The development and establishment of a heat inactivated preparation of Mycobacterium tuberculosis (H37Rv) as the first international standard for nucleic acid amplification techniques.

A need to develop an inactivated Mycobacterium tuberculosis (Mtb) preparation, to be used as a DNA standard, as an important and urgent requirement for Tuberculosis (TB) diagnostics standardization was identified in 2018. A candidate material was generated using a heat inactivated culture of Mtb strain H37Rv. A lyophilised preparation was evaluated for its suitability as an International Standard for molecular detection of Mtb DNA in an international collaborative study. Together with the use of quantitative PCR assays and rapid diagnostic tests, this candidate standard was demonstrated to be fit-for-purpose. Based on the results from this collaborative study, it is proposed this lyophilised heat inactivated Mtb preparation (NIBSC code: 20/152) to be established by the World Health Organization Expert Committee on Biological Standardization, as the First WHO International Standard for Mycobacterium tuberculosis (H37Rv) for nucleic acid amplification techniques with an assigned unitage of 6.3 log10 or 2 × 106 International Units per vial. The intended uses of this IS are for calibration of secondary or in-house reference preparations used in the assays for the molecular detection of Mtb DNA. It may also be used for assay validation and monitoring the performance in terms of limit of detection of rapid diagnostic tests.

Natural Killer Anti-Tumor Activity Can Be Achieved by In Vitro Incubation With Heat-Killed BCG.

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.

BCG Therapy of Bladder Cancer Stimulates a Prolonged Release of the Chemoattractant CXCL10 (IP10) in Patient Urine.

Background: Intra-vesical instillation of Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is an effective therapy for high-grade non-muscle invasive bladder cancer (NMIBC), which provokes a local immune response resulting in 70% of patients free of relapse after three years. Because non-responder patients usually have a bad prognosis, the early identification of treatment failure is crucial. We hypothesized that, if an effective immune response was taking place in the bladder, soluble factors would be released to the urine many days after BCG instillations. Methods: An extensive panel of cytokines and chemokines released into the urine seven days after every BCG instillation was screened in a cohort of NMIBC patients over three years. Results: The determinations of the urinary concentrations of cytokines, chemokines, and creatinine showed that increasing concentrations of C-X-C motif chemokine 10 (CXCL10) also known as interferon-inducible protein 10 (IP10) could be detected during the six-week induction cycle of BCG-treated patients released into the urine by CD14+ cells. In vitro, CXCL10 facilitated the recruitment of effector immune cells after the BCG-mediated upregulation of CXCR3 in both T- and natural killer (NK)-cells. Conclusions: The high concentrations of chemokine detected one week after the encounter with mycobacteria suggest that the CXCL10 axis might be related to the intensity of the immune anti-tumor response.

Cross-laboratory evaluation of multiplex bead assays including independent common reference standards for immunological monitoring of observational and interventional human studies.

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.

Characterization of a human anti-tumoral NK cell population expanded after BCG treatment of leukocytes.

Immunotherapy, via intra-vesical instillations of 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, is believed to induce a local immune response that eliminates these tumors, but the detailed mechanisms of action of this therapy are not well understood. Here, we have studied the phenotype and function of the responding lymphocyte populations as well as the spectrum of cytokines and chemokines produced in an in vitro model of human peripheral blood mononuclear cells (PBMCs) co-cultured with BCG. Natural killer (NK) cell activation was a prominent feature of this immune response and we have studied the expansion of this lymphocyte population in detail. We show that, after BCG stimulation, CD56dim NK cells proliferate, upregulate CD56, but maintain the expression of CD16 and the ability to mediate ADCC. CD56bright NK cells also contribute to this expansion by increasing CD16 and KIR expression. These unconventional CD56bright cells efficiently degranulated against bladder cancer cells and the expansion of this population required the release of soluble factors by other immune cells in the context of BCG. Consistent with these in vitro data, a small, but significant increase in the intensity of CD16 expression was noted in peripheral blood CD56bright cells from bladder cancer patients undergoing BCG therapy, that was not observed in patients treated with mitomycin-C instillations. These observations suggest that activation of NK cells may be an important component of the anti-tumoral immune response triggered by BCG therapy in bladder cancer.

Development of a custom pentaplex sandwich immunoassay using Protein-G coupled beads for the Luminex® xMAP® platform.

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.

The establishment of sub-strain specific WHO Reference Reagents for BCG vaccine.

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.

An assessment of thermal stability of Clostridium difficile toxoid formulations.

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.

Report of an International collaborative study to establish the first WHO reference reagents for BCG vaccines of three different sub-strains.

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.

WHO Informal Consultation on standardization and evaluation of BCG vaccines Geneva, Switzerland 22-23 September 2009.

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.

Report of an international collaborative study to evaluate the suitability of multiplex PCR as an identity assay for different sub-strains of BCG vaccine.

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.

Report of an international collaborative study to establish the suitability of using modified ATP assay for viable count of BCG vaccine.

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.

Preclinical laboratory evaluation of a bivalent Staphylococcus aureus saccharide-exotoxin A protein conjugate 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) immunoassay as an in vitro alternative assay for identity and confirmation of potency.

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 "stomatin" gene and protein in overhydrated hereditary stomatocytosis.

In overhydrated hereditary stomatocytosis (OHSt), Coomassie- and silver-stained polyacrylamide gels show an apparently complete deficit of the 32-kDa membrane protein, stomatin. We have used an antistomatin antibody to examine peripheral blood films, bone marrow, splenic tissue, and hepatic tissue from these patients by immunocytochemistry. This technique revealed that, in fact, some red cells did show positive stomatin immunoreactivity; and consistent with this result, Western blot analysis of the red cell membranes confirmed that about one twentieth to one fiftieth of the normal amount of stomatin was in fact present. Flow cytometry, combining immunoreactive quantitation of stomatin expression with thiazole orange staining for reticulocytes, showed that in OHSt, it was the young cells that had more stomatin. Magnetic-activated cell separation studies, using beads to which an anti-transferrin receptor antibody was conjugated, confirmed that in OHSt there was a correspondence between expression of stomatin and the transferrin receptor. Immunocytochemistry and Western blotting revealed that in OHSt patients, the protein was present in spleen, liver, neutrophils, platelets, monocytes, and about 50% of the peripheral lymphocytes, with the same distribution as in healthy controls. Neither Southern blots, nor direct sequencing of multiple subclones of the cDNA, nor sequencing of amplicons from genomic DNA revealed any significant abnormality in stomatin gene sequence in these patients. The deficiency of stomatin from red cells appears to be due to a loss of stomatin from these red cells on maturation in the bone marrow and in the circulation.

Evaluation of meningococcal C oligosaccharide conjugate vaccines by size-exclusion chromatography/multi-angle laser light scattering.

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.

Physico-chemical and immunological examination of the thermal stability of tetanus toxoid conjugate vaccines.

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.