In spite of the huge global morbidity and mortality inflicted by malaria, an effective and practical vaccine against this disease has not yet been achieved. Early studies on immunization against sporozoite-induced rodent malaria resulted in close to 100% protection when mice were immunized with Plasmodium berghei sporozoites irradiated to sufficient levels and the immunized mice were subsequently challenged with non-irradiated sporozoites [1, 2].
Nevertheless, immunization with attenuated P. berghei sporozoites via routes other than intravenous (IV) was found to be far less protective. Thus, even after five immunizations, mice immunized by intramuscular (IM), intraperitoneal (IP) or intradermal (ID) routes were protected only 32%, 26% and 24%, respectively, in contrast to 95% protection after IV immunization . In another P. berghei study done with similar protocols, IM immunization resulted in only 11% protection, although addition of albumin to the immunizing inoculum raised this to 42%; in contrast, IV immunization yielded 100% protection .
Because sporozoite suspensions used for immunization were heavily contaminated with microorganisms and mosquito components, it was clear that such immunization trials by IV injection could not be directly extended to humans. An alternate approach, however, allowed irradiated mosquitoes to directly inoculate attenuated sporozoites into hosts, the mosquitoes thereby acting as vehicles of immunization. This approach was first established with rodent malaria  and then extended to the first successful human vaccination trial against P falciparum malaria . A compendium of subsequent human vaccination trials with this approach showed that when sufficient numbers of mosquitoes were used for immunization, greater than 90% of volunteers were completely protected against challenge by bite of infected mosquitoes [7, 8]. Recent progress by this group, under the auspices of the biopharmaceutical company Sanaria, has permitted the raising of large numbers of mosquitoes infected with Plasmodium falciparum sporozoites, the purification of these sporozoites sufficient to render them acceptable for human vaccination, and the successful freeze-preservation of the attenuated sporozoites. Trials are currently underway to attempt to vaccinate humans by syringe injection of these sporozoites [9, 10].
A central question for any human trials relates to an appropriate route of immunization. It had long been assumed that most sporozoites injected by mosquitoes rapidly reach the blood, after which they travel to the liver for further development. Thus, there was a supposition that sporozoite inoculation by mosquitoes mimicked IV inoculation of sporozoites by syringe. But studies have shown that most if not all mosquito-injected sporozoites are deposited into avascular portions of the skin and sub-cutaneous tissues and that sporozoites then use gliding motility to reach blood vessels to travel to the liver [11, 12], or enter lymph vessels to travel to local draining lymph nodes . This has led to the possibility that inoculation of isolated sporozoites directly into the skin by syringe might successfully replicate the recognized successful approach of allowing mosquitoes to inoculate attenuated sporozoites into skin.
Accordingly, the Plasmodium yoelii rodent malaria system was used to explore this approach and investigate ways of further enhancing the protective immunogenicity of ID-injected, attenuated sporozoites. The P. yoelii system is far superior to the P. berghei system in the infectivity of sporozoites, appearing to be similar in that respect to the human malarias. Indeed, others have contended that "the P. yoelii system has accurately predicted the success or failure of every approach to malaria vaccination that has been tested in humans" . A recent publication reported that it took at least four ID immunizations with a total of 6,000 P. yoelii sporozoites to equal the protective immunity that could be accomplished with only three doses and a total of only 2,250 sporozoites administered IV . Because of the practical value of achieving protective immunity with fewer doses of sporozoites, the current study tested whether a substantially higher number of sporozoites given in only two doses ID might result in protection equivalent to that obtained with IV immunization.
The main goal was to assess the protective immunogenicity of attenuated P. yoelii sporozoites injected by syringe into the skin. ID immunization with P. yoelii sporozoites was found to give far better protection than had been observed in previous attempts using non-IV routes of administration to immunize with P. berghei sporozoites [3, 4]. Furthermore, administering larger numbers of P. yoelii sporozoites ID was able to give a degree of protective immunity equivalent to what had been reported for ID immunization with twice the number of immunizing doses .
Finally, because many sporozoites injected by mosquitoes remain in the skin and either deteriorate  or differentiate  and because such sporozoites may be involved in induction of the immune response , two methods were tested to possibly enhance the immune response with adjuvants in conjunction with ID-administration of sporozoites; a) topical application of the toll-like receptor (TLR) agonist Imiquimod, as previously done for immunization studies using sub-unit vaccines against malaria , b) "tape-stripping" (TS), a procedure known to disrupt the stratum corneum and enhance the immunogenicity of ID-injected antigens . All ID-injection procedures were found to lead to levels of protection not significantly different from that which had been observed after IV immunization.