Systems vaccinology and big data in the vaccine development chain.

Systems vaccinology has proven a fascinating development in the last decade. Where traditionally vaccine development has been dominated by trial and error, systems vaccinology is a tool that provides novel and comprehensive understanding if properly used. Data sets retrieved from systems-based studies endorse rational design and effective development of safe and efficacious vaccines. In this review we first describe different omics-techniques that form the pillars of systems vaccinology. In the second part, the application of systems vaccinology in the different stages of vaccine development is described. Overall, this review shows that systems vaccinology has become an important tool anywhere in the vaccine development chain.

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.

In vitro alternative for reactogenicity assessment of outer membrane vesicle based vaccines.

Intrinsic or added immune activating molecules are key for most vaccines to provide desired immunity profiles but may increase systemic reactogenicity. Regulatory agencies require rabbit pyrogen testing (RPT) for demonstration of vaccine reactogenicity. Recently, the monocyte activation test (MAT) gained popularity as in vitro alternative, yet this assay was primarily designed to test pyrogen-free products. The aim was to adjust the MAT to enable testing of pyrogen containing vaccines in an early stage of development where no reference batch is yet available. The MAT and RPT were compared for assessing unknown safety profiles of pertussis outer membrane vesicle (OMV) vaccine candidates to those of Bexsero as surrogate reference vaccine. Pertussis OMVs with wild-type LPS predominantly activated TLR2 and TLR4 and were more reactogenic than Bexsero. However, this reactogenicity profile for pertussis OMVs could be equalized or drastically reduced compared to Bexsero or a whole-cell pertussis vaccine, respectively by dose changing, modifying the LPS, intranasal administration, or a combination of these. Importantly, except for LPS modified products, reactogenicity profiles obtained with the RPT and MAT were comparable. Overall, we demonstrated that this pertussis OMV vaccine candidate has an acceptable safety profile. Furthermore, the MAT proved its applicability to assess reactogenicity levels of pyrogen containing vaccines at multiple stages of vaccine development and could eventually replace rabbit pyrogen testing.

Naturally circulating pertactin-deficient Bordetella pertussis strains induce distinct gene expression and inflammatory signatures in human dendritic cells.

Respiratory infections caused by Bordetella pertussis are reemerging despite high pertussis vaccination coverage. Since the introduction of the acellular pertussis vaccine in the late twentieth century, circulating B. pertussis strains increasingly lack expression of the vaccine component pertactin (Prn). In some countries, up to 90% of the circulating B. pertussis strains are deficient in Prn. To better understand the resurgence of pertussis, we investigated the response of human monocyte-derived dendritic cells (moDCs) to naturally circulating Prn-expressing (Prn-Pos) and Prn-deficient (Prn-Neg) B. pertussis strains from 2016 in the Netherlands. Transcriptome analysis of moDC showed enriched IFNα response-associated gene expression after exposure to Prn-Pos B. pertussis strains, whereas the Prn-Neg strains induced enriched expression of interleukin- and TNF-signaling genes, as well as other genes involved in immune activation. Multiplex immune assays confirmed enhanced proinflammatory cytokine secretion by Prn-Neg stimulated moDC. Comparison of the proteomes from the Prn-Pos and Prn-Neg strains revealed, next to the difference in Prn, differential expression of a number of other proteins including several proteins involved in metabolic processes. Our findings indicate that Prn-deficient B. pertussis strains induce a distinct and stronger immune activation of moDCs than the Prn-Pos strains. These findings highlight the role of pathogen adaptation in the resurgence of pertussis as well as the effects that vaccine pressure can have on a bacterial population.

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.

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.

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.

Meta-Analysis of Pulmonary Transcriptomes from Differently Primed Mice Identifies Molecular Signatures to Differentiate Immune Responses following Bordetella pertussis Challenge.

Respiratory infection with Bordetella pertussis leads to severe effects in the lungs. The resulting immunity and also immunization with pertussis vaccines protect against disease, but the induced type of immunity and longevity of the response are distinct. In this study the effects of priming, by either vaccination or infection, on a subsequent pathogen encounter were studied. To that end, three postchallenge transcriptome datasets of previously primed mice were combined and compared to the responses in unprimed control mice. In total, 205 genes showed different transcription activity. A coexpression network analysis assembled these genes into 27 clusters, combined into six groups with overlapping biological function. Local pulmonary immunity was only present in mice with infection-induced immunity. Complement-mediated responses were more prominent in mice immunized with an outer membrane vesicle pertussis vaccine than in mice that received a whole-cell pertussis vaccine. Additionally, 46 genes encoding for secreted proteins may serve as markers in blood for the degree of protection (Cxcl9, Gp2, and Pla2g2d), intensity of infection (Retnla, Saa3, Il6, and Il1b), or adaptive recall responses (Ighg, C1qb). The molecular signatures elucidated in this study contribute to better understanding of functional interactions in challenge-induced responses in relation to pertussis immunity.

Transcriptome signature for dampened Th2 dominance in acellular pertussis vaccine-induced CD4(+) T cell responses through TLR4 ligation.

Current acellular pertussis (aP) vaccines promote a T helper 2 (Th2)-dominated response, while Th1/Th17 cells are protective. As our previous study showed, after adding a non-toxic TLR4 ligand, LpxL1, to the aP vaccine in mice, the Bordetella pertussis-specific Th2 response is decreased and Th1/Th17 responses are increased as measured at the cytokine protein level. However, how this shift in Th response by LpxL1 addition is regulated at the gene expression level remains unclear. Transcriptomics analysis was performed on purified CD4(+) T cells of control and vaccinated mice after in vitro restimulation with aP vaccine antigens. Multiple key factors in Th differentiation, including transcription factors, cytokines, and receptors, were identified within the differentially expressed genes. Upregulation of Th2- and downregulation of follicular helper T cell-associated genes were found in the CD4(+) T cells of both aP- and aP+LpxL1-vaccinated mice. Genes exclusively upregulated in CD4(+) T cells of aP+LpxL1-vaccinated mice included Th1 and Th17 signature cytokine genes Ifng and Il17a respectively. Overall, our study indicates that after addition of LpxL1 to the aP vaccine the Th2 component is not downregulated at the gene expression level. Rather an increase in expression of Th1- and Th17-associated genes caused the shift in Th subset outcome.

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.

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.

Molecular signatures of the evolving immune response in mice following a Bordetella pertussis infection.

Worldwide resurgence of pertussis necessitates the need for improvement of pertussis vaccines and vaccination strategies. Since natural infections induce a longer-lasting immunity than vaccinations, detailed knowledge of the immune responses following natural infection can provide important clues for such improvement. The purpose was to elucidate the kinetics of the protective immune response evolving after experimental Bordetella pertussis (B. pertussis) infection in mice. Data were collected from (i) individual analyses, i.e. microarray, flow cytometry, multiplex immunoassays, and bacterial clearance; (ii) twelve time points during the infection; and (iii) different tissues involved in the immune responses, i.e. lungs, spleen and blood. Combined data revealed detailed insight in molecular and cellular sequence of events connecting different phases (innate, bridging and adaptive) of the immune response following the infection. We detected a prolonged acute phase response, broad pathogen recognition, and early gene signatures of subsequent T-cell recruitment in the lungs. Activation of particular transcription factors and specific cell markers provided insight into the time course of the transition from innate towards adaptive immune responses, which resulted in a broad spectrum of systemic antibody subclasses and splenic Th1/Th17 memory cells against B. pertussis. In addition, signatures preceding the local generation of Th1 and Th17 cells as well as IgA in the lungs, considered key elements in protection against B. pertussis, were established. In conclusion, molecular and cellular immunological processes in response to live B. pertussis infection were unraveled, which may provide guidance in selecting new vaccine candidates that should evoke local and prolonged protective immune responses.