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1.
PLoS Pathog ; 14(5): e1007034, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29742161

RESUMO

Malaria transmission remains high in Sub-Saharan Africa despite large-scale implementation of malaria control interventions. A comprehensive understanding of the transmissibility of infections to mosquitoes may guide the design of more effective transmission reducing strategies. The impact of P. falciparum sexual stage immunity on the infectious reservoir for malaria has never been studied in natural settings. Repeated measurements were carried out at start-wet, peak-wet and dry season, and provided data on antibody responses against gametocyte/gamete antigens Pfs48/45 and Pfs230 as anti-gametocyte immunity. Data on high and low-density infections and their infectiousness to anopheline mosquitoes were obtained using quantitative molecular methods and mosquito feeding assays, respectively. An event-driven model for P. falciparum sexual stage immunity was developed and fit to data using an agent based malaria model infrastructure. We found that Pfs48/45 and Pfs230 antibody densities increased with increasing concurrent gametocyte densities; associated with 55-70% reduction in oocyst intensity and achieved up to 44% reduction in proportions of infected mosquitoes. We showed that P. falciparum sexual stage immunity significantly reduces transmission of microscopic (p < 0.001) but not submicroscopic (p = 0.937) gametocyte infections to mosquitoes and that incorporating sexual stage immunity into mathematical models had a considerable impact on the contribution of different age groups to the infectious reservoir of malaria. Human antibody responses to gametocyte antigens are likely to be dependent on recent and concurrent high-density gametocyte exposure and have a pronounced impact on the likelihood of onward transmission of microscopic gametocyte densities compared to low density infections. Our mathematical simulations indicate that anti-gametocyte immunity is an important factor for predicting and understanding the composition and dynamics of the human infectious reservoir for malaria.


Assuntos
Malária/transmissão , Glicoproteínas de Membrana/imunologia , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/imunologia , Animais , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Doenças Transmissíveis/transmissão , Culicidae , Humanos , Insetos Vetores , Malária Falciparum/genética , Malária Falciparum/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Plasmodium falciparum/imunologia , Plasmodium falciparum/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
2.
Nat Commun ; 9(1): 1498, 2018 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-29643330

RESUMO

The original version of this Article contained errors in Fig. 3. In panel a, bars from a chart depicting the percentage of antibody-positive individuals in non-infectious and infectious groups were inadvertently included in place of bars depicting the percentage of infectious individuals, as described in the Article and figure legend. However, the p values reported in the Figure and the resulting conclusions were based on the correct dataset. The corrected Fig. 3a now shows the percentage of infectious individuals in antibody-negative and -positive groups, in both the PDF and HTML versions of the Article. The incorrect and correct versions of Figure 3a are also presented for comparison in the accompanying Publisher Correction as Figure 1.The HTML version of the Article also omitted a link to Supplementary Data 6. The error has now been fixed and Supplementary Data 6 is available to download.

3.
Nat Commun ; 9(1): 558, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29422648

RESUMO

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes.


Assuntos
Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Plasmodium falciparum , Adulto , Idoso , Idoso de 80 Anos ou mais , Burkina Faso/epidemiologia , Camarões/epidemiologia , Estudos de Casos e Controles , Feminino , Gâmbia/epidemiologia , Humanos , Imunoglobulina G/sangue , Malária Falciparum/sangue , Masculino , Pessoa de Meia-Idade
4.
Microb Cell Fact ; 16(1): 97, 2017 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-28569168

RESUMO

BACKGROUND: The sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies. We have investigated a downstream purification process of a sub-unit (R0.6C) fragment representing the C-terminal 6-Cys domain of Pfs48/45 (6C) genetically fused to the R0 region (R0) of asexual stage Glutamate Rich Protein expressed in Lactococcus lactis. RESULTS: A series of R0.6C fusion proteins containing features, which aim to increase expression levels or to facilitate protein purification, were evaluated at small scale. None of these modifications affected the overall yield of recombinant protein. Consequently, R0.6C with a C-terminal his tag was used for upstream and downstream process development. A simple work-flow was developed consisting of batch fermentation followed by two purification steps. As such, the recombinant protein was purified to homogeneity. The composition of the final product was verified by HPLC, mass spectrometry, SDS-PAGE and Western blotting with conformation dependent antibodies against Pfs48/45. The recombinant protein induced high levels of functional TB antibodies in rats. CONCLUSIONS: The established production and purification process of the R0.6C fusion protein provide a strong basis for further clinical development of this candidate transmission blocking malaria vaccine.


