Nobilon International BV

Boxmeer, Netherlands

Nobilon International BV

Boxmeer, Netherlands
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Verheul R.J.,University Utrecht | Hagenaars N.,University Utrecht | Van Es T.,VUmc | Van Gaal E.V.B.,University Utrecht | And 10 more authors.
European Journal of Pharmaceutical Sciences | Year: 2012

Recently we reported that reacetylation of N,N,N-trimethyl chitosan (TMC) reduced the adjuvant effect of TMC in mice after intranasal (i.n.) administration of whole inactivated influenza virus (WIV) vaccine. The aim of the present study was to elucidate the mechanism of this lack of adjuvanticity. Reacetylated TMC (TMC-RA, degree of acetylation 54%) was compared with TMC (degree of acetylation 17%) at six potentially critical steps in the induction of an immune response after i.n. administration in mice. TMC-RA was degraded in a nasal wash to a slightly larger extent than TMC. The local i.n. distribution and nasal clearance of WIV were similar for both TMC types. Fluorescently labeled WIV was taken up more efficiently by Calu-3 cells when formulated with TMC-RA compared to TMC and both TMCs significantly reduced transport of WIV over a Calu-3 monolayer. Murine bone-marrow derived dendritic cell activation was similar for plain WIV, and WIV formulated with TMC-RA or TMC. The inferior adjuvant effect in mice of TMC-RA over that of TMC might be caused by a slightly lower stability of TMC-RA-WIV in the nasal cavity, rather than by any of the other factors studied in this paper. © 2011 Elsevier B.V. All rights reserved.


Hagenaars N.,University Utrecht | Mania M.,University Utrecht | de Jong P.,University Utrecht | Que I.,Leiden University | And 9 more authors.
Journal of Controlled Release | Year: 2010

The nose is a promising immunization site and intranasal (i.n.) vaccination studies with whole inactivated influenza virus (WIV) adjuvanted with N,N,N-trimethylchitosan (TMC-WIV) have shown promising results. In this study, the influence of TMC on the i.n. delivery of WIV was studied in mice by comparing the nasal residence time and the specific location in the nasal cavity of WIV and TMC-WIV. Additionally, the local toxicity profile of the WIV formulations was assessed. In vivo fluorescence imaging was used to study the nasal residence time and the fate of the bulk vaccine in mice that received vaccines fluorescently labeled with IRDye800CW®. An immunohistochemical (IHC) staining method for nasal cross-sections was developed to visualize the antigen in the nasal cavity. Therefore, mice were sacrificed at different time points after vaccination with various vaccine formulations and nasal cross-sections were made. The local toxicity was assessed using hematoxylin and eosin staining for the nasal cross-sections. No significant differences in the nasal residence time between WIV and TMC-WIV were observed. However, IHC revealed a striking difference in the location and distribution of WIV in the nasal cavity. When formulated as plain WIV, positive staining was mainly found in the nasal cavity, presumably in mucus blobs. TMC-coated WIV, on the other hand, was mostly present as a thin layer on the epithelial surfaces of the naso- and maxilloturbinates. This difference in staining pattern correlates with the observed differences in immunogenicity of these two vaccines and indicates that TMC-WIV results in a much closer interaction of WIV with the epithelial surfaces than WIV alone, potentially leading to enhanced uptake and induction of immune responses. This study further shows that both WIV and TMC-WIV formulations induce minimal local toxicity. Taken altogether, these results provide more insight in the mode of action and safety of TMC and justify further research to develop TMC-adjuvanted nasal vaccines. © 2010 Elsevier B.V.


Van Kessel G.,Center for Mathematical science Europe | Geels M.J.,Nobilon International BV | de Weerd S.,Nobilon International BV | Buijs L.J.,Nobilon International BV | And 6 more authors.
Vaccine | Year: 2012

