Patricia de la Vega

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.

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.

Induction in Humans of CD8 + and CD4 + T Cell and Antibody Responses by Sequential Immunization with Malaria DNA and Recombinant Protein

Vaccine-induced protection against diseases like malaria, AIDS, and cancer may require induction of Ag-specific CD8+ and CD4+ T cell and Ab responses in the same individual. In humans, a recombinant Plasmodium falciparum circumsporozoite protein (PfCSP) candidate vaccine, RTS,S/adjuvant system number 2A (AS02A), induces T cells and Abs, but no measurable CD8+ T cells by CTL or short-term (ex vivo) IFN-γ ELISPOT assays, and partial short-term protection. P. falciparum DNA vaccines elicit CD8+ T cells by these assays, but no protection. We report that sequential immunization with a PfCSP DNA vaccine and RTS,S/AS02A induced PfCSP-specific Abs and Th1 CD4+ T cells, and CD8+ cytotoxic and Tc1 T cells. Depending upon the immunization regime, CD4+ T cells were involved in both the induction and production phases of PfCSP-specific IFN-γ responses, whereas, CD8+ T cells were involved only in the production phase. IFN-γ mRNA up-regulation was detected in both CD45RA− (CD45RO+) and CD45RA+CD4+ and CD8+ T cell populations after stimulation with PfCSP peptides. This finding suggests CD45RA+ cells function as effector T cells. The induction in humans of the three primary Ag-specific adaptive immune responses establishes a strategy for developing immunization regimens against diseases in desperate need of vaccines.

Identification and Characterization of the Protective Hepatocyte Erythrocyte Protein 17 kDa Gene of Plasmodium yoelii, homolog of Plasmodium falciparum Exported Protein 1

We recently reported the discovery of a 17-kDa Plasmodium yoelii protein expressed in infected hepatocytes and erythrocytes, P. yoelii hepatocyte erythrocyte protein 17 (PyHEP17), and have demonstrated that this protein is a target of protective antibodies and T cells. Here, we report the identification and characterization of the gene encoding this protein and reveal that it is composed of two exons. Immunization of mice with PyHEP17 plasmid DNA induces antibodies, cytotoxic T lymphocytes, and protective immunity directed against the infected hepatocyte. Based on extensive sequence homology, expression pattern, and antigenic cross-reactivity, the Plasmodium falciparum homolog of PyHEP17 is identified as the protein known as exported protein-1 (PfExp-1), also called antigen 5.1, circumsporozoite related antigen, or QF116. Identity between PyHEP17 and PfExp-1 is 37% at the amino acid level (60/161 residues), mapping primarily to two regions within the second exon of 73% (16/22 residues) and 71% (25/35 residues) identity. On this basis, PfExp-1 is proposed as an important component of pre-erythrocytic human malaria vaccines.

First-in-human evaluation of genetically attenuated Plasmodium falciparum sporozoites administered by bite of Anopheles mosquitoes to adult volunteers

BACKGROUND Immunization with genetically engineered, attenuated malaria parasites (GAP) that arrest during liver infection confers sterile protection in mouse malaria models. A first generation Plasmodium falciparum GAP (Pf p52(-)/p36(-) GAP) was previously generated by deletion of two pre-erythrocytic stage-expressed genes (P52 and P36) in the NF54 strain. METHODS A first-in-human, proof-of-concept, safety and immunogenicity clinical trial in six human volunteers was conducted. Exposure consisted of delivery of Pf p52(-)/p36(-) GAP sporozoites via infected Anopheles mosquito bite with a five-bite/volunteer exposure followed by an approximately 200-bite exposure/volunteer one month later. RESULTS The exposures were well tolerated with mild to moderate local and systemic reactions. All volunteers remained blood stage negative after low dose exposure. Five volunteers remained blood stage negative after high dose exposure. One volunteer developed peripheral parasitemia twelve days after high dose exposure. Together the findings indicate that Pf p52(-)/p36(-) GAP was severely but not completely attenuated. All six volunteers developed antibodies to CSP. Furthermore, IFN-γ responses to whole sporozoites and multiple antigens were elicited in 5 of 6 volunteers, with both CD4 and CD8 cell cytokine production detected. CONCLUSION Severe attenuation and favorable immune responses following administration of a first generation Pf p52(-)/p36(-) GAP suggests that further development of live-attenuated strains using genetic engineering should be pursued.

Molecular mechanism of host specificity in Plasmodium falciparum infection: role of circumsporozoite protein.

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.

