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Natural Killer cell receptors allow this heterogeneous immune population to efficiently fight both tumors and infection, so their use as immunotherapy agents is an active field of research. Cytokine activation, particularly by myeloid cell-derived IL15, can induce potent NK anti-tumor responses. While studying the mechanism of action of intravesical instillations of Bacille Calmette-Guérin (BCG) as therapy for patients with high risk non-muscle invasive bladder cancer, we showed that BCG can activate a cytotoxic CD56bright NK cell population which efficiently recognized bladder cancer cells. This pioneer immunotherapy provides an invaluable model to understand the role of different immune populations in tumor elimination. However, during the propagation of BCG worldwide a large number of genetically diverse BCG substrains developed. Here, we investigated the capacity of different BCG substrains to promote NK cell activation and confirmed that they were able to activate lymphocytes. Tice, Connaught and Moreau were the substrains with a stronger NK activation effect as measured by CD56 upregulation. Surprisingly, dead mycobacteria also stimulated PBMC cultures and we further demonstrate here that subcellular fractions of BCG-Tice, in the absence of live mycobacteria, could also induce an NK cell response. Lipids from BCG-Tice, but not from Mycobacterium bovis, stimulated NK cell activation and degranulation, whereas the aqueous fraction of either bacteria did not activate lymphocytes. However, delipidated BCG-Tice bacteria were able to activate effector cells (CD3+CD56+ and NK, CD3-CD56+). These data demonstrate that different components of mycobacteria can stimulate different immune subpopulations resulting in phenotypes suitable for cancer elimination.

Immunotherapy, via intravesical instillations of Bacillus Calmette-Guérin (BCG) is the therapy of choice for patients with high risk non-muscle invasive bladder cancer. The subsequent recruitment of lymphocytes and myeloid cells, as well as the release of cytokines and chemokines, induces a local immune response that contributes to eliminate these tumours. The history of BCG development resulted in a large number of genetically diverse BCG substrains which could stimulate the immune system in different ways. Here, while investigating the capacity of different BCG substrains to promote the activation of NK cells, we confirmed that all the evaluated substrains could activate a cytotoxic CD56bright NK cell population which efficiently degranulated against bladder cancer cells; Tice, Connaught and Moreau were the substrains having a stronger effect. Dead mycobacteria also stimulated PBMC cultures and we demonstrate that subcellular fractions of BCG-Tice could contribute to the induction of this NK cell response. Lipids from BCG-Tice, but not from Mycobacterium bovis, stimulated NK cell activation and degranulation, however the aqueous fraction of either bacteria did not activate lymphocytes. Delipidated BCG-Tice activated effector cells (CD3+CD56+ and NK). These data suggest that different immune subpopulations could be stimulated using different fractions of mycobacteria for cancer elimination.

© 2018 van Meijgaarden et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Multiplex assays are increasingly applied to analyze multicomponent signatures of human immune responses, including the dynamics of cytokine and chemokine production, in observational as well as interventional studies following treatment or vaccination. However, relatively limited information is available on the performance of the different available multiplex kits, and comparative evaluations addressing this important issue are lacking. Study design To fill this knowledge gap we performed a technical comparison of multiplex bead assays from 4 manufacturers, each represented by 3 different lots, and with the assays performed by 3 different laboratories. To cross compare kits directly, spiked samples, biological samples and a newly made reference standard were included in all assays. Analyses were performed on 324 standard curves to allow for evaluation of the quality of the standard curves and the subsequent interpretation of biological specimens. Results Manufacturer was the factor which contributed most to the observed variation whereas variation in lots, laboratory or type of detection reagent contributed minimally. Inclusion of a common reference standard allowed us to overcome observed differences in cytokine and chemokine levels between manufacturers. Conclusions We strongly recommend using multiplex assays from the same manufacturer within a single study and across studies that are likely to compare results in a quantitative manner. Incorporation of common reference standards, and application of the same analysis method in assays can overcome many analytical biases and thus could bridge comparison of independent immune profiling (e.g. vaccine immunogenicity) studies. With these recommendations taken into account, the multiplex bead assays performed as described here are useful tools in capturing complex human immune-signatures in observational and interventional studies. FP7 EURIPRED (FP7-INFRA-2012 Grant Agreement No. 312661 to HMcS, HMD, THMO, MMH) and EC HORIZON2020 TBVAC2020 (Grant Agreement No. 643381EC to HMcS, HMD, THMO).

