George P. Lomonossoff

Use of viral vectors for vaccine production in plants

The small size of plant viral genomes, the ease with which they can be manipulated, and the simplicity of the infection process is making the viral vectors an attractive alternative to the transgenic systems for the expression of foreign proteins in plants. One use of these virus expression systems is for vaccine production. There are two basic types of viral system that have been developed for the production of immunogenic peptides and proteins in plants: epitope presentation and polypeptide expression systems. In this review, we discuss advances made in this field.

Extremely High-Level and Rapid Transient Protein Production in Plants without the Use of Viral Replication

Plant-based overexpression of heterologous proteins has attracted much interest and development in recent years. To date, the most efficient vectors have been based on RNA virus-derived replicons. A system based on a disabled version of cowpea mosaic virus RNA-2 has been developed, which overcomes limitations on insert size and introduces biocontainment. This system involves positioning a gene of interest between the 5′ leader sequence and 3′ untranslated region (UTR) of RNA-2, thereby emulating a presumably stable mRNA for efficient translation. Thus far, the sequence of the 5′ UTR has been preserved to maintain the ability of the modified RNA-2 to be replicated by RNA-1. However, high-level expression may be achieved in the absence of RNA-1-derived replication functions using Agrobacterium-mediated transient transformation. To investigate those features of the 5′ UTR necessary for efficient expression, we have addressed the role of two AUG codons found within the 5′ leader sequence upstream of the main initiation start site. Deletion of an in-frame start codon upstream of the main translation initiation site led to a massive increase in foreign protein accumulation. By 6 d postinfiltration, a number of unrelated proteins, including a full-size IgG and a self-assembling virus-like particle, were expressed to >10% and 20% of total extractable protein, respectively. Thus, this system provides an ideal vehicle for high-level expression that does not rely on viral replication of transcripts.

Use of Viral Replicons for the Expression of Genes in Plants

Autonomously replicating virus-based vectors have been investigated as a means of introducing heterologous genes into plants. This approach has a number of potential advantages over stable genetic transformation, particularly in terms of speed and levels of expression that can be obtained. Several groups of plant viruses, with genomes consisting of both DNA and RNA, have been investigated as possible gene vectors. In the case of DNA viruses, it has generally been possible to identify nonessential regions of the genome that can be replaced by foreign sequences. However, there appear to be limitations on the size of insert which can be tolerated. In the case of RNA viruses, replacement of viral sequences usually has a drastic effect on the viability. However, in several cases it has proved possible to substantially increase the size of the viral genome by the direct insertion of additional sequences while still retaining the ability of the viruses to multiply and spread in plants. These RNA virus-based systems appear to have the greatest potential as gene vectors.

A Serine Carboxypeptidase-Like Acyltransferase Is Required for Synthesis of Antimicrobial Compounds and Disease Resistance in Oats

Serine carboxypeptidase-like (SCPL) proteins have recently emerged as a new group of plant acyltransferases. These enzymes share homology with peptidases but lack protease activity and instead are able to acylate natural products. Several SCPL acyltransferases have been characterized to date from dicots, including an enzyme required for the synthesis of glucose polyesters that may contribute to insect resistance in wild tomato (Solanum pennellii) and enzymes required for the synthesis of sinapate esters associated with UV protection in Arabidopsis thaliana. In our earlier genetic analysis, we identified the Saponin-deficient 7 (Sad7) locus as being required for the synthesis of antimicrobial triterpene glycosides (avenacins) and for broad-spectrum disease resistance in diploid oat (Avena strigosa). Here, we report on the cloning of Sad7 and show that this gene encodes a functional SCPL acyltransferase, SCPL1, that is able to catalyze the synthesis of both N-methyl anthraniloyl- and benzoyl-derivatized forms of avenacin. Sad7 forms part of an operon-like gene cluster for avenacin synthesis. Oat SCPL1 (SAD7) is the founder member of a subfamily of monocot-specific SCPL proteins that includes predicted proteins from rice (Oryza sativa) and other grasses with potential roles in secondary metabolism and plant defense.

