HK1260786A1

HK1260786A1 - Rotavirus-like particle production in plants

Info

Publication number: HK1260786A1
Application number: HK19120825.5A
Authority: HK
Inventors: M-A‧达奥斯特; N‧兰德里; P-O‧拉沃伊; 新井正明; 浅原尚美; D‧L‧R‧穆特法; I‧I‧希泽洛斯; E‧P‧里比基
Original assignee: 田边三菱制药株式会社; 麦迪卡格公司
Priority date: 2012-05-11
Filing date: 2019-03-12
Publication date: 2019-12-20

Description

Production of rotavirus-like particles in plants

The application is a divisional application of Chinese patent application No.201380024759.7(PCT application number is PCT/CA2013/050364) with the application date of 2013, 5, 10 and the name of 'generation of rotavirus-like particles in plants'.

Technical Field

The present invention relates to the production of rotavirus structural proteins in plants. More specifically, the invention relates to the production of viroid particles comprising rotavirus structural protein in plants.

Background

Rotavirus infection is a global problem, mainly affecting children under five years of age. It causes severe gastroenteritis and in the worst case death.

Rotaviruses are members of the Reoviridae (Reoviridae) family (rotaviruses), which infect the gastrointestinal system and the respiratory tract. The name stems from the rotavirus appearance of the virion when viewed by negative contrast electron microscopy (FIG. 1 a; prior art). Rotaviruses are generally spherical in shape and are named for the outer and inner shells or their double-shelled capsid structure. The outer shell diameter was about 70nm and the inner shell diameter was about 55nm, respectively. The double-shelled capsid of the rotavirus surrounds a core that includes an inner protein shell and a genome. The rotavirus genome consists of a double-stranded RNA fragment encoding at least 11 rotavirus proteins.

The dsRNA encodes six structural proteins (VP) and six non-structural proteins (NSP) (FIG. 1 c; prior art). Structural proteins include VP1, VP2, VP3, VP4, VP6, and VP7 (FIG. 1 b; prior art). The three concentric layers were formed by assembly of VP2, VP6, and VP7, respectively, with VP4 forming a "spike" (spike) on the surface of the viral structure. NSPs are synthesized within infected cells and function in multiple parts of the replication cycle or interact with some of the host proteins to affect pathogenesis or immune response to infection (Greenberg and Estes, 2009).

VP2 is a 102kDa protein and is the most abundant protein in the viral core. It forms the innermost structural protein layer and provides the backbone for the correct assembly of components of the viral core and the transcription enzymes (Lawton, 2000). VP1 is the largest viral protein of 125kDa, which functions as the RNA-dependent polymerase of rotavirus, producing a core replication intermediate, and binds to VP2 at the vertex of its icosahedron (Varani and Allain, 2002; Vende et al, 2002). VP3 (a 98kDa protein) also binds directly to the viral genome, acting as a mRNA capping enzyme (capping enzyme) that adds a 5' end cap structure to the viral mRNA. VP1 forms a complex with VP3 that attaches to the outer 5-fold apex of the VP2 coat layer (Angel, 2007). VP6 is a 42kDa protein that forms the mesocapsid of the viral core, which is the major capsid protein and accounts for more than 50% of the total proteinaceous material of the viral particle (Gonz a lez et al, 2004; Estes, 1996). It is essential for gene transcription and can function in the encapsulation of rotavirus RNA by anchoring VP1 to VP2 in the core, as observed in bluetongue virus (bluetongue virus), another member of the reoviridae family. It also determines the classification of rotaviruses into five groups (a to E), with group a most frequently infecting humans (Palombo, 1999). VP6 in rotavirus group a has at least four Subgroups (SG): SG I, SG II, SG (I + II) and SG non- (I + II), depending on the presence or absence of SG-specific epitopes. Groups B and C lack antigens common to group a, but are also known to infect humans, while group D only infects animals, e.g., chickens and cows (Thongprachum, 2010).

The two outer capsid proteins, VP7, glycoprotein (G) at 37kDa and protease sensitive VP4(P) at 87kDa, define the serotype of the virus. These two proteins induce a neutralizing antibody response and are thus used to classify rotavirus serotypes into a dual-naming system, depending on the G-P antigen combination (e.g., G1P [8] or G2P [4]) (Sanchez-Padilla et al, 2009). The VP4 protein dimerizes to form 60 spikes on the viral coat, which are directly involved in the initial stages of host cell entry. The spike protein comprises a cleavage site at amino acid (aa) position 248. Once infected, it is cleaved by the protease trypsin, thereby yielding VP5(529aa, 60kDa) and VP8(246aa, 28kDa) (Denisova et al, 1999). This process enhances virus infectivity (cell attachment and invasion of host cells) and stabilizes spike structure (Glass, 2006). The VP7 glycoprotein forms the third or outer layer of the virus. Currently, 27G and 35P genotypes are known (Greenbergand Estes, 2009). VP4 and VP7 are the main antigens involved in virus neutralization and are important targets for vaccine development (Dennehy, 2007).

In infected mammalian cells, rotavirus undergoes a unique form of morphogenesis to form an intact trilayer VP2/6/4/7 virus particle (Lopez et al, 2005). The three-layered capsid is a very stable complex that enables it to fecal-oral spread and deliver the virus to the small intestine, where it infects non-dividing differentiated intestinal epithelial cells near the tip of the villus (Greenberg and Estes, 2009). First, the whole virus was attached to sialic acid independent receptors via 60 VP4 dimer spikes on the virus surface (Lundgren and Svensson, 2001). The 60 VP4 dimer spikes on the virus surface allow the virus to attach to these cellular receptors. VP4 is susceptible to proteolytic cleavage by trypsin, which results in conformational changes that expose additional attachment sites on the surface of the glycoprotein for interaction with a range of co-receptors.

However, the multi-step attachment and entry process is not well understood, but the virus is delivered across the host cell membrane. The VP7 outer capsid (also involved in the entry process) is removed in the process and double-layer particles (DLP) are delivered to the cytoplasm in vesicles (fig. 2; prior art). DLP breaks away from vesicles and enters non-membrane bound cytoplasmic inclusions (cytoplasmicinclusions). Early transcription through the genome of VP1 begins in the granule, so that the dsRNA is never exposed to the cytoplasm. RNA replication and core formation occur in these non-membrane bound cytoplasmic contents. The primary (+) RNA is then transported into the cytoplasm and used as a template for viral protein synthesis. VP4 is produced in the cytosol and transported to the Rough Endoplasmic Reticulum (RER), and VP7 is secreted into the RER. VP2 is produced and assembled with VP6 in the cytosol of the virion and subsequently budded (bud) into the RER compartment, during which a transient membrane envelope is obtained (Lopez et al, 2005; Tian et al, 1996). In RER, the transient membrane envelope of the virus particle is removed and replaced by VP4 and VP7 protein monomers with critical involvement of the rotavirus glycoprotein NSP 4(Tian et al, 1996; Lopez et al, 2005; Gonzalez et al, 2000). NSP4 functions as an intracellular receptor in the ER membrane and binds to newly produced subviral particles and possibly also to the spike protein VP4(Tian et al, 1996). NSP4 is also toxic to humans and is a causative agent of diarrhea. Subsequently, intact, mature particles are secreted by golgi transfer from the RER to the plasma membrane (Lopez et al, 2005).

A number of different approaches have been taken to produce rotavirus vaccines suitable for protecting the population against rotavirus of various serotypes. These include various Jennerian approaches, the use of live attenuated viruses, the use of viroid particles, nucleic acid vaccines and viral subunits as immunogens. There are two oral vaccines currently available on the market, however, in some developing countries these vaccines are less effective due to the variation of the virus strain and the presence of other pathogens.

U.S. patent nos. 4,624,850, 4,636,385, 4,704,275, 4,751,080, 4,927,628, 5,474,773 and 5,695,767 each describe various rotavirus vaccines and/or methods for making them. A commonality common to the members of this group is that each of these vaccines relies on the use of whole virus particles to produce the final rotavirus vaccine. In view of the long-term need for effective multivalent vaccines, it is clear that this body of work has only partially succeeded in addressing the need for such vaccines.

Unlike traditional vaccine production methods, advances in the field of molecular biology have allowed the expression of individual rotavirus proteins. Crawford et al (J Virol.1994 September; 68(9): 5945-5952) cloned VP2, VP4, VP6 and VP7 encoding the major capsid proteins into a baculovirus expression system and expressed each protein in insect cells. Co-expression of different combinations of rotavirus major structural proteins results in the formation of stable virus-like particles (VLPs). Co-expression of VP2 and VP6 alone or with VP4 resulted in the production of VP2/6VLP or VP2/4/6VLP, which are similar to double-layered rotavirus particles. Co-expression of VP2, VP6 and VP7 (with or without VP4) produced three-layered VP2/6/7 VLPs or VP2/4/6/7 VLPs, which are similar to natural infectious rotavirus particles. Each VLP maintained the structural and functional characteristics of the native particle (as determined by particle electron microscopy), the presence of non-neutralizing and neutralizing epitopes on VP4 and VP7, and the hemagglutination activity of VP2/4/6/7 VLPs.

Vaccine candidates generated from viroid particles of different protein composition have shown potential as subunit vaccines. O' Neal et al ("Rotavirus viruses-like Particles added to the Mucosally induced protective Immunity," J.virology, 71(11):8707-8717(1997)) show that when VLPs containing VP2 and VP6 or VLPs containing VP2, VP6 and VP7 are Administered to mice with and without the addition of cholera toxin, they induce protective Immunity in immunized mice, whereas protection is more effective when each VLP is Administered with Cholera Toxin (CT).

The core-like particles (CLPs) and VLPs are also used for immunizing cows, Fernandez et al ("Passive Immunity to Bovine rotaviruses in New born calcium Fed Colostrum Supplements From cow Immunity with Recombinant SA11 rotaviruses core-like particles (CLPs) or viruses-like particles (VLPs) vaccines," Vaccine,16(5):507-516 (1998)). In this study, the ability of CLPs to produce passive immunity with VLPs was studied. The study group summarized: VLPs are more effective than CLPs in inducing passive immunity.

Plants are increasingly being used for large-scale production of recombinant proteins. For example, US 2003/0175303 discloses the expression of recombinant rotavirus structural proteins VP6, VP2, VP4 or VP7 in stably transformed tomato plants.

Saldana et al expressed VP2 and VP6 in the cytoplasm of tomato plants using cauliflower mosaic virus (CaMV)35S promoter and recombinant Agrobacterium (A. tumefaciens) (Saldana et al, 2006). Electron microscopy studies showed that a small fraction of the particles had been assembled into 2/6 VLPs. A protective immune response was detected in the mice and this could be due to unassembled VP to some extent. Each protein has been shown to elicit an immune response in mice, as in the case of VP8 and VP6 (Zhou et al, 2010).

Matsumura et al (2002) first reported the expression and assembly of bovine rotavirus A VP6 in transgenic potato plants. In their studies, they used transgenic potato plants regulated by the cauliflower mosaic virus (CaMV)35S promoter and recombinant Agrobacterium tumefaciens (Agrobacterium tumefaciens) carrying the VP6 gene. Proteins were expressed, purified and subjected to immunological studies. The immune response in adult mice showed the presence of the VP6 antibody in the serum. However, they showed no evidence of the assembled VP6 protein. It may be a simple monomer or trimer (which may elicit an immune response in mice). Work of another panel showed assembly of VP6 in tobacco (nicotianabentanamiana) using a Potato Virus X (PVX) vector (O' Brien et al, 2000). When the VP6 protein is expressed in plants, it was found that it only assembles when fused to PVX rod proteins (PVX protein rods). Once cleavage has occurred, VP6 assembles into the icosahedral VLPs as observed by Marusic et al (2001) in a similar study on HIV-PVX. This result may suggest that rotavirus proteins may require additional factors or potentiation to form VLPs.

Production of VLPs is a challenging task due to the need for both synthesis and assembly of one or more recombinant proteins. This is true for VLPs of rotaviruses (which are RNA viruses with capsids formed from 1860 monomers of four different proteins). For the production of VLPs, simultaneous expression and assembly of two to three recombinant proteins is necessary. These include 120 molecules of VP2 (inner layer), 780 molecules of VP6 (middle layer) and 780 molecules of glycoprotein VP7 (outer layer), eventually forming a bilayer or trilayer particle. In addition, the production of most VLPs requires the simultaneous expression and assembly of several recombinant proteins, which in the case of Rotavirus Like Particles (RLP) needs to occur in a single host cell.

Recent studies have shown that codon-optimized human rotavirus VP6 is successfully expressed in Chenopodium quinoa (Chenopodium amaranthicolor) using a Beetroot Black Spot Virus (BBSV) mediated expression system. The protein is engineered as a replacement for the open reading frame of the BBSV coat protein. Oral immunization of female BALB/c mice with plant-based VP6 protein induced high titers of anti-VP 6 mucosal IgA and serum IgG (Zhou et al, 2010). However, this group did not mention whether the VP6 protein was assembled into VLPs or particles.

Rotavirus VP7 has also been successfully expressed in tobacco plants and it was shown to maintain its neutralizing immune response in mice (Yu and Langridge, 2001). Another study using transgenic potato plants to express VP7 showed that the VP7 gene was stable in transformed plants for more than 50 generations. VP7 protein from passage 50 induced both protective as well as neutralizing antibodies in adult mice (Li et al, 2006).

Yang et al (Yang Y M, Li X, Yang H et al, 2011) co-expressed three rotavirus capsid proteins VP2, VP6 and VP7 of group A RV (P8G 1) into tobacco plants and studied the expression level of these proteins and the formation and immunogenicity of rotavirus-like particles. VLPs were purified from transgenic tobacco plants and analyzed by electron microscopy and Western blotting. The Yang et al results show that the plant-derived VP2, VP6 and VP7 proteins self-assemble into 2/6 or 2/6/7 rotavirus particles with a diameter of 60-80 nm.

Disclosure of Invention

The present invention relates to the production of rotavirus structural proteins in plants. More specifically, the invention also relates to the production of viroid particles comprising rotavirus structural protein in plants.

According to the present invention, there is provided a method (a) for producing Rotavirus Like Particles (RLP) in plants, said method comprising:

a) introducing into the plant, part of a plant, or plant cell a first nucleic acid comprising a first regulatory region active in the plant and operatively linked to a first nucleotide sequence encoding a first rotavirus structural protein, a second nucleic acid comprising a second regulatory region active in the plant and operatively linked to a second nucleotide sequence encoding a second rotavirus structural protein, and a third nucleic acid comprising a third regulatory region active in the plant and operatively linked to a third nucleotide sequence encoding a third rotavirus structural protein,

b) growing said plant, plant part, or plant cell under conditions that allow transient expression of said first, second, and third nucleic acids, thereby producing RLP.

Furthermore, in step a), a fourth nucleic acid comprising a fourth regulatory region active in a plant and operatively linked to a fourth nucleotide sequence encoding a fourth rotavirus structural protein may be introduced into the plant, part of a plant or plant cell, and expressed when the plant, part of a plant or plant cell is grown in step b).

As described in the above method (a), the first rotavirus structural protein may be VP2, the second rotavirus structural protein may be VP6, and the third rotavirus structural protein may be VP4 or VP 7. Furthermore, the fourth rotavirus structural protein may be VP7 or VP 4. The protease may be co-expressed in the plant.

The present invention also provides a method (B) of producing a Rotavirus Like Particle (RLP), the method comprising:

a) introducing into a plant, part of a plant or plant cell a nucleic acid comprising a regulatory region active in plants and operatively linked to a first nucleotide sequence encoding one or more rotavirus structural protein,

b) growing the plant, plant part, or plant cell under conditions that allow transient expression of the first nucleic acid, thereby producing RLP.

The method (B) as described above may further include: introducing in step a) a second nucleic acid comprising a second regulatory region active in the plant and operatively linked to a second nucleotide sequence encoding one or more rotavirus structural protein, and expressing the second nucleic acid when the plant, part of a plant or plant cell is grown in step b).

The method (B) as described above may further include: introducing into the plant in step a) a third nucleic acid comprising a third regulatory region active in the plant and operatively linked to a third nucleotide sequence encoding one or more rotavirus structural protein, and expressing the third nucleic acid when the plant, part of a plant or plant cell is grown in step b).

Furthermore, in method (a) or (B), an additional nucleic acid may be expressed in the plant, part of a plant or plant cell, and wherein the additional nucleic acid comprises a regulatory region active in the plant and operatively linked to a nucleotide sequence encoding a suppressor of silencing.

The codon usage of the nucleotide sequence may be adjusted to preferred human codon usage, increased GC content, or a combination thereof.

Rotavirus structural proteins may include truncated native or non-native signal peptides. The non-native signal peptide may be a protein disulfide isomerase signal (PDI) peptide.

The first, second, third or fourth nucleotide sequence, or a combination thereof, can be operatively linked to a cowpea mosaic virus (CPMV) regulatory region.

The method (a) or (B) as described above may further include the steps of:

c) harvesting said plant, part of a plant or plant cell, and

d) purifying RLP from said plant, plant part or plant cell.

VP4 may be processed or cleaved using trypsin, trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin during the harvesting or purification steps in method (a) or (B) to produce VP5 and VP 8.

RLP can range in size from 70-100nm and can be purified in the presence of calcium.

The present invention provides RLP produced by the method (A) or (B) as described above. The RLP produced may comprise at least VP4 rotavirus structural protein. VP4 can be cleaved into VP5 and VP8 using proteases, e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin. The protease may be co-expressed in the plant or added during harvesting, purification, or both. Further, the RLP generated by the method (a) or (B) may be a double-layer RLP and/or a triple-layer RLP.

In addition, the present invention provides nucleotide sequences. The nucleotide sequence encoding VP2 may comprise a nucleotide sequence identical to that shown as SEQ id no: 13. SEQ ID NO: 14 or SEQ ID NO: 45 is 80% to 100% identical. The nucleotide sequence encoding VP6 may comprise a nucleotide sequence identical to that shown as SEQ ID NO: 17. SEQ ID NO: 18 or SEQ ID NO: 46 between 80% and 100% identity. The nucleotide sequence encoding VP7 may comprise a nucleotide sequence identical to that shown as SEQ ID NO: 19. 20, 48, 49, 52, 53, 54 or 57 from 80% to 100% identity. Also, the nucleotide sequence encoding VP4 may comprise a nucleotide sequence identical to SEQ ID NO: 15. 16, 47, 50 or 51, or a pharmaceutically acceptable salt thereof, wherein the nucleotide sequence is 80% to 100% identical. In addition, VP2 may be encoded by a polynucleotide comprising a sequence identical to SEQ ID NO: 1or SEQ ID NO: 25 from 80% to 100% identity to the amino acid sequence defined in seq id no. VP6 may be encoded by a polynucleotide comprising a sequence identical to SEQ ID NO: 3 or SEQ ID NO: 31 between 80% and 100% identical to the amino acid sequence defined in claim 31. VP7 may be encoded by a polynucleotide comprising a sequence identical to SEQ ID NO: 4. 39, 43 or 59, or a pharmaceutically acceptable salt thereof. VP4 may be encoded by a polynucleotide comprising a sequence corresponding to seq id NO: 2or SEQ ID NO: 36. 33, or a sequence encoding from 80% to 100% identity of an amino acid sequence as defined herein. The one or more rotavirus structural proteins may be VP2, VP4, VP6 and/or VP 7. VP4 can be processed into VP5 and VP 8. The one or more rotavirus structural proteins may be selected from rotavirus strain G9P [6], rotavirus A WA strain, rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain and rotavirus SA11 strain.

In method (a) as described above, the first, second or third nucleic acid sequence, or combination thereof, may comprise a regulatory region active in the plant operably linked to one or more comovirus (comovirus) enhancers, to one or more amplification elements and to a nucleotide sequence encoding a rotavirus structural protein, and wherein a fourth nucleic acid encoding a replicase may be introduced into the plant, portion of a plant or plant cell.

In method (B) as described above, the first, second, third or fourth nucleic acid sequence or combination thereof may comprise a regulatory region active in the plant operably linked to one or more comovirus (comovirus) enhancers, to one or more amplification elements and to a nucleotide sequence encoding a rotavirus structural protein, and wherein a fifth nucleic acid encoding a replicase may be introduced into the plant, portion of a plant or plant cell.

