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.

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.

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.

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.

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.

Una vacuna de vesículas de membrana externa de Vibrio cholerae es aún una promesa: Caracterización proteómica / Outer membrane vesicle vaccine from Vibrio cholerae is still a promise: Proteomic characterization

Las epidemias de cólera afectan a un gran número de países africanos, asiáticos y del Caribe. Los cambios climatológicos y las constantes migraciones hacen que esta enfermedad se extienda, por lo que resulta necesario disponer de vacunas protectoras. En el presente trabajo se caracterizó una nueva vacuna de vesículas de membrana externa (VMEs) obtenidas de Vibrio cholerae O1 biotipo El Tor serotipo Ogawa cepa C7258, en el Instituto Finlay de vacunas (Cuba), a través de métodos proteómicos. Se identificaron 53 proteínas presentes en las VME (4 proteínas por banda electroforética) separadas por electroforesis unidimensional (1D) y digeridas con tripsina. Los fragmentos obtenidos fueron separados por cromatografía líquida de alta resolución (HPLC) acoplada a espectrometría de masa, secuenciados e identificados mediante bases de datos de proteínas Swiss-Prot y TrEMBL. El patrón proteico obtenido presentó algunas de las proteínas (12 proteínas citoplasmáticas y 5 proteínas de membrana externa) sugeridas dentro del proteoma de buena calidad para candidatos vacunales. Se estudiaron las mejores condiciones para la separación de las proteínas a través de electroforesis bidimensional. Las VME evaluadas cuentan con una composición fundamentada en proteínas necesarias para garantizar una respuesta inmune que proteja contra Vibrio cholerae O1 biotipo El Tor serotipo Ogawa.

Cholera epidemics affect a large number of African, Asian and Caribbean countries. The climate changes and the constant migrations cause this disease to spread, making it is necessary to obtain protective vaccines. In the present work, a new vaccine of outer membrane vesicles (OMV) from V. cholerae O1 El Tor biotype Ogawa serotype strain C7258 at Finlay Institute of vaccines (Cuba) was characterized by proteomic methods. A total of 53 proteins present in the OMV (approximate ratio of 4 proteins by electrophoresis band) were identified, separated by one dimension electrophoresis and digested by tripsin method. The fragments were separated by high performance liquid chromatography (HPLC) coupled to mass spectrometry, sequenced and identified, using Swiss-Prot and TrEMBL protein databases. The pattern showed some proteins (12 cytoplasmic proteins and 5 outer membrane proteins) suggested within the highest quality proteome for vaccine candidate. The best conditions for proteins separation by two dimension electrophoresis were studied. The OMV composition was based on proteins described to the immunity response and protection against V. cholerae O1 El Tor biotype Ogawa serotype.

As epidemias de cólera afetam um grande número de países africanos, asiáticos e caribenhos. As mudanças climáticas e as constantes migrações fazem com que esta doença se espalhe, portanto é necessário ter vacinas protectoras. No presente trabalho, uma nova vacina de vesículas de membrana externa (VMEs) obtidas de Vibrio cholerae 01 biotipo El Tor sorotipo Ogawa cepa C7258, no Instituto de Vacinas Finlay (Cuba), através de métodos proteômicos. Foram identificadas 53 proteínas presentes nas VME (4 proteínas por banda eletroforética) separadas por eletroforese unidimensional (1D) e digeridas com tripsina. Os fragmentos obtidos foram separados por cromatografia de alta resolução (HPLC) acoplada a espectrometria de massa, sequenciados e identificados usando bancos de dados de proteínas Swiss-Prot e TrEMBL. O padrão proteico obtido apresentou algumas das proteínas (12 proteínas citoplasmáticas e 5 proteínas de membrana externa) sugeridas dentro do proteoma de boa qualidade para candidatos vacinais. As melhores condições para a separação de proteínas através de eletroforese bidimensional foram estudadas. As VME avaliados possuem uma composição baseada em proteínas necessárias para garantir uma resposta imune que proteja contra Vibrio cholerae O1 biotipo El Tor sorotipo Ogawa.

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.

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.

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.

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

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.

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

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.

Proteomic analysis of rat plasma following transient focal cerebral ischemia.

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.

Bioanalysis of meningococcal vaccines.

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.

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

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.