Stimulation of neutralizing antibodies to human immunodeficiency virus type 1 in three strains of mice immunized with a 22 amino acid peptide of gp41 expressed on the surface of a plant virus

A plant virus, cowpea mosaic virus, expressing a 22 amino acid peptide 731-752 of the gp41 glycoprotein of human immunodeficency virus type 1 (HIV-1 IIIB), was shown previously to stimulate HIV-1 cross reactive neutralizing antibodies in adult C57/BL6 mice. Here some parameters concerning the stimulation of HIV-1-specific neutralizing and ELISA antibody have been determined in adult C57/BL6, C3H/He-mg and BALB/c mice. Two injections per mouse of all CPMV-HIV/1 doses tested (100, 10 and 1 microgram chimera which contained, respectively, 1700, 170 and 17 ng HIV peptide per injection) stimulated a strong serum neutralizing antibody response in all mice. One hundred micrograms or 10 micrograms CPMV-HIV/1 per injection gave 99% neutralization of HIV-1 IIIB in C8166 cells at a serum dilution of 1/200, whereas sera from mice immunized with 1 microgram per injection neutralized virus to 97%, 79% and 63% at a 1/200 dilution of serum from C3H/He-mg, C57/BL6 and BALB/c mice, respectively. Restimulation of these mice with the same immunogen dose marginally increased the neutralization titres. The longevity of the neutralizing antibody response increased as the immunogen dose decreased, and was dependent on the strain of mouse, in the order C57/BL6C3H/He-mg BALB/c. Re-immunization with a third injection improved the longevity of the antibody response. All mice immunized with 100 micrograms CPMV-HIV/1 responded with ELISA antibody to the gp41 peptide bound in solid phase. Ten micrograms stimulated ELISA antibody in some but not all mice, while mice immunized with 1 microgram had no detectable ELISA antibody. This synthesis of ELISA antibody decreased > or = 230-fold over the range of immunogen doses tested but, in the same mice, the neutralizing antibody response decreased only twofold, showing an unusual bias to production of the latter. Neutralizing antibodies were thus stimulated at a lower immunogen dose than ELISA antibodies. Antibody which was affinity purified using the free gp41 peptide gave a good ELISA titre but did not neutralize HIV-1, suggesting that the neutralizing antibody is recognizing a conformational epitope on the gp41 oligomer.

Association of Phosphatidylinositol 3-Kinase with Nuclear Transcription Sites in Higher Plants

The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function. Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase encoded by the human VPS34 homolog has been equally implicated in the control of intracellular vesicle traffic. The plant VPS34 homolog also is required for normal growth and development, and although a role for PtdIns 3-kinase in vesicle trafficking is likely, it has not been established. In this study, we have shown that considerable PtdIns 3-kinase activity is associated with the internal matrix of nuclei isolated from carrot suspension cells. Immunocytochemical and confocal laser scanning microscopy studies using the monoclonal antibody JIM135 (John Innes Monoclonal 135), raised against a truncated version of the soybean PtdIns 3-kinase, SPI3K-5p, revealed that this kinase appears to have a distinct and punctate distribution within the plant nucleus and nucleolus. Dual probing of root sections with JIM135 and anti–bromo-UTP antibodies, after in vitro transcription had been allowed to proceed in the presence of bromo-UTP, showed that SPI3K-5p associates with active nuclear and nucleolar transcription sites. These findings suggest a possible link between PtdIns 3-kinase activity and nuclear transcription in plants.

Plant viral genes in DNA idiotypic vaccines activate linked CD4+ T-cell mediated immunity against B-cell malignancies

DNA delivery of tumor antigens can activate specific immune attack on cancer cells. However, antigens may be weak, and immune capacity can be compromised. Fusion of genes encoding activating sequences to the tumor antigen sequence facilitates promotion and manipulation of effector pathways. Idiotypic determinants of B-cell tumors, encoded by the variable region genes, are clone-specific tumor antigens. When assembled as single-chain Fv (scFv) alone in a DNA vaccine, immunogenicity is low. Previously, we found that fusion of a sequence from tetanus toxin (fragment C; FrC) promoted anti-idiotypic protection against lymphoma and myeloma. We have now investigated an alternative fusion gene derived from a plant virus, potato virus X coat protein, a primary antigen in humans. When fused to scFv, the self-aggregating protein generates protection against lymphoma and myeloma. In contrast to scFv–FrC, protection against lymphoma is mediated by CD4+ T cells, as is protection against myeloma. Plant viral proteins offer new opportunities to activate immunity against linked T-cell epitopes to attack cancer.

Cowpea mosaic virus-based chimaeras: Effects of inserted peptides on the phenotype, host range, and transmissibility of the modified viruses

Expression of foreign peptides on the surface of cowpea mosaic virus particles leads to the creation of chimaeras with a variety of phenotypes and yields. Two factors were shown to be particularly significant in determining the properties of a given chimaera: the length of the inserted sequence and its isoelectric point. The deleterious effect of high isoelectric point on the ability of chimeras to produce a systemic infection occurs irrespective of the site of insertion of the peptide. Ultrastructural analysis of tissue infected with chimaeras with different phenotypes showed that all produced particles with a tendency to aggregate, irrespective of the size or isoelectric point of the insert. Host range and transmission studies revealed that the expression of a foreign peptide did not (1) alter the virus host range, (2) increase the rate of transmission by beetles or through seed, or (3) change the insect vector specificity. These findings have implications for both the utility and the biosafety of Cowpea mosaic virus-based chimaeras.

CPMV-DOX delivers

The plant virus, Cowpea mosaic virus (CPMV), is developed as a carrier of the chemotherapeutic drug doxorubicin (DOX). CPMV-DOX conjugate, in which eighty DOX molecules are covalently bound to external surface carboxylates of the viral nanoparticle (VNP), shows greater cytotoxicity than free DOX toward HeLa cells when administered at low dosage. At higher concentrations, CPMV-DOX cytotoxicity is time-delayed. The CPMV conjugate is targeted to the endolysosomal compartment of the cells, in which the proteinaceous drug carrier is degraded and the drug released. This study is the first demonstrating the utility of CPMV as a drug delivery vehicle.

Structure-Based Engineering of an Icosahedral Virus for Nanomedicine and Nanotechnology

A quintessential tenet of nanotechnology is the self-assembly of nanometer-sized components into devices. Biological macromolecular systems such as viral particles were found to be suitable building blocks for nanotechnology for several reasons: viral capsids are extremely robust and can be produced in large quantities with ease, the particles self-assemble into monodisperse particles with a high degree of symmetry and polyvalency, they have the propensity to form arrays, and they offer programmability through genetic and chemical engineering. Here, we review the recent advances in engineering the icosahedral plant virus Cowpea mosaic virus (CPMV) for applications in nano-medicine and -technology. In the first part, we will discuss how the combined knowledge of the structure of CPMV at atomic resolution and the use of chimeric virus technology led to the generation of CPMV particles with short antigenic peptides for potential use as vaccine candidates. The second part focuses on the chemical addressability of CPMV. Strategies to chemically attach functional molecules at designed positions on the exterior surface of the viral particle are described. Biochemical conjugation methods led to the fabrication of electronically conducting CPMV particles and networks. In addition, functional proteins for targeted delivery to mammalian cells were successfully attached to CPMV. In the third part, we focus on the utilization of CPMV as a building block for the generation of 2D and 3D arrays. Overall, the potential applications of viral nanobuilding blocks are manifold and range from nanoelectronics to biomedical applications.