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Martin J.,UK National Institute for Biological Standards and Control | Milne C.,European Directorate for the Quality of Medicines | Minor P.,UK National Institute for Biological Standards and Control | Chumakov K.,U.S. Food and Drug Administration | And 3 more authors.
Vaccine | Year: 2011

Oral poliomyelitis vaccine (OPV) is a critical part of the polio eradication programme. A high number of doses are administered each year with an impact on billions of citizens worldwide. It is therefore essential that written standards concerning OPV are up to date and widely available. The World Health Organization (WHO) publishes technical guidance on the quality, safety and efficacy of vaccines intended to assist national regulatory authorities (NRAs), national control laboratories (NCLs) and manufacturers. As part of its programme, on 20-22 July 2010 WHO convened a working group meeting to initiate the revision of the WHO recommendations on the production and control of OPV as presently outlined in the Technical Reports Series (TRS) issues Nos. 904 and 910 [1,2]. The attendees included experts from academia, NRAs/NCLs and industry involved in the study, manufacture, and authorization and testing/release of OPV from countries around the world including representatives from China, the European Union, Indonesia, Japan, Mexico, and the USA. The objective was to review the state of knowledge concerning production and control of OPV, with a focus on neurovirulence testing, to determine how the existing guidelines should be updated and what recommendations should be made for the future. The outcomes of this meeting will be taken into consideration in future revision of the WHO TRS. © 2011.


Zhang C.X.Y.,Canadian Food Inspection Agency | Zhang C.X.Y.,University of Ottawa | Creskey M.C.,Center for Vaccine Evaluation | Cyr T.D.,Center for Vaccine Evaluation | And 5 more authors.
Proteomics | Year: 2013

This study aimed to identify proteins exposed on the surface of Listeria monocytogenes cells for diagnostic reagent development. Brief trypsin treatment of L. monocytogenes cells followed by peptide separation and identification by nano-LC and online-MS/MS was performed. In parallel, as a negative control, proteins secreted into the digest buffer as well as proteins from cell lysis were identified. One hundred and seventy-four proteins were identified in at least two of three trials in either the negative control or during cell digest. Nineteen surface, 21 extracellularly secreted, 132 cytoplasmic, and two phage proteins were identified. Immunofluorescence microscopy of L. monocytogenes cells revealed the surface localization of two potential candidates for L. monocytogenes isolation and detection: lipoprotein LMOf2365_0546 and PBPD1 (LMOf2365_2742). In this report, we present the first data set of surface-exposed L. monocytogenes proteins currently available. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000035.© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Behrensdorf-Nicol H.A.,Paul Ehrlich Institute | Bonifas U.,Paul Ehrlich Institute | Isbrucker R.,Center for Vaccine Evaluation | Ottiger H.,Institute of Virology and Immunology IVI | And 4 more authors.
Biologicals | Year: 2014

Tetanus vaccines contain detoxified tetanus neurotoxin. In order to check for residual toxicity, the detoxified material (toxoid) has to be tested in guinea pigs. These tests are time-consuming and raise animal welfare issues. In line with the "3R" principles of replacing, reducing and refining animal tests, the "binding and cleavage" (BINACLE) assay for detection of active tetanus neurotoxin has been developed as a potential alternative to toxicity testing in animals. This invitro test system can discriminate well between toxic and detoxified toxin molecules based on their receptor-binding and proteolytic characteristics.Here we describe an international study to assess the transferability of the BINACLE assay. We show that all participating laboratories were able to successfully perform the assay. Generally, assay variability was within an acceptable range. A toxin concentration-dependent increase of assay signals was observed in all tests. Furthermore, participants were able to detect low tetanus neurotoxin concentrations close to the estimated invivo detection limit.In conclusion, the data from this study indicate that the methodology of the BINACLE assay seems to be robust, reproducible and easily transferable between laboratories. These findings substantiate our notion that the method can be suitable for the routine testing of tetanus toxoids. © 2014 The International Alliance for Biological Standardization.


PubMed | King Abdulaziz University, Public Health Agency of Canada, University of Ottawa, Center for Vaccine Evaluation and 3 more.
Type: Journal Article | Journal: Journal of immunology (Baltimore, Md. : 1950) | Year: 2014

CD40L, a key regulator of the immune system, was studied as both a targeting ligand and a molecular adjuvant in nucleoprotein (NP)-based host defense against influenza in mouse models with different genetic backgrounds. Adenoviral vectors secreting NP-CD40L fusion protein (denoted as rAd-SNP40L) afforded full protection of immunocompetent and immunocompromised mice (CD40L(-/-) and CD4(-/-)) against lethal influenza infection. Mechanistically, rAd-SNP40L preferentially induced early and persistent B cell germinal center formation, and accelerated Ig isotype-switching and Th1-skewed, NP-specific Ab response. Moreover, it drastically augmented primary and memory NP-specific CTL activity and polyfunctional CD8(+) T cells. The markedly enhanced nonneutralizing Abs and CTLs significantly reduced viral burdens in the lungs of mice upon lethal virus challenge. Data generated from CD40L(-/-) and CD4(-/-) mice revealed that the protection was indeed CD40L mediated but CD4(+) T cell independent, demonstrating the viability of the fusion Ags in protecting immunodeficient hosts. Notably, a single dose of rAd-SNP40L completely protected mice from lethal viral challenge 4 mo after immunization, representing the first report, to our knowledge, on NP in conjunction with a molecular adjuvant inducing a robust and long-lasting memory immune response against influenza. This platform is characterized by an increased in vivo load of CD40-targeted Ag upon the secretion of the fusion protein from adenovirus-infected cells and may represent a promising strategy to enhance the breadth, durability, and potency of Ag-specific immune responses.


Hong A.,Public Health Agency of Canada | Han D.D.,Capital Medical University | Wright C.J.,Public Health Agency of Canada | Burch T.,Public Health Agency of Canada | And 12 more authors.
Biochemical and Biophysical Research Communications | Year: 2012

We identified the interaction between HBV X (HBx) protein and the oncogene AIB1 (amplified in breast cancer 1). A serine/proline motif (SSPSPS) in HBx was found to be required for the interaction. Two LXD motifs [LLXX(X)L, X means any amino acids], LLRNSL and LLDQLHTLL in AIB1 were also found to be involved in the HBx-AIB1 interaction. The HBx-AIB1 interaction was important for the activation of NFκB signal transduction, the HBx mutant that did not interact with AIB1showed dramatically lower NFκB activation activity than the WT HBx. These findings contribute to the new understanding on signal transduction activation mechanisms of HBx. © 2012.


Gao J.,Center for Vaccine Evaluation | Johnston G.M.,Dalhousie University | Lavergne M.R.,University of British Columbia | McIntyre P.,Dalhousie University
Journal of Palliative Care | Year: 2011

Classification and regression tree (CART) analysis was used to identify subpopulations with lower palliative care program (PCP) enrolment rates. CART analysis uses recursive partitioning to group predictors. The PCP enrolment rate was 72 percent for the 6,892 adults who died of cancer from 2000 and 2005 in two counties in Nova Scotia, Canada. The lowest PCP enrolment rates were for nursing home residents over 82 years (27 percent), a group residing more than 43 kilometres from the PCP (31 percent), and another group living less than two weeks after their cancer diagnosis (37 percent). The highest rate (86 percent) was for the 2,118 persons who received palliative radiation. Findings from multiple logistic regression (MLR) were provided for comparison. CART findings identified low PCP enrolment subpopulations that were defined by interactions among demographic, social, medical, and health system predictors. © 2011 Institut universitaire de gériatrie de Montréal.


Creskey M.C.,Center for Vaccine Evaluation | Li C.,National Institutes for Food and Drug Control | Wang J.,National Institutes for Food and Drug Control | Girard M.,Center for Vaccine Evaluation | And 6 more authors.
Vaccine | Year: 2012

Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1μg/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS E, a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures. © 2012.


Lorbetskie B.,Center for Vaccine Evaluation | Wang J.,Center for Vaccine Evaluation | Gravel C.,Center for Vaccine Evaluation | Allen C.,Center for Vaccine Evaluation | And 4 more authors.
Vaccine | Year: 2011

A previously described reversed-phase HPLC (RP-HPLC) method based on fast separations on a non-porous silica stationary phase [1] was optimized and qualified for the quantitative determination of hemagglutinin (HA) in influenza vaccine preparations. Optimization of the gradient elution conditions led to improved separation of the HA1 subunit from other vaccine constituents. The sensitivity of the method was significantly increased by using native fluorescence detection, resulting in an approximately 10-fold increase as compared to UV-vis detection. This enabled the elimination of the concentration step described in the original method and allowed direct analysis of vaccine preparations. The method was qualified for linearity, range, limit of detection, limit of quantitation and precision. Overall, it was found to be linear over the range of 2.5-100μg. HA/mL for all subtypes examined. This range covered 50-150% of the concentration found for individual strains in seasonal influenza vaccines and in the pandemic H1N1 vaccine. The limit of detection and limit of quantitation for each subtype were found to be suitable for the method's intended purpose and compared well to values found by the single radial immunodiffusion (SRID). The repeatability of the method gave RSD values below 5% for both retention time and peak areas. As expected for intermediate precision, larger RSD values for peak area were obtained but were below 10% and deemed acceptable.The RP-HPLC results were compared to Western blot analysis using a HA universal antibody for a set of 15 monovalent A/California H1N1 preparations and showed good correlation. Similarly, the quantitative nature of the RP-HPLC method was assessed in relation to the SRID assay currently used for the determination of the HA content in bulk antigen and final vaccine preparations. Thus, for a series of 23 monovalent A/Brisbane/59/2007 H1N1 bulks, ranging between 12.7 and 15.9μg. HA/mL by SRID, the RP-HPLC values were found to be in very good agreement, ranging between 11.9 and 14.1μg. HA/mL (n=5) for five determinations carried out on 5 different days.During the 2009-10 H1N1 influenza pandemic the quantitative RP-HPLC method was used alongside several other test methods for the analysis of pandemic H1N1 vaccine preparations that included bulk antigen and final vaccines. The HA content of vaccines formulated at 15 or 30μg/mL was measured by RP-HPLC and SRID and results showed that the HA content determined by RP-HPLC correlated well to that determined by SRID and to values determined by Western blot. Overall, the results provided further evidence of the usefulness of RP-HPLC for the detection and quantitation of the HA content once a reference standard has been established. © 2011 Elsevier Ltd.


A previously described reversed-phase HPLC (RP-HPLC) method based on fast separations on a non-porous silica stationary phase [1] was optimized and qualified for the quantitative determination of hemagglutinin (HA) in influenza vaccine preparations. Optimization of the gradient elution conditions led to improved separation of the HA1 subunit from other vaccine constituents. The sensitivity of the method was significantly increased by using native fluorescence detection, resulting in an approximately 10-fold increase as compared to UV-vis detection. This enabled the elimination of the concentration step described in the original method and allowed direct analysis of vaccine preparations. The method was qualified for linearity, range, limit of detection, limit of quantitation and precision. Overall, it was found to be linear over the range of 2.5-100 g HA/mL for all subtypes examined. This range covered 50-150% of the concentration found for individual strains in seasonal influenza vaccines and in the pandemic H1N1 vaccine. The limit of detection and limit of quantitation for each subtype were found to be suitable for the methods intended purpose and compared well to values found by the single radial immunodiffusion (SRID). The repeatability of the method gave RSD values below 5% for both retention time and peak areas. As expected for intermediate precision, larger RSD values for peak area were obtained but were below 10% and deemed acceptable. The RP-HPLC results were compared to Western blot analysis using a HA universal antibody for a set of 15 monovalent A/California H1N1 preparations and showed good correlation. Similarly, the quantitative nature of the RP-HPLC method was assessed in relation to the SRID assay currently used for the determination of the HA content in bulk antigen and final vaccine preparations. Thus, for a series of 23 monovalent A/Brisbane/59/2007 H1N1 bulks, ranging between 12.7 and 15.9 g HA/mL by SRID, the RP-HPLC values were found to be in very good agreement, ranging between 11.9 and 14.1 g HA/mL (n=5) for five determinations carried out on 5 different days. During the 2009-10 H1N1 influenza pandemic the quantitative RP-HPLC method was used alongside several other test methods for the analysis of pandemic H1N1 vaccine preparations that included bulk antigen and final vaccines. The HA content of vaccines formulated at 15 or 30 g/mL was measured by RP-HPLC and SRID and results showed that the HA content determined by RP-HPLC correlated well to that determined by SRID and to values determined by Western blot. Overall, the results provided further evidence of the usefulness of RP-HPLC for the detection and quantitation of the HA content once a reference standard has been established.


PubMed | Center for Vaccine Evaluation
Type: Journal Article | Journal: Vaccine | Year: 2012

Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 g/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS(E), a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures.

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