John B. Sacci

A proteomic view of the Plasmodium falciparum life cycle.

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.

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.

CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes.

The success of immunization with irradiated sporozoites is unparalleled among the current vaccination approaches against malaria, but its mechanistic underpinnings have yet to be fully elucidated. Using a model mimicking natural infection by Plasmodium yoelii, we delineated early events governing the development of protective CD8+ T-cell responses to the circumsporozoite protein. We demonstrate that dendritic cells in cutaneous lymph nodes prime the first cohort of CD8+ T cells after an infectious mosquito bite. Ablation of these lymphoid sites greatly impairs subsequent development of protective immunity. Activated CD8+ T cells then travel to systemic sites, including the liver, in a sphingosine-1-phosphate (S1P)-dependent fashion. These effector cells, however, no longer require bone marrow–derived antigen-presenting cells for protection; instead, they recognize antigen on parenchymal cells—presumably parasitized hepatocytes. Therefore, we report an unexpected dichotomy in the tissue restriction of host responses during the development and execution of protective immunity to Plasmodium.

Improving Protective Immunity Induced by DNA-Based Immunization: Priming with Antigen and GM-CSF-Encoding Plasmid DNA and Boosting with Antigen-Expressing Recombinant Poxvirus

Intramuscular immunization with a naked DNA plasmid expressing the Plasmodium yoelii circumsporozoite protein (pPyCSP) protects mice against challenge with P. yoelii sporozoites. This protection can be improved either by coadministration of a plasmid expressing murine GM-CSF (pGMCSF) or by boosting with recombinant poxvirus expressing the PyCSP. We now report that combining these two strategies, by first mixing the priming dose of pPyCSP with pGMCSF and then boosting with recombinant virus, can substantially increase vaccine effectiveness. Not only were immune responses and protection improved but the pPyCSP dose could be lowered from 100 μg to 1 μg with little loss of immunogenicity after boost with recombinant poxvirus. Comparing mice primed by the 1-μg doses of pPyCSP plus 1 μg pGMCSF with mice primed by 1-μg doses of pPyCSP alone, the former were better protected (60% vs 0) and had higher concentrations of Abs (titers of 163, 840 vs 5, 120 by indirect fluorescent Ab test against sporozoites), more ex vivo CTL activity (25% vs 7% specific lysis), and more IFN-γ-secreting cells by enzyme-linked immunospot assay (1460 vs 280 IFN-γ spot-forming cells/106 cells). Priming with plasmid vaccine plus pGMCSF and boosting with recombinant poxviruses strongly improves the immunogenicity and protective efficacy of DNA vaccination and allows for significant reduction of dose.

Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.

Safety and Clinical Outcome of Experimental Challenge of Human Volunteers with Plasmodium falciparum-Infected Mosquitoes: An Update

BACKGROUND Challenge of volunteers by the bites of membrane-fed anopheline mosquitoes infected with Plasmodium falciparum was reported in 1986. In 1997, an analysis of experience with 118 volunteers indicated that mosquito inoculation of P. falciparum could be a safe, well-tolerated, reproducible, and efficient method of challenge. METHODS We reviewed the records of 47 volunteers challenged at our institution with the NF54 isolate of P. falciparum between 1998 and 2002. We also reviewed data from 17 published studies of experimental challenge conducted since 1996. RESULTS At our institution, the time to onset of first symptoms (incubation period) was 8.9 days, and the time to first detectable parasitemia on blood smear (prepatent period) was 10.5 days. All volunteers became symptomatic. Most symptoms were mild to moderate, although 21% of volunteers had at least 1 severe symptom. None developed complicated or severe malaria, and all were cured. Laboratory assessments demonstrated modest, short-term abnormalities typical of malaria. Review of 17 published studies demonstrated that an additional 367 volunteers received experimental challenge safely with similar outcomes. CONCLUSIONS In total, data from 532 volunteers demonstrate that experimental challenge is safe and results in predictable incubation and prepatent periods. Our findings support the continued use of this method for testing efficacy of vaccines and drugs against P. falciparum.

