High-level expression of the malaria blood-stage vaccine candidate Plasmodium falciparum apical membrane antigen 1 and induction of antibodies that inhibit erythrocyte invasion.
ABSTRACT: Apical membrane antigen 1 (AMA-1) is a highly promising malaria blood-stage vaccine candidate that has induced protection in rodent and nonhuman primate models of malaria. Authentic conformation of the protein appears to be essential for the induction of parasite-inhibitory antibody responses. Here we have developed a synthetic gene with adapted codon usage to allow expression of Plasmodium falciparum FVO strain AMA-1 (PfAMA-1) in Pichia pastoris. In addition, potential N-glycosylation sites were changed, exploiting the lack of conservation of these sites in Plasmodium, to obtain high-level secretion of a homogeneous product, suitable for scale-up according to current good manufacturing procedures. Purified PfAMA-1 displayed authentic antigenic properties, indicating that the amino acid changes had no deleterious effect on the conformation of the protein. High-titer antibodies, raised in rabbits, reacted strongly with homologous and heterologous P. falciparum by immunofluorescence. In addition, purified immunoglobulin G from immunized animals strongly inhibited invasion of red blood cells by homologous and, to a somewhat lesser extent, heterologous P. falciparum.
Project description:This Phase 1/2a study evaluated the safety, immunogenicity, and efficacy of an experimental malaria vaccine comprised of the recombinant Plasmodium falciparum protein apical membrane antigen-1 (AMA-1) representing the 3D7 allele formulated with either the AS01B or AS02A Adjuvant Systems.After a preliminary safety evaluation of low dose AMA-1/AS01B (10 microg/0.5 mL) in 5 adults, 30 malaria-naïve adults were randomly allocated to receive full dose (50 microg/0.5 mL) of AMA-1/AS01B (n = 15) or AMA-1/AS02A (n = 15), followed by a malaria challenge. All vaccinations were administered intramuscularly on a 0-, 1-, 2-month schedule. All volunteers experienced transient injection site erythema, swelling and pain. Two weeks post-third vaccination, anti-AMA-1 Geometric Mean Antibody Concentrations (GMCs) with 95% Confidence Intervals (CIs) were high: low dose AMA-1/AS01B 196 microg/mL (103-371 microg/mL), full dose AMA-1/AS01B 279 microg/mL (210-369 microg/mL) and full dose AMA-1/AS02A 216 microg/mL (169-276 microg/mL) with no significant difference among the 3 groups. The three vaccine formulations elicited equivalent functional antibody responses, as measured by growth inhibition assay (GIA), against homologous but not against heterologous (FVO) parasites as well as demonstrable interferon-gamma (IFN-gamma) responses. To assess efficacy, volunteers were challenged with P. falciparum-infected mosquitoes, and all became parasitemic, with no significant difference in the prepatent period by either light microscopy or quantitative polymerase chain reaction (qPCR). However, a small but significant reduction of parasitemia in the AMA-1/AS02A group was seen with a statistical model employing qPCR measurements.All three vaccine formulations were found to be safe and highly immunogenic. These immune responses did not translate into significant vaccine efficacy in malaria-naïve adults employing a primary sporozoite challenge model, but encouragingly, estimation of parasite growth rates from qPCR data may suggest a partial biological effect of the vaccine. Further evaluation of the immunogenicity and efficacy of the AMA-1/AS02A formulation is ongoing in a malaria-experienced pediatric population in Mali.www.clinicaltrials.gov NCT00385047.
Project description:Apical membrane antigen 1 (AMA-1), an asexual blood-stage antigen of Plasmodium falciparum, is an important candidate for testing as a component of a malaria vaccine. This study investigates the nature of diversity in the Plasmodium chabaudi adami homolog of AMA-1 and the impact of that diversity on the efficacy of the recombinant antigen as a vaccine against challenge with a heterologous strain of P. chabaudi. The nucleotide sequence of the AMA-1 gene from strain DS differs from the published 556KA sequence at 79 sites. The large number of mutations, the nonrandom distribution of both synonymous and nonsynonymous mutations, and the nature of both the codon changes and the resulting amino acid substitutions suggest that positive selection operates on the AMA-1 gene in regions coding for antigenic sites. Protective immune responses induced by AMA-1 were strain specific. Immunization of mice with the refolded ectodomain of DS AMA-1 provided partial protection against challenge with virulent DS (homologous) parasites but failed to protect against challenge with avirulent 556KA (heterologous) parasites. Passive immunization of mice with rabbit antibodies raised against the same antigen had little effect on heterologous challenge but provided significant protection against the homologous DS parasites.
