RESUMEN
At least a million people, mainly African children under 5 years old, still die yearly from malaria, and the burden of disease and death has increased. Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is one of the most promising blood-stage malarial vaccine candidates. However, the allelic polymorphism observed in this protein is a potential stumbling block for vaccine development. To overcome the polymorphism- and strain-specific growth inhibition in vitro, we previously showed in a rabbit model that vaccination with a mixture of two allelic forms of PfAMA1 induced parasite growth-inhibitory antisera against both strains of P. falciparum parasites in vitro. In the present study, we have established that, in contrast to a single-allele protein, the antigen mixture elicits primarily antibodies recognizing antigenic determinants common to the two antigens, as judged by an antigen reversal growth inhibition assay (GIA). We also show that a similar reactivity pattern occurs after immunization of mice. By contrast, sera from rhesus monkeys do not distinguish the two alleles when tested by an enzyme-linked immunosorbent assay or by GIA, regardless of whether the immunogen is a single AMA1 protein or the mixture. This is the first report that a malarial vaccine candidate induced different specificities of functional antibodies depending on the animal species immunized. These observations, as well as data available on human immune responses in areas of endemicity, suggest that polymorphism in the AMA1 protein may not be as formidable a problem for vaccine development as anticipated from studies with rabbits and mice.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Alelos , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Formación de Anticuerpos , Especificidad de Anticuerpos , Antígenos de Protozoos/administración & dosificación , Ensayo de Inmunoadsorción Enzimática/métodos , Macaca mulatta , Malaria Falciparum/parasitología , Proteínas de la Membrana/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Pichia , Polimorfismo Genético , Proteínas Protozoarias/administración & dosificación , Conejos , Especificidad de la EspecieRESUMEN
UNLABELLED: A Phase 1 dose escalating study was conducted in malaria naïve adults to assess the safety, reactogenicity, and immunogenicity of the blood stage malaria vaccine BSAM2/Alhydrogel®+ CPG 7909. BSAM2 is a combination of the FVO and 3D7 alleles of recombinant AMA1 and MSP1(42), with equal amounts by weight of each of the four proteins mixed, bound to Alhydrogel®, and administered with the adjuvant CPG 7909. Thirty (30) volunteers were enrolled in two dose groups, with 15 volunteers receiving up to three doses of 40 µg total protein at Days 0, 56, and 180, and 15 volunteers receiving up to three doses of 160 µg protein on the same schedule. Most related adverse events were mild or moderate, but 4 volunteers experienced severe systemic reactions and two were withdrawn from vaccinations due to adverse events. Geometric mean antibody levels after two vaccinations with the high dose formulation were 136 µg/ml for AMA1 and 78 µg/ml for MSP1(42). Antibody responses were not significantly different in the high dose versus low dose groups and did not further increase after third vaccination. In vitro growth inhibition was demonstrated and was closely correlated with anti-AMA1 antibody responses. A Phase 1b trial in malaria-exposed adults is being conducted. TRIAL REGISTRATION: Clinicaltrials.gov NCT00889616.
Asunto(s)
Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Vacunación/métodos , Adolescente , Adulto , Hidróxido de Aluminio/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Eritrocitos/parasitología , Femenino , Estudios de Seguimiento , Cefalea/inducido químicamente , Humanos , Vacunas contra la Malaria/uso terapéutico , Malaria Falciparum/parasitología , Malaria Falciparum/prevención & control , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteína 1 de Superficie de Merozoito/genética , Proteína 1 de Superficie de Merozoito/inmunología , Persona de Mediana Edad , Oligodesoxirribonucleótidos/inmunología , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Resultado del Tratamiento , Vacunación/efectos adversos , Adulto JovenRESUMEN
Enzyme linked immunosorbent assay (ELISA) has been widely used to measure antibody titers for evaluating the immunogenicity of a vaccine. However, there is as yet no generally accepted way of expressing the ELISA results in the case of experimental vaccines, since there is usually no uniform standard. Both end point and single dilution methods have significant disadvantages. In this paper, we obtained reproducible data with fewer dilutions of samples by addition of serially diluted standard serum to each ELISA plate. Since this ELISA method gives reliable antibody titer with less labor than other methods, it can strongly support vaccine development.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Vacunas contra la Malaria/inmunología , Animales , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Pfs25 and Pvs25, surface proteins of mosquito stage of the malaria parasites P. falciparum and P. vivax, respectively, are leading candidates for vaccines preventing malaria transmission by mosquitoes. This single blinded, dose escalating, controlled Phase 1 study assessed the safety and immunogenicity of recombinant Pfs25 and Pvs25 formulated with Montanide ISA 51, a water-in-oil emulsion. METHODOLOGY/PRINCIPAL FINDINGS: The trial was conducted at The Johns Hopkins Center for Immunization Research, Washington DC, USA, between May 16, 2005-April 30, 2007. The trial was designed to enroll 72 healthy male and non-pregnant female volunteers into 1 group to receive adjuvant control and 6 groups to receive escalating doses of the vaccines. Due to unexpected reactogenicity, the vaccination was halted and only 36 volunteers were enrolled into 4 groups: 3 groups of 10 volunteers each were immunized with 5 microg of Pfs25/ISA 51, 5 microg of Pvs25/ISA 51, or 20 microg of Pvs25/ISA 51, respectively. A fourth group of 6 volunteers received adjuvant control (PBS/ISA 51). Frequent local reactogenicity was observed. Systemic adverse events included two cases of erythema nodosum considered to be probably related to the combination of the antigen and the adjuvant. Significant antibody responses were detected in volunteers who completed the lowest scheduled doses of Pfs25/ISA 51. Serum anti-Pfs25 levels correlated with transmission blocking activity. CONCLUSION/SIGNIFICANCE: It is feasible to induce transmission blocking immunity in humans using the Pfs25/ISA 51 vaccine, but these vaccines are unexpectedly reactogenic for further development. This is the first report that the formulation is associated with systemic adverse events including erythema nodosum. TRIAL REGISTRATION: ClinicalTrials.gov NCT00295581.
Asunto(s)
Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Vacunas contra la Malaria/efectos adversos , Vacunas contra la Malaria/inmunología , Manitol/análogos & derivados , Ácidos Oléicos/administración & dosificación , Proteínas Protozoarias/efectos adversos , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/inmunología , Adolescente , Adulto , Animales , Antígenos de Protozoos/química , Antígenos de Superficie/química , Transmisión de Enfermedad Infecciosa , Femenino , Humanos , Vacunas contra la Malaria/química , Malaria Falciparum/prevención & control , Malaria Falciparum/transmisión , Malaria Vivax/prevención & control , Malaria Vivax/transmisión , Masculino , Manitol/administración & dosificación , Manitol/química , Persona de Mediana Edad , Ácidos Oléicos/química , Plasmodium falciparum/inmunología , Plasmodium vivax/inmunología , Proteínas Protozoarias/química , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/químicaRESUMEN
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.
RESUMEN
Recombinant apical membrane antigen 1 (AMA1) is a leading vaccine candidate for Plasmodium falciparum malaria, as antibodies against recombinant P. falciparum AMA1 (PfAMA1) interrupt merozoite invasion into erythrocytes. In order to investigate the role of posttranslational modification in modulating the functional immune response to recombinant AMA1, two separate alleles of PfAMA1 (FVO and 3D7), in which native N-glycosylation sites have been mutated, were produced using Escherichia coli and a Pichia pastoris expression system. Recombinant Pichia pastoris AMA1-FVO (PpAMA1-FVO) and PpAMA1-3D7 are O-linked glycosylated, and 45% of PpAMA1-3D7 is nicked, though all four recombinant molecules react with conformation-specific monoclonal antibodies. To address the immunological effect of O-linked glycosylation, we compared the immunogenicity of E. coli AMA1-FVO (EcAMA1-FVO) and PpAMA1-FVO antigens, since both molecules are intact. The effect of antigen nicking was then investigated by comparing the immunogenicity of EcAMA1-3D7 and PpAMA1-3D7. Our data demonstrate that there is no significant difference in the rabbit antibody titer elicited towards EcAMA1-FVO and PpAMA1-FVO or to EcAMA1-3D7 and PpAMA1-3D7. Furthermore, we have demonstrated that recombinant AMA1 (FVO or 3D7), whether expressed and refolded from E. coli or produced from the Pichia expression system, is equivalent and mimics the functionality of the native protein in in vitro growth inhibition assay experiments. We conclude that in the case of recombinant AMA1, the E. coli- and P. pastoris-derived antigens are immunologically and functionally equivalent and are unaffected by the posttranslational modification resulting from expression in these two systems.
Asunto(s)
Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/inmunología , Vacunas Sintéticas/inmunología , Secuencia de Aminoácidos , Animales , Escherichia coli/genética , Datos de Secuencia Molecular , Pichia/genética , Conejos , Proteínas Recombinantes/inmunologíaRESUMEN
Apical membrane antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. A phase 1 trial was conducted with 30 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of recombinant proteins based on sequences from the FVO and 3D7 clones of Plasmodium falciparum. The proteins were expressed in Pichia pastoris and adsorbed on Alhydrogel. Ten volunteers in each of three dose groups (5 mug, 20 mug, and 80 mug) were vaccinated in an open-label study at 0, 28, and 180 days. The vaccine was well tolerated, with pain at the injection site being the most commonly observed reaction. Anti-AMA1 immunoglobulin G (IgG) was detected by enzyme-linked immunosorbent assay (ELISA) in 15/28 (54%) volunteers after the second immunization and in 23/25 (92%) after the third immunization, with equal reactivity to both AMA1-FVO and AMA1-3D7 vaccine components. A significant dose-response relationship between antigen dose and antibody response by ELISA was observed, and the antibodies were predominantly of the IgG1 isotype. Confocal microscopic evaluation of sera from vaccinated volunteers demonstrated reactivity with P. falciparum schizonts in a pattern similar to native parasite AMA1. Antigen-specific in vitro inhibition of both FVO and 3D7 parasites was achieved with IgG purified from sera of vaccinees, demonstrating biological activity of the antibodies. To our knowledge, this is the first AMA1 vaccine candidate to elicit functional immune responses in malaria-naive humans, and our results support the further development of this vaccine.