The optimization of helper T lymphocyte (HTL) function in vaccine development

Helper T lymphocyte (HTL) responses play an important role in the induction of both humoral and cellular immune responses. Therefore, HTL epitopes are likely to be a crucial component of prophylactic and immunotherapeutic vaccines. For this reason, Pan DR helper T cell epitopes (PADRE), engineered to bind most common HLA-DR molecules with high affinity and act as powerful immunogens, were developed. Short linear peptide constructs comprising PADRE andPlasmodium-derived B cell epitopes induced antibody responses comparable to more complex multiple antigen peptides (MAP) constructs in mice. These antibody responses were composed mostly of the IgG subclass, reactive against intact sporozoites, inhibitory of schizont formation in liver invasion assays, and protective against sporozoite challenge in vivo. The PADRE HTL epitope has also been shown to augment the potency of vaccines designed to stimulate a cellular immune response. Using a HBV transgenic murine model, it was found that CTL tolerance was broken by PADRE-CTL epitope lipopeptide, but not by a similar construct containing a conventional HTL epitope. There are a number of prophylactic vaccines that are of limited efficacy, require multiple boosts, and/or confer protection to only a fraction of the immunized population. Also, in the case of virally infected or cancerous cells, new immunotherapeutic vaccines that induce strong cellular immune responses are desirable. Therefore, optimization of HTL function by use of synthetic epitopes such as PADRE or pathogen-derived, broadly crossreactive epitopes holds promise for a new generation of highly efficacious vaccines.

Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship.

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.

Monoclonal Antibody MG96 Completely Blocks Plasmodium yoelii Development in Anopheles stephensi

ABSTRACT In spite of research efforts to develop vaccines against the causative agent of human malaria, Plasmodium falciparum, effective control remains elusive. The predominant vaccine strategy focuses on targeting parasite blood stages in the vertebrate host. An alternative approach has been the development of transmission-blocking vaccines (TBVs). TBVs target antigens on parasite sexual stages that persist within the insect vector, anopheline mosquitoes, or target mosquito midgut proteins that are presumed to mediate parasite development. By blocking parasite development within the insect vector, TBVs effectively disrupt transmission and the resultant cascade of secondary infections. Using a mosquito midgut-specific mouse monoclonal antibody (MG96), we have partially characterized membrane-bound midgut glycoproteins in Anopheles gambiae and Anopheles stephensi. These proteins are present on the microvilli of midgut epithelial cells in both blood-fed and unfed mosquitoes, suggesting that the expression of the protein is not induced as a result of blood feeding. MG96 exhibits a dose-dependent blocking effect against Plasmodium yoelii development in An. stephensi. We achieved 100% blocking of parasite development in the mosquito midgut. Preliminary deglycosylation assays indicate that the epitope recognized by MG96 is a complex oligosaccharide. Future investigation of the carbohydrate epitope as well as gene identification should provide valuable insight into the possible mechanisms of ookinete attachment and invasion of mosquito midgut epithelial cells.

Effect on antibody and T-cell responses of mixing five GMP-produced DNA plasmids and administration with plasmid expressing GM-CSF

One potential benefit of DNA vaccines is the capacity to elicit antibody and T-cell responses against multiple antigens at the same time by mixing plasmids expressing different proteins. A possible negative effect of such mixing is interference among plasmids regarding immunogenicity. In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone. The mixture induced higher levels of antibodies against whole parasites than did the individual plasmids, but was associated with a decrease in antibodies to individual P. falciparum proteins. T-cell responses were in general decreased by administration of the mixture. Immune responses to individual plasmids and mixtures were generally higher in inbred mice than in outbreds. In inbred BALB/c and C57BL/6 mice, coadministration of a plasmid expressing murine granulocyte–macrophage colony-stimulating factor (mGM-CSF), increased antibody and T-cell responses, but in outbred CD-1 mice, coadministration of mGM-CSF was associated with a decrease in antibody responses. Such variability in data from studies in different strains of mice underscores the importance of genetic background on immune response and carefully considering the goals of any preclinical studies of vaccine mixtures planned for human trials.