Project description:A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites. The MSP1(42) gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P. falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits. The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P. falciparum FVO erythrocytic-stage malaria parasites. The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP1(42) (FVO) as a positive control. Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E. coli MSP1(42) were significantly higher than those induced by the baculovirus-expressed antigen. None of the six monkeys that were vaccinated with the E. coli MSP1(42) antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia. Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP1(42), but not the MSP1(42) protein itself or the EGF-like domain 1 fragment. Soluble MSP1(42) (FVO) expressed in E. coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria.
Project description:BACKGROUND:The safety and immunogenicity of PfAMA1, adjuvanted with Alhydrogel(®) was assessed in malaria-experienced Malian adults. The malaria vaccine, PfAMA1-FVO [25-545] is a recombinant protein Pichia pastoris-expressed AMA-1 from Plasmodium falciparum FVO clone adsorbed to Alhydrogel(®), the control vaccine was tetanus toxoid produced from formaldehyde detoxified and purified tetanus toxin. METHODS:A double blind randomized controlled phase 1 study enrolled and followed 40 healthy adults aged 18-55 years in Bandiagara, Mali, West Africa, a rural setting with intense seasonal transmission of P. falciparum malaria. Volunteers were randomized to receive either 50 µg of malaria vaccine or the control vaccine. Three doses of vaccine were given on Days 0, 28 and 56, and participants were followed for 1 year. Solicited symptoms were assessed for seven days and unsolicited symptoms for 28 days after each vaccination. Serious adverse events were assessed throughout the study. The titres of anti-AMA-1 antibodies were measured by ELISA and P. falciparum growth inhibition assays were performed. RESULTS:Commonest local solicited adverse events were the injection site pain and swelling more frequent in the PfAMA1 group. No vaccine related serious adverse events were reported. A significant 3.5-fold increase of anti-AMA-1 IgG antibodies was observed in malaria vaccine recipients four weeks after the third immunization compared to the control group. CONCLUSION:The PfAMA1 showed a good safety profile. Most adverse events reported were of mild to moderate intensity. In addition, the vaccine induced a significant though short-lived increase in the anti-AMA1 IgG titres. Registered on www.clinicaltrials.gov with the number NCT00431808.
Project description:OBJECTIVES:To assess the safety and immunogenicity of two vaccines, MSP1(42)-FVO/Alhydrogel and MSP1(42)-3D7/Alhydrogel, targeting blood-stage Plasmodium falciparum parasites. DESIGN:A Phase 1 open-label, dose-escalating study. SETTING:Quintiles Phase 1 Services, Lenexa, Kansas between July 2004 and November 2005. PARTICIPANTS:Sixty healthy malaria-naïve volunteers 18-48 y of age. INTERVENTIONS:The C-terminal 42-kDa region of merozoite surface protein 1 (MSP1(42)) corresponding to the two allelic forms present in FVO and 3D7 P. falciparum lines were expressed in Escherichia coli, refolded, purified, and formulated on Alhydrogel (aluminum hydroxide). For each vaccine, volunteers in each of three dose cohorts (5, 20, and 80 microg) were vaccinated at 0, 28, and 180 d. Volunteers were followed for 1 y. OUTCOME MEASURES:The safety of MSP1(42)-FVO/Alhydrogel and MSP1(42)-3D7/Alhydrogel was assessed. The antibody response to each vaccine was measured by reactivity to homologous and heterologous MSP1(42), MSP1(19), and MSP1(33) recombinant proteins and recognition of FVO and 3D7 parasites. RESULTS:Anti-MSP1(42) antibodies were detected by ELISA in 20/27 (74%) and 22/27 (81%) volunteers receiving three vaccinations of MSP1(42)-FVO/Alhydrogel or MSP1(42)-3D7/Alhydrogel, respectively. Regardless of the vaccine, the antibodies were cross-reactive to both MSP1(42)-FVO and MSP1(42)-3D7 proteins. The majority of the antibody response targeted the C-terminal 19-kDa domain of MSP1(42), although low-level antibodies to the N-terminal 33-kDa domain of MSP1(42) were also detected. Immunofluorescence microscopy of sera from the volunteers demonstrated reactivity with both FVO and 3D7 P. falciparum schizonts and free merozoites. Minimal in vitro growth inhibition of FVO or 3D7 parasites by purified IgG from the sera of the vaccinees was observed. CONCLUSIONS:The MSP1(42)/Alhydrogel vaccines were safe and well tolerated but not sufficiently immunogenic to generate a biologic effect in vitro. Addition of immunostimulants to the Alhydrogel formulation to elicit higher vaccine-induced responses in humans may be required for an effective vaccine.
