AIDSWEEKLY Plus, 01 April 1996 issue; Published by Charles Henderson, Publisher. Editorial & Publishing Office: P.O. Box 5528, Atlanta, GA 30307-0528 / Telephone: (800) 633-4931; Subscription Office: P.O. Box 830409, Birmingham, AL 35283-0409 / FAX: (205) 995-1588
Daniel J. DeNoon, Senior Editor

Pharmaceutical plants of the future may literally be just that.

By using genetically engineered plant viruses, researchers now have the ability to turn vegetables into efficient factories for producing vaccine antigens.

"The productivity of this system is really quite impressive," said Paul B. Rodgers, director of development for Axis Genetics Ltd., Cambridge, England. "Ten milligrams of purified particles are produced per plant in two to three weeks."

Rodgers described vaccine and therapeutic protein production in plants in an address to the Cambridge Healthtech Institute's Vaccines: New Technologies and Applications conference, held March 18-20, 1996, in McLean, Virginia.

The protocol is relatively simple:

* Appropriate plant viruses are genetically engineered to express the desired peptides or proteins. Plant viruses currently being utilized are cowpea mosaic virus, potato virus X, tomato bushy stunt virus, and tobacco mosaic virus.

* Typically, two leaves of a plant are inoculated with the engineered virus.

* The plant grows new leaves.

* The new leaves are harvested and virus particles are extracted in a process that includes grinding the leaves, centrifuging the raw extract, and precipitation with polyethylene glycol.

* The virus particles are used to infect new plants.

* Large numbers of new plants are grown.

* Chimeric virions are extracted and purified.

The newest technique being developed to permit plant production of entire proteins, is termed "overcoat" technology.

This still-experimental method uses the rod-shaped potato virus X PVX). Rodgers noted that PVX has an RNA core surrounded by a single coat protein.

"The 25 kDa coat protein of PVX was fused, through a special linker peptide of 16-18 amino acids, to a high- molecular-weight foreign reporter protein - the green fluorescent protein (GFP) from a jellyfish, Aequorea victoria," Rodgers and colleagues wrote in a supplement to his presentation.

"Serendipitously, the 55 kDa fusion protein could still assemble to form PVX particles which were not only longer (because of the added 900 nucleotides of GFP-coding RNA) but also twice as wide as normal PVX."

Rodgers said that other foreign proteins - including large polypeptide epitopes from pathogens - can be fused to PVX coat proteins in similar overcoats.

Further developed is what Rodgers referred to as "epicoat" technology for the expression of foreign peptides on the surfaces of icosahedral plant viruses.

This technique uses the cowpea mosaic virus (CPMV). This RNA virus has a 28-30 nm diameter and consists of two RNA strands, one coding for regulatory proteins and one coding for structural proteins.

By linking foreign genes to a specific site in the sequence, 60 copies of the desired peptides are expressed on peaks dotting the surface of the virus particle.

"The largest polypeptide which has been expressed so far using the epicoat is 36 amino acids in length," Rodgers noted.

By weight, the foreign peptide represents 3 percent of the chimeric CPMV particle.

To test the immunogenicity of antigen-expressing CPMV, Rodgers described an experiment in which CPMV was used to express an epitope from the HIV-1[IIIB] gp41 transmembrane glycoprotein.

Mice inoculated with 100 (micro)g of chimeric CPMV (containing 3 (micro)g of the HIV epitope) with alum adjuvant developed a strong neutralizing antibody response. These antibodies also recognized heterologous gp41 from the RF (70 percent neutralization) and SF2 (60 percent neutralization) strains of HIV-1.

In another experiment, the CPMV system was used to express the 17-amino-acid MEV epitope of canine parvovirus (CPV).

Mink were inoculated with either 100 (micro)g (low dose) or 1 mg (high dose) of chimeric CPMV. While all six untreated control animals died within seven days of oronasal challenge with virulent CPV, none of the 12 vaccinated animals developed symptoms.

Four of the six animals in the low-dose group became infected as indicated by viral shedding, but none developed symptoms. Only transient viral shedding was seen in two of the six animals in the high-dose group.

Rodgers said that a wide range of epitopes have been expressed in CPMV chimeric virus particles. These include:

* Viral epitopes from HIV-1 gp41, HIV-1 gp120, human rhinovirus, foot and mouth disease virus, and canine parvovirus.

* Bacterial epitopes from Staphylococcus aureus up to 33 amino acids in length.

* Mammalian epitopes from hormones and from colon-cancer cells.

* Fungal epitopes.

* Protozoan epitopes from Plasmodium falciparum.

Further recommending the chimeric virus particles (CVPs) are their stability:

* Genetic stability, as demonstrated after 20 passages in culture of CVP-HIV-1.

* Thermal stability: the thermal inactivation point of wild- type CPMV is 65(degree)C.

* Acid stability, as demonstrated with CVP-HIV-1 culture at pH 1 for one hour at 37(degree)C.

* Protease stability, as demonstrated with CVP-HIV-1 culture with pepsin at pH 2 for one hour at 37(degree)C.

Rodgers also touted the safety of the CVP technology.

"The carrier particles are non-infectious to mammals, and contamination by adventitious agents is not a major concern for a plant-based production process," he noted in his presentation supplement. "A wide range of vaccine administration routes is available: parenteral and nasal (purified particles), oral (formulated crude leaf extracts), and edible (whole or homogenized leaves, fruits, or vegetable tissues."

Rodgers said that the epicoat and overcoat techniques represent a platform technology for his firm.

"This will give us the ability to develop a wide range of therapeutics," he said.

Copyright (c) 1995 - Charles Henderson, Publisher. All rights Reserved. Permission to reproduce granted to AEGIS by Charles W. Henderson. Authorization to reproduce for personal use granted granted by C. W. Henderson, Publisher, provided that the fee of US$4.50 per copy, per page is paid directly to the Copyright Clearance Center, 27 Congress Street, Salem, Massachusetts 01970, USA.

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Copyright © 1996 - Charles Henderson, Publisher. All rights Reserved. Permission to reproduce granted to AEGIS by Charles W. Henderson. Authorization to reproduce for personal use granted granted by C. W. Henderson, Publisher, provided that the fee of US$4.50 per copy, per page is paid directly to the Copyright Clearance Center, 27 Congress Street, Salem, Massachusetts 01970, USA.