Further, according to the present invention, there is provided a method (C) for producing Rotavirus Like Particles (RLP) in plants, the method comprising:

a) introducing into a plant or part of a plant a nucleic acid comprising a regulatory region active in plants and operatively linked to a nucleotide sequence encoding one or more rotavirus structural protein,

b) growing said plant, plant part, or parts thereof under conditions that allow transient expression of said first nucleic acid, thereby producing RLP.

Furthermore, a second nucleic acid comprising a second regulatory region active in the plant and operatively linked to a second nucleotide sequence encoding one or more rotavirus structural protein can be introduced into the plant or part of the plant, and wherein the second nucleic acid is expressed when the plant or part of the plant is grown in step b).

Furthermore, a third nucleic acid comprising a third regulatory region active in the plant and operatively linked to a third nucleotide sequence encoding one or more rotavirus structural protein can be introduced into the plant or part of the plant, and wherein the third nucleic acid is expressed when the plant or part of the plant is grown in step b).

Method (C) as described above may further comprise the steps of harvesting the plants and extracting the RLP.

The one or more rotavirus structural protein of method (C) may be rotavirus protein VP2, VP4 or VP 6. The one or more rotavirus structural protein encoded by the first or second nucleotide sequence may be VP 2or VP 6. The one or more rotavirus structural protein encoded by the third nucleotide sequence may be VP 4. VP4 can be cleaved to produce VP5 and VP 8.

The first, second or third nucleotide sequence may also encode, comprise, or encode and comprise one or more compartment targeting sequences and/or amplification elements. The one or more compartment targeting sequences direct one or more rotavirus structural proteins to the Endoplasmic Reticulum (ER), chloroplast, plastid, or apoplast of a plant cell. The compartment targeting sequence may encode an apoplast signal peptide or a plastid signal peptide.

The invention also provides a method (D) of producing a Rotavirus Like Particle (RLP), the method comprising:

a) providing a plant or part of a plant comprising a nucleic acid comprising a regulatory region active in the plant and operatively linked to a nucleotide sequence encoding one or more rotavirus structural protein,

b) growing the plant, portion of a plant, or plant cell under conditions that allow transient expression of the nucleic acid, thereby producing RLP.

Furthermore, the plant or plant part of method (D) may further comprise:

i) a second nucleic acid comprising a second regulatory region active in plants and operably linked to a second nucleotide sequence encoding one or more rotavirus structural protein,

ii) a second and a third nucleic acid, wherein the second nucleic acid comprises a second regulatory region active in plants and operatively linked to a second nucleotide sequence encoding one or more rotavirus structural protein and the third nucleic acid comprises a third regulatory region active in plants and operatively linked to a third nucleotide sequence encoding one or more rotavirus structural protein,

wherein the second nucleic acid, or the second and third nucleic acids, are expressed when the plant or part of a plant is grown in step b).

The one or more structural proteins in method (D) may be rotavirus protein VP2, VP4 or VP 6. The one or more rotavirus structural protein encoded by the first or second nucleotide sequence may be VP 2or VP 6. The one or more rotavirus structural protein encoded by the third nucleotide sequence may be VP 4. VP4 can be cleaved into VP5 and VP8 using proteases, e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin. The protease may be co-expressed in the plant or added during harvesting, purification, or both.

The present invention provides RLP produced by the method (A), the method (B), the method (C), the method (D), or a combination thereof as described above. The RLP may comprise one or more rotavirus structural proteins, which may comprise plant-specific N-glycans or modified N-glycans.

The present invention includes compositions comprising an effective amount of an RLP prepared by method (A), method (B), method (C), method (D), or a combination thereof, as just described, for inducing an immune response, and a pharmaceutically acceptable carrier.

The invention also includes a method of inducing immunity to rotavirus infection in a subject, the method comprising administering to the subject RLP as just described. RLP can be administered to a subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

The present invention also provides plant material comprising RLP produced by method (a), method (B), method (C), method (D), or a combination thereof, as described above. Plant material may be used to induce immunity to rotavirus infection in a subject. Plant materials can also be blended as food supplements.

In the methods described above (methods A, B, C or D), the plant or part of the plant may also be applied with another nucleic acid sequence encoding a suppressor of silencing or may also comprise another nucleic acid sequence encoding a suppressor of silencing.

Furthermore, the present invention provides a method (E) for producing a rotavirus structural protein in a plant, which comprises:

a) introducing into a plant or part of a plant a nucleic acid comprising a regulatory region active in a plant and operatively linked to a nucleotide sequence encoding one or more rotavirus structural protein;

b) incubating the plant or plant part under conditions that allow transient expression of the nucleic acid, thereby producing one or more rotavirus structural protein.

This summary is not intended to describe all features of the present invention.

The present invention relates to the following items:

item 1, a method of producing a Rotavirus Like Particle (RLP) in a plant, part of a plant, or plant cell, the method comprising:

a) introducing into the plant, part of a plant or plant cell a first nucleic acid comprising a first regulatory region active in the plant and operatively linked to a first nucleotide sequence encoding a first rotavirus structural protein, a second nucleic acid comprising a second regulatory region active in the plant and operatively linked to a second nucleotide sequence encoding a second rotavirus structural protein, and a third nucleic acid comprising a third regulatory region active in the plant and operatively linked to a third nucleotide sequence encoding a third rotavirus structural protein,

b) growing said plant, plant part, or plant cell under conditions that allow transient expression of said first, second, and third nucleic acids, thereby producing said RLP.

Item 2, the method according to item 1, wherein in step a) a fourth nucleic acid comprising a fourth regulatory region active in the plant and operatively linked to a fourth nucleotide sequence encoding a fourth rotavirus structural protein is introduced into the plant, part of a plant or plant cell, and the fourth nucleic acid is expressed when the plant, part of a plant or plant cell is grown in step b).

Item 3 the method of item 1, wherein the first rotavirus structural protein is VP2, the second rotavirus structural protein is VP6, and the third rotavirus structural protein is VP 4.

Item 4 the method of item 1, wherein the first rotavirus structural protein is VP2, the second rotavirus structural protein is VP6, and the third rotavirus structural protein is VP 7.

Item 5 the method of item 2, wherein the first rotavirus structural protein is VP2, the second rotavirus structural protein is VP6, the third rotavirus structural protein is VP4, and the fourth rotavirus structural protein is VP 7.

Item 6, the method of item 1or 2, wherein an additional nucleic acid is expressed in the plant, part of a plant or plant cell, and wherein the additional nucleic acid comprises a regulatory region active in the plant and operatively linked to a nucleotide sequence encoding a suppressor of silencing.

Item 7, the method of item 1or 2, wherein the codon usage of the nucleotide sequence is adjusted to a preferred human codon usage, an increased GC content, or a combination thereof.

Item 8, the method of item 1or 2, wherein the rotavirus structural protein comprises a truncated, native or non-native signal peptide.

Item 9, the method of item 8, wherein the non-native signal peptide is a protein disulfide isomerase signal (PDI) peptide.

Item 10, the method of item 1, wherein the first, second, or third nucleotide sequence, or a combination thereof, is operatively linked to a cowpea mosaic virus (CPMV) regulatory region.

Item 11, the method of item 2, wherein the first, second, third or fourth nucleotide sequence, or a combination thereof, is operatively linked to a cowpea mosaic virus (CPMV) regulatory region.

Item 12, the method of item 1or 2, further comprising the steps of:

c) harvesting said plant, part of a plant or plant cell, and

d) purifying said RLP from said plant, plant part or plant cell, wherein said RLP has a size in the range of 70-100 nm.

Item 13, the method of item 12, wherein the RLP is purified in the presence of calcium.

Item 14, an RLP produced by the method of item 1or 2, wherein the RLP comprises at least a VP4 rotavirus structural protein.

Item 15, an RLP produced by the method of item 3, wherein the RLP is a dual-layer RLP.

Item 16, the RLP produced by the method of item 4 or 5, wherein the RLP is a three-layered RLP.

Item 17, the method of item 1or 2, wherein the rotavirus structural protein is selected from rotavirus strain G9P [6], rotavirus a WA strain, rotavirus a vaccine USA/Rotarix-a41CB052A/1988/G1P1A [8] strain and rotavirus SA11 strain.

Item 18, the method of any one of items 3, 4 and 5, wherein the nucleotide sequence encoding VP2 comprises a nucleotide sequence identical to a sequence defined by SEQ ID NO: 13. SEQ ID NO: 14 or SEQ ID NO: 45 of the nucleotide sequence defined by seq id No. 80% to 100% identity.

Item 19, the method of any one of items 3, 4 and 5, wherein the nucleotide sequence encoding VP6 comprises a nucleotide sequence identical to a sequence defined by SEQ ID NO: 17. SEQ ID NO: 18 or SEQ ID NO: 46, or a nucleotide sequence defined by the formula (i) from 80% to 100% identity.

Item 20, the method according to any one of items 4 and 5, wherein the nucleotide sequence encoding VP7 comprises a nucleotide sequence identical to a sequence defined by seq id NO: 19. 20, 48, 49, 52, 53, 54 or 57, or a pharmaceutically acceptable salt thereof.

Item 21, the method according to any one of items 3 and 5, wherein the nucleotide sequence encoding VP4 comprises a nucleotide sequence identical to a sequence defined by seq id NO: 15. 16, 47, 50 or 51, or a nucleotide sequence defined by the amino acid sequence of seq id No. 80% to 100% identity.

Item 22, the method of any one of items 3, 4 and 5, wherein VP2 is encoded by an amino acid sequence comprising an amino acid sequence identical to SEQ ID NO: 1or SEQ ID NO: 25, or a pharmaceutically acceptable salt thereof, and from 80% to 100% identity of an amino acid sequence defined by seq id no.

Item 23, the method of any one of items 3, 4 and 5, wherein VP6 is encoded by an amino acid sequence comprising an amino acid sequence identical to SEQ ID NO: 3 or SEQ ID NO: 31 from 80% to 100% identity of the amino acid sequence defined.

Item 24, the method according to any one of items 4 and 5, wherein VP7 is encoded by an amino acid sequence comprising an amino acid sequence identical to SEQ ID NO: 4. 39, 43 or 59, or a pharmaceutically acceptable salt thereof.

Item 25, the method according to any one of items 3 and 5, wherein VP4 is encoded by an amino acid sequence comprising an amino acid sequence identical to SEQ ID NO: 2or SEQ ID NO: 36, and 80% to 100% identity of the amino acid sequence defined by 36.

Item 26, the method according to item 1, wherein the first, second or third nucleic acid sequence or combination thereof comprises a regulatory region active in the plant operatively linked to one or more comovirus enhancers, to one or more amplification elements, and to a nucleotide sequence encoding a rotavirus structural protein, and wherein a fourth nucleic acid encoding a replicase is introduced into the plant, portion of a plant or plant cell.

Item 27, the method according to item 2, wherein the first, second, third or fourth nucleic acid sequence or combination thereof comprises a regulatory region active in the plant operatively linked to one or more comovirus enhancers, to one or more amplification elements, and to a nucleotide sequence encoding a rotavirus structural protein, and wherein a fifth nucleic acid encoding a replicase is introduced into the plant, portion of a plant or plant cell.

Item 28, the method of item 1, wherein the first, second, or third nucleotide sequence, or a combination thereof, is further operatively linked to a compartment targeting sequence.

Item 29, the method of item 2, wherein the first, second, third, or fourth nucleotide sequence, or a combination thereof, is further operatively linked to a compartment targeting sequence.

Item 30, a method of producing a Rotavirus Like Particle (RLP) in a plant, portion of a plant, or plant cell, the method comprising:

a) introducing into a plant, part of a plant or plant cell a nucleic acid comprising a regulatory region active in said plant and operatively linked to a first nucleotide sequence encoding one or more rotavirus structural protein,

b) growing said plant, plant part, or plant cell under conditions that allow transient expression of said first nucleic acid, thereby producing said RLP.

Item 31, the method of item 30, wherein in step a) a second nucleic acid comprising a second regulatory region active in the plant and operatively linked to a second nucleotide sequence encoding one or more rotavirus structural protein is introduced into the plant, part of a plant or plant cell, and the second nucleic acid is expressed when the plant, part of a plant or plant cell is cultivated in step b).

Item 32, the method of item 31, wherein in step a) a third nucleic acid is introduced into the plant, part of a plant, or plant cell, said third nucleic acid comprising a third regulatory region active in the plant and operatively linked to a third nucleotide sequence encoding one or more rotavirus structural protein, and said third nucleic acid is expressed when the plant, part of a plant, or plant cell is grown in step b).

Item 33, the method of any one of items 30-32, wherein the one or more rotavirus structural protein is selected from the group comprising VP2, VP4, VP6 and VP 7.

Item 34, the method of any one of items 30 to 33, wherein an additional nucleotide sequence is expressed in the plant, part of a plant, or plant cell, the additional nucleotide sequence encoding a suppressor of silencing, and the additional nucleotide sequence is operably linked to a regulatory region active in the plant.

Item 35 the method of any one of items 30 to 34, wherein the one or more rotavirus structural protein is from rotavirus strain G9P [6 ].

Item 36, the method of any one of items 30-35, wherein the nucleotide sequence is further operatively linked to a compartment targeting sequence.

Item 37, the method of any one of items 28, 29 and 36, wherein the compartment targeting sequence directs the one or more rotavirus structural protein to the Endoplasmic Reticulum (ER), chloroplast, plastid or apoplast of the plant cell.

Item 38, the method of item 37, wherein the compartment targeting sequence encodes an apoplast signal peptide or a plastid signal peptide.

Item 39, the method of any of items 30-38, further comprising the steps of:

c) harvesting said plant, part of a plant or plant cell, and

Item 40, a method of producing a Rotavirus Like Particle (RLP) in a plant, portion of a plant, or plant cell, the method comprising:

a) providing a plant, part of a plant or plant cell comprising a nucleic acid comprising a regulatory region active in said plant and operatively linked to a nucleotide sequence encoding one or more rotavirus structural protein;

b) growing said plant, plant part, or plant cell under conditions that allow transient expression of said nucleic acid, thereby producing said RLP.

Item 41, a method of producing a Rotavirus Like Particle (RLP) in a plant, portion of a plant, or plant cell, the method comprising:

a) providing a plant, part of a plant, or plant cell comprising into the plant, part of a plant, or plant cell a first nucleic acid comprising a first regulatory region active in the plant and operatively linked to a first nucleotide sequence encoding a first rotavirus structural protein, a second nucleic acid comprising a second regulatory region active in the plant and operatively linked to a second nucleotide sequence encoding a second rotavirus structural protein, and a third nucleic acid comprising a third regulatory region comprising a third nucleotide sequence active in the plant and operatively linked to a third nucleotide sequence encoding a third rotavirus structural protein; and

Item 42, the method of item 41, wherein in step a) the plant, part of a plant or plant cell is provided with a fourth nucleic acid comprising a fourth regulatory region active in the plant and operatively linked to a fourth nucleotide sequence encoding a fourth rotavirus structural protein, and the fourth rotavirus structural protein is expressed when the plant, part of a plant or plant cell is cultivated in step b).

Item 43, the RLP produced by the method of any one of items 30 to 42, wherein the RLP comprises at least VP4 rotavirus structural protein.

Item 44, a composition comprising an effective dose of an RLP according to any one of items 14, 15, 16, and 43 for inducing an immune response in a subject, and a pharmaceutically acceptable carrier.

Item 45, a method of inducing immunity to a rotavirus infection in a subject comprising administering a composition of item 44.

Item 46 the method of item 45, wherein the composition is administered to the subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

Item 47, plant material comprising RLP produced by the method according to any one of items 1 to 13 and 17 to 42.

Item 48, a method of producing a rotavirus structural protein in a plant, portion of a plant or plant cell comprising:

a) introducing into said plant, part of a plant or plant cell a nucleic acid comprising a regulatory region active in said plant and operatively linked to a nucleotide sequence encoding one or more rotavirus structural protein;

b) incubating the plant, portion of a plant, or plant cell under conditions that allow transient expression of the nucleic acid, thereby producing one or more rotavirus structural protein.

Item 49 the method of item 48, wherein the nucleotide sequence is further operably linked to a compartment targeting sequence.

Item 50, the method of item 48, wherein the one or more rotavirus structural protein is VP2, VP4, VP6 or VP 7.

Drawings

These and other features of the present invention will become more apparent from the following description made with reference to the accompanying drawings, in which:

figure 1 shows rotavirus structure and gene-protein partitioning. (A) Transmission electron microscopy of rotavirus particles (scale bars indicate 100 nm). (B) The organization of viral capsid proteins, which includes the interior, the middle and the exterior. (C) A rotavirus dsRNA fragment arranged by size and function. The dsRNA (D) can be isolated by polyacrylamide gel electrophoresis. The protein in (C) is represented by the dsRNA fragment in (D). The pictures are from: crawford et al, 1997(A), SwissInstitute of Bioinformatics,2008(B) and Greenberg and Estes,2009 (D).

FIG. 2 shows cellular entry and replication of rotavirus. When rotavirus enters the cell, VP4 and VP7 are lost, thereby forming a double-layer particle (DLP). Transcription of the dsRNA begins, resulting in translation of VP2, VP4, VP6, and VP 7. A core of progeny that have replicase activity is produced in the virus factory (also known as the virus stock). Late-phase transcription occurs in these progeny cores. Around the virus factories (virus factories), these cores are coated with VP6, thus forming immature DLPs that bud and cross the endoplasmic reticulum membrane, obtaining a transient lipid membrane modified by ER-resident viral glycoproteins NSP4 and VP 7; these coated particles also contain VP 4. When the particles move towards the inside of the ER retention vesicles (cisternae), the transient lipid membrane and the nonstructural protein NSP4 are lost, while the viral surface proteins VP4 rearrange with VP7 to form the outermost viral protein layer, resulting in mature infectious trilayer particles (see Swiss Institute of biologics (ViralZone): ViralZone. expass. org/ViralZone/all _ by _ specs/107. html).

FIG. 3 shows Agrobacterium vectors pTRAc, pTRAkc-rbcs1-cTP and pTRAkc-ERH. P35SS, CaMV 35S promoter with repetitive transcriptional enhancer; CHS, the 5' untranslated region of chalcone synthase (chalcon synthase); pA35S, CaMV 35S polyadenylation signal; SAR, the backbone binding region of the tobacco Rb7 gene (scaffold attachment region); left and right borders of LB and RB, T-DNA integration; ColE1ori, origin of replication of escherichia coli (e.coli); RK2ori, the origin of replication of Agrobacterium; bla, ampicillin/carbenicillin resistant bla gene; LPH, signal peptide sequence from the heavy chain of murine mAb 24; his6, 6 × His tag sequence; SEKDEL, ER-retention signal sequence; rbcS1-cTP, chloroplast transit peptide sequence from Rubisco small subunit gene (rbcS1) of potato (Solanum tuberosum); npt II, kanamycin-resistant npt II gene; pnos and pAnos, the promoter and polyadenylation signal of nopaline synthase (nopaline synthase) gene (Maclean et al, 2007).

Figure 4 shows an overview of the rotavirus cloning and infiltration (infiltration) procedure.

FIG. 5 shows an apoplast protein extraction scheme. (A) Illustrative of the location of plant cells and apoplast. VP protein is expressed in the cytosol and targets the apoplast (red arrow). (B) After the time trial, plant leaves were vacuum infiltrated with PBS (1) and placed in a perforated spin column (2) and then centrifuged in a 2ml microcentrifuge tube (Eppendorf) to collect the juice (3).

FIG. 6 shows a Western blot analysis of rotavirus VP6 protein expression in the leaf cell compartment of plants over 7 days. A mouse anti-rotavirus VP6 antibody (1:5000) was used to probe the membrane. (+) and (-) denote expression with or without suppressor of silencing, respectively. The red line indicates the position of the VP6 protein (. about.40 kDa) in the various samples analyzed. The expression and extraction efficiency of VP6 is optimal in the cytoplasm.

Figure 7 shows Western blots demonstrating the respective expression of histidine-tagged rotavirus proteins in the cytoplasm of leaves of the present nicotiana benthamiana (n.benthamiana) plant on day 3. + ve-bacterially expressed rotavirus VP 2; m-molecular weight markers; VP-rotavirus capsid protein. Infiltration with VP7 resulted in yellowing of leaf (b).

