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In the present study, IL-9 was initially identified as a strong regulator of blood glucose in diabetic db/db mice through a hydrodynamic gene delivery screening effort. IL-9 belongs to the common gamma-chain binding cytokine family and has classically been linked to asthma and type 2 immune responses. Alongside the very clear dosing effects of mouse IL-9 on fasting blood glucose, the more classical effects of IL-9, including IL-5 plasma increases and eosinophilia, were also observed in the db/db mice. An expansion of epididymal white adipose tissue macrophages was shown with IL-9-, but not IL-5 dosing. Inhibition of endogenous IL-5, with an anti-IL5 mAb, attenuated the IL-9 effects on eosinophils but not macrophages. Interestingly, suppression of glucagon levels following IL-9 dosing was a general observation in several diabetic mouse studies. The addition of anti-IL-5 mAb to IL-9 dosing did not affect glucagon levels in the db/db mouse model, and an association between eosinophil/macrophage changes and blood glucose could not be established. To examine the translation of these interesting IL-9 effects to higher species, a recombinant protracted human IL-9 dosed to low streptozotocin induced diabetic mini-pigs (2-week study) , showed increased numbers of circulating eosinophils, but no significantly reduced levels of glucagon or plasma glucose. Finally, an 11-week treatment study in type 2 diabetic cynomolgus monkeys was performed. Eosinophilia was observed as an indicator of IL-9 receptor engagement, but several glycaemic parameters and glucagon levels were unaffected throughout the study. Conclusion: The pronounced glycaemic effects observed for IL-9 in a diabetic mouse model was not found in diabetic minipigs and non-human primates highlighting the complexity and need for further studies of the translation of immuno-metabolic phenotypes. Disclosure T.Guo: Employee; Novo Nordisk A/S. K.S.Frederiksen: Employee; Novo Nordisk A/S. E.Straarup: Employee; Novo Nordisk A/S. J.Lindroos christensen: Employee; Novo Nordisk A/S. G.Bolt: Employee; Novo Nordisk A/S. P.Lisby nørby: Employee; Novo Nordisk A/S. W.Zhe: Employee; Novo Nordisk A/S. T.A.Pedersen: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. M.Ferreras: Employee; Novo Nordisk A/S. K.Sass-ørum: Employee; Novo Nordisk A/S.

Essentials Factor (F)VIII with an intermediate-length B-domain showed higher levels in murine gene therapy. FVIII with different B-domain lengths were analysed. FVIII variants with B-domains between 186 and 240 amino acids (aa) have extended half-life in mice. Reduced cell binding of FVIII with a 237aa B-domain may explain the extended half-life. SUMMARY: Background Factor VIII consists of the A1-domain, A2-domain, B-domain, A3-domain, C1-domain, and C2-domain. FVIII with an intermediate-length B-domain of 226 amino acids (aa) has previously been evaluated in murine gene therapy studies. Objective To characterize FVIII with intermediate-length B-domains in vitro and in vivo in F8-knockout (KO) mice. Methods and results FVIII molecules with B-domains of 186-240aa had longer half-lives in F8-KO mice than FVIII molecules with shorter or longer B-domains. FVIII with a B-domain containing the 225 N-terminal aa fused to the 12 C-terminal aa of the wild-type B-domain (FVIII-237) had a 1.6-fold extended half-life in F8-KO mice as compared with FVIII with a 21aa B-domain (FVIII-21). The in vitro and in vivo activity of FVIII-237 were comparable to those of FVIII-21, as was binding to von Willebrand factor. Cell binding to LDL receptor-related protein 1 (LRP-1)-expressing cells was markedly reduced for FVIII-237 as compared with FVIII-21, whereas the affinity for LRP-1 was not reduced in surface plasmon resonance (SPR) studies. FVIII-21 cell binding and internalization could be inhibited by a fragment consisting of the 226 N-terminal aa of the FVIII B-domain, and SPR analysis suggested that this B-domain fragment might bind with weak affinity to FVIII-21. Conclusion Reduced cell binding of FVIII-237 might explain the observed extended half-life in F8-KO mice. This may contribute to the increased FVIII levels measured in murine gene therapy studies using FVIII constructs with similar B-domain lengths.

UNLABELLED Essentials Von Willebrand factor (VWF) stabilizes factor VIII (FVIII) and prevents its premature clearance. Rat anatomical and hepatocellular distribution studies assessed the VWF effect on FVIII clearance. Hepatocytes and liver sinusoidal endothelial cells play a key role in FVIII clearance. Anatomical and hepatocellular distribution of FVIII is independent of high-affinity VWF binding. ABSTRACT Background Von Willebrand factor (VWF) stabilizes factor VIII in the circulation and prevents its premature clearance. Objective To study the effects of VWF on FVIII clearance in rats with endogenous VWF. Methods Anatomical and hepatocellular distribution studies were performed in rats following intravenous administration of glycoiodinated recombinant FVIII (rFVIII) and a FVIII variant, FVIII-Y1680F, lacking high-affinity VWF binding. Radioactivity was quantified in organs, and in distinct liver cell populations. The role of VWF binding was also studied by immunohistochemical staining of rat livers perfused ex vivo with rFVIII alone or with a FVIII-binding VWF fragment. Results The liver was the predominant organ of rFVIII distribution, and a radioactivity peak was also observed in the intestines, suggesting FVIII secretion to the bile by hepatocytes. In the liver, ~60% of recovered radioactivity was associated with hepatocytes, 32% with liver sinusoidal endothelial cells (LSECs), and 9% with Kupffer cells (KCs). When calculated per cell, 1.5-fold to 3-fold more radioactivity was associated with LSECs than with hepatocytes. The importance of hepatocytes and LSECs was confirmed by immunohistochemical staining; strong staining was seen in LSECs, and less intense, punctate staining in hepatocytes. Minor staining in KCs was observed. Comparable anatomical and hepatocellular distributions were observed with rFVIII and FVIII-Y1680F, and the presence of the VWF fragment, D'D3A1, did not change the FVIII staining pattern in intact livers. Conclusions The present data support FVIII clearance via the liver, with hepatocytes and LSECs playing a key role. High-affinity VWF binding did not alter the anatomical or hepatocellular distribution of FVIII.

Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.

Recombinant factor VIIa (rFVIIa, NovoSeven) is currently the only bypassing agent produced by recombinant technology for the treatment of haemophiliacs whose disease is complicated by inhibitory antibodies. In addition, recombinant production of FVIIa has made it widely available for a variety of purposes and accelerated the growth of our knowledge about FVIIa by generating an abundance of clinical and biochemical data. This fascinating molecule has turned out to be a safe haemostatic agent with great potential in the clinic and has inspired the generation of improved variants currently in (pre-)clinical testing. The present review describes the structural and functional aspects of FVIIa, followed by sections dealing with the manufacturing, therapeutic mechanism of action, clinical development and experience with rFVIIa.

SUMMARY A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg(-1). Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.

The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+) binding to a loop (residues 210-220) in the protease domain. Structural analysis revealed that Ca(2+) may enhance the activity by attenuating electrostatic repulsion of Glu(296) and/or by facilitating interactions between the loop and Lys(161) in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca(2+)-binding loop appears to suffice for some residual interaction with Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to abrogation of the interaction with the N-terminal tail. However, Ca(2+) attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca(2+)-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca(2+)-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization.

SummaryCoagulation factorVII (FVII) is a vitamin K-dependent glycoprotein that undergoes extensive post-translational modification prior to secretion. Secretion of FVII proteins from producer cells is a slow process. To identify bottlenecks for the transport of FVII through the secretory pathway of FVII-producing cells, we analysed the processing of intracellular FVII by pulse-chase of FVII producing CHO cells followed by radioimmuno precipitation, SDS-PAGE, and autoradiography. FVII was coprecipitated with GRP78 and vice versa for at least three hours after synthesis of the labelled FVII, suggesting that nascent FVII is retained in the endoplasmic reticulum (ER). Judged from barium citrate precipitation assay, gamma-carboxylation of the pulse-labelled FVII was a slow process requiring several hours and seemed to be the most important bottleneck in the intracellular processing of FVII. Nevertheless, FVII was not released from the cells immediately after gamma-carboxylation. Gamma-carboxylated FVII accumulated in the cells and migrated as a band with reduced mobility compared to uncarboxylated FVII. This shift in migration was caused by N-glycan processing in the Golgi complex. Thus, the release of FVII from producer cells is delayed by at least two bottlenecks. The major bottleneck appears to be gamma-carboxylation, which determines the rate of transport of FVII out of the ER. Another bottleneck retains FVII in the cells after processing of the N-glycans into complex chains. Cells with an intact gamma-carboxylation machinery appear to posses mechanisms that protect nascent FVII from intracellular degradation and keep FVII in the ER until it is gamma-carboxylated.

We examined the consequences of isolation and adaptation to Vero cells for the receptorbinding haemagglutinin (H) gene of four syncytia-forming isolates of canine distemper virus (CDV) and of a dolphin morbillivirus isolate. A Vero-adapted CDV isolate exhibited biased hypermutation, since 11 out of 12 nucleotide differences to other isolates from the same epidemic were U-C transitions. Most of these transitions appeared to have taken place during in vitro cultivation. Previously, biased hypermutation in morbilliviruses has almost exclusively been described for subacute sclerosing panencephalitis and measles inclusion body encephalitis, which are rare measles virus brain infections. Amino acid changes in the H proteins were not required for Vero cell adaptation, suggesting that Vero cells express receptors for wild-type morbilliviruses. This strongly indicate the existence of other morbillivirus receptors than CD46 and CDw150.

Coagulation factor VIII (FVIII) is one of the most complex biopharmaceuticals due to the large size, poor protein stability and extensive post-translational modifications. As a consequence, efficient production of FVIII in mammalian cells poses a major challenge, with typical yields two to three orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII productivities was selected for RNA sequencing analysis. The analysis showed distinct differences in F8 RNA composition between the clones. The exogenous F8-dhfr transcript was found to make up the most abundant transcript in the present clones. No correlation was seen between F8 mRNA levels and the measured FVIII productivity. It was found that three MTX resistant, nonproducing clones had different truncations of the F8 transcripts. We find that by using deep sequencing, in contrast to microarray technology, for determining the transcriptome from CHO transfectants, we are able to accurately deduce the mature mRNA composition of the transgene and identify significant truncations that would probably otherwise have remained undetected.

Vaccine strains of measles virus (MV) use CD46 as receptor and downregulate CD46 from the surface of infected cells. MVs isolated and passaged on B-lymphoid cells (wild-type MVs) seem to use another receptor and do not downregulate CD46. In the present study, we found that isolation of MV on human or marmoset B-lymphoid cells did not alter the MV haemagglutinin (H) protein relative to that in the patient. The wild-type isolates were adapted to the human epithelial HEp-2 cell line or the monkey fibroblast Vero cell line. All HEp-2 cell adapted viruses and 1 out of 4 Vero cell adapted viruses acquired the capacity to use CD46 as receptor, as measured by their ability to infect murine cells expressing human CD46. Adaptation to CD46 receptor usage was coupled to substitution of amino acid 481 of the MV H protein from asparagine to tyrosine but not to CD46 downregulation. The present study demonstrates that CD46 receptor usage can be induced by adaptation of wild-type MV to cells that do not express a wild-type receptor and suggests that a similar mechanism acted on the progenitor viruses of the present MV vaccine strains during their isolation and attenuation.

The fusion (F) glycoprotein of respiratory syncytial virus (RSV) is synthesized as a nonfusogenic precursor protein (F(0)), which during its migration to the cell surface is activated by cleavage into the disulfide-linked F(1) and F(2) subunits. In the present study, soluble secreted human furin produced by a recombinant baculovirus cleaved RSV F(0) into proteins the size of F(1) and F(2). Furthermore, cleavage of F(0) was partially inhibited in the furin defective LoVo cell line, in calcium depleted HEp-2 cells, and in HEp-2 cells treated with the furin inhibitor decanoyl-R-V-K-R-chloromethylketon. These findings strongly suggest an important role for furin in activation of the RSV F protein. The F(0) protein could not be detected on the surface of cells, in which F protein activation was inhibited, and RSV particles did not appear to be released from these cells. It thus seems that in contrast to the F proteins of most other paramyxoviruses, the RSV F(0) protein is very inefficient in reaching the cell surface or is unable to reach the cell surface and therefore cannot be incorporated into virus particles.

To characterize the variability of recent field isolates of canine distemper virus (CDV) from different hosts and geographical areas, we conducted nucleotide sequence analysis of the gene encoding the haemagglutinin (H), the attachment protein of this virus. Pronounced differences between field isolates were revealed in comparison to the Convac and Onderstepoort vaccine strains. The diversity of CDV appeared to exceed that determined for measles virus. Phylogenetic analysis also separated the field isolates of CDV from the vaccine strains and provided evidence for the existence of different contemporary genotypes of CDV. Isolates from a Greenlandic sledge dog and a Siberian seal formed a distinct lineage. The remaining isolates formed a group. This group contained two European isolates from mink and ferret, a single lineage comprising three European dog isolates, and another separate lineage of North American isolates from dog, javelina, raccoon and captive leopards.

