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van Els C.A.C.M.,National Institute for Public Health and the Environment | Corbiere V.,Free University of Colombia | Smits K.,Free University of Colombia | van Gaans-van den Brink J.A.M.,National Institute for Public Health and the Environment | And 7 more authors.
Frontiers in Immunology | Year: 2014

CD4+ T cells are prominent effector cells in controlling Mycobacterium tuberculosis (Mtb) infection but may also contribute to immunopathology. Studies probing the CD4+ T cell response from individuals latently infected with Mtb or patients with active tuberculosis using either small or proteome-wide antigen screens so far revealed a multi-antigenic, yet mostly invariable repertoire of immunogenic Mtb proteins. Recent developments in mass spectrometry-based proteomics have highlighted the occurrence of numerous types of post-translational modifications (PTMs) in proteomes of prokaryotes, including Mtb. The well-known PTMs in Mtb are glycosylation, lipidation, or phosphorylation, known regulators of protein function or compartmentalization. Other PTMs include methylation, acetylation, and pupylation, involved in protein stability. While all PTMs add variability to the Mtb proteome, relatively little is understood about their role in the anti-Mtb immune responses. Here,we reviewMtb protein PTMs and methods to assess their role in protective immunity against Mtb. © 2014 van Els, Corbière, Smits, vanGaans-van den Brink, Poelen, Mascart, Meiring and Locht. Source


Thomassen Y.E.,Institute for Translational Vaccinology | Bakker W.A.M.,Institute for Translational Vaccinology
Vaccine | Year: 2015

Polio is expected to be eradicated within only a few years from now. Upon polio eradication, the use of oral polio vaccines, which can cause circulating and virulent vaccine derived polio viruses, will be stopped. From this moment onwards, inactivated polio vaccines (IPV) will be used for worldwide vaccination against polio. An increased demand for IPV is thus anticipated. As a result, process development studies regarding the IPV production process, developed in the 1960s, have intensified. Studies on yield optimization aiming at costs reduction as well as the use of alternative polio viruses, which are more biosafe for manufacturing, are actual. Here our strategy to setup a new IPV production process using attenuated Sabin polio virus strains is presented. Moreover, aspects on reduction of the costs of goods and the impact of process optimization on sIPV costs are reviewed. © 2015 Elsevier Ltd. Source


Pupo E.,Institute for Translational Vaccinology | Hamstra H.-J.,National Institute for Public Health and the Environment | Meiring H.,Institute for Translational Vaccinology | Van Der Ley P.,Institute for Translational Vaccinology
Journal of Biological Chemistry | Year: 2014

Engineering the lipopolysaccharide (LPS) biosynthetic pathway offers the potential to obtain modified derivatives with optimized adjuvant properties. Neisseria meningitidis strain H44/76 was modified by expression of the pagL gene encoding lipid A 3-O-deacylase from Bordetella bronchiseptica and by inactivation of the lgtB gene encoding the terminal oligosaccharide galactosyltransferase. Mass spectrometry analysis of purified mutant LPS was used for detailed compositional analysis of all present molecular species. This determined that the modified LPS was mainly pentaacylated, demonstrating high efficiency of conversion from the hexaacyl to the 3-O-deacylated form by heterologous lipid A 3-O-deacylase (PagL) expression. MS analyses also provided evidence for expression of only one major oligosaccharide glycoform, which lacked the terminal galactose residue as expected from inactivation of the lgtB gene. The immunomodulatory properties of PagL-deacylated LPS were compared with another pentaacyl form obtained from an lpxL1- mutant, which lacks the 2′ secondary acyl chain. Although both LPS mutants displayed impaired capacity to induce production of the pro-inflammatory cytokine IL-6 in the monocytic cell line Mono Mac 6, induction of the Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β-dependent chemokine interferon-γ-induced protein 10 was largely retained only for the lgtB-/pagL+ mutant. Removal of remaining hexaacyl species exclusively present in lgtB-/pagL+ LPS demonstrated that these minor species potentiate but do not determine the activity of this LPS. These results are the first to indicate a qualitatively different response of human innate cells to pentaacyl lpxL1- and pagL+ LPS and show the importance of detailed structure-function analysis when working with modified lipid A structures. The pagL+ LPS has significant potential as immune modulator in humans. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Thomassen Y.E.,Institute for Translational Vaccinology | Rubingh O.,Institute for Translational Vaccinology | Wijffels R.H.,Wageningen University | van der Pol L.A.,Institute for Translational Vaccinology | Bakker W.A.M.,Institute for Translational Vaccinology
Vaccine | Year: 2014

Vero cells were grown adherent to microcarriers (Cytodex 1; 3gL-1) using animal component free media in stirred-tank type bioreactors. Different strategies for media refreshment, daily media replacement (semi-batch), continuous media replacement (perfusion) and recirculation of media, were compared with batch cultivation. Cell densities increased using a feed strategy from 1×106 cellsmL-1 during batch cultivation to 1.8, 2.7 and 5.0×106 cellsmL-1 during semi-batch, perfusion and recirculation, respectively.The effects of these different cell culture strategies on subsequent poliovirus production were investigated. Increased cell densities allowed up to 3 times higher d-antigen levels when compared with that obtained from batch-wise Vero cell culture. However, the cell specific d-antigen production was lower when cells were infected at higher cell densities. This cell density effect is in good agreement with observations for different cell lines and virus types. From the evaluated alternative culture methods, application of a semi-batch mode of operations allowed the highest cell specific d-antigen production.The increased product yields that can easily be reached using these higher cell density cultivation methods, showed the possibility for better use of bioreactor capacity for the manufacturing of polio vaccines to ultimately reduce vaccine cost per dose. Further, the use of animal-component-free cell- and virus culture media shows opportunities for modernization of human viral vaccine manufacturing. © 2014. Source


Westdijk J.,Institute for Translational Vaccinology | Koedam P.,Bilthoven Biologicals | Barro M.,Global Vaccines Inc. | Barro M.,Health-U | And 7 more authors.
Vaccine | Year: 2013

Six different adjuvants, each in combination with inactivated polio vaccine (IPV) produced with attenuated Sabin strains (sIPV), were evaluated for their ability to enhance virus neutralizing antibody titres (VNTs) in the rat potency model. The increase of VNTs was on average 3-, 15-, 24-fold with adjuvants after one immunization (serotypes 1, 2, and 3, respectively). Also after a boost immunization the VNTs of adjuvanted sIPV were on average another 7-20-27 times higher than after two inoculations of sIPV without adjuvant. The results indicate that it is feasible to increase the potency of inactivated polio vaccines by using adjuvants. © 2013 Elsevier Ltd. Source

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