Robert Z. Maigetter

Characterization of highly purified, inactivated HIV-1 particles isolated by anion exchange chromatography

This report characterizes inactivated, gp120 depleted, HIV-1 particles purified by an anion exchange chromatography production process. This antigen formulated with incomplete Freunds adjuvant constitutes Remune, which is being evaluated in a phase III clinical endpoint trial to determine the effect of this immune-based therapy on clinical progression of HIV-1 seropositive patients. Multiple production lots of the inactivated HIV-1 antigen strain HZ321, isolated by anion exchange chromatography, exhibit purity of > 95% by gel filtration. These findings are corroborated by thin section electron microscopy showing a homogenous field of intact particles. Analyses of the purified virus particles for protein, lipid, carbohydrate and RNA show structural retention of the envelope proteins, lipid bilayer and core components after large scale processing. The qualitative identification of at least 85% of total HIV-1 protein is determined by ELISA, Western blot, HPLC and amino acid sequencing analyses. Quantitative values are assigned to 50% of these proteins. The data confirm the presence of virally encoded proteins p6, p7, pI15, p17, p24, p32, pI39Gag, gp41, pp55Gag, p66/51, Vpr, Vif and Nef. Excellent consistency between production lots and equivalency to HIV-1 preparations purified by sucrose density gradient sedimentation has been established for protein and lipid composition, and overall purity. These findings further establish that non-viral encoded proteins and lipids are integral structural components of the intact virion and are not contaminants unique to a particular isolation method. The data confirm the presence of multicomponent antigens in the viral particles for stimulating a broad HIV-1 specific immune response. Finally, the work demonstrates that the two inactivation procedures (beta-propiolactone and gamma irradiation), which achieve efficient viral inactivation meeting US FDA guidelines, do not damage the protein antigens of the viral particles.

Antigenic analysis of the epstein-barr virus major membrane antigen (gp350/220) expressed in yeast and mammalian cells: Implications for the development of a subunit vaccine

The Epstein-Barr virus (EBV) major surface membrane antigen, gp350/220, was expressed in recombinant yeast cells and in several recombinant mammalian cell lines. Each of the expressed proteins was analyzed for its ability to bind to a panel of anti-gp350/220 monoclonal antibodies and to a series of anti-EBV positive human sera. The antigens also were used as immunogens for the immunization of rabbits. Each expressed protein was found to be unique both in its pattern of reactivity to the various antibodies and in the spectrum of antibody induced following animal immunization. These results suggest that cell-specific post-translational modifications critically influence the antigenic presentation of the expressed proteins. Nonetheless, all of the mammalian cell-derived versions of the membrane antigen were found capable of inducing EBV-specific neutralizing antibodies.

Production of hepatitis B surface antigen (preS2 + S) by high-cell density cultivations of a recombinant yeast.

A high-cell density bioprocess has been developed for the production of hepatitis B surface protein (preS2 + S) by recombinant yeast. This fed-batch process utilizes a growth medium containing yeast extract, soy peptone and glucose which was fed at a constant rate to maintain cells in a respiratory state. Cell densities of up to 60 g l-1 dry weight were achieved, which represented a 6-fold increase over those from batch bioprocesses. This increase in cell mass was attained without compromising specific activity; therefore, volumetric productivities of six times those of batch bioprocesses were achieved.

Fermentation of recombinant yeast producing hepatitis B surface antigen

SummaryFermentations were performed to determine parameters affecting the expression of hepatitis B surface antigen (HBsAg) in the yeastSaccharomyces cerevisiae containing the HBsAg gene. These studies emphasized inereasing both the relative abundance (HBsAg: cell mass) and total production of HBsAg. Specific activity was increased 70-fold when cells were grown in shake flasks containing nonselective rather than selective medium. The addition of adenine, ammonium sulfate or glucose to the complex medium reduced the production of antigen. Results similar to those achieved in shake flasks were obtained when the growth was performed in fermenters. A nutrient addition system was employed to increase the production of cells and HBsAg. The addition of glucose to the culture medium increased cell mass 6-fold but decreased the production of antigen. This imbalance was corrected by supplementing the glucose with complex nutrients.

High-cell-density fermentation studies of a recombinantE. coli that expresses atrial natriuretic factor

SummaryStudies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.

Harvesting recombinant microbial cells using crossflow filtration

A contained, crossflow filtration (CFF) membrane system is described for harvesting Saccharomyces cerevisiae and Escherichia coli cells. This system is portable and can be cleaned and sanitized in place. Low- and high-cell density (LCD, HCD) fermentations of recombinant cells in 10- to 200-l volumes were used as the starting material. LCD fermentations, up to 8.3 g l-1 dry weight (dcw) of S. cerevisiae, with volumes of 10 to 200 l were harvested and diafiltered in 0.5 and 1.5 h, respectively. HCD 200-l fermentations of S. cerevisiae (47-63 g l-1 dcw) were harvested and diafiltered in approximately 2 h. E. coli fermentations, LCD and HCD (up to 16.2 g l-1 dcw), of 200-l volumes were harvested and diafiltered in 2.3 h while employing 14 and 75 ft2 of membrane area, respectively. Using hollow fiber or flat sheet membranes from different sources, cell harvesting times were less than 2.5 h. These studies demonstrate that CFF is an efficient method for harvesting and diafiltering recombinant S. cerevisiae and E. coli cells from fermentation broth.

Galactose-regulated expression of hepatitis B surface antigen by a recombinant yeast

SummaryA yeast expressing the hepatitis B surface antigen (HBsAg) gene under the control of a regulated promoter was grown in batch and fed-batch modes. In batch fermentations, the addition of galactose caused the rapid production of HBsAg. Cells grown in fed-batch mode did not produce HBsAg unless the conditioned medium was replaced prior to induction.

Regulated production of recombinant echistatin by yeast

SummaryThe α-mating factor pre-pro-leader sequence under the regulation of theGAL10 promoter was used to direct the secretion of echistatin by recombinant yeasts. Optimization of the culture medium and host strain increased the productivity of shake flask cultures twenty-fold to 8 mg/L. In fermentors, the production of echistatin was greater than 40 mg/L.

Production of cytotoxic proteins in Escherichia coli: a fermentation process for producing enzymatically active HIV-1 protease

SummaryTwo fermentation processes for the tryptophan-regulated expression of active HIV protease (HIV-1 prt) in Escherichia coli are described. Since overexpression of HIV-1 prt results in cell death, stringent control of product expression was necessary to attain high enzyme levels. Such control was achieved by separation of growth and production phases in a two-step process or by implementation of nutrient feed in a one-step process. When the two-stage process was used, soluble product was detectable only when induction occurred at low culture density (A550 < 3.5). Short induction periods of 1–2 h and rapid harvesting were necessary to recover active product. Similar results were obtained when the single-stage process was operated at 37°C; however, cultivation and induction at 28°C resulted in active enzyme formation following induction at increased cell density (A550=10).

Studies on the chemical composition of lipopolysaccharide from Neisseria meningitidis group B.

A lipopolysaccharide was isolated from Neisseria meningitidis group B by phenol/water extraction and purified by differential ultracentrifugation. This preparation exhibited endotoxic properties as shown by the limulus-lysate assay. Mild acid hydrolysis of the lipopolysaccharides yielded a lipid A fraction and a polysaccharide fraction. The lipid A fraction contained fatty acids, phosphorus and glucosamine. Analysis of the polysaccharide fraction revealed the presence of glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid and phosphorus. There was no heptose.