Publications by authors named "Jun X Wheeler"

26 Publications

  • Page 1 of 1

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

Anal Biochem 2021 Oct 10:114413. Epub 2021 Oct 10.

National Institute for Biological Standards and Control, South Mimms, Potters Bar, EN6 3QG, UK. Electronic address:

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 SRM-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.
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http://dx.doi.org/10.1016/j.ab.2021.114413DOI Listing
October 2021

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

Vaccine 2020 02 12;38(6):1431-1435. Epub 2019 Dec 12.

National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.

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.
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http://dx.doi.org/10.1016/j.vaccine.2019.11.082DOI Listing
February 2020

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

J Biol Chem 2019 12 14;294(51):19616-19634. Epub 2019 Nov 14.

Division of Biotherapeutics, The National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom

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 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 .
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http://dx.doi.org/10.1074/jbc.RA119.010637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926469PMC
December 2019

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

Sci Rep 2019 05 2;9(1):6843. Epub 2019 May 2.

Lydia Becker Institute of Immunology and Inflammation, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, M13 9PL, UK.

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.
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http://dx.doi.org/10.1038/s41598-019-43139-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497663PMC
May 2019

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

Stem Cells Dev 2019 05 20;28(9):565-578. Epub 2019 Mar 20.

1 Myocardial Function, National Heart and Lung Institute, Imperial College, London, United Kingdom.

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.
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http://dx.doi.org/10.1089/scd.2018.0210DOI Listing
May 2019

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

Infect Immun 2019 03 21;87(3). Epub 2019 Feb 21.

Centre for Inflammation and Tissue Repair, UCL Respiratory, Division of Medicine, University College Medical School Rayne Institute, London, United Kingdom

Current vaccination against 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 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 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 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 .
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http://dx.doi.org/10.1128/IAI.00846-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386546PMC
March 2019

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

Anal Biochem 2018 11 6;560:50-55. Epub 2018 May 6.

National Institute for Biological Standards and Control, South Mimms, Potters Bar, EN6 3QG, UK.

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.
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http://dx.doi.org/10.1016/j.ab.2018.05.006DOI Listing
November 2018

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

Biologicals 2018 Mar 1;52:59-66. Epub 2018 Feb 1.

Division of Advanced Therapies, National Institute for Biological Standards and Control, Blanche Lane, Potters Bar, EN6 3QG, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.biologicals.2017.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910304PMC
March 2018

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

Microbiology (Reading) 2016 Feb 16;162(2):364-375. Epub 2015 Dec 16.

Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, M13 9PTUK.

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.
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http://dx.doi.org/10.1099/mic.0.000227DOI Listing
February 2016

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

Vaccine 2015 Aug 15;33(36):4586-93. Epub 2015 Jul 15.

Division of Virology, National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom. Electronic address:

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.
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http://dx.doi.org/10.1016/j.vaccine.2015.06.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550476PMC
August 2015

Identification of peptide sequences as a measure of Anthrax vaccine stability during storage.

Hum Vaccin Immunother 2014 17;10(6):1669-81. Epub 2014 Mar 17.

Division of Bacteriology; National Institute for Biological Standards and Control; Hertfordshire, UK.

The UK anthrax vaccine is an alum precipitate of a sterile filtrate of Bacillus anthracis Sterne culture (AVP). An increase in shelf life of AVP from 3 to 5 years prompted us to investigate the in vivo potency and the antigen content of 12 batches with a shelf life of 6.4 to 9.9 years and one bulk with a shelf life of 23.8 years. All batches, except for a 9.4-year-old batch, passed the potency test. Mass spectrometry (MS) and in-gel difference 2-dimensional gel electrophoresis (DIGE) were used to examine antigens of the pellet and supernatant of AVP. The pellet contained proteins with a MW in excess of 15 kDa. DIGE of desorbed proteins from the pellet revealed that with aging, 19 spots showed a significant change in size or intensity, a sign of protein degradation. MS identified 21 proteins including protective antigen (PA), enolase, lethal factor (LF), nucleoside diphosphate kinase, edema factor, and S-layer proteins. Fifteen proteins were detected for the first time including metabolic enzymes, iron binding proteins, and manganese dependent superoxide dismutase (MnSOD). The supernatant contained131 peptide sequences. Peptides representing septum formation inhibitor protein and repeat domain protein were most abundant. Five proteins were shared with the pellet: 2,3,4,5-tetrahydropyridine-6-dicarboxylate N-succinyltransferase, enolase, LF, MnSOD, and PA. The number of peptide sequences increased with age. Peptides from PA and LF appeared once batches exceeded their shelf life by 2 and 4 years, respectively. In conclusion, changes in antigen content resulting from decay or desorption only had a limited effect on in vivo potency of AVP. The presence of PA and LF peptides in the supernatant can inform on the age and stability of AVP.
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http://dx.doi.org/10.4161/hv.28443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4185962PMC
June 2015

Identification of vaccine antigens using integrated proteomic analyses of surface immunogens from serogroup B Neisseria meningitidis.

J Proteomics 2014 Apr 21;101:63-76. Epub 2014 Feb 21.

National Institute for Biological Standards and Control, Medicine and Healthcare Products Regulatory Agency, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom. Electronic address:

Unlabelled: Meningococcal surface proteins capable of evoking a protective immune response are candidates for inclusion in protein-based vaccines against serogroup B Neisseria meningitidis (NmB). In this study, a 2-dimensional (2-D) gel-based platform integrating surface and immune-proteomics was developed to characterize NmB surface protein antigens. The surface proteome was analyzed by differential 2-D gel electrophoresis following treatment of live bacteria with proteinase K. Alongside, proteins recognized by immune sera from mice challenged with live meningococci were detected using 2-D immunoblots. In combination, seventeen proteins were identified including the well documented antigens PorA, OpcA and factor H-binding protein, previously reported potential antigens and novel potential immunogens. Results were validated for the macrophage infectivity potentiator (MIP), a recently proposed NmB vaccine candidate. MIP-specific antisera bound to meningococci in whole-cell ELISA and facilitated opsonophagocytosis and deposition of complement factors on the surface of meningococcal isolates of different serosubtypes. Cleavage by proteinase K was confirmed in western blots and shown to occur in a fraction of the MIP expressed by meningococci suggesting transient or limited surface exposure. These observations add knowledge for the development of a protein NmB vaccine. The proteomic workflow presented here may be used for the discovery of vaccine candidates against other pathogens.

Biological Significance: This study presents an integrated proteomic strategy to identify proteins from N. meningitidis with desirable properties (i.e. surface exposure and immunogenicity) for inclusion in subunit vaccines against bacterial meningitis. The effectiveness of the method was demonstrated by the identification of some of the major meningococcal vaccine antigens. Information was also obtained about novel potential immunogens as well as the recently described potential antigen macrophage infectivity potentiator which can be useful for its consideration as a vaccine candidate. Additionally, the proteomic strategy presented in this study provides a generic 2-D gel-based platform for the discovery of vaccine candidates against other bacterial infections.
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http://dx.doi.org/10.1016/j.jprot.2014.02.013DOI Listing
April 2014

Proteomic analysis of rat plasma following transient focal cerebral ischemia.

Biomark Med 2011 Dec;5(6):837-46

National Institute for Biological Standards & Control, Health Protection Agency, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, UK.

Aim: This study aimed to identify plasma protein changes in a rat model of ischemic stroke using a proteomic approach.

Materials & Methods: Four male Sprague-Dawley rats (3-6 months old) were subjected to 90 min of left middle cerebral artery occlusion under anesthesia with 1.5% isoflurane in O(2)/air followed by 24-h reperfusion. Blood samples (~100 µl) were collected at baseline, at the end of 90-min middle cerebral artery occlusion and at 24-h postreperfusion. Brain injuries were assessed by MRI at 24-h postreperfusion. Quantitative comparison of global plasma protein expression was performed using 2D differential in-gel electrophoresis. Differentially expressed protein spots were identified using peptide sequencing tandem mass spectrometry.

Results: These rats had clear brain infarction in the left hemisphere detected by MRI. Thirty-three protein spots of plasma samples were differentially expressed following focal cerebral ischemia/reperfusion. These protein spots belonged to eight proteins. Six of them (α2-macroglobulin, complement C3, inter-α- trypsin inhibitor heavy chain H3, serum albumin, haptoglobin and transthyretin), which are a class of acute-phase proteins, changed significantly.

Conclusion: This study describes the responses of young rats to focal cerebral ischemia and suggests that future studies should use aged animals to better mimic the clinical ischemic stroke setting.
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http://dx.doi.org/10.2217/bmm.11.89DOI Listing
December 2011

Bioanalysis of meningococcal vaccines.

Bioanalysis 2010 Feb;2(2):343-61

Bioanalytical and Vaccine Research, Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.

Meningococcal meningitis is feared because of the rapid onset of severe disease from mild symptoms and, therefore, is an important target for vaccine research. Five serogroups, defined by the structures of their capsular polysaccharides, are responsible for the vast majority of disease. Protection against four of these five serogroups can be obtained with polysaccharide or glycoconjugate vaccines, in which fragments of the capsular polysaccharides attached to a carrier protein generate anticarbohydrate immune responses, whilst protection against group B disease requires protein immunogens, often presented in vesicles containing outer membrane proteins. Glycoconjugate vaccines are now an established technology, but outer-membrane protein vaccines are still under development and present significant challenges. This review discusses physicochemical approaches to the characterization and quality control of these vaccines, as well as highlighting the problems and differences in vaccine design required for protection against different serogroups of the same species of pathogen.
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http://dx.doi.org/10.4155/bio.09.181DOI Listing
February 2010

Characterization of meningococcal serogroup B outer membrane vesicle vaccines from strain 44/76 after growth in different media.

Vaccine 2010 Apr 25;28(18):3211-8. Epub 2010 Feb 25.

National Institute for Biological Standards and Control, Health Protection Agency, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.

In this study, we evaluated the effect of the growth medium on the composition and immunogenicity of meningococcal outer membrane vesicle (OMV) vaccines after cultivation of the Norwegian serogroup B 44/76 vaccine strain in either Frantz' or modified Catlin-6 media (MC.6M). Differential proteomic analysis revealed that 97% of the OMV proteins maintained the same levels in the two preparations. However, a number of differentially expressed proteins, including TdfH, OpcA, OMP NMB0088, hypothetical NMB2134, lipoprotein NMB1126/1164 and NspA, increased significantly in OMVs produced from bacteria grown in the MC.6M. Together with increased lipopolysaccharide levels, the increased expression of these proteins was associated with significantly higher serum bactericidal titres in mice immunized with the MC.6M OMV vaccine. The high resolution two-dimensional separation of the OMVs on a large-format gel across a pH range of 3-11 resolved around 2000 protein spots from which 75 proteins were identified by mass spectrometry.
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http://dx.doi.org/10.1016/j.vaccine.2010.02.023DOI Listing
April 2010

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

Proteomics 2009 Mar;9(6):1727-30

Laboratory for Molecular Structure, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire, UK.

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.
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http://dx.doi.org/10.1002/pmic.200800563DOI Listing
March 2009

Quantitation of haemagglutinin in H5N1 influenza viruses reveals low haemagglutinin content of vaccine virus NIBRG-14 (H5N1).

Vaccine 2008 Dec;26(51):6550-4

Division of Virology, National Institute for Biological Standards and Control, Blanche Lane, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.

The assessment of potential candidate influenza vaccine viruses includes a number of factors. Growth properties of the virus and yield of antigen, specifically the haemagglutinin (HA), are of key importance. The recently developed H5N1 candidate vaccine virus NIBRG-14 (with HA and NA genes derived from the clade 1 virus A/Viet Nam/1194/2004 in an A/Puerto Rico/8/34 background) has been suggested to yield low amounts of antigen. While investigating the antigen yield of H5N1 vaccine viruses, we found that accurate quantitation of the HA content of some H5N1 viruses was difficult due to the migration characteristics of the proteins on SDS-PAGE gels. The HA1 and HA2 bands co-migrated with nucleoprotein (NP) and matrix protein (M1) respectively, preventing accurate analysis. We have developed an accurate way of quantitating HA from these H5N1 viruses by introducing a deglycosylation step to the standard protocol. Using this method, we showed reproducibly that the low yield of NIBRG-14 is, at least in part, due to a lower than usual content of HA in virus preparations. This was also found to be the case for the parent wild type A/Viet Nam/1194/2004 virus.
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http://dx.doi.org/10.1016/j.vaccine.2008.09.050DOI Listing
December 2008

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

Biologicals 2008 Nov 8;36(6):383-92. Epub 2008 Aug 8.

National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.

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.
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http://dx.doi.org/10.1016/j.biologicals.2008.06.003DOI Listing
November 2008

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

Hum Vaccin 2008 Jan-Feb;4(1):23-30. Epub 2007 Jul 29.

Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, UK.

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.
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http://dx.doi.org/10.4161/hv.4.1.4806DOI Listing
August 2008

Proteomic analysis of the response of the human neutrophil-like cell line NB-4 after exposure to anthrax lethal toxin.

Proteomics Clin Appl 2007 Oct 11;1(10):1266-79. Epub 2007 Sep 11.

Laboratory of Molecular Structure, National Institute for Biological Standards and Control, Potters Bar, UK.

We used 2-D DIGE to analyze the early response of NB-4 cells, a human promyelotic leukemia cell line, exposed to lethal toxin from Bacillus anthracis at the proteome level. After a 2 h exposure, cells were still viable and 43% of spots (n = 1042) showed a significant change in protein level. We identified 59 spots whose expression had changed significantly, and these reflected cytoskeleton damage, mitochondrial lysis and endoplasmic reticulum stress. Actin filament assembly was disrupted as evidenced by an increase in both actin subunits and phosphorylated cofilin, whilst levels of tropomyosin, tropomodulin and actin related protein 2/3 complex subunit decreased. Lower levels of ATP synthase subunits and mitochondrial inner membrane protein were identified as markers of mitochondrial lysis. Levels of various stress response proteins rose and, uniquely, levels of Ca(2+) binding proteins such as translationally controlled tumor protein rose and hippocalcin-like protein 1 decreased. This response may have mitigated effects brought about by mitochondrial lysis and endoplasmic reticulum stress, and delayed or prevented apoptosis in NB-4 cells. These results resemble findings of similar proteomics studies in murine macrophages, although quantitative differences were observed.
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http://dx.doi.org/10.1002/prca.200700074DOI Listing
October 2007

Exploring the proteome of meningococcal outer membrane vesicle vaccines.

Proteomics Clin Appl 2007 Sep 17;1(9):1198-210. Epub 2007 Aug 17.

Laboratory of Molecular Structure, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, UK.

Neisseria meningitidis, one of the principal causes of bacterial meningitis and septicemia, continues to present a challenge for vaccine developers. While significant progress has been made in the development and implementation of conjugate vaccines, which are based on the capsular polysaccharide of the organism, this approach has failed to produce a vaccine against organisms expressing a serogroup B capsule. The completion of the first meningococcal genome sequences in 2000 provided new ways of meeting this challenge. One approach has been to learn more about meningococcal biology and pathogenesis through exploring its proteome. This article reviews the results of ten recent studies of the meningococcal proteome and compares the different methodologies employed. Not surprisingly, given the renewed impetus to develop a comprehensive vaccine and the continuing clinical development of outer membrane vesicle vaccines, many of these studies focus on the proteome of the outer membrane fraction. As in other areas of proteome research, the direct comparison of data from different studies is hampered by the lack of standardization of separation technologies and data formats. Nevertheless, proteomic analysis, especially when combined with detailed knowledge of meningococcal population structures, represents a powerful tool in the development of vaccines against this important pathogen.
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http://dx.doi.org/10.1002/prca.200700204DOI Listing
September 2007

Proteomics analysis of cellular components in lentiviral vector production using Gel-LC-MS/MS.

Proteomics Clin Appl 2007 Feb 22;1(2):224-30. Epub 2007 Jan 22.

Laboratory for Molecular Structure, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK.

Among the gene therapy vectors developed to date, lentiviral vectors persist in the host and are therefore best suited for long-term gene transfer and gene-replacement therapies. Human immunodeficiency virus (HIV)-1-based lentiviral vector products are currently produced by transient transfection, filtration and ultracentrifugation, and the quality of the resultant vector product is variable. We reason that by identifying host proteins co-produced with viral vectors we may better understand the mechanism of viral vector production, and improve the safety and quality of the resultant products. Our LC-MS/MS studies identified both viral vector proteins and host proteins, including nuclear proteins, elongation factors and chaperone and heat shock proteins (HSP), and confirmed the presence of known HIV-incorporated proteins, e.g. elongation factor-1α, HSP70 and Histone 2A, demonstrating the capability and viability of LC-MS/MS methods for the proteomic analysis of highly complex samples. Evaluation of the functions of the identified components is in progress to understand their implication for product quality and safety. These studies support the development of improved production and characterisation methods and advance the clinical application of lentiviral vector-based products.
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http://dx.doi.org/10.1002/prca.200600522DOI Listing
February 2007

Characterization of the protein content of a meningococcal outer membrane vesicle vaccine by polyacrylamide gel electrophoresis and mass spectrometry.

Hum Vaccin 2005 Mar-Apr;1(2):80-4. Epub 2005 Mar 2.

Division of Bacteriology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK.

The development and evaluation of outer membrane vesicles as vaccines against meningococcal disease has been carried out for more than two decades. Although such vaccines have limitations and are not widely licensed, they continue to be used to disrupt clonal outbreaks caused by group B meningococci and a wealth of information is now available from large-scale clinical studies. One dimensional polyacrylamide gel electrophoresis and semi-quantitative measurement of the major proteins is one method used to evaluate and control these products. However, it is often difficult to determine exactly which bands on a one dimensional gel correspond to the key antigens whose presence must be demonstrated for control and lot release. We have therefore carried out mass spectrometric analyses of outer membrane vesicle vaccine samples to definitively identify the bands containing seven key antigens: Omp85, FetA, PorA, PorB, RmpM, OpcA and NspA. An additional 33 proteins present in the vaccine were also identified and this information will be useful both for future quality control and for the interpretation of data from vaccine trials.
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http://dx.doi.org/10.4161/hv.1.2.1651DOI Listing
October 2006

Proteomic analysis of a meningococcal outer membrane vesicle vaccine prepared from the group B strain NZ98/254.

Proteomics 2006 Jun;6(11):3400-13

Department of Bacteriology, National Institute for Biological Standards and Control, South Mimms, Hertfordshire, UK.

In the absence of a suitable carbohydrate-based vaccine, outer membrane vesicle (OMV) vaccines have been used to disrupt outbreaks of serogroup B meningococcal disease for more than 20 years. Proteomic technology provides physical methods with the potential to assess the composition and consistency of these complex vaccines. 2-DE, combined with MS, were used to generate a proteome map of an OMV vaccine, developed to disrupt a long-running outbreak of group B disease in New Zealand. Seventy four spots from the protein map were identified including the outer membrane protein (OMP) antigens: PorA, PorB, RmpM and OpcA. Protein identification indicates that, in addition to OMPs, OMV vaccines contain periplasmic, membrane-associated and cytoplasmic proteins. 2-D-DIGE technology highlighted differences between preclinical development batches of vaccines from two different manufacturers.
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http://dx.doi.org/10.1002/pmic.200500821DOI Listing
June 2006

The human heart proteome: Two-dimensional maps using narrow-range immobilised pH gradients.

Electrophoresis 2006 Apr;27(8):1547-55

National Heart & Lung Institute, Imperial College School of Medicine, Heart Science Centre, Harefield Hospital, Harefield, Middlesex, UK.

The analysis of complex proteomes is undertaken using a variety of techniques and technologies such as 2-DE, surface-enhanced laser desorption ionisation, and various types of MS. In order to overcome the complexities of protein expression in discrete proteomes, sample fractionation has become an important aspect of proteomic experiments. The use of narrow-range IPGs (nrIPGs) is of special importance using the 2-DE proteomics workflow, since an enhanced visualisation of a given proteome is achieved through an improved physical separation and resolution of proteins. The work described in this paper presents a series of protein maps of the human heart left ventricle proteome that have been generated using nrIPGs for the first, IEF, dimension of 2-DE. A total of 374 gel spots were excised from seven different pH gradients, covering the range pH 3-10, giving rise to a total of 388 identifications from 110 unique proteins. Using Gene Ontologies (GOs), the identified proteins were found to be associated with 97 types of GO Process, 144 types of GO Function, and 54 types of GO Component. It is hoped that the maps presented in this paper will be of use to other researchers for reference purposes.
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http://dx.doi.org/10.1002/elps.200500777DOI Listing
April 2006
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