Infection with human influenza virus leads to serious respiratory disease. Vaccination is the most common and effective prophylactic measure to prevent influenza. Influenza vaccine manufacturing and release is controlled by the correct determination of the potency-defining haemagglutinin (HA) content. This determination is historically done by single radial immunodiffusion (SRID), which utilizes a statistical slope-ratio model to estimate the actual HA content. In this paper we describe the development and qualification of a parallel line model for analysis of HA quantification by SRID in cell culture-derived whole virus final monovalent and trivalent influenza vaccines. We evaluated plate layout, sample randomization, and validity of data and statistical model. The parallel line model was shown to be robust and reproducible. The precision studies for HA content demonstrated 3.8-5.0% repeatability and 3.8%-7.9% intermediate precision. Furthermore, system suitability criteria were developed to guarantee long-term stability of this assay in a regulated production environment. SRID is fraught with methodological and logistical difficulties and the determination of the HA content requires the acceptance of new and modern release assays, but until that moment, the described parallel line model represents a significant and robust update for the current global influenza vaccine release assay. © 2011 Elsevier Ltd.


Heldens J.,Nobilon International BV | Heldens J.,Merck Sharpe and Dohme Animal Health BV | Hulskotte E.,Merck Sharpe and Dohme Oss BV | Voeten T.,Nobilon International BV | And 9 more authors.
Vaccine | Year: 2014

Live attenuated influenza vaccine (LAIV) offers the promise of inducing a variety of immune responses thereby conferring protection to circulating field strains. LAIVs are based on cold adapted and temperature sensitive phenotypes of master donor viruses (MDVs) containing the surface glycoprotein genes of seasonal influenza strains. Two types of MDV lineages have been described, the Ann Arbor lineages and the A/Leningrad/17 and B/USSR/60 lineages. Here the safety and immunogenicity of a Madin Darby Canine Kidney - cell culture based, intranasal LAIV derived from A/Leningrad/17 and B/USSR, was evaluated in healthy influenza non-naive volunteers 18-50 years of age. In a double-blind, randomized, placebo-controlled design, single escalating doses of 1×105, 1×106, or 1×107 tissue culture infectious dose 50% (TCID50) of vaccine containing each of the three influenza virus re-assortants recommended by the World Health Organization for the 2008-2009 season were administered intranasally. A statistically significant geometric mean increase in hemagglutination inhibition titer was reached for influenza strain A/H3N2 after immunization with all doses of LAIV. For the A/H1N1 and B strains, the GMI in HI titer did not increase for any of the doses. Virus neutralization antibody titers showed a similar response pattern. A dose-response effect could not be demonstrated for any of the strains, neither for the HI antibody nor for the VN antibody responses. No influenza like symptoms, no nasal congestions, no rhinorrhea, or other influenza related upper respiratory tract symptoms were observed. In addition, no difference in the incidence or nature of adverse events was found between vaccine and placebo treated subjects. Overall, the results indicated that the LAIV for nasal administration is immunogenic (i.e. able to provoke an immune response) and safe both from the perspective of the attenuated virus and the MDCK cell line from which it was derived, and it warrants further development. © 2014 Elsevier Ltd.


Van Remmerden Y.,Netherlands Vaccine Institute | Xu F.,Netherlands Vaccine Institute | Van Eldik M.,Netherlands Vaccine Institute | Heldens J.G.M.,Nobilon International BV | And 5 more authors.
Virology Journal | Year: 2012

Background: Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable. Methods. Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described. Results: The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed. Conclusions: The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale) vaccine clinical trials. © 2012 van Remmerden et al.; licensee BioMed Central Ltd.


Rudenko L.,BioDiem Ltd. | van den Bosch H.,Nobilon International BV | Kiseleva I.,BioDiem Ltd. | Mironov A.,Microgen Federal State Company | And 3 more authors.
Vaccine | Year: 2011

In February 2009, Nobilon granted the World Health Organization (WHO) a non-exclusive licence to develop, register, manufacture, use and sell seasonal a pandemic live attenuated influenza vaccine (LAIV) produced on embryonated chicken eggs. WHO was permitted to grant sub-licences to vaccine manufacturers in developing countries within the framework of its influenza vaccine technology transfer initiative. In parallel, the Institute of Experimental Medicine (IEM), Russia, concluded an agreement with WHO for the supply of Russian LAIV reassortants for use by these manufacturers.Also in 2009, IEM carried out a study on a novel A/17/California/2009/38 (H1N1) pandemic LAIV candidate derived from the pandemic-related A/California/07/2009 (H1N1) influenza virus and the attenuated A/Leningrad/134/17/57 (H2N2) master donor virus, using routine reassortant technique in embryonated chicken eggs. Following successful preclinical studies in eggs and in ferrets, a double-blind, controlled, randomized clinical trial was carried out in immunologically naïve study participants between 12-18 and 18-60 years old. Collectively, the immunogenicity data (haemagglutinin inhibition test, ELISA and cytokine tests for the detection of memory T cells) support the use of a single dose of the pandemic H1N1 LAIV in 12-60 year olds. The outcome of the studies showed no significant adverse reactions attributable to the vaccine, and suggests that the vaccine is as safe and immunogenic as seasonal influenza vaccines. Importantly, it was clearly demonstrated that reliance on the HAI assay alone is not recommended for testing LAIV.To date, via the licence agreement with WHO, the H1N1 LAIV has been transferred to the Government Pharmaceutical Organization in Thailand, the Serum Institute of India, and the Zhejiang Tianyuan Bio-Pharmaceutical Co., Ltd. in China. © 2011 Elsevier Ltd.


Jans J.,Radboud University Nijmegen | Vissers M.,Radboud University Nijmegen | Heldens J.G.M.,Radboud University Nijmegen | Heldens J.G.M.,Nobilon International BV | And 4 more authors.
Reviews in Medical Virology | Year: 2014

SUMMARY: RSV infections are a major burden in infants less than 3months of age. Newborns and infants express a distinct immune system that is largely dependent on innate immunity and passive immunity from maternal antibodies. Antibodies can regulate immune responses against viruses through interaction with Fc gamma receptors leading to enhancement or neutralization of viral infections. The mechanisms underlying the immunomodulatory effect of Fc gamma receptors on viral infections have yet to be elucidated in infants. Herein, we will discuss current knowledge of the effects of antibodies and Fc gamma receptors on infant innate immunity to RSV. A better understanding of the pathogenesis of RSV infections in young infants may provide insight into novel therapeutic strategies such as vaccination. © 2013 John Wiley & Sons, Ltd.


Hilgers L.A.,Nobilon International BV
Methods in molecular biology (Clifton, N.J.) | Year: 2010

To develop novel adjuvants for use in humans, the efficacy/toxicity (E/T) ratio of experimental products in large animal species can be investigated. The test model included two intramuscular immunizations in pigs at 3 weeks interval and analysis of immune responses and local reactions 1 week after the second injection. The antigen used to determine adjuvant activity was a well-defined, purified, viral glycoprotein that without adjuvant induces low immune responses and no detectable local reactions. Efficacy was determined by measuring ELISA and virus-neutralizing antibody titres. Toxicity was determined by necropsy and estimating size and severity of local reactions to each treatment. The persistence of the side effects was deduced from the difference in the local reaction 4 weeks after the first and 1 week after the second injection. For graphic representation of E/T ratios, toxicity was expressed in arbitrary units and plotted against antibody titre. The graphs provided insight into dose- and structure-response relationships and enabled the stepwise optimization of adjuvant candidates.


Metz S.W.,Wageningen University | Geertsema C.,Wageningen University | Martina B.E.,Erasmus University Rotterdam | Andrade P.,Wageningen University | And 4 more authors.
Virology Journal | Year: 2011

Background: Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results: Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions: Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections. © 2011 Metz et al; licensee BioMed Central Ltd.


PubMed | Nobilon International BV
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2010

To develop novel adjuvants for use in humans, the efficacy/toxicity (E/T) ratio of experimental products in large animal species can be investigated. The test model included two intramuscular immunizations in pigs at 3 weeks interval and analysis of immune responses and local reactions 1 week after the second injection. The antigen used to determine adjuvant activity was a well-defined, purified, viral glycoprotein that without adjuvant induces low immune responses and no detectable local reactions. Efficacy was determined by measuring ELISA and virus-neutralizing antibody titres. Toxicity was determined by necropsy and estimating size and severity of local reactions to each treatment. The persistence of the side effects was deduced from the difference in the local reaction 4 weeks after the first and 1 week after the second injection. For graphic representation of E/T ratios, toxicity was expressed in arbitrary units and plotted against antibody titre. The graphs provided insight into dose- and structure-response relationships and enabled the stepwise optimization of adjuvant candidates.

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