Figure 8 shows the expression of rotavirus VP2(a) and VP4(b) targeting multiple leaf cell compartments of nicotiana benthamiana (n. The proteins were probed using chicken anti-rotavirus sera (1:2000) against VP2 and VP4, respectively. cTp-chlorophyll body; ER-endoplasmic reticulum; pTRAc-cytoplasm; a-apoplast; negative control (-ve) -plants infiltrated with suppressor silencing only; (a) positive control (+ ve) -bacteria-expressed VP2 in (b) -bacteria-expressed VP4 in (a); (-and +) with or without a suppressor of silencing; m-molecular weight markers. The arrows indicate the position of the protein bands of interest.

FIG. 9 shows a Western blot analysis of day 3 extracts of VP2/6/4 co-expressed in the cytoplasm of lamina of Nicotiana benthamiana (N.benthamiana). Proteins were probed with a mixture of chicken anti-rotavirus serum (anti-VP 2(1/5000) and anti-VP 4(1/5000)) and mouse anti-VP 6 antibody (1: 5000). Infiltration of recombinant Agrobacterium was accomplished with silencing suppressors. Negative control (-ve) -whole plant infiltrated with suppressor of silencing only; m-molecular weight markers.

Figure 10 shows an electron micrograph of day 3 cytoplasmic extracted rotavirus protein stained with uranyl acetate. (a) A negative protein sample extract with a suppressor of silencing; (b) VP6 protein extract; (c) VP2/6 protein extract and (d) VP2/6/4 protein extract. The scale bar is 200 nm. All RLP tested were between 70-100nm in diameter. (b) The arrow in (b) indicates the VP6 sheath/pad (sheath/mat). (C) Arrow in (a) represents an example of aRLP. All proteins are expressed in the presence of a suppressor of silencing. All proteins were captured with mouse anti-VP 6 antibody (1: 2000).

FIG. 11 shows sucrose gradient purification of co-expressed VP2/6 and VP2/6/4 (a). Dot blots of sucrose gradient purified VP2/6(b) and VP2/6/4 (c). The protein extract was loaded onto a sucrose gradient (10-60%) and ultracentrifuged. Fractions were analyzed by detection with (b) mouse anti-VP 6 antibody (1:5000) and (c) chicken anti-VP 2 and VP4 serum (1: 5000).

FIG. 12 shows a Western blot analysis (a) of the fraction VP2/6, a photograph of a Coomassie stained gel on SDS-PAGE of fractions 16 and 17 of fraction VP2/6(b), and a Western blot analysis of fractions 16 and 17 (C). Mouse anti-VP 6(1:5000) and chicken anti-VP 2 serum (1:5000) were used in Western blot (a) and mouse anti-VP 6 alone (1:5000) was used in (c). (a) And negative control (-ve) -bacteria-expressed VP4 in (c), negative control (-ve) -plants impregnated with sirtuins and purified with sucrose gradients in (b); crude-unpurified VP2/6 extract; (a) the positive control (+ ve) -bacteria-expressed VP2 of (a), (b) and the positive control (+ ve) -plant-expressed VP6 of (c); VP6-SF 9-known concentrations of VP6 protein expressed in SF9 insect cells. The arrows indicate the protein bands in question.

FIG. 13 shows the determination of total soluble protein in the fractions of VP2/6 purified by sucrose density gradient. (a) IgG standard curve, (b) -absorbance readings of fractions read at 750 nm. Points of interest are: fractions 16 to 19.

FIG. 14 shows a sucrose density gradient analysis of cytoplasmic co-expressed VP2/6/4 fractions. The raw absorbance readings were read at 750nm to verify the protein peak previously detected on the spot blot of VP 2/6/4.

FIG. 15 shows a transmission electron micrograph of sucrose density gradient purified VP2/6 particles. (a) And (b) both show two different cross-sections observed on the copper mesh. All RLPs detected have a diameter between 70-100 nm. The sample was captured with a mouse anti-VP 6 antibody (1: 2000). The scale bars represent 200 nm.

FIGS. 16A-1 through 16A-6 show the amino acid sequence (SEQ ID NO: 1) and nucleotide sequence (SEQ ID NO: 13 and 14) of rotavirus VP 2. FIGS. 16B-1 through 16B-5 show the amino acid sequence (SEQ ID NO: 2) and nucleotide sequence (SEQ ID NO: 15 and 16) of rotavirus VP 4. FIGS. 16C-1 to 16C-3 show the amino acid sequence (SEQ ID NO: 3) and nucleotide sequence (SEQ ID NO: 17 and 18) of rotavirus VP 6. FIGS. 16D-1 to 16D-3 show the amino acid sequence (SEQ ID NO: 4) and nucleotide sequence (SEQ ID NO: 19 and 20) of rotavirus VP 7.

FIG. 17A shows the nucleotide sequence of primer IF-WA _ VP2(opt). s1+3c (SEQ ID NO: 21). FIG. 17B shows the nucleotide sequence of primer IF-WA _ VP2(opt). s1-4r (SEQ ID NO: 22). Figure 17C shows a schematic representation of construct 1191. The SacII and StuI restriction enzyme sites used for plasmid linearization are annotated on the schematic.

FIG. 18 shows the nucleotide sequence of construct 1191 from the left t-DNA border to the right t-DNA border (underlined) (SEQ ID NO: 23). 2X35S/CPMV-HT/NOS with plastocyanin-P19-plastocyanin suppressor expression cassette.

FIG. 19 shows the nucleotide sequence (SEQ ID NO: 45) encoding VP2(opt) from rotavirus A WA strain.

FIG. 20 shows the amino acid sequence of VP2 from rotavirus A WA strain (SEQ ID NO: 25).

Figure 21 shows a schematic of construct No. 1710.

Fig. 22A shows a schematic of construct 193. The SacII and StuI restriction enzyme sites used for plasmid linearization are annotated on the schematic. FIG. 22B shows the nucleotide sequence of construct 193 (SEQ ID NO: 26). Construct 193 is shown from the left t-DNA border to the right t-DNA border (underlined). 2X35S/CPMV-HT/NOS entered the BeYDV (m) + replicase amplification System in a manner to carry the plastocyanin-P19-plastocyanin silencer expression cassette.

FIG. 23 shows the nucleotide sequence of expression cassette 1710 (SEQ ID NO: 27). Expression cassette No. 1710 is shown from the 2X35S promoter to the NOS terminator. VP2(opt) from rotavirus A WA strain is underlined.

Figure 24 shows a schematic representation of construct 1711.

FIG. 25A shows the nucleotide sequence (SEQ ID NO: 28) of primer IF-WA _ VP6(opt). s1+3 c. FIG. 25B shows the nucleotide sequence of primer IF-WA _ VP6(opt). s1-4r (SEQ ID NO: 29). FIG. 25c shows expression cassette 1713 (SEQ ID NO: 30) from the 2X35S promoter to the NOS terminator. VP6(opt) from rotavirus A WA strain is underlined. FIG. 25d shows the nucleotide sequence (SEQ ID NO: 46) encoding VP6(opt) from rotavirus A WA strain.

FIG. 26 shows the amino acid sequence of VP6 from rotavirus A WA strain (SEQ ID NO: 31).

Figure 27 shows a schematic of construct 1713.

FIG. 28 shows the nucleotide sequence from the 2X35S promoter to the NOS terminator of expression cassette No. 1714 (SEQ ID NO: 32). VP6(opt) from rotavirus A WA strain is underlined.

Figure 29 shows a schematic representation of construct 1714.

FIG. 30A shows the nucleotide sequence (SEQ ID NO: 33) of the primer IF-Rtx _ VP4(opt). s1+3 c. FIG. 30B shows the nucleotide sequence (SEQ ID NO: 34) of primer IF-Rtx _ VP4(opt). s1-4 r.

FIG. 31A shows the nucleotide sequence from the 2X35S promoter to the NOS terminator of expression cassette No. 1731 (SEQ ID NO: 35). VP4(opt) from rotavirus A Rotarix strain is underlined. FIG. 31B shows the optimized coding sequence (SEQ ID NO: 47) of rotavirus A VP4 from RVA/Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A [8 ]. FIG. 31C shows the nucleotide sequence of expression cassette 1730 from the 2X35S promoter to the NOS terminator (SEQ ID NO: 44). VP4(opt) from rotavirus A Rotarix strain is underlined.

FIG. 32 shows the amino acid sequence of VP4 from rotavirus A Rotarix strain (SEQ ID NO: 36).

FIG. 33A shows a schematic representation of construct No. 1730. FIG. 33B shows a schematic representation of construct No. 1731.

FIG. 34A shows the nucleotide sequence (SEQ ID NO: 37) of primer IF-Rtx _ VP7(opt). s1+3 c. FIG. 34B shows the nucleotide sequence of primer IF-Rtx _ VP7(opt). s1-4r (SEQ ID NO: 38). FIG. 34C shows the nucleotide sequence of expression cassette No. 1733 from the 2X35S promoter to the NOS terminator. VP7 from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain is underlined (SEQ ID NO: 24). FIG. 34D shows the nucleotide sequence (SEQ ID NO: 48) encoding VP7 from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8 ]. FIG. 34E shows the optimized coding sequence (SEQ ID NO: 54) of rotavirus A VP7 from RVA/Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A [8 ].

FIG. 35 shows the amino acid sequence of VP7(SEQ ID NO: 39) from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain.

FIG. 36 shows a schematic representation of construct No. 1733.

FIG. 37 shows the nucleotide sequence (SEQ ID NO: 40) of the primer IF-Rtx _ VP7(opt). s2+4 c.

Figure 38 shows a schematic representation of construct 1192. The SacII and StuI restriction enzyme sites used for plasmid linearization are annotated on the schematic.

FIG. 39 shows the nucleotide sequence of construct 1192 from the left t-DNA border to the right t-DNA border (underlined) (SEQ ID NO: 41). 2X35S/CPMV-HT/PDISP/NOS with plastocyanin-P19-plastocyanin suppressor expression cassette is shown.

FIG. 40A shows the nucleotide sequence from the 2X35S promoter to the NOS terminator of expression cassette 1735 (SEQ ID NO: 42). PDISP/VP7(opt) from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain is indicated by underlining. FIG. 40B shows the nucleotide sequence (SEQ ID NO: 49) encoding PDISP/VP7(opt) from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain.

FIG. 41 shows the amino acid sequence of PDISP/VP 7(SEQ ID NO: 43) from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8 ].

FIG. 42 shows a schematic representation of construct No. 1735.

FIG. 43A shows the coding sequence of rotavirus AVP 4(SEQ ID NO: 50) from RVA/Simian-tc/ZAF/SA11-H96/1958/G3P5B [2] strain. FIG. 43B shows the optimized coding sequence (SEQ ID NO: 51) of rotavirus A VP4 from RVA/Simian-tc/ZAF/SA11-H96/1958/G3P5B [2] strain. FIG. 43C shows the coding sequence of rotavirus A VP7(SEQ ID NO: 52) from RVA/Simian-tc/ZAF/SA11-H96/1958/G3P5B [2] strain. FIG. 43D shows the optimized coding sequence (SEQ ID NO: 53) of rotavirus AVP7 from RVA/Simian-tc/ZAF/SA11-H96/1958/G3P5B [2] strain.

FIG. 44A shows the nucleotide sequence (SEQ ID NO: 55) of primer IF-TrSP + Rtx _ VP7(opt). s1+3 c. FIG. 44B shows the nucleotide sequence (SEQ ID NO: 56) of primer IF-Rtx _ VP7(opt). s1-4 r. FIG. 44C shows the nucleotide sequence of the optimized coding sequence of rotavirus A VP7 from RVA/Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A [8] (SEQ ID NO: 57). FIG. 44D shows the nucleotide sequence of expression cassette 1734 from the 2X35S promoter to the NOS terminator (SEQ ID NO: 58). VP7 from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain is underlined. FIG. 44E shows the amino acid sequence of TrSp-VP7 from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] (SEQ ID NO: 59). FIG. 44F shows a schematic representation of construct No. 1734.

Figure 45 shows the purification of rotavirus like particles comprising VP2 and VP6 by iodixanol (iodixanol) density gradient centrifugation. FIG. 45A is a Coomassie stained SDS-PAGE analysis of the load before centrifugation and fractions 1 to 10 (fraction 1 at the bottom of the tube). Arrows show the location of rotavirus antigens. FIG. 45B is a Western blot analysis of the same fractions as in (A) using rabbit polyclonal anti-rotavirus antibody. FIG. 45C is a Western blot analysis of the same fractions as in (A) using rabbit polyclonal anti-VP 2 antibody.

Figure 46 shows purification of rotavirus like particles comprising VP2, VP6 and VP7 by iodixanol density gradient centrifugation. FIG. 46A is a Coomassie stained SDS-PAGE analysis of the load before centrifugation and fractions 1 to 10 (fraction 1 at the bottom of the tube). Arrows show the location of rotavirus antigens. FIG. 46B is the same Western blot analysis using rabbit polyclonal anti-rotavirus antibody as fraction (A). FIG. 46C is a Western blot analysis using rabbit polyclonal anti-VP 7 antibody identical to the fractions in (A).

Figure 47 shows purification of rotavirus particles comprising VP2, VP4, VP6 and VP7 by iodixanol density gradient centrifugation. FIG. 47A is a Coomassie stained SDS-PAGE analysis of the load before centrifugation and fractions 1 to 10 (fraction 1 at the bottom of the tube). Arrows show the location of rotavirus antigens. FIG. 47B is a Western blot analysis of the same fractions as in (A) using rabbit polyclonal anti-rotavirus antibody. FIG. 47C is a Western blot analysis of the same fractions as in (A) using rabbit polyclonal anti-VP 7 antibody.

Figure 48 shows the assessment of VP4 content in purified rotavirus particles comprising VP2, VP4, VP6 and VP7 by anti-VP 4 specific ELISA.

Figure 49 shows cryoelectron microscopy images of purified rotavirus-like particles comprising VP2 and VP6 (left hand portion) and VP2, VP4, VP6 and VP7 (right hand portion).

Detailed Description

The following is a description of preferred embodiments.

The present invention relates to a rotavirus like particle (VLP) comprising one or more rotavirus structural proteins (i.e. rotavirus like particle, rotavirus VLP or RLP) and a method of producing a Rotavirus Like Particle (RLP) in a plant. Thus, a Rotavirus Like Particle (RLP) may comprise one or more rotavirus structural proteins. RLP may be bi-layered or tri-layered.

The present invention provides, in part, methods for producing Rotavirus Like Particles (RLP) in plants. The method may comprise introducing into a plant or part of a plant one or more nucleic acids comprising a regulatory region active in the plant and operatively linked to a nucleotide sequence encoding one or more rotavirus structural protein. This is followed by growing the plant or plant part under conditions which allow transient expression of the nucleic acid, thereby producing RLP.

In addition, the present invention provides, in part, methods of producing rotavirus particle (RLP) -like vaccine candidates in plants. The method may include: introducing into the plant, part of a plant, or plant cell a first nucleic acid comprising a first regulatory region active in the plant and operatively linked to a first nucleotide sequence encoding a first rotavirus structural protein, a second nucleic acid comprising a second regulatory region active in the plant and operatively linked to a second nucleotide sequence encoding a second rotavirus structural protein, and a third nucleic acid comprising a third regulatory region active in the plant and operatively linked to a third nucleotide sequence encoding a third rotavirus structural protein. This is followed by growing the plant, plant part or plant cell under conditions that allow transient expression of the first, second and third nucleic acids, thereby producing RLP. The RLP may be single-layered, double-layered, or triple-layered.

"rotavirus structural protein" may mean all or part of the rotavirus structural protein sequence isolated from a rotavirus, which is present in any natural or variant rotavirus strain or isolate. Thus, the term rotavirus structural protein or the like includes variants of the native rotavirus structural protein sequence produced by mutation during the viral life cycle or in response to selective stress (e.g., pharmacotherapy, expansion of host cell tropism or infectivity, etc.). Recombinant techniques can also be used to produce these rotavirus structural protein sequences and variants thereof, as will be appreciated by those skilled in the art.

In addition, structural proteins may include capsid proteins (e.g., VP2 and VP6) and/or surface proteins (e.g., VP 4). The structural protein may also include, for example, VP 7.

Non-limiting examples of rotavirus structural proteins are the rotavirus proteins VP2, VP4, VP6 and VP7, and fragments of VP2, VP4, VP6 and VP 7. Non-limiting examples of fragments of VP2, VP4, VP6 and VP7, or VP2, VP4, VP6 and VP7 proteins that can be used according to the present invention include those VP2, VP4, VP6 and VP7 proteins from rotavirus strain G9P [6], rotavirus A WA strain, rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain and rotavirus SA11 strain.

An example of VP2 structural protein is shown in SEQ ID NO: 1 and SEQ ID NO: 25, but this should not be considered as limiting. In addition, VP2 structural proteins may include SEQ ID NO: 1. SEQ ID NO: 25, or sequences having at least about 90-100% sequence similarity thereto (including any percent similarity within these ranges, such as sequence similarities of 91, 92, 93, 94, 95, 96, 97, 98, 99% thereto). In addition, the VP2 structural protein may be represented by SEQ ID NO: 13. 14, 25 or 45, or by a sequence having at least about 80-100% sequence similarity thereto (including any percent similarity within these ranges, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto).

An example of VP4 structural protein is shown in SEQ ID NO: 2 and SEQ ID NO: 36, but this should not be considered as limiting. In addition, VP4 structural proteins may include SEQ ID NO: 2. SEQ ID NO: 36, or sequences having at least about 90-100% sequence similarity thereto (including any percent similarity within these ranges, such as sequence similarities of 91, 92, 93, 94, 95, 96, 97, 98, 99% thereto). In addition, the VP4 structural protein may be represented by SEQ ID NO: 15. 16, 47, 50, or 51, or by a sequence having at least about 80-100% sequence similarity thereto (including any percent similarity within these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto).

An example of VP6 structural protein is shown in SEQ ID NO: 3 and SEQ ID NO: 31, but this should not be considered as limiting. In addition, VP6 structural proteins may include SEQ ID NO: 3. SEQ ID NO: 31, or sequences having at least about 90-100% sequence similarity thereto (including any percent similarity within these ranges, such as sequence similarities of 91, 92, 93, 94, 95, 96, 97, 98, 99% thereto). In addition, the VP6 structural protein may be represented by SEQ ID NO: 17. 18 or 46, or by a sequence having at least about 80-100% sequence similarity thereto (including any percent similarity within these ranges, e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto).

An example of VP7 structural protein is shown in SEQ ID NO: 4 and SEQ ID NO: 39, but this should not be considered as limiting. In addition, VP7 structural proteins may include SEQ ID NO: 4. SEQ ID NO: 39 and SEQ ID NO: 43, or sequences having at least about 90-100% sequence similarity thereto (including any percent similarity within these ranges, e.g., having 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto). In addition, the VP7 structural protein may be represented by SEQ ID NO: 19. 20, 48, 49, 52, 53, or 54, or by a sequence having at least about 80-100% sequence similarity thereto (including any percent similarity within these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto).

Amino acid sequence similarity or identity can be calculated by using the BLASTP and TBLASTN programs, which employ the BLAST (basic local alignment search tool) 2.0 algorithm. Techniques for calculating amino acid sequence similarity or identity are well known to those skilled in the art, and the use of the BLAST algorithm is described in ALTSCHOL et al (1990, J mol. biol.215:403-410) and ALTSCHOL et al (1997, Nucleic Acids Res. 25: 3389-3402).

The term "viroid particle (VLP)" or "VLP" refers to a structure that self-assembles and comprises one or more structural proteins (such as, for example, rotavirus structural proteins, such as, but not limited to, VP2, VP4, VP6, and/or VP7 structural proteins). VLPs comprising rotavirus structural proteins may also be referred to as "rotavirus VLPs", "rotavirus-like particles (RVLP)", "rotavirus-like particles (RLP)", "rotavirus-like particles", "RVLP", or "RLP". VLPs or RLP are generally morphologically and antigenically similar to the virions produced in infection, but lack sufficient genetic information to replicate and are thus non-infectious. VLPs may be produced in suitable host cells, including plant host cells. After extraction from the host cell and isolation and further purification under suitable conditions, the VLPs can be purified to intact structures. The RLP may be a single layer, a double layer, or a triple layer RLP. The monolayer of RLP can be obtained by expressing a rotavirus structural protein (e.g., VP 2or VP 6). Bilayer RLP can be obtained by expressing two rotavirus structural proteins (e.g., by co-expressing both VP2 and VP6, for example), which can be performed with or without VP 4. Triple-layered RLP can be obtained by simultaneous expression of at least three rotavirus structural proteins (e.g., co-expression of VP2, VP6, and VP7), which can be performed with or without VP 4. Co-expression of VP4 resulted in spiked particles, which were similar to native rotavirus. VP4 can be processed or cleaved to produce VP5 and VP 8. Such processing can occur within the host using endogenous proteases or by co-expression of suitable proteases (e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin). Alternatively, VP4 may be processed during any step of the RLP extraction scheme or after RLP purification by addition of a suitable protease (e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin) to produce VP5 and VP 8.

