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

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. Pertussis or whooping cough, caused by the gram-negative bacterium Bordetella pertussis, remains endemic even in highly vaccinated population

Antibody Specificity Following a Recent 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

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

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

Immunological Signatures after <i>Bordetella pertussis</i> Infection Demonstrate Importance of Pulmonary Innate Immune Cells

<div><p>Effective immunity against <i>Bordetella pertussis</i> 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 <i>B</i>. <i>pertussis</i> 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 <i>B</i>. <i>pertussis</i>. These responses comprised of innate makers, such as <i>Clca3</i>, <i>Retlna</i>, <i>Glycam1</i>, <i>Gp2</i>, and <i>Umod</i>, 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 <i>B</i>. <i>pertussis</i>.</p></div

Serum cytokine profiles and percentage of splenic neutrophils following <i>B</i>. <i>pertussis</i> challenge in unprotected and protected mice.

<p>(A) The serum concentrations of 33 cytokines were analyzed before and after a <i>B</i>. <i>pertussis</i> challenge in unprotected (lighter blue bars) and protected (dark blue and red bars) mice, as indicated. Concentrations of CXCL13, CCL11, CXCL1, G-CSF, IL-6, CXCL10, and IL-13 serum were significantly altered and represented as mean concentrations of individual values (n = 3). Significant values were calculated by one-way ANOVA with multiple comparison compared to the pre-challenge level (D0) of unprotected mice or protected mice (* = <i>p</i><0.05, ** = <i>p</i><0.01, and *** = <i>p</i><0.001). (B) The percentage of Gr1⁺ cells (neutrophils) was determined over time in the spleen of unprotected and protected mice by using Flow cytometry (* = <i>p</i><0.05).</p

Naturally circulating pertactin-deficient 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

Serum and pulmonary antibody profiles in unprotected and protected mice following <i>B</i>. <i>pertussis</i> challenge.

<p>Serum IgA, IgG, and IgG subclass responses specific for (A) OMV, (B) Ptx, (C) Prn, (D) FHA, and (E) Fim2/3 were determined by using a MIA. Data were obtained in naive mice and protected mice prior to challenge (D0) and 14 days post infection (p.i.) or 14 days post-challenge (p.c.) (n = 3/time point). (F) Pulmonary IgA responses against these antigens were determined on the same time points. * = p<0.05 experimental group versus unprotected group (D0), + = p<0.05 protected group (D0) versus protected group (14 days p.c.). (G) The kinetics of the anti-OMV IgA antibody formation in lung lysates were analyzed at more time points (n = 3/time point) and expressed in fluorescence intensity (F.I.). **** = p<0.0001, challenged unprotected or protected group versus unprotected or protected group (day 0), ++ and +++ = p<0.01 and p<0.001 unprotected group versus protected group (for each time point). (H) Western blot on separated <i>B</i>. <i>pertussis</i> B1917 proteins was performed with pooled lung lysates (1:50) of unprotected and protected mice prior to challenge (D0), and of protected mice 14 days p.c. with IR800-labeled secondary antibody. Left panel shows whole protein range (260kDa-<10kDa) of <i>B</i>. <i>pertussis</i> lysate. Right panel shows more detailed separation of the 110-60kDa protein range. Antigen identification for Vag8 and LPS is depicted.</p

Pulmonary gene expression profiles of genes related to T-cells, B-cells, MHC-I and II and novel genes in protected and unprotected mice following <i>B</i>. <i>pertussis</i> challenge.

<p>Expression profiles of genes in the lungs related to (A) T-cells, (B) B-cells, (C) and antigen presentation by MHC-I and MHC-II were selected according to GO-BP terms, KEGG pathways and text mining. In addition, genes with unknown or poorly understood function that showed interesting gene expression profiles in protected mice were listed as (D) novel genes. Additionally, the color codes of the six clusters from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164027#pone.0164027.g002" target="_blank">Fig 2</a> are added.</p