Assuntos
Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/imunologia , Imunogenicidade da Vacina/imunologia , Lactococcus lactis/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Animais , Vacinas Bacterianas/isolamento & purificação , Reatores Biológicos , Lactococcus lactis/genética , Plasmodium falciparum/química , Plasmodium falciparum/imunologia , Subunidades Proteicas/biossíntese , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Ratos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação
5.
Pharm Res ; 34(9): 1970-1983, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28646324

RESUMO

PURPOSE: Production and characterization of a chimeric fusion protein (GMZ2'.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2'.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite. METHODS: GMZ2'.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS. RESULTS: CP-GMZ2'.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2'.10C. CP-GMZ2'.10C and IP-GMZ2'.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus Pfs48/45 was analysed by tandem mass spectrometry and was established for GMZ2'.10C and two reference fusion proteins encompassing similar parts of Pfs48/45. CONCLUSION: GMZ2'.10C, combining GMZ2' and correctly-folded Pfs48/45 can be produced by the Lactoccus lactis P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of Pfs48/45 was revealed experimentally, providing an important guideline for employing the Pfs48/45 antigen in vaccine design.


Assuntos
Antígenos de Protozoários/uso terapêutico , Vacinas Antimaláricas/uso terapêutico , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/uso terapêutico , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Clonagem Molecular , Humanos , Lactococcus lactis/genética , Vacinas Antimaláricas/química , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Plasmodium falciparum/química , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Estabilidade Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Ratos Wistar , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico
6.
Vaccine ; 35(24): 3249-3255, 2017 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-28479181

RESUMO

Carbohydrate fatty acid sulphate esters (CFASEs) formulated in a squalane-in-water emulsion are effective adjuvants for humoral responses to a wide range of antigens in various animal species but rise in body temperature and local reactions albeit mild or minimal hampers application in humans. In rabbits, body temperature increased 1°C one day after intramuscular (IM) injection, which returned to normal during the next day. The effect increased with increasing dose of CFASE but not with the number of injections (up to 5). Antigen enhanced the rise in body temperature after booster immunization (P<0.01) but not after priming. Synthetic CFASEs are mixtures of derivatives containing no sulphate, one or multiple sulphate groups and the monosulphate derivatives (CMS) were isolated, incorporated in a squalane in-water emulsion and investigated. In contrast to CFASE, CMS adjuvant did not generate rise in body temperature or local reactions in rabbits immunized with a purified, recombinant malaria chimeric antigen R0.10C. In comparison to alum, CMS adjuvant revealed approximately 30-fold higher antibody titres after the first and >100-fold after the second immunization. In ferrets immunized with 7.5µg of inactivated influenza virus A/H7N9, CMS adjuvant gave 100-fold increase in HAI antibody titres after the first and 25-fold after the second immunisation, which were 10-20-fold higher than with the MF59-like AddaVax adjuvant. In both models, a single immunisation with CMS adjuvant revealed similar or higher titres than two immunisations with either benchmark, without detectable systemic and local adverse effects. Despite striking chemical similarities with monophospholipid A (MPL), CMS adjuvant did not activate human TLR4 expressed on HEK cells. We concluded that the synthetic CMS adjuvant is a promising candidate for poor immunogens and single-shot vaccines and that rise in body temperature, local reactions or activation of TLR4 is not a pre-requisite for high adjuvanticity.


