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.     

Stabilization of HAC1 Influenza Vaccine by Spray Drying: Formulation Development and Process Scale-Up

Purpose

Stable vaccines with long shelf lives and reduced dependency on the cold chain are ideal for stockpiling and rapid deployment during public emergencies, including pandemics. Spray drying is a low-cost process that has potential to produce vaccines stable at a wide range of temperatures. Our aim was to develop a stable formulation of a recombinant H1N1 influenza hemagglutinin vaccine candidate and take it to pilot-scale spray-drying production.

Methods

Eight formulations containing different excipients were produced and assayed for antigen stability, powder characteristics, and immunogenicity after storage at a range of temperatures, resulting in the identification of four promising candidates. A pilot-scale spray-drying process was then developed for further testing of one formulation.

Results

The pilot-scale process was used to reproducibly manufacture three batches of the selected formulation with yields >90%. All batches had stable physical properties and in vitro potency for 6 months at temperatures from ?20°C to +50°C. Formulations stored for 3 months elicited immunogenic responses in mice equivalent to a frozen lot of bulk vaccine used as a stability control.

Conclusions

This study demonstrates the feasibility of stabilizing subunit vaccines using a spray-drying process and the suitability of the process for manufacturing a candidate product.

     

Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253-275) and nucleoprotein (N protein) (amino acids 404-418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.     

Expression of alfalfa mosaic virus coat protein in tobacco mosaic virus (TMV) deficient in the production of its native coat protein supports long-distance movement of a chimeric TMV

Alfalfa mosaic virus (AlMV) coat protein is involved in systemic infection of host plants, and a specific mutation in this gene prevents the virus from moving into the upper uninoculated leaves. The coat protein also is required for different viral functions during early and late infection. To study the role of the coat protein in long-distance movement of AlMV independent of other vital functions during virus infection, we cloned the gene encoding the coat protein of AlMV into a tobacco mosaic virus (TMV)-based vector Av. This vector is deficient in long-distance movement and is limited to locally inoculated leaves because of the lack of native TMV coat protein. Expression of AlMV coat protein, directed by the subgenomic promoter of TMV coat protein in Av, supported systemic infection with the chimeric virus in Nicotiana benthamiana, Nicotiana tabacum MD609, and Spinacia oleracea. The host range of TMV was extended to include spinach as a permissive host. Here we report the alteration of a host range by incorporating genetic determinants from another virus.     

High-affinity RNA-binding domains of alfalfa mosaic virus coat protein are not required for coat protein-mediated resistance.

A virus-based vector was used for the transient expression of the alfalfa mosaic virus coat protein (CP) gene in protoplasts and plants. The accumulation of wild-type CP conferred strong protection against subsequent alfalfa mosaic virus infection, enabling the efficacy of CP mutants to be determined without developing transgenic plants. Expression of the CP mRNA alone without CP accumulation conferred weaker protection against infection. The activity of the N-terminal mutant CPs in protection did not correlate with their activities in genome activation. The activity of a C-terminal mutant suggested that encapsidation did not have a role in protection. Our results indicate that interaction of the CP with alfalfa mosaic virus RNA is not important in protection, thereby leaving open the possibility that interactions with host factors lead to protection.     

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.     

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.     

Clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond

In the last few years, plants have become an increasingly attractive platform for recombinant protein production. This builds on two decades of research, starting with transgenic approaches to develop oral vaccines in which antigens or therapeutics can be delivered in processed plant biomass, and progressing to transient expression approaches whereby high yields of purified targets are administered parenterally. The advantages of plant-based expression systems include high scalability, low upstream costs, biocontainment, lack of human or animal pathogens, and ability to produce target proteins with desired structures and biological functions. Using transgenic and transient expression in whole plants or plant cell culture, a variety of recombinant subunit vaccine candidates, therapeutic proteins, including monoclonal antibodies, and dietary proteins have been produced. Some of these products have been tested in early phase clinical trials, and show safety and efficacy. Among those are mucosal vaccines for diarrheal diseases, hepatitis B and rabies; injectable vaccines for non-Hodgkin's lymphoma, H1N1 and H5N1 strains of influenza A virus, and Newcastle disease in poultry; and topical antibodies for the treatment of dental caries and HIV. As lead plant-based products have entered clinical trials, there has been increased emphasis on manufacturing under current Good Manufacturing Practice (cGMP) guidelines, and the preparation and presentation to the relevant government agencies of regulatory packages.     

N-terminal basic amino acids of alfalfa mosaic virus coat protein involved in the initiation of infection

Alfalfa mosaic virus coat protein or its messenger RNA is required in the inoculum for virus infection. The N-terminus of the coat protein is required for activity; thus, changes were made in the amino acid sequence of this region. Six coat protein mutants were tested for activity in virus infection assays in protoplasts. A coat protein mutant in which N-terminal residues 3-19 were absent was inactive; whereas, a mutant in which residues 3-11 were absent (CP deltaN9) still had 73% of wild-type activity. Substitution of alanine for the basic residues at positions 14, 17, and 18 in full-length coat protein and in CP deltaN9 resulted in mutant proteins that were inactive in infection. Thus, one, two, or three of these basic residues in CP are required for activity.     

The green revolution: plants as heterologous expression vectors

Agrobacterium tumefaciens mediated gene transfer into the plant genome laid the groundwork for new procedures aimed at crop improvement, including resistance to pathogens, increased product yield, modified oil content, and resistance to environmental stress conditions. New developments in molecular plant virology have led to the generation of plant-based systems for transient expression of foreign sequences using plant virus vectors. In the last decade both transgenic plants and plant virus vectors have been used increasingly to produce a wide range of biomedical reagents, including vaccine antigens, in a safe and economically feasible manner. These new plant-based technologies have enormous potential for a variety of applications, including the oral delivery of vaccine antigens.