A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus

Background

Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the ‘bush-meat’ trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.

Methodology/Principal Findings

We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a ‘proof-of-concept’ for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8⁺ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP_CTL). MCMV/ZEBOV-NP_CTL induced high levels of long-lasting (>8 months) CD8⁺ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection.

Conclusions/Significance

This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for ‘disseminating’ CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.     

Miliolipora species (Foraminifera,Miliolina) from the Rhaetian Dachstein Limestone of Karavanke Mts (Slovenia): Palaeoecological and palaeobiogeographic implications

The Carnian-Rhaetian genus Miliolipora (Soritoidea, Milioliporidae) is characterised by a coarsely perforated porcelaneous wall and a quinqueloculinoid arrangement of semi-tubular chambers. Miliolipora tamarae nov. sp. has been documented in the Rhaetian Dachstein reef limestone of Mt. Begunj??ica (Karavanke Mts., northern Slovenia). This new species differs from Miliolipora cuvillieri Brönnimann and Zaninetti in its costate outer chambers and evolute coiling. Both species are abundant in an oncoid rudstone/grapestone facies located immediately behind the central reef zone. In this depositional context, Miliolipora spp. were subjected to mechanical sorting and are believed to have been transported a short distance from their habitat. The costae and the overall less rounded shape of the test helped to stabilize M. tamarae nov. sp. on the sea floor. Both species were widely spread in the Tethyan realm confirming the broad palaeobiogeographic distribution of the Late Triassic foraminifera.     

High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions

Celiac disease is an immune-mediated disorder in which mucosal autoantibodies to the enzyme transglutaminase 2 (TG2) are generated in response to the exogenous antigen gluten in individuals who express human leukocyte antigen HLA-DQ2 or HLA-DQ8 (ref. 3). We assessed in a comprehensive and nonbiased manner the IgA anti-TG2 response by expression cloning of the antibody repertoire of ex vivo-isolated intestinal antibody-secreting cells (ASCs). We found that TG2-specific plasma cells are markedly expanded within the duodenal mucosa in individuals with active celiac disease. TG2-specific antibodies were of high affinity yet showed little adaptation by somatic mutations. Unlike infection-induced peripheral blood plasmablasts, the TG2-specific ASCs had not recently proliferated and were not short-lived ex vivo. Altogether, these observations demonstrate that there is a germline repertoire with high affinity for TG2 that may favor massive generation of autoreactive B cells. TG2-specific antibodies did not block enzymatic activity and served as substrates for TG2-mediated crosslinking when expressed as IgD or IgM but not as IgA1 or IgG1. This could result in preferential recruitment of plasma cells from naive IgD- and IgM-expressing B cells, thus possibly explaining why the antibody response to TG2 bears signs of a primary immune response despite the disease chronicity.     

New approach in studies of microalgal cell lysis

A new approach to the studies of the microalgal cell lysis by utilizing a combination of two complementary methods is presented. Delayed fluorescence (DF) is a measure of the living algal biomass, detecting only cells with active photosynthesis. Thermal lens spectrometry (TLS) detects the total pigment amount released from lysed cells. Both methods select for photosynthetic organisms, reducing possible background from other sources (e.g. heterotrophic bacteria, zooplankton, and abiotic substances). The DF/TLS method was tested with a laboratory Skeletonema costatum culture exposed to a geometric dilution series of the lysing factor poly- APS. The exposure resulted in similar EC₅0 values for DF intensity, TLS and dissolved esterase activity of 0.8±0.2, 1.77±0.35, and 1.25±0.1 mg poly-APS l⁻¹, respectively. The combined DF/TLS method enabled a rapid evaluation of the living vs. dead cells without any sample pretreatment or manipulation.     

Efficacy of healthy weight loss program in obesity treatment: Croatian experience

We evaluated the efficiency of a six-month outpatient weight loss treatment program combining healthy diet, fat reduction, psychological counseling, exercise, and orlistat treatment, by measuring body weight and levels of cardiovascular risk factors in 476 subjects with BMI over 30 or 28 with increased blood pressure, cholesterol, and sugar at the baseline and at the end of program. After four weeks of adjustment to a mild low-calorie diet (1600 kcal/day) and counseling, subjects started receiving orlistat (120 mg TID). The mean weight loss after 6 months was 10.9%. Systolic pressure dropped by 6.7%, diastolic by 4.2%, fasting blood glucose by 10.1%, and total cholesterol by 9.8%. Only 9 subjects (7.8%) poorly tolerated the treatment. More men than women were able to maintain the achieved weight loss six months after the program (70.6% vs. 58.3%, respectively). The healthy weight loss program was an efficient approach to obesity treatment.     

Expression and purification of glycine N-methyltransferases in Escherichia coli

Expression and purification of recombinant mouse, rat, and human glycine N-methyltransferases (GNMTs) in pTYB and pET expression vectors was done in order to prepare the proteins for structure studies of the enzymes from different sources. When human and mouse GNMTs were expressed in pTYB vector as a fusion protein with intein and the chitin binding domain, an unusual cleavage of intein was found. This cleavage takes place at two sites near the N-terminus of intein. This resulted in the appearance of an abnormal GNMT protein after on-column cleavage of the fusion protein, which could not be separated from normal GNMT. For this reason expression of mouse, rat, and human GNMTs was done in the pET-17b expression vector, resulting in the expression of soluble protein at about 20-40mg/L of culture. A new procedure for GNMT isolation after expression in the pET vector was developed. This included only two steps, ammonium sulfate precipitation and ion-exchange chromatography, and resulted in preparations containing 95-97% pure protein. All expressed proteins were tetrameric with molecular weights of 130kDa as determined by size-exclusion chromatography. Activity in Tris buffer at pH 9 of mouse, rat, and human GNMTs was found to be 255, 260, and 540U/mg, respectively. This implies that expressed and purified GNMT proteins are biologically active and suitable for biochemical and structural studies.     

A bacterial selection for the directed evolution of pyruvate aldolases

A novel bacterial in vivo selection for pyruvate aldolase activity is described. Pyruvate kinase deficient cells, which lack the ability to biosynthetically generate pyruvate, require supplementation of exogenous pyruvate when grown on ribose. Supplementation with pyruvate concentrations as low as 50 microM rescues cell growth. A known substrate of the KDPG aldolases, 2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (KHPB), also rescues cell growth, consistent with retroaldol cleavage by KDPG aldolase and rescue through pyruvate release. An initial round of selection against 2-keto-4-hydroxyoctonate (KHO), a nonsubstrate for wild-type aldolase, produced three mutants with intriguing alterations in protein sequence. This selection system allows rapid screening of mutant enzyme libraries and facilitates the discovery of enzymes with novel substrate specificities.     

5-methyltetrahydrofolate is bound in intersubunit areas of rat liver folate-binding protein glycine N-methyltransferase

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr5, Leu207, and Met215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.