Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae

Fu, Jeffrey; VanDusen, William J.; Kolodin, D. Garrett; O'Keefe, Donald O.; Herber, Wayne K.; George, Hugh A.

doi:10.1002/(sici)1097-0290(19960305)49:5<578::aid-bit11>3.0.co;2-6

Cited by 13 publications

(6 citation statements)

References 33 publications

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“…In contrast, the hepatitis B vaccine lacks the ability to induce durable antibody titres, even after a prime-boost-boost regimen and protection seems, in this case, to depend on memory B cells rather than on long-lived plasma cells [14, 29, 30]. The HBV vaccine is composed of the HBsAg, presented, at low density (100–120 HBsAg per VLP), on the surface of non-rigid lipid particles [31, 32], and it has been proposed that HBV particles represent a sub-optimal virus-like epitope display compared to HPV L1 VLPs [33]. Accordingly, since it appears that the RTS,S vaccine is protective only while antibodies are circulating, HBsAg VLPs (i.e., the hepatitis B vaccine) are likely a sub-optimal platform for displaying the CSP antigen.…”

Section: Discussionmentioning

confidence: 99%

Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses

Janitzek

Matondo

Thrane

et al. 2016

Malar J

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BackgroundMalaria, caused by Plasmodium falciparum, continues to have a devastating impact on global health, emphasizing the great need for a malaria vaccine. The circumsporozoite protein (CSP) is an attractive target for a malaria vaccine, and forms a major component of RTS,S, the most clinically advanced malaria vaccine. The clinical efficacy of RTS,S has been moderate, yet has demonstrated the viability of a CSP-based malaria vaccine. In this study, a vaccine comprised of the full-length CSP antigen presented on a virus-like particle (VLP) is produced using a split-intein conjugation system (SpyTag/SpyCatcher) and the immunogenicity is tested in mice.MethodsFull-length 3d7 CSP protein was genetically fused at the C-terminus to SpyCatcher. The CSP-SpyCatcher antigen was then covalently attached (via the SpyTag/SpyCatcher interaction) to Acinetobacter phage AP205 VLPs which were modified to display one SpyTag per VLP subunit. To evaluate the VLP-display effect, the immunogenicity of the VLP vaccine was tested in mice and compared to a control vaccine containing AP205 VLPs plus unconjugated CSP.ResultsFull-length CSP was conjugated at high density (an average of 112 CSP molecules per VLP) to AP205 SpyTag-VLPs. Vaccination of mice with the CSP Spy-VLP vaccine resulted in significantly increased antibody titres over a course of 7 months as compared to the control group (2.6-fold higher at 7 months after immunization). Furthermore, the CSP Spy-VLP vaccine appears to stimulate production of IgG2a antibodies, which has been linked with a more efficient clearing of intracellular parasite infection.ConclusionThis study demonstrates that the high-density display of CSP on SpyTag-VLPs, significantly increases the level and quality of the vaccine-induced humoral response, compared to a control vaccine consisting of soluble CSP plus AP205 VLPs. The SpyTag-VLP platform utilized in this study constitutes a versatile and rapid method to develop highly immunogenic vaccines. It might serve as a generic tool for the cost-effective development of effective VLP-vaccines, e.g., against malaria.

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Section: Discussionmentioning

confidence: 99%

Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses

Janitzek

Matondo

Thrane

et al. 2016

Malar J

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“…An unbaffled 250 mL Erlenmeyer shake flask culture was prepared with a chemically defined medium (Fu et al, 1995) with 4% dextrose and 1% (v/v) inoculum from a thawed S. cerevisiae ISB seed stock. The cultures were incubated for approximately 24 h at 28-C, 250 rpm.…”

Section: Data Acquisition Systemmentioning

confidence: 99%

Twenty‐four‐well plate miniature bioreactor high‐throughput system: Assessment for microbial cultivations

Isett¹,

George²,

Herber³

et al. 2007

Biotech & Bioengineering

120

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High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (micro)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the micro-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6 +/- 2.4, 46.5 +/- 4.6, 51.6 +/- 3.7, and 56.1 +/- 1.6 h(-1) at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively.

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“…The hepatitis vaccine is composed of a VLP based on the hepatitis B surface antigen (HBsAg). It can be produced recombinantly in yeast, such as Saccharomyces cerevisiae [17][18][19][20][21] or mammalian cells [22]. It buds from the endoplasmic reticulum as a 22 nm lipoprotein ( Fig.…”

Section: Introductionmentioning

confidence: 99%

A monolith purification process for virus-like particles from yeast homogenate

Burden

Jin

Podgornik

et al. 2012

Journal of Chromatography B

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Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. The monolith structure provides a more open pore structure to improve accessibility for these materials and better mass transport from convective flow and reduced pressure drops. To examine the performance of this format for bioprocessing we selected the challenging capture of a VLP from clarified yeast homogenate. Using a recombinant Saccharomyces cerevisiae host it was found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity was shown to be three-fold superior for the monolith with equivalent 90% recovery of the VLP. To understand the impact of the crude feed material confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. It was seen that the lipid formed a layer on top of the column, even after regeneration of the column with isopropanol, resulting in increasing pressure drops with the number of operational cycles. Removal of the lipid pre-column significantly reduces the amount and rate of this fouling process. Using Amberlite/XAD-4 beads around 70% of the lipid was removed, with a loss of VLP around 20%. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11 mg/mL column to 0.25 mg/mL column.

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Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae

Cited by 13 publications

References 33 publications

Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses

Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses

Twenty‐four‐well plate miniature bioreactor high‐throughput system: Assessment for microbial cultivations

A monolith purification process for virus-like particles from yeast homogenate

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