DMV, dolphin morbillivirus, a paramyxovirus of uncertain origin recently emerged in Mediterranean dolphins. This study presents the complete nucleotide sequence of the hemagglutinin (H) gene including the gene boundaries. The single open reading frame of the DMV H gene encodes a protein of 604 residues which exhibits overall sequence characteristics similar to the H genes of other morbilliviruses. When compared to its closest homologues, measles virus (MV) and rinderpest virus (RPV), DMV has, respectively, 44 and 46% of amino acid residues in identical positions. The primary sequence of the DMV H protein is markedly less conserved than that of the fusion protein. The comparative data at the genomic level correspond with cross-neutralization studies with different morbilliviruses. Retrospective serogical studies dating back to 1983 indicate DMV-like infections in whales of the eastern Atlantic. The presented data support and extend previous studies suggesting that this novel morbillivirus is one of the phylogenetically oldest morbilliviruses known to circulate today. The relationship of DMV and established morbilliviruses to the newly emerged candidate morbillivirus infecting horse and man is discussed.

Mutations abolishing or substantially reducing coagulation factor IX (FIX) function lead to hemophilia B, a hereditary bleeding disorder that can be managed by replacement therapy using plasma-derived or recombinant FIX. For prophylaxis, 2weekly infusions are traditionally recommended. Thus, a modified FIX protein with prolonged circulatory half-life allowing less frequent infusions may improve patient convenience and compliance. Several strategies for reducing clearance of FIX have been described. Recombinant FIX compounds prolonged by glycoPEGylation [1], genetic fusion to albumin [2] or genetic fusion to the IgG Fc domain [3] are currently in clinical development. In the present study, we investigated the possibility of prolonging FIX by introducing new N-glycans. Previous studies suggest a possible role for N-glycans in determining the clearance of FIX. Enzymatic removal of N-glycans increased clearance of wild-type recombinant FIX [4,5], and a correlation between N-glycan sialic acid content and terminal half-life of recombinant FIX has been reported [6]. N-glycosylation is anintracellular processcarriedoutbythe oligosaccharyl transferase complex, which glycosylates asparagine residues in N-X-S/T motifs of nascent proteins. Thus, N-glycans can be added to a recombinant protein by introducing mutations that establish N-glycosylation motifs in the amino acid chain. This strategy has previously proven efficient in prolonging the circulatory half-lives of erythropoietin, follicle stimulating hormone, interferon alpha and growth hormone [7].

Morbilliviruses have been isolated from stranded dolphins and porpoises. The present paper describes the cloning and sequencing of the porpoise morbillivirus (PMV) F gene and of the dolphin morbillivirus (DMV) M and F genes and their flanking regions. The gene order of the DMV genome appeared to be identical to that of other morbilliviruses, A genomic untranslated region of 837 nucleotides was found between the translated DMV M and F gene regions. The predicted DMV M protein were highly conserved with those of other morbilliviruses. Both the deduced PMV and DMV F 0 proteins exhibited three major hydrophobic regions as well as a cysteine rich region, a leucine zipper motif and a cleavage motif allowing cleavage of the F 0 protein into F 1 and F 2 subunits. Apparently the DMV F 0 cleavage motif was not modified by adaptation of DMV to Vero cells. The predicted PMV and DMV F proteins were 94% identical. Comparisons with the corresponding sequences of other morbilliviruses demonstrated that the cetacean morbillivirus does not derive from any known morbillivirus but represents an independent morbillivirus lineage.

In 1990 an epidemic caused by a morbillivirus was noticed among Mediterranean dolphins. RNA was extracted from the tissues of dolphins and from cell cultures infected with a corresponding dolphin morbillivirus isolate. By nucleic acid hybridization this RNA was compared to RNA extracted from animal tissue or cell cultures infected with canine distemper virus (CDV), phocine distemper virus (PDV) or measles virus (MV). The presence of morbillivirus RNA in the dolphin tissue was demonstrated. Morbillivirus N, P, M and F gene mRNAs were detected in the RNA from dolphin morbillivirus infected cells. These mRNA species seemed to be of approximately the same size as the corresponding mRNA species of CDV, PDV and MV. The results of the comparison demonstrated that the dolphin morbillivirus is genetically different from CDV, PDV and MV. No indication of a close relationship between the dolphin isolate and either CDV, PDV or MV was found.

