Distinctive Responses in an In Vitro Human Dendritic Cell-Based System upon Stimulation with Different Influenza Vaccine Formulations

Vaccine development relies on testing vaccine candidates in animal models. However, results from animals cannot always be translated to humans. Alternative ways to screen vaccine candidates before clinical trials are therefore desirable. Dendritic cells (DCs) are the main orchestrators of the immune system and the link between innate and adaptive responses. Their activation by vaccines is an essential step in vaccine-induced immune responses. We have systematically evaluated the suitability of two different human DC-based systems, namely the DC-cell line MUTZ-3 and primary monocyte-derived DCs (Mo-DCs) to screen immunopotentiating properties of vaccine candidates. Two different influenza vaccine formulations, whole inactivated virus (WIV) and subunit (SU), were used as model antigens as they represent a high immunogenic and low immunogenic vaccine, respectively. MUTZ-3 cells were restricted in their ability to respond to different stimuli. In contrast, Mo-DCs readily responded to WIV and SU in a vaccine-specific way. WIV stimulation elicited a more vigorous induction of activation markers, immune response-related genes and secretion of cytokines involved in antiviral responses than the SU vaccine. Furthermore, Mo-DCs differentiated from freshly isolated and freeze/thawed peripheral blood mononuclear cells (PBMCs) showed a similar capacity to respond to different vaccines. Taken together, we identified human PBMC-derived Mo-DCs as a suitable platform to evaluate vaccine-induced immune responses. Importantly, we show that fresh and frozen PBMCs can be used indistinctly, which strongly facilitates the routine use of this system. In vitro vaccine pre-screening using human Mo-DCs is thus a promising approach for evaluating the immunopotentiating capacities of new vaccine formulations that have not yet been tested in humans

Innate responses induced by whole inactivated virus or subunit influenza vaccines in cultured dendritic cells correlate with immune responses in vivo

Vaccine development involves time-consuming and expensive evaluation of candidate vaccines in animal models. As mediators of both innate and adaptive immune responses dendritic cells (DCs) are considered to be highly important for vaccine performance. Here we evaluated how far the response of DCs to a vaccine in vitro is in line with the immune response the vaccine evokes in vivo. To this end, we investigated the response of murine bone marrow-derived DCs to whole inactivated virus (WIV) and subunit (SU) influenza vaccine preparations. These vaccine preparations were chosen because they differ in the immune response they evoke in mice with WIV being superior to SU vaccine through induction of higher virus-neutralizing antibody titers and a more favorable Th1-skewed response phenotype. Stimulation of DCs with WIV, but not SU vaccine, resulted in a cytokine response that was comparable to that of DCs stimulated with live virus. Similarly, the gene expression profiles of DCs treated with WIV or live virus were similar and differed from that of SU vaccine-treated DCs. More specifically, exposure of DCs to WIV resulted in differential expression of genes in known antiviral pathways, whereas SU vaccine did not. The stronger antiviral and more Th1-related response of DCs to WIV as compared to SU vaccine correlates well with the superior immune response found in mice. These results indicate that in vitro stimulation of DCs with novel vaccine candidates combined with the assessment of multiple parameters, including gene signatures, may be a valuable tool for the selection of vaccine candidates

Innate Responses Induced by Whole Inactivated Virus or Subunit Influenza Vaccines in Cultured Dendritic Cells Correlate with Immune Responses In Vivo

Vaccine development involves time-consuming and expensive evaluation of candidate vaccines in animal models. As mediators of both innate and adaptive immune responses dendritic cells (DCs) are considered to be highly important for vaccine performance. Here we evaluated how far the response of DCs to a vaccine in vitro is in line with the immune response the vaccine evokes in vivo. To this end, we investigated the response of murine bone marrow-derived DCs to whole inactivated virus (WIV) and subunit (SU) influenza vaccine preparations. These vaccine preparations were chosen because they differ in the immune response they evoke in mice with WIV being superior to SU vaccine through induction of higher virus-neutralizing antibody titers and a more favorable Th1-skewed response phenotype. Stimulation of DCs with WIV, but not SU vaccine, resulted in a cytokine response that was comparable to that of DCs stimulated with live virus. Similarly, the gene expression profiles of DCs treated with WIV or live virus were similar and differed from that of SU vaccine-treated DCs. More specifically, exposure of DCs to WIV resulted in differential expression of genes in known antiviral pathways, whereas SU vaccine did not. The stronger antiviral and more Th1-related response of DCs to WIV as compared to SU vaccine correlates well with the superior immune response found in mice. These results indicate that in vitro stimulation of DCs with novel vaccine candidates combined with the assessment of multiple parameters, including gene signatures, may be a valuable tool for the selection of vaccine candidates

Intestinal microbiota and mucosal IgA

Het lichaam van mens en dier wordt bevolkt door heel veel verschillende bacteriën. Ondanks de aanwezigheid van deze bacteriën worden we meestal niet ziek. Soms echter, raken de darmen chronisch geïnfecteerd en dit kan ziekten tot gevolg hebben. De Ziekte van Crohn is hiervan een voorbeeld. De ziekte komt steeds vaker voor in westerse samenle­vingen. Maaike Stoel onderzocht de rol die IgA, een bepaald type antistof, speelt in het evenwicht tussen de darmbacteriën en het gespecialiseerde afweersysteem van de darmen. Dit mucosale immuunsysteem heeft een belangrijke functie om het systemische immuunsys­teem af te schermen van de darmbacteriën. Hierbij speelt IgA een grote rol door bijvoor­beeld het voorkomen van het hechten van bacteriën aan het epitheel van de darm.

Rat salivary gland reveals a more restricted IgA repertoire than ileum

Secretory IgA is the most abundantly produced Ig in different mucosal tissues, such as the gastrointestinal tract and the salivary glands. These mucosal tissues are considered to be part of the common mucosal immune system. The specificity and immunoglobulin (19) V-H gene repertoire of the IgA producing cells of both tissues is still largely unknown. To investigate the diversity of the antibody repertoire of IgA producing cells at different mucosal effector sites, we analysed used Ig VH genes by H-CDR3 spectrotyping and V-H gene sequencing of both ileum and salivary gland IgA producing cells of PVG rats. Both types of tissues showed a limited diversity for the two major VH gene families, J558 and PC7183. The salivary gland showed even less diversity than the ileum of the same rat. Cloning and sequencing of used IgA VH genes confirmed the very restricted usage Of VH genes since multiple sets of clonally related sequences in both types of tissues were found. More clones were found in salivary gland than in ileum and both tissues did not have shared VDJ joining regions. IgA derived from salivary gland used germline or near germline VH genes, whereas the ileal VH genes contained more mutations. Furthermore, clonal evolution patterns from all analyzed VH gene sequences of the salivary gland IgA producing cells show mainly randomly acquired somatic mutations, in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer's patches. Our results imply that IgA producing cells in the salivary gland are neither induced at the same place nor selected in the same way as the IgA producing cells in the ileum. The function of the IgA secreted by salivary gland is very likely a first line of defense with (near) germline encoded IgA, whereas in the intestine the majority of utilized IgA VH genes show evidence of somatic hypermutation. (c) 2007 Elsevier Ltd. All rights reserved

Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat

We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of the Brown Norway (BN) rat (assembly V3.4; Rat Genomic Sequence Consortium). In addition to known variable region genes, we found 12 novel previously unidentified functional IGHV genes and 1 novel functional IGHD gene. In total, the variable region of the rat IGH locus is composed of at least 353 unique IGHV genes, 21 IGHD genes, and 5 IGHJ genes, of which 131, 14, and 4 are potentially functional genes, respectively. Of all species studied so far, the rat seems to have the highest number of functional IGHV genes in the genome. Rat IGHV genes can be classified into 13 IGHV families based on nucleotide sequence identity. The variable region of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization. Like the mouse, members of the various IGHV gene families are more or less grouped together on the genome, albeit some members of IGHV gene families are found intermingled with each other. In the rat, the largest IGHV gene families are IGHV1, IGHV2, and IGHV5. The overall conclusion is that the genomic organization of the variable region of the rat IGH locus is strikingly similar to that of the mouse, illustrating the close evolutionary relationship between these two species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00251-010-0448-x) contains supplementary material, which is available to authorized users