Transcriptome and proteome analysis of innate immune responses to inactivated Leptospira and bivalent Leptospira vaccines in canine 030-D cells.

Mandatory potency testing of Leptospira vaccine batches relies partially on in vivo procedures, requiring large numbers of laboratory animals. Cell-based assays could replace in vivo tests for vaccine quality control if biomarkers indicative of Leptospira vaccine potency are identified. We investigated innate immune responsiveness induced by inactivated L. interrogans serogroups Canicola and Icterohaemorrhagiae, and two bivalent, non-adjuvanted canine Leptospira vaccines containing the same serogroups. First, the transcriptome and proteome analysis of a canine monocyte/macrophage 030-D cell line stimulated with Leptospira strains, and vaccine B revealed more than 900 DEGs and 23 DEPs in common to these three stimuli. Second, comparison of responses induced by vaccine B and vaccine D revealed a large overlap in DEGs and DEPs as well, suggesting potential to identify biomarkers indicative of Leptospira vaccine quality. Because not many common DEPs were identified, we selected seven molecules from the identified DEGs, associated with pathways related to innate immunity, of which CXCL-10, IL-1ß, SAA, and complement C3 showed increased secretion upon stimulation with both Leptospira vaccines. These molecules could be interesting targets for development of biomarker-based assays for Leptospira vaccine quality control in the future. Additionally, this study contributes to the understanding of the mechanisms by which Leptospira vaccines induce innate immune responses in the dog.

Mass Spectrometry-Based Quantification of the Antigens in Aluminum Hydroxide-Adjuvanted Diphtheria-Tetanus-Acellular-Pertussis Combination Vaccines.

Vaccines undergo stringent batch-release testing, most often including in-vivo assays for potency. For combination vaccines, such as diphtheria-tetanus-pertussis (DTaP), chemical modification induced by formaldehyde inactivation, as well as adsorption to aluminum-based adjuvants, complicates antigen-specific in-vitro analysis. Here, a mass spectrometric method was developed that allows the identification and quantitation of DTaP antigens in a combination vaccine. Isotopically labeled, antigen-specific internal standard peptides were employed that permitted absolute quantitation of their antigen-derived peptide counterparts and, consequently, the individual antigens. We evaluated the applicability of the method on monovalent non-adjuvanted antigens, on final vaccine lots and on experimental vaccine batches, where certain antigens were omitted from the drug product. Apart from the applicability for final batch release, we demonstrated the suitability of the approach for in-process control monitoring. The peptide quantification method facilitates antigen-specific identification and quantification of combination vaccines in a single assay. This may contribute, as part of the consistency approach, to a reduction in the number of animal tests required for vaccine-batch release.

Proteomic analysis of chicken bone marrow-derived dendritic cells in response to an inactivated IBV + NDV poultry vaccine.

Inactivated poultry vaccines are subject to routine potency testing for batch release, requiring large numbers of animals. The replacement of in vivo tests for cell-based alternatives can be facilitated by the identification of biomarkers for vaccine-induced immune responses. In this study, chicken bone marrow-derived dendritic cells were stimulated with an inactivated vaccine for infectious bronchitis virus and Newcastle disease virus, as well as inactivated infectious bronchitis virus only, and lipopolysaccharides as positive control, or left unstimulated for comparison with the stimulated samples. Next, the cells were lysed and subjected to proteomic analysis. Stimulation with the vaccine resulted in 66 differentially expressed proteins associated with mRNA translation, immune responses, lipid metabolism and the proteasome. For the eight most significantly upregulated proteins, mRNA expression levels were assessed. Markers that showed increased expression at both mRNA and protein levels included PLIN2 and PSMB1. Stimulation with infectious bronchitis virus only resulted in 25 differentially expressed proteins, which were mostly proteins containing Src homology 2 domains. Stimulation with lipopolysaccharides resulted in 118 differentially expressed proteins associated with dendritic cell maturation and antimicrobial activity. This study provides leads to a better understanding of the activation of dendritic cells by an inactivated poultry vaccine, and identified PLIN2 and PSMB1 as potential biomarkers for cell-based potency testing.