Assuntos
Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/química , Ésteres/efeitos adversos , Ésteres/imunologia , Imunidade Humoral , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/síntese química , Animais , Anticorpos Antivirais/sangue , Temperatura Corporal , Carboidratos/administração & dosagem , Carboidratos/efeitos adversos , Carboidratos/química , Carboidratos/imunologia , Composição de Medicamentos , Ésteres/administração & dosagem , Ésteres/química , Ácidos Graxos/administração & dosagem , Ácidos Graxos/efeitos adversos , Ácidos Graxos/química , Ácidos Graxos/imunologia , Furões/imunologia , Células HEK293 , Testes de Inibição da Hemaglutinação , Humanos , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Injeções Intramusculares , Lipídeo A/análogos & derivados , Lipídeo A/química , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Polissorbatos/administração & dosagem , Coelhos , Esqualeno/administração & dosagem , Esqualeno/imunologia , Receptor 4 Toll-Like/imunologia , Vacinação
7.
J Nanobiotechnology ; 14: 30, 2016 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-27117585

RESUMO

BACKGROUND: Virus-like particles (VLPs) represent a significant advance in the development of subunit vaccines, combining high safety and efficacy. Their particulate nature and dense repetitive subunit organization makes them ideal scaffolds for display of vaccine antigens. Traditional approaches for VLP-based antigen display require labor-intensive trial-and-error optimization, and often fail to generate dense antigen display. Here we utilize the split-intein (SpyTag/SpyCatcher) conjugation system to generate stable isopeptide bound antigen-VLP complexes by simply mixing of the antigen and VLP components. RESULTS: Genetic fusion of SpyTag or SpyCatcher to the N-terminus and/or C-terminus of the Acinetobacter phage AP205 capsid protein resulted in formation of stable, nonaggregated VLPs expressing one SpyCatcher, one SpyTag or two SpyTags per capsid protein. Mixing of spy-VLPs with eleven different vaccine antigens fused to SpyCatcher or SpyTag resulted in formation of antigen-VLP complexes with coupling efficiencies (% occupancy of total VLP binding sites) ranging from 22-88 %. In mice, spy-VLP vaccines presenting the malaria proteins Pfs25 or VAR2CSA markedly increased antibody titer, affinity, longevity and functional efficacy compared to corresponding vaccines employing monomeric proteins. The spy-VLP vaccines also effectively broke B cell self-tolerance and induced potent and durable antibody responses upon vaccination with cancer or allergy-associated self-antigens (PD-L1, CTLA-4 and IL-5). CONCLUSIONS: The spy-VLP system constitutes a versatile and rapid method to develop highly immunogenic VLP-based vaccines. Our data provide proof-of-concept for the technology's ability to present complex vaccine antigens to the immune system and elicit robust functional antibody responses as well as to efficiently break B cell self-tolerance. The spy-VLP-system may serve as a generic tool for the cost-effective development of effective VLP-vaccines against both infectious- and non-communicable diseases and could facilitate rapid and unbiased screening of vaccine candidate antigens.


Assuntos
Vacinas de Partículas Semelhantes a Vírus/imunologia , Acinetobacter/imunologia , Animais , Antígenos de Bactérias/imunologia , Linfócitos B/imunologia , Bacteriófagos/imunologia , Proteínas do Capsídeo/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinação/métodos
8.
Vaccine ; 34(19): 2207-15, 2016 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-26994314

RESUMO

A subunit vaccine targeting both transmission and pathogenic asexual blood stages of Plasmodium falciparum, i.e., a multi-stage vaccine, could be a powerful tool to combat malaria. Here, we report production and characterization of the recombinant protein GMZ2.6C, which contains a fragment of the sexual-stage protein Pfs48/45-6C genetically fused to GMZ2, an asexual vaccine antigen in advanced clinical development. To select the most suitable vaccine formulation for downstream clinical studies, GMZ2.6C was tested with various immune modulators in different adjuvant formulations (stable emulsions, liposomes, and alum) in C57BL/6 mice. Some, but not all, formulations containing either the synthetic TLR4 agonist GLA or SLA elicited the highest parasite-specific antibody titers, the greatest IFN-γ responses in CD4+ TH1 cells, and the highest percentage of multifunctional CD4+ T cells expressing IFN-γ and TNF in response to GMZ2.6C. Both of these agonists have good safety records in humans.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Receptor 4 Toll-Like/agonistas , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Feminino , Imunidade Celular , Imunidade Humoral , Interferon gama/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Plasmodium falciparum , Proteínas de Protozoários/imunologia
9.
Sci Rep ; 6: 20440, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26861587

RESUMO

Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3-5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites.


