Co-expression of multiple target proteins in plants from a tobacco mosaic virus vector using a combination of homologous and heterologous subgenomic promoters.

To co-express multiple target proteins, we engineered a single-component chimeric tobacco mosaic virus (TMV)-based vector containing homologous and heterologous capsid protein subgenomic RNA promoters. Delivery of this vector into Nicotiana benthamiana plants via agroinfiltration resulted in co-expression of two reporter genes within a single cell. Furthermore, co-expression of a host-specific antisense RNA or a silencing suppressor protein from this vector augmented the accumulation of green fluorescent protein or a vaccine antigen, hemagglutinin from avian influenza virus A/Vietnam/1194/04. These findings suggest that this chimeric vector utilizing the homologous and heterologous subgenomic TMV promoters has a potential for high-level production of multiple therapeutic proteins including monoclonal antibodies.

Production of recombinant proteins in clonal root cultures using episomal expression vectors.

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities.

The potential of plant virus vectors for vaccine production.

Plants viruses are versatile vectors that allow the rapid and convenient production of recombinant proteins in plants. Compared with production systems based on transgenic plants, viral vectors are easier to manipulate and recombinant proteins can be produced more quickly and in greater yields. Over the last few years, there has been much interest in the development of plant viruses as vectors for the production of vaccines, either as whole polypeptides or epitopes displayed on the surface of chimeric viral particles. Several viruses have been extensively developed for vaccine production, including tobacco mosaic virus, potato virus X and cowpea mosaic virus. Vaccine candidates have been produced against a range of human and animal diseases, and in many cases have shown immunogenic activity and protection in the face of disease challenge. In this review, we discuss the advantages of plant virus vectors, the development of different viruses as vector systems, and the immunological experiments that have demonstrated the principle of plant virus-derived vaccines.

Deletions within the 3' non-translated region of Alfalfa mosaic virus RNA4 do not affect replication but significantly reduce long-distance movement of chimeric Tobacco mosaic virus.

Alfalfa mosaic virus (AlMV) RNAs 1 and 2 with deletions in their 3' non­translated regions (NTRs) have been previously shown to be encapsidated into virions by coat protein (CP) expressed from RNA3, indicating that the 3' NTRs of RNAs 1 and 2 are not required for virion assembly. Here, we constructed various mutants by deleting sequences within the 3' NTR of AlMV subgenomic (sg) RNA4 (same as of RNA3) and examined the effect of these deletions on replication and translation of chimeric Tobacco mosaic virus (TMV) expressing AlMV sgRNA4 from the TMV CP sg promoter (Av/A4) in tobacco protoplasts and Nicotiana benthamiana plants. While the Av/A4 mutants were as competent as the wild-type Av/A4 in RNA replication in protoplasts, their encapsidation, long-distance movement and virus accumulation varied significantly in N. benthamiana. These data suggest that the 3' NTR of AlMV sgRNA4 contains potential elements necessary for virus encapsidation.

A novel two-component Tobacco mosaic virus-based vector system for high-level expression of multiple therapeutic proteins including a human monoclonal antibody in plants.

Expression of multiple therapeutic proteins from Tobacco mosaic virus (TMV)-based vectors was not successful when plants were coinoculated with a mixture of two TMV vectors engineered to express two foreign genes individually. Here, we have engineered and developed a defective RNA (dRNA)-based TMV vector (dRT-V) that utilizes two components of the same virus, with the dRNA component depending on the helper virus for replication. Agrobacterium-mediated coinoculation of Nicotiana benthamiana plants with both components of the dRT-V resulted in high-level expression of a human growth hormone and a lichenase-fused lethal factor protein of Bacillus anthracis. Furthermore, both heavy and light chains were expressed and assembled into a monoclonal antibody (mAb) specific to the protective antigen of B. anthracis, and the average yield of the purified antibody obtained was 120 mg/kg of fresh tissue. Our data suggest that dRT-V has a potential for rapid, cost-effective, large-scale manufacturing of multiple therapeutic proteins including mAbs in response to any biological emergencies.

Plant-produced human growth hormone shows biological activity in a rat model.

Plants have been shown to be efficient systems for expressing a wide range of recombinant proteins from various origins. Here, using a plant virus-based expression vector to produce human growth hormone (hGH) in Nicotiana benthamiana plants, we demonstrate, for the first time, that the plant-produced hGH (pphGH) is biologically active in a hypophysectomized rat model. We observed an average weight gain of approximately 17 g per animal in a group of 10 animals that were injected subcutaneously with pphGH with 60 microg/dose for 10 days. With the increasing demand for hGH, accompanied with the need to make this recombinant protein available to a wider population at a more reasonable cost, plants provide a feasible alternative to current production platforms.

Plant-derived hemagglutinin protects ferrets against challenge infection with the A/Indonesia/05/05 strain of avian influenza.