Each rotavirus structural protein has different properties and sizes, and the amounts required to assemble the RLP vary. The terms "rotavirus VLP", "rotavirus-like virus particle (RVLP)", "rotavirus-like virus particle (RLP)", "rotavirus-like virus particle", "RVLP" or "RLP" refer to a virus-like particle (VLP) comprising one or more rotavirus structural proteins. Examples of rotavirus structural proteins may include, but are not limited to, VP2, VP4 (or VP5 and VP8), VP6, and VP7 structural proteins.

The present invention also provides methods for producing RLP in plants, wherein a first nucleic acid (first nucleic acid) encoding a first rotavirus structural protein (e.g., VP 2or VP6 protein) is co-expressed with a second nucleic acid encoding a second rotavirus structural protein (e.g., VP6 or VP2 protein). In addition, a third nucleic acid encoding a third rotavirus structural protein (e.g., VP4 or VP7) can be co-expressed with the first and second nucleic acids, thereby co-expressing the first, second, and third nucleic acids in a plant. The first nucleic acid, the second nucleic acid, and the third nucleic acid may be introduced into the plant in the same step, or may be introduced into the plant sequentially. By co-expressing a nucleic acid encoding a suitable protease (e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin), VP4 may be processed or cleaved in a host to produce VP5 and VP 8. Alternatively, VP4 may be processed during any step of RLP extraction or after RLP purification by adding a satisfiable (sapiable) protease (e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin).

In addition, a plant expressing the first nucleic acid (encoding the first rotavirus structural protein), the second nucleic acid (encoding the second rotavirus structural protein), and the third nucleic acid (encoding the third rotavirus structural protein) can be further transformed with a fourth nucleic acid encoding a fourth rotavirus structural protein (e.g., VP7 or VP4 protein) such that the first, second, third, and fourth nucleic acids are co-expressed in the plant. By co-expressing a nucleic acid encoding a suitable protease (e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin), VP4 may be processed or cleaved in a host to produce VP5 and VP 8. Alternatively, VP4 may be processed during any step of RLP extraction or after RLP purification by adding a satisfiable protease (e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin).

Furthermore, a first plant expressing a first nucleic acid encoding one or more rotavirus structural protein (e.g., VP 2or VP6 protein) can be crossed with a second plant expressing a second nucleic acid encoding one or more rotavirus structural protein (e.g., without limitation, VP6 or VP2 protein) to produce progeny plants (third plants) co-expressing the first and second nucleic acids encoding VP2 and VP6, respectively, or VP6 and VP2, respectively. In addition, third plants expressing first and second nucleic acids encoding VP2 and VP6, respectively, or VP6 and VP2, respectively, can be crossed with fourth plants expressing third nucleic acids encoding one or more rotavirus structural proteins (such as, but not limited to, VP4 or VP7) to produce further progeny plants (fifth plants) co-expressing first, second and third nucleic acids encoding VP2, VP6, and VP4 or VP7, respectively. VP4 can be processed or cleaved in plants to produce VP5 and VP8 using a host protease, or by co-expressing a nucleic acid encoding a suitable protease (e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin) in one of the first, second, third or fourth plants. Alternatively, VP4 may be processed during any step of RLP extraction or after RLP purification by adding a satisfiable protease (e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin).

As described in more detail below, RLP can be produced in plants by expressing a nucleic acid (a first nucleic acid) encoding one or more rotavirus structural proteins (such as, but not limited to, VP2, VP6, or VP 7). A second nucleic acid encoding a second rotavirus structural protein (such as, but not limited to, VP7, VP6, or VP2) can be co-expressed in a plant. In addition, a third nucleic acid encoding a third rotavirus structural protein (such as, but not limited to, VP6, VP7, or VP2) can be co-expressed in plants. The nucleic acid, the second nucleic acid and the third nucleic acid may be introduced into the plant in the same step, or they may be introduced into the plant sequentially. The nucleic acid, the second nucleic acid and the third nucleic acid may be introduced into the plant in a transient manner or in a stable manner.

Furthermore, a plant expressing a first nucleic acid encoding a first rotavirus structural protein (e.g., VP2 protein) can be transformed with a second nucleic acid encoding a second rotavirus structural protein (e.g., without limitation, VP6 or VP7) such that both the first nucleic acid and the second nucleic acid are co-expressed in the plant. The plant may be further transformed with a third nucleic acid encoding a third rotavirus structural protein (such as, but not limited to, VP7 or VP 6).

Alternatively, plants expressing VP6 or VP7 protein (second nucleic acid) may be transformed with a first nucleic acid encoding VP2 protein, such that both the first nucleic acid and the second nucleic acid are co-expressed in the plant. The plant may be further transformed with a third nucleic acid encoding a third rotavirus structural protein (such as, but not limited to, VP7 or VP 6).

Additionally, plants expressing first and second nucleic acids encoding first and second rotavirus structural proteins (e.g., VP2 and VP6 proteins) can be transformed with a third nucleic acid encoding a third rotavirus structural protein (e.g., VP4 or VP 7). By co-expressing nucleic acids encoding suitable proteases (e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin), VP4 can be processed or cleaved to produce VP5 and VP 8. Alternatively, VP4 may be processed during any step of RLP extraction or after RLP purification by addition of a satisfiable protease (e.g., trypsin-like protease, serine protease, chymotrypsin-like protease, subtilisin).

The present invention also provides a method of producing RLP in a plant, the method comprising introducing into a plant, part of a plant, or plant cell one or more nucleic acids encoding one or more rotavirus structural protein operably linked to a regulatory region active in a plant, and one or more compartment targeting sequences and/or amplification elements. The plant, part of a plant, or plant cell is then cultured under conditions that allow expression of the one or more nucleic acids, thereby producing RLP. The one or more rotavirus structural proteins can be fragments of VP2, VP4 (or VP5 and VP8), VP6, VP7, VP2, VP4 (or VP5 and VP8), VP6, VP7, or combinations thereof.

The present invention also provides RLP comprising one or more rotavirus structural proteins such as, but not limited to, VP2, VP4 (or VP5 and VP8), VP6, VP7, or a combination thereof. RLP may be generated by one or more methods as provided by the present invention.

The presence of RLP may be detected using any suitable method, such as density gradient centrifugation or size exclusion chromatography. The structure and size of the RLP can be assessed, for example, by electron microscopy or by size exclusion chromatography.

For size exclusion chromatography, total soluble protein can be extracted from plant tissue by homogenizing a sample of freeze-squeezed plant material in extraction buffer (Polytron), and removing insoluble material by centrifugation. Precipitation with ice cold acetone or PEG may also be beneficial. Quantifying the soluble protein and passing the extract through Sephacryl^TMColumns, e.g. Sephacryl^TMAnd (S500) column. Blue Dextran 2000 can be used as a calibration standard. After chromatography, the fractions may be further analyzed by immunoblotting to determine the protein complement of the fractions.

For example, the separated fraction may be a supernatant (if by centrifugation, sedimentation or precipitation) or a filtrate (if by filtration) and enriched with proteins or suprastructure proteins, such as, for example, nanotubes, nanospheres, or higher order, higher molecular weight particles, such as monolayer (sl), bilayer (dl), or trilayer (tl) RLP.

The separated fractions may be further processed by, for example, additional centrifugation steps, precipitation, chromatography steps (e.g., size exclusion chromatography, ion exchange chromatography, affinity chromatography), tangential flow filtration, or combinations thereof, to separate, purify, concentrate, or combinations thereof, proteins, suprastructure proteins, or higher order particles. The presence of purified proteins, suprastructure proteins or higher order particles (such as RLP) can be confirmed by, for example, native or SDS-PAGE, immunoassay using an appropriate detection antibody, capillary electrophoresis, electron microscopy, or any other method apparent to one skilled in the art.

The RLP produced according to the invention may be purified or partially purified from plants, parts of plants, or plant material, or it may be administered as an oral vaccine using methods known to those skilled in the art.

RLP purification can include gradient centrifugation, e.g., sucrose, iodixanol, OptiPrep^TMOr cesium chloride (CsCl) density gradients can be used to purify or partially purify RLP from the biomass of transformed plants. As shown, for example, in fig. 45, an iodixanol step gradient or iodixanol continuous gradient may be used to purify RLP and/or expressed rotavirus structural protein.

Calcium (Ca2+) concentration has been shown to be important for the conversion of triple-layer particles (TLP) to double-layer particles (DLP) and is strain dependent (see, e.g., Martin et al, Journal of Virology, Jan 2002, which is incorporated herein by reference). Complete loss of outer capsid proteins from TLP (TLP decapping) is at [ Ca²⁺]Occurs in the nanomolar range. Thus, purification and/or extraction of RLP may be performed in the presence of calcium and a gradient centrifugation step may be performed in the presence of calcium, e.g., in the presence of CaCl₂Is performed in the case of (1). For example, the concentration of CaCl2 may be between 1mM and 1000mM, or may be any amount therebetween, e.g., 2,3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 2mM5. 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 50, 600, 650, 700, 750, 800, 850, 900, 950mM or any amount therebetween.

Minimal processing can be performed on the plant or plant pieces. The term "minimally processed" refers to plant material, e.g., a plant or portion thereof, containing the protein of interest and/or RLP that is partially purified to obtain a plant extract, homogenate, plant homogenate fraction, or the like (i.e., minimally processed). Partial purification may include, but is not limited to, disrupting the plant cell structure, thereby producing a composition comprising soluble plant components, as well as insoluble plant components (which may be separated by, for example, but not limited to, centrifugation, filtration, or a combination thereof). In this regard, proteins secreted in the intercellular spaces of the leaves or other tissues can be readily obtained using vacuum or centrifugal extraction, or the tissues can be extracted under pressure, by tumbling or grinding or the like, to squeeze or release the proteins from the intercellular spaces. Minimal processing may also include the preparation of crude extracts of soluble proteins, as these preparations may have negligible contamination from secondary plant products. In addition, minimal processing may include aqueous extraction of soluble proteins from the leaves followed by precipitation with any suitable salt. Other methods may include large scale maceration and juice extraction to enable the extract to be used directly. Any suitable method (e.g., mechanical extraction or biochemical extraction) may be used to purify or extract RLP.

One or more rotavirus structural proteins can be synthesized in amounts up to 2g per kilogram fresh weight of the plant. For example, the amount of synthetic structural protein may be between 1 and 2g per kg fresh weight of the plant, or any amount therebetween, such as 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2g per kg fresh weight, or any amount therebetween. For example, structural proteins can be synthesized in amounts of up to 1.54g per kg fresh weight of plant.

Furthermore, RLP can be synthesized in amounts of up to 1.5g per kg of fresh weight of the plant. For example, the amount of RLP synthesized may be between 0.5 and 1.5g, or any amount therebetween, per kg of fresh weight of the plant, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5g per kg of fresh weight of the plant. For example, RLP can be synthesized in amounts of up to 1.1g per kg of fresh plant weight.

The size (i.e., diameter) of the RLP defined above, which is typically between 50 and 110nm, or any size therebetween, can be measured by, for example, Dynamic Light Scattering (DLS) or Electron Microscopy (EM) techniques. For example, the size of the complete RLP structure can be in the range of about 70nm to about 110nm, or any size in between, e.g., 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, or any size in between.

The invention also provides nucleic acids comprising a nucleotide sequence encoding one or more rotavirus structural protein operably linked to a regulatory region active in plants. The nucleotide sequence may be optimized for, e.g., human codon usage or plant codon usage. In addition, one or more rotavirus structural protein can be operably linked to one or more amplification element. In addition, one or more rotavirus structural protein can be operably linked to one or more compartment targeting sequences. The one or more rotavirus structural proteins encoded by the nucleotide sequence may be, for example, VP2, VP4, VP6 or VP 7. Furthermore, the one or more rotavirus structural proteins encoded by said nucleotide sequence may be from, for example, any rotavirus of group a to group G, but more preferably from group a. Furthermore, one or more rotavirus structural proteins encoded by said nucleotide sequence may be from any rotavirus strain having a genotype of any combination of G-and P-types from G1 to G27 and from P1 to P34 (more preferably from G1 to G19 and from P1 to P27), including but not limited to G1P [8], G2P [4], G2P [8], G3P [8], G4P [8], G9P [6], G9P [8], rotavirus WA a strain, rotavirus a vaccine USA/Rotarix-a41CB052A/1988/G1P1A [8] or rotavirus SA11 strain.

For nucleic acid sequences as referred to in the present invention, the nucleic acid sequence may be "substantially homologous", "substantially similar" or "substantially identical" to one or more nucleotide sequences as defined herein or the complement of a nucleotide sequence, if the nucleic acid sequence hybridizes under stringent hybridization conditions with said sequence or with the complement of said sequence. For a plurality of sequences, when at least about 70%, or between 70% and 100%, or any amount therebetween (e.g., 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100%, or any amount therebetween) of nucleotides match over a given length of the nucleotide sequence, the sequences are "substantially homologous," "substantially similar," or "substantially identical," provided that the homologous sequences exhibit one or more properties of the sequence or of the encoded product as described herein.

For example, the invention provides isolated polynucleotides comprising nucleic acids encoding one or more rotavirus structural protein that differs from a nucleotide sequence such as SEQ ID NO: 13. 14, 15, 16,17, 18, 19, 20, 45, 46, 47, 49, 50, 51, 52, 53, 54 has at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any amount therebetween. The polynucleotide may be a polynucleotide that has been codon optimized by any method known in the art.

Furthermore, the present invention provides RLP comprising a rotavirus structural protein encoded, for example, by a nucleic acid which hybridizes with, for example, SEQ ID NO: 13. 14, 15, 16,17, 18, 19, 20, 45, 46, 47, 49, 50, 51, 52, 53, 54 has at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any amount therebetween of identity.

Nucleotide sequence comparison programs can be used to determine the sequence similarity or identity as provided within DNASIS (using parameters such as, but not limited to, GAP penalty of 5, # top diagonal of 5, fixed GAP penalty of 10, k-tuple of 2, floating GAP of 10, and window size of 5). However, other methods of alignment of sequences for comparison are known in the art, such as the algorithm of Smith & Waterman (1981, adv. Appl. Math.2:482), the algorithm of Needleman & Wunsch (J.mol. biol.48:443,1970), the algorithm of Pearson & Lipman (1988, Proc. Nat' l. Acad. Sci. USA 85:2444) and the computerized implementation of these algorithms (GAP, BESTFIT, FASTA and BLAST, available through NIH), or by Manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology, Ausubel et al eds. 1995 support), or by Southern or Northern hybridization under stringent conditions (see, Southern or Northern hybridization methods described by Man et al in cloning (A Laboratory), Harboury, 1982). Preferably, substantially homologous sequences exhibit a sequence similarity of at least about 80%, and most preferably at least about 90%, over a given length of the molecule.

An example of one such stringent hybridization condition may be hybridization in 4 XSSC at 65 ℃ overnight (about 16-20 hours), followed by a wash at 0.1 XSSC for 1 hour at 65 ℃, or two washes at 0.1 XSSC for 20 or 30 minutes at 65 ℃. Alternatively, an exemplary stringent hybridization condition may be hybridization in 50% formamide, 4 XSSC at 42 ℃ overnight (16-20 hours), followed by washing at 65 ℃ for 1 hour with 0.1 XSSC, or washing at 65 ℃ twice with 0.1 XSSC for 20 or 30 minutes each; alternatively, hybridization may be carried out overnight (16-20 hours) at 65 ℃ in Church phosphate aqueous buffer (7% SDS; 0.5M NaPO4 buffer, pH 7.2; 10mM EDTA) and washing twice with 0.1 XSSC, 0.1% SDS at 50 ℃ for 20 or 30 minutes each, or twice with 2 XSSC, 0.1% SDS at 65 ℃ for 20 or 30 minutes each (for unique sequence regions).

Nucleic acids encoding rotavirus structural polypeptides can be described as "rotavirus nucleic acid", "rotavirus nucleotide sequence", "rotavirus nucleic acid" or "rotavirus nucleotide sequence". For example, viroid particles comprising one or more rotavirus structural protein or rotavirus structural polypeptide may be described as "rotavirus VLP", "RVLP" or "RLP", but this should not be considered as limiting.

Many organisms show a preference for using a particular codon in an extended peptide chain to encode a particular amino acid insertion. Codon preference or codon usage (difference in codon usage between organisms) is taken up by the degeneracy of the genetic code and is well documented in many organisms. Codon bias is often correlated with the translation efficiency of messenger RNA (mRNA), which in turn is believed to depend on the nature of the codon to be translated and the availability of a particular transfer RNA (tRNA) molecule, among other things. The dominance of the selected tRNA in the cell is typically a reflection of the codons most commonly used in peptide synthesis. Thus, genes can be adjusted based on codon optimization to achieve optimal gene expression in a given organism. The process of optimizing the nucleotide sequence encoding the heterologously expressed protein may be an important step in increasing the expression yield. Optimization requirements may include steps to improve the ability of the host to produce the foreign protein.

"codon optimization" is defined as: the nucleic acid sequence is modified to enhance expression in a cell of interest by replacing at least one, more than one, or a significant number of codons of the native sequence with codons that may be more commonly used or most commonly used in a gene of another organism or species. Various species show a particular preference for certain codons for a particular amino acid.

The invention includes synthetic polynucleotide sequences that have been codon optimized, e.g., sequences that have been optimized for human codon usage or plant codon usage. The codon optimized polynucleotide sequence may then be expressed in a plant. More specifically, sequences optimized for human codon usage or plant codon usage may be expressed in plants. Without wishing to be bound by theory, it is believed that sequences optimized for human codons increase the guanine-cytosine content (GC content) of the sequence and improve expression yield in plants.

It is known in the art that different codon optimization techniques exist for improving the translation kinetics of a translationally inefficient protein coding region. These techniques rely primarily on identifying codon usage for a particular host organism. If a particular gene or sequence is to be expressed in the organism, the coding sequence for those genes and sequences will be modified so that codons for the sequence of interest are replaced with codons more commonly used in host organisms.

Rotavirus structural proteins or polypeptides may be expressed in expression systems comprising virus-based DNA or RNA expression systems such as, but not limited to, cowpea mosaic virus (comovirus) based expression cassettes and geminivirus based amplification elements.

The expression system as described herein may comprise an expression cassette based on bipartite virus (bipartite virus) or virus with bipartite genomes. For example, the bipartite virus may be of the Comoviridae family (Comoviridae family). Genera of the Comovirus family include Comovirus (Comovirus), helminthovirus (Nepovirus), leguminous virus (Fabavirus), prunus filing virus (Cheravirus) and satwavirus (Sadwavirus). The comovirus includes cowpea mosaic virus (CPMV), cowpea severe mosaic virus (CPSMV), pumpkin mosaic virus (SqMV), Red Clover Mottle Virus (RCMV), Bean Pod Mottle Virus (BPMV), turnip ring spot virus (TuRSV), broad bean true mosaic virus (BBtMV), Broad Bean Staining Virus (BBSV), and radish mosaic virus (RaMV). Examples of cowpea mosaic virus RNA-2 sequences comprising an enhancer element that may be used in aspects of the invention include, but are not limited to: CPMV RNA-2(GenBank accession No. NC _003550), RCMV RNA-2(GenBank accession No. NC _003738), BPMV RNA-2(GenBank accession No. NC _003495), CPSMV RNA-2(GenBank accession No. NC _003544), SqMV RNA-2(GenBank accession No. NC _003800), TuRSV RNA-2(GenBank accession No. NC _013219.1), BBtMV RNA-2(GenBank accession No. gu810904), BBSV RNA2(GenBank accession No. fj028650), and RaMV (GenBank accession No. NC _ 003800).

