RESUMO
BACKGROUND: Previous studies have shown that the baculovirus-vectored vaccine based on the "baculovirus dual expression system (BDES)" is an effective vaccine delivery platform for malaria. However, a point of weakness remaining for use of this vaccine platform in vivo concerns viral inactivation by serum complement. In an effort to achieve complement resistance, the gene encoding the human decay-accelerating factor (hDAF) was incorporated into the BDES malaria vaccine expressing the Plasmodium falciparum circumsporozoite protein (PfCSP). RESULTS: The newly-developed BDES vaccine, designated BDES-sPfCSP2-Spider, effectively displayed hDAF and PfCSP on the surface of the viral envelope, resulting in complement resistance both in vitro and in vivo. Importantly, upon intramuscular inoculation into mice, the BDES-sPfCSP2-Spider vaccine had a higher protective efficacy (60%) than that of the control vaccine BDES-sPfCSP2-Spier (30%) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. CONCLUSION: DAF-shielded BDES-vaccines offer great potential for development as a new malaria vaccine platform against the sporozoite challenge.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos CD55/genética , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Vacinação/métodos , Animais , Baculoviridae/genética , Baculoviridae/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Ratos , Esporozoítos/imunologia , Inativação de VírusRESUMO
BACKGROUND: The approach of using transgenic rodent malaria parasites to assess the immune system's response to antigenic targets from a human malaria parasite has been shown to be useful for preclinical evaluation of new vaccine formulations. The transgenic Plasmodium berghei parasite line [PvCSP(VK210)/Pb] generated previously expresses the full-length circumsporozoite protein (CSP) VK210 from Plasmodium vivax. The transgenic parasite expresses one of the two most common alleles of CSP, defined by nine amino acids at the central repeat region of this protein. In the present study, a transgenic P. berghei parasite line [PvCSP(VK247)/Pb] expressing the full-length PvCSP(VK247), which is the alternative common allele, was generated and characterized. METHODS: The P. berghei expressing full-length PvCSP(VK247) was generated and examined its applicability to CSP-based vaccine research by examining its biological characteristics in mosquitoes and mice. RESULTS: Similar to PvCSP(VK210)/Pb, PvCSP(VK247)/Pb developed normally in mosquitoes and produced infectious sporozoites equipped to generate patent infections in mice. Invasion of HepG2 cells by PvCSP(VK247)/Pb sporozoites was inhibited by an anti-PvCSP(VK247) repeat monoclonal antibody (mAb), but not by an anti-PvCSP(VK210) repeat mAb. CONCLUSIONS: These two transgenic parasites thus far can be used to evaluate the potential efficacy of PvCSP-based vaccine candidates encompassing the two major genetic variants in preclinical trials.
Assuntos
Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Organismos Geneticamente Modificados/imunologia , Plasmodium berghei/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Animais , Vacinas Antimaláricas/administração & dosagem , Camundongos , Organismos Geneticamente Modificados/genética , Plasmodium berghei/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Resultado do TratamentoRESUMO
A multistage malaria vaccine targeting the pre-erythrocytic and sexual stages of Plasmodium could effectively protect individuals against infection from mosquito bites and provide transmission-blocking (TB) activity against the sexual stages of the parasite, respectively. This strategy could help prevent malaria infections in individuals and, on a larger scale, prevent malaria transmission in communities of endemicity. Here, we describe the development of a multistage Plasmodium vivax vaccine which simultaneously expresses P. vivax circumsporozoite protein (PvCSP) and P25 (Pvs25) protein of this species as a fusion protein, thereby acting as a pre-erythrocytic vaccine and a TB vaccine, respectively. A new-concept vaccine platform based on the baculovirus dual-expression system (BDES) was evaluated. The BDES-Pvs25-PvCSP vaccine displayed correct folding of the Pvs25-PvCSP fusion protein on the viral envelope and was highly expressed upon transduction of mammalian cells in vitro. This vaccine induced high levels of antibodies to Pvs25 and PvCSP and elicited protective (43%) and TB (82%) efficacies against transgenic P. berghei parasites expressing the corresponding P. vivax antigens in mice. Our data indicate that our BDES, which functions as both a subunit and DNA vaccine, can offer a promising multistage vaccine capable of delivering a potent antimalarial pre-erythrocytic and TB response via a single immunization regimen.
Assuntos
Portadores de Fármacos , Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Plasmodium vivax/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Baculoviridae/genética , Transmissão de Doença Infecciosa/prevenção & controle , Feminino , Vetores Genéticos , Malária/transmissão , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium berghei/genética , Plasmodium berghei/imunologia , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
A photo-sensitive amino acid analogue was introduced into an outer membrane lipoprotein, Pal, and then subjected to photo-crosslinking with the lipoprotein-specific ABC transporter LolCDE. Pal crosslinked to LolE but not LolC in vivo despite that both are structurally similar membrane subunits. LolCDE liganded with Pal containing the photo-sensitive amino acid analogue was isolated and subjected to in vitro photo-crosslinking. LolE was found to be the binding site for Pal. ATP binding to LolD decreased the LolE-Pal crosslinking by decreasing their hydrophobic interaction. ATP hydrolysis in the presence of LolA completely abolished the LolE-Pal crosslinking and, concomitantly, generated a new LolA-Pal crosslinked product.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Benzofenonas/química , Western Blotting , Cromatografia de Afinidade , Códon de Terminação , Reagentes de Ligações Cruzadas/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/isolamento & purificação , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/química , Processos Fotoquímicos , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
During rice (Oryza sativa L.) seed development, the primary endosperm nucleus undergoes a series of divisions without cytokinesis, producing a multinucleate cell, known as a syncytium. After several rounds of rapid nuclear proliferation, the syncytium ceases to undergo mitosis; thereafter, the syncytium is partitioned into individual cells by a specific type of cytokinesis called cellularization. The transition between syncytium and cellularization is important in determining the final seed size and is a model for studying the cell cycle and cytokinesis. The involvement of cyclin-dependent kinase (CDK) inhibitors (CKIs) in cell cycle control was investigated here during the transition between syncytium and cellularization. It was found that one of the rice CKIs, Orysa;KRP3, is strongly expressed in the caryopsis at 2 d after flowering (DAF), and its expression is significantly reduced at 3 DAF. The other CKI transcripts did not show such a shift at 2 DAF. In situ hybridization analysis revealed that Orysa;KRP3 is expressed in multinucleate syncytial endosperm at 2 DAF, but not in cellularized endosperm at 3 DAF. Two-hybrid assays showed that Orysa;KRP3 binds Orysa;CDKA;1, Orysa;CDKA;2, Orysa;CycA1;1, and Orysa;CycD2;2. By contrast, Orysa;CDKB2;1 and Orysa;CycB2;2 do not show binding to Orysa;KRP3. Orysa;KRP3 was able to rescue yeast premature cell division due to the dominant positive expression of mutant rice CDKA;1 indicating that Orysa;KRP3 inhibited rice CDK. These data suggest that Orysa;KRP3 is involved in cell cycle control of syncytial endosperm.
