Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants

Key message

The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements.

Abstract

Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.

     

Heterocapsa psammophila sp. nov. (Peridiniales, Dinophyceae), a new sand-dwelling marine dinoflagellate

A new armored dinoflagellate species, Heterocapsa psammophila Tamura, Iwataki et Horiguchi sp. nov. is described from Kenmin‐no‐hama beach, Hiroshima, Japan using light and electron microscopy. This dinoflagellate possesses the typical thecal plate arrangement of the genus Heterocapsa, Po, cp, 5′, 3a, 7′′, 6c, 5s, 5′′′, 2′′′′; and the 3‐D body scales of Heterocapsa on the plasma membrane. The cell shape is ovoidal. The spherical nucleus and the pyrenoid are situated in the hypotheca and the epitheca, respectively. The ultrastructure of H. psammophila is typical of dinoflagellates and the pyrenoid is invaginated by cytoplasmic tubules. H. psammophila is distinguished from all other hitherto‐described Heterocapsa species by the cell shape, the relative position of the nucleus and pyrenoid and the structure of the body scale. The habitat and behavior of this new species in culture suggest that the organism is truly a sand‐dwelling species.     

Purification and subunit structure of rat-liver phosphoprotein phosphatase, whose molecular weight is 260000 by gel filtration (phosphatase IB)

1. Phosphoprotein phosphatase IB is a form of rat liver phosphoprotein phosphatase, distinguished from the previously studied phosphoprotein phosphatase II [Tamura et al. (1980) Eur. J. Biochem. 104, 347-355] by earlier elution from DEAE-cellulose, by higher molecular weight on gel filtration (260000) and by lower activity toward phosphorylase alpha. This enzyme was purified to apparent homogeneity by chromatography on DEAE-cellulose, aminohexyl--Sepharose-4B, histone--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200. 2. The molecular weight of purified phosphatase IB was 260000 by gel filtration and 185000 from S20,W and Stokes' radius. Using histone phosphatase activity as the reference for comparison, the phosphorylase phosphatase activity of purified phosphatase IB was only one-fifth that of phosphatase II. 3. Sodium dodecyl sulfate gel electrophoresis revealed that phosphatase IB contains three types of subunit, namely alpha, beta and gamma, whose molecular weights are 35000, 69000 and 58000, respectively. The alpha subunit is identical to the alpha subunit of phosphatase II. While the beta subunit is also identical or similar to the beta subunit of phoshatase II, the gamma subunit appears to be unique to phosphatase IB. 4. When purified phosphatase IB was treated with 2-mercaptoethanol at -20 degrees C, the enzyme was dissociated to release the catalytically active alpha subunit. Along with this dissociation, there was a 7.4-fold increase in phosphorylase phosphatase activity; but histone phosphatase activity increased only 1.6-fold. The possible functions of the gamma subunit are discussed in relation to this activation of enzyme.     

Cell type-specific activation of metabolism reveals that beta-cell secretion suppresses glucagon release from alpha-cells in rat pancreatic islets

Abnormal glucagon secretion is often associated with diabetes mellitus. However, the mechanisms by which nutrients modulate glucagon secretion remain poorly understood. Paracrine modulation by beta- or delta-cells is among the postulated mechanisms. Herein we present further evidence of the paracrine mechanism. First, to activate cellular metabolism and thus hormone secretion in response to specific secretagogues, we engineered insulinoma INS-1E cells using an adenovirus-mediated expression system. Expression of the Na+-dependent dicarboxylate transporter (NaDC)-1 resulted in 2.5- to 4.6-fold (P < 0.01) increases in insulin secretion in response to various tricarboxylic acid cycle intermediates. Similarly, expression of glycerol kinase (GlyK) increased insulin secretion 3.8- or 4.2-fold (P < 0.01) in response to glycerol or dihydroxyacetone, respectively. This cell engineering method was then modified, using the Cre-loxP switching system, to activate beta-cells and non-beta-cells separately in rat islets. NaDC-1 expression only in non-beta-cells, among which alpha-cells are predominant, caused an increase (by 1.8-fold, P < 0.05) in glucagon secretion in response to malate or succinate. However, the increase in glucagon release was prevented when NaDC-1 was expressed in whole islets, i.e., both beta-cells and non-beta-cells. Similarly, an increase in glucagon release with glycerol was observed when GlyK was expressed only in non-beta-cells but not when it was expressed in whole islets. Furthermore, dicarboxylates suppressed basal glucagon secretion by 30% (P < 0.05) when NaDC-1 was expressed only in beta-cells. These data demonstrate that glucagon secretion from rat alpha-cells depends on beta-cell activation and provide insights into the coordinated mechanisms underlying hormone secretion from pancreatic islets.     

Structure–activity-relationship studies on dihydrofuran-fused perhydrophenanthrenes as an anti-Alzheimer’s disease agent

As an extended study on development of anti-Alzheimer’s disease agent, we newly synthesized various dihydrofuran-fused perhydrophenanthrenes via o-quinodimethane chemistry. This study revealed that the introduction of carbon side-chain on 8-position or removal of the acetal moiety on 3-position arose a cytotoxicity on rat cortical neurons. On the other hand, the ethereal or thio-ethereal substituent on 8-position enhanced the elongation effect on Aβ-damaged neurons. The necessity of the cyano group on 10b position was also proved in this structure–activity-relationship study.