A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions

International audienceRabies virus strain production in Vero cells grown on Cytodex 1 in a 2 L stirred tank bioreactor and in a medium free of components of human or animal origin (VP-SFM) is described. Cell banking procedure in VP-SFM supplemented with an animal components free mixture (10%DMSO + 0.1%methylcellulose) was reported and cell growth after revitalization was assessed. Vero cells exhibited growth performances (specific growth rate and cell division number) similar to that obtained in serum containing medium. To design a scalable process that is totally free of animal-derived substances, two proteases: TrypLE Select and Accutase, were assessed as an alternative to trypsin which is routinely used for cell passage. Growth performance of Vero cells grown in VP-SFM and MEM + 10% fetal calf serum (FCS) over four passages and subcultivated with either TrypLE Select or Accutase was evaluated. TrypLE Select showed the best performance in terms of specific growth rate and cell division number. Kinetics of cell growth and rabies virus production (LP2061/Vero strain) were investigated in spinner flask and in a 2L bioreactor. In spinner flask, a maximal cell density level of 1.85 x 10(6) cells/mL was achieved when the cells were grown in VP-SFM on 2 g/L Cytodex 1. Cell infection experiments conducted at an MOI of 0.3 and without the medium exchange step, typically needed for serum containing rabies virus production, resulted in a maximal virus titer equal to 2 x 10(7) (Fluorescent Focus Unit) FFU/mL. In stirred tank bioreactor, Vero cell growth in VP-SFM on 3 g/L Cytodex 1 was shown to be sensitive to the aeration mode. Sparging the culture was detrimental for cell growth, whereas cell density level was greatly enhanced when only headspace aeration was used. A cell density level of 2.6 x 10(6) cells/mL was obtained when the cells were grown on 3 g/L Cytodex I and in batch culture mode. Cell infection at an MOI of 0.1 without any medium exchange, yielded a maximal rabies virus titer of 2.4 x 10(7) FFU/mL. Furthermore, Vero cell growth in a 2 L bioreactor using recirculation culture mode during cell proliferation step and perfusion for virus multiplication phase was investigated. In comparison to batch culture, a higher cell density level that was equal to 5 x 10(6) cells/mL was reached. Cell infection under conditions similar to batch culture, resulted in a maximal virus titer equal to 1.38 x 10(8) FFU/mL. The potency of the pooled inactivated virus harvests showed an activity of 2.58 IU/mL which was comparable to that obtained in serum supplemented medium. (C) 2007 Elsevier Ltd. All rights reserved

Functional categories of differentially expressed genes between the <i>ptxP1</i> and <i>ptxP3</i> strains.

<p>The gene count (absolute number of genes) of differentially expressed genes per category (blue). And the gene count of differentially expressed genes relative to the total number of genes (red) present in the genomes of the analyzed strains.</p

Differentially expressed genes in <i>ptxP3</i> vs. <i>ptxP1 Bordetella pertussis</i> strains.

<p>Differentially expressed genes in <i>ptxP3</i> vs. <i>ptxP1 Bordetella pertussis</i> strains.</p

Role of the <i>ptxP3</i> and <i>ptxP1</i> mutations and the genetic background of <i>ptxP1</i> and <i>ptxP3</i> strains in colonizing the trachea (A) and lungs (B) in mice.

<p>Mice were intranasally infected with the wildtype <i>ptxP1</i> strain, the <i>ptxP3</i> strain, isogenic strains carrying the <i>ptxP3</i> allele in the <i>ptxP1</i> genetic background (P1 gb:<i>ptxP3</i>) or the <i>ptxP1</i> allele in the <i>ptxP3</i> genetic background (P3 gb:<i>ptxP1</i>). CFUs were determined in the trachea and lungs four days post-infection. The mean is indicated by a thin line. P-values (uncorrected for multiple tests) were shown if greater than 0.05. The experiment was performed two times representative result is shown.</p

Genome-Wide Gene Expression Analysis of <i>Bordetella pertussis</i> Isolates Associated with a Resurgence in Pertussis: Elucidation of Factors Involved in the Increased Fitness of Epidemic Strains

<div><p><i>Bordetella pertussis (B. pertussis)</i> is the causative agent of whooping cough, which is a highly contagious disease in the human respiratory tract. Despite vaccination since the 1950s, pertussis remains the most prevalent vaccine-preventable disease in developed countries. A recent resurgence pertussis is associated with the expansion of <i>B. pertussis</i> strains with a novel allele for the pertussis toxin (ptx) promoter <i>ptxP3</i> in place of resident <i>ptxP1</i> strains. The recent expansion of <i>ptxP3</i> strains suggests that these strains carry mutations that have increased their fitness. Compared to the <i>ptxP1</i> strains, <i>ptxP3</i> strains produce more Ptx, which results in increased virulence and immune suppression. In this study, we investigated the contribution of gene expression changes of various genes on the increased fitness of the <i>ptxP3</i> strains. Using genome-wide gene expression profiling, we show that several virulence genes had higher expression levels in the <i>ptxP3</i> strains compared to the <i>ptxP1</i> strains. We provide the first evidence that wildtype <i>ptxP3</i> strains are better colonizers in an intranasal mouse infection model. This study shows that the <i>ptxP3</i> mutation and the genetic background of <i>ptxP3</i> strains affect fitness by contributing to the ability to colonize in a mouse infection model. These results show that the genetic background of <i>ptxP3</i> strains with a higher expression of virulence genes contribute to increased fitness.</p></div

Relative differences in gene expression between <i>ptxP3</i> and <i>ptxP1</i> strains, as detected by microarrays and Q-PCR.

<p>Relative differences in gene expression between <i>ptxP3</i> and <i>ptxP1</i> strains, as detected by microarrays and Q-PCR.</p

Genes from the vir-core regulon were significantly upregulated in the <i>ptxP3</i> strains compared to the <i>ptxP1</i> strains.

<p>Genes from the vir-core regulon were significantly upregulated in the <i>ptxP3</i> strains compared to the <i>ptxP1</i> strains.</p

Surface display of a borrelial lipoprotein on meningococcal outer membrane vesicles

Outer Membrane Vesicles (OMVs) are gaining attention as vaccine candidates. The successful expression of heterologous antigens in OMVs, with the OMV functioning both as adjuvant and delivery vehicle, has greatly enhanced their vaccine potential. Since there are indications that surface exposed antigens might induce a superior immune response, targeting of heterologous antigens to the OMV surface is of special interest. Several systems for surface display of heterologous antigens on OMVs have been developed. However, these systems have not been used to display lipidated membrane-associated proteins known as lipoproteins, which are emerging as key targets for protective immunity. We were therefore interested to see whether we could express a foreign lipoprotein on the outer surface of OMVs. When outer surface protein A (OspA), a borrelial surface-exposed lipoprotein, was expressed in meningococci, it was found that although OspA was present in OMVs, it was no longer surface-exposed. Therefore, a set of fusions of OspA to different regions of factor H binding protein (fHbp), a meningococcal surface-exposed lipoprotein, were designed and tested for their surface-exposure. An N-terminal part of fHbp was found to be necessary for the successful surface display of OspA on meningococcal OMVs. When mice were immunized with this set of OMVs, an OspA-specific antibody response was only elicited by OMVs with clearly surface-exposed OspA, strengthening the idea that the exact positioning of an antigen in the OMV affects the immune response. This method for the surface display of heterologous lipoproteins on OMVs is a step forward in the development of OMVs as a vaccine platfor