The fragments of the bipartite cowpea mosaic virus RNA genome refer to RNA-1 and RNA-2. RNA-1 encodes a protein involved in replication, while RNA-2 encodes a protein required for cell-cell movement as well as two capsid proteins. Any suitable cowpea mosaic virus-based cassette may be used, including CPMV, CPSMV, SqMV, RCMV or BPMV, for example, the expression cassette may be based on CPMV.

An "expression cassette" refers to a nucleotide sequence comprising a nucleic acid of interest under the control of and operably (operatively) linked to a suitable promoter or other regulatory element for transcription of the nucleic acid of interest in a host cell.

The expression system may also comprise amplification elements from geminivirus (geminivirus), for example from bean yellow dwarf virus (BeYDV). The BeYDV belongs to the genus Mastrevelires (which is adapted to dicotyledonous plants). BeYDV is a single-stranded circular DNA genome (monoprotite) virus and it can replicate to very high copy numbers by a rolling circle mechanism. BeYDV-derived DNA replicon vector systems have been used for rapid, high-yield protein production in plants.

As used herein, the term "amplification element" refers to a nucleic acid fragment comprising at least a portion of one or more Long Intergenic Regions (LIRs) or long intergenic repeats (long intergenic regions) of a geminivirus genome. As used herein, "long intergenic region" or "long intergenic repeat" refers to a region of the long intergenic region that contains a Rep binding site that is capable of mediating excision and replication by geminivirus Rep proteins. In some aspects, a nucleic acid fragment comprising one or more LIRs can further comprise a short intergenic region or a Small Intergenic Region (SIR) of the geminivirus genome. As used herein, "short intergenic region" or "small intergenic region" refers to the complementary strand (short IR (SIR) of masslevirus). Any suitable geminivirus derived amplification element may be used herein. See, e.g., WO 2000/20557; WO 2010/025285; zhang x. et al (2005, Biotechnology and b bioengineering, vol.93,271-279), Huang z. et al (2009, Biotechnology and b bioengineering, vol.103, 706-714), Huang z. et al (2009, Biotechnology and b bioengineering, vol.106, 9-17); the above documents are incorporated herein by reference). If more than one LIR is used in the construct, e.g., two LIRs, then the promoter, CMPV-HT region and nucleic acid sequence of interest and terminator are enclosed by each of the two LIRs. In addition, the amplification elements can be derived, for example, from the sequences disclosed in Halley-Stott et al, (2007) Archives of Virology 152:1237-1240, which is registered with Gen Bank accession number DQ458791, which is incorporated herein by reference. The nucleic acid fragment comprising LIR is the linked nucleotides 2401-2566 and 1-128. The nucleic acid fragment containing SIR is nucleotide 1154 ~ 1212.

As described herein, co-delivery of a bean yellow dwarf virus (BeYDV) -derived vector and a Rep/RepA donor vector by agrobacterium infiltration of tobacco seemer's (Nicotiana benthamiana) leaves resulted in efficient replicon amplification and robust protein production.

The cowpea mosaic virus-based expression cassette and the geminivirus derived amplification element may be comprised on different vectors, or the components may be comprised in one vector. If two vectors are used, the first and second vectors may be introduced into the plant cell simultaneously or separately.

Viral replicases may also be included in expression systems as described herein to increase expression of a nucleic acid of interest. A non-limiting example of a replicase is the BeYDV replicase (pREP110) encoding BeYDV Rep and RepA (C2/C1; Huang et al, 2009, Biotechnol. Bioeng.103, 706-714; incorporated herein by reference). Another non-limiting example of a replicase is disclosed in Halley-Stott et al (2007) Archives of Virology 152:1237-1240, which is registered with Gen Bank accession number DQ458791, which is incorporated herein by reference. The nucleic acid fragment comprising the C1: C2 gene is nucleotides 1310 to 2400.

"Co-expression" means that two or more nucleotide sequences are expressed in a plant and in the same tissue of the plant at approximately the same time. However, the nucleotide sequences need not be expressed at exactly the same time. Instead, two or more nucleotide sequences are expressed in such a way that the encoded products have an opportunity to interact. Two or more nucleotide sequences may be co-expressed using a transient expression system, wherein the two or more sequences are introduced into the plant at about the same time under conditions in which both sequences are expressed. Alternatively, a platform plant comprising one of the nucleotide sequences may be transformed in a stable manner using other sequences encoding proteins of interest (e.g., one or more rotavirus structural proteins) introduced into the platform plant in a transient manner.

Proper folding of a protein may be important for protein stability, multimer formation, RLP formation and function. The folding of a protein may be affected by one or more factors including, but not limited to: the sequence of the protein, the relative abundance of the protein, the degree of intracellular crowding, the availability of cofactors that can bind to or are transiently associated with the folded, partially folded or unfolded protein. In addition, compartments or sub-compartments within the plant expressing the protein may affect the folding and expression levels of the protein.

Expression of one or more rotavirus structural proteins can be targeted to specific plant cell compartments and/or sub-compartments in transgenic plants by agro-infiltration. A compartment or sub-compartment may for example be a plastid, Endoplasmic Reticulum (ER), chloroplast or apoplast. Without wishing to be bound by theory, compartment or sub-compartment targeting may increase accumulation of protein into the targeted compartment or sub-compartment beyond cytoplasmic accumulation. Compartment or sub-compartment accumulation may protect the protein from degradation by proteases present in the cytoplasm and/or allow it to accumulate to higher concentrations without affecting the function of the plant cell.

Thus, the expression cassette or vector may be altered to render it suitable for directing the vector or rotavirus structural protein or polypeptide expressed from the vector to a desired compartment or sub-compartment in a plant.

For example, the expression cassette or vector may be altered to render it suitable for targeting to plastids by allowing the expressed rotavirus structural protein or polypeptide to include a moiety capable of interacting with the thylakoid membrane of a plastid (in particular, the transport mechanism of the thylakoid membrane). This interaction may result in the import of rotavirus structural protein or polypeptide from the cytoplasm where it is expressed into the plastid. Without wishing to be bound by theory, the import mechanism from the cytoplasm may be important for proper folding of the protein. It will be appreciated that the expression cassette or vector may be altered so that it is suitable to target the plastid itself to a transformed state, and that expression of the rotavirus structural protein or polypeptide may occur entirely within the plastid.

The term "targeting sequence" means that the targeting sequence may be included in a vector or expression cassette. Such targeting sequences can be translated into peptides that direct the vector or its product to a target compartment or sub-compartment (e.g., plastid) in a plant. For example, plastid signal peptides (also referred to in the art as "plastid transit peptides") for targeting proteins to plastids are known in the art. A non-limiting example of a plastid transit peptide that can be used is rbcs 1-cTP. A suitable example of a chloroplast transit peptide sequence is the Rubisco small subunit gene (rbcS1) from, for example, potato (Solanum tuberosum).

Thus, the rotavirus structural protein or polypeptide may include a signal peptide of the same origin or heterologous to the rest of the polypeptide or protein. The term "signal peptide" is well known in the art and generally refers to a short (about 5-30 amino acids) amino acid sequence, usually found at the N-terminus of a polypeptide, that can direct translocation of a newly translated polypeptide to a particular organelle, facilitating the localization of a particular domain of a polypeptide chain relative to other domains. As non-limiting examples, the signal peptide may target translocation of the protein to the endoplasmic reticulum, and/or assist in the positioning of the N-terminal proximal domain of the nascent polypeptide relative to the membrane anchoring domain, thereby facilitating cleavage and folding of the mature protein (e.g., rotavirus structural protein, although this should not be considered as a limitation).

The Signal Peptide (SP) may be native to the protein or viral protein, or the signal peptide may be heterologous with respect to the primary sequence of the protein or viral protein to be expressed. For example, the natural signal peptide of rotavirus structural protein can be used to express rotavirus structural protein in a plant system.

The signal peptide may also be non-native, e.g., a native structural protein from a protein, viral protein or virus other than rotavirus protein, or a polypeptide from a plant, animal or bacterium. A non-limiting example of a signal peptide that can be used is the protein disulfide isomerase (PDI SP) of alfalfa (alfalfalfalfa) (nucleotides 32-103 of accession No. Z11499). In addition, the signal peptide may be deleted completely or truncated. Truncation, or various part-of-speech forms thereof, refers to the deletion of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any amount therebetween, of an amino acid residue from the signal peptide. Preferably, the truncated amino acid residue is contiguous and the truncation occurs from the second methionine onward.

The invention thus provides rotavirus structural proteins comprising a native, non-native signal peptide or a truncated signal peptide, such as, for example, VP2, VP4, VP6 and/or VP7, and nucleic acids encoding these rotavirus structural proteins.

One or more genetic constructs of the invention may be expressed in any suitable plant host transformed with a nucleotide sequence, construct or vector as described herein. Examples of suitable hosts include, but are not limited to, agricultural crops including alfalfa, rapeseed, Brassica (Brassica spp.), corn, Nicotiana (Nicotiana spp.), potato, ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, cotton, and the like.

The nucleotide sequence encoding the rotavirus structural protein can be transferred into a plant host using 1, 2,3, 4 or 5 binary plasmid vectors. Thus, each binary plasmid vector may comprise 1, 2,3, 4 or 5 nucleotide sequences encoding a rotavirus structural protein.

One or more genetic constructs of the invention may also comprise a 3' untranslated region. The 3' untranslated region refers to a portion of a gene that comprises a DNA fragment that contains a polyadenylation signal and any other regulatory signals that enable mRNA processing or gene expression to proceed efficiently. Polyadenylation signals are generally characterized by allowing the efficient addition of a polyadenylation chain to the 3' end of the mRNA precursor. Polyadenylation signals are usually recognized by the presence of homology to the standard form 5 'AATAAA-3', although variations are not uncommon. Non-limiting examples of suitable 3 'regions are the 3' transcribed untranslated regions containing the polyadenylation signal of the following genes: agrobacterium (Agrobacterium) tumorigenic (Ti) plasmid genes (e.g., nopaline synthase (NOS) gene), plant genes (e.g., soybean storage protein gene), genes of the ribulose-1, 5-bisphosphate carboxylase small subunit (ssRUBISCO; US 4962028, incorporated herein by reference), promoters for regulating plastocyanin expression as described in US7125978, incorporated herein by reference.

If desired, one or more genetic constructs of the invention may also comprise other enhancers (translational or transcriptional enhancers). Enhancers may be located 5 'or 3' to the sequence to be transcribed. Enhancer regions are well known to those skilled in the art and may include the ATG start codon, adjacent sequences (adjacencies), and the like. The initiation codon, when present, can be in a state that is in-frame ("in-frame") with the reading frame of the coding sequence to provide for proper translation of the transcribed sequence.

The constructs of the present invention can be introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, and the like. For a review of these techniques, see, e.g., Weissbach and Weissbach, Methods for Plant Molecular Biology, academic Press, New York VIII, pp421-463 (1988); geierson and Corey, Plant Molecular Biology, 2 nd edition (1988); and Mikiand Iyer, fundametals of Gene Transfer In Plants, In Plants Metabolism, 2 nd edition, DT. Dennis, DH Turpin, DD Lefebry, DB Layzell (eds), Addison Wesley, LangmansLtd. London, pp.561-579 (1997). Other methods include direct DNA uptake, use of liposomes, electroporation (e.g., using protoplasts), microinjection, microprojectile bombardment or whiskers (whiskers), and vacuum infiltration (vacumminfiltation). See, e.g., Bilang et al (Gene 100:247-250(1991)), Scheid et al (mol.Gen.Genet.228:104-112, 1991), Guerche et al (Plant Science 52:111-116,1987), Neuhause et al (the or.appl Gene.75: 30-36,1987), Klein et al (Nature 327:70-73 (1987)); howell et al (Science 208:1265,1980), Horsch et al (Science 227:1229-1231,1985), DeBlock et al (Plant Physiology 91:694-701, 1989), Methods for Plant Molecular Biology (Weissbach and Weissbach, Inc.,1988), Methods in Plant Molecular Biology (Schuler and Zielinski, Academic Press, 1989), Liu and Lomonosoff (J Virol method, 105:343-348,2002), U.S. Pat. No. 4945050; 5036006, respectively; and 5100792, U.S. patent application Ser. No.08/438666, filed 5/10.1995, and U.S. patent application Ser. No.07/951715, filed 9/25.1992 (all of which are incorporated herein by reference).

Transient expression

Without wishing to be bound by theory, the protein concentration and ratio of different rotavirus structural proteins may be important for the efficiency of RLP assembly. Thus, the multiplicity and timing of infection may be important to control protein concentration in plants and overall assembly efficiency of RLP.

The constructs of the invention may be transiently expressed in a plant or plant part. Transient expression systems that rely on extrachromosomal (epichromosomal) expression of recombinant Agrobacterium (Agrobacterium tumefaciens) in plants, parts of plants or plant cells may be used to express rotavirus structural proteins targeted to multiple cellular compartments or sub-compartments. The instantaneous expression system allows high production speeds to be achieved. Furthermore, a large amount of protein is available within a few days after infiltration of the recombinant Agrobacterium into the plant (Rybicki, 2010; Fischer et al, 1999). Long gene sequences can also be expressed and allow more than one gene to be expressed simultaneously in the same cell, allowing for efficient assembly of multimeric proteins (Lombardi et al, 2009).

The nucleotide sequence encoding the rotavirus structural protein may be transferred to a plant host to 1, 2,3, 4 or 5 transformed Agrobacterium (Agrobacterium tumefaciens) strains.

However, during transient expression, post-transcriptional gene silencing may limit expression of heterologous proteins in plants. Co-expression of silencing suppressors (such as, but not limited to, Nss from Tomato spotted wilt virus) can be used to counteract the specific degradation of transgenic mRNA (Brigneti et al, 1998). Alternative suppressor of silencing are well known in the art and may be used as described herein (Chiba et al, 2006, Virology346: 7-14; which is incorporated herein by reference), such as, but not limited to, HcPro, TEV-p1/HC-Pro (tobacco etch virus-p 1/HC-Pro), BYV-p21, p19 of tomato bushy stunt virus (TBSV p19), capsid protein of tomato shriveling virus (TCV-CP), 2b of cucumber mosaic virus (CMV-2b), p25 of potato virus X (PVX-p25), p11 of potato virus M (PVM-p11), p11 of potato virus S (PVS-p11), plt 6 of blueberry blight virus (BScV-pl6), p23 of citrus tachyphylos (CTV-p23), combined grape vine leaf roll virus (GLV-p 24-24) and GLV-24, P10 of grapevine virus A (GVA-P10), P14 of grapevine virus B (GVB-pl4), P10 of angelica latent virus (HLV-P10) or P16 of garlic common latent virus (GCLV-P16). Thus, suppressor of silencing (e.g., HcPro, TEV-p1/HC-Pro, BYV-p21, TBSV p19, TCV-CP, CMV-2b, PVX-p25, PVM-p11, PVS-p11, BScV-p16, CTV-p23, GLRaV-2p24, GBV-p14, HLV-p10, GCLV-p16, or GVA-p10) can be co-expressed with one or more rotavirus structural proteins (e.g., VP2, VP4, VP6, or a combination thereof) to further ensure high levels of protein production in a plant or portion of a plant.

The present invention also provides a method as described above, wherein the further (second, third, fourth or fifth) nucleotide sequence is expressed in the plant and the further (second, third, fourth or fifth) nucleotide sequence encoding a suppressor of silencing is operably linked to a further (second, third, fourth or fifth) regulatory region active in plants. The nucleotide sequence encoding a suppressor of silencing may be, for example, Nss, HcPro, TEV-p1/HC-Pro, BYV-p21, TBSV p19, TCV-CP, CMV-2b, PVX-p25, PVM-p11, PVS-p11, BScV-p16, CTV-p23, GLRaV-2p24, GBV-p14, HLV-p10, GCLV-p16 or GVA-p 10.

The constructs of the invention can be expressed using transient expression Methods, as described below (see Liu and lomonossoff,2002, Journal of viral Methods, 105: 343-348; incorporated herein by reference). Alternatively, vacuum-based transient expression methods can be used, as described by Kapila et al, 1997 (which is incorporated herein by reference). These methods may include, for example, but are not limited to, methods of agrobacterium inoculation (Agro-inoculation) or Agro-infiltration (Agro-infiltration), syringe infiltration (syringing-infiltration), but other transient methods may also be used as described above. By agrobacterium inoculation, agrobacterium infiltration, or syringe infiltration, a mixture of agrobacterium containing the desired nucleic acid enters the interstitial space of a tissue, e.g., leaves, aerial parts of a plant (including stems, leaves, and flowers), other parts of a plant (stems, roots, flowers), or the entire plant. After crossing the epidermis, the Agrobacterium infects the t-DNA copy and transfers it to the cell. t-DNA is transcribed in an episomal manner and mRNA is translated, resulting in the production of the protein of interest in the infected cell, but penetration of t-DNA into the nucleus is transient.

useful selectable markers include enzymes that provide resistance to chemicals, such as antibiotics, e.g., gentamicin, hygromycin, kanamycin, or herbicides, such as glufosinate (phosphinothricin), glyphosate, chlorsulfuron, and the like.

Transgenic plants, plant cells or seeds containing the genetic constructs of the invention are also considered part of the invention. Methods for regenerating whole plants from plant cells are also known in the art. Generally, transformed plant cells are cultured in a suitable medium, which may comprise a selective agent (such as an antibiotic), wherein a selectable marker is used to aid in the identification of the transformed plant cells. Once callus is formed, the shoots can be induced to form according to known methods by using appropriate plant hormones and transferred to rooting medium to regenerate the plants. The plant may then be used to establish repeated generations, which may be achieved from seeds or using techniques of vegetative reproduction. Transgenic plants can also be produced without the use of tissue culture.

In the present application, the use of the terms "regulatory region", "regulatory element" or "promoter" refers to a portion of a nucleic acid that is typically (but not always) upstream of the protein coding region of a gene, which may comprise DNA or RNA, or both DNA and RNA. This may allow expression of a gene of interest when the regulatory region is active and in a state of operative association with or operatively linked to the gene of interest. Regulatory elements may be capable of mediating organ specificity, or controlling development or temporal gene activation. A "regulatory region" may comprise a promoter element, a core promoter element that exhibits basal promoter activity, an element that has inducibility in response to an external stimulus, an element that mediates promoter activity, such as a negative regulatory element or a transcriptional enhancer. As used herein, "regulatory region" may also comprise elements that are active after transcription, for example, regulatory elements that regulate gene expression, such as translational and transcriptional enhancers, translational and transcriptional repressors, upstream activating sequences, and mRNA instability determinants. Several of these latter elements may be located in proximity to the coding region.

In the context of the present disclosure, the term "regulatory element" or "regulatory region" typically denotes a DNA sequence located generally (but not always) upstream (5') of the coding sequence of a structural gene, which controls the expression of the coding region by providing recognition for RNA polymerase and/or other factors required for transcription to start at a specific site. However, it will be appreciated that other nucleotide sequences or sequences located 3' to introns may also contribute to the regulation of expression of the coding region of interest. An example of a regulatory element that provides recognition of RNA polymerase or other transcription factor to ensure initiation at a particular site is a promoter element. Most, but not all, eukaryotic promoter elements contain a TATA box, a conserved nucleic acid sequence comprising an adenosine and thymidine nucleotide base pair (which is typically located about 25 base pairs upstream of the transcription start site). Promoter elements include the basic promoter element responsible for transcription initiation, as well as other regulatory elements (as listed above) that modify gene expression.

There are several types of regulatory regions, including those that are developmentally regulated, inducible, or constitutive. The differentially expressed regulatory regions of a developmentally regulated or control gene under its control are activated within certain organs or organ tissues at specific times during development of that organ or tissue. However, some developmentally regulated regulatory regions may preferably be active in certain organs or tissues at a particular developmental stage, they may also be active in a developmentally regulated manner, or they may also be present at basal levels in other organs or tissues within the plant. Examples of tissue-specific regulatory regions (e.g., see-specific regulatory regions) include napin promoter and cruciferin promoter (Rask et al, 1998, J.plant Physiol.152: 595-599; Bilodeau et al, 1994, Plant Cell 14: 125-130). Examples of leaf-specific promoters include the plastocyanin promoter (see US7,125,978, which is incorporated herein by reference).

