Recombinant subunit vaccines are an efficient strategy to meet the demands of a possible influenza pandemic, because of rapid and scalable production. However, vaccines made from recombinant hemagglutinin (HA) subunit protein are often of low potency, requiring high dose or boosting to generate a sustained immune response. We have improved the immunogenicity of a plant-made HA vaccine by chemical conjugation to the surface of the Tobacco mosaic virus (TMV) which is non infectious in mammals. We have previously shown that TMV is taken up by mammalian dendritic cells and is a highly effective antigen carrier. In this work, we tested several TMV-HA conjugation chemistries, and compared immunogenicity in mice as measured by anti-HA IgG titers and hemagglutination inhibition (HAI). Importantly, pre-existing immunity to TMV did not reduce initial or boosted titers. Further optimization included dosing with and without alum or oil-in water adjuvants. Surprisingly, we were able to stimulate potent immunogenicity and HAI titers with a single 15µg dose of HA as a TMV conjugate. We then evaluated the efficacy of the TMV-HA vaccine in a lethal virus challenge in mice. Our results show that a single dose of the TMV-HA conjugate vaccine is sufficient to generate 50% survival, or 100% survival with adjuvant, compared with 10% survival after vaccination with a commercially available H1N1 vaccine. TMV-HA is an effective dose-sparing influenza vaccine, using a single-step process to rapidly generate large quantities of highly effective flu vaccine from an otherwise low potency HA subunit protein.

Tobacco Mosaic virus (TMV) coat protein is well known for its ability to self-assemble into supramolecular nanoparticles, either as protein discs or as rods originating from the ~300 bp genomic RNA origin-of-assembly (OA). We have utilized TMV self-assembly characteristics to create a novel Flock House virus (FHV) RNA nanoparticle. FHV encodes a viral polymerase supporting autonomous replication of the FHV genome, which makes it an attractive candidate for viral transgene expression studies and targeted RNA delivery into host cells. However, FHV viral genome size is strictly limited by native FHV capsid. To determine if this packaging restriction could be eliminated, FHV was adapted to express enhanced green fluorescent protein (GFP), to allow for monitoring of functional FHV RNA activity. Then TMV OA was introduced in six 3' insertion sites, with only site one supporting functional FHV GFP expression. To create nanoparticles, FHV GFP-OA modified genomic RNA was mixed in vitro with TMV coat protein and monitored for encapsidation by agarose electrophoresis and electron microscopy. The production of TMV-like rod shaped nanoparticles indicated that modified FHV RNA can be encapsidated by purified TMV coat protein by self-assembly. This is the first demonstration of replication-independent packaging of the FHV genome by protein self-assembly.

Everyone appreciates the irony of using tobacco plants to cure cancer. (1) Recently featured in a populist Wall Street Journal article, (2) the use of plants to produce medicinal products was presented as novel, even though we are decades into development of numerous products for specific medical applications (reviewed extensively in (3, 4)). Though the tobacco plant and its relatives offer a qualified set of advantages for producing complex biologicals, and in many cases overcome problems that plague traditional expression systems, FDA licensed products derived from bioengineered plants have yet to appear in the marketplace. Despite a difficult beginning, recent advances in plant biotechnology have been as cutting edge as those in the fields of molecular biology and chemical engineering, which now position the field for a new level of commercial relevance. The basis for this review is a description of the first FDA qualified parenterally administered vaccine clinical trial using a plant derived product. We have confirmed in this trial that plant proteins can be qualified to the same level as biologicals from other sources, and are safe when given as injected vaccines. Most importantly though, immune responses to plant proteins were seen in 66% of cancer patients, and these responses were to the desired antigenic determinants, not to xenogenic plant antigens. Problems and solutions that arose during the development of a safe and effective human vaccine are discussed.

Tularemia is a fatal human disease caused by Francisella tularensis, a Gram-negative encapsulated coccobacillus bacterium. Due to its low infectious dose, ease of aerosolized transmission, and lethal effects, the CDC lists F. tularensis as a Category A pathogen, the highest level for a potential biothreat agent. Previous vaccine studies have been conducted with live attenuated, inactivated, and subunit vaccines, which have achieved partial or full protection from F. tularensis live vaccine strain (LVS) challenge, but no vaccine has been approved for human use. We demonstrate the improved efficacy of a multi-antigen subunit vaccine by using Tobacco Mosaic virus (TMV) as an antigen carrier for the F. tularensis SchuS4 proteins DnaK, OmpA, SucB and Tul4 (DOST). The magnitude and quality of immune responses were compared after mice were immunized by subcutaneous or intranasal routes of administration with a TMV-DOST mixture, with or without four different adjuvants. Immune responses varied in magnitude and isotype profile, by antigen, by route of administration, and by protection in an F. tularensis LVS challenge model of disease. Interestingly, our analysis demonstrates an overwhelming IgG2 response to SucB after intranasal dosing, as well as a robust cellular response, which may account for the improved two-dose survival imparted by the tetravalent vaccine, compared to a previous study that tested efficacy of TMV-DOT. Our study provides evidence that potent humoral, cellular and mucosal immunity can be achieved by optimal antigen combination, delivery, adjuvant and appropriate route of administration, to improve vaccine potency and provide protection from pathogen challenge.