Therapeutic use of recombinant GH typically involves daily sc injections. We examined the possibilities for prolonging the in vivo circulation of GH by introducing N-glycans. Human GH variants with a single potential N-glycosylation site (N-X-S/T) introduced by site-directed mutagenesis were expressed in HEK293 cells. In a scan of 15 different positions for N-glycosylation sites, four positions (amino acids 93, 98, 99, and 101) were efficiently utilized and did not influence GH in vitro activity. A GH variant (3N-GH) with all these sites was produced in CHOK1SV cells and contained up to three N-glycans. Two pools of 3N-GH were purified and separated according to their charge by anion-exchange chromatography. Anion-exchange HPLC revealed that the N-glycans in the two pools were very similar except for the extent of sialylation. Both 3N-GH pools circulated longer in rats than wild-type GH. The terminal half-life of 3N-GH after iv injection was 24-fold prolonged compared with wild-type GH for the pool with the most pronounced sialylation, 13-fold prolonged for the less sialylated pool, and similar to the wild-type for desialylated 3N-GH. The less sialylated 3N-GH pool exhibited a profound pharmacodynamic effect in GH-deficient rats. Over a 4-d period, a single injection of 3N-GH induced a stronger IGF-I response and a larger increase in body weight than daily injections with wild-type GH. Thus, N-glycans can prolong the in vivo circulation and enhance the pharmacodynamic effect of GH. Sialic acids seem to play a pivotal role for the properties of glycosylated GH.

SummaryHuman coagulation factor VII (FVII) has two N-glycosylation sites (N145 and N322) and two O-glycosylation sites (S52 and S60). In transiently transfected COS-7 cells, all combinations of N- and O-glycosylation knock-out mutations reduced the release of FVII to the medium. Pulse-chase analysis of CHO-K1 cell lines expressing recombinant FVII demonstrated that virtually all wild-type FVII synthesized was secreted from the cells, whereas both N- and O- glycosylation knock-out mutations induced partial intracellular degradation of the synthesized FVII. Likewise, two thirds of the FVII synthesized in vitamin K-depleted and warfarin-treated CHO cells was degraded intracellularly, demonstrating the importance of gamma-carboxylation for the secretion of FVII. The furin inhibitor decanoyl-R-V-K-R-chloromethylketone inhibited propeptide cleavage, but FVII with propeptide appeared to be secreted equally well as FVII without propeptide. Propeptide cleavage was not inhibited by vitamin K depletion and warfarin treatment, suggesting that for FVII, correct gamma-carboxylation is not required for optimal processing of the propeptide. In conclusion, all post-translational modifications of FVII except propeptide cleavage were important for complete secretion of the synthesized FVII and to avoid intracellular degradation. Thus, the extensive post-translational modification of FVII seems critical for the intracellular stability of the protein and is required for keeping the protein in the secretory pathway.

We examined the consequences of isolation and adaptation to Vero cells for the receptorbinding haemagglutinin (H) gene of four syncytia-forming isolates of canine distemper virus (CDV) and of a dolphin morbillivirus isolate. A Vero-adapted CDV isolate exhibited biased hypermutation, since 11 out of 12 nucleotide differences to other isolates from the same epidemic were U-C transitions. Most of these transitions appeared to have taken place during in vitro cultivation. Previously, biased hypermutation in morbilliviruses has almost exclusively been described for subacute sclerosing panencephalitis and measles inclusion body encephalitis, which are rare measles virus brain infections. Amino acid changes in the H proteins were not required for Vero cell adaptation, suggesting that Vero cells express receptors for wild-type morbilliviruses. This strongly indicate the existence of other morbillivirus receptors than CD46 and CDw150.

The influence of measles virus (MV) infection on gene expression by human peripheral blood mononuclear cells (PBMCs) was examined with cDNA microarrays. The mRNA levels of more than 3000 cellular genes were compared between uninfected PBMCs and cells infected with either the Edmonston MV strain or a wild-type MV isolate. The MV-induced upregulation of individual genes identified by microarray analyses was confirmed by RT–PCR. In the present study, a total of 17 genes was found to be upregulated by MV infection. The Edmonston strain grew better in the PBMC cultures than the wild-type MV, and the Edmonston strain was a stronger inducer of the upregulated host cell genes than the wild-type virus. The anti-apoptotic B cell lymphoma 3 (Bcl-3) protein and the transcription factor NF-κB p52 subunit were upregulated in infected PBMCs both at the mRNA and at the protein level. Several genes of the interferon system including that for interferon regulatory factor 7 were upregulated by MV. The genes for a number of chaperones, transcription factors and other proteins of the endoplasmic reticulum stress response were also upregulated. These included the gene for the pro-apoptotic and growth arrest-inducing CHOP/GADD153 protein. Thus, the present study demonstrated the activation by MV of cellular mechanisms and pathways that may play a role in the pathogenesis of measles.

During the last decade, various zinc salts have been used against the common cold syndrome, which is known to be initiated by respiratory viruses, particularly rhinoviruses. Using rhinovirus as the challenge virus, we investigated whether zinc salts (Zn) could potentiate the antiviral action of native human leukocyte interferon (HuIFN-alpha) and rHuIFN-gamma. We found that HuIFN-alpha was potentiated tenfold at rather low levels of IFN activity (0.6-0.8 U/ml), resulting in 100% protection. Zn alone gave only marginal protection, if any. In contrast to HuIFN-alpha, rHuIFN-gamma directly increased the cytopathic effect of rhinovirus at low levels (2 U/ml) but protected the cells at higher IFN levels (5-20 U/ml). No potentiation was seen with Zn. HuIFN-beta protected against rhinovirus at the same doses as used with HuIFN-alpha, but in contrast to HuIFN-alpha, no potentiation was noted.

The envelope of measles virus (MV) particles contains two viral glycoproteins, the haemagglutinin (H) and the fusion (F) protein, which together induce the entry of MV into cells. In the present study, we investigated the role of oligosaccharide processing for the function and antigenicity of the MV glycoproteins by means of glycosidase inhibitors. Golgi α-mannosidase inhibitors (1-deoxymannojirimycin and swainsonine) prevented the oligosaccharides on the MV glycoproteins from obtaining Endo H resistance, but that did not appear to influence in vitro MV infections, indicating that conversion of oligosaccharide chains into the complex form was not required for the function of the MV glycoproteins. The α-glucosidase inhibitor castanospermine (CSP) quantitatively reduced the production of infectious MV particles in cells infected with both vaccine strain and wild-type MV. CSP reduced the detection of the MV F protein by certain monoclonal antibodies (MAbs) that appeared to recognize nonlinear epitopes. CSP also inhibited syncytium formation in MV infected cells, but did not affect MV induced CD46 downregulation, suggesting that CSP primarily influenced the F protein. We propose that CSP induces aberrant folding of MV glycoproteins in a manner that influences their function and antigenicity.

Background and novelty Coagulation Factor VIII (FVIII) is an essential cofactor in the blood coagulation cascade. Inability to produce functional FVIII results in haemophilia A which can be treated with recombinant FVIII [1]. Chinese Hamster Ovary (CHO) cells are the most used cell line for producing complex biopharmaceuticals due to its ability to perform complex post-translational modifications. When mammalian cells overexpress a protein like FVIII they will adapt by regulating various proteins and pathways to support synthesis/production of this protein. Yields of FVIII produced in CHO are low and for this reason a greater understanding of what constitute a high producing cell line is desired. In this study a full transcriptome analysis was undertaken in order to analyze the differences between high and low producers of FVIII

A morbillivirus of uncertain origin recently killed hundreds of Mediterranean dolphins. This is the first report of the nucleotide and deduced amino acid sequence of a dolphin morbillivirus (DMV) gene. The sequence of the nucleocapsid (N) gene including boundaries was determined. When the DMV N gene coding region was compared with the corresponding sequences of other morbilliviruses a distant evolutionary relationship between these viruses and DMV was apparent. Phylogenetic analysis of the sequence data provided further evidence that DMV is not closely related to any known morbillivirus, whereas phocine distemper virus exhibits a relatively close relationship to canine distemper virus.

