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Second generation anthrax vaccines focus on the use of recombinant protective antigen (rPA) to elicit a strong, toxin neutralizing antibody responses in immunized subjects. The main difference between the rPA vaccines compared to the current licensed vaccine, anthrax vaccine absorbed (AVA), is the rPA vaccines are highly purified preparations of only rPA. These second generation rPA vaccines strive to elicit strong immune responses with substantially fewer doses than AVA while provoking less side effects. Many of the rPA candidates have shown to be effective in pre-clinical studies, but most of the second generation molecules have stability issues which reduce their efficacy over time. These stability issues are evident even under refrigerated conditions and thus emphasis has been directed to stabilizing the rPA molecule and determining an optimized final formulation. Stabilization of vaccines for long-term storage is a major challenge in the product development life cycle. The effort required to identify suitable formulations can be slow and expensive. The ideal storage for stockpiled vaccines would allow the candidate to withstand years of storage at ambient temperatures. The Fraunhofer Center for Molecular Biotechnology is developing a plant-produced rPA vaccine candidate that shows instability when stored under refrigerated conditions in a solution, as is typical for rPA vaccines. Increased stability of our plant-produced rPA vaccine candidate was achieved in a spray dried powder formulation that could eliminate the need for conventional cold chain allowing greater confidence to stockpile vaccine for civilian and military biodefense., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance.

MAIN CONCLUSION : A RhoA-derived peptide fused to carrier molecules from plants showed enhanced biological activity of in vitro assays against respiratory syncytial virus compared to the RhoA peptide alone or the synthetic RhoA peptide. A RhoA-derived peptide has been reported for over a decade as a potential inhibitor of respiratory syncytial virus (RSV) infection both in vitro and in vivo and is anticipated to be a promising alternative to monoclonal antibody-based therapy against RSV infection. However, there are several challenges to furthering development of this antiviral peptide, including improvement in the peptide’s bioavailability, development of an efficient delivery system and identification of a cost-effective production platform. In this study, we have engineered a RhoA peptide as a genetic fusion to two carrier molecules, either lichenase (LicKM) or the coat protein (CP) of Alfalfa mosaic virus. These constructs were introduced into Nicotiana benthamiana plants using a tobacco mosaic virus-based expression vector and targets purified. The results demonstrated that the RhoA peptide fusion proteins were efficiently expressed in N. benthamiana plants, and that two of the resulting fusion proteins, RhoA-LicKM and RhoA2-FL-d25CP, inhibited RSV growth in vitro by 50 and 80 %, respectively. These data indicate the feasibility of transient expression of this biologically active antiviral RhoA peptide in plants and the advantage of using a carrier molecule to enhance target expression and efficacy.

Despite decades of effort battling against malaria, the disease is still a major cause of morbidity and mortality. Transmission-blocking vaccines (TBVs) that target sexual stage parasite development could be an integral part of measures for malaria elimination. In the 1950s, Huff et al. first demonstrated the induction of transmission-blocking immunity in chickens by repeated immunizations with Plasmodium gallinaceum-infected red blood cells. Since then, significant progress has been made in identification of parasite antigens responsible for transmission-blocking activity. Recombinant technologies accelerated evaluation of these antigens as vaccine candidates, and it is possible to induce effective transmission-blocking immunity in humans both by natural infection and now by immunization with recombinant vaccines. This chapter reviews the efforts to produce TBVs, summarizes the current status and advances and discusses the remaining challenges and approaches., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Asymmetrical flow field-flow fractionation (afFFF) was used to investigate the properties of a plant-produced anthrax toxin protective antigen, pp-PA83. The afFFF fractogram consisted of two main peaks with molar masses similar to the molecular mass of pp-PA83 monomer. afFFF carrier solutions strongly influenced the ratio and the intensity of the two main peaks. These differences indicate that conformation changes in the pp-PA83 molecule occurred during the afFFF analysis. Similar fractograms were obtained for different pp-PA83 formulations when the afFFF carrier solution and the protein formulation were the same (or very similar). The data show that in specific cases, afFFF could be used to study protein conformation and document the importance of studying the influence of the carrier solution on afFFF., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell.