Project description:BACKGROUND: The Plasmodium falciparum chimeric protein PfCP-2.9 is a promising asexual-stage malaria vaccine evaluated in clinical trials. This chimeric protein consists of two cysteine-rich domains: domain III of the apical membrane antigen 1 (AMA-1 [III]) and the C-terminal region of the merozoite surface protein 1 (MSP1-19). It has been reported that the fusion of these two antigens enhanced their immunogenicity and antibody-mediated inhibition of parasite growth in vitro. METHODS: The 15N-labeled and 13C/15N-labeled PfCP-2.9 was produced in Pichia pastoris for nuclear magnetic resonance (NMR) structure analysis. The chemical shift assignments of PfCP-2.9 were compared with those previously reported for the individual domains (i.e., PfAMA-1(III) or PfMSP 1-19). The two-dimensional spectra and transverse relaxation rates (R2) of the PfMSP1-19 alone were compared with that of the PfCP-2.9. RESULTS: Confident backbone assignments were obtained for 122 out of 241 residues of PfCP-2.9. The assigned residues in PfCP-2.9 were very similar to those previously reported for the individual domains. The conformation of the PfMSP1-19 in different constructs is essentially the same. Comparison of transverse relaxation rates (R2) strongly suggests no weak interaction between the domains. CONCLUSIONS: These data indicate that the fusion of AMA-1(III) and MSP1-19 as chimeric protein did not change their structures, supporting the use of the chimeric protein as a potential malaria vaccine.
Project description:BACKGROUND:Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is one of leading blood stage malaria vaccine candidates. However, genetic variation and antigenic diversity identified in global PfAMA-1 are major hurdles in the development of an effective vaccine based on this antigen. In this study, genetic structure and the effect of natural selection of PfAMA-1 among Myanmar P. falciparum isolates were analysed. METHODS:Blood samples were collected from 58 Myanmar patients with falciparum malaria. Full-length PfAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. PfAMA-1 sequence of each isolate was sequenced. Polymorphic characteristics and effect of natural selection were analysed with using DNASTAR, MEGA4, and DnaSP programs. Polymorphic nature and natural selection in 459 global PfAMA-1 were also analysed. RESULTS:Thirty-seven different haplotypes of PfAMA-1 were identified in 58 Myanmar P. falciparum isolates. Most amino acid changes identified in Myanmar PfAMA-1 were found in domains I and III. Overall patterns of amino acid changes in Myanmar PfAMA-1 were similar to those in global PfAMA-1. However, frequencies of amino acid changes differed by country. Novel amino acid changes in Myanmar PfAMA-1 were also identified. Evidences for natural selection and recombination event were observed in global PfAMA-1. Among 51 commonly identified amino acid changes in global PfAMA-1 sequences, 43 were found in predicted RBC-binding sites, B-cell epitopes, or IUR regions. CONCLUSIONS:Myanmar PfAMA-1 showed similar patterns of nucleotide diversity and amino acid polymorphisms compared to those of global PfAMA-1. Balancing natural selection and intragenic recombination across PfAMA-1 are likely to play major roles in generating genetic diversity in global PfAMA-1. Most common amino acid changes in global PfAMA-1 were located in predicted B-cell epitopes where high levels of nucleotide diversity and balancing natural selection were found. These results highlight the strong selective pressure of host immunity on the PfAMA-1 gene. These results have significant implications in understanding the nature of Myanmar PfAMA-1 along with global PfAMA-1. They also provide useful information for the development of effective malaria vaccine based on this antigen.
