
strains. The genetic diversity of P. falciparum
poses significant challenges to vaccine development 
, and the development of a heterologous challenge model will permit assessment of the ability of vaccine candidates to provide protection against genetically diverse parasites and of anti-malarial drugs to prevent malaria caused by P. falciparum
of differing drug sensitivities.
The data from this trial demonstrate that aseptic sporozoites can transmit malaria. This is the first step toward developing and assessing the infectivity of aseptic, purified, cryopreserved P. falciparum sporozoites administered by needle and syringe to infect volunteers rather than relying on the bite of a mosquito. This would allow institutions without the capability of rearing infectious mosquitoes to safely, and reliably conduct malaria challenge trials, and such a study is being planned. Ultimately, our goal is to have multiple strains of P. falciparum parasites that are cryopreserved and packaged for transport to be used by institutions worldwide for testing malaria vaccines and pharmacologic agents.