Corrected and Republished from: "A Novel, Multiple-Antigen Pneumococcal Vaccine Protects against Lethal Streptococcus pneumoniae Challenge".

Current vaccination against Streptococcus pneumoniae uses vaccines based on capsular polysaccharides from selected serotypes and has led to nonvaccine serotype replacement disease. We have investigated an alternative serotype-independent approach, using multiple-antigen vaccines (MAV) prepared from S. pneumoniae TIGR4 lysates enriched for surface proteins by a chromatography step after culture under conditions that induce expression of heat shock proteins (Hsp; thought to be immune adjuvants). Proteomics and immunoblot analyses demonstrated that, compared to standard bacterial lysates, MAV was enriched with Hsps and contained several recognized protective protein antigens, including pneumococcal surface protein A (PspA) and pneumolysin (Ply). Vaccination of rodents with MAV induced robust antibody responses to multiple serotypes, including nonpneumococcal conjugate vaccine serotypes. Homologous and heterologous strains of S. pneumoniae were opsonized after incubation in sera from vaccinated rodents. In mouse models, active vaccination with MAV significantly protected against pneumonia, while passive transfer of rabbit serum from MAV-vaccinated rabbits significantly protected against sepsis caused by both homologous and heterologous S. pneumoniae strains. Direct comparison of MAV preparations made with or without the heat shock step showed no clear differences in protein antigen content and antigenicity, suggesting that the chromatography step rather than Hsp induction improved MAV antigenicity. Overall, these data suggest that the MAV approach may provide serotype-independent protection against S. pneumoniae.

Quantitation of thrombin-activatable fibrinolysis inhibitor in human plasma by isotope dilution mass spectrometry.

Measurement of Thrombin-activatable fibrinolysis inhibitor (TAFI) in human plasma is dependent on reproducible assays. To date, standards for measuring TAFI are frequently calibrated relative to pooled normal human plasma and arbitrarily assigned a potency of 100% TAFI, despite variation in TAFI concentrations between plasma pools. Alternatively, TAFI calibrators can be assigned a value in SI units but the approach used for value assignment is not consistent and furthermore, if purified TAFI is used to determine TAFI concentration in plasma, may be adversely affected by matrix effects. A TAFI plasma standard in mass units with traceability to the SI unit of mass is desirable. We report here the establishment of a quantitative mass spectrometry method for TAFI in plasma. Traceability is obtained by reference to calibrators that consist of blank plasma spiked with a defined amount of purified TAFI, value assigned by amino acid analysis. The calibrators are run alongside the samples, using the same preparation steps and conditions; an acetonitrile assisted tryptic digestion and multi-dimensional liquid chromatography (LC) separation followed by MRM-MS analysis. We measured the TAFI quantitatively in human plasma with reproducibility, reliability and precision, and demonstrated the applicability of this approach for value assigning a common reference standard.

The impact of thioredoxin reduction of allosteric disulfide bonds on the therapeutic potential of monoclonal antibodies.

Therapeutic mAbs are used to manage a wide range of cancers and autoimmune disorders. However, mAb-based treatments are not always successful, highlighting the need for a better understanding of the factors influencing mAb efficacy. Increased levels of oxidative stress associated with several diseases are counteracted by the activities of various oxidoreductase enzymes, such as thioredoxin (Trx), which also reduces allosteric disulfide bonds in proteins, including mAbs. Here, using an array of in vitro assays, we explored the functional effects of Trx-mediated reduction on the mechanisms of action of six therapeutic mAbs. We found that Trx reduces the interchain disulfide bonds of the mAbs, after which they remain intact but have altered function. In general, this reduction increased antigen-binding capacity, resulting in, for example, enhanced tumor necrosis factor (TNF) neutralization by two anti-TNF mAbs. Conversely, Trx reduction decreased the antiproliferative activity of an anti-tyrosine kinase-type cell-surface receptor HER2 mAb. In all of the mAbs, Fc receptor binding was abrogated by Trx activity, with significant loss in both complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) activity of the mAbs tested. We also confirmed that without alkylation, Trx-reduced interchain disulfide bonds reoxidize, and ADCC activity is restored. In summary, Trx-mediated reduction has a substantial impact on the functional effects of an mAb, including variable effects on antigen binding and Fc function, with the potential to significantly impact mAb efficacy in vivo.