The global spread of highly pathogenic avian influenza virus (H5N1 subtype) has promoted efforts to develop human vaccines against potential pandemic outbreaks. However, current platforms for influenza vaccine production are cumbersome, limited in scalability and often require the handling of live infectious virus. We describe the production of hemagglutinin from the A/Indonesia/05/05 strain of H5N1 influenza virus by transient expression in plants, and demonstrate the immunogenicity and protective efficacy of the vaccine candidate in animal models. Immunization of mice and ferrets with plant-derived hemagglutinin elicited serum hemagglutinin-inhibiting antibodies and protected the ferrets against challenge infection with a homologous virus. This demonstrates that plant-derived H5 HA is immunogenic in mice and ferrets, and can induce protective immunity against infection with highly pathogenic avian influenza virus. Plants could therefore be suitable as a platform for the rapid, large-scale production of influenza vaccines in the face of a pandemic.

Recent progress in the development of plant derived vaccines.

Recombinant subunit vaccines have been with us for the last 30 years and they provide us with the unique opportunity to choose from the many available production systems that can be used for recombinant protein expression. Plants have become an attractive production platform for recombinant biopharmaceuticals and vaccines have been at the forefront of this new and expanding industry sector. The particular advantages of plant-based vaccines in terms of cost, safety and scalability are discussed in the light of recent successful clinical trials and the likely impact of plant systems on the vaccine industry is evaluated.

Development of a new cucumber mosaic virus-based plant expression vector with truncated 3a movement protein.

We have developed a Cucumber mosaic virus (CMV)-based expression vector for the production of heterologous proteins in plants. Cell-to-cell movement of CMV is dependent on the presence of coat protein (CP). Previous studies have shown that deletion of 33 amino acids (aa) from the carboxy-terminus of the 3a movement protein facilitates cell-to-cell movement that is independent of CP. The CMV-based expression vector that we have designed utilizes this truncated 3a protein, allowing the expression of target genes from the strong CP subgenomic promoter and without the need for providing CP in trans for cell-to-cell spread. Using this vector we achieved expression levels of ~450 mg/kg leaf tissue of green fluorescent protein (GFP) when the vector was delivered into Nicotiana benthamiana plants by agroinfiltration. Human growth hormone (hGH), on the other hand, accumulated to ~170 mg/kg of leaf tissue when the same approach was used to deliver the vector.

A plant-produced influenza subunit vaccine protects ferrets against virus challenge.

BACKGROUND: Influenza A viruses are of major concern for public health, causing worldwide epidemics associated with high morbidity and mortality. Vaccines are critical for protection against influenza, but given the recent emergence of new strains with pandemic potential, and some limitations of the current production systems, there is a need for new approaches for vaccine development. OBJECTIVE: To demonstrate the immunogenicity and protective efficacy of plant-produced influenza antigens. Method We engineered, using influenza A/Wyoming/3/03 (H3N2) as a model virus, the stem and globular domains of hemagglutinin (HA) produced in plants as fusions to a carrier protein and used purified antigens with and without adjuvant for ferret immunization. RESULTS: These plant-produced antigens were highly immunogenic and conferred complete protection against infection in the ferret challenge model. The addition of plant-produced neuraminidase was shown to enhance the immune response in ferrets. CONCLUSIONS: Plants can be used as a production vehicle for vaccine development against influenza. Domains of HA can generate protective immune responses in ferrets.

Immunogenicity of a subunit vaccine against Bacillus anthracis.

The current approved vaccine against anthrax is based on protective antigen (PA) of Bacillus anthracis, requires six injections over an 18-month period and has a known history of side effects. Therefore, there is significant effort towards developing an improved vaccine against B. anthracis. Here we separately engineered and expressed domain 4 of PA (PAD4) and domain 1 of lethal factor (LFD1) as fusions to lichenase (LicKM), a thermostable enzyme from Clostridium thermocellum, and transiently expressed these fusions in Nicotiana benthamiana. Plant-produced antigens were combined and immunogenicity was evaluated in mice. All animals that received the experimental vaccine developed high antibody titers that were predominantly IgG1 and were able to neutralize the effects of LeTx in vitro.

A launch vector for the production of vaccine antigens in plants.

Historically, most vaccines have been based on killed or live-attenuated infectious agents. Although very successful at immunizing populations against disease, both approaches raise safety concerns and often have limited production capacity. This has resulted in increased emphasis on the development of subunit vaccines. Several recombinant systems have been considered for subunit vaccine manufacture, including plants, which offer advantages both in cost and in scale of production. We have developed a plant expression system utilizing a 'launch vector', which combines the advantageous features of standard agrobacterial binary plasmids and plant viral vectors, to achieve high-level target antigen expression in plants. As an additional feature, to aid in target expression, stability and purification, we have engineered a thermostable carrier molecule to which antigens are fused. We have applied this launch vector/carrier system to engineer and express target antigens from various pathogens, including, influenza A/Vietnam/04 (H5N1) virus.

Expression of Aedes trypsin-modulating oostatic factor on the virion of TMV: A potential larvicide.

We report the engineering of the surface of the tobacco mosaic virus (TMV) virion with a mosquito decapeptide hormone, trypsin-modulating oostatic factor (TMOF). The TMV coat protein (CP) was fused to TMOF at the C terminus by using a read-through, leaky stop codon that facilitated expression of CP and chimeric CP-TMOF (20:1 ratio) that were coassembled into virus particles in infected Nicotiana tabacum. Plants that were infected with the hybrid TMV RNA accumulated TMOF to levels of 1.3% of total soluble protein. Infected tobacco leaf discs that were fed to Heliothis virescens fourth-instar larvae stunted their growth and inhibited trypsin and chymotrypsin activity in their midgut. Purified CP-TMOF virions fed to mosquito larvae stopped larval growth and caused death. Because TMV has a wide host range, expressing TMV-TMOF in plants can be used as a general method to protect them against agricultural insect pests and to control vector mosquitoes.

Odontoglossum ringspot virus host range restriction in Nicotiana sylvestris maps to the replicase gene.

SUMMARY The experimental host range of Odontoglossum ringspot virus (ORSV), a member of the tobamoviruses, includes several species of Nicotiana, but not N. sylvestris. However, ORSV was able to replicate in protoplasts from N. sylvestris leaves. By using the green fluorescent protein (GFP) as a marker inserted into ORSV, it was found that a small number of single epidermal cells became infected in mechanically inoculated leaves, but the virus did not move cell to cell. The ORSV movement protein (MP) and coat protein (CP) were examined for their ability to effect movement by substitution into Tobacco mosaic virus (TMV) hybrids. Both proteins and the 3' non-translated region (NTR) of ORSV allowed movement of TMV hybrids in N. sylvestris. These results suggested that the inability of ORSV to move in N. sylvestris was due to the replicase gene or the 5'NTR. One possibility was that the replicase gene could indirectly affect movement by failing to produce subgenomic (sg) RNAs for expression of MP or CP, but this appeared not to be the case as ORSV replicated and produced MP and CP sgRNAs, both of which were translated in N. sylvestris protoplasts. Additionally, genomic RNA was encapsidated into virions in N. sylvestris protoplasts. Because the 5'NTR permitted efficient replication and production of replicase proteins, these findings suggest that the replicase of ORSV is responsible for the defect in cell-to-cell movement of ORSV in N. sylvestris.

The p23 protein of citrus tristeza virus controls asymmetrical RNA accumulation.

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb positive-stranded RNA genome that is organized into 12 open reading frames (ORFs) with the 10 3' genes expressed via a nested set of nine or ten 3'-coterminal subgenomic mRNAs (sgRNAs). Relatively large amounts of negative-stranded RNAs complementary to both genomic and sgRNAs accumulate in infected cells. As is characteristic of RNA viruses, wild-type CTV produced more positive than negative strands, with the plus-to-minus ratios of genomic and sgRNAs estimated at 10 to 20:1 and 40 to 50:1, respectively. However, a mutant with all of the 3' genes deleted replicated efficiently, but produced plus to minus strands at a markedly decreased ratio of 1 to 2:1. Deletion analysis of 3'-end genes revealed that the p23 ORF was involved in asymmetric RNA accumulation. A mutation which caused a frameshift after the fifth codon resulted in nearly symmetrical RNA accumulation, suggesting that the p23 protein, not a cis-acting element within the p23 ORF, controls asymmetric accumulation of CTV RNAs. Further in-frame deletion mutations in the p23 ORF suggested that amino acid residues 46 to 180, which contained RNA-binding and zinc finger domains, were indispensable for asymmetrical RNA accumulation, while the N-terminal 5 to 45 and C-terminal 181 to 209 amino acid residues were not absolutely required. Mutation of conserved cysteine residues to alanines in the zinc finger domain resulted in loss of activity of the p23 protein, suggesting involvement of the zinc finger in asymmetric RNA accumulation. The absence of p23 gene function was manifested by substantial increases in accumulation of negative-stranded RNAs and only modest decreases in positive-stranded RNAs. Moreover, the substantial decrease in the accumulation of negative-stranded coat protein (CP) sgRNA in the presence of the functional p23 gene resulted in a 12- to 15-fold increase in the expression of the CP gene. Apparently the excess negative-stranded sgRNA reduces the availability of the corresponding positive-stranded sgRNA as a messenger. Thus, the p23 protein controls asymmetric accumulation of CTV RNAs by downregulating negative-stranded RNA accumulation and indirectly increases expression of 3' genes.