Transgenic tomato fruit expressing plant-optimized respiratory syncytial virus f antigen

Abstract

The present disclosure relates to transgenic tomato plants, which have been transformed to produce a fragment of respiratory syncytial virus (RSV) F protein, and the plant-optimized plasmids that produce such transgenic tomato plants. In some aspects, the transgenic tomato plant expresses the ectodomain of RSV F protein. In certain embodiments, the fragment of RSV F protein comprises the ectodomain of RSV F protein, and the p27 region of the RSV F protein is replaced with a sequence-specific linker region comprising the amino acid sequence of GSGSGRSLG. Also described are compositions comprising an extraction from a fruit of the transgenic tomato plant, wherein the extraction from the fruit of the transgenic tomato plant comprises the fragment of RSV F protein.

Description

TRANSGENIC TOMATO FRUIT EXPRESSING PLANT-OPTIMIZEDRESPIRATORY SYNCYTIAL VIRUS F ANTIGENRELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. provisional patent application 63 / 674,218, filed July 22, 2024, titled “TRANSGENIC TOMATO FRUIT VACCINE COMPOSITIONS FOR MUNCOSAL DELIVERY” the entirety of the disclosure of which is hereby incorporated by this reference.SEQUENCE LISTING

[0002] In accordance with 37 C.F.R. § 1.831, the present specification makes reference to a Sequence Listing submitted electronically in the form of an XML file (entitled “149WO- PCT.xml”, created on July 22, 2025, 55,701 bytes in size). The entire contents of the Sequence Listing are herein incorporated by reference in their entirety, with the intention that, upon publication (including issuance), this incorporated Sequence Listing will be inserted in the published document immediately before the claims.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under 2026281 awarded by the National Science Foundation. The government has certain rights in the invention.TECHNICAL FIELD

[0004] The present disclosure relates to transgenic tomato fruit vaccine compositions against respiratory syncytial virus.BACKGROUND

[0005] Respiratory syncytial virus (RSV) is an enveloped RNA virus of the genus Pneumovirus. RSV has two main antigenic glycoproteins, F protein and G protein. Additional structural proteins include a major nucleocapsid protein (N), a nucleocapsid phosphoprotein (P), a large nucleocapsid protein (L), an envelope matrix protein (M), and a glycoprotein (22,000 Daltons). A virally encoded protein of about 9,500 Daltons and other small proteinsare also known to be in infected cells. Infections with RSV affect all segments of the respiratory tract and are usually associated with fever, cough, runny nose, and fatigue.

[0006] RSV is one of the most important pathogens of infancy and early childhood. Surveys of children hospitalized with RSV show mortality rates of 0.1-2.5%. The annual infant hospitalization cost in the U.S. related to RSV infection is around $2 billion. In recent years, increased incidences of RSV infections have been observed in the elderly, particularly, those living in nursing homes and other group settings. Up to two-thirds of the infected elderly develop pneumonia. According to the U.S. Centers for Disease Control and Prevention, each year in the U.S., RSV leads to approximately 60,000-160,000 hospitalizations and 6,000- 10,000 deaths among adults 65 years of age and older.

[0007] Re-infection with RSV is also common because natural infection confers only temporary protection against the disease. The immunity elicited by primary infection declines rapidly and cannot prevent reinfection although the severity of the disease may decrease. This is also a major difficulty in developing an effective RSV vaccine.

[0008] Another obstacle in the development of an RSV vaccine candidates is eliciting the incorrect immune response. One of the earlier RSV vaccine candidates exacerbated RSV disease and was withdrawn from use. Several live attenuated forms of RSV have been proposed for treatment of RSV infection, but there are limitations imposed by such approaches. For example, live infection does not provide full immunity and single point mutations may revert to wild type with undesirable consequences. Accordingly, a need exists for a better way to treat RSV.SUMMARY OF THE DISCLOSURE

[0009] A transgenic tomato plant transformed to express at least a fragment of Respiratory Syncytial Virus (RSV) F protein is described. In some aspects, the fragment of RSV F protein is the ectodomain of RSV F protein. In some embodiments, the ectodomain of RSV F protein lacks the p27 region. For example, the fragment of RSV F protein comprises the ectodomain of RSV F protein, and the p27 region of the RSV F protein is replaced with a sequence-specific linker region comprising the amino acid sequence of GSGSGRSLG. In particular embodiments, the transgenic tomato plant is a tomato plant transformed with pKEAM- BAFd27. In certain embodiments, the ectodomain of RSV F protein is expressed as a fusion protein with the transmembrane and cytosolic domains of human immunodeficiency virus(HIV) gp41. In particular embodiments, the transgenic tomato plant is a tomato plant transformed with pKEAM-BAFd27-gp41.

[0010] Accordingly, the plant expression vector for producing transgenic tomato plants that express RSV F protein and / or express a fragment of RSV F protein are described. For the vector to express RSV F protein in a transgenic tomato plant, the plant expression vector comprises a first nucleic acid sequence encoding barley alpha-amylase signal peptide and a second nucleic acid sequence encoding a modified RSV F protein lacking RSV F protein native signal peptide region. The modified RSV F protein encoded in the second nucleic acid sequence further comprises a p27 region replaced an amino acid sequence of GSGSGRSLG. For example, the plant expression vector is pKEAM-BAFd27, with a nucleic acid sequence set forth in SEQ ID NO. 1. In some embodiments, the vector to express a fragment of RSV F protein is pKEAM- BAFd27-gp41, with a nucleic acid sequence set forth in SEQ ID NO. 2.

[0011] Also described is a composition comprising an extraction from a fruit of the transgenic tomato plant described herein. The extraction from the fruit of the transgenic tomato plant comprises the RSV F protein or a fragment thereof. In some aspects, the fruit extraction comprises the RSV F protein in a trimeric state. In some embodiments, the composition comprises a pharmaceutically acceptable carrier or excipient. In some aspects, the extraction from the fruit of the transgenic tomato plant is a puree of the fruit of the transgenic tomato plant. In some embodiments, the extraction from the fruit of the transgenic tomato plant is a freeze-dried puree of the fruit of the transgenic tomato plant.

[0012] In some embodiments, the composition comprises a first isolate from the fruit of the transgenic tomato plant comprising a fragment of RSV F protein; and a second isolate from the fruit of the transgenic tomato plant comprising tomatine. In certain embodiments, the vaccine composition further comprises a source of tomatine. The source of tomatine may be from a non-fruit part of the transgenic tomato plant or non-transgenic tomato plant or from a tomato fruit of a non-transgenic plant. The non-fruit part of the transgenic tomato plant may be, for example, a leaf.

[0013] In other embodiments, the composition comprises a first isolate from the fruit of the transgenic tomato plant comprising a fragment of RSV F protein; and a second isolate from the non-fruit part of the transgenic tomato plant comprising tomatine.

[0014] In some aspects, the composition comprises at least 100 pg of fragment of RSV F protein. For example, the composition comprises at least 120 pg of fragment of RSV F protein, at least 150 pg of fragment of RSV F protein, at least 160 pg of fragment of RSV F protein, atleast 170 pg of fragment of RSV F protein, 180 pg of fragment of RSV F protein, at least 190 pg of fragment of RSV F protein, at least 200 pg of fragment of RSV F protein, or at least 210 pg of fragment of RSV F protein.

[0015] In certain implementations, the composition is in a dosage form selected from the group consisting of: a liquid, a paste, a bar, a cake, a powder, a granulate, a chewable, a tablet, a capsule, a lozenge, a fast-melting tablet or wafer, and a sublingual tablet. In some embodiments, the composition is a liquid. In some aspects, where the tomato fruit material is a paste, the dosage form is a paste. In particular embodiments, the composition is freeze-dried. In other aspects, the vaccine composition further comprises a pharmaceutically acceptable carrier, wherein the composition is formulated for administration intranasally, for example, in the form of a nasal spray. In yet other aspects, the composition is formulated for administration sublingually. Thus, the vaccine composition may further comprise a pharmaceutically acceptable carrier to formulation the composition for sublingual administration.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 depicts, in accordance with certain embodiments, RSV F protection expression levels in fruits from transformed Lizzano cultivar of tomato at various ripening stages. Transformation of the tomato plants is facilitated by Agrobacterium tumefaciens directed to constitutive expression (pVG 100) or fruit-specific expression (pVG 102). Abbreviations in figure: control (CTL-Av), pVG 100 (lOOL-Av), pVG 102 (102L-Av).

[0017] FIG. 2 depicts, in accordance with certain embodiments, RSV F protection expression levels in fruits from transformed Sugary cultivar of tomato at various ripening stages. Transformation of the tomato plants is facilitated by Agrobacterium tumefaciens directed to constitutive expression (pVG 100) or fruit-specific expression (pVG 102). Abbreviations in figure: control (CTL-Av), pVG 100 (lOOL-Av), pVG 102 (102L-Av).

[0018] FIG. 3 depicts, in accordance with certain embodiments, RSV F protection expression levels in fruits from transformed Sweet Chelsea cultivar of tomato at various ripening stages. Transformation of the tomato plants is facilitated by Agrobacterium tumefaciens directed to constitutive expression (pVG 100) or fruit-specific expression (pVG 102). Abbreviations in figure: control (CTL-Av), pVG 100 (lOOL-Av), pVG 102 (102L-Av).

[0019] FIG. 4 depicts, in accordance with certain embodiments, the vector map of pKEAM- BAFd27-gp41, the sequence of which is set forth in SEQ ID NO. 2.

[0020] FIG. 5 depicts, in accordance with certain embodiments, the vector map of pKEAM- BAFd27, the sequence of which is set forth in SEQ ID NO. 1.

[0021] FIG. 6 depicts, in accordance with certain embodiments, a Western blot detecting the presence of RSV F protein presence in tomato fruit extracts. Left Lane: 6 pg RSV-F reference standard (Sino Biological) added to untransformed tomato fruit extract. Lane 2: Extract of fruit from tomato plant transformed with pKEAM-BAFd27-gp41. Lane 3 : extract of untransformed tomato fruit. Samples were electrophoresed in 2% SDS sample buffer without reducing agent and were not heated before loading. Proteins were transferred to a membrane and probed with anti-F antibody (Meridian Life Sciences, Inc.). The expected positions of F trimers, full-length Fo, Fi, and F2 fragments are marked. Based on the reference-curve, the RSV-F concentration of the transformed tomato extract was 15.45 pg / g fresh fruit.

[0022] FIG. 7 depicts, in accordance with certain embodiments, the vector map of pVG 100, the sequence of which is set forth in SEQ ID NO. 3.

[0023] FIG. 8 depicts, in accordance with certain embodiments, the vector map of pVG 102, the sequence of which is set forth in SEQ ID NO. 4.DETAILED DESCRIPTION

[0024] Detailed aspects and applications of the disclosure are described below in the drawings and detailed description of the disclosure. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.

[0025] In the following description, and for the purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the various aspects of the disclosure. It will be understood, however, by those skilled in the relevant arts, that the present disclosure may be practiced without these specific details. It should be noted that there are many different and alternative configurations, devices, and technologies to which the disclosed disclosures may be applied. The full scope of the disclosures is not limited to the examples that are described below.

[0026] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a step” includes reference to one or more of such steps.

[0027] This disclosure is directed transgenic tomato plants expressing at least a fragment of Respiratory Syncytial Virus (RSV) F protein and plant-expression vectors used in producingsuch transgenic plants. In some aspects, the fragment of RSV F protein is the ectodomain of RSV F protein. In some embodiments, the ectodomain of RSV F protein lacks the p27 region. For example, the fragment of RSV F protein expressed in transgenic tomato plants comprises the ectodomain of RSV F protein, and the p27 region of the RSV F protein is replaced with a sequence-specific linker region comprising the amino acid sequence of GSGSGRSLG. In some embodiments, the transgenic tomato plant is transformed with pKEAM-BAFd27 (sequence set forth in SEQ ID NO. 1). In certain embodiments, the ectodomain of RSV F protein is expressed as a fusion protein with a fragment of human immunodeficiency virus (HIV) gp41. In particular embodiments, the transgenic tomato plant is a tomato plant transformed with pKEAM- BAFd27-gp41 (sequence set forth in SEQ ID NO. 2) .

[0028] The fragment of gp41 comprises the N-terminal ectodomain of the protein. In some embodiments, the fragment of gp41 comprises the transmembrane domain and cytosolic domain of the gp41 protein. In some aspects, the fragment of gp41 consists of the transmembrane domain and cytosolic domain of the gp41 protein. In other aspects, the fragment of gp41 is dgp41. In some embodiments, the fragment of gp41 comprises the MPER, transmembrane domain, and cytosolic domain of the gp41 protein. In particular embodiments, the fragment of gp41 consists of the MPER, transmembrane domain, and cytosolic domain of the gp41 protein. Accordingly, in some implementations, the C-terminus ectodomain of the antigen is linked to the N-terminus of the transmembrane domain of the gp41 protein. In other embodiments, the C-terminus of the transmembrane domain of the antigen is linked to the N- terminus of the cytosolic domain of the gp41 protein.

[0029] A pharmaceutical or therapeutic composition comprising an extraction from the fruit of the transgenic tomato plant described herein is also disclosed. The extraction from the fruit of the transgenic tomato plant comprises the fragment of RSV F protein. In some aspects, the extraction from the fruit of the transgenic tomato comprises the fragment of RSV F protein in a trimeric state. In some embodiments of the composition, the extraction from the fruit of the transgenic tomato plant is a puree of the fruit of the transgenic tomato plant. The extraction of from the fruit of the transgenic tomato plant may be any stage of tomato development, for example, when the fruit has turned yellow, orange, or red. In particular implementations where the transgenic tomato plant produces red tomatoes when ripe, for example of a cultivar selected from Lizzano, Sugary, and Sweet Chelsea, the extraction is from yellow-colored, orangecolored, or red-colored fruit.

[0030] In one aspect, the composition comprises a first isolate from the fruit of the transgenic tomato plant comprising a fragment of RSV F protein and a second isolate from the fruit of the transgenic tomato plant comprising tomatine. In some embodiments, the vaccine composition further comprises a source of tomatine. In particular embodiments, the source of tomatine is also from the transgenic tomato plant. The source of tomatine may be from the tomato fruit or a non-fruit part of the transgenic tomato plant. In other embodiments, the source of tomatine is from a tomato fruit of a non-transgenic plant. In another aspects, the composition comprises a first isolate from the fruit of the transgenic tomato plant comprising a fragment of RSV F protein; and a second isolate from the non-fruit part of the transgenic tomato plant comprising tomatine.

[0031] In a particular embodiment, the extraction from the fruit of the transgenic tomato plant comprises tomatine and a fragment of RSV F protein. In some aspects, the extraction from the fruit of the transgenic tomato plant is a puree of the fruit of the transgenic tomato plant. In some embodiments, the extraction from the fruit of the transgenic tomato plant is a freeze-dried puree of the fruit of the transgenic tomato plant.

[0032] In some aspects, the composition comprises at least 100 pg of fragment of RSV F protein. For example, the composition comprises at least 150 pg of fragment of RSV F protein, at least 160 pg of fragment of RSV F protein, at least 170 pg of fragment of RSV F protein, 180 pg of fragment of RSV F protein, at least 190 pg of fragment of RSV F protein, at least 200 pg of fragment of RSV F protein, or at least 210 pg of fragment of RSV F protein. In some aspects, the concentration of tomatine in the composition is no more than 5 mg / mL, no more 5 mg / g, no more than 2.5 mg / mL, or no more than 2.5 mg / g.

[0033] In some implementations, the composition is formulated for intranasal, oral, or sublingual administration. In certain implementations, the vaccine composition is in a dosage form selected from the group consisting of a liquid, a paste, a bar, a cake, a powder, a granulate, a chewable, a tablet, a capsule, a lozenge, a fast-melting tablet or wafer, and a sublingual tablet. In some embodiments, the composition is a liquid. In some aspects, where the tomato fruit material is a paste, the dosage form is a paste. In particular embodiments, the composition is freeze-dried. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or additive. The additive may be selected from a stabilizer, a preservative, a filler, an excipient, and / or an adjuvant that enhances the immune response generated by the composition. In some aspects, the composition comprises a pharmaceutically acceptable carrier, wherein the composition is formulated for administration intranasally, for example, inthe form of a nasal spray. In other aspects, the composition comprises a pharmaceutically acceptable carrier or additive and is formulated for sublingual administration.EXAMPLES

[0034] The present disclosure is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.Example 1. Exemplary Protocol for Transformation of Tomato Plants

[0035] Agrobacterium used to facilitate gene transfer in tomato plants is transformed with either pVG 100 (with 35S promotor, constitutive expression) (FIG. 7) or pVG 102 (with E4ZE8 promoter, fruit-specific expression) (FIG. 8). Specifically, Electro-competent Agrobacterium tumefaciens LBA4404 cells were electroporated with pVG 100 (106 ng / pL) or pVG 102 (120 ng / pL). The transformed A. tumefaciens are then exposed to seeds from three cherry tomato cultivars (Lizzano, Sugary, and Sweet Chelsea).A. Preparing plant material (Day 0)1. Sterilize seeds from each tomato cultivar with 70% ethanol for 1 min.2. Treat sterilized seeds with 20% Clorox™ for each cultivar (stock: 5.25% NaClO, 5: 1 dilution giving a final concentration of 1.05% NaClO) for 10 min. Each cultivar is handled separately.3. Rinsed seeds 3 times with sterile distilled H2O for a period of 2-3 min per rinse.4. Germinate seeds on TS medium solidified with 8 g agar / L (autoclaved approx. 100 mL medium in each glass vessel of 473-mL size, 135 mm height 77 mm diameter with polypropylene screw-caps) for 8 days at 16-hr photoperiod (to use for cotyledon and hypocotyl explants).5. For planting, four jars were used for each cultivar (3 seeds / jar x 4 jars = 12seeds / cultivar). Each jar was planted with three seeds on a triangle, keeping away from the wall of the jar. One of the 4 jars was kept on the window-sill, while the rest were kept under the light box. Light intensity under the 2’x3’ light table was approx. 60 pmols^m'2.

[0036] The whole procedure is conducted under a sterile biosafety (laminar-flow) cabinet using sterile utensils.B. Day before transformation (Day 7)

[0037] Inoculate yeast extract peptone (YEP) broth with original plates (with 4 days old cultures) of electroporated tumefaciens LBA4404: transformed tumefaciens was placed into 500 mL liquid YEP containing 100 mg / L streptomycin and 50 mg / L kanamycin. The culture is then grown overnight on orbital shaker (150 rpm) at 28°C.C. Transformation day (Day 8)1. Transfer approx. 40 mL overnight A tumefaciens culture into 50-mL sterile tubes (12 tubes total per gene construct) and centrifuged for 30-40 min at 3000 rpm (3750 ref) to produce a pellet.2. Resuspend the pellet with 25 mL Agrobacterium minimal medium (AS medium; RL medium containing 40 mg / L acetosyringone to increase permeability) and grow for 1 hr at 28°C on orbital shaker (150 rpm).3. Take ODeoo reading of A. tumefaciens resuspension (0.9-1.1, GeneQuant Pro spectrophotometer).4. Dilute the A. tumefaciens resuspension in a sterile 50-mL tube with AS medium to produce ODeoo of approx. 0.5. Undiluted A. tumefaciens resuspension can be stored at -20°C for later transformation (for example, transformation of 1 month old plants).5. Using a sterile scalpel, cut off seedling tops to collect hypocotyls and well-shaped (not curly) cotyledons. Separated real leaves and shoot apexes (if present) from the petioles, and processed all plant parts, including roots for incubation.Hypocotyls, petioles, shoot-apexes, and roots i. Separate hypocotyls from cotyledons and put hypocotyls, petioles, shootapexes, and roots into small amount of RL medium in 100x25 mm Petri-plate. ii. Cut all plant parts into 3-5 mm segments. iii. Placed plant part segments in separate Petri-plate with 30-40 mL diluted A. tumefaciens resuspension in sterile 100 mL beakers capped with foil.Cotyledons & real leaves i. Tips and petioles were cut off from the cotyledons and real leaves.ii. Placed prepared cotyledons, leaves, tips, and petioles in small amount of RL medium in Petri-plates. Make sure to cut cotyledons and leaves into 3-5 mm segments. iii. Placed in 30-40 mL diluted A. tumefaciens resuspension in sterile 100 mL beakers capped with foil.6. Submerged all explants in diluted A. tumefaciens resuspension in sterile 100 mL beakers and gently swirl (alternatively, put on slow shaker at 50 rpm) for 30 min for inoculation of explants.7. Blot-dried the explants on sterile paper towels.8. Placed explants partially submerged on TCC (co-cultivation) medium on sterile 150x15 mm Petri-dishes for 48 hr (10 days total taken up to now) in dark at 25°C.D. 48 hrs after transformation (Day 10)1. Wash hypocotyl and cotyledon explants separately 2-3 times with RL medium + 300 mg timentin / L (added after autoclaving). Blot-dry with sterile paper towels.2. Partially submerge the explants on TSR (shoot generation) medium on Petri-dishes for 1 week with 16 hr photoperiod (17 days total). Light intensity is approx. 60 pmol / m2 / sec at 25°C.3. Re-position the explants on TSR by pressing down gently to achieve full contact with the medium and continue to incubate for 3 weeks (38 days total).4. Subculture all explants on fresh TSR or TDZ (explants were divided 50:50 into each shooting medium) in 8 oz (237 mL) 4.5”x4.5”xl.5”(height) polystyrene culture vessels (Stericon™, PhytoTechnology Laborotories, KS) with 16 hr photoperiod (20 W cool -white fluorescent light tubes, light intensity = 60 pmol / m2 / sec) at 25°C. Grow shoots until 1-2 cm long.5. Excise the 1-2 cm tall shoots and place on TR or lAA’ MS media for rooting (contains 50 mg / L kanamycin for secondary selection).6. Transfer plants with well-developed roots to small pots (3”x3”x4”) with soil.7. Place small pots in a growth chamber and cover pot with clear plastic for one week for acclimatization (over 45 days total including time for T).8. Transfer plants to permanent pots (9” diameter x 12” tall round pots) and grow in greenhouse for further growth and fruiting.Example 2, Quantification of RSV F Protein Fragment Production Through Ripening Stages of Tomato Fruit

[0038] Estimation of RSV-F protein per g of fruit from transgenic tomato plants was made using double antibody sandwich ELSA. Yellow, orange, and red fruits of control, pVG 100 and pVG 102 plants of Lizzano, Sugary, and Sweet Chelsea cultivars were harvested 1-2 days prior to ELISA and stored in the freezer until use.1. Experimental Materials

[0039] Buffer for control tomato extract: 5x coating buffer (CB). The table provides the recipe for preparing the coating buffer. pH of the coating buffer was not adjusted.

[0040] Buffer solution for antibodies: phosphate buffered saline (PBS) + 0.05% Tween 20 (PBST). 75 pL of buffered antigen or antibody solution was added per well unless otherwise specified.2. Reference Curve

[0041] Three red control tomatoes from each cultivar were homogenized in 1 : 1.5 (w / v) of 5x CB and used for each dilution of RSV-F standard, to obtain the reference curve. The range of the reference curve is 0-10 pg / mL, with 0 pg / mL, 0.5 pg / mL, 1 pg / mL, 2.5 pg / mL, 5 pg / mL, and 10 pg / mL being the specific concentrations used to generate the reference curve. The 10 pg / mL dilution was prepared by dissolving 20 pL of 100 pg / mL RSV F stock in 180 pL lx CB.3. Sample Collection

[0042] Two fruits from a collection / mix for each cultivar were homogenized to obtain tomato extracts from yellow, orange, and red fruits of control (CT), pVG 100 (100) and pVG 102 (102) plants of Lizzano, Sugary, or Sweet Chelsea cultivars. This plan results in 4 biological repetitions for each treatment, with two technical reps for plating (Plates: Thermo Sci., Immulon-IB flat bottom).4. ELISA Protocol1. Each well of the plates were added with 75 pl of antigen (commercial RSV F protein as the standard or transgenic tomato extracts) following the plate plan. The antigen standards were prepared and used as described above. Tomato extracts were homogenized in 5x CB, 1 :3 w / v.2. The plates were covered with plastic wrap and placed on a tray with some damp paper towels on the bottom. The plates were incubated overnight at 4°C.3. The plates were washed five times with PBST (plates 1 & 2) or DIWT (plates 3 & 4) and tap-dried on a paper towel.4. Aliquots of 75 pL / well of mouse monoclonal antibody (diluted 1 : 1000, in PBST) were added to all four plates. All the plates were covered as in step (2), and incubated for 2 hr at 37°C.5. The plates were washed five times using PBST (plates 1 & 2) or DIWT (plates 3 & 4) and tap-dried on a paper towel.6. The antibody-enzyme conjugate (RAM PCAb-AP) was diluted 1 : 1000, in PBST, and added 75 pl to each test well. All the plates were covered as in step (2) and incubated at 37°C for 2 hour.7. The plates were washed and dried as described in step (5).8. The substrate was prepared just before use by adding p-nitrophenyl phosphate disodium hexahydrate (pNPP) to the substrate buffer (one 5 mg pNPP tablet in 10 ml of buffer).9. Aliquots of 100 pl of prepared substrate were added to each test well of all four plates.10. The plates were covered as in step (2) above and incubated in the dark at room temperature for 1 hour.11. The absorbance was read using a spectrophotometer at 405 nm. Readings were taken up to 2 days if needed, depending on the rate of color development.

[0043] FIGs. 1-3 present the ELISA results for the Lizzano, Sugary, or Sweet Chelsea cultivars respectively. As shown in the figures, the selection of constitutive versus fruit-specific Ti plasmids for transforming the agrobacterium did not significantly change the protein expression level of RSV F protein fragment in the fruit of transgenic tomato plants. Interestingly, the Sugary and Sweet Chelsea cultivars had in the most consistent level of RSV F protein fragment expression in the fruit regardless of ripeness. The highest yield of RSV Fprotein fragment expression was in red colored tomato fruit from the Lizzano cultivar transformed with Agrobacteria transformed with the pVG 100 plasmid.Example 3, Stabilization of RSV F Protein Expression in Tomato Plants

[0044] Tomato cultivars Lizzano, Sugary, and Sweet Chelsea were transformed several different plant-optimized RSV-F gene-constructs created to enable enhanced expression of RSV F protein in the fruits. The following vectors were used for separate transformation procedures:• pKEAM-BAFd27: Produces a plant-optimized RSV F protein with the 27-aa furin cleavage site replaced with a short linker “GSGSGRSLG”. The native signal peptide was also replaced, with that from barley alpha amylase.• pKEAM-BAFd27-gp41 : Produces a fusion protein of a fragment of HIV gp41 and the RSV antigen. The RSV F protein’s Fd27 ectodomain was modified to be preceded by the barley alpha-amylase signal peptide and fused to the HIV gp41 transmembrane (TM) domain and C-terminal cytosolic domain.

[0045] Tomato fruits were sampled from independent transformants and processed for nondenaturing SDS-PAGE and Western blot. The fruits were extracted in 0.05% TBST. Samples were electrophoresed in 2% SDS sample buffer without reducing agent and were not heated before loading. Proteins were transferred to a membrane and probed with anti-F antibody (Meridian Life Sciences, Inc.).

[0046] FIG. 6 depicts an exemplary Western blot. Based on the reference-curve, the RSV-F concentration of the transformed tomato extract was 15.45 pg / g fresh fruit. The average concentration of plants transformed with the original RSV-F construct was 9.85 pg / g fresh fruit. Therefore, transformation with the pKEAM constructs results in over 50% increase in the RSV-F expression levels in fruits compared with the original RSV-F constructs.

[0047] One transgenic tomato line that expressed F protein at high level has been identified (FIG. 6). The plants were transformed with pKEAM-BAFd27-gp41. Apparent trimers at relative MW -160 kDa for Fd27-gp41, and -120 kDa for the F reference standard were detected. Note that the Fd27-gp41 fusion protein has an expected MW 72 kDa, while native F is -61 kDa, which accounts for the observed difference in dimer and trimer sizes. Smaller F fragments accumulated, with the most abundant -37 kDa.

Claims

CLAIMSWhat is claimed:

1. A transgenic tomato plant transformed to express Respiratory Syncytial Virus (RSV) F protein or a fragment thereof, wherein: the expressed RSV F protein comprises a p27 region replaced with a sequence-specific linker region comprising the amino acid sequence of GSGSGRSLG; and / or the expressed fragment of RSV F protein is the ectodomain of the RSV F protein.

2. The transgenic tomato plant of claim 1, wherein the transgenic tomato plant expresses pKEAM-BAFd27 (SEQ ID NO. 1) thereby expressing RSV F protein with its p27 region replaced with the sequence-specific linker region comprising the amino acid sequence of GSGSGRSLG.

3. The transgenic tomato plant of claim 1, wherein the transgenic tomato plant expresses pKEAM-BAFd27-gp41 (SEQ ID NO. 2).

4. The transgenic tomato plant of claim 1, wherein the fragment of RSV F protein is expressed as a fusion protein with a transmembrane domain and a cytosolic domain of human immunodeficiency virus (HIV) gp41, wherein the C-terminus of the fragment of RSV F protein is linked the N-terminus of the transmembrane domain of the gp41 protein.

5. The transgenic tomato plant of any one of claim 1-4, wherein the plant is a cultivar selected from the group consisting of: Lizzano, Sugary, and Sweet Chelsea.

6. The transgenic plant of any one of claims 1-4, wherein fruit produced by the transgenic plant expresses the RSV F protein.

7. A plant expression vector for expressing Respiratory Syncytial Virus (RSV) F protein, wherein the plant expression vector comprises: a first nucleic acid sequence encoding barley alpha-amylase signal peptide; and a second nucleic acid sequence encoding a modified RSV F protein lacking RSV F protein native signal peptide region and comprising a p27 region replaced an amino acid sequence of GSGSGRSLG.

8. The plant expression vector of claim 7, wherein the plant expression vector has the sequence set forth in SEQ ID NO. 1.

9. A plant expression vector for expressing a fragment of Respiratory Syncytial Virus (RSV) F protein having the sequence set forth in SEQ ID NO. 2.

10. A composition comprising:an extraction from a fruit of the transgenic tomato plant of any one of claims 1-6, wherein the extraction from the fruit of the transgenic tomato plant comprises the fragment of RS V F protein; and optionally a pharmaceutically acceptable carrier or excipient.

11. The composition of claim 10, wherein the extraction from the fruit of the transgenic tomato comprises the fragment of RSV F protein in a trimeric state.

12. The composition of claim 10 or 11, wherein the extraction from the fruit of the transgenic tomato plant is a puree of the fruit of the transgenic tomato plant.

13. The composition of claim 10 or 11, wherein the extraction from the fruit of the transgenic tomato plant further comprises tomatine.

14. The composition of claim 13, wherein the composition comprises: a first isolate from the fruit of the transgenic tomato plant comprising a fragment of RSV F protein; and a second isolate from the fruit of the transgenic tomato plant comprising tomatine.

15. The composition of claim 10 or 11, further comprising a source of tomatine.

16. The composition of claim 15, wherein the source of tomatine is from a non-fruit part of the transgenic tomato plant or non-transgenic tomato plant or from a tomato fruit of a non- transgenic plant.

17. The composition of claim 16, wherein the composition comprises: a first isolate from the fruit of the transgenic tomato plant comprising a fragment of RSV F protein; and a second isolate from the non-fruit part of the transgenic tomato plant comprising tomatine.

18. The composition of any one of claims 10-17, wherein the composition is in a dosage form selected from the group consisting of: a liquid, a paste, a bar, a cake, a powder, a granulate, a chewable, a tablet, a capsule, a lozenge, a fast-melting tablet or wafer, and a sublingual tablet.

19. The composition of any one of claims 10-17, wherein the composition is formulated for intranasal administration.

20. The composition of any one of claims 10-17, wherein the composition is formulated for oral administration.

Images