Development of a monoclonal antibody sandwich ELISA for the determination of antigen content and quality in diphtheria vaccines.

At present, quality control of diphtheria vaccines by both manufacturers and national control laboratories relies heavily on in vivo assays to confirm potency. As part of the VAC2VAC project we have developed a monoclonal antibody (mAb) enzyme-linked immunosorbent assay (ELISA) to measure the relative amount and quality of diphtheria toxoid (DTxd) in diphtheria-tetanus based vaccines and believe this test has the potential to play a key role in a control strategy no longer including an in vivo potency test. The mAb ELISA is highly specific, has good dilutional linearity, and is suitable for detecting DTxd in a range of different human vaccine products. We demonstrate the ability of the assay to discriminate between batches of different content and quality using vaccine batches that were prepared to contain differing amounts of DTxd or were altered by exposure to heat or oxidative stress. We also demonstrate successful transfer of the method to other laboratories and show that different diphtheria antigen materials may be able to serve as a reference antigen for local standardization of the method. The assay is ideally suited for incorporation into a consistency approach for routine diphtheria vaccine quality control testing and may be suitable to serve as the stability indicating test in replacement of the current in vivo potency test.

Diphtheria vaccines help to protect against diphtheria infection. Currently, animal tests are used to ensure the potency of such vaccines. Since these tests were first introduced, there have been improvements in non-animal technologies that can be used to ensure consistent production of potent vaccine batches. To demonstrate that a new batch of diphtheria vaccine is consistent with a previous batch of known potency, the quality and amount of the component that stimulates the immune response upon vaccination must be assessed in comparison. We have developed an assay that can measure the quality of a range of different diphtheria vaccine product types. The assay is very specific and reliable, and different laboratories obtained comparable results, showing that the assay is suited for routine use. Once validated by manufacturers and recognized by regulators, this assay will greatly reduce the number of animals needed for batch release of diphtheria vaccines.

Development of a multiplex-based immunoassay for the characterization of diphtheria, tetanus and acellular pertussis antigens in human combined DTaP vaccines.

Routine batch quality testing before vaccine release, notably for potency evaluation, still relies on animal use for several animal and human vaccines. In this context, the VAC2VAC project is a public-private consortium of 22 partners funded by EU whose the main objective is to reduce the number of animal used for batch testing by developing immunoassays that could be implemented for routine potency assessment of vaccines. This paper focused on the development of a Luminex-based multiplex assay to monitor the consistency of antigen quantity and quality throughout the production process of DTaP vaccines from two human vaccine manufacturers. Indepth characterized monoclonal antibody pairs were used for development and optimization of the Luminex assay with non-adsorbed and adsorbed antigens and with complete vaccine formulations from both manufacturers. The multiplex assay demonstrated good specificity, reproducibility and absence of cross-reactivity. Analysis of over and underdosed formulations, heat and H2O2-degraded products as well as batch to batch consistency of vaccines from both manufacturers brought the proof of concept for a future application of the multiplex immunoassay as a useful tool in the frame of DTaP vaccine quality control.

Potential business model for a European vaccine R&D infrastructure and its estimated socio-economic impact.

Background: Research infrastructures are facilities or resources that have proven fundamental for supporting scientific research and innovation. However, they are also known to be very expensive in their establishment, operation and maintenance. As by far the biggest share of these costs is always borne by public funders, there is a strong interest and indeed a necessity to develop alternative business models for such infrastructures that allow them to function in a more sustainable manner that is less dependent on public financing. Methods: In this article, we describe a feasibility study we have undertaken to develop a potentially sustainable business model for a vaccine research and development (R&D) infrastructure. The model we have developed integrates two different types of business models that would provide the infrastructure with two different types of revenue streams which would facilitate its establishment and would be a measure of risk reduction. For the business model we are proposing, we have undertaken an ex ante impact assessment that estimates the expected impact for a vaccine R&D infrastructure based on the proposed models along three different dimensions: health, society and economy. Results: Our impact assessment demonstrates that such a vaccine R&D infrastructure could achieve a very significant socio-economic impact, and so its establishment is therefore considered worthwhile pursuing. Conclusions: The business model we have developed, the impact assessment and the overall process we have followed might also be of interest to other research infrastructure initiatives in the biomedical field.

Listeria monocytogenes isolates from Cornu aspersum snails: Whole genome-based characterization and host-pathogen interactions in a snail infection model.

Even though Listeria monocytogenes is an extensive-studied foodborne pathogen, genome analysis of isolates from snails that may represent a reservoir of L. monocytogenes are still scarce. Here, we use whole-genome sequencing (WGS) to assess the genomic diversity of hypervirulent, virulent and non-virulent phenotypes of 15 L. monocytogenes isolated from snails to unveil their survival, virulence, and host-pathogen mechanisms of interactions in a snail infection model. Most of isolates (66.7%) were characterized as multidrug resistant (MDR) and belonged to clonal complexes (CCs) which are strongly associated with cases of human infection. All isolates contained intact genes associated with invasion and infection while hypervirulent isolates are adapted to host environment, possessing genes which are involved in teichoic acid biosynthesis, peptidoglycan modification and biofilm formation, correlating with their tolerance to haemolymph plasma phenotype and biofilm formation ability. A snail infection model showed that hypervirulent isolates triggered programmed host cell death pathway by increasing up to 30% the circulating apoptotic hemocytes in combination with induced nitrate production and reactive oxygen species (ROS) generation in snails' haemolymph. In contrast, the administration of the non-virulent strain which possesses a truncated mogR gene that regulates flagellar motility gene expression led only to an increase of necrotic non-apoptotic cells. Overall, this study provides significant insights into the genetic diversity of L. monocytogenes from snails, the genomic features of them linked to their hypervirulent/non-virulent phenotype, and the mechanisms of host-pathogen interactions.

Development and validation of a targeted LC-MS/MS quantitation method to monitor cell culture expression of tetanus neurotoxin during vaccine production.

The tetanus neurotoxin (TeNT) is one of the most toxic proteins known to man, which prior to the use of the vaccine against the TeNT producing bacteria Clostridium tetani, resulted in a 20% mortality rate upon infection. The clinical detrimental effects of tetanus have decreased immensely since the introduction of global vaccination programs, which depend on sustainable vaccine production. One of the major critical points in the manufacturing of these vaccines is the stable and reproducible production of high levels of toxin by the bacterial seed strains. In order to minimize time loss, the amount of TeNT is often monitored during and at the end of the bacterial culturing. The different methods that are currently available to assess the amount of TeNT in the bacterial medium suffer from variability, lack of sensitivity, and/or require specific antibodies. In accordance with the consistency approach and the three Rs (3Rs), both aiming to reduce the use of animals for testing, in-process monitoring of TeNT production could benefit from animal and antibody-free analytical tools. In this paper, we describe the development and validation of a new and reliable antibody free targeted LC-MS/MS method that is able to identify and quantify the amount of TeNT present in the bacterial medium during the different production time points up to the harvesting of the TeNT just prior to further upstream purification and detoxification. The quantitation method, validated according to ICH guidelines and by the application of the total error approach, was utilized to assess the amount of TeNT present in the cell culture medium of two TeNT production batches during different steps in the vaccine production process prior to the generation of the toxoid. The amount of TeNT generated under different physical stress conditions applied during bacterial culture was also monitored.

Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Batch release testing for human and veterinary tetanus vaccines still relies heavily on methods that involve animals, particularly for potency testing. The quantity and quality of tetanus antigen present in these products is of utmost importance for product safety and clinical effect. Immunochemical methods that measure consistency of antigen content and quality, potentially as an indicator of potency, could be a better choice and negate the need for an in vivo potency test. These immunochemical methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against tetanus with a view to select antibodies that can be used for development of an in vitro potency immunoassay. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined using an in-house cell-based neutralisation assay to support prior in vivo potency data that was available for some, but not all, of the antibodies. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Characterisation of diphtheria monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Immunoassays are used for routine potency assessment of several vaccines, in some cases having been specifically developed as alternatives to in vivo potency tests. These methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against diphtheria with a view to select antibodies that can be used for development of an in vitro potency immunoassay for diphtheria vaccines. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined by a cell-based toxin neutralisation test and diphtheria toxin-domain recognition was determined by Western blotting. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Recommendations of the VAC2VAC workshop on the design of multi-centre validation studies.

Within the Innovative Medicines Initiative 2 (IMI 2) project VAC2VAC (Vaccine batch to vaccine batch comparison by consistency testing), a workshop has been organised to discuss ways of improving the design of multi-centre validation studies and use the data generated for product-specific validation purposes. Moreover, aspects of validation within the consistency approach context were addressed. This report summarises the discussions and outlines the conclusions and recommendations agreed on by the workshop participants.

Influence of maternal vaccination against diphtheria, tetanus, and pertussis on the avidity of infant antibody responses to a pertussis containing vaccine in Belgium.

Maternal antibodies induced by vaccination during pregnancy cross the placental barrier and can close the susceptibility gap to pertussis in young infants up to the start of primary immunization. As not only the quantity but also the quality of circulating antibodies is important for protection, we assessed whether maternal immunization affects the avidity of infant vaccine-induced IgG antibodies, in the frame of a prospective clinical trial on pregnancy vaccination in Belgium. Infants born from Tdap (Boostrix®) vaccinated (N = 55) and unvaccinated (N = 26) mothers were immunized with a hexavalent pertussis containing vaccine (Infanrix Hexa®) at 8, 12 and 16 weeks, followed by a fourth dose at 15 months of age. Right before and one month after this fourth vaccine dose, the avidity of IgG antibodies against diphtheria toxin (DT), tetanus toxin (TT), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (Prn) was determined using 1.5 M ammonium thiocyanate as dissociating agent. In both groups, antibody avidity was moderate for TT, PT, FHA and Prn and low for DT after priming. After a fourth dose, antibody avidity increased significantly to high avidity for TT and PT, whereas it remained moderate for FHA and Prn and low for DT. The avidity correlated positively with antibody level in both study groups, yet not significantly for PT. When comparing both study groups, only PT-specific antibodies showed significantly lower avidity in infants born from vaccinated than from unvaccinated mothers after the fourth vaccine dose. The clinical significance of lower avidity of vaccine induced infant antibodies after maternal vaccination, if any, needs further investigation.

Serologic screening for 13 infectious agents in roe deer (Capreolus capreolus) in Flanders.

INTRODUCTION: In order to investigate the role of roe deer in the maintenance and transmission of infectious animal and human diseases in Flanders, we conducted a serologic screening in 12 hunting areas. MATERIALS AND METHODS: Roe deer sera collected between 2008 and 2013 (n=190) were examined for antibodies against 13 infectious agents, using indirect enzyme-linked immunosorbent assay, virus neutralisation, immunofluorescence, or microagglutination test, depending on the agent. RESULTS AND DISCUSSION: High numbers of seropositives were found for Anaplasma phagocytophilum (45.8%), Toxoplasma gondii (43.2%) and Schmallenberg virus (27.9%), the latter with a distinct temporal distribution pattern following the outbreak in domestic ruminants. Lower antibody prevalence was found for Chlamydia abortus (6.7%), tick-borne encephalitis virus (5.1%), Neospora caninum (4.8%), and Mycobacterium avium subsp paratuberculosis (4.1%). The lowest prevalences were found for Leptospira (1.7%), bovine viral diarrhoea virus 1 (1.3%), and Coxiella burnetii (1.2%). No antibodies were found against Brucella sp., bovine herpesvirus 1, and bluetongue virus. A significant difference in seroprevalence between ages (higher in adults >1 year) was found for N. caninum. Four doubtful reacting sera accounted for a significant difference in seroprevalence between sexes for C. abortus (higher in females). CONCLUSIONS: Despite the more intensive landscape use in Flanders, the results are consistent with other European studies. Apart from maintaining C. abortus and MAP, roe deer do not seem to play an important role in the epidemiology of the examined zoonotic and domestic animal pathogens. Nevertheless, their meaning as sentinels should not be neglected in the absence of other wild cervid species.

Trends in genotype frequency resulting from breeding for resistance to classical scrapie in Belgium (2006 ˜ 2011).

In sheep, susceptibility to scrapie is mainly determined by codons 136, 154, and 171 of the PRNP gene. Five haplotypes are usually present (ARR, ARQ, ARH, AHQ, and VRQ). The ARR haplotype confers the greatest resistance to classical scrapie while VRQ renders animals most susceptible. In 2004, the European Union implemented a breeding program that promotes selection of the ARR haplotype while reducing the incidence of VRQ. From 2006 to 2011 in Belgium, frequency for the ARR/ARR genotypes increased from 38.3% to 63.8% (n = 6,437), the ARQ haplotype diminished from 21.1% to 12.9%, and the VRQ haplotype decreased from 2.0% to 1.7%. The status of codon 141, a determinant for atypical scrapie, was also evaluated. Out of 27 different breeds (n = 5,163), nine were abundant. The ARR/ARR frequency increased in eight of these nine major breeds. The selection program has had a major impact on the ARR haplotype frequency in Belgium. However, the occurrence of atypical scrapie represents a critical point for this program that warrants the continuous monitoring of scrapie. Additionally, genotype frequencies among the breeds varied greatly. Texel, a breed that is common in Belgium, can still be selected for due to its average ARR frequency.

Spatiotemporal dynamics of Puumala hantavirus in suburban reservoir rodent populations.

The transmission of pathogens to susceptible hosts is dependent on the vector population dynamics. In Europe, bank voles (Myodes glareolus) carry Puumala hantavirus, which causes nephropathia epidemica (NE) in humans. Fluctuations in bank vole populations and epidemics in humans are correlated but the main factors influencing this relationship remain unclear. In Belgium, more NE cases are reported in spring than in autumn. There is also a higher incidence of human infections during years of large vole populations. This study aimed to better understand the link between virus prevalence in the vector, vole demography, habitat quality, and human infections. Three rodent populations in different habitats bordering Brussels city, Belgium, were studied for two years. The seroprevalence in voles was influenced first by season (higher in spring), then by vole density, vole weight (a proxy for age), and capture site but not by year or sex. Moreover, voles with large maximal distance between two captures had a high probability for Puumala seropositivity. Additionally, the local vole density showed similar temporal variations as the number of NE cases in Belgium. These results showed that, while season was the main factor influencing vole seroprevalence, it was not sufficient to explain human risks. Indeed, vole density and weight, as well as the local habitat, were essential to understanding the interactions in these host-pathogen dynamics. This can, in turn, be of importance for assessing the human risks.

Tick-borne encephalitis virus seropositive dog detected in Belgium: screening of the canine population as sentinels for public health.

Tick-borne encephalitis virus (TBEV) is an important emerging tick-borne viral infection of humans and dogs in Europe. Currently, TBEV surveillance is virtually nonexistent in Belgium, which is considered nonendemic. A commercial enzyme-linked immunosorbent assay (ELISA) was adapted for the detection of TBEV-specific IgG-antibodies in canine sera. Serum samples of Belgian dogs were obtained from three diagnostic laboratories from Northern (n=688) and Southern Belgium (n=192). ELISA-positive and borderline samples were subjected to a TBEV rapid fluorescent focus inhibition confirmation test. One dog was confirmed TBEV seropositive. Several ELISA-positive and borderline sera underwent seroneutralization and hemagglutinin inhibition tests to rule out West Nile and Louping Ill viruses, but tested negative. The clinical history of the seropositive dog could not explain beyond doubt where and when TBEV infection was acquired. Further surveillance is necessary to determine whether this dog remains a single travel-related case or whether it represents an early warning of a possible future emergence of TBEV.

No H- and L-type cases in Belgium in cattle diagnosed with bovine spongiform encephalopathy (1999-2008) aging seven years and older.

BACKGROUND: The bovine spongiform encephalopathy (BSE) epidemic presented homogeneity of the phenotype. This classical BSE (called C-type) was probably due to the contamination of the food chain by a single prion strain. However, due to the active surveillance and better techniques, two rare variants of BSE have been recently reported in different continents without a clear correlation to the BSE epidemic. These emerging types behave as different strains of BSE and were named H-type and L-type according to the high and low molecular mass of the unglycosylated fragment of their proteinase K resistant prion protein (PrPres). In these types, the proportion of the un-, mono- and di-glycosylated fragments of PrP (glycoprofile) is also atypical and represents an effective diagnostic parameter. This study evaluated the presence of such types in bovine of 7 years and older in Belgium. RESULTS: The Belgian BSE archive contained 41 bovines of at least 7 years of age. The biochemical features of their PrPres were analyzed by Western blot with five antibodies recognising different regions of PrPres, from N- to C-terminus: 12B2, 9A2, Sha31, SAF84 and 94B4. All antibodies clearly detected PrPres except 12B2 antibody, which is specific for N-terminal region 101-105, a PrP region that is only retained in H-types. The glycoprofiles did correspond to that of C-type (with more than 55% of diglycosylated PrPres using antibody 94B4). Therefore, all cases have the features of C-type BSE. CONCLUSIONS: This study supports that, among the BSE cases of 7 years and older identified in Belgium, none was apparently of the H- or L- type. This is consistent with the very rare occurrence of atypical BSE and the restricted dimension of Belgium. These results shed some light on the worldwide prevalence of atypical BSE.

Association between habitat and prevalence of hantavirus infections in bank voles (Myodes glareolus) and wood mice (Apodemus sylvaticus).

In order to determine the habitat preferred by Myodes (before Clethrionomys) glareolus and the corresponding Puumala hantavirus seroprevalence in those habitats, we captured rodents simultaneously in three significantly different habitats. We compared trapping success and presence of virus per habitat during an ongoing epidemic in order to test the hypothesis of a density-dependent seroprevalence. Our study showed that bank vole population density, as well as Puumala virus seroprevalence, were habitat dependent. Apodemus sylvaticus was found more vulnerable for deteriorating habitat conditions than M. glareolus and could play a role as vehicle for Puumala virus and as mediator for inter- and conspecific virus transmission.

Recording strategies and selection potential of feed intake measured using the X-ray method in rainbow trout.

This study examines the way long-term feed intake should be recorded accurately for selective breeding purposes, and estimates selection potential in feed intake using the X-ray method to record individual daily feed intake in rainbow trout (Oncorhynchus mykiss). The analysis showed that the point estimates of daily feed intake displayed low repeatabilities (r = 0.09-0.32). This indicates that a minimum of three repeated records were needed to accurately record average feed intake at a fixed age. To effectively breed for feed intake over the whole growing period, it is necessary to determine average feed intake at different ages, since there were only moderate phenotypic and genetic correlations between average daily feed intake recorded at 140 g, 750 g and 2000 g wet mass. Heritability for average daily feed intake was low (average h(2) = 0.10), indicating that modest genetic changes can be obtained in response to selection. It was concluded that selection to genetically change long-term feed intake can be successful, yet repeated observations at several life stages are needed to ensure the accuracy of feed intake estimates and the efficiency of selection.