Assuntos
Anopheles/metabolismo , Proteínas de Insetos/metabolismo , Plasmodium falciparum/fisiologia , Animais , Anopheles/crescimento & desenvolvimento , Anopheles/parasitologia , Suscetibilidade a Doenças , Inativação Gênica , Immunoblotting , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/deficiência , Insetos Vetores/parasitologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Oocistos/metabolismo , Oocistos/parasitologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNA
10.
Malar J ; 14: 443, 2015 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-26552428

RESUMO

BACKGROUND: Plasmodium falciparum is transmitted from person to person by Anopheles mosquitoes after completing its sexual reproductive cycle within the infected mosquito. An efficacious vaccine holds the potential to interrupt development of the parasite in the mosquito leading to control and possibly eradication of malaria. A multi-component, R0.10C, was developed comprising P. falciparum glutamate-rich protein (R0) fused in frame to a correctly folded fragment of Pfs48/45 (10C). Here, a series of novel adjuvants were screened for their ability to elicit transmission-blocking (TB) antibodies. METHODS: The recombinant fusion protein R0.10C was produced in Lactococcus lactis and purified by affinity-chromatography on a monoclonal antibody (mAb 85RF45.1) against a major epitope for TB antibodies (epitope 1) harboured on R0.10C. Immune-purified R0.10C was mixed with a series of adjuvants and tested in mice and rats. RESULTS: In general, all R0.10C formulations elicited high levels of antibodies recognizing native Pfs48/45 in macrogametes/zygotes. TB activity of anti-R0.10C antisera was assessed in the standard membrane-feeding assay (SMFA). Potency of different adjuvant/R0.10C combinations was tested in mice and rats using aluminium hydroxide (Alum), Alum with micellar and emulsion formulations of a synthetic TLR4 agonist, Glucopyranosyl Lipid Adjuvant (GLA), stable emulsion (SE)/GLA, AbISCO-100 and Freund's adjuvant (as reference). All formulations produced high antibody titres recognizing the native Pfs48/45 protein in macrogametes/zygotes. Interestingly, the GLA-Alum combination adjuvant was the most potent inducer of TB antibodies based on serum collected after two immunizations. In agreement with previous observations, biological activity in the SMFA correlated well with the level of anti-Pfs48/45 antibodies. CONCLUSION: The combined data provide a strong basis for entering the next phase of clinical grade R0.10C production and testing.


Assuntos
Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/genética , Vacinas Protozoárias/farmacologia , Adjuvantes Imunológicos/farmacologia , Hidróxido de Alumínio/farmacologia , Animais , Anticorpos Antiprotozoários/sangue , Emulsões/farmacologia , Feminino , Glucosídeos/farmacologia , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Lipídeo A/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Protozoários/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saponinas/farmacologia
11.
J Infect ; 71(1): 117-27, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25869538

RESUMO

OBJECTIVES: Pfs48/45 and Pfs230 are Plasmodium falciparum sexual stage proteins and promising malaria transmission-blocking vaccine candidates. Antibody responses against these proteins may be naturally acquired and target antigens may be under selective pressure. This has consequences for the future evaluation of vaccine immunogenicity and efficacy in populations naturally exposed to malaria. METHODS: We determined naturally acquired antibody responses to the recombinant proteins Pfs48/45-10C and Pfs230-230CMB in children from three malaria endemic settings in Ghana, Tanzania and Burkina Faso. We also examined genetic polymorphisms in the P. falciparum gene pfs48/45. RESULTS: Antibody prevalence was 1.1-18.2% for 10C and 6.7-18.9% for 230CMB. In Burkina Faso we observed evidence of an age-dependent acquisition pattern for both 10C (p < 0.001) and 230CMB (p = 0.031). Membrane feeding assays on a separate dataset demonstrated an association between functional transmission reducing activity and antibody prevalence for both 10C (p = 0.017) and 230CMB (p = 0.049). 17 single nucleotide polymorphisms were found in pfs48/45 (from 126 samples), with 5 non-synonymous SNPs in the Pfs48/45 10C region. CONCLUSIONS: We conclude there are naturally acquired antibody responses to both vaccine candidates which have functional relevance by reducing the transmissibility of infected individuals. We identified genetic polymorphisms, in pfs48/45 which exhibited geographical specificity.


Assuntos
Anticorpos Antiprotozoários/sangue , Formação de Anticorpos , Antígenos de Protozoários/imunologia , Malária Falciparum/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Burkina Faso , Criança , Estudos Transversais , Feminino , Gana , Humanos , Masculino , Glicoproteínas de Membrana/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Tanzânia
12.
Vaccine ; 33(16): 1981-6, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25728318

RESUMO

The sexual stage Pfs48/45 antigen is a well-established lead candidate for a transmission blocking (TB) vaccine because of its critical role in parasite fertilization. We have recently produced the carboxy-terminal 10C-fragment of Pfs48/45 containing three known epitopes for TB antibodies as a chimera with the N-terminal region of GLURP (R0). The resulting fusion protein elicited high titer TB antibodies in rodents. To increase the relatively low yield of correctly folded Pfs48/45 we have generated a series of novel chimera truncating the 10C-fragments to 6 cysteine residues containing sub-units (6C). All constructs harbor the major epitope I for TB antibodies. One of these sub-units (R0.6Cc), produced high yields of correctly folded conformers, which could be purified by a simple 2-step procedure. Purified R0.6Cc was stable and elicits high titer TB antibodies in rats. The yield, purity and stability of R0.6Cc allows for further clinical development.


Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Epitopos/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Epitopos/química , Epitopos/genética , Expressão Gênica , Malária Falciparum/transmissão , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia
13.
Hum Vaccin Immunother ; 11(1): 124-32, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25483525

RESUMO

Malaria transmission blocking vaccines (TBV) directed against proteins expressed on sexual stages of Plasmodium falciparum in the mosquito midgut are considered an effective means to reduce malaria transmission. Antibodies induced by TBV block sporogonic development in the mosquito, and thus transmission to the next human host. The Pfs25 protein, expressed on the surface of gametes, zygotes and ookinetes, is one of the primary targets for TBV development. Using a plant virus-based transient expression system, we have successfully produced Pfs25 fused to a modified lichenase (LicKM) carrier in Nicotiana benthamiana, purified and characterized the protein (Pfs25-FhCMB), and evaluated this vaccine candidate in animal models for the induction of transmission blocking antibodies. Soluble Pfs25-FhCMB was expressed in plants at a high level, and induced transmission blocking antibodies that persisted for up to 6 months post immunization in mice and rabbits. These data demonstrate the potential of the new malaria vaccine candidate and also support feasibility of expressing Plasmodium antigens in a plant-based system.


Assuntos
Anticorpos Antiprotozoários/sangue , Transmissão de Doença Infecciosa/prevenção & controle , Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Proteínas de Protozoários/imunologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Potyvirus/genética , Proteínas de Protozoários/genética , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Fatores de Tempo , Nicotiana/genética , Nicotiana/metabolismo , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
14.
Vaccine ; 32(22): 2623-30, 2014 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-24662702

RESUMO

Effective control and eventual eradication of malaria drives the imperative need for clinical development of a malaria vaccine. Asexual parasite forms are responsible for clinical disease and death while apathogenic gametocytes are responsible for transmission from man to mosquito. Vaccines that combine antigens from both stages may provide direct protection and indirect benefit by reducing the force of infection. We constructed a chimeric antigen composed of a fragment of the Plasmodium falciparum (Pf) glutamate-rich protein fused in frame to a correctly folded fragment of Pfs48/45. The chimera was produced in Lactococcus lactis and induced robust antibody responses in rodents to the individual components. Specific antibodies showed strong transmission blocking activity against multiple Pf-strains in the standard membrane feeding assay and functional activity against asexual stages in the antibody dependent cellular inhibition assay. The combined data provide a strong rationale for entering the next phase of clinical grade production and testing.


Assuntos
Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Glicoproteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Animais , Anopheles , Anticorpos Antiprotozoários/sangue , Formação de Anticorpos , Soros Imunes/imunologia , Imunoglobulina G/sangue , Plasmodium falciparum , Ratos Wistar , Proteínas Recombinantes/imunologia , Vacinas de Subunidades Antigênicas/imunologia
15.
Sci Rep ; 3: 3418, 2013 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-24301557

RESUMO

Mosquito feeding assays are important in evaluations of malaria transmission-reducing interventions. The proportion of mosquitoes with midgut oocysts is commonly used as an outcome measure, but in natural low intensity infections the effect of oocyst non-rupture on mosquito infectivity is unclear. By identifying ruptured as well as intact oocysts, we show that in low intensity P. falciparum infections i) 66.7-96.7% of infected mosquitoes experienced oocyst rupture between 11-21 days post-infection, ii) oocyst rupture led invariably to sporozoite release, iii) oocyst rupture led to salivary gland infections in 97.8% of mosquitoes, and iv) 1250 (IQR 313-2400) salivary gland sporozoites were found per ruptured oocyst. These data show that infectivity can be predicted with reasonable certainty from oocyst prevalence in low intensity infections. High throughput methods for detecting infection in whole mosquitoes showed that 18s PCR but not circumsporozoite ELISA gave a reliable approximation of mosquito infection rates on day 7 post-infection.


Assuntos
Culicidae/fisiologia , Culicidae/parasitologia , Oocistos/fisiologia , Oocistos/parasitologia , Esporozoítos/fisiologia , Animais , Feminino , Insetos Vetores/parasitologia , Insetos Vetores/fisiologia , Malária/parasitologia , Plasmodium falciparum , Prevalência , Glândulas Salivares/parasitologia , Glândulas Salivares/fisiologia , Esporozoítos/parasitologia
16.
PLoS One ; 8(11): e79538, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24260245

RESUMO

Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based 'launch' vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20-30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the 'launch' vector technology for the production of VLP-based recombinant vaccines against infectious diseases.


Assuntos
Anticorpos Bloqueadores/imunologia , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Animais , Antígenos de Protozoários/imunologia , Camundongos , Proteínas de Protozoários/imunologia , Proteínas Recombinantes
17.
Proc Natl Acad Sci U S A ; 110(19): 7862-7, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23599283

RESUMO

Volunteers immunized under chloroquine chemoprophylaxis with Plasmodium falciparum sporozoites (CPS) develop complete, long-lasting protection against homologous sporozoite challenge. Chloroquine affects neither sporozoites nor liver-stages, but kills only asexual forms in erythrocytes once released from the liver into the circulation. Consequently, CPS immunization exposes the host to antigens from both preerythrocytic and blood stages, and induced immunity might target either of these stages. We therefore explored the life cycle stage specificity of CPS-induced protection. Twenty-five malaria-naïve volunteers were enrolled in a clinical trial, 15 of whom received CPS immunization. Five immunized subjects and five controls received a sporozoite challenge by mosquito bites, whereas nine immunized and five control subjects received an i.v. challenge with P. falciparum-infected erythrocytes. The latter approach completely bypasses preerythrocytic stages, enabling a direct comparison of protection against either life cycle stage. CPS-immunized subjects (13 of 14) developed anticircumsporozoite antibodies, whereas only one volunteer generated minimal titers against typical blood-stage antigens. IgG from CPS-immunized volunteers did not inhibit asexual blood-stage growth in vitro. All CPS-immunized subjects (5 of 5) were protected against sporozoite challenge. In contrast, nine of nine CPS-immunized subjects developed parasitemia after blood-stage challenge, with identical prepatent periods and blood-stage multiplication rates compared with controls. Intravenously challenged CPS-immunized subjects showed earlier fever and increased plasma concentrations of inflammatory markers D-dimer, IFN-γ, and monokine induced by IFN-γ than i.v. challenged controls. The complete lack of protection against blood-stage challenge indicates that CPS-induced protection is mediated by immunity against preerythrocytic stages. However, evidence is presented for immune recognition of P. falciparum-infected erythrocytes, suggesting memory responses unable to generate functional immunity.


Assuntos
Cloroquina/uso terapêutico , Vacinas Antimaláricas/uso terapêutico , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Esporozoítos/imunologia , Adolescente , Adulto , Animais , Anopheles , Antígenos de Protozoários/imunologia , Antimaláricos/uso terapêutico , Eritrócitos/parasitologia , Humanos , Cinética , Malária Falciparum/tratamento farmacológico , Resultado do Tratamento , Adulto Jovem
18.
PLoS One ; 8(2): e56863, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23451100

