No abstract

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.

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. Plasmodium vivax is currently the most widely distributed human malaria parasite, with an "at risk" population in 2010 of almost 3 billion people (a third of the global population) and approximately 100 to 300 million clinical cases each year (1, 2). Several factors, including (i) the recent appearance of chloroquine-resistant P. vivax, (ii) the lack of alternatives to primaquine for attacking the dormant liver-stage hypnozoites, and (iii) increasing global temperatures caused by climate change, raise concerns about increases in the risk of severe P. vivax disease (3-6). Although the importance of P. vivax vaccines is recognized, the lack of long-term in vitro culture systems in red blood cells and suitable animal models as well as the complex life cycle of this parasite has hindered advances in the development of a potent vaccine (7,8).The development of malaria vaccines has been focused mostly on single antigens from different stages of the parasite life cycle: (i) the pre-erythrocytic stages (including the liver stages), (ii) the asexual blood stages, and (iii) the mosquito sexual stages, where antigens expressed on the gametocyte, gamete, zygote, or ookinete are targeted to prevent transmission from the human hosts to the mosquito vectors (9). There are concerns that the single-stage vaccine may not be effective because of sequence variability among different parasite isolates, host genetic restriction of immune responses to specific epitopes, and short-lived protective immunity induced by some single-antigen vaccines (10). Therefore, a multistage vaccine, which targets several antigens exp...

Anti-malarial transmission-blocking vaccines (TBVs) aim to inhibit the transmission of Plasmodium from humans to mosquitoes by targeting the sexual/ookinete stages of the parasite. Successful use of such interventions will subsequently result in reduced cases of malarial infection within a human population, leading to local elimination. There are currently only five lead TBV candidates under examination. There is a consequent need to identify novel antigens to allow the formulation of new potent TBVs. Here we describe the design and evaluation of a potential TBV (BDES-PbPSOP12) targeting Plasmodium berghei PSOP12 based on the baculovirus dual expression system (BDES), enabling expression of antigens on the surface of viral particles and within infected mammalian cells. In silico studies have previously suggested that PSOP12 (Putative Secreted Ookinete Protein 12) is expressed within the sexual stages of the parasite (gametocytes, gametes and ookinetes), and is a member of the previously characterized 6-Cys family of plasmodial proteins. We demonstrate that PSOP12 is expressed within the sexual/ookinete forms of the parasite, and that sera obtained from mice immunized with BDES-PbPSOP12 can recognize the surface of the male and female gametes, and the ookinete stages of the parasite. Immunization of mice with BDES-PbPSOP12 confers modest but significant transmission-blocking activity in vivo by active immunization (53.1% reduction in oocyst intensity, 10.9% reduction in oocyst prevalence). Further assessment of transmission-blocking potency ex vivo shows a dose-dependent response, with up to a 76.4% reduction in intensity and a 47.2% reduction in prevalence observed. Our data indicates that PSOP12 in Plasmodium spp. could be a potential new TBV target candidate, and that further experimentation to examine the protein within human malaria parasites would be logical.