Abstract 1182 VWF protects FVIII from clearance in the circulation and is believed to ensure location of platelets to the site of injury. However, it is unknown if binding of FVIII to VWF has a role in localizing and thereby also facilitating the effect of FVIII in vivo. In the present study, a FVIII variant, FVIII-Y1680F, lacking the high affinity binding to VWF (Leyet et al. JCB 1991; 15; 740) was used to evaluate the binding of FVIII to VWF in clot formation hemophilia A mice in vivo. Binding of the FVIII variant to immobilized VWF was evaluated by surface plasmon resonance showing a 50–25 fold reduction in the Kd for FVIII-Y1680F compared to wt FVIII. Furthermore, pharmacokinetic studies in hemophilia A mice indicated that FVIII-Y1680F is basically devoid of VWF binding in vivo. The circulating half-life decreased from 7–8 hours for wt FVIII to 0.5 hours for FVIII-Y1680F (see figure) which is comparable to the half-life of wt FVIII in VWF knockout mice (0.5 hours). Using a chromogenic assay the specific activity of FVIII-Y1680F was 9200 IU/mg similar to that of wt FVIII confirming normal activity FVIII-Y1680F after cleavage with thrombin and removal of VWF. As the short half-life may influence the haemostatic effect of FVIII-Y1680F in vivo, a 40 kDa PEG moiety was attached to the O-glycan in the B-domain of the FVIII variant. This re-establishes the circulating half-life (6.1 hours) to that of wt FVIII without affecting the specific activity in vitro. The haemostatic effect of 40K-O-PEG FVIII-Y1680F was subsequently used to investigate if high affinity VWF binding of FVIII influences its haemostatic effect in vivo. The acute haemostatic effect of 40K-O-PEG-FVIII-Y1680F was compared to wt FVIII (Advate®) in the tail bleeding model in hemophilia A mice at doses equivalent to the 50% of the maximal effect and at maximal efficacy (20 and 280 IU/kg; Elm et al., Hemophilia 2011 epub). The blood loss was significantly reduced at both doses with comparable effect of 40K-O-PEG-FVIII-Y1680F and wt FVIII (see see figure, * indicates significant differences compared to vehicle treated hemophilia A mice). This indicates that the lack of VWF binding does not interfere with the haemostatic properties of FVIII in this particular model. To further support these data, the haemostatic effect of 40K-O-PEG-FVIII-Y1680F was tested in the FeCl3 injury model (2.5, 5 and 10 IU/kg) in hemophilia A mice. No clot formation was observed in vehicle mice and 40K-O-PEG-FVIII-Y1680F normalized dose dependently the clot formation time comparable to wt FVIII. In conclusion, the current data suggest that the haemostatic effect of FVIII in vivo is not dependent on high affinity binding of FVIII to VWF. Disclosures: Holmberg: Novo Nordisk A/S: Employment. Kjalke:Novo Nordisk A/S: Employment. Karpf:Novo NOrdisk A/S: Employment. Hilden:Novo Nordisk A/S: Employment. Pelzer:Novo Nordisk A/S: Employment. Koefoed-Hansen:Novo Nordisk A/S: Employment. Johnsen:Novo Nordisk A/S: Employment. Thim:Novo Nordisk A/S: Employment. Karlsson:Novo Nordisk A/S: Employment. Jespersgaard:Novo Nordisk A/S: Employment. Bolt:Novo Nordisk: Employment. Stennicke:Novo Nordisk A/S: Employment.

The nucleotide sequence of the phosphoprotein (P) gene of a dolphin morbillivirus (DMV) isolate was determined. Like those of other morbilliviruses the DMV P gene encoded P and C proteins in overlapping open reading frames and V protein by editing the P gene transcript. Among P mRNA based clones the editing site variants GGGC, GGGG, GAGC and GGGGGGC predicting a P protein, and the variants GGGGC and GGGGGG predicting a V protein, were found. Surprisingly, the three variants GGGC, GGGG and GAGC were also found among clones generated from genomic RNA of the DMV isolate. Thus, more than one viral genome type appeared to be present in cells infected with the DMV isolate. By a similar analysis of the virus genomes in the tissue from which the DMV isolate was obtained, only the GGGC type was found, indicating that the GGGG and GAGC types arose during adaptation of the virus to growth in cell cultures. No editing site variants likely to have arisen by editing the GAGC type were encountered, and it remains ot be determined whether mRNA encoding V protein can be transcribed from genomes with this editing site. Using antisera raised against the common N terminus and unique C termini of the predicted P and V proteins, the in vivo expression of these proteins was demonstrated.