An inducible regulatory region is a regulatory region that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducing agent. In the absence of an inducing agent, the DNA sequence or gene will not be transcribed. Typically, a protein factor that specifically binds to an inducible regulatory region to activate transcription may exist in an inactive form, which is then converted to an active form either directly or indirectly by an inducing agent. However, the protein factor may also be absent. The inducer may be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound, or a physiological stress applied directly by heat, cold, salt or toxic elements or indirectly by the action of a pathogen or disease agent such as a virus. The plant cells containing the inducible regulatory region may be exposed to the inducer by externally applying the inducer to the cells or plant, for example, by spraying, watering, heating, or the like. Inducible regulatory elements can be derived from Plant genes or non-Plant genes (e.g., Gatz, C. and Lenk, LR.P.,1998, Trends Plant Sci.3, 352-358; which is incorporated by reference). Examples of possible inducible promoters include, but are not limited to, tetracycline-inducible promoters (Gatz, C.,1997, Ann.Rev. Plant physiol.plant mol.biol.48, 89-108; incorporated by reference), steroid-inducible promoters (Aoyama.T.and Chua, N.H.,1997, Plant 1.2,397-404; incorporated by reference) and ethanol-inducible promoters (salt, M.G. et al, 1998, Plant Journal 16, 127-132; Caddick, M.X. et al, 1998, Nature Biotech.16,177-180, incorporated by reference), cytokinin-induced IB6 and CKI 1 genes (Brandstatter, I.and K.ieber,1.1, 1998, Plant 10, 1009-1019; Kamotto, T.T., Sci 985, 196985; incorporated by reference, DR-5; Cell growth-induced by Cell growth element, 1973; Cell 369; incorporated by reference).

Constitutive regulatory regions direct gene expression continuously in various parts of a plant and throughout plant development. Examples of known constitutive regulatory elements include promoters associated with the following genes or transcripts: CaMV 35S transcripts (Odell et al, 1985, Nature,313:810-812), rice actin 1(Zhang et al, 1991, Plant Cell,3:1155-1165), actin 2(An et al, 1996, Plant J., 10:107-121) or tms 2(U.S.5,428,147, which is incorporated herein by reference), and triosephosphate isomerase 1(Xu et al, 1994, Plant Physiol.106:459-467) genes, maize ubiquitin 1 genes (Cornejo et al, 1993, Plant mol.biol.29:637-646), Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al, 1995, Plant mol.biol.29:637-646), and tobacco translation initiation factor 4A genes (Mandell et al, 1995, Plant mol.biol.29: 995-1004).

The term "constitutive" as used herein does not necessarily indicate that a gene under the control of a constitutive regulatory region is expressed at the same level in all cell types, but means that the gene is expressed in a wide range of cell types, although differences in abundance are often observed. Constitutive regulatory elements may be coupled to other sequences to further enhance transcription and/or translation of the nucleotide sequences to which they are operably linked. For example, the CPMV-HT system is derived from the untranslated region of cowpea mosaic virus (CPMV) and exhibits enhanced translation of the relevant coding sequence. "native" means that the nucleic acid or amino acid sequence is naturally occurring, or is "wild-type". By "operably linked" is meant that particular sequences (e.g., coding regions and regulatory elements of interest) interact, directly or indirectly, to perform a desired function, such as mediating or regulating gene expression. The interaction of the operably linked sequences may be mediated, for example, by a protein that interacts with the operably linked sequences.

RLP produced in plants can induce the structural protein of rotavirus VP7, which contains plant-specific N-glycans. Accordingly, the present invention also provides RLP comprising VP7 with plant specific N glycans.

In addition, modifications to N-glycans in plants are known (see, e.g., U.S. 60/944,344; which is incorporated herein by reference), and VP7 with modified N-glycans can be produced. VP7 comprising a modified glycosylation pattern (e.g., N-glycans with reduced fucosylation, xylosylation, or both fucosylation and xylosylation) may be obtained, or VP7 (wherein the protein lacks fucosylation, xylosylation, or both, and comprises increased galactosylation) may be obtained with a modified glycosylation pattern. Furthermore, modulation of post-translational modifications (e.g. addition of terminal galactose) may result in reduced fucosylation and xylosylation of expressed VP7 compared to wild type plants expressing VP 7.

for example, and not to be considered as limiting, synthesis of VP7 with a modified glycosylation pattern can be achieved by co-expressing VP7 together with a nucleotide sequence encoding a β -1.4 galactosyltransferase (GalT) (such as, but not limited to, mammalian GalT or human GalT, but GalT from other sources can also be used). the catalytic domain of GalT can also be fused to the CTS domain (i.e., cytoplasmic tail, transmembrane domain, stem region) of N-acetylglucosaminyltransferase (GNT1) to produce a GNT1-GalT hybrid, and this hybrid can be co-expressed with VP 7. VP7 can also be co-expressed with a nucleotide sequence encoding N-acetylglucosaminyltransferase III (GnT-III) (such as, but not limited to, mammalian GnT-III or human GnT-III, and GnT-III from other sources can also be used.

Accordingly, the present invention also provides RLP comprising VP7 with modified N glycans.

Without wishing to be bound by theory, the presence of plant N-glycans on VP7 may stimulate an immune response by promoting binding of VP7 to antigen presenting cells. Saint-Jore-Dupas et al (2007) have proposed the use of plant N-glycans to stimulate an immune response.

The invention is further illustrated in the following examples.

Examples

Example 1

Expression of rotavirus proteins and production of VLPs in leaves of Nicotiana benthamiana (N.benthamiana) plants

The following assay used rotavirus capsid proteins from the G9P [6] rotavirus strain and evaluated whether rotavirus-like particles were formed in multiple compartments of leaf cells of Nicotiana benthamiana (N. Co-expression of VP2 and VP6 and co-expression of various combinations of VP2, VP6, VP7 and VP4 in tobacco plant leaves were investigated.

Materials and methods

Plasmid construction

plant codon optimized rotavirus cDNA to VP2, VP4, VP6 and VP7 was provided by Geneart, Germany according to the manufacturer's instructions, plastid DNA was transformed into DH5- α chemically competent escherichia coli (e^TMLucigen). In this study, a novel binary Agrobacterium provided by Rainer Fischer (Fraunhofer institute for Molecular Biology and Applied Ecology, IME, Germany) was usedVectors pTRAc (cytoplasmic), pTRAkc-rbcs1-cTP (chloroplast targeted), and pTRAkc-ERH (endoplasmic reticulum targeted). Another vector, pTRAkc-A (apoplast), was derived from modification of pTRAkc-ERH at multiple cloning sites by Restriction Enzyme (RE) digestion at NcoI and XhoI sites (FIG. 3). This removes the histidine tag and KDEL sequences (which retain the protein in the ER). Alternatively, the protein is targeted to the apoplast.

for direct cloning of DNA into pTRAc, pTRAkc-rbcs-cTP and pTRAkc-A, RE digestion of each vector was performed at the AflIII/XhoI, MluI/XhoI and NcoI/XhoI sites, respectively, cloning of the DNA in the vector was performed according to standard protocols, followed by transformation into chemically competent E.coli (E.coli) DH5- α cells (E.clononi)^TMLucigen) selected recombinant colonies were confirmed by colony PCR for cloning in pTRAkc-ERH NotI restriction enzyme sites were added by PCR amplification in place of the stop codon of each of the four rotavirus cDNAs the cDNA amplification using the primers detailed in Table 1 PCR reaction conditions included denaturation at 95 ℃ for 5 minutes followed by 5 cycles of denaturation at 95 ℃ for 30 seconds, annealing at 52 ℃ for 1 minute and elongation at 72 ℃ for 1.5 minutes, additional 20 cycles of 95 ℃ for 30 seconds, 57 ℃ for 1 minute, 72 ℃ for 1.5 minutes and 72 ℃ for 5 minutes, then the amplified fragments were cloned into pGEM-T-Easy (Promega) according to the manufacturer's instructions, transformed in chemically competent E.coli (E.coli DH) 5- α (E.coli)^TMLucigen). Colony PCR was then performed on the selected colonies, as was the other three constructs.

Table 1: rotavirus cDNA primer for ER vector cloning

pGEM-VP DNA from positive colonies was sequenced to verify the fidelity of PCR digestion of DNA with NcoI/NotI and cloning of the appropriate DNA fragments into pTRAkc-ERH at NcoI and NotI sites to form pTRAkc-ERH-VP. then transformation into E.coli (E.coli) DH5- α cells was performed as previously.

Agrobacterium transformation

Agrobacterium (Agrobacterium tumefaciens) GV3101 strain was supplied by professor Rainer Fischer (Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany) and made electrocompetent as described before (Shen and Forde, 1989). in a 0.1 cmelectopy cuvette (BioRad. TM.), 300ng of the isolated rotavirus pTRA-VP construct was mixed with 100. mu.l of electrocompetent GV3101 cells, followed by electroporation in a GenePulser (BioRad) in the setting of 1.8Kv, 25. mu.F and 200. omega. for 1 hour at 27 ℃ in 900. mu.l LB, after which the transformants were incubated with 50. mu.g/ml carbenicillin (carb), 30. mu.g/ml kanamycin (kanamycin) and 50. mu.g/ml) in 35f for 1 hour, and the transformants were checked for the presence of the relevant transformants in a PCR plate for the presence of the transformants and the transformants were checked for the presence of the transformants in the transformants of the transformants, the transformants were checked for the transformants in the transformants of the transformants (Agrobacterium strain Escherichia coli) by incubation in the presence of the plasmid DNA and the plasmid DNA which were made in the plasmid DNA which was transferred in the plasmid was transferred in the medium (Escherichia coli strain).

Infiltration with recombinant Agrobacterium

Agrobacterium (A. tumefaciens) LBA4404(pBIN-NSs) used in this study was obtained from MarcelPrins (Laboratory of Virology, Wageningen University, Binnenhaven, Netherlands). It contains NSs silencing suppressors found in Tomato Spotted Wilt Virus (TSWV). Recombinant Agrobacterium from glycerol stocks (pTRA-VPs) were grown overnight at 27 ℃ in LB with 50. mu.g/ml carb, 30. mu.g/ml kan and 50. mu.g/ml rif. Then, the recombinant Agrobacterium was mixed with each of LBA4404(pBIN-NSs)Each was inoculated into an induction medium (LB, 10mM 2- (N-morpholino) ethanesulfonic acid MES, 2mM MgSO₄20 μ M acetosyringone, 50 μ g/ml carb, 30 μ g/ml kan and 50 μ g/ml rif, and pH 5.6).

The cultures were incubated overnight at 27 ℃. Agrobacterium cells were collected by centrifugation at 4000rpm for 5 minutes at 4 ℃ and then resuspended in 2ml of infiltration medium (10mM MES, 10mM MgCl)₂3% sucrose, pH 5.6, 200 μ M acetosyringone and sterile water). The optical density (OD600) of the cells was verified and diluted with infiltration medium to obtain an OD600 of 0.25. For each pTRA-VP construct, LBA4404 was mixed with recombinant agrobacterium to a final OD600 of 0.5. For co-expression studies, each construct was added to a total OD600 of 0.5, e.g., VP2-0.25 and VP6-0.25, until the mixture OD600 was equal to 0.5. Acetosyringone used in induction and infiltration medium helps activation of vir genes in agrobacterium.

The wounded plant cells release phenolic compounds, which are detected by Vir a and Vir G genes in agrobacterium, which subsequently lead to induction of protein expression in the host cell (Zupan, j. et al, 2000). Then, the cells were cultured at room temperature for 1 hour to allow acetosyringone to induce the vir genes. Three-week-old wild-type nicotiana benthamiana (n.benthamiana) plants were infiltrated with recombinant agrobacterium expressing VP protein. This involves vacuum infiltration of the entire plant or injection of recombinant Agrobacterium (pTRA-VP) into the abaxial air space on the ventral side of the plant leaf. Recombinant Agrobacterium was infiltrated with or without the suppressor of silencing LBA4404 (pBIN-NSs).

Initially, 2ml of agrobacterium infiltration medium suspension was injected into each plant using one syringe per construct. In a seven day time trial, one plant was used per construct. Co-expression of rotavirus proteins was also performed, in which VP2, VP6 and VP4 were simultaneously expressed in the cytoplasm of leaves of the present Nicotiana benthamiana (N.benthamiana) plant. Combination studies were performed for the combination of VP2/6 and the combination of VP 2/6/4. VP4 "spike" proteins can bind to VP6 and therefore there is a possibility that they may be added to RLP structures. The VP7 clone was attempted, but proved to be problematic in terms of host cell toxicity. Recombinant VP7 agrobacterium killed leaf cells within one day after infiltration. Several methods were tried to avoid this, such as soaking the plants at a low temperature of 17 ℃ and soaking after day 3 and/or day 5 of the timekeeping trial. Likewise, VP7 was omitted in co-expression studies due to its toxic nature in tobacco plants.

Protein extraction

Either whole leaves or two leaf disks were harvested for each construct and ground in liquid nitrogen. The ground leaf material was resuspended in sterile PBS containing complete protease inhibitor (EDTA-free; Roche). Then, it was centrifuged at 13000rpm for 5 minutes, and the pellet (plant leaf material) was discarded. Then, 100. mu.l of each construct was mixed with 5 XSDS-PAGE loading buffer and boiled at 95 ℃ for 2 minutes, ready for further analysis on SDS-PAGE gels and Western blots. The remaining samples were stored at-20 ℃ until use. FIG. 4 shows an overview of the cloning and infiltration protocol for rotavirus cDNA.

Apoplast protein extraction

An additional extraction protocol was performed on the apoplast construct pTRAkc-a. The apoplast is the free diffusion space between the plasma membrane and the cell wall of the plant cell (fig. 5 a). Proteins expressed in the cytoplasm have export sequences that target them to the apoplast, so they accumulate in the apoplast. In the subsequent extraction protocol, whole leaves from each extraction day were vacuum or injection infiltrated with sterile PBS containing complete protease inhibitors. For vacuum infiltration, individual plant leaves were suspended in PBS and placed in a vacuum tank at 100mbar for 10 minutes. The leaf was then rolled up and gently placed in a spin column with a hole in the bottom (similar to Qiagen spin columns) (FIG. 5b 2). The holes allow fluid to pass easily through the vane but not allow solid vane matter to pass. The spin columns were placed in 2ml microcentrifuge tubes (Eppendorf tubes) and centrifuged at 4000rpm for 15 minutes (FIG. 5b 3). The filtrates were collected and protein loading dye for SDS-PAGE gel and Western blot analysis was added to each 100. mu.l filtrate sample.

Western blot and Coomassie staining

Western blots and Coomassie blue stained SDS-PAGE gels were used as described previously. In Western blotting, mouse anti-rotavirus VP6 antibody (US Biologicals) (1:5000), anti-mouse histidine-tag antibody (His-tag antibody) were used(1:2000), chicken anti-VP 2, and chicken anti-VP 4 sera (1:2000) probed for each individual protein. Proteins were quantified by scanning the band density using coomassie blue stained SDS-PAGE gels using Syngene gel imaging system.

Electron microscope

To determine whether the expressed proteins are assembled into RLPs, Transmission Electron Microscopy (TEM) analysis of the immunocapture particles was performed on day 3 of expression of cytoplasmic expressed VP6, VP2/6 and VP2/6/4, all in the presence of the suppressor of silencing NSS. Glow-discharge carbon/copper grids were placed on 20. mu.l mouse anti-rotavirus VP6 antibody (1:5000) for 5 minutes and then washed 3 times with sterile distilled water. Then, the mesh was placed on 10 μ l of the protein extract and left for 2 minutes, followed by washing again 3 times with sterile distilled water. Finally, the mesh was floated for 1 minute in 20 μ l of 2% uranyl acetate and then observed under TEM (Zeiss 912 OMEGA energy filtration projection electron microscope, University of cap Town).

For samples isolated from sucrose gradients, sucrose must first be removed by dialysis before immunocapture on a copper mesh. If not removed, sucrose crystals inhibit the definitive observation of the sample under TEM as sucrose forms crystals on the mesh, thereby disrupting the structure of the bound carbon and material. The sucrose grade was placed in a 10000MW dialysis cassette and dialyzed in sterile PBS containing 0.4M NaCl for 4 hours, then the buffer was changed and stirred at 4 ℃ overnight. Since the volume increases with dialysis, the protein sample needs to be concentrated. The samples were vacuum freeze dried for 3 hours and resuspended in 2ml sterile PBS, ready for further analysis.

Sucrose gradient purification of RLP

Initially, the plant protein extract is filtered through a microporous cloth to remove solid plant matter. In each 40ml tube, a sucrose gradient from 10 to 60% sucrose was established by generating six layers of 5ml sucrose dissolved in sterile PBS (pH 7.4). The clarified protein sample was then loaded at the top of each gradient column in a volume of 5 to 10 ml.

Ultracentrifugation (SWTi28 Pontoon rotor, Beckmann Coulter) was carried out at 150000 g for 1 hour 30 minutes at 4 ℃. At the end of centrifugation, 2ml fractions were collected from the bottom of each column by tube puncture. Dot blot analysis was then performed to determine the fraction with the protein of interest. For each fraction, 1 μ l of sample was loaded onto a mesh on a nitrocellulose membrane, which was then blocked with BSA blocking buffer. Then, Western blot analysis was performed in a conventional manner. The protein VP6 was probed with mouse anti-V6 antibody (1:5000), or the other two proteins were probed with chicken anti-VP 2 and VP4 sera (1: 5000).

Total soluble protein assay

Total Soluble Protein (TSP) was determined by Bradford assay. This assay was performed to compare the level of protein accumulation of cytoplasmic co-expressed VP 2/6. Protein IgG (1.43mg/ml stock) was used as a standard in serial dilutions. Mu.l of standard and sample were added to a clean dry microtiter plate, respectively. Total soluble protein reagents A and B were added according to the manufacturer's instructions (Bio-Rad Dc protein assay). All experiments were performed in triplicate. Absorbance readings at 750nm were recorded using a microplate reader (Bio-tek PowerWave XS).

Results

Expression of VP6 in the leaf cell compartment of plants

VP6 was expressed with or without a suppressor of silencing and was targeted to all cellular compartments (FIG. 6; (line labeled VP6, at about 42 kDa)). In the cytoplasm, the protein was expressed from the first day of the chronology test, and the accumulation of the protein in the cytoplasm gradually increased during the 1 week test period (fig. 6 a). In the ER, protein accumulation was clearly observed only on day 3 (fig. 6 b). This protein is shown as a higher band size (approximately 11kDa larger) than the other proteins. This may be due to the 6-histidine tag added at the C-terminus of the protein as well as the cleavage site (reference to the pProEx vector sequence).

Protein accumulation in chloroplasts occurred between day 1 and day 3 (fig. 6 "chlorophyll"). Since the protein is not detectable in the absence of a suppressor, the suppressor has an effect on the protein. No protein was expressed on days 5 and 7. As in the ER, apoplast had the best protein accumulation between day 3 and day 5 of the time trial (fig. 6 "apoplast"), while it was completely absent on days 1 and 7. The suppressor of silencing has a positive effect especially on day 3, which results in higher levels of protein detection compared to the case without suppressor of silencing. It was also found that two bands were observed at the about 40kDa marker, probably due to cleavage of the signal tag on the VP6 protein.

At day 3, ER, chloroplasts, and apoplast all showed the highest protein expression, with the most protein accumulated in the presence of the suppressor of silencing. The cytoplasm is optimal in terms of protein accumulation since it shows high and gradually increased protein expression throughout the timing trial.

Expression of histidine-tagged rotavirus proteins in the cytoplasm

Four rotavirus VPs were cloned into an additional vector (pTRAc-HT). The vector includes a 6-histidine tag of a protein targeted to the cytoplasm, and if an antibody to the protein of interest is not available, detection can be easily performed by using an anti-histidine-tag antibody. In our case, only VP6 has commercially available antibodies, so while waiting for serum, we tried this procedure for early detection of all proteins. The same holds true for VP6 expression and facilitates our attempts for other proteins.

Western blot results of the extracts at day 3 in the 7 day timing trial showed: VP2, VP4 and VP6 were successfully expressed (FIG. 7 a). In order to obtain expression of VP7 in plants, various techniques were attempted. However, plants infiltrated with VP7 showed leaf yellowing starting from day 1 and progressed to wilting during the course of the timekeeping trial (fig. 7 b). Protein expression was not detected under these conditions, even after day 1 of infiltration when the plants still looked reasonably good.

Expression of VP2 and VP4 in plants

VP2 and VP were infiltrated in the leaves of the present nicotiana tabacum (n. Since we could not obtain any positive clones in escherichia coli (e.coli), we could not express VP2 targeted to the apoplast vector. However, this protein was successfully expressed and targeted to all other 3 compartments (fig. 8 a). Chicken anti-VP 2 and anti-VP 4 sera (1:2000) were used in Western blot analysis of the extracts. As shown in FIGS. 8a and 8b, respectively, the VP2 and VP4 bands are visible just below the 100kDa marker (protein band indicated by arrows). For VP2, it appears to be best expressed in the cytoplasm and ER, while for VP4 it is best in the cytoplasm and apoplast. The suppressor of silencing has no significant effect on the expression of the protein. As can be seen from the Western blot, it only slightly increased expression in the VP2ER construct, not as much in the others. The VP4 construct was expressed in the presence of a suppressor of silencing.

Co-expression of VP2/6 and VP2/6/4 in the cytoplasm

The cytoplasm appeared to be optimal for expression of the rotavirus capsid protein and showed the highest extraction efficiency. Thus, all further expression work was done with proteins targeted to the cytoplasm.

VP2 and VP6 have been shown to form RLP with a protective immunogenic response in mice, and therefore, co-expression of VP2/6 and VP2/6/4 in the cytoplasm was investigated. The day 3 extracts of co-expressed VP2/6/4 were examined by Western blotting using anti-VP 2 with anti-VP 4 serum (1/5000) and mouse anti-VP 6 antibody (1:5000) (FIG. 9). As previously determined, VP6 expression was very high, but VP2 and VP4 expression was very low, as seen by the very faint bands at the 100kDa marker. This may be due to co-expression, which results in more host cell resources being used in the overexpression of VP6, leaving less resources for VP2 and/or VP 4. It is also not readily determinable if the detected band is either of VP2 and VP4, or either of the two proteins. A very distinct band above 130kDa is likely to be dimerized VP6 protein. The band visible at the 55kDa marker is most likely the bulk of the plant enzyme Rubisco.

Cytoplasmic expressed VP6 and co-expressed VP2/6 and VP2/6/4 were subjected to transmission electron microscopy analysis to examine the protein particles and assembled RLP (FIG. 10). This also determined whether VP2 and/or VP4 were indeed successfully co-expressed. When expressed alone, VP6 assembles to form a protein sheath, as shown by the arrows in fig. 10 b. Upon addition of VP2, the particles assembled to form RLP (FIG. 10 c). VP2 functions as a backbone protein that enables other proteins to assemble and ultimately form a complete rotavirus structure. VP6 binds to VP2 as well, but the VP4 structure in co-expressed VP2/6/4 is not easily determined. The electron micrograph in FIG. 10d may be a simple assembly of VP2/6 particles. It has been shown that VP4 binds to VP6 during protein assembly, and this occurs prior to VP7 binding. It is possible that these VP4 structures are unstable and may detach from the RLP structures during the preparation procedure for electron microscopy.

Sucrose gradient purification of VP2/6 and VP2/6/4

VP2/6 and VP2/6/4 were purified on a sucrose gradient of 10 to 60% sucrose (FIG. 11 a). Fractions of 2ml were collected from the bottom of each tube and probed with mouse anti-VP 6 antibody and/or chicken anti-VP 2 and VP4 sera to determine fractions containing the protein. For VP2/6, proteins were found in fractions 16 and 17, as they were positive on the blot for VP6 protein (FIG. 11 b). In all fractions, positive results were shown by the analysis of the VP2/6/4 blot using chicken anti-VP 2 and VP4 sera. This may be due to the high level of protein detection background by chicken serum. However, as seen in fig. 11c, the spot intensities were highest in fractions 17 and 18, probably due to the higher concentration of the protein of interest in these fractions.

Summarizing these results (fig. 11b and 11c) it was found that rotavirus proteins are present in the range of fractions 16 to 20.

Western blot and Coomassie staining of fractions

Western blotting and SDS-PAGE of the co-expressed VP2/6 was performed to verify the presence of VP2 and VP6 proteins in fractions 13 to 20. Western blot analysis of VP6 protein probed with mouse anti-VP 6 antibody was positive in fractions 16 through 20 (FIG. 12a, bottom arrow and FIG. 12 c). VP2 protein detected with chicken anti-VP 2 serum was detected in fractions 17 to 20 (fig. 12a, top arrow). In previous co-expression studies, VP2 has been shown to express less than VP6, which is also shown in figure 12a of the present invention, where the VP2 protein band is less intense than VP 6.

Fractions 16 and 17, previously confirmed by dot blot to contain co-expressed VP2/6 of VP6 protein (FIG. 11b), were electrophoresed on SDS-PAGE gels. A known concentration of the protein, VP6 expressed by SF9 insect cells (0.91. mu.g/. mu.l), was included to determine the concentration of VP2/6 crude protein (FIGS. 11b and 11 c). This was done by density scanning of the crude protein bands (lanes marked as crude protein) using the Syngene gel imaging system, which thereby enabled us to determine the amount of VP2/6 per kg of leaf material. The protein yield was found to be about 1.54g/kg Fresh Weight (FW). 1.1mg of purified RLP (1.1g/kg) was obtained from 1g of plant material.

Total soluble protein assay for VP2/6

Total Soluble Protein (TSP) was determined on the co-expressed VP2/6 fraction to determine the relative amount of VP2/6 protein (FIG. 13). By using IgG standards, the protein concentrations of fractions 17 and 18 were calculated to be 0.538mg/ml and 1.012mg/ml, respectively (fig. 13 a). Protein bands corresponding to VP2/6 in these fractions were calculated by density scanning on a Syngene gel imaging system and found to be about 0.108mg/ml and 0.202mg/ml, respectively.

Thus, VP2/6 in fractions 17 and 18 each had approximately 20% TSP. The majority of RLP in the sucrose column was found to be between 15 and 25% sucrose, corresponding to fraction 15 to about fraction 20, at which time a sudden peak was recorded on the graph and then resolved. The density differences of the various materials in the extract enable us to isolate and thereby purify the protein of interest. SDS-PAGE gels stained with Coomassie blue showed only one prominent band, indicating that the protein was relatively pure (FIG. 12 b).

Purified TEM of VP2/6

The purified VP2/6 fractions were pooled together and dialyzed against high salt PBS to remove sucrose, then observed on a transmission electron microscope. TEM was performed to determine purity and to check whether RLP remained intact after the purification procedure. As shown in fig. 15, most of the background material, which is composed mainly of host cell products (fig. 10b, c and d), is removed, leaving RLP behind. Most RLP remain intact, but some appear to lose shape, which may be caused by distortion due to conditions on the EM net.

Preliminary analysis of rotavirus structural protein expression in leaf discs of Nicotiana benthamiana (N.benthamiana)

This preliminary analysis focused on the expression of rotavirus structural proteins VP2(SEQ ID NO: 1), VP4(SEQ ID NO: 2), VP6(SEQ ID NO: 3) and VP7(SEQ ID NO: 4) in the lamina of Nicotiana benthamiana (N.benthamiana) as an exemplary host expression system. The rotavirus strain selected by the present invention is G9P [6] strain which is mainly epidemic in south Africa and other sub-Saharan regions. An RLP vaccine targeting this strain would help to reduce the burden of disease in sub-saharan africa.

Transient expression systems mediated by Agrobacterium were used in this assay. Transient expression, in contrast to transgenic expression, allows rapid expression of proteins in a relatively short time, where the rotavirus capsid protein gene is not integrated in the host chromosome. Most of the protein was expressed and accumulated to a detectable amount on day 3 when the recombinant agrobacterium infiltrated the leaves of nicotiana benthamiana (n. As shown below, successful expression of several rotavirus structural proteins was observed in the plant leaf cell compartment, including VP2, VP4 and VP6, as detailed in table 2:

table 2: expression of rotavirus VP protein in multiple leaf cell compartments

0-no expression 1-expressed

No expression of glycoprotein VP7 was observed, probably due to its toxic effect on plant cells. Notably, VP7, which contained its native signal peptide, was used in this preliminary study. Infiltration was also attempted on day 3 during the co-expression assay. This attempt was made to see if the protein was expressed and assembled with VP2 and VP6 shortly thereafter to form RLP. The toxic properties of recombinant VP7 observed in this study have been described previously (Williams et al, 1995; McCorquuaodale, 1987; Arias et al, 1986).

VP7 expression studies in transgenic potatoes have been reported in the past (Li et al, 2006; Choi et al, 2005; Wu et al, 2003). Choi et al (2005) used simian rotavirus VP7, while Li et al and Wu et al (Li et al, 2006; Wu et al, 2003) used human group A G1VP 7. The results described herein used human rotavirus G9VP 7.

VP2 expresses and targets all compartments except apoplast, because we cannot clone the appropriate cDNA and the time limit only allows us to make several attempts before doing so with other constructs. It was noted that the expression level of VP2 was significantly lower in all compartments. In a past study cited in Saldana et al, 2006, the following conclusions were drawn: although mRNA was detected in plant cells, VP2 whose sequence was optimized for expression in plants was unlikely to be expressed. However, they successfully expressed in tomato plant cells using synthetic DNA. The difficulty in expression of VP2 is probably due to incorrect translation of mRNA or to mRNA containing certain sequence motifs which destabilize plant cells (Kawaguchi and Bailey-Serres, 2002). Evidence of low expression levels of VP2 compared to VP6 has been found in plant and insect cell expression studies by Mena et al (2006), Saldana et al (2006), Vieira et al (2005) and Labbete et al (1991).

The outer capsid protein VP4, which forms spikes on the surface of the viral particle structure, is expressed and targeted for accumulation in the cytoplasm, ER and apoplast. No protein accumulation was detected in the chloroplasts. On Western blots, protein expression levels for VP4 were lower than seen for VP6, as observed for VP 2. This protein has a trypsin cleavage site, which will produce two proteins: VP5 and VP 8. The following may occur: the local trypsin in the lamina of nicotiana benthamiana (n.benthamiana) cleaves some proteins as they are produced, resulting in lower levels of intact VP4 concentration accumulated in a given compartment. This protein has been shown to be the major neutralizing antigen, but there have been several attempts to clone the whole protein for vaccine development (Khodabandehloo et al, 2009; Mahajan et al, 1995; Nishikawa et al, 1989). However, several studies in insect cell and yeast expression systems have shown expression of the VP5 or VP8 subunit of VP4 (Andre et al, 2006; Favacho et al, 2006; Kovacs-Nolan et al, 2001). To date, this study was the first study showing whole protein expression in plant expression systems.

VP6 was expressed in all compartments, where overexpression was observed in the cytoplasm and protein accumulation was observed in this compartment from day 1 to day 7. This is in contrast to some literature which indicates that protease activity and gene silencing will reduce or prevent the accumulation of foreign proteins in the cytoplasm (Fischer et al, 2004). Furthermore, considering the correct pH conditions, VP6 is known to self-assemble into tubular or helical particles, much like those observed in our studies (fig. 9b) (Estes et al, 1987). VP6 makes up approximately 50% of the viral core and is therefore the main antigen for rotavirus vaccine development. The results obtained above enabled us to further study the co-expression of VP2, VP6 and VP4 in the cytoplasm.

When co-expressed in the cytoplasm, VP2 and VP6 assemble to form RLP. Very high protein yields between 1.27-1.54g/kg FW from transient expression systems were observed. When purified on a sucrose column, the amount of VP retained is 1.1g/kg FW. This yield is comparable to the yield obtainable for the production of antibody IgG in Nicotiana benthamiana (N.benthamiana) using a transient expression system, up to 1.5g/kg FW (Vetzina et al, 2009). Saldana et al (2006) is the only group known to date to successfully co-express rotavirus VP2 and VP6 in transgenic tomato plants and reach about 1% total soluble protein level. The assembly of VP2/6 in insect cell expression systems has been well documented (Vieira et al, 2005; O' Brien et al, 2000). These VP2/6 RLPs have also been shown to provide protective immunity against rotavirus infection (Zhou et al, 2011; Saldana et al, 2006). Therefore, we produced VP2/6RLP in a plant expression system as a suitable candidate for the development of subunit rotavirus vaccines.

VP2/6/4 was also co-expressed and detected. The first visible peak in the total protein absorbance reading of the co-expressed protein (FIG. 14, fraction 16) was probably assembled VP2/6/4, but no RLP was detected when the fraction was observed under TEM. The observed protein peak may be due to accumulation of VP4 monomer or its respective VP5 and VP8 subunits. When examined under TEM, the second peak (fraction 18) showed RLP structures very similar to those observed in the VP2/6 sample. However, Crawford et al have previously reported that VP4 was not observed under TEM, and that VP2/6/4 and VP2/6/4/7 particles had similar structure and diameter under TEM (Crawford 1994). We have made the same observations for VP2/6/7RLP, VP2/6/4/7 RLP, and VP2/6RLP, which all look similar under conventional TEM.

Example 2

Construct

A-2X35S/CPMV-HT/RVA (WA) VP2(opt)/NOS (construct No. 1710)

The optimized sequence encoding VP2 from rotavirus A WA strain WAs cloned into the 2X35S-CPMV-HT-NOS expression system in a plasmid containing the Plasto _ pro/P19/Plasto _ ter expression cassette using the PCR-based method described below. Fragments comprising the VP2 coding sequence were amplified using the optimized VP2 gene sequence (FIG. 19, SEQ ID NO: 45) as template, primers IF-WA _ VP2(opt) s1+3c (FIG. 17A, SEQ ID NO: 21) and IF-WA _ VP2(opt) s1-4r (FIG. 17B, SEQ ID NO: 22). For sequence optimization, the VP2 protein sequence (Genbank accession number CAA33074) was reverse translated (backstrate) and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned into the 2X35S/CPMV-HT/NOS expression system using the In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct No. 1191 (fig. 17C) was digested with SacII and StuI restriction enzymes and the linearized plasmid was used for In Fusion assembly reactions. Construct No. 1191 is an acceptor plasmid intended for "In Fusion" cloning of the gene of interest In the CPMV-HT based expression cassette. It also introduces a gene construct for co-expressing the TBSV P19 silencing suppressor under the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and FIG. 18 shows this sequence from the left t-DNA border to the right t-DNA border (SEQ ID NO: 23). The resulting construct is numbered 1710 (FIG. 23, SEQ ID NO: 27). FIG. 20 shows the amino acid sequence of VP2 from rotavirus A strain WA (SEQ ID NO: 25). Fig. 21 shows a schematic of plasmid 1710.

B-2X35S/CPMV-HT/RVA (WA) VP2(opt)/NOS to BeYDV (m) + replicase amplification System (construct coding) No. 1711)

The optimized sequence encoding VP2 from rotavirus A WA strain WAs cloned into 2X35S/CPMV-HT/NOS containing BeYDV (m) + replicase amplification system in a plasmid containing the Plasto _ pro/P19/Plasto _ ter expression cassette using the PCR-based method described below. A fragment comprising the coding sequence for VP2 WAs amplified using the optimized VP2 gene sequence (SEQ ID NO: 45) as template, using primers IF-WA _ VP2(opt) s1+3c (FIG. 17A, SEQ ID NO: 21) and IF-WA _ VP2(opt) s1-4r (FIG. 17B, SEQ ID NO: 22). For sequence optimization, the VP2 protein sequence (Genbank accession number CAA33074) was reverse translated and optimized for human codon usage, GC content, and mRNA structure. The PCR product was cloned into the BeYDV (m) amplification system In the 2X35S/CPMV-HT/NOS expression cassette using the In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct 193 (fig. 22A) was digested with SacII and StuI restriction enzymes and the linearized plasmid was used for In Fusion assembly reactions. Construct No. 193 is a recipient plasmid intended for "In Fusion" cloning of a gene of interest into the BeYDV (m) amplification system In a CPMV-HT based expression cassette. It also introduced a gene construct for co-expression of the TBSV P19 silencing suppressor under the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and FIG. 22B shows this sequence from the left t-DNA border to the right t-DNA border (SEQ ID NO: 26). The resulting construct is numbered 1711 (FIG. 23, SEQ ID NO: 27). FIG. 20 shows the amino acid sequence of VP2 from rotavirus A strain WA (SEQ ID NO: 25). Figure 24 shows a schematic of plasmid 1711.

C-2X35S/CPMV-HT/RVA (WA) VP6(opt)/NOS (construct No. 1713)

The optimized sequence encoding VP6 from rotavirus A WA strain WAs cloned into the 2X35S-CPMV-HT-NOS expression system in a plasmid containing the Plasto _ pro/P19/Plasto _ ter expression cassette using the PCR-based method described below. A fragment comprising the coding sequence of VP6 WAs amplified using the optimized VP6 gene sequence (SEQ ID NO: 46) as template, using primers IF-WA _ VP6(opt) s1+3c (FIG. 25a, SEQ ID NO: 28) and IF-WA _ VP6(opt) s1-4r (FIG. 25b, SEQ ID NO: 29). For sequence optimization, the VP6 protein sequence (Genbank accession AAA47311) was reverse translated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned into the 2X35S/CPMV-HT/NOS expression system using the In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct No. 1191 (fig. 17C) was digested with SacII and StuI restriction enzymes and the linearized plasmid was used for the InFusion assembly reaction. Construct No. 1191 is an acceptor plasmid intended for "In Fusion" cloning of the gene of interest In the CPMV-HT based expression cassette. It also introduces a gene construct for co-expressing the TBSV P19 silencing suppressor under the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and FIG. 18 shows this sequence from the left t-DNA border to the right t-DNA border (SEQ ID NO: 23). The resulting construct is numbered 1713 (FIG. 25c, SEQ ID NO: 30). FIG. 26 shows the amino acid sequence of VP6 from rotavirus A strain WA (SEQ ID NO: 31). Figure 27 shows a schematic of plasmid 1713.

D-2X35S/CPMV-HT/RVA (WA) VP6(opt)/NOS to BeYDV (m) + replicase amplification System (construct coding) No. 1714)

The optimized sequence encoding VP6 from rotavirus A WA strain WAs cloned into 2X35S/CPMV-HT/NOS containing BeYDV (m) + replicase amplification system in a plasmid containing the Plasto _ pro/P19/Plasto _ ter expression cassette using the PCR-based method described below. A fragment comprising the coding sequence for VP6 WAs amplified using the optimized VP6 gene sequence (SEQ ID NO: 46) as template, using primers IF-WA _ VP6(opt) s1+3c (FIG. 25a, SEQ ID NO: 28) and IF-WA _ VP6(opt) s1-4r (FIG. 25b, SEQ ID NO: 29). For sequence optimization, the VP6 protein sequence (Genbank accession AAA47311) was reverse translated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned into the BeYDV (m) amplification system In the 2X35S/CPMV-HT/NOS expression cassette using the In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct 193 (fig. 22A) was digested with SacII and StuI restriction enzymes and the linearized plasmid was used for In Fusion assembly reactions. Construct No. 193 is a recipient plasmid intended for "In Fusion" cloning of a gene of interest into the BeYDV (m) amplification system In a CPMV-HT based expression cassette. It also introduced a gene construct for co-expression of the TBSV P19 silencing suppressor under the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and FIG. 22B shows this sequence from the left t-DNA border to the right t-DNA border (SEQ ID NO: 26). The resulting construct was numbered 1714 (FIG. 28, SEQ ID NO: 32). FIG. 26 shows the amino acid sequence of VP6 from rotavirus A strain WA (SEQ ID NO: 31). Figure 29 shows a schematic of plasmid 1714.

C-2X35S/CPMV-HT/RVA (Rtx) VP4(opt)/NOS (construct No. 1730)

The optimized sequence encoding VP4 from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain was cloned into 2X35S/CPMV-HT/NOS in a plasmid containing the Plasto _ pro/P19/Plasto _ ter expression cassette using the PCR-based method described below. A fragment comprising the VP4 coding sequence was amplified using the optimized VP4 gene sequence (FIG. 31B, SEQ ID NO: 47) as template, using primers IF-Rtx _ VP4(opt) s1+3c (FIG. 30A, SEQ ID NO: 33) and IF-Rtx _ VP4(opt) s1-4r (FIG. 30B, SEQ ID NO: 34). For sequence optimization, the VP4 protein sequence (Genbank accession number AEX30660) was reverse translated and optimized for human codon usage, GC content and mRNA structure. The PCR products were cloned into the 2X35S/CPMV-HT/NOS expression cassette using the In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct No. 1191 (fig. 18, SEQ ID NO: 23) was digested with SacII and StuI restriction enzymes and the linearized plasmid was used for In Fusion assembly reactions. Construct No. 1191 is an acceptor plasmid intended for the gene of interest for the "In Fusion" clone In the CPMV-HT based expression cassette. It also introduces a gene construct for co-expression of the TBSV P19 silencing suppressor under both the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and FIG. 18 shows this sequence from the left t-DNA border to the right t-DNA border (SEQ ID NO: 23). The resulting construct was numbered 1730 (FIG. 31C, SEQ ID NO: 50). FIG. 32 shows the amino acid sequence of VP4(SEQ ID NO: 36) from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8 ]. FIG. 33A shows a schematic of plasmid 1730.

E-2X35S/CPMV-HT/RVA (Rtx) VP4(opt)/NOS to BeYDV (m) + replicase amplification System (construct) Number 1731)

The optimized sequence encoding VP4 from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain was cloned into 2X35S/CPMV-HT/NOS containing BeYDV (m) + replicase expression system in a plasmid containing the Plasto _ pro/P19/Plasto _ ter expression cassette using the PCR-based method described below. A fragment comprising the sequence encoding VP4 was amplified using the optimized VP4 gene sequence (SEQ ID NO: 47) as template, using primers IF-Rtx _ VP4(opt) s1+3c (FIG. 30A, SEQ ID NO: 33) and IF-Rtx _ VP4(opt) s1-4r (FIG. 30B, SEQ ID NO: 34). For sequence optimization, the VP4 protein sequence (Genbank accession number AEX30660) was reverse translated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned into the BeYDV (m) amplification system In the 2X35S/CPMV-HT/NOS expression cassette using the In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct 193 (fig. 22A) was digested with SacII and StuI restriction enzymes and the linearized plasmid was used for In Fusion assembly reactions. Construct No. 193 is a recipient plasmid intended for "In Fusion" cloning of a gene of interest into the BeYDV (m) amplification system In a CPMV-HT based expression cassette. It also introduced a gene construct for co-expression of a suppressor of TBSV P19 silencing under the promoter and terminator of the alfalfa plastocyanin gene. The backbone is a pCAMBIA binary plasmid, and FIG. 22B shows this sequence from the left t-DNA border to the right t-DNA border (SEQ ID NO: 26). The resulting construct was numbered 1731 (FIG. 31, SEQ ID NO: 35). FIG. 32 shows the amino acid sequence of VP4(SEQ ID NO: 36) from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8 ]. FIG. 33B shows a schematic representation of plasmid 1731.

F-2X35S/CPMV-HT/RVA (Rtx) VP7(opt)/NOS (construct No. 1733)

The optimized sequence encoding VP7 with its native signal peptide from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain was cloned into the 2X35S-CPMV-HT-NOS expression system in a plasmid containing the Plasto _ pro/P19/Plasto _ ter expression cassette using the PCR-based method described below. A fragment comprising the coding sequence of VP7 was amplified using the optimized VP7 gene sequence (SEQ ID NO: 54) as template, and the primers IF-Rtx _ VP7(opt) s1+3c (FIG. 34A, SEQ ID NO: 37) and IF-Rtx _ VP7(opt) s1-4r (FIG. 34B, SEQ ID NO: 38). For sequence optimization, the VP7 protein sequence (Genbank accession number AEX30682) was reverse translated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned into the 2X35S/CPMV-HT/NOS expression system using the In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct No. 1191 (fig. 17C) was digested with SacII and StuI restriction enzymes and the linearized plasmid was used for In Fusion assembly reactions. Construct No. 1191 is an acceptor plasmid intended for "In Fusion" cloning of the gene of interest In the CPMV-HT based expression cassette. It also introduces a gene construct for co-expressing the TBSV P19 silencing suppressor under the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and FIG. 18 shows this sequence from the left t-DNA border to the right t-DNA border (SEQ ID NO: 23). The resulting construct was numbered 1733 (FIG. 34C, SEQ ID NO: 24). FIG. 35 shows the amino acid sequence of VP7 with the native signal peptide from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] (SEQ ID NO: 39). FIG. 36 shows a schematic representation of plasmid 1733.

D-2X35S/CPMV-HT/TrSp-RVA (Rtx) VP7(opt)/NOS (construct No. 1734)

The optimized sequence of VP7 with the truncated form of the native signal peptide from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain was cloned into the 2X35S-CPMV-HT-NOS expression system in a plasmid containing the Plasto _ pro/P19/Plasto _ ter expression cassette using the PCR-based method described below. A fragment comprising the sequence encoding VP7 was amplified using the optimized VP7 gene sequence (corresponding to nt 88-981, SEQ ID NO: 57 in FIG. 44C) as template, using primers IF-TrSP + Rtx _ VP7(opt). s1+3C (FIG. 44A, SEQ ID NO: 55) and IF-Rtx _ VP7(opt). s1-4r (FIG. 44B, SEQ ID NO: 56). For sequence optimization, the VP7 protein sequence (Genbank accession number AEX30682) was reverse translated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned into the 2X35S/CPMV-HT/NOS expression system using the In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct No. 1191 (fig. 17C) was digested with SacII and StuI restriction enzymes and the linearized plasmid was used for In Fusion assembly reactions. Construct No. 1191 is an acceptor plasmid intended for "InFusion" cloning of the gene of interest in the CPMV-HT based expression cassette. It also introduced a gene construct for co-expression of the TBSV P19 silencing suppressor under the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and FIG. 18 shows this sequence from the left t-DNA border to the right t-DNA border (SEQ ID NO: 23). The resulting construct was numbered 1734 (FIG. 44D, SEQ ID NO: 58). FIG. 44E shows the amino acid sequence of VP7 with a truncated signal peptide from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] (SEQ ID NO: 59). FIG. 44F shows a schematic representation of plasmid 1734.

G-2X35S/CPMV-HT/PDISP/RVA (WA) VP7(opt)/NOS to BeYDV (m) + replicase amplification System (construct) Build number 1735)

Using the following PCR-based method, the sequence encoding VP7 from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain was cloned into the 2X35S-CPMV-HT-PDISP-NOS expression system in a plasmid containing the Plasto _ pro/P19/Plasto _ ter expression cassette. Fragments comprising the VP7 coding sequence without his wild-type signal peptide were amplified using the optimized VP7 gene sequence (SEQ ID NO: 54) as template, using primers IF-Rtx _ VP7(opt). s2+4c (FIG. 37A, SEQ ID NO: 40) and IF-Rtx _ VP7(opt). s1-4r (FIG. 34B, SEQ ID NO: 38). For sequence optimization, the VP7 protein sequence (Genbank accession number AEX30682) was reverse translated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned In-frame with the alfalfa PDI signal peptide into the 2X35S/CPMV-HT/NOS expression cassette using the In-Fusion cloning system (Clontech, MountainView, Calif.). Construct 1192 (fig. 38) was digested with SacII and StuI restriction enzymes and the linearized plasmid was used for In Fusion assembly reaction. Construct No. 1192 is an acceptor plasmid intended for "In Fusion" cloning of a gene of interest In frame with the alfalfa PDI signal peptide In a CPMV-HT based expression cassette. It also introduced a gene construct for co-expression of the TBSV P19 silencing suppressor under the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and the t-DNA border of the sequence is shown from left to right in FIG. 39 (SEQ ID NO: 41). The resulting construct was numbered 1735 (FIG. 40, SEQ ID NO: 42). FIG. 41 shows the amino acid sequence of PDISP/VP 7(SEQ ID NO: 43) from rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strain. Fig. 42 shows a schematic of plasmid 1735.

TABLE 3 description of synthetic genes for the production of RLP.

Optimized sequences were modified to favor preferred human codon usage and increase GC content.

TABLE 4 description of constructs assembled and tested for RLP production

Wild-type signal peptide, SpPDI: plant-derived signal peptide cloned from alfalfa protein disulfide isomerase gene, TrSp: a truncated wild-type signal peptide, TrSp, starting from the second Met in WtSp (M30).

Optimized indicates that the sequence has been optimized based on codon usage, GC content and RNA structure.

Example 3

Assembly of Gene constructs and Agrobacterium transformation

All plasmids (including plasmids 1710, 1713, 1730 and 1734) were used to transform Agrobacterium tumefaciens (AGL 1; ATCC, Manassas, VA 20108, USA) by electroporation (Mattanovich et al, 1989, Nucleic Acid Res.17:6747), or alternatively, heat shock could be performed using competent cells prepared by CaCl2 (XU et al, 2008, Plant Methods 4). The integrity of the plasmid in the resulting agrobacterium (a. tumefaciens) strain was confirmed by restriction mapping. The agrobacterium (a. tumefaciens) strain transformed with a given binary plasmid is named AGL 1/"plasmid number". For example, an agrobacterium (a. tumefaciens) strain transformed with construct No. 1710 was named "AGL 1/1710".

Preparation, inoculation, agroinfiltration and harvesting of plant biomass

Tobacco Sedum Semtschaticum (Nicotianabenthamiana) plants were grown from seeds in flat ground filled with commercial peat moss substrate. Plants were grown in the greenhouse under a 16/8 light cycle and a temperature schedule of 25 deg.C during the day/20 deg.C at night. 3 weeks after sowing, individual plants were picked up, transplanted into pots and allowed to grow for a further 3 weeks in the greenhouse under the same environmental conditions.

Agrobacterium transfected with each construct were cultured in plant-derived LB medium supplemented with 10mM 2- (N-morpholine) ethanesulfonic acid (MES) and 50. mu.g/ml kanamycin pH 5.6 until they reached an OD600 between 0.6 and 2.5. Agrobacterium suspensions were mixed to achieve the appropriate ratio for each construct and infiltration medium (10mM MgCl)₂And 10mM MES pH 5.6) to 2.5 XOD 600. Agrobacterium (A. tumefaciens) suspensions were stored overnight at 4 ℃. On the day of infiltration, the batch culture was diluted with infiltration medium in 2.5 suspension volumes and allowed to warm prior to use. Whole plants of Nicotiana benthamiana (N. benthamiana) were inverted for 2 minutes in a bacterial suspension in an air-tight stainless steel jar under vacuum of 20-40 Torr. After infiltration, the plants were returned to the greenhouse and cultured for a period of 3-12 days until harvest. The harvested biomass was kept in a frozen state (-80 ℃) until used for particle purification.

Extraction and purification of rotavirus-like particles

With 3 volumes of extraction buffer (TNC: 10mM Tris pH7.4, 140mM NaCl, 10mM CaCl)₂) Protein is extracted from the frozen biomass by mechanical extraction in a blender. The slurry was filtered through a macroporous nylon filter to remove large debris and centrifuged at 5000g for 5 minutes at 4 ℃. The supernatant was collected and centrifuged again at 5000g for 30 minutes (4 ℃) to remove other debris. The supernatant was depth filtered and ultrafiltered, and the filtrate was centrifuged at 75000g for 20 minutes (4 ℃) to concentrate the rotavirus like particles. The particles containing the particles were resuspended in 1/12 of the volume of TNC and insoluble material was removed by centrifugation at 5000g for 5 minutes. On a microporous clothThe supernatant was filtered before loading it onto an iodixanol density gradient.

Density gradient centrifugation was performed as follows. Tubes containing a step gradient from 5% to 45% iodixanol were prepared and covered with a filtered extract containing rotavirus like particles. The gradient was centrifuged at 120000g for 4 h (4 ℃). After centrifugation, 1ml fractions were collected from bottom to top and analyzed by coomassie stained SDS-PAGE and Western blot. To remove iodixanol from the fractions selected for further analysis, the selected fractions were centrifuged at 75000g for 20 minutes (4 ℃) and the precipitated particles were resuspended in fresh TNC buffer.

SDS-PAGE and immunoblot analysis

Protein concentrations were determined by BCA protein assay (Pierce Biochemicals, Rockport IL). Proteins were separated by SDS-PAGE under reducing or non-reducing conditions and stained with Coomassie blue. The stained gel was scanned and densitometric analysis was performed using ImageJ software (NIH).

For immunoblot analysis, electrophoresed proteins were electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (Roche diagnostics Corporation, Indianapolis, IN). Prior to immunoblot analysis, membranes were blocked with 5% skim milk in Tris buffer (TBS-T) and 0.1% Tween-20 at 4 ℃ for 16-18 hours.

Immunoblot analysis was performed by incubating the appropriate antibodies (table 5) at 2 μ g/ml in 2% skim milk in TBS-tween 200.1%. The respective secondary antibodies used for the chemiluminescence detection were diluted in 2% skim milk in TBS-tween 200.1% as shown in table 5. The immunocompetent complexes were detected by chemiluminescence using luminol (Roche diagnostics corporation) as a substrate. By using EZ-LinkActivation peroxidase conjugation kit (Pierce, Rockford, IL) for horseradish peroxidase-conjugation of human IgG antibodies.

Table 5: electrophoresis conditions, antibodies and dilutions for immunoblot analysis of rotavirus antigens.

anti-VP 4 enzyme-linked immunosorbent assay (ELISA)

U-bottom 96-well microtiter plates were coated with a mouse monoclonal anti-VP 4 (profound by Koki Taniguchi) diluted 1: 100000-fold in 10mM PBS pH7.4 (phosphate buffered saline), 150mM NaCl at 4 ℃ for 16-18 hours. After incubation, plates were washed three times with 10mM PBS pH7.4, 1M NaCl containing 0.1% Tween-20 and blocked with 5% BSA in 10mM PBS pH7.4, 150mM NaCl containing 0.1% Tween-20 at 37 ℃ for 1 hour. After the blocking step, plates were washed three times with 10mM PBS pH7.4, 1M NaCl containing 0.1% Tween-20. Samples were added and the plates were incubated at 37 ℃ for 1 hour. Then, 10mM PBS containing 0.1% Tween-20 pH7.4, 1M NaCl, 1mM CaCl₂、0.5mM MgCl₂The plates were washed 3 times. The wash buffer remained the same for all remaining wash steps, and the plates were incubated for 10 minutes at room temperature during the third wash before complete removal of the wash solution. Add 10mM PBS containing 0.1% Tween-20 pH7.4, 150mM NaCl, 1mM CaCl₂、0.5mM MgCl₂Anti-rotavirus rabbit polyclonal antibody diluted 1:10000 times in 3% BSA (profound by professor Koki Taniguchi) and plates incubated at 37 ℃ for 1 hour. Then, the plate was washed 3 times, and added in containing 0.1% Tween-20 10mM PBS pH7.4, 150mM NaCl, 1mM CaCl₂Horseradish peroxidase-conjugated goat anti-rabbit antibody (111-035-144, Jackson Immunoresearch, West Grove, PA) diluted 1:5000 fold in 3% BSA in (1) and plates incubated for 1 hour at 37 ℃. The plate was washed 3 times. After the final wash, the plates were incubated with SureBlue TMB peroxidase substrate (KPL, Gaithersburg, MD) for 20 minutes at room temperature. The reaction was stopped by adding 1N HCl and Mul was usedA450 value was measured with a tiskanAscent microplate reader (Thermo Scientific, Waltham, Mass.).

Production of rotavirus-like particles comprising VP2 and VP6

Rotavirus-like particles comprising VP2 and VP6 were produced by transient expression in Nicotiana benthamiana (Nicotiana benthamiana). Plants were agroinfiltrated with an agrobacterium inoculum comprising a 1:1 mixture of AGL1/1710 and AGL1/1713 and incubated for 7 days prior to harvest. Rotavirus-like particles are purified from biomass using the methods described in the materials and methods section. The first ten fractions from the bottom of the tube were analyzed by coomassie stained SDS-PAGE after centrifugation of the clarified extract on a iodixanol density gradient. As shown in figure 45A, rotavirus antigens (VP2 and VP6) were found predominantly in density-gradient fractions 2 and 3, with a concentration of iodixanol of about 35%, at which rotavirus-like particles are expected to be found. Very little plant protein contamination was found in these fractions. Western blot analysis of the fractions with anti-rotavirus hyperimmune rabbit serum and polyclonal rabbit anti-VP 2 antibody confirmed the identity of VP2 and VP6 in the density gradient fractions (fig. 45B and 45C). Fractions 2 and 3 were combined, iodixanol removed by high speed centrifugation and resuspension, and the purified particles were sent to cryoelectron microscopy (NanoImagingServices inc., La Jolla, CA) to confirm the assembly of VP2 with VP6 into rotavirus particle-like particles. As shown in figure 49 (left panel), cryoelectron microscopy (cryoEM) images of VP2/VP6 particles confirmed the correct assembly of antigen into rotavirus-like particles.

Production of rotavirus-like particles comprising VP2, VP6 and VP7

Rotavirus-like particles comprising VP2, VP6 and VP7 were produced by transient expression in Nicotiana benthamiana. Agrobacterium plants were infiltrated with an Agrobacterium inoculum comprising a 1:1:1 mixture of AGL1/1710, AGL1/1713, AGL1/1734 and incubated for 7 days prior to harvest. Rotavirus-like particles are purified from biomass using the methods described in the materials and methods section. The first ten fractions from the bottom of the tube were analyzed using coomassie stained SDS-PAGE after centrifugation of the clarified extract on an iodixanol density gradient. As shown in figure 46A, rotavirus antigens (VP2, VP6 and VP7) were found predominantly in density-gradient fractions 2 and 3, with a concentration of ioxatol of about 35%, at which rotavirus-like particles are expected to be found. Very little plant protein contamination was found in these fractions. Western blot analysis of the fractions with anti-rotavirus hyperimmune rabbit serum and polyclonal rabbit anti-VP 7 antibody confirmed the identity of VP6 and VP7 in the density gradient fractions (fig. 46B and 46C).

Production of rotavirus-like particles comprising VP2, VP4, VP6 and VP7

Rotavirus-like particles comprising VP2, VP4, VP6 and VP7 were produced by transient expression in Nicotiana benthamiana (Nicotiana benthamiana). Plants were agroinfiltrated with an Agrobacterium inoculum of a 1:1:1:1 mixture comprising AGL1/1710, AGL1/1730, AGL1/1713, AGL1/1734 and incubated for 7 days prior to harvest. Rotavirus-like particles are purified from biomass using the methods described in the materials and methods section. After centrifugation of the clarified extract on a iodixanol density gradient, the first ten fractions from the bottom of the tube were analyzed using coomassie-stained SDS-PAGE. As shown in figure 47A, 3 of the 4 rotavirus antigens (VP2, VP6 and VP7) were observed, which were found predominantly in fraction 3 of the density gradient with a concentration of iodixanol of about 35%, at which rotavirus-like particles were expected to be found. Very little plant protein contamination was found in these fractions. No detectable level of VP4 was expected in the coomassie stained gel, since VP4 was not observed when the same analysis was performed on purified human rotavirus particles. Western blot analysis of the fractions with anti-rotavirus hyperimmune rabbit serum and polyclonal rabbit anti-VP 7 antibody confirmed the identity of VP6 and VP7 in the density gradient fractions (fig. 47B and 47C). Iodixanol was removed from fraction 3 by high speed centrifugation and re-suspension and the purified particles were analyzed by ELISA to confirm the presence of VP 4. The results shown in figure 48 clearly show that ELISA specifically recognizes VP4, since negative control particles comprising VP2/VP6 and VP7 only resulted in background signal levels. In contrast, analysis of 3 different batches of purified particles comprising VP2, VP4, VP6 and VP7 antigens showed strong and uniform signals when tested under the same conditions. Purified VP2/VP4/VP6/VP7RLP was sent to a cryoelectron microscope for analysis (NanoImaging services inc., La Jolla, CA) to confirm that the four antigens were assembled into particles resembling rotavirus particles. As shown in fig. 49 (right panel), cryoelectron microscopy (cryoEM) images of VP2/VP4/VP6/VP7 particles confirmed the correct assembly of the antigen into rotavirus-like particles.

Table 6 lists sequences provided in various embodiments of the present invention.

Table 6: sequence description of sequence identifier

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All cited documents are incorporated herein by reference.

The invention has been described with respect to one or more embodiments. It will be apparent, however, to one skilled in the art that numerous changes and modifications can be made without departing from the scope of the invention as defined in the claims.

Claims

1. A method of producing a Rotavirus Like Particle (RLP) in a plant, part of a plant or plant cell, the method comprising:

2. A Rotavirus Like Particle (RLP) produced in a plant, part of a plