RESUMEN
There is a remarkable heterogeneity in the functional profile (quality) of T cell responses. Importantly, the magnitude and/or quality of a response required for protection may be different depending on the infection. Here, we assessed the capacity of different Toll like receptor (TLR)-binding compounds to influence T helper cell (Th)1 and CD8+ T cell responses when used as adjuvants in nonhuman primates (NHP) with HIV Gag as a model antigen. NHP were immunized with HIV Gag protein emulsified in Montanide ISA 51, an oil-based adjuvant, with or without a TLR7/8 agonist, a TLR8 agonist, or the TLR9 ligand cytosine phosphate guanosine oligodeoxynucleotides (CpG ODN), and boosted 12 wk later with a replication-defective adenovirus-expressing HIV-Gag (rAD-Gag). Animals vaccinated with HIV Gag protein/Montanide and CpG ODN or the TLR7/8 agonist had higher frequencies of Th1 responses after primary immunization compared to all other vaccine groups. Although the rAD-Gag boost did not elevate the frequency of Th1 memory cytokine responses, there was a striking increase in HIV Gag-specific CD8+ T cell responses after the boost in all animals that had received a primary immunization with any of the TLR adjuvants. Importantly, the presence and type of TLR adjuvant used during primary immunization conferred stability and dramatically influenced the magnitude and quality of the Th1 and CD8+ T cell responses after the rAD-Gag boost. These data provide insights for designing prime-boost immunization regimens to optimize Th1 and CD8+ T cell responses.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Memoria Inmunológica/efectos de los fármacos , Manitol/análogos & derivados , Ácidos Oléicos/administración & dosificación , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Animales , Linfocitos T CD8-positivos/inmunología , Islas de CpG/inmunología , Citocinas/inmunología , Productos del Gen gag/administración & dosificación , Productos del Gen gag/inmunología , Inmunización , Macaca mulatta , Manitol/administración & dosificación , Manitol/inmunología , Ácidos Oléicos/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , Células TH1/inmunología , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 8/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunologíaRESUMEN
Hookworms digest hemoglobin from erythrocytes via a proteolytic cascade that begins with the aspartic protease, APR-1. Ac-APR-1 from the dog hookworm, Ancylostoma caninum, protects dogs against hookworm infection via antibodies that neutralize enzymatic activity and interrupt blood-feeding. Toward developing a human hookworm vaccine, we expressed both wild-type (Na-APR-1(wt)) and mutant (Na-APR-1(mut)-mutagenesis of the catalytic aspartic acids) forms of Na-APR-1 from the human hookworm, Necator americanus. Refolded Na-APR-1(wt) was catalytically active, and Na-APR-1(mut) was catalytically inactive but still bound substrates. Vaccination of canines with Na-APR-1(mut) and heterologous challenge with A. caninum resulted in significantly reduced parasite egg burdens (P=0.034) and weight loss (P=0.022). Vaccinated dogs also had less gut pathology, fewer adult worms, and reduced blood loss compared to controls but these did not reach statistical significance. Vaccination with Na-APR-1(mut) induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Na-APR-1(wt) and APR-1 orthologues from three other hookworm species that infect humans. IgG1 against Na-APR-1(mut) was the most prominently detected antibody in sera from people resident in high-transmission areas for N. americanus, indicating that natural boosting may occur in exposed humans. Na-APR-1(mut) is now a lead antigen for the development of an antihematophagy vaccine for human hookworm disease.