As exigências cognitivas e a complexidade do trabalho para o agricultor urbano: um estudo de caso / The cognitive demands and the complexity of work for the urban farmers: a case study

Resumo O trabalho na agricultura vem aumentando as exigências impostas ao trabalhador com o uso de tecnologias. Porém, para atividades agrícolas onde o incremento do uso de tecnologias é limitado, como na agricultura urbana orgânica, pouco se sabe sobre a carga cognitiva da atividade. Atualmente, não há registros de estudos sobre a carga cognitiva de trabalhadores da agricultura urbana orgânica, mesmo porque esse ainda é um movimento tímido. A importância deste estudo reside no fato de aumentar o conhecimento e as informações sobre esta população para fomentar o desenvolvimento da área. O objetivo deste artigo foi relatar as exigências cognitivas impostas a estes trabalhadores de forma a identificar a complexidade do seu trabalho. Para isso, realizou-se um estudo de caso com agricultores urbanos que utilizam práticas orgânicas, comercializam seus produtos e fazem dessa sua atividade econômica principal. Os achados comprovam que a carga cognitiva é elevada, contribuindo para um trabalho complexo para estes agricultores. Assim, a importância em ampliar os estudos para validar as informações encontradas e contribuir para a construção de políticas públicas efetivas é urgente.

Abstract Agricultural work has been increasing the demands placed on workers with the use of technologies. However, for agricultural activities, in which the increase in the use of technologies is limited, such as organic urban agriculture, little is known about the cognitive load of this activity. Currently, there are no records of studies on the cognitive load of workers in organic urban agriculture, since this is still a timid movement. The importance of this study lies in the fact that it increases the knowledge and information about this population to encourage the development of the area. The purpose of this article was to report the cognitive requirements imposed on these workers to identify the complexity of their work. A case study was conducted with urban farmers that use organic practices, market their products and make of this their main economic activity. The findings prove that the cognitive load is high, contributing to a complex job for these farmers. Thus, the importance of expanding the studies to validate the information found and contribute to the construction of effective public policies is urgent.

The Role of Virulence Proteins in Protection Conferred by Bordetella pertussis Outer Membrane Vesicle Vaccines.

The limited protective immunity induced by acellular pertussis vaccines demands development of novel vaccines that induce broader and longer-lived immunity. In this study, we investigated the protective capacity of outer membrane vesicle pertussis vaccines (omvPV) with different antigenic composition in mice to gain insight into which antigens contribute to protection. We showed that total depletion of virulence factors (bvg(-) mode) in omvPV led to diminished protection despite the presence of high antibody levels. Antibody profiling revealed overlap in humoral responses induced by vaccines in bvg(-) and bvg(+) mode, but the potentially protective responses in the bvg(+) vaccine were mainly directed against virulence-associated outer membrane proteins (virOMPs) such as BrkA and Vag8. However, deletion of either BrkA or Vag8 in our outer membrane vesicle vaccines did not affect the level of protection. In addition, the vaccine-induced immunity profile, which encompasses broad antibody and mixed T-helper 1, 2 and 17 responses, was not changed. We conclude that the presence of multiple virOMPs in omvPV is crucial for protection against Bordetella pertussis. This protective immunity does not depend on individual proteins, as their absence or low abundance can be compensated for by other virOMPs.

[Results of lack of policies to encourage urban organic farming: a case study in two Brazilian cities]. / O reflexo da ausência de políticas de incentivo à agricultura urbana orgânica: um estudo de caso em duas cidades no Brasil.