A Novel, Multiple-Antigen Pneumococcal Vaccine Protects against Lethal Streptococcus pneumoniae Challenge.

Current vaccination against Streptococcus pneumoniae uses vaccines based on capsular polysaccharides from selected serotypes and has led to nonvaccine serotype replacement disease. We have investigated an alternative serotype-independent approach, using multiple-antigen vaccines (MAV) prepared from S. pneumoniae TIGR4 lysates enriched for surface proteins by a chromatography step after culture under conditions that induce expression of heat shock proteins (Hsp; thought to be immune adjuvants). Proteomics and immunoblot analyses demonstrated that, compared to standard bacterial lysates, MAV was enriched with Hsps and contained several recognized protective protein antigens, including pneumococcal surface protein A (PspA) and pneumolysin (Ply). Vaccination of rodents with MAV induced robust antibody responses to multiple serotypes, including nonpneumococcal conjugate vaccine serotypes. Homologous and heterologous strains of S. pneumoniae were opsonized after incubation in sera from vaccinated rodents. In mouse models, active vaccination with MAV significantly protected against pneumonia, while passive transfer of rabbit serum from MAV-vaccinated rabbits significantly protected against sepsis caused by both homologous and heterologous S. pneumoniae strains. Direct comparison of MAV preparations made with or without the heat shock step showed no clear differences in protein antigen content and antigenicity, suggesting that the chromatography step rather than Hsp induction improved MAV antigenicity. Overall, these data suggest that the MAV approach may provide serotype-independent protection against S. pneumoniae.

Quantification of Müllerian Inhibiting Substance/Anti-Müllerian Hormone polypeptide by isotope dilution mass spectrometry.

Measurement of serum concentrations of Müllerian inhibiting substance (MIS), also known as anti-Müllerian Hormone (AMH) by immunoassay is gaining clinical acceptance and widespread use for the diagnosis of ovarian conditions and for prediction of the response to ovarian stimulation protocols as part of assisted reproductive therapies. Provision of an International Standard to harmonize immunoassay methods is required. It is desirable for the content of a future International Standard to be assigned in mass units for consistency with the units reported by current methods. Isotope dilution mass spectrometry (IDMS), a physicochemical method with traceability to the SI (Système International d'Unités) unit of mass, is a candidate approach to provide orthogonal data to support this mass assignment. Here, we report on the development of an IDMS method for quantitation of AMH using three peptides from different regions of the AMH monomer as surrogates for the measurement of AMH. We show the sensitivity and linearity of the standard peptides and demonstrate the reproducibility and consistency of the measurement amongst the three peptides for determining the AMH content in buffered preparations and in trial preparations of recombinant AMH, lyophilised in the presence of an excess of bovine casein.

Mass spectrometry analysis reveals differences in the host cell protein species found in pseudotyped lentiviral vectors.

Lentiviral vectors (LVs) have been successfully used in clinical trials showing long term therapeutic benefits. Studying the role of cellular proteins in lentivirus HIV-1 life cycle can help understand virus assembly and budding, leading to improvement of LV production for gene therapy. Lentiviral vectors were purified using size exclusion chromatography (SEC). The cellular protein composition of LVs produced by two different methods was compared: the transient transfection system pseudotyped with the VSV-G envelope, currently used in clinical trials, and a stable producer cell system using a non-toxic envelope derived from cat endogenous retrovirus RD114, RDpro. Proteins of LVs purified by size exclusion chromatography were identified by tandem mass spectrometry (MS/MS). A smaller number of cellular protein species were detected in stably produced vectors compared to transiently produced vector samples. This may be due to the presence of co-purified VSV-G vesicles in transiently produced vectors. AHNAK (Desmoyokin) was unique to RDpro-Env vectors. The potential role in LV particle production of selected proteins identified by MS analysis including AHNAK was assessed using shRNA gene knockdown technique. Down-regulation of the selected host proteins AHNAK, ALIX, and TSG101 in vector producer cells did not result in a significant difference in vector production.

Dissection of the function of the RmpM periplasmic protein from Neisseria meningitidis.

RmpM is a periplasmic protein from Neisseria meningitidis that comprises an N-terminal domain (residues 1-47) and a separate globular C-terminal domain (residues 65-219) responsible for binding to peptidoglycan. Here we show, through the use of size exclusion chromatography and pull-down assays, that a recombinant N-terminal fragment of RmpM binds to both the major outer membrane porins, PorA and PorB. Analysis by semi-native SDS-PAGE established that both recombinant full-length RmpM and an N-terminal fragment, but not the C-terminal peptidoglycan-binding domain, were sufficient to stabilize the PorA and PorB oligomeric complexes. Evidence from binding assays indicated that the meso-diaminopimelate moiety plays an important role in peptidoglycan recognition by RmpM. Site-directed mutagenesis showed that two highly conserved residues, Asp120 and Arg135, play an important role in peptidoglycan binding. The yield of outer membrane vesicles, which have been used extensively as a vaccine against N. meningitidis, was considerably higher in an N. meningitidis strain expressing a truncated N-terminal fragment of RmpM (ΔC-term rmpM) than in the WT strain. The native oligomeric state of the PorA/PorB complexes was maintained in this strain. We conclude that the dual functions of RmpM are independent, and that it is possible to use this knowledge to engineer a strain with higher yield of outer membrane vesicles, whilst preserving PorA and PorB, which are key protective antigens, in their native oligomeric state.

Factor H binding protein (fHbp)-mediated differential complement resistance of a serogroup C Neisseria meningitidis isolate from cerebrospinal fluid of a patient with invasive meningococcal disease.

During an outbreak of invasive meningococcal disease (IMD) at the University of Southampton, UK, in 1997, two Neisseria meningitidis serogroup C isolates were retrieved from a student ('Case'), who died of IMD, and a close contact ('Carrier') who, after mouth-to-mouth resuscitation on the deceased, did not contract the disease. Genomic comparison of the isolates demonstrated extensive nucleotide sequence identity, with differences identified in eight genes. Here, comparative proteomics was used to measure differential protein expression between the isolates and investigate whether the differences contributed to the clinical outcomes. A total of six proteins were differentially expressed: four proteins (methylcitrate synthase, PrpC; hypothetical integral membrane protein, Imp; fructose-1,6-bisphosphate aldolase, Fba; aldehyde dehydrogenase A, AldA) were upregulated in the Case isolate, while one protein (Type IV pilus-associated protein, PilC2) was downregulated. Peptides for factor H binding protein (fHbp), a major virulence factor and antigenic protein, were only detected in the Case, with a single base deletion (ΔT366) in the Carrier fHbp causing lack of its expression. Expression of fHbp resulted in an increased resistance of the Case isolate to complement-mediated killing in serum. Complementation of fHbp expression in the Carrier increased its serum resistance by approximately 8-fold. Moreover, a higher serum bactericidal antibody titre was seen for the Case isolate when using sera from mice immunized with Bexsero (GlaxoSmithKline), a vaccine containing fHbp as an antigenic component. This study highlights the role of fHbp in the differential complement resistance of the Case and the Carrier isolates. Expression of fHbp in the Case resulted in its increased survival in serum, possibly leading to active proliferation of the bacteria in blood and death of the student through IMD. Moreover, enhanced killing of the Case isolate by sera raised against an fHbp-containing vaccine, Bexsero, underlines the role and importance of fHbp in infection and immunity.

