Stephen J. Streatfield

Virus-like particles as a highly efficient vaccine platform: Diversity of targets and production systems and advances in clinical development

Abstract Virus-like particles (VLPs) are a class of subunit vaccines that differentiate themselves from soluble recombinant antigens by stronger protective immunogenicity associated with the VLP structure. Like parental viruses, VLPs can be either non-enveloped or enveloped, and they can form following expression of one or several viral structural proteins in a recombinant heterologous system. Depending on the complexity of the VLP, it can be produced in either a prokaryotic or eukaryotic expression system using target-encoding recombinant vectors, or in some cases can be assembled in cell-free conditions. To date, a wide variety of VLP-based candidate vaccines targeting various viral, bacterial, parasitic and fungal pathogens, as well as non-infectious diseases, have been produced in different expression systems. Some VLPs have entered clinical development and a few have been licensed and commercialized. This article reviews VLP-based vaccines produced in different systems, their immunogenicity in animal models and their status in clinical development.

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.

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.

Corn as a production system for human and animal vaccines

The synthesis of selected antigens in plants and their oral delivery has great potential for reducing the costs of vaccine production and administration. The application of this technology requires antigen concentrations in final plant material to be uniform to ensure consistent dosing. In addition, antigen levels should be such as to allow the volume of each dose, containing a set amount of antigen, to be practical for oral delivery. Here, we demonstrate that the Lt-B protein of enterotoxigenic E. coli is evenly distributed in defatted corn germ prepared from transgenic grain. Furthermore, the choice of sub-cellular location for Lt-B affects accumulation of the protein in excess of four orders of magnitude.

A corn-based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine.

Abstract Recombinant plant expression systems offer a means to produce large quantities of selected antigens for subunit vaccines. Cereals are particularly well-suited expression vehicles since the expressed proteins can be stored at relatively high concentrations for extended periods of time without degradation and dry seed can be formulated into oral vaccines suitable for commercial applications. A subunit vaccine candidate directed against porcine transmissible gastroenteritis virus and expressed in corn seed has been developed for oral delivery to swine. Here, we show that this vaccine, when administered to previously sensitized gilts, can boost neutralizing antibody levels in the animals’ serum, colostrum and milk. Thus, this vaccine candidate is effective at boosting lactogenic immunity and is appropriate to pursue through large-scale field trials preceding commercialization.

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.

A Plant-Produced Pfs25 VLP Malaria Vaccine Candidate Induces Persistent Transmission Blocking Antibodies against Plasmodium falciparum in Immunized Mice

Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based ‘launch’ vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20–30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the ‘launch’ vector technology for the production of VLP-based recombinant vaccines against infectious diseases.

Plant production systems for vaccines.

Plants offer an attractive alternative for the production and delivery of subunit vaccines. Various antigens have been expressed at sufficiently high levels in plants to render vaccine development practical. An increasing body of evidence demonstrates that these plant-produced antigens can induce immunogenic responses and confer protection when delivered orally. Plant-based vaccines are relatively inexpensive to produce and production can be rapidly scaled up. There is also the potential for oral delivery of these vaccines, which can dramatically reduce distribution and delivery costs. Here we describe the technology to develop plant-based vaccines, review their advantages and discuss potential roadblocks and concerns over their commercialization. We also speculate on likely future developments with these vaccines and on their potential impact in the realms of human and animal health.

A novel plant-produced Pfs25 fusion subunit vaccine induces long-lasting transmission blocking antibody responses.

Malaria transmission blocking vaccines (TBV) directed against proteins expressed on sexual stages of Plasmodium falciparum in the mosquito midgut are considered an effective means to reduce malaria transmission. Antibodies induced by TBV block sporogonic development in the mosquito, and thus transmission to the next human host. The Pfs25 protein, expressed on the surface of gametes, zygotes and ookinetes, is one of the primary targets for TBV development. Using a plant virus-based transient expression system, we have successfully produced Pfs25 fused to a modified lichenase (LicKM) carrier in Nicotiana benthamiana, purified and characterized the protein (Pfs25-FhCMB), and evaluated this vaccine candidate in animal models for the induction of transmission blocking antibodies. Soluble Pfs25-FhCMB was expressed in plants at a high level, and induced transmission blocking antibodies that persisted for up to 6 months post immunization in mice and rabbits. These data demonstrate the potential of the new malaria vaccine candidate and also support feasibility of expressing Plasmodium antigens in a plant-based system.

Analysis of the maize polyubiquitin-1 promoter heat shock elements and generation of promoter variants with modified expression characteristics.

The maize polyubiquitin-1(Ubi-1) promoter is one of a few select promoters used to express foreign genes in monocots, such that recombinant proteins can be produced at commercially viable levels. Modifying the activity, specificity and responsiveness of such promoters provides a means to achieve desired levels and patterns of expression of genes encoding target products. Ubi-1 is constitutively expressed but is further induced by heat shock. The promoter contains two overlapping sequences with similarity to defined heat shock elements and we show that these sequences are also present upstream of the Ubi-1 homologue isolated from teosinte. Both the maize and teosinte promoters can mediate a heat shock response in transgenic maize. We have dissected the overlapping maize Ubi-1 promoter heat shock elements and demonstrate that the 3′ element is required to mediate a heat shock response. The Ubi-1 promoter is particularly active in tissues consisting of rapidly dividing cells, and within the seed it is strongly biased towards driving expression in the embryo. However, replacement of the heat shock elements with a trimer of a basic domain/leucine zipper factor binding site of a pea lectin promoter shifts the balance in seed expression towards the endosperm. The Ubi-1 variants described here differ in their overall activity in the seed, but they all show potential for driving high levels of heterologous gene expression in maize.