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.

Annually influenza virus infections are responsible for hospitalization and mortality, especially in high risk groups. Constant antigenic changes in seasonal influenza viruses resulted from antigenic shifts and antigenic drifts, enable emerging of novel virus subtypes that may reduce current vaccine efficacy and impose the continuous revision of vaccine component. Currently available vaccines are usually limited by their production processes in terms of rapid adaptation to new circulating subtypes in high quantities meeting the global demand. Thus, new approaches to rapidly manufacture high yields of influenza vaccines are required. New technologies to reach maximal protection with minimal vaccine doses also need to be developed. In this study, we evaluated the systemic and local immunogenicity of a new double-adjuvanted influenza vaccine administered at the site of infection, the respiratory tract. This vaccine combines a plant-produced H1N1 influenza hemagglutinin antigen (HAC1), a silica nanoparticle-based (SiO₂) drug delivery system and the mucosal adjuvant candidate bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Mice were vaccinated by intratracheal route with HAC1/SiO₂ or HAC1/c-di-GMP (single-adjuvanted vaccine) or HAC1/SiO₂/c-di-GMP (double-adjuvanted vaccine) and evaluated for target-specific immune responses, such as hemagglutination inhibition and hemagglutinin-specific IgG titers, as well as local antibody (IgG and IgA) titers in the bronchoalveolar lavage (BAL). Furthermore, the HAC1-specific T-cell re-stimulation potential was assessed using precision-cut lung slices (PCLS) of vaccinated mice. The double-adjuvanted vaccine induced high systemic antibody responses comparable to the systemic vaccination control. In addition, it induced local IgG and IgA responses in the BAL. Furthermore, HAC1 induced a local T-cell response demonstrated by elevated IL-2 and IFN-γ levels in PCLS of c-di-GMP-vaccinated mice upon re-stimulation. Overall, the present study showed the potential of the double-adjuvanted vaccine to induce systemic humoral immune responses in intratracheally vaccinated mice. Furthermore, it induced a strong mucosal immune response, with evidence of antigen-primed T-cells in the lung., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Novel influenza viruses continue to pose a potential pandemic threat worldwide. In recent years, plants have been used to produce recombinant proteins, including subunit vaccines. A subunit influenza vaccine, HAC1, based on recombinant hemagglutinin from the 2009 pandemic A/California/04/2009 (H1N1) strain of influenza virus, has been manufactured using a plant virus-based transient expression technology in Nicotiana benthamiana plants and demonstrated to be immunogenic and safe in pre-clinical studies (Shoji et al., 2011)., Methods: A first-in-human, Phase 1, single-center, randomized, placebo-controlled, single-blind, dose escalation study was conducted to investigate safety, reactogenicity and immunogenicity of an HAC1 formulation at three escalating dose levels (15 μg, 45 μg and 90 μg) with and without Alhydrogel(®), in healthy adults 18-50 years of age (inclusive). Eighty participants were randomized into six study vaccine groups, a saline placebo group and an approved monovalent H1N1 vaccine group. Recipients received two doses of vaccine or placebo (except for the monovalent H1N1 vaccine cohort, which received a single dose of vaccine, later followed by a dose of placebo)., Results: The experimental vaccine was safe and well tolerated, and comparable to placebo and the approved monovalent H1N1 vaccine. Pain and tenderness at the injection site were the only local solicited reactions reported following vaccinations. Nearly all adverse events were mild to moderate in severity. The HAC1 vaccine was also immunogenic, with the highest seroconversion rates, based on serum hemagglutination-inhibition and virus microneutralization antibody titers, in the 90 μg non-adjuvanted HAC1 vaccine group after the second vaccine dose (78% and 100%, respectively)., Conclusions: This is the first study demonstrating the safety and immunogenicity of a plant-produced subunit H1N1 influenza vaccine in healthy adults. The results support further clinical investigation of the HAC1 vaccine as well as demonstrate the feasibility of the plant-based technology for vaccine antigen production., (Copyright © 2014. Published by Elsevier Ltd.)

The increased worldwide awareness of seasonal and pandemic influenza, including pandemic H1N1 virus, has stimulated interest in the development of economic platforms for rapid, large-scale production of safe and effective subunit vaccines. In recent years, plants have demonstrated their utility as such a platform and have been used to produce vaccine antigens against various infectious diseases. Previously, we have produced in our transient plant expression system a recombinant monomeric hemagglutinin (HA) protein (HAC1) derived from A/California/04/09 (H1N1) strain of influenza virus and demonstrated its immunogenicity and safety in animal models and human volunteers. In the current study, to mimic the authentic HA structure presented on the virus surface and to improve stability and immunogenicity of the HA antigen, we generated trimeric HA by introducing a trimerization motif from a heterologous protein into the HA sequence. Here, we describe the engineering, production in Nicotiana benthamiana plants, and characterization of the highly purified recombinant trimeric HA protein (tHA-BC) from A/California/04/09 (H1N1) strain of influenza virus. The results demonstrate the induction of serum hemagglutination inhibition antibodies by tHA-BC and its protective efficacy in mice against a lethal viral challenge. In addition, the immunogenic and protective doses of tHA-BC were much lower compared with monomeric HAC1. Further investigation into the optimum vaccine dose and/or regimen as well as the stability of trimerized HA is necessary to determine whether trimeric HA is a more potent vaccine antigen than monomeric HA.

The potential use of Bacillus anthracis as a bioterrorism weapon threatens the security of populations globally, requiring the immediate availability of safe, efficient and easily delivered anthrax vaccine for mass vaccination. Extensive research efforts have been directed toward the development of recombinant subunit vaccines based on protective antigen (PA), the principal virulence factor of B. anthracis. Among the emerging technologies for the production of these vaccine antigens is our launch vector-based plant transient expression system. Using this system, we have successfully engineered, expressed, purified and characterized full-length PA (pp-PA83) in Nicotiana benthamiana plants using agroinfiltration. This plant-produced antigen elicited high toxin neutralizing antibody titers in mice and rabbits after two vaccine administrations with Alhydrogel. In addition, immunization with this vaccine candidate protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. The vaccine effects were dose-dependent and required the presence of Alhydrogel adjuvant. In addition, the vaccine antigen formulated with Alhydrogel was stable and retained immunogenicity after two-week storage at 4°C, the conditions intended for clinical use. These results support the testing of this vaccine candidate in human volunteers and the utility of our plant expression system for the production of a recombinant anthrax vaccine.

Prevention of infectious diseases by vaccination is often limited because of the lack of safe, effective, and accessible vaccines. Traditional vaccines are expensive and require special conditions for storage, distribution, and administration. Plants have potential for large-scale production of a variety of inexpensive and highly effective recombinant proteins for biomedical and pharmaceutical applications, including subunit vaccines. There are several approaches for the production of vaccine antigens in plants, including transient expression systems based on Agrobacterium delivery of binary vectors or plant viral vectors, stable transgenic plants, and plant cell or tissue cultures. Axenic plant cultures maintained under defined physical and chemical conditions appear to be an attractive production platform when target proteins need to be synthesized in a fully controlled environment. Hairy root cultures meet the criteria for such a system. Hairy root cultures, generated from edible plants and producing target antigens, provide a potential approach for the development of vaccines for oral delivery. With this approach, there are no protein extraction and purification costs and the active biomolecule is protected by the plant cell wall during passage through the upper gastrointestinal tract. This allows for gradual release of antigen at mucosal surfaces in the gut. Lyophilized hairy root cultures expressing vaccine antigens can be stored at ambient temperature for extended periods of time, which should facilitate storage and distribution, ultimately allowing for large populations to be vaccinated.

Rapid production of influenza vaccine antigen is an important challenge when a new pandemic occurs. Production of recombinant antigens in plants is a quick, cost effective and up scalable new strategy for influenza vaccine production. In this study, we have characterized a recombinant influenza haemagglutinin antigen (HAC1) that was derived from the 2009 pandemic H1N1 (pdmH1N1) virus and expressed in tobacco plants. Volunteers vaccinated with the 2009 pdmH1N1 oil-in-water adjuvanted vaccine provided serum and lymphocyte samples that were used to study the immunogenic properties of the HAC1 antigen in vitro. By 7 d post vaccination, the vaccine fulfilled the licensing criteria for antibody responses to the HA detected by haemagglutination inhibition and single radial hemolysis. By ELISA and ELISPOT analysis we showed that HAC1 was recognized by specific serum antibodies and antibody secreting cells, respectively. We conducted a kinetic analysis and found a peak of serum HAC1 specific antibody response between day 14 and 21 post vaccination by ELISA. We also detected elevated production of IL-2 and IFNγ and low frequencies of CD4(+) T cells producing single or multiple Th1 cytokines after stimulating PBMCs (peripheral blood mononuclear cells) with the HAC1 antigen in vitro. This indicates that the antigen can interact with T cells, although confirming an effective adjuvant would be required to improve the T-cell stimulation of plant based vaccines. We conclude that the tobacco derived recombinant HAC1 antigen is a promising vaccine candidate recognized by both B- and T cells.

Background: Influenza virus is a globally important respiratory pathogen that causes a high degree of annual morbidity and mortality. Significant antigenic drift results in emergence of new, potentially pandemic, virus variants. The best prophylactic option for controlling emerging virus strains is to manufacture and administer pandemic vaccines in sufficient quantities and to do so in a timely manner without impacting the regular seasonal influenza vaccine capacity. Current, egg-based, influenza vaccine production is well established and provides an effective product, but has limited capacity and speed., Objectives: To satisfy the additional global demand for emerging influenza vaccines, high-performance cost-effective technologies need to be developed. Plants have a potential as an economic and efficient large-scale production platform for vaccine antigens., Methods: In this study, a plant virus-based transient expression system was used to produce hemagglutinin (HA) proteins from the three vaccine strains used during the 2008-2009 influenza season, A/Brisbane/59/07 (H1N1), A/Brisbane/10/07 (H3N2), and B/Florida/4/06, as well as from the recently emerged novel H1N1 influenza A virus, A/California/04/09., Results: The recombinant plant-based HA proteins were engineered and produced in Nicotiana benthamiana plants within 2 months of obtaining the genetic sequences specific to each virus strain. These antigens expressed at the rate of 400-1300 mg/kg of fresh leaf tissue, with >70% solubility. Immunization of mice with these HA antigens induced serum anti-HA IgG and hemagglutination inhibition antibody responses at the levels considered protective against these virus infections., Conclusions: These results demonstrate the feasibility of our transient plant expression system for the rapid production of influenza vaccine antigens., (© 2011 Blackwell Publishing Ltd.)

Influenza is a prevalent, highly contagious and sometimes fatal respiratory disease. Vaccination provides an effective approach to control the disease, but because of frequent changes in the structure of the major surface proteins, there is great need for a technology that permits rapid preparation of new forms of the vaccine each year in sufficient quantities. Recently, using a safe, simple, time- and cost-effective plant viral vector-based transient expression system, the hemagglutinin antigen of H1N1 influenza A strain (HAC1), an H1N1 influenza vaccine candidate, has been produced in Nicotiana benthamiana plants. As a step toward the generation of a commercially viable subunit influenza vaccine, we developed HAC1 formulations in the presence and absence of an aluminum salt adjuvant (Alhydrogel(®)), analyzed their properties, and assessed immunogenicity in an animal model. Biophysical properties of HAC1 were evaluated using several spectroscopic and light scattering techniques as a function of pH and temperature combined with data analysis using an empirical phase diagram approach. Excipients that were potent stabilizers of the recombinant protein were identified using intrinsic fluorescence spectroscopy. The adsorptive capacity and thermal stability of the protein on the surface of Alhydrogel(®) were then examined in the presence and absence of selected stabilizers using UV absorbance after centrifugation and intrinsic fluorescence spectroscopy, respectively. Immunogenicity studies conducted in mice demonstrated that the highest level of serum immune responses (hemagglutination-inhibiting antibody titers), with a 100% seropositive rates, were induced by HAC1 in the presence of Alhydrogel(®), and this response was elicited regardless of the solution conditions of the formulation.

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.

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.

H5N1 avian influenza continues to be a potential pandemic threat. Several vaccine candidates based on potentially pandemic influenza strains and antiviral drugs have been tested in preclinical and clinical studies. The data obtained so far have shown some promise, but have also revealed some shortcomings with both of these approaches. We have identified and characterized an H5N1 neuraminidasespecific monoclonal antibody which specifically inhibits N1 neuraminidase activity of highly pathogenic avian influenza (HPAI) strains from clades 1 and 2. We have also shown the protective efficacy of this antibody in animal challenge models using homologous virus. Specific and effective inhibition of N1 NA could make this mAb a useful therapeutic tool in the treatment of human infection, in particular with oseltamivirand zanamivir-resistant strains of HPAI.

Malaria is a serious and sometimes fatal mosquito-borne disease caused by a protozoan parasite. Each year, it is estimated that over one million people are killed by malaria, yet the disease is preventable and treatable. Developing vaccines against the parasite is a critical component in the fight against malaria and these vaccines can target different stages of the pathogen's life cycle. We are targeting sexual stage proteins of P. falciparum which are found on the surface of the parasite reproductive cells present in the mosquito gut. Antibodies against these proteins block the progression of the parasite's life cycle in the mosquito, and thus block transmission to the next human host. Transmission blocking vaccines are essential to the malaria eradication program to ease the disease burden at the population level. We have successfully produced multiple versions of the Pfs25 antigen in a plant virus-based transient expression system and have evaluated these vaccine candidates in an animal model. The targets are expressed in plants at a high level, are soluble and most importantly, generate strong transmission blocking activity as determined by a standard membrane feeding assay. These data demonstrate the feasibility of expressing Plasmodium antigens in a plant-based system for the economic production of a transmission blocking vaccine against malaria.

We have produced the recombinant intracellular domain of human IA-2 (IA-2ic), a diabetes-associated autoantigen, in plants. This was achieved by transient expression using agroinfiltration of Nicotiana benthamiana plants. The resulting plant-derived IA-2ic had the expected size, reacted with polyclonal and monoclonal antibodies specific to human IA-2ic and competitively inhibited radiolabeled IA-2ic in an immunoprecipitation assay. The expression level of recombinant IA-2ic was estimated to be 0.5% of the total soluble protein (TSP). Transient expression in plants has the potential to produce a large amount of human IA-2ic protein at low cost in a short period of time.

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.

We have developed a plant virus-mediated transgene activation (VMTA) system that utilizes a viral expression vector to present the inducer. The concept was tested using two well characterized components: (i) an artificial promoter based on the yeast GAL4 upstream activating sequence and the minimal TATA element of Cauliflower Mosaic Virus 35S RNA promoter, and (ii) a transcriptional activator (TA) consisting of a fusion between the GAL4 DNA binding domain and the Herpes simplex virus VP16 activation domain. The TA was expressed under the control of the subgenomic promoter of a Tobacco Mosaic Virus-based expression vector. The VMTA system was functional in transient Agroinfiltration assays with the reporter gene beta-glucuronidase, the intracellular domain of the diabetes associated autoimmune antigen, IA-2ic, and with the anti-tetanus antibody 9F12. Transgenic lines harboring the reporter gene were also examined. The VMTA system displayed tight transcriptional control in both transient assays and in transgenic Nicotiana benthamiana plants carrying the TA-inducible reporter.