Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Plasmablasts generated during repeated dengue infection are virus glycoprotein-specific and bind to multiple virus serotypes.

Dengue virus immune protection is specific to the serotype encountered and is thought to persist throughout one's lifetime. Many serotype cross-reactive memory B cells isolated from humans with previous dengue infection are specific for the nonstructural and the prM structural viral proteins, and they can enhance infection in vitro. However, plasmablasts circulating in enormous numbers during acute secondary infection have not been studied. In this study, we analyzed single plasmablasts from two patients by sorting the cells for Ig sequence analysis and for recombinant expression of Abs. In contrast to memory B cells, most plasmablast-derived Abs bound to the structural E protein of dengue, and protection experiments in mice revealed that virus serotypes encountered during past infections were neutralized more efficiently than were the serotypes of the current infection. Together with genetic analyses, we show evidence that plasmablasts in dengue patients are a polyclonal pool of activated E protein-specific memory B cells and that their specificity is not representative of the serum Abs secreted by long-lived plasma cells in the memory phase. These results contribute to the understanding of the phenomenon of original antigenic sin in dengue.

Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK.

Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here, we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules, we have identified SB203580, a specific p38 MAP kinase inhibitor, as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations <10 microM, induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers, so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly, transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly, cardiomyogenesis was strongly inhibited at SB203580 concentrations > or =15 microM. Thus, modulation of the p38MAP kinase pathway, in combination with factors released by END2 cells, plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process.

Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells.

Many applications of human embryonic stem cells (hESCs) will require fully defined growth and differentiation conditions including media devoid of fetal calf serum. To identify factors that control lineage differentiation we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-1, which also suppressed cardiomyocyte differentiation, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-1 effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC differentiation. Relatively high levels of the neural ectoderm marker Sox1 were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6-10-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of >10% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.

Plasmablasts During Acute Dengue Infection Represent a Small Subset of a Broader Virus-specific Memory B Cell Pool.

Dengue is endemic in tropical countries worldwide and the four dengue virus serotypes often co-circulate. Infection with one serotype results in high titers of cross-reactive antibodies produced by plasmablasts, protecting temporarily against all serotypes, but impairing protective immunity in subsequent infections. To understand the development of these plasmablasts, we analyzed virus-specific B cell properties in patients during acute disease and at convalescence. Plasmablasts were unrelated to classical memory cells expanding in the blood during early recovery. We propose that only a small subset of memory B cells is activated as plasmablasts during repeat infection and that plasmablast responses are not representative of the memory B cell repertoire after dengue infection.

Dengue serotype cross-reactive, anti-e protein antibodies confound specific immune memory for 1 year after infection.

Dengue virus has four serotypes and is endemic globally in tropical countries. Neither a specific treatment nor an approved vaccine is available, and correlates of protection are not established. The standard neutralization assay cannot differentiate between serotype-specific and serotype cross-reactive antibodies in patients early after infection, leading to an overestimation of the long-term serotype-specific protection of an antibody response. It is known that the cross-reactive response in patients is temporary but few studies have assessed kinetics and potential changes in serum antibody specificity over time. To better define the specificity of polyclonal antibodies during disease and after recovery, longitudinal samples from patients with primary or secondary DENV-2 infection were collected over a period of 1 year. We found that serotype cross-reactive antibodies peaked 3 weeks after infection and subsided within 1 year. Since secondary patients rapidly produced antibodies specific for the virus envelope (E) protein, an E-specific ELISA was superior compared to a virus particle-specific ELISA to identify patients with secondary infections. Dengue infection triggered a massive activation and mobilization of both naïve and memory B cells possibly from lymphoid organs into the blood, providing an explanation for the surge of circulating plasmablasts and the increase in cross-reactive E protein-specific antibodies.

Susceptibility and response of human blood monocyte subsets to primary dengue virus infection.

Human blood monocytes play a central role in dengue infections and form the majority of virus infected cells in the blood. Human blood monocytes are heterogeneous and divided into CD16(-) and CD16(+) subsets. Monocyte subsets play distinct roles during disease, but it is not currently known if monocyte subsets differentially contribute to dengue protection and pathogenesis. Here, we compared the susceptibility and response of the human CD16(-) and CD16(+) blood monocyte subsets to primary dengue virus in vitro. We found that both monocyte subsets were equally susceptible to dengue virus (DENV2 NGC), and capable of supporting the initial production of new infective virus particles. Both monocyte subsets produced anti-viral factors, including IFN-α, CXCL10 and TRAIL. However, CD16(+) monocytes were the major producers of inflammatory cytokines and chemokines in response to dengue virus, including IL-1ß, TNF-α, IL-6, CCL2, 3 and 4. The susceptibility of both monocyte subsets to infection was increased after IL-4 treatment, but this increase was more profound for the CD16(+) monocyte subset, particularly at early time points after virus exposure. These findings reveal the differential role that monocyte subsets might play during dengue disease.

Dengue virus activates polyreactive, natural IgG B cells after primary and secondary infection.

BACKGROUND: Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection. METHODOLOGY/PRINCIPAL FINDINGS: We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4-7 days after fever onset was more than 50% even after primary infection. CONCLUSIONS/SIGNIFICANCE: Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and "innate specificities" seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development.

Up-scaling single cell-inoculated suspension culture of human embryonic stem cells.

We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only approximately 44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to approximately 60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (>86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines.

Cardiomyocyte enrichment from human embryonic stem cell cultures by selection of ALCAM surface expression.

AIMS: The production of a homogenous population of human cardiomyocytes that can be expanded in vitro may facilitate development of replacement tissue lost as a result of cardiac disease and injury. MATERIALS AND METHODS: We evaluated the utility of activated leukocyte cell-adhesion molecule, CD166 (ALCAM) expression as a marker for isolating cardiomyocytes from differentiating cultures of human embryonic stem cells (hESCs). Using RT-qPCR, immunohistochemistry and DNA methylation studies, we evaluated the developmental age of hESC-derived cardiomyocytes. RESULTS AND CONCLUSIONS: We demonstrate that cardiomyocytes derived from hESC cultures express ALCAM and that this surface antigen can be used to select a population of differentiated cells that are enriched for cardiomyocytes. Expression of contractile proteins and ion channels, and DNA methylation patterns, suggest that ALCAM-enriched cardiomyocytes have an embryonic phenotype. Selected cardiomyocyte populations survive sorting, adhere to collagen-coated tissue culture plastic and proliferate in short-term culture. Long-term in vitro survival of cardiomyocytes was achieved by culturing cells in 3D aggregates.