Quantification of FAM20A in human milk and identification of calcium metabolism proteins.

BACKGROUND: FAM20A, a recently discovered protein, is thought to have a fundamental role in inhibiting ectopic calcification. Several studies have demonstrated that variants of FAM20A are causative for the rare autosomal recessive disorder, enamel-renal syndrome (ERS). ERS is characterized by defective mineralization of dental enamel and nephrocalcinosis suggesting that FAM20A is an extracellular matrix protein, dysfunction of which causes calcification of the secretory epithelial tissues. FAM20A is a low-abundant protein that is difficult to detect in biofluids such as blood, saliva, and urine. Thus, we speculated the abundance of FAM20A to be high in human milk, since the secretory epithelium of lactating mammary tissue is involved in the secretion of highly concentrated calcium. Therefore, the primary aim of this research is to describe the processes/methodology taken to quantify FAM20A in human milk and identify other proteins involved in calcium metabolism. METHOD: This study used mass spectrometry-driven quantitative proteomics: (1) to quantify FAM20A in human milk of three women and (2) to identify proteins associated with calcium regulation by bioinformatic analyses on whole and milk fat globule membrane fractions. RESULTS: Shotgun MS/MS driven proteomics identified FAM20A in whole milk, and subsequent analysis using targeted proteomics also successfully quantified FAM20A in all samples. Combination of sample preparation, fractionation, and LC-MS/MS proteomics analysis generated 136 proteins previously undiscovered in human milk; 21 of these appear to be associated with calcium metabolism. CONCLUSION: Using mass spectrometry-driven proteomics, we successfully quantified FAM20A from transitional to mature milk and obtained a list of proteins involved in calcium metabolism. Furthermore, we show the value of using a combination of both shotgun and targeted driven proteomics for the identification of this low abundant protein in human milk.

Measurement of surface protein antigens, PorA and PorB, in Bexsero vaccine using quantitative mass spectrometry.

Bexsero is a multivalent vaccine containing outer membrane vesicles (OMV) derived from Neisseria meningitidis group B strain NZ98/254 and three recombinant meningococcal proteins, Neisserial adhesin A, Heparin binding antigen and factor H binding protein. OMV production relies on the growth of large-scale cultures of N. meningitidis under controlled conditions. Changes to environmental factors, such as temperature, pH, nutrient availability and trace elements, can impact the growth rate of the meningococcus. Furthermore outer membrane expression levels vary in response to the environmental milieu, thus any changes in environmental conditions can result in changes in OMV protein content. This makes consistent production of OMVs challenging and the ability to measure the protein content of the final product is desirable to ensure product quality. The aim of this work was to develop a mass spectrometry (MS) method for measuring the porin proteins and to evaluate this approach for assessing the batch consistency of Bexsero vaccine. Using isotope dilution MS, we measured the PorA and PorB content in 75 lots of Bexsero vaccine. PorA ranged from 4.0 to 5.95 µg/dose with an average of 4.8 µg/dose. PorB ranged from 5.4 to 8.7 µg/dose with an average of 6.5 µg/dose. This is the first description of the quantitative characterisation of adjuvanted Bexsero vaccine drug product at the final stage of the production process, once the aluminium adjuvanted vaccine has been packaged into syringes, to assess manufacturing consistency. The significance of our findings to quality control in the future is discussed.

Comparison of two combinations of cyanine dyes for prelabelling and gel electrophoresis.

We report the use of IC-OSu ethyl-Cy3 and ethyl-Cy5 N-hydroxysuccinimide ester (NHS) cyanine dyes, which have similar chemical properties as the CyDye DIGE fluor minimal dyes for pre-electrophoresis labelling. Multiple sample analyses in different laboratories indicate that the use of IC-OSu ethyl-Cy3 and ethyl-Cy5 NHS ester cyanine dyes produces equivalent results to those obtained with DIGE CyDyes, and allows sample multiplexing and accurate quantitation for differential proteome analysis.

