Pf Sporozoites Research Articles

Poly(I:C) is an effective adjuvant for antibody and multi-functional CD4+ T cell responses to Plasmodium falciparum circumsporozoite protein (CSP) and αDEC-CSP in non human primates

Development of a fully effective vaccine against the pre-erythrocytic stage of malaria infection will likely require induction of both humoral and cellular immune responses. Protein based vaccines can elicit such broad-based immunity depending on the adjuvant and how the protein is formulated. Here to assess these variables, non human primates (NHP) were immunized three times with Plasmodium falciparum (Pf) circumsporozoite protein (CSP) or CSP cloned into MG38, a monoclonal antibody that targets DEC-205 (αDEC-CSP), an endocytic receptor on dendritic cells (DCs). Both vaccines were administered with or without poly(I:C) as adjuvant. Following three immunizations, the magnitude and quality of cytokine secreting CD4+ T cells were comparable between CSP + poly(I:C) and αDEC-CSP + poly(I:C) groups with both regimens eliciting multi-functional cytokine responses. However, NHP immunized with CSP + poly(I:C) had significantly higher serum titers of CSP-specific IgG antibodies and indirect immunofluorescent antibody (IFA) titers against Pf sporozoites. Furthermore, sera from both CSP or αDEC-CSP + poly(I:C) immunized animals limited sporozoite invasion of a hepatocyte cell line (HC04) in vitro. To determine whether CSP-specific responses could be enhanced, all NHP primed with CSP or αDEC-CSP + poly(I:C) were boosted with a single dose of 150,000 irradiated Pf sporozoites (PfSPZ) intravenously. Remarkably, boosting had no effect on the CSP-specific immunity. Finally, immunization with CSP + poly-ICLC reduced malaria parasite burden in the liver in an experimental mouse model. Taken together, these data showing that poly(I:C) is an effective adjuvant for inducing potent antibody and Th1 immunity with CSP based vaccines offers a potential alternative to the existing protein based pre-erythrocytic vaccines.

Profiling humoral immune responses to P. falciparum infection with protein microarrays

A complete description of the serological response following exposure of humans to complex pathogens is lacking and approaches suitable for accomplishing this are limited. Here we report, using malaria as a model, a method which elucidates the profile of antibodies that develop after natural or experimental infection or after vaccination with attenuated organisms, and which identifies immunoreactive antigens of interest for vaccine development or other applications. Expression vectors encoding 250 Plasmodium falciparum (Pf) proteins were generated by PCR/recombination cloning; the proteins were individually expressed with >90% efficiency in Escherichia coli cell-free in vitro transcription and translation reactions, and printed directly without purification onto microarray slides. The protein microarrays were probed with human sera from one of four groups which differed in immune status: sterile immunity or no immunity against experimental challenge following vaccination with radiation-attenuated Pf sporozoites, partial immunity acquired by natural exposure, and no previous exposure to Pf. Overall, 72 highly reactive Pf antigens were identified. Proteomic features associated with immunoreactivity were identified. Importantly, antibody profiles were distinct for each donor group. Information obtained from such analyses will facilitate identifying antigens for vaccine development, dissecting the molecular basis of immunity, monitoring the outcome of whole-organism vaccine trials, and identifying immune correlates of protection.

Plasmodium falciparum: Effect of radiation on levels of gene transcripts in sporozoites

Humans immunized by the bites of irradiated Plasmodium falciparum (Pf) sporozoite-infected mosquitoes are protected against malaria. Radiation attenuates the sporozoites preventing them from fully developing and replicating in hepatocytes, but the effects of radiation on gene expression in sporozoites are unknown. We used RT-PCR (35 cycles of PCR followed by densitometry) to assess the expression of ten genes in Pf sporozoites, and in sporozoites irradiated with 15,000 cGy. Irradiation reduced expression substantially (>60%) of two DNA repair genes; moderately (30–60%) of PfUIS3, the Pf orthologue of PbUIS3, a gene up-regulated in Plasmodium berghei sporozoites and of a third DNA repair gene; and minimally (<30%) of the Pf18S ribosomal RNA, PfCSP, PfSSP2/TRAP, and PfCELTOS genes. Irradiation increased expression of PfSPATR minimally. PfLSA1 RNA was not detectable in sporozoites. These results establish that radiation of sporozoites affects gene expression levels and provide the foundation for studies to identify specific genes involved in attenuation and protective immunity.

Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine.

Annually, malaria causes >300 million clinical cases and 1 million deaths, is responsible for the loss of >1% of gross domestic product (GDP) in Africa and is a serious concern for travelers. An effective vaccine could have a dramatic impact on the disease. For 20 years, scientists have tried to develop modern, recombinant 'subunit' malaria vaccines. This has been difficult. In fact, there is only one recombinant protein vaccine on the market for any disease, and no vaccines based on synthetic peptides, recombinant viruses, recombinant bacteria or DNA plasmids. Most vaccines are based on attenuated or inactivated whole pathogens or material derived directly from the infectious agent. It is in that context that our recent report summarizing the protection of humans with attenuated Plasmodium falciparum (Pf) sporozoites produced at four different sites over 25 years is important. In studies utilizing live mosquitoes as the vaccine delivery mechanism, there was complete protection against malaria in 93% of volunteers (13/14) and 94% of challenges (33/35). Sanaria's goal is to develop and commercialize a non-replicating, metabolically active Pf sporozoite vaccine. Three practical questions must be addressed before manufacturing for clinical trials: (1) can one administer the vaccine by a route that is clinically practical; (2) can one produce adequate quantities of sporozoites; and (3) can sporozoites be produced with the physical characteristics that meet the regulatory, potency and safety requirements of regulatory authorities? Once these questions have been answered, Sanaria will demonstrate that the vaccine protects >90% of human recipients against experimental challenge with Pf sporozoites, can be produced with an efficiency that makes it economically feasible, and protects >90% of African infants and children from infection, and has severe morbidity and mortality. By producing a vaccine for travelers, Sanaria will provide the infrastructure, regulatory foundation and funds necessary to speed licensure, manufacturing and deployment of the vaccine for the infants and children who need it most.

Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites.

During 1989-1999, 11 volunteers were immunized by the bites of 1001-2927 irradiated mosquitoes harboring infectious sporozoites of Plasmodium falciparum (Pf) strain NF54 or clone 3D7/NF54. Ten volunteers were first challenged by the bites of Pf-infected mosquitoes 2-9 weeks after the last immunization, and all were protected. A volunteer challenged 10 weeks after the last immunization was not protected. Five previously protected volunteers were rechallenged 23-42 weeks after a secondary immunization, and 4 were protected. Two volunteers were protected when rechallenged with a heterologous Pf strain (7G8). In total, there was protection in 24 of 26 challenges. These results expand published findings demonstrating that immunization by exposure to thousands of mosquitoes carrying radiation-attenuated Pf sporozoites is safe and well tolerated and elicits strain-transcendent protective immunity that persists for at least 42 weeks.