Characterization and evaluation of a recombinant hepatitis B vaccine expressed in yeast defective for N-linked hyperglycosylation

Kniskern, Peter J.; Hagopian, Arpi; Burke, P; Schultz, Loren D.; Montgomery, Donna L.; Hurni, William M.; Ip, C.Yu.; Schulman, C A; Maigetter, Robert Z.; Wampler, D E; Kubek, Dennis J.; Sitrin, Robert D.; West, David J.; Ellis, Ronald W.; Miller, William J.

doi:10.1016/0264-410x(94)90339-5

Cited by 30 publications

(17 citation statements)

References 11 publications

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“…cerevisiae has been widely used for the expression of recombinant antigens (Dargeviciute et al, 2002;Gozar et al, 1998;Kniskern et al, 1994;Lowe et al, 1997;Stowers et al, 2001). The advantages of S. cerevisiae as a host organism are varied, including its GRAS status and the capacity to carry out post-translational processing.…”

Section: Discussionmentioning

confidence: 99%

“…However, hyperglycosylation of some recombinant proteins and low levels of secretion remain a problem. Kniskern et al (1994) tackled the problem of hyperglycosylation in a yeast expressed hepatitis B vaccine by using a yeast strain defective for N-glycosylation. In the case of VP8*, the single potential N-glycosylation site is not naturally glycosylated, since VP4 does not enter the endoplasmic reticulum.…”

Section: Discussionmentioning

confidence: 99%

“…As an eukaryotic organism S. cerevisiae can perform post-translational processing, leading to proteins with a conformation similar to the one adopted in higher eukaryotic cells, a very important characteristic since in some cases recognition antigen-antibody is conformational (Stowers et al, 2001), and permitting the intracellular formation of virus-like particles (VLP) (Lowe et al, 1997). Finally, the hyperglycosylation that in some cases may affect yeast-expressed recombinant proteins can be reduced using strains deficient in N-glycosylation (Kniskern et al, 1994). …”

Section: Introductionmentioning

confidence: 97%

“…It is a GRAS organism that can be easily genetically manipulated and grown cheaply in large-scale industrial fermenters. Accordingly, S. cerevisiae has been used for the secretion or intracellular expression of recombinant antigens from hepatitis B virus (Kniskern et al, 1994;Valenzuela et al, 1982), hantavirus (Dargeviciute et al, 2002), Plasmodium falciparum (Gozar et al, 1998;Stowers et al, 2001), and papillomavirus (Lowe et al, 1997) among others. As an eukaryotic organism S. cerevisiae can perform post-translational processing, leading to proteins with a conformation similar to the one adopted in higher eukaryotic cells, a very important characteristic since in some cases recognition antigen-antibody is conformational (Stowers et al, 2001), and permitting the intracellular formation of virus-like particles (VLP) (Lowe et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

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Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

Andrés

Rodrı́guez-Dı́az

Buesa

et al. 2005

Biotech & Bioengineering

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The VP8* fragment from the rotavirus spike protein was expressed as a fusion protein with two different cell wall proteins of Saccharomyces cerevisiae, Icwp (Ssr1p) and Pir4, to achieve cell wall targeting or secretion to the growth medium of the fusion proteins. Two different host strains were used for the expression of the fusion proteins, a standard S. cerevisiae strain and a mnn9 glycosylation deficient strain, the later to reduce hyper-glycosylation. The Icwp-VP8* fusion could only be detected in the growth medium, indicating that the presence of the VP8* moiety interferes with the anchorage of Icwp to the cell wall. In the case of the Pir4-VP8* fusion proteins, we achieved cell wall targeting or secretion depending on how the gene fusion had been performed. In all cases, the fusion proteins expressed in the mnn9 strain showed a reduced level of glycosylation. Mice were inoculated intraperitoneally either with Pir4-VP8* or Icwp-VP8* fusion proteins purified from the growth medium of mnn9 strains expressing them or with whole cells of an mnn9 strain expressing a Pir4-VP8 fusion protein on its cell walls. Hundred percent of mice inoculated with the Pir4-VP8* fusion protein and 25% of those inoculated with the Icwp-VP8* fusion protein showed high titers of anti-VP8* antibodies. No specific immune response was detected in those mice inoculated with whole cells. Finally, susceptibility to rotavirus infection of the offspring of immunized dams was determined and protection was found in a percentage of approximately 60% with respect to the control group.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

Andrés

Rodrı́guez-Dı́az

Buesa

et al. 2005

Biotech & Bioengineering

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“…In fact, as long as viability remains stable during storage, it is unlikely that other characteristics of the cell bank (i.e. recombinant expression system) would require further verification at intervals during cryopreserved storage to meet regulatory requirements [11].The mnn9 strain of Saccharomyces cerevisiae, such as that developed for the manufacture of a recombinant vaccine protein [12] presents a challenge for testing the viability of inocula intended for manufacturing. The strain, chosen for its inability to hypermannosylate potential vaccine antigens [12], grows as a population of clumps of up to dozens of cells each.…”

Section: Introductionmentioning

confidence: 99%

Assessing the viability of a clumpy mnn9 strain of Saccharomyces cerevisiae used in the manufacture of recombinant pharmaceutical proteins

Duncan

Gallagher

McKerral

et al. 2004

J IND MICROBIOL BIOTECHNOL

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Demonstration of the viability of cryopreserved cell bank used to make a biopharmaceutical product is an important indicator of the ability to consistently manufacture over a long period of time, and is mandated in regulatory guidances. A mnn9 strain of Saccharomyces cerevisiae, chosen for its inability to hypermannosylate vaccine antigens, has a clumpy growth tendency due to the inactivation of the gene MNN9 (wild-type), complicating the interpretation of conventional viability measurements useful for single cells. Therefore, two growth-based measurements as well as staining by a membrane-impermeable dye were examined for their ability to reflect changes in viability of a clumpy mnn9 (defective) strain. The cell clumps proved to be stable to mixing, and variability of agar-plate-based viable counts (VC) of undisrupted suspensions of this clumpy mnn9 strain was consistent with variability observed for cell banks of a non-clumpy MNN9 strain. Both the VC and the growth times in an oxygen-sensing broth-based microplate assay corresponded well with shake-flask growth times for a set of stressed and unstressed samples, although the correlation was highest between the two broth-based systems. Counts of trypan-blue-stained cells within clumps also increased with time of stress, suggesting that this method could be adapted as a simple index of viability as well.

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Glycosylation of human α1-antitrypsin inSaccharomyces cerevisiae and methylotrophic yeasts

Kang

Sohn

Choi

et al. 1998

Yeast

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Human α1‐antitrypsin (α1‐AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α1‐AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α1‐AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α1‐AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α1‐AT. The human α1‐AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α1‐AT in the methylotrophic yeasts appeared to be relatively shorter than those of α1‐AT in S. cerevisiae. The α1‐AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α1‐AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein. © 1998 John Wiley & Sons, Ltd.

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Characterization and evaluation of a recombinant hepatitis B vaccine expressed in yeast defective for N-linked hyperglycosylation

Cited by 30 publications

References 11 publications

Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

Assessing the viability of a clumpy mnn9 strain of Saccharomyces cerevisiae used in the manufacture of recombinant pharmaceutical proteins

Glycosylation of human α1-antitrypsin inSaccharomyces cerevisiae and methylotrophic yeasts

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