Project description:BACKGROUND:In this study, seven adjuvants were compared for use with Plasmodium falciparum DiCo-Apical Membrane Antigen 1 (Pf-DiCo-AMA1), with the aim to identify an ideal adjuvant which yields high antibody titres and potentially broadens the responses in clinical trials. The following adjuvant formulations were evaluated: SE, SE-GLA, Liposomes, Liposomes-GLA, CoVaccine HT™, ImSaVac-P and ImSaVac-P o/w. The study was performed in rabbits, which were immunized with FVO-AMA1 in combination with one of the seven adjuvants. Antibody levels (humoral responses) and functional activity of the antibodies induced against malaria vaccine candidate AMA1 were evaluated. Thus, in this study the ideal adjuvant is expected to induce high functional antibody levels, a long-lived response, and a broad cross-strain activity. RESULTS:AMA1 formulated in all adjuvants was immunogenic. However, the magnitude of the immune responses differed between the seven adjuvants. The highest IgG levels were observed for the CoVaccine HT™ group, this was statistically significant for all four AMA1 variants versus all other adjuvant groups. No differences were observed in the breadth of the humoral response, i.e., increased recognition of AMA1 variants. Also, Growth Inhibition Activity (GIA) for both Plasmodium falciparum strains (FCR3 - homologous to FVO AMA1 protein and NF54 - heterologous to FVO AMA1 protein) were significantly higher in the CoVaccine HT™ group as compared to the other adjuvant groups. CONCLUSIONS:In brief, all seven vaccine - adjuvant formulations were immunogenic. The magnitude of the immune responses differed between the seven adjuvants. No statistically significant differences were observed in the breadth of the humoral response, nor in longevity of the response. Nevertheless, AMA1 formulated in CoVaccine HT™ appeared as the best adjuvant for use in clinical trials.
Project description:A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.
Project description:BACKGROUND:Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a promising candidate antigen for a blood-stage malaria vaccine. However, antigenic variation and diversity of PfAMA-1 are still major problems to design a universal malaria vaccine based on this antigen, especially against domain I (DI). Detail understanding of the PfAMA-1 gene polymorphism can provide useful information on this potential vaccine component. Here, general characteristics of genetic structure and the effect of natural selection of DIs among Bioko P. falciparum isolates were analysed. METHODS:214 blood samples were collected from Bioko Island patients with P. falciparum malaria between 2011 and 2017. A fragment spanning DI of PfAMA-1 was amplified by nested polymerase chain reaction and sequenced. Polymorphic characteristics and the effect of natural selection were analysed using MEGA 5.0, DnaSP 6.0 and Popart programs. Genetic diversity in 576 global PfAMA-1 DIs were also analysed. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 program. RESULTS:131 different haplotypes of PfAMA-1 were identified in 214 Bioko Island P. falciparum isolates. Most amino acid changes identified on Bioko Island were found in C1L. 32 amino acid changes identified in PfAMA-1 sequences from Bioko Island were found in predicted RBC-binding sites, B cell epitopes or IUR regions. Overall patterns of amino acid changes of Bioko PfAMA-1 DIs were similar to those in global PfAMA-1 isolates. Differential amino acid substitution frequencies were observed for samples from different geographical regions. Eight new amino acid changes of Bioko island isolates were also identified and their three-dimensional protein structural consequences were predicted. Evidence for natural selection and recombination event were observed in global isolates. CONCLUSIONS:Patterns of nucleotide diversity and amino acid polymorphisms of Bioko Island isolates were similar to those of global PfAMA-1 DIs. Balancing natural selection across DIs might play a major role in generating genetic diversity in global isolates. Most amino acid changes in DIs occurred in predicted B-cell epitopes. Novel sites mapped on a three dimensional structure of PfAMA-1 showed that these regions were located at the corner. These results may provide significant value in the design of a malaria vaccine based on this antigen.