RESUMO

BACKGROUND: We conducted a prospective study in a cohort of short-term travelers assessing the incidence rate of anti-circumsporozoite seroconversion, adherence to chemoprophylaxis, symptoms of malaria during travel, and malaria treatment abroad. METHODS: Adults were recruited from the travel clinic of the Public Health Service Amsterdam. They kept a structured daily travel diary and donated blood samples before and after travel. Blood samples were serologically tested for the presence of Plasmodium falciparum anti-circumsporozoite antibodies. RESULTS: Overall, the incidence rate (IR) of anti-circumsporozoite seroconversion was 0.8 per 100 person-months. Of 945 travelers, 620 (66%) visited high-endemic areas and were advised about both chemoprophylaxis and preventive measures against mosquito bites. Most subjects (520/620 = 84%) took at least 75% of recommended prophylaxis during travel. Travel to Africa, use of mefloquine, travel duration of 14-29 days in endemic areas, and concurrent use of DEET (N,N-diethyl-meta-toluamide) were associated with good adherence practices. Four travelers without fever seroconverted, becoming anti-circumsporozoite antibody-positive. All four had been adherent to chemoprophylaxis; two visited Africa, one Suriname, one India. Ten subjects with fever were tested for malaria while abroad and of these, three received treatment. All three were adherent to chemoprophylaxis and tested negative for anti-circumsporozoite antibodies. CONCLUSION: Travel to Africa, using mefloquine, travel duration of 14-29 days in endemic areas, and use of DEET were associated with good adherence to chemoprophylaxis. The combination of chemoprophylaxis and other preventive measures were sufficient to protect seroconverting travelers from clinical malaria. Travelers who were treated for malaria abroad did not seroconvert.


Assuntos
Antimaláricos/uso terapêutico , Quimioprevenção , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/patogenicidade , Viagem , Adolescente , Adulto , Anticorpos Antiprotozoários/sangue , Feminino , Humanos , Malária Falciparum/sangue , Malária Falciparum/imunologia , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/imunologia , Estudos Prospectivos , Adulto Jovem
19.
PLoS One ; 7(8): e42821, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22936993

RESUMO

INTRODUCTION: In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes. METHODS: WE COMPARED TWO COMMONLY USED MOSQUITO FEEDING ASSAY PROCEDURES: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum. RESULTS: 930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94-2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68-2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52-0.70). CONCLUSIONS: Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.


Assuntos
Anopheles/parasitologia , Malária Falciparum/transmissão , Plasmodium falciparum/patogenicidade , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Insetos Vetores , Masculino , Pessoa de Meia-Idade , Adulto Jovem
20.
Infect Immun ; 79(12): 4957-64, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21969000

RESUMO

Acquisition of immunity to Plasmodium falciparum sexual stages is a key determinant for reducing human-mosquito transmission by preventing the fertilization and the development of the parasite in the mosquito midgut. Naturally acquired immunity against sexual stages may therefore form the basis for the development of transmission-blocking vaccines, but studies conducted to date offer little in the way of consistent findings. Here, we describe the acquisition of antigametocyte immune responses in malaria-exposed individuals in Burkina Faso. A total of 719 blood samples were collected in a series of three cross-sectional surveys at the start, peak, and end of the wet season. The seroprevalence of antibodies with specificity for the sexual stage antigens Pfs48/45 and Pfs230 was 2-fold lower (22 to 28%) than that for an asexual blood stage antigen glutamate-rich protein (GLURP) (65%) or for the preerythrocytic stage antigen circumsporozoite protein (CSP) (54%). The youngest children responded at frequencies similar to those for all four antigens but, in contrast with the immune responses to GLURP and CSP that increased with age independently of season and area of residence, there was no evidence for a clear age dependence of responses to Pfs48/45 and Pfs230. Anti-Pfs230 antibodies were most prevalent at the peak of the wet season (P < 0.001). Our findings suggest that naturally acquired immunity against Pfs48/45 and Pfs230 is a function of recent exposure rather than of cumulative exposure to gametocytes.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Malária Falciparum/imunologia , Glicoproteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Anticorpos Antiprotozoários/biossíntese , Burkina Faso/epidemiologia , Criança , Pré-Escolar , Estudos Transversais , Humanos , Lactente , Malária Falciparum/epidemiologia , Malária Falciparum/transmissão , Estações do Ano , Estudos Soroepidemiológicos , Adulto Jovem
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