Multiple Antigen Peptide Vaccines against Plasmodium falciparum Malaria

ABSTRACT The multiple antigen peptide (MAP) approach is an effective method to chemically synthesize and deliver multiple T-cell and B-cell epitopes as the constituents of a single immunogen. Here we report on the design, chemical synthesis, and immunogenicity of three Plasmodium falciparum MAP vaccines that incorporated antigenic epitopes from the sporozoite, liver, and blood stages of the life cycle. Antibody and cellular responses were determined in three inbred (C57BL/6, BALB/c, and A/J) strains, one congenic (HLA-A2 on the C57BL/6 background) strain, and one outbred strain (CD1) of mice. All three MAPs were immunogenic and induced both antibody and cellular responses, albeit in a somewhat genetically restricted manner. Antibodies against MAP-1, MAP-2, and MAP-3 had an antiparasite effect that was also dependent on the mouse major histocompatibility complex background. Anti-MAP-1 (CSP-based) antibodies blocked the invasion of HepG2 liver cells by P. falciparum sporozoites (highest, 95.16% in HLA-A2 C57BL/6; lowest, 11.21% in BALB/c). Furthermore, antibodies generated following immunizations with the MAP-2 (PfCSP, PfLSA-1, PfMSP-142, and PfMSP-3b) and MAP-3 (PfRAP-1, PfRAP-2, PfSERA, and PfMSP-142) vaccines were able to reduce the growth of blood stage parasites in erythrocyte cultures to various degrees. Thus, MAP-based vaccines remain a viable option to induce effective antibody and cellular responses. These results warrant further development and preclinical and clinical testing of the next generation of candidate MAP vaccines that are based on the conserved protective epitopes from Plasmodium antigens that are widely recognized by populations of divergent HLA types from around the world.

Disruption of the Plasmodium falciparum liver‐stage antigen‐1 locus causes a differentiation defect in late liver‐stage parasites

The malaria parasite Plasmodium falciparum infects humans and first targets the liver where liver‐stage parasites undergo pre‐erythrocytic replication. Liver‐stage antigen‐1 (LSA‐1) is currently the only identified P. falciparum protein for which expression is restricted to liver stages. Yet, the importance of LSA‐1 for liver‐stage parasite development remains unknown. Here we deleted LSA‐1 in the NF54 strain of P. falciparum and analysed the lsa‐1‐ parasites throughout their life cycle. lsa‐1‐ sporozoites had normal gliding motility and invasion into hepatocytes. Six days after infection of a hepatocytic cell line, lsa‐1‐ parasites exhibited a moderate phenotype with an ∼50% reduction of late liver‐stage forms when compared with wild type. Strikingly, lsa‐1‐ parasites growing in SCID/Alb‐uPA mice with humanized livers showed a severe defect in late liver‐stage differentiation and exo‐erythrocytic merozoite formation 7 days after infection, a time point when wild‐type parasites develop into mature merozoites. The lsa‐1‐ parasites also showed aberrant liver‐stage expression of key parasite proteins apical membrane antigen‐1 and circumsporozoite protein. Our data show that LSA‐1 plays a critical role during late liver‐stage schizogony and is thus important in the parasite transition from the liver to blood. LSA‐1 is the first P. falciparum protein identified to be required for this transitional stage of the parasite life cycle.

Clinical trial in healthy malaria-naïve adults to evaluate the safety, tolerability, immunogenicity and efficacy of MuStDO5, a five-gene, sporozoite/hepatic stage Plasmodium falciparum DNA vaccine combined with escalating dose human GM-CSF DNA

When introduced in the 1990s, immunization with DNA plasmids was considered potentially revolutionary for vaccine development, particularly for vaccines intended to induce protective CD8 T cell responses against multiple antigens. We conducted, in 1997−1998, the first clinical trial in healthy humans of a DNA vaccine, a single plasmid encoding Plasmodium falciparum circumsporozoite protein (PfCSP), as an initial step toward developing a multi-antigen malaria vaccine targeting the liver stages of the parasite. As the next step, we conducted in 2000–2001 a clinical trial of a five-plasmid mixture called MuStDO5 encoding pre-erythrocytic antigens PfCSP, PfSSP2/TRAP, PfEXP1, PfLSA1 and PfLSA3. Thirty-two, malaria-naïve, adult volunteers were enrolled sequentially into four cohorts receiving a mixture of 500 μg of each plasmid plus escalating doses (0, 20, 100 or 500 μg) of a sixth plasmid encoding human granulocyte macrophage-colony stimulating factor (hGM-CSF). Three doses of each formulation were administered intramuscularly by needle-less jet injection at 0, 4 and 8 weeks, and each cohort had controlled human malaria infection administered by five mosquito bites 18 d later. The vaccine was safe and well-tolerated, inducing moderate antigen-specific, MHC-restricted T cell interferon-γ responses but no antibodies. Although no volunteers were protected, T cell responses were boosted post malaria challenge. This trial demonstrated the MuStDO5 DNA and hGM-CSF plasmids to be safe and modestly immunogenic for T cell responses. It also laid the foundation for priming with DNA plasmids and boosting with recombinant viruses, an approach known for nearly 15 y to enhance the immunogenicity and protective efficacy of DNA vaccines.