Proteomic Analysis Reveals Temporal Changes in Protein Expression in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes In Vitro.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold great promise for regenerative medicine and in vitro screening. Despite displaying key cardiomyocyte phenotypic characteristics, they more closely resemble fetal/neonatal cardiomyocytes, and further characterization is necessary. By combining the use of tandem mass tags to label cell lysates, followed by multiplexing, we have determined the effects of short-term (30 day) in vitro culture on hiPSC-CM protein expression. We found that hiPSC-CM exhibit temporal changes in global protein expression; alterations in protein expression were pronounced during the first 2 weeks following thaw and dominated by reductions in proteins associated with protein synthesis and ubiquitination. Between 2 and 4 weeks, proceeding thaw alterations in protein expression were dominated by metabolic pathways, indicating a potential temporal metabolic shift from glycolysis toward oxidative phosphorylation. Time-dependent changes in proteins associated with cardiomyocyte contraction, excitation-contraction coupling, and metabolism were detected. While some were associated with expected functional outcomes in terms of morphology or electrophysiology, others such as metabolism did not produce the anticipated maturation of hiPSC-CM. In several cases, a predicted outcome was not clear because of the concerted changes in both stimulatory and inhibitory pathways. Nevertheless, clear development of hiPSC-CM over this time period was evident.

Immunogenicity profiling of protein antigens from capsular group B Neisseria meningitidis.

Outer membrane vesicle (OMV)- based vaccines have been used to provide strain-specific protection against capsular group B Neisseria meningitidis infections, but the full breadth of the immune response against the components of the OMV has not been established. Sera from adults vaccinated with an OMV vaccine were used to screen 91 outer membrane proteins (OMPs) incorporated in an antigen microarray panel. Antigen-specific IgG levels were quantified pre-vaccination, and after 12 and 18 weeks. These results were compared with IgG levels from mice vaccinated with the same OMV vaccine. The repertoires of highly responding antigens in humans and mice overlapped, but were not identical. The highest responding antigens to human IgG comprised four integral OMPs (PorA, PorB, OpcA and PilQ), a protein which promotes the stability of PorA and PorB (RmpM) and two lipoproteins (BamC and GNA1162). These observations will assist in evaluating the role of minor antigen components within OMVs in providing protection against meningococcal infection. In addition, the relative dominance of responses to integral OMPs in humans emphasizes the importance of this subclass and points to the value of maintaining conformational epitopes from integral membrane proteins in vaccine formulations.

Physicochemical and biological assays for quality control of biopharmaceuticals: interferon alpha-2 case study.

A selection of physicochemical and biological assays were investigated for their utility in detecting changes in preparations of Interferon alpha-2a and Interferon alpha-2b (IFN-alpha 2a, IFN-alpha 2b), which had been subjected to stressed conditions, in order to create models of biopharmaceutical products containing product-related impurities. The stress treatments, which included oxidation of methionine residues and storage at elevated temperatures for different periods of time, were designed to induce various degrees of degradation, aggregation or oxidation of the interferon. Biological activity of the stressed preparations was assessed in three different in vitro cell-based bioassay systems: a late-stage anti-proliferative assay and early-stage assays measuring reporter gene activation or endogenous gene expression by quantitative real time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Relevant physicochemical methods such as SDS-PAGE, reverse phase (RP) chromatography, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), proved their complementarity in detecting structural changes in the stressed preparations which were reflected by reductions in biological activity.

Characterization of the key antigenic components and pre-clinical immune responses to a meningococcal disease vaccine based on Neisseria lactamica outer membrane vesicles.

Serogroup B strains are now responsible for over 80% of meningococcal disease in the UK and no suitable vaccine is available that confers universal protection against all serogroup B strains. Neisseria lactamica shares many antigens with the meningococcus, except capsule and the surface protein PorA. Many of these antigens are thought to be responsible for providing cross-protective immunity to meningococcal disease. We have developed an N. lactamica vaccine using methods developed for meningococcal outer membrane vesicle (OMV) vaccines. The major antigenic components were identified by excision of 11 major protein bands from an SDS-PAGE gel, followed by mass spectrometric identification. These bands contained at least 22 proteins identified from an unassembled N. lactamica genome, 15 of which having orthologues in published pathogenic Neisseria genomes. Western blotting revealed that most of these bands were immunogenic, and antibodies to these proteins generally cross-reacted with N. meningitidis proteins. Sera from mice and rabbits immunized with either N. lactamica or N. meningitidis OMVs produced comparable cross-reactive ELISA titres against OMVs prepared from a panel of diverse meningococcal strains. Mice immunized with either N. meningitidis or N. lactamica OMVs showed no detectable serum bactericidal activity against the panel of target strains except N. meningitidis OMV sera against the homologous strain. Similarly, rabbit antisera to N. lactamica OMVs elicited little or no bactericidal antibodies against the panel of serogroup B meningococcal strains. However, such antisera did mediate opsonophagocytosis, suggestingthat this may did mediate opsonophagocytosis, suggesting that this may be a mechanism by which this vaccine protects in a mouse model of meningococcal bacteraemia.

Typing complex meningococcal vaccines to understand diversity and population structure of key vaccine antigens.

Background: Protein-conjugate capsular polysaccharide vaccines can potentially control invasive meningococcal disease (IMD) caused by five (A, C, W, X, Y) of the six IMD-associated serogroups.  Concerns raised by immunological similarity of the serogroup B capsule to human neural cell carbohydrates, meant that 'serogroup B substitute' vaccines target more variable subcapsular protein antigens.  A successful approach using outer membrane vesicles (OMVs) as major vaccine components had limited strain coverage. In 4CMenB (Bexsero ®), recombinant proteins have been added to ameliorate this problem.  Methods: Scalable, portable, genomic techniques were used to investigate the Bexsero ® OMV protein diversity in meningococcal populations. Shotgun proteomics identified 461 proteins in the OMV, defining a complex proteome. Amino acid sequences for the 24 proteins most likely to be involved in cross-protective immune responses were catalogued within the PubMLST.org/neisseria database using a novel OMV peptide Typing (OMVT) scheme. Results: Among these proteins there was variation in the extent of diversity and association with meningococcal lineages, identified as clonal complexes (ccs), ranging from the most conserved peptides (FbpA, NEISp0578, and putative periplasmic protein, NEISp1063) to the most diverse (TbpA, NEISp1690).  There were 1752 unique OMVTs identified amongst 2492/3506 isolates examined by whole-genome sequencing (WGS). These OMVTs were grouped into clusters (sharing ≥18 identical OMVT peptides), with 45.3% of isolates assigned to one of 27 OMVT clusters. OMVTs and OMVT clusters were strongly associated with cc, genogroup, and Bexsero ® antigen variants, demonstrating that combinations of OMV proteins exist in discrete, non-overlapping combinations associated with genogroup and Bexsero ® Antigen Sequence Type. This highly structured population of IMD-associated meningococci is consistent with strain structure models invoking host immune and/or metabolic selection. Conclusions: The OMVT scheme facilitates region-specific WGS investigation of meningococcal diversity and is an open-access, portable tool with applications for vaccine development, especially in the choice of antigen combinations, assessment and implementation.

Comparison of single radial immunodiffusion, SDS-PAGE and HPLC potency assays for inactivated influenza vaccines shows differences in ability to predict immunogenicity of haemagglutinin antigen.

The current gold-standard potency test for inactivated influenza vaccines is the single radial immunodiffusion (SRD) assay. A number of alternative potency tests for inactivated influenza vaccines have been proposed in recent years. Evaluation of these new potency tests commonly involves comparison with SRD, in order to ascertain that the new method obtains values that correlate with those measured by the standard potency test. Here, we extended comparison of two methods, reverse-phase HPLC and SDS-PAGE, with SRD by assessing the methods' capacity to detect loss of potency induced by various deliberate treatments of vaccine samples. We demonstrate that neither of these methods detected the loss of potency observed by SRD; importantly, neither SDS-PAGE nor reverse-phase HPLC reflected results from mouse experiments that showed decreased immunogenicity and protection in vivo. These results emphasise the importance of assessing the stability-indicating nature, ie the ability to measure loss of vaccine potency, of any potential new potency assay.

Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans.

Plants are potential hosts for the expression of recombinant glycoproteins intended for therapeutic purposes. However, N-glycans of mammalian glycoproteins produced in transgenic plants differ from their natural counterparts. The use of the endoplasmic reticulum (ER)-retention signal has been proposed to restrict glycosylation of plantibodies to only high-mannose-type N-glycans. Furthermore, little is known about the influence of plant development and growth conditions on N-linked glycosylation. Here, we report a detailed N-glycosylation profiling study of CB.Hep1, a mouse IgG2b monoclonal antibody (mAb) against hepatitis B surface antigen (HBsAg) currently expressed in tobacco plants (Nicotiana tabacum L.). The KDEL ER-retention signal was fused to the C-terminal of both light and heavy chains. The structures of the N-linked glycans of this mAb produced in transgenic tobacco plants at various growth stages were analysed by high-performance liquid chromatography (HPLC) profiling techniques and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and compared with those of murine origin. The high-mannose-type oligosaccharides accounted for more than 80% of the total N-glycans, with Man7GlcNAc2 being the most abundant species. Some complex N-glycans bearing xylose and small amounts of oligosaccharides with both xylose and fucose were identified. No appreciable differences were detected when comparing glycosylation at different leaf ages, e.g. from seedling leaves up to 8 weeks old and top or basal leaves of mature plants, or between leaves, stems and whole plants. A strict retention of glycoproteins to ER by the use of the tetrapeptide KDEL was not sufficient, even though the majority of the resulting N-glycosylation was of the high-mannose type. It is highly likely to be dependent on other factors, which are most probably protein specific.

In vitro and in vivo growth alter the population dynamic and properties of a Jeryl Lynn mumps vaccine.

Mumps vaccines are live attenuated viruses. They are known to vary in effectiveness, degree of attenuation and adverse event profile. However, the underlying reasons are poorly understood. We studied two closely related mumps vaccines which originate from the same attenuated Jeryl Lynn-5 strain but have different efficacies. Jeryl Lynn-Canine Kidney (JL-CK), produced on primary canine kidney cells, is less effective than RIT4385, which is produced on chicken embryo fibroblasts. JL-CK and RIT4385 could be distinguished by a number of in vitro and in vivo properties. JL-CK produced heterogeneous, generally smaller plaques than RIT4385, but gave 100-fold higher titres when grown in cells and showed a higher degree of hydrocephalus formation in neonatal rat brains. Sanger sequencing of JL-CK identified 14 regions of heterogeneity throughout the genome. Plaque purification of JL-CK demonstrated the presence of five different Jeryl Lynn-5 variants encompassing the 14 mutations. One JL-CK mutation was associated with a small plaque phenotype, the effects of the others in vitro or in vivo were less clear. Only 4% of the JL-CK population corresponded to the parental Jeryl Lynn-5 strain. Next generation sequencing of JL-CK and virus before and after growth in cell lines or neonatal rat brains showed that propagation in vitro or in vivo altered the population dramatically. Our findings indicate that growth of JL-CK in primary canine kidney cells resulted in the selection of a mixture of mumps virus variants that have different biological properties compared to the parent Jeryl Lynn-5 virus. We also report three previously unknown heterogenic regions within the N gene of the RIT4385 vaccine.