As the latest addition to the sub-strain specific WHO Reference Reagents of BCG vaccine, an international collaborative study was completed to evaluate the suitability of a candidate BCG Moreau-RJ sub-strain as a WHO Reference Reagent of BCG vaccine. This follows the recent replacement of the WHO 1st International Reference Preparation for BCG vaccine, by three sub-strain specific WHO Reference Reagents of BCG vaccine (Danish 1331, Tokyo 172-1 and Russian BCG-I) in order to complete the coverage of most predominant sub-strains used for BCG vaccine production and distribution for use worldwide. The study used cultural viable count and modified ATP assays to quantify the preparation and multiplex PCR to confirm the identity of the sub-strain. The establishment of this WHO Reference Reagent of BCG vaccine of Moreau-RJ sub-strain was approved by the WHO Expert Committee on Biological Standardization meeting in October 2012. This preparation is available for distribution by NIBSC-MHRA, UK. The data from real-time stability monitoring demonstrated that these Reference Reagents of BCG vaccine are very stable in storage condition at −20°C. They serve as the valuable source of BCG Reference Reagents for use as comparators (1) for viability assays (such as cultural viable count and modified ATP assays); (2) for in vivo assays (such as the absence of virulent mycobacteria, dermal reactivity and protection assays) in the evaluation of candidate TB vaccines in non-clinical models; (3) for identity assays using molecular biology techniques.

Multiplex bead-based assays have many advantages over ELISA, particularly for the analyses of large quantities of samples and/or precious samples of limited volume. Although many commercial arrays covering multitudes of biologically significant analytes are available, occasionally the development of custom arrays is necessary. Here, the development of a custom pentaplex sandwich immunoassay using Protein G-coupled beads, for analysis using the Luminex® xMAP® platform, is described. This array was required for the measurement of candidate biomarkers of vaccine safety in small volumes of mouse sera. Optimisation of this assay required a stepwise approach: testing cross-reactivity of the antibody pairs, the development of an in-house serum diluent buffer as well as heat-inactivation of serum samples to prevent interference from matrix effects. We then demonstrate the use of this array to analyse inflammatory mediators in mouse serum after immunisation. The work described here exemplifies how Protein G-coupled beads offer a flexible and robust approach to develop custom multiplex immunoassays, which can be applied to a range of analytes from multiple species.

Strains of Clostridium difficile produce toxins A and B that can cause diarrhoea and pseudomembranous colitis. Currently, there is no preventative therapy for this infection but antibodies to the toxins provide protection, therefore a toxoid-based vaccine is needed. To evaluate thermal stability, a lyophilized and liquid formulation of toxoids A and B were stored at a range of temperatures for 5 weeks. Changes in toxoid structures and immune responses in an animal model before and after the incubation period were assessed. The structural integrity and the immune responses to liquid formulations were affected when stored at 56°C but the lyophilized formulation was thermally stable and same treatment did not result in significant loss of immunological responses when immunized in an animal model.

The WHO First International Reference Preparation for BCG vaccine is over forty years old and is no longer available for distribution due to stock depletion and its significant loss of viability. International consultations identified a demand for replacement with sub-strain specific BCG preparations. An International collaborative study was carried out to evaluate three candidates for WHO Reference Reagent for BCG vaccine of Danish 1331, Russian BCG-I and Tokyo 172-1 sub-strains. These candidates were quantified for viability using both cultural viable count and modified ATP assays. The proposal for the establishment of these First WHO Reference Reagents for BCG vaccines was discussed in the WHO Expert Committee on Biological Standardization meeting, October 2009.

As part of the World Health Organisation (WHO) initiative to update the current requirements for BCG vaccine a collaborative study was carried out to establish the robustness, reproducibility and the suitability of the modified ATP assay. This assay was developed by Statens Serum Institut, Denmark, as a potential replacement of the method for detection of viable counts of BCG vaccine which is routinely used as a quality control test for lot release. Two BCG preparations, of same strain but different production methods, were tested. For each preparation, two different storage conditions of -20 or 37 degrees C were used in order to establish the suitability of this assay for testing heat-treated BCG vaccine as in the temperature stability test. The lyophilised BCG samples were tested using the ATP reagents from the same source and same principle of testing but some procedural modifications were allowed to accommodate different equipment and resource availability in different laboratories. Data from four laboratories showed that the heat-treated BCG samples contained significantly lower ATP content per sample than the untreated control stored at -20 degrees C. Three laboratories gave consistent mean ATP contents, especially for control samples, even with variations in testing protocol. The present study showed that this modified ATP assay is very robust and can be reproducible. Once the correlation of cultural viable count and ATP content of a BCG vaccine product has been established, this rapid alternative assay may be used to monitor BCG viable count. Due to the fact that this study was small, further investigation is planned. A collaborative study will be carried out using this modified ATP assay in parallel with the cultural viable count method in the establishment of the replacement of the WHO International Reference Preparation of BCG vaccine.

A bivalent, unadjuvanted conjugate vaccine composed of Staphylococcus aureus capsular polysaccharides type 5 and 8 (T5 and T8 PS) conjugated to a novel carrier protein, the mutant nontoxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA), has been the subject of recent clinical trials. A program of preclinical laboratory evaluation was carried out in support of the clinical trials conducted by the National Vaccine Evaluation Consortium. This involved physical chemical characterization and limited assessment of toxicity and immunogenicity. The carrier protein showed good stability and its conformation was essentially maintained when conjugated. The T5- and T8-rEPA conjugates were of a size range (1-3 x 10(6) g/ mol) consistent with polysaccharide conjugates. Fluorescence spectroscopy and molecular sizing showed good batch-to-batch consistency. Although all batches of final fill preparations elicited positive immune responses in the mouse model with three schedule doses of 0.25 microg of each T5/8 conjugate per dose, the mouse serum IgG response to T8 PS varied from batch to batch. Storage temperature at 37 degrees C or below or with repetitive temperature fluctuations did not significantly affect the IgG responses to T5 or T8 PS. Storage at 56 degrees C, however, diminished the mouse serum IgG response to T5 PS. The conformation of the conjugated protein and size of the conjugates correlated well with mouse immunogenicity in the thermal stability samples; significant unfolding of the protein and downshifts in molecular size of the conjugate were only observed when stored at 56 degrees C. The relatively high stability of the novel carrier protein when conjugated to large polysaccharides makes this an attractive candidate carrier protein for other conjugate vaccines. When assayed for serum IgG concentration, the bivalent T5/ 8 conjugate was found to evoke an IgG response well over the threshold value of 10 microg/ ml anti-T5 and -T8 IgG established for the ELISA immunogenicity assay.

Tuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

The hereditary stomatocytoses are a group of dominant haemolytic anaemias that show two main features: invaginated, 'stomatocytic' morphology; and a membrane leak to the univalent cations Na and K. A patient with the most severe variant of these conditions was reported to show a defect in an in vitro process of ATP-dependent endocytic vesiculation (ADEV), which is found in normal red cells. We have examined this endocytosis process in 11 leaky red cell pedigrees available to us in the UK. ADEV in broken membranes was absent only in the two most severely affected, 'overhydrated' pedigrees studied, both of which showed a deficiency in the membrane raft protein, stomatin. The process was present, although typically diminished by about 10-20% compared with normal red cells, in all others. The cross-linker dimethyl adipimate (DMA), which could correct the cation leak in some of these patients, also corrected the ADEV defect in the same patients. In those patients in whom DMA had no effect on the ion leak, ADEV was not absent. In normal cells, this process of vesiculation was inhibited by inhibitors of membrane 'raft' function, by an antistomatin antibody and by vanadate and N-ethyl maleimide, but not by inhibitors of a number of kinases. These data highlight the heterogeneity of these conditions. A mechanism is discussed by which a defect in raft-based endocytosis could lead to the exaggerated surface exposure of an ion channel, which could then function constitutively, i.e. 'leak'.

The thermal stability of meningococcal C (MenC)- and Haemophilus influenzae b (Hib)-tetanus toxoid (TT) conjugate vaccines was investigated using spectroscopic and chromatographic techniques and immunogenicity assays in animal models. In this stability study, both the bulk concentrate and final fills were incubated at -20, 4, 23, 37 or 55 degrees C for 5 weeks or subjected to cycles of freeze-thawing. The structural stability, hydrodynamic size and molecular integrity of the treated vaccines were monitored by circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopic techniques, size exclusion chromatography (FPLC-SEC), and high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Only storage at 55 degrees C for 5 weeks caused some slight unfolding and modification in the tertiary structure of the carrier protein in the MenC-TT conjugate. Substantial loss of saccharide content from the MenC conjugates was observed at 37 and 55 degrees C. Unexpectedly, the experimental immunogenicity of MenC-TT vaccine adsorbed to Alhydrogel was significantly reduced only by repeated freeze-thawing, but not significantly decreased by thermal denaturation. Neither the molecular integrity nor the immunogenicity of the lyophilised Hib-TT vaccines was significantly affected by freeze-thawing or by storage at high temperature. In conclusion, the MenC- and Hib-TT conjugate vaccines were relatively stable when stored at higher temperatures, though when MenC-TT vaccine was adsorbed to Alhydrogel, it was more vulnerable to repeated freeze-thawing. When compared with CRM(197) conjugate vaccines studied previously using similar techniques, the tetanus toxoid conjugates were found to have higher relative thermal stability in that they retained immunogenicity following storage at elevated temperatures.

We describe two British families with similar, dominantly-inherited, temperature-related variants of hereditary stomatocytosis, consistent with the original description of 'cryohydrocytosis'. The cells show a 5-6-fold increase in passive permeability at 37 degrees C with abnormal intracellular Na and K levels at 15-20 and 60-65 mmol/(l cells) respectively Marked temperature effects were evident: lysis of red cells on storage in the cold was blatant and when whole heparinized blood was stored at room temperature, It accumulated in the plasma, producing 'pseudohyperkalaemia', Studies of the temperature dependence of passive permeability showed that the minimum in the passive permeability, which is seen in normal cells at 8-10 degrees C, was shifted up to 23 degrees C in these abnormal cells, such that the permeability at 0 degrees C exceeded that at 37 degrees C. The abnormal temperature dependence in these genetically abnormal red cells strongly resembles that seen in normal cells when suspended in media in which either Na or Cl has been replaced by an organic cation or anion: it could be said these cells had a genetic mutation that somehow rendered the cell resistant to the stabilizing action of NaCl at low temperatures.

A family with an unusual form of hereditary stomatocytosis is described. The affected members showed a mild, dominantly-inherited, haemolytic anaemia with intracellular Na and K levels of 41-48 and 44-53 mmol/(l cells) respectively. This anaemia was associated with marked 'pseudohyperkalaemia': that is, loss of K from red cells on storage at room temperature. At 37 degrees C, 'leak' tracer flux rates (assessed as the ouabain + bumetanide-resistant K fluxes) showed a roughly 5-fold acceleration compared to normal, and an abnormal temperature dependence with a shallow slope between 37 and 20 degrees C (mean Q10 (ratio of reaction rates at temperature T and T - 10) over this interval, 1.6; normal 2.2). The pseudohyperkalaemia could be attributed to the disparity between pump and leak at 20 degrees C. This is an identical mechanism to that previously shown for the haemato logically trivial condition, 'familial pseudohyperkalaemia. No protein or lipid abnormality was found in the membrane of these cells.

The current World Health Organization Requirements for BCG vaccine are in need of revision to address the diversity of sub-strains used for production, potential improvements of quality control assays for lot release, and the establishment of sub-strain specific Reference Reagents. A consultation meeting was organized to discuss issues regarding the standardization and evaluation of BCG vaccines in the forum of regulators, BCG vaccine manufacturers, developers of selected new live tuberculosis (TB) vaccines and researchers. The development of new recombinant BCG and live attenuated TB vaccines and the characterisation of different BCG sub-strains using state-of-the-art technologies were also reviewed. The objective of the meeting was to revise and update the current recommendations focused on the scope, terminology, manufacturing issues, and the incorporation of new reference reagents and new quality control tests.

Current methods for the identification of BCG vaccine in quality control settings involve acid-fast staining with microscopic examination. However, this method is unable to distinguish the many different sub-strains of BCG, or to differentiate BCG strains from virulent members of the Mycobacterium tuberculosis complex. A multiplex PCR (mPCR) which uses six target regions in mycobacteria has been developed to identify specific sub-strains of BCG. This study reports the findings from an international collaborative study to assess the accuracy, robustness and reproducibility of this mPCR method to differentiate BCG sub-strains. The method was found to fulfil these criteria successfully and was able to distinguish BCG sub-strains in vaccine preparations. The majority of the participants in the study generated the expected PCR product profiles indicating the method is also robust.

Spectroscopic methods were used to detect modifications in the structures of CRM197, the mutant diphtheria toxin, and meningococcal C capsular oligosaccharide following their conjugation and incubation at various temperatures. Meningococcal C oligosaccharide-CRM197 conjugate vaccines obtained from two different manufacturers were incubated at -20, 4, 23, 37 or 55 degrees C for 5 weeks or subjected to ten cycles of freeze-thawing. The CRM197 carrier protein and the saccharide components of the treated vaccines were monitored by CD and NMR spectroscopic techniques. CD data indicated incubation temperature-dependent conformational changes in the carrier protein from vaccine A. Modifications appeared in both secondary and tertiary structures of the conjugated CRM(197) when incubated at 23 degrees C or above. This was characteristic of the 'open' conformation previously observed for this protein component. The NMR spectra also indicated modification of the structure of the conjugated CRM197 component of vaccine A when incubated at 23 degrees C or above, but failed to show any modification in the conjugated oligosaccharide. On the other hand, the structure of the oligosaccharide chains in vaccine B appeared to be degraded following incubation at 55 degrees C, even though the thermal effect on the conjugated CRM197 was less apparent. Repeated freeze-thawing did not affect the CD or NMR spectra. In conclusion, the two meningococcal C oligosaccharide-CRM197 conjugate vaccines were stable when stored at their recommended temperatures, but were differently affected by elevated temperatures. The conjugates differ in their conjugation chemistry, attachment positions, oligosaccharide chain length and loading, as well as recommended pH and storage buffer, and their different stability properties can probably be attributed to a combination of these factors.

In this stability study, meningococcal C-CRM(197) conjugate vaccines from two different manufacturers that differ in oligosaccharide chain length, number of conjugation sites, conjugation chemistry, manufacturing process and formulation were used. Both the bulk concentrated and final fill preparations were incubated at -20, 4, 23, 37 or 55 degrees C for 5 weeks or subjected to ten cycles of freeze-thawing. The structural stability, hydrodynamic size and integrity of the treated vaccines were monitored by size exclusion chromatography (FPLC-SEC), high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and fluorescence spectroscopy techniques. The data showed that the structural stability of the oligosaccharide chains and of the protein carrier varied between the two conjugates. The experimental immunogenicity was not severely affected by repeated freeze-thawing, incubation at -20 or 4 degrees C, but one developed conformational changes in the protein carrier when incubated at 23 degrees C or above, although the integrity of the oligosaccharide structure was maintained. This was not associated with any reduction in primary IgG or IgM antibody responses to meningococcal C polysaccharide. In the other conjugate vaccine, exposure to 55 degrees C resulted in the release of a substantial proportion of free saccharide that was accompanied by significant reduction in both IgG and IgM antibody responses to immunisation in the model system. In conclusion, the two meningococcal C-CRM(197) conjugate vaccines were stable when stored at the recommended temperatures, although their structural stability and subsequent immunogenicity were influenced by their conjugation chemistry and formulation.

Response to Letter Is Pseudomonas aeruginosa exotoxin A a good carrier protein for conjugate vaccines?Gerald Pier

The mean molecular masses of three different meningococcal C saccharide (MenC)-protein conjugate vaccines and their constituent proteins were estimated using HPLC size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) and refractive-index (RI) detection (SEC/MALLS). Chromatography of two CRM(197) conjugates (MenC-CRM(197)-A and MenC-CRM(197)-B) and one tetanus toxoid (TT) conjugate (MenC-TT) was performed in PBS, pH 7.4, on TSK-Gel (TosoHaas) analytical columns [CRM(197) is a non-catalytic cross-reacting mutant (CRM) of diphtheria toxin]. Analysis of the light-scattering signal measured at 18 angles simultaneously, using the RI signal as a measure of concentration, gave absolute weight-average-molecular-mass (M(w)) values for the CRM(197) conjugates as follows: MenC-CRM(197)-A, approximately 75,000 g x mol(-1) and MenC-CRM(197)-B, approximately 350,000 g x mol(-1), suggesting that MenC-CRM(197)-A is a monomer (one carrier protein per conjugate molecule), while MenC-CRM(197)-B is largely composed of conjugates containing three or four CRM(197) molecules. The MenC-TT conjugate eluted as a two-component system with (M(w)) of 1.63 x 10(6) and 395,000 g x mol(-1), suggesting that some cross-linked complexes contain up to six TT molecules. Comparison of results from MALLS/RI with those obtained using UV detection highlights the differences in size and relative composition of the various subpopulations of the MenC conjugates that can be obtained using different detection systems.