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Anticuerpos Antihelmínticos/uso terapéutico , Cisteína Endopeptidasas/inmunología , Infecciones por Uncinaria/prevención & control , Necator americanus/inmunología , Ancylostomatoidea/inmunología , Animales , Anticuerpos Antihelmínticos/administración & dosificación , Perros , Infecciones por Uncinaria/terapia , Humanos , Intestinos/parasitología , Resultado del Tratamiento , Vacunación/métodos , Vacunas/farmacología , Vacunas/uso terapéutico , Pérdida de PesoRESUMEN
CpG oligodeoxynucleotides are potent immunostimulants. For parenterally delivered alum-based vaccines, the immunostimulatory effect of CpG depends on the association of the CpG and antigen to the alum. We describe effects of buffer components on the binding of CPG 7909 to aluminum hydroxide (Alhydrogel), assays for measuring binding of CPG 7909 to alum and CPG 7909 induced dissociation of antigen from the alum. Free CPG 7909 is a potent inducer of IP-10 in mice. However the lack of IP-10 production from formulations containing bound CPG 7909 suggested that CPG 7909 does not rapidly dissociate from the alum after injection. It also suggests that IP-10 assays are not a good basis for potency assays for alum-based vaccines containing CPG 7909.
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Hidróxido de Aluminio/química , Densitometría/métodos , Espectrometría de Masas/métodos , Oligodesoxirribonucleótidos/análisis , Vacunas/química , Compuestos de Alumbre/química , Animales , Antígenos/química , Tampones (Química) , Quimiocina CXCL10 , Quimiocinas CXC/sangre , Ratones , Oligodesoxirribonucleótidos/química , Fosfatos/químicaRESUMEN
Residual host cell protein impurities in recombinant proteins intended for human use must be accurately quantified to help establish their safety. We describe a novel means of host cell protein quantitation, in which a slot blot system was employed together with scanning laser densitometry to allow picogram level sensitivity in detection of residual host cell proteins in unpurified fermentation products and final purified bulk samples. Two allelic forms of merozoite surface protein 1, a promising malaria vaccine candidate antigen currently undergoing evaluation in clinical trials, were expressed in E. coli as clinical grade proteins, refolded, and carried through several chromatographic purification steps. Several lots of these proteins were analyzed with this generic quantitative assay that uses rat polyclonal antibodies generated against soluble and insoluble E. coli proteins. The assay had a detection range of 6.1-1562 ng/mL, with a detection limit of 6.1 ng/mL, comparable to reported ELISA-based methods. This assay proved simple yet very sensitive and accurate, giving highly reproducible results. Thus it is suitable for evaluating host cell protein levels in clinical grade recombinant proteins expressed in E. coli.
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Proteínas de Escherichia coli/inmunología , Immunoblotting/métodos , Vacunas Sintéticas/normas , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/análisis , Proteína 1 de Superficie de Merozoito/inmunología , Proteína 1 de Superficie de Merozoito/aislamiento & purificación , Proteína 1 de Superficie de Merozoito/normas , Ratas , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificaciónAsunto(s)
Antígenos Bacterianos/análisis , Densitometría/métodos , Contaminación de Medicamentos , Pruebas Inmunológicas/métodos , Proteínas Recombinantes/aislamiento & purificación , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Escherichia coli/genética , Humanos , Rayos Láser , Proteínas Recombinantes/genéticaRESUMEN
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
CpG oligodeoxynucleotides are potent immunostimulants. In this study, CPG 7909 was formulated with the recombinant Plasmodium falciparum protein AMA1-C1 adsorbed to Alhydrogel (aluminum hydroxide) and used to immunize mice. Mice receiving free CPG 7909 in a separate same site injection to the AMA1-C1/Alhydrogel had the same antibody responses as mice receiving AMA1-C1/Alhydrogel alone. For mice immunized with CPG 7909 bound to the AMA1-C1/Alhydrogel formulation, there was a bell shaped CPG 7909 dose-response curve with the highest antibody response co-incident with the concentration of CPG 7909 that saturated binding to the Alhydrogel. At a higher CPG 7909 dose where 74% was unbound, there was no enhancement of response over AMA1-C1/Alhydrogel alone. Our results suggest that the adjuvant effects of CpGs are optimal when adsorbed to Alhydrogel and highlight the need for careful characterization of the vaccine formulation.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Malaria/inmunología , Oligodesoxirribonucleótidos/inmunología , Plasmodium falciparum/inmunología , Animales , Relación Dosis-Respuesta Inmunológica , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunoglobulina G/sangre , RatonesRESUMEN
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
This article provides a perspective on vaccine development for neglected tropical diseases in the nonprofit setting, with particular emphasis on recombinant protein vaccines. The Human Hookworm Vaccine Initiative is discussed as a model product development public-private partnership, and the major challenges are covered that accompany antigen selection, gene cloning, fermentation and purification process development, assay development, vaccine formulation and testing and clinical evaluation for those developing vaccines, especially against neglected tropical diseases, in the nonprofit sector. Throughout this perspective, special emphasis is placed on the growing promise that product development public-private partnerships hold for developing vaccines for the world's poorest people.
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Presupuestos , Organizaciones sin Fines de Lucro/economía , Medicina Tropical/economía , Vacunas/economía , Presupuestos/métodos , Presupuestos/tendencias , Humanos , Medicina Tropical/métodos , Vacunas/síntesis química , Vacunas/uso terapéuticoRESUMEN
Plasmodium vivax is responsible for the majority of malaria cases outside of Africa, and results in substantial morbidity. Transmission blocking vaccines are a potentially powerful component of a multi-faceted public health approach to controlling or eliminating malaria. We report the first phase 1 clinical trial of a P. vivax transmission blocking vaccine in humans. The Pvs25H vaccine is a recombinant protein derived from the Pvs25 surface antigen of P. vivax ookinetes. The protein was expressed in Saccharomyces cerevisiae, purified, and adsorbed onto Alhydrogel. Ten volunteers in each of three dose groups (5, 20, or 80 microg) were vaccinated by intramuscular injection in an open-label study at 0, 28 and 180 days. No vaccine-related serious adverse events were observed. The majority of adverse events causally related to vaccination were mild or moderate in severity. Injection site tenderness was the most commonly observed adverse event. Anti-Pvs25H antibody levels measured by ELISA peaked after the third vaccination. Vaccine-induced antibody is functionally active as evidenced by significant transmission blocking activity in the membrane feeding assay. Correlation between antibody concentration and degree of inhibition was observed. Pvs25H generates transmission blocking immunity in humans against P. vivax demonstrating the potential of this antigen as a component of a transmission blocking vaccine.
Asunto(s)
Antígenos de Protozoos/uso terapéutico , Antígenos de Superficie/uso terapéutico , Vacunas contra la Malaria/uso terapéutico , Malaria Vivax/prevención & control , Malaria Vivax/transmisión , Adolescente , Adulto , Animales , Antígenos de Protozoos/efectos adversos , Antígenos de Superficie/efectos adversos , Culicidae/inmunología , Culicidae/parasitología , Relación Dosis-Respuesta Inmunológica , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Esquemas de Inmunización , Vacunas contra la Malaria/efectos adversos , Masculino , Persona de Mediana Edad , Dinámicas no Lineales , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/uso terapéuticoRESUMEN
Montanide ISA 720 is an experimental adjuvant, formulated as water-in-oil emulsions, that induces high antibody titers in several animal species. It has been used in human vaccine trials with malaria and HIV vaccines. The heightened response is likely due, in part, to the formation of a depot at the injection site. However, post-formulation modifications were seen with seven proteins tested during storage of ISA 720 formulations at 37 degrees C for 1 week and two proteins stored longer at 4 degrees C. Potency studies in mice, in which the stored vaccines were diluted into placebo emulsions for appropriate dosing, indicated that this instability could lead to loss of immunogenicity in the post-injection depot, limiting the allowable storage time of preformed vaccines. We describe point-of-injection formulation for ISA 720 vaccines that meets the requirement for in vitro stability. For preformed vaccines, addition of glycine or glycylglycine prevented antigen modification on storage at 37 degrees C, providing a potential way of stabilizing antigen/ISA 720 formulations for in vitro storage and the post-injection depot.
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Adyuvantes Inmunológicos/normas , Antígenos/inmunología , Manitol/análogos & derivados , Ácidos Oléicos/normas , Vacunas/química , Vacunas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos/administración & dosificación , Antígenos/química , Antígenos de Protozoos/inmunología , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Emulsiones , Ensayo de Inmunoadsorción Enzimática , Femenino , Manitol/administración & dosificación , Manitol/inmunología , Manitol/normas , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Ácidos Oléicos/administración & dosificación , Ácidos Oléicos/inmunología , Control de Calidad , Proteínas Recombinantes , Temperatura , Vacunas/administración & dosificaciónRESUMEN
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.
Asunto(s)
Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Vacunas Sintéticas/inmunología , Adulto , Animales , Anticuerpos Antiprotozoarios/sangre , Femenino , Humanos , Inmunoglobulina G/sangre , Vacunas contra la Malaria/efectos adversos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
The budding yeast Saccharomyces cerevisiae has been used to express the recombinant protein Pvs25H, currently the only candidate transmission-blocking vaccine against Plasmodium vivax malaria. This molecule contains four epidermal growth factor-like domains and is expressed as at least two stable monomeric forms with different physicochemical properties. Pvs25H-A is apparently homogeneous and seems to have a correct disulfide bond structure. By contrast, Pvs25H-B is produced as a heterogeneous population of molecules, some of which are associated with an as yet unidentified chromophore, and it contains both internal and N-terminal cleavages. We report here a procedure for successfully separating these two forms with a process suitable for clinical production of this antigen.
Asunto(s)
Antígenos de Protozoos/química , Antígenos de Protozoos/aislamiento & purificación , Antígenos de Superficie/química , Antígenos de Superficie/aislamiento & purificación , Antígenos/química , Antígenos/aislamiento & purificación , Vacunas contra la Malaria/química , Vacunas contra la Malaria/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Western Blotting , Cromatografía , Cromatografía Líquida de Alta Presión , Ensayos Clínicos como Asunto , Disulfuros , Fermentación , Humanos , Malaria/prevención & control , Datos de Secuencia Molecular , Plasmodium vivax/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismoRESUMEN
In previously published studies, Saccharomyces cerevisiae recombinant protein expression systems have been employed to express the malaria parasite antigen Pfs25, a candidate transmission-blocking vaccine antigen against Plasmodium falciparum malaria. However, despite having been in two Phase 1 trials, the recombinant Pfs25 so produced (previously called TBV25H) exists as a mixture of two monomeric protein conformational forms, Pfs25H-A and Pfs25H-B. In this study, we optimized the expression and purification of the two Pfs25H conformers in S. cerevisiae, and characterized their biochemical and antigenic properties, immunogenicities, and transmission-blocking activities. Pfs25H-A is apparently homogeneous, and has the correct conformation as measured by monoclonal antibody recognition. It is, however, expressed at a low yield of only 0.19mg/l. By contrast, Pfs25H-B is produced as a heterogeneous population of molecules that do not seem to have the correct conformation. Nonetheless, both forms appear equally effective in their ability to produce transmission-blocking antibodies in mice. To address the low yield seen with S. cerevisiae, we also expressed Pfs25 in Pichia pastoris. P. pastoris is apparently superior to S. cerevisiae in producing higher yield, immunologically more potent, biologically more active Pfs25H-A.
Asunto(s)
Ensayos Clínicos como Asunto , Vacunas contra la Malaria/genética , Vacunas contra la Malaria/uso terapéutico , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Pichia/metabolismo , Proteínas Protozoarias/biosíntesis , Secuencia de Aminoácidos , Animales , Ensayos Clínicos como Asunto/métodos , Culicidae/inmunología , Culicidae/parasitología , Femenino , Regulación Fúngica de la Expresión Génica/inmunología , Humanos , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/inmunología , Malaria Falciparum/transmisión , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pichia/inmunología , Plásmidos/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/aislamiento & purificación , Saccharomyces cerevisiae/inmunología , Saccharomyces cerevisiae/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , Vacunas Sintéticas/biosíntesis , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéuticoRESUMEN
Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P. falciparum have been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P. falciparum strains is unknown. We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P. falciparum clones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage.