Jill van Kessel

Novel vaccine formulations against pertussis offer earlier onset of immunity and provide protection in the presence of maternal antibodies

Whooping cough is a respiratory illness most severe in infants and young children. While the introduction of whole-cell (wP) and acellular pertussis (aP) vaccines has greatly reduced the burden of the disease, pertussis remains a problem in neonates and adolescents. New vaccines are needed that can provide early life and long-lasting protection of infants. Vaccination at an early age, however, is problematic due to the interference with maternally derived antibodies (MatAbs) and the bias towards Th2-type responses following vaccination. Here we report the development of a novel vaccine formulation against pertussis that is highly protective in the presence of MatAbs. We co-formulated pertussis toxoid (PTd) and filamentous hemagglutinin (FHA) with cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN), cationic innate defense regulator (IDR) peptide and polyphosphazene (PP) into microparticle and soluble vaccine formulations and tested them in murine and porcine models in the presence and absence of passive immunity. Vaccines composed of the new adjuvant formulations induced an earlier onset of immunity, higher anti-pertussis IgG2a and IgA titers, and a balanced Th1/Th2-type responses when compared to immunization with Quadracel(®), one of the commercially available vaccines for pertussis. Most importantly, the vaccines offered protection against challenge infection in the presence of passively transferred MatAbs.

Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells

Real-time quantitative PCR (RT-qPCR) is a critical tool used to evaluate changes in gene expression. The precision of this tool is reliant upon the selection of reference genes whose expression remains unaltered in culture conditions and following stimulation. Stably expressed reference genes are used to normalize data so observed changes in expression are not due to artifacts but rather reflect physiological changes. In this study, we examined the expression stability of the porcine genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), eukaryotic elongation factor 1 gamma-like protein (eEF1), ribosomal protein L19 (RPL19), beta-actin (ACTB) and ATP synthase mitochondrial F0 complex (ATP5G1) in peripheral blood mononuclear cells (PBMCs), monocytes, monocyte-derived dendritic cells (MoDCs), blood isolated dendritic cells (BDCs) and T cells with or without stimulation with lipolysaccharide (LPS). An M value was used as a measure of gene stability as determined using geNORM software. Recommendations for the use of reference genes include using GAPDH and B-actin in PBMCs: RPL19 and SDHA in T cells; RPL19 and B-actin in monocytes; RPL-19 and SDHA in BDCs: and RPL-19 and ATP5GA in MoDCs.

Differential activation and maturation of two porcine DC populations following TLR ligand stimulation

Dendritic cells (DCs) are at the interface of innate and adaptive immune responses. Once activated via triggering of their pattern recognition receptors (PRRs), they acquire a mature state and migrate to the lymph nodes where they activate T cells and direct the immune response. Compounds that trigger PRRs are potential vaccine adjuvants, hence in this study we stimulated two porcine DC populations, namely monocyte-derived DCs (MoDCs) and blood DCs (BDCs), with a broad range of toll-like receptors (TLRs) ligands and assessed the activation/maturation state of these porcine DCs. In order to determine if TLR ligands would have an effect on porcine DCs, we characterized the expression of TLRs and demonstrated that MoDCs and BDCs expressed the same set of TLRs but at different levels. Of the TLR ligands examined, lipopolysaccharide (LPS) and poly I:C were the most potent activators of MoDCs, inducing the up-regulation of co-stimulatory molecules CD80/86 and the chemokine receptor CCR7, and production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)alpha. The most effective in inducing BDCs activation were LPS and class A CpG oligodeoxynucleotide (ODN), resulting in up-regulation of chemokine receptor (CCR)7 and down-regulation of CCR2 and CCR5, production of IL-12p40, and expression of a broad range of chemokines that were able to attract porcine immune cells.

A comparison between isolated blood dendritic cells and monocyte-derived dendritic cells in pigs

Various dendritic cell (DC) populations exist that differ in phenotype and ability to present antigen to T cells. For example, plasmacytoid DCs (pDCs) are less potent T cell activators compared with conventional DCs (cDCs). Here, we compared porcine blood DCs (BDCs), containing pDCs and cDCs, and monocyte‐derived DCs (MoDC), consisting of cDCs, in their phenotype, ability to uptake antigen, activation and maturation and their ability to present antigen to autologous T cells. Pigs represent an important animal model, whose immune system in many respects closely resembles that of humans. For example, the distribution of Toll‐like receptors is similar to that of humans, in contrast to that of mice. Here we demonstrate that both populations endocytose foreign material. Following lipopolysaccharide stimulation, CD80/86 and chemokine receptor (CCR)7 expression was increased in both populations as was the expression of the chemokine ligands (CCL)‐2, CCL‐4, CCL‐20 and CXCL‐2. Although basal and post‐stimulation protein concentrations of interleukins 6 and 8 and tumour necrosis factor‐α were higher in MoDCs, protein concentrations showed a higher fold increase in BDCs. Antigen‐specific proliferation of autologous T cells was induced by MoDCs and BDCs. Interestingly, while MoDCs induced stronger proliferation in naive T cells, no difference in proliferation was observed when primed T cells were studied. These results demonstrate that isolated porcine BDCs are highly responsive to stimulation with lipopolysaccharide and are functionally able to drive primed T‐cell proliferation to the same extent as MoDCs.

Induction, regulation and physiological role of IL-17 secreting helper T-cells isolated from PBMC, thymus, and lung lymphocytes of young pigs.

Interleukin-17 (IL-17) producing cells, referred to as Th17, have recently emerged as a third subset of the T helper (Th) cell family. Studies in mice have demonstrated that Th17 cells and their associated cytokines are involved in several autoimmune diseases and host defense against infection. Murine Th17 cells differentiate from naïve CD4(+) T-cells in the presence of TGFβ and IL-6, however, there are contradicting reports as to the role of TGFβ in the differentiation of human Th17 cells and very little is known about these cells in other animals. We report here the presence of IL-17 secreting lymphocytes in the lung and peripheral blood of pigs. The cDNA of porcine IL-17 gene was cloned and sequenced from activated lung lymphocytes and PBMC from piglets. A 17kDa recombinant protein was expressed and purified both under denaturing and native conditions from E. coli BL21 cells. Furthermore, we demonstrate that TGFβ in the presence of IL-6 and/or IL-1β induces in vitro differentiation of Th17 cells from naïve porcine CD4(+) thymocytes.

Porcine Neonatal Blood Dendritic Cells, but Not Monocytes, Are More Responsive to TLRs Stimulation than Their Adult Counterparts

The neonatal immune system is often considered as immature or impaired compared to the adult immune system. This higher susceptibility to infections is partly due to the skewing of the neonatal immune response towards a Th2 response. Activation and maturation of dendritic cells (DCs) play an important role in shaping the immune response, therefore, DCs are a target of choice for the development of efficient and protective vaccine formulations able to redirect the neonatal immune response to a protective Th1 response. As pigs are becoming more important for vaccine development studies due to their similarity to the human immune system, we decided to compare the activation and maturation of a subpopulation of porcine DCs in adult and neonatal pigs following stimulation with different TLR ligands, which are promising candidates for adjuvants in vaccine formulations. Porcine blood derived DCs (BDCs) were directly isolated from blood and consisted of a mix of conventional and plasmacytoid DCs. Following CpG ODN (TLR9 ligand) and imiquimod (TLR7 ligand) stimulation, neonatal BDCs showed higher levels of expression of costimulatory molecules and similar (CpG ODN) or higher (imiquimod) levels of IL-12 compared to adult BDCs. Another interesting feature was that only neonatal BDCs produced IFN-α after TLR7 or TLR9 ligand stimulation. Stimulation with CpG ODN and imiquimod also induced enhanced expression of several chemokines. Moreover, in a mixed leukocyte reaction assay, neonatal BDCs displayed a greater ability to induce lymphoproliferation. These findings suggest that when stimulated via TLR7 or TLR9 porcine DCs display similar if not better response than adult porcine DCs.

c-di-GMP Enhances Protective Innate Immunity in a Murine Model of Pertussis

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.

Protective Role of Passively Transferred Maternal Cytokines against Bordetella pertussis Infection in Newborn Piglets

ABSTRACT Maternal vaccination represents a potential strategy to protect both the mother and the offspring against life-threatening infections. This protective role has mainly been associated with antibodies, but the role of cell-mediated immunity, in particular passively transferred cytokines, is not well understood. Here, using a pertussis model, we have demonstrated that immunization of pregnant sows with heat-inactivated bacteria leads to induction of a wide range of cytokines (e.g., tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], interleukin-6 [IL-6], IL-8, and IL-12/IL-23p40) in addition to pertussis-specific antibodies. These cytokines can be detected in the sera and colostrum/milk of vaccinated sows and subsequently were detected at significant levels in the serum and bronchoalveolar lavage fluid of piglets born to vaccinated sows together with pertussis-specific antibodies. In contrast, active vaccination of newborn piglets with heat-inactivated bacteria induced high levels of specific IgG and IgA but no cytokines. Although the levels of antibodies in vaccinated piglets were comparable to those of passively transferred antibodies, no protection against Bordetella pertussis infection was observed. Thus, our results demonstrate that a combination of passively transferred cytokines and antibodies is crucial for disease protection. The presence of passively transferred cytokines/antibodies influences the cytokine secretion ability of splenocytes in the neonate, which provides novel evidence that maternal immunization can influence the newborns cytokine milieu and may impact immune cell differentiation (e.g., Th1/Th2 phenotype). Therefore, these maternally derived cytokines may play an essential role both as mediators of early defense against infections and possibly as modulators of the immune repertoire of the offspring.

Intrauterine vaccination induces a dose-sensitive primary humoral response with limited evidence of recall potential

Induction of the local mucosal immune system within the reproductive tract is widely considered to be a key component in the development of effective prophylactic vaccines to control the spread of sexually transmitted infections. Here, we examine the capacity of the upper reproductive tract to act as a site of immune induction following.

The Importance of Animal Models in the Development of Vaccines

Efficient translation of basic vaccine research into clinical therapies greatly depends upon the availability of appropriate animal models. Testing novel vaccine candidates in animal models is a critical step in the development of modern vaccines. Animal models are being used to assess the quality and quantity of the immune response, to identify the optimal route of delivery and formulation, to determine protection from infection and disease transmission, and to evaluate the safety and toxicity of the vaccine formulation. Animal models help to make the translation from basic research to clinical application, and they often allow prediction of the vaccine potential, which helps in predicting the financial risks for vaccine manufacturers. Choosing an appropriate animal model has become increasingly important for the field, as each model has its own advantages and disadvantages. In this review, the criteria for selecting the right animal model, the advantages and disadvantages of various animal models, as well as the future needs for animal models are being discussed.