Urban organic farming is an important stimulus for cities' sustainable development, contributing to food security, environmental improvement, social inclusion, and income generation. This study aimed to assess the main difficulties faced by urban organic farmers in cities where there are no policies to encourage urban farming. Semi-structured interviews were applied from July to December 2018 with seven urban organic farmers who had adopted this practice as their profession. The results were examined with content analysis, detecting such gaps as lack of trained labor, lack of adequate machinery and equipment for this practice in small areas, and insufficient financing, all of which were similar to family organic farming. It is thus essential to expand policies to promote urban organic farming, addressing these issues to decrease the barriers and stimulate the profession.

Intranasal immunization with outer membrane vesicle pertussis vaccine confers broad protection through mucosal IgA and Th17 responses.

A vaccine based on outer membrane vesicles of pertussis (omvPV) is protective in a mouse-challenge model and induces a broad antibody and mixed Th1/Th2/Th17 response against multiple antigens following subcutaneous immunization. However, this route did not result in mucosal immunity and did not prevent nasopharyngeal colonization. In this study, we explored the potential of intranasal immunization with omvPV. Only intranasal immunization induced strong mucosal immune responses that encompasses enhanced pulmonary and nasal IgA antibody levels, mainly directed against Vag8 and LPS. Furthermore, high numbers of IgA- and IgG-producing plasma cells were detected as well as lung-resident IgA memory B-cells. Finally, only intranasal immunization induced pulmonary Th1/Th17-related cytokine responses. The magnitude and type of systemic immunity was comparable between both routes and included high systemic IgG antibody levels, strong IgG-producing plasma cell responses, memory B-cells residing in the spleen and systemic Th1/Th2/Th17-related cytokine responses. Importantly, only intranasal immunization prevented colonization in both the lungs and the nasal cavity. In conclusion, intranasal omvPV immunization induces mucosal IgA and Th17-mediated responses without influencing the systemic immunity profile. These responses resulted in prevention of Bordetella pertussis colonization in the respiratory tract, including the nasal cavity, thereby potentially preventing transmission.

Work in organic farming: an overview / O trabalho na agricultura orgânica: uma visão geral

ABSTRACT: In addition to the expansion of sustainability with crop rotation and use of residues from the property itself, the main characteristics of organic agriculture are the extinction of the use of chemicals and producing food free of these contaminants. This review aimed to find evidence that substantiates the improvement of the health and working conditions of farmers working with this model of agriculture. To this end, the ProKnow-C instrument was used, which recommends seven steps to select and evaluate the publications found. Exclusion criteria included studies related to the consumption of these products, school feeding, management, and cultivation techniques or studies conducted with non-organic farmers. After the exclusion, 11 documents remained for content analysis. Results of these studies indicated that when compared to conventional agriculture, there is an increase in cognitive load due to the variety of tasks; however, no mental suffering was identified. Despite the absence of appropriate technology and technical assistance, there is an increase in job satisfaction and an improvement of the health of the family as a whole, improving the quality of life. Additionally, the results of this review pointed to the gaps of research that can be carried out, such as longitudinal studies on organic agriculture, social interactions, environmental working conditions, and studies in the technological area. Finally, the results evinced that organic agriculture has a positive impact on the health of workers. Thus, the need for empirical research is important to develop innovations that improve work in organic agriculture.

RESUMO: A agricultura orgânica tem como principal característica a extinção do uso de produtos químicos gerando alimentos livres desses contaminantes, além de ampliar a sustentabilidade com rotação de culturas e uso de resíduos oriundos da própria propriedade. O objetivo desta revisão foi encontrar evidências que comprovem a melhoria das condições de saúde e trabalho de agricultores que trabalham com esse modelo de agricultura. Para tanto, foi utilizado o instrumento ProKnow-C que preconiza 7 etapas para selecionar e avaliar as publicações encontradas. Os critérios de exclusão foram estudos ligados ao consumo desses produtos, alimentação escolar, técnicas de manejo e cultivo ou estudos realizados com agricultores não orgânicos. Após, 11 documentos restaram para a análise do conteúdo. Como resultados, os estudos apontaram que, quando comparado à agricultura convencional, há aumento da carga cognitiva devido à variedade das tarefas, no entanto não identificou-se sofrimento mental. Apesar da ausência de tecnologia apropriada e de assistência técnica, há um aumento da satisfação com o trabalho, melhoria da saúde da família como um todo, melhorando a qualidade de vida. Ainda como resultados, esta revisão aponta as lacunas de pesquisas que podem ser realizadas, como estudos longitudinais sobre a agricultura orgânica, as interações sociais, as condições ambientais de trabalho, e estudos na área tecnológica. Enfim, concluem que a agricultura orgânica interfere positivamente na saúde dos trabalhadores. Portanto, a necessidade de pesquisas empíricas é iminente para desenvolver inovações que aprimorem o trabalho na agricultura orgânica.

O reflexo da ausência de políticas de incentivo à agricultura urbana orgânica: um estudo de caso em duas cidades no Brasil / Results of lack of policies to encourage urban organic farming: a case study in two Brazilian cities / El reflejo de la ausencia de políticas de incentivo a la agricultura urbana orgánica: un estudio de caso en dos ciudades en Brasil

A agricultura urbana orgânica é um importante estímulo para o desenvolvimento sustentável das cidades, contribuindo para a segurança alimentar, melhoria do meio ambiente, inclusão social e geração de renda. O objetivo deste estudo foi caracterizar as principais dificuldades que o agricultor urbano orgânico enfrenta em municípios onde não há políticas de incentivo à agricultura urbana. Para isso, entrevistas semiestruturadas foram aplicadas entre julho e dezembro de 2018, com sete agricultores urbanos orgânicos que adotam a prática como profissão. Os resultados foram examinados conforme análise de conteúdo e apontaram carências como a ausência de mão de obra capacitada, falta de máquinas e equipamentos adequados à prática em pequenos espaços e insuficiência de recursos financeiros, insuficiências semelhantes às da agricultura familiar orgânica. Portanto, é fundamental a ampliação de políticas de fomento à agricultura urbana orgânica, contemplando tais aspectos no sentido de diminuir os obstáculos e estimular a profissão.

Urban organic farming is an important stimulus for cities' sustainable development, contributing to food security, environmental improvement, social inclusion, and income generation. This study aimed to assess the main difficulties faced by urban organic farmers in cities where there are no policies to encourage urban farming. Semi-structured interviews were applied from July to December 2018 with seven urban organic farmers who had adopted this practice as their profession. The results were examined with content analysis, detecting such gaps as lack of trained labor, lack of adequate machinery and equipment for this practice in small areas, and insufficient financing, all of which were similar to family organic farming. It is thus essential to expand policies to promote urban organic farming, addressing these issues to decrease the barriers and stimulate the profession.

La agricultura urbana orgánica es un importante estímulo para el desarrollo sostenible de las ciudades, contribuyendo a la seguridad alimentaria, mejora del medio ambiente, inclusión social y generación de renta. El objetivo de este estudio fue caracterizar las principales dificultades que el agricultor urbano orgánico enfrenta en municipios donde no existen políticas de incentivo para la agricultura urbana. Para eso, se realizaron entrevistas semiestructuradas entre julio y diciembre de 2018, con siete agricultores urbanos orgánicos que adoptan la práctica como profesión. Los resultados fueron examinados según análisis de contenido y apuntaron carencias como la ausencia de mano de obra capacitada, falta de máquinas y equipamientos adecuados para la práctica en pequeños espacios e insuficiencia de recursos financieros, insuficiencias semejantes a la de la agricultura familiar orgánica. Por lo tanto, es fundamental la ampliación de políticas de fomento a la agricultura urbana orgánica, contemplando tales aspectos, con el fin de disminuir los obstáculos y estimular la profesión.

Antibody Specificity Following a Recent Bordetella pertussis Infection in Adolescence Is Correlated With the Pertussis Vaccine Received in Childhood.

Bordetella (B.) pertussis resurgence affects not only the unvaccinated, but also the vaccinated population. Different vaccines are available, however, it is currently unknown whether the type of childhood vaccination has an influence on antibody responses following a B. pertussis infection later in life. Therefore, the study aim was to profile serum antibody responses in young adults with suspected B. pertussis infections, immunized during childhood with either whole-cell (wPV) or monocomponent acellular pertussis (aPV) vaccines. Serum anti-pertussis toxin (PTx) IgG antibody levels served as an indicator for a recent B. pertussis infection. Leftover sera from a diagnostic laboratory from 36 Danish individuals were included and divided into four groups based on immunization background (aPV vs. wPV) and serum anti-PTx IgG levels (- vs. +). Pertussis-specific IgG/IgA antibody levels and antigen specificity were determined by using multiplex immunoassays (MIA), one- and two-dimensional immunoblotting (1 & 2DEWB), and mass spectrometry. Besides enhanced anti-PTx levels, wPV(+) and aPV(+) groups showed increased IgG and IgA levels against pertactin, filamentous hemagglutinin, fimbriae 2/3, and pertussis outer membrane vesicles (OMV). In the wPV(-) and aPV(-) groups, only low levels of anti-OMV antibodies were detected. 1DEWB demonstrated that antibody patterns differed between groups but also between individuals with the same immunization background and anti-PTx levels. 2DWB analysis for serum IgG revealed 133 immunogenic antigens of which 40 were significantly different between groups allowing to differentiate wPV(+) and aPV(+) groups. Similarly, for serum IgA, 7 of 47 immunogenic protein spots were significantly different. This study demonstrated that B. pertussis infection-induced antibody responses were distinct on antigen level between individuals with either wPV or aPV immunization background. Importantly, only 2DEWB and not MIA could detect these differences indicating the potential of this method. Moreover, in individuals immunized with an aPV containing only PTx in childhood, the infection-induced antibody responses were not limited to PTx alone.

Development of a thermostable spray dried outer membrane vesicle pertussis vaccine for pulmonary immunization.

Worldwide resurgence of whooping cough calls for improved, next-generation pertussis vaccines that induce broad and long-lasting immunity. A mucosal pertussis vaccine based on outer membrane vesicles (omvPV) is a promising candidate. Further, a vaccine that is stable outside the cold chain would be of substantial advantage for worldwide distribution and application. A vaccine formulated as a powder could both stabilize the vaccine as well as make it suitable for pulmonary vaccination. To that end, we developed a spray dried omvPV with improved stability compared to the liquid omvPV formulation. Spray drying did not affect the structural integrity of the omvPV. The antigenicity of Vag8, a major antigen in omvPV was diminished slightly and an altered tryptophan fluorescence indicated some changes in protein structure. However, when administered via the pulmonary route in mice after reconstitution, spray dried omvPV showed comparable immune responses and protection against challenge with live B. pertussis as liquid omvPV. Mucosal IgA and Th17 responses were established in addition to broad systemic IgG and Th1/Th17 responses, indicating the induction of an effective immunity profile. Overall, a spray dried omvPV was developed that maintained effective immunogenic properties and has an improved storage stability.

Reporter cell lines for detection of pertussis toxin in acellular pertussis vaccines as a functional animal-free alternative to the in vivo histamine sensitization test.

Detoxified pertussis toxin (pertussis toxoid) is a major antigen in acellular pertussis vaccines. Testing these vaccines on the presence of residual pertussis toxin (PTx) and reversion to toxicity is performed by the regulatory required in vivo Histamine Sensitization test (HIST). Lack of mechanistic understanding of the HIST, technical handicaps and animal welfare concerns, have promoted the development of alternative methods. As the majority of the cellular effects of PTx depend on its ability to activate intracellular pathways involving cAMP, the in vitro cAMP-PTx assay was developed. Although this assay could be used to detect PTx activity, it lacked sensitivity and robustness for use in a quality control setting. In the present study, novel reporter cell lines (CHO-CRE and A10-CRE) were generated that stably express a reporter construct responsive to changes in intracellular cAMP levels. These reporter cell lines were able to detect PTx in a concentration-dependent manner when combined with fixed amounts of forskolin. The CHO-CRE cell line enabled detection of PTx in the context of a multivalent vaccine containing aP, with a sensitivity equal to the HIST. However, the sensitivity of the A10-CRE cells was insufficient for this purpose. The experiments also suggest that the CHO-CRE reporter cell line might be suitable for assessment of cellular effects of PTd reverted to PTx. The CHO-CRE reporter cell line provides a platform that meets the criteria for specificity and sensitivity and is a promising in vitro model with potential to replace the HIST.

Bordetella pertussis outer membrane vesicle vaccine confers equal efficacy in mice with milder inflammatory responses compared to a whole-cell vaccine.

The demand for improved pertussis vaccines is urgent due to the resurgence of whooping cough. A deeper understanding of the mode of action of pertussis vaccines is required to achieve this improvement. The vaccine-induced effects of a candidate outer membrane vesicle vaccine (omvPV) and a classical protective but reactogenic whole cell vaccine (wPV) were comprehensively compared in mice. The comparison revealed essential qualitative and quantitative differences with respect to immunogenicity and adverse effects for these vaccines. Both vaccines stimulated a mixed systemic Th1/Th2/Th17 response. Remarkably, omvPV evoked higher IgG levels, lower systemic pro-inflammatory cytokine responses and enhanced splenic gene expression than wPV. The omvPV-induced transcriptome revealed gene signatures of the IFN-signaling pathway, anti-inflammatory signatures that attenuate LPS responses, anti-inflammatory metabolic signatures, and IgG responses. Upon intranasal challenge, both immunized groups were equally efficient in clearing Bordetella pertussis from the lungs. This study importantly shows that immunization with omvPV provides a milder inflammatory responses but with equal protection to bacterial colonization and induction of protective antibody and Th1/Th17 type immune responses compared to wPV. These results emphasize the potential of omvPV as a safe and effective next-generation pertussis vaccine.

Immunological Signatures after Bordetella pertussis Infection Demonstrate Importance of Pulmonary Innate Immune Cells.

Effective immunity against Bordetella pertussis is currently under discussion following the stacking evidence of pertussis resurgence in the vaccinated population. Natural immunity is more effective than vaccine-induced immunity indicating that knowledge on infection-induced responses may contribute to improve vaccination strategies. We applied a systems biology approach comprising microarray, flow cytometry and multiplex immunoassays to unravel the molecular and cellular signatures in unprotected mice and protected mice with infection-induced immunity, around a B. pertussis challenge. Pre-existing systemic memory Th1/Th17 cells, memory B-cells, and mucosal IgA specific for Ptx, Vag8, Fim2/3 were detected in the protected mice 56 days after an experimental infection. In addition, pre-existing high activity and reactivation of pulmonary innate cells such as alveolar macrophages, M-cells and goblet cells was detected. The pro-inflammatory responses in the lungs and serum, and neutrophil recruitment in the spleen upon an infectious challenge of unprotected mice were absent in protected mice. Instead, fast pulmonary immune responses in protected mice led to efficient bacterial clearance and harbored potential new gene markers that contribute to immunity against B. pertussis. These responses comprised of innate makers, such as Clca3, Retlna, Glycam1, Gp2, and Umod, next to adaptive markers, such as CCR6+ B-cells, CCR6+ Th17 cells and CXCR6+ T-cells as demonstrated by transcriptome analysis. In conclusion, besides effective Th1/Th17 and mucosal IgA responses, the primary infection-induced immunity benefits from activation of pulmonary resident innate immune cells, achieved by local pathogen-recognition. These molecular signatures of primary infection-induced immunity provided potential markers to improve vaccine-induced immunity against B. pertussis.

Incompatibility of lyophilized inactivated polio vaccine with liquid pentavalent whole-cell-pertussis-containing vaccine.

A hexavalent vaccine containing diphtheria toxoid, tetanus toxoid, whole cell pertussis, Haemophilius influenza type B, hepatitis B and inactivated polio vaccine (IPV) may: (i) increase the efficiency of vaccination campaigns, (ii) reduce the number of injections thereby reducing needlestick injuries, and (iii) ensure better protection against pertussis as compared to vaccines containing acellular pertussis antigens. An approach to obtain a hexavalent vaccine might be reconstituting lyophilized polio vaccine (IPV-LYO) with liquid pentavalent vaccine just before intramuscular delivery. The potential limitations of this approach were investigated including thermostability of IPV as measured by D-antigen ELISA and rat potency, the compatibility of fluid and lyophilized IPV in combination with thimerosal and thimerosal containing hexavalent vaccine. The rat potency of polio type 3 in IPV-LYO was 2 to 3-fold lower than standardized on the D-antigen content, suggesting an alteration of the polio type 3 D-antigen particle by lyophilization. Type 1 and 2 had unaffected antigenicity/immunogenicity ratios. Alteration of type 3 D-antigen could be detected by showing reduced thermostability at 45°C compared to type 3 in non-lyophilized liquid controls. Reconstituting IPV-LYO in the presence of thimerosal (TM) resulted in a fast temperature dependent loss of polio type 1-3 D-antigen. The presence of 0.005% TM reduced the D-antigen content by ∼20% (polio type 2/3) and ∼60% (polio type 1) in 6h at 25°C, which are WHO open vial policy conditions. At 37°C, D-antigen was diminished even faster, suggesting that very fast, i.e., immediately after preparation, intramuscular delivery of the conceived hexavalent vaccine would not be a feasible option. Use of the TM-scavenger, l-cysteine, to bind TM (or mercury containing TM degradation products), resulted in a hexavalent vaccine mixture in which polio D-antigen was more stable.

Measuring Poliovirus Antigenicity by Surface Plasmon Resonance. Application for Potency Indicating Assays.

The D-antigen ELISA is the commonly accepted test for release of inactivated poliovirus containing vaccines. However, this test has a few drawbacks regarding the many variations in the method to quantify the D-unit. The result may depend on method and reagents used which makes standardization of inactivated polio vaccines, based on D-units, to a real challenge. This chapter describes a surface plasmon resonance based method to quantify D-units. The advantage of the calibrated D-antigen assay is the decrease in test variations because no labels, [no incubation times] and no washing steps are necessary. For standardization of both IPV and Sabin IPV, the calibration free concentration analysis could be an improvement as compared to ELISA or other SPR methods because this method combines quantity (particle concentration) and quality (antigenicity) in one assay.

Immunoproteomic Profiling of Bordetella pertussis Outer Membrane Vesicle Vaccine Reveals Broad and Balanced Humoral Immunogenicity.

The current resurgence of whooping cough is alarming, and improved pertussis vaccines are thought to offer a solution. Outer membrane vesicle vaccines (omvPV) are potential vaccine candidates, but omvPV-induced humoral responses have not yet been characterized in detail. The purpose of this study was to determine the antigen composition of omvPV and to elucidate the immunogenicity of the individual antigens. Quantitative proteome analysis revealed the complex composition of omvPV. The omvPV immunogenicity profile in mice was compared to those of classic whole cell vaccine (wPV), acellular vaccine (aPV), and pertussis infection. Pertussis-specific antibody levels, antibody isotypes, IgG subclasses, and antigen specificity were determined after vaccination or infection by using a combination of multiplex immunoassays, two-dimensional immunoblotting, and mass spectrometry. The vaccines and infection raised strong antibody responses, but large quantitative and qualitative differences were measured. The highest antibody levels were obtained by omvPV. All IgG subclasses (IgG1/IgG2a/IgG2b/IgG3) were elicited by omvPV and in a lower magnitude by wPV, but not by aPV (IgG1) or infection (IgG2a/b). The majority of omvPV-induced antibodies were directed against Vag8, BrkA, and LPS. The broad and balanced humoral response makes omvPV a promising pertussis vaccine candidate.