Imaging mosquito transmission of Plasmodium sporozoites into the mammalian host: immunological implications.

The malaria infection is initiated in mammals by injection of the sporozoite stage of the parasite through the bite of Plasmodium-infected, female Anopheles mosquitoes. Sporozoites are injected into extravascular portions of the skin while the mosquito is probing for a blood source. Sporozoite gliding motility allows them to locate and penetrate blood vessels of the dermis or subcutaneous tissues; once in the blood, they reach the liver, within which they continue their development. Some of the injected parasites invade dermal lymph vessels and travel to the proximal draining lymphatic node, where they interact with host immunocytes. The host responds to viable or attenuated sporozoites with antibodies directed against the immunodominant circumsporozoite protein (CSP), as well as against other sporozoite proteins. These CSP antibodies can inhibit the numbers of sporozoites injected by mosquitoes and the motility of those injected into the skin. This first phase of the immune response is followed by cell-mediated immunity involving CD8 T-cells directed against the developing liver stage of the parasite. This review discusses the early history of imaging studies, and focuses on the role that imaging has played in enabling a better understanding of both the induction and effector functions of the immune responses against sporozoites.

Intradermal immunization of mice with radiation-attenuated sporozoites of Plasmodium yoelii induces effective protective immunity.

BACKGROUND: Intravenous injection of mice with attenuated Plasmodium berghei sporozoites induces sterile immunity to challenge with viable sporozoites. Non-intravenous routes have been reported to yield poor immunity. Because intravenous immunization has been considered to be unacceptable for large scale vaccination of humans, assessment was made of the results of intradermal immunization of mice with Plasmodium yoelii, a rodent malaria parasite whose infectivity resembles that of human malaria. METHODS: Mice were immunized with two injections of isolated, radiation-attenuated P. yoelii sporozoites, either by intravenous (IV) or intradermal (ID) inoculation. In an attempt to enhance protective immunogenicity of ID-injections, one group of experimental mice received topical application of an adjuvant, Imiquimod, while another group had their injections accompanied by local "tape-stripping" of the skin, a procedure known to disrupt the stratum corneum and activate local immunocytes. Challenge of immunized and non-immunized control mice was by bite of sporozoite-infected mosquitoes. Degree of protection among the various groups of mice was determined by microscopic examination of stained blood smears. Statistical significance of protection was determined by a one-way ANOVA followed by Tukey's post hoc test. RESULTS: Two intravenous immunizations produced 94% protection to mosquito bite challenge; intradermal immunization produced 78% protection, while intradermal immunization accompanied by "tape-stripping" produced 94% protection. There were no statistically significant differences in degree of protective immunity between immunizations done by intravenous versus intradermal injection. CONCLUSIONS: The use of a sub-microlitre syringe for intradermal injections yielded excellent protective immunity. ID-immunization with large numbers of radiation-attenuated P. yoelii sporozoites led to levels of protective immunity comparable to those achieved by IV-immunization. It remains to be determined whether an adjuvant treatment can be found to substantially reduce the numbers of attenuated sporozoites required to achieve a strong protective immunity with as few doses as possible for possible extension to immunization of humans.

Initiation of Plasmodium sporozoite motility by albumin is associated with induction of intracellular signalling.

Malaria infection is initiated when a mosquito injects Plasmodium sporozoites into a mammalian host. Sporozoites exhibit gliding motility both in vitro and in vivo. This motility is associated with the secretion of at least two proteins, circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Both derive from micronemes, which are organelles that empty out of the apical end of the sporozoite. Sporozoite motility can be initiated in vitro by albumin added to the medium. To investigate how albumin functions in this process, we studied second messenger signalling within the sporozoite. Using pharmacological activators and inhibitors, we have concluded that gliding motility is initiated when albumin interacts with the surface of the sporozoite and that this leads to a signal transduction cascade within the sporozoite, including the elevation of intracellular cAMP, the modulation of sporozoite motility by Ca(2+) and the release of microneme proteins.

Reflections on early malaria vaccine studies, the first successful human malaria vaccination, and beyond.

Advances towards protective vaccines against malaria were made feasible by the development of a rodent model of mammalian malaria that allowed production of all stages of the malaria parasite for study. Investigations with sporozoites (the stage transmitted by mosquitoes in their saliva) demonstrated that immunization with radiation-attenuated sporozoites could produce a solid, sterile immunity, first shown in studies with mice and later with human volunteers. Protective immune mechanisms involve anti-sporozoite antibodies that immobilize sporozoites injected into the skin by mosquitoes, followed by CD4+ and CD8+ T-cells acting against liver stage parasites produced by sporozoites that have escaped antibody-based immunity and invaded hepatocytes. Two alternative approaches now being used in human trials are immunization with intact, attenuated sporozoites vs. immunization with "sub-unit" vaccines based on immunogenic components of sporozoites or liver stage parasites. In addition to immunization against these pre-erythrocytic stages, encouraging progress is being made on immunization against blood stage parasites and on immunization for production of transmission-blocking antibodies. There is reason to be optimistic that one or more of the approaches will work on a large scale, and that a multi-stage vaccine may be able to combine several of these approaches in a sequential immunological assault against the malaria parasite as it progresses through its stages.

Re-ingestion of Plasmodium berghei sporozoites after delivery into the host by mosquitoes.

Malaria-infected mosquitoes feeding on a mammalian host inject sporozoites into the skin to induce a malaria infection. The numbers of sporozoites ultimately able to reach the liver may be important determinants of the characteristics of the ensuing blood infection. Because feeding mosquitoes not only inject sporozoites into the host but concomitantly ingest blood to obtain their bloodmeal, some sporozoites are re-ingested by the feeding mosquito. We studied transmission of fluorescent Plasmodium berghei sporozoites injected into mice by Anopheles stephensi mosquitoes and found that the numbers of sporozoites re-ingested by mosquitoes are comparable to numbers previously reported to be delivered directly into mice. Thus, re-ingestion of sporozoites likely plays a significant role in transmission dynamics of malaria by mosquitoes, and may account for the failure of some sporozoite-infected mosquitoes to induce a blood infection.

Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes.

Previous studies have shown that mosquitoes inject Plasmodium sporozoites into avascular portions of the skin of their rodent host rather than directly into the blood circulation. Then, over time, these sporozoites move into the circulation, from where they reach the liver to initiate a malaria infection. By use of intravital microscopy of the skin, we present direct morphological evidence of mosquito probing that introduces sporozoites into avascular tissue, of the migration of these sporozoites through the dermis and into blood vessels, and of the role of anti-sporozoite antibodies in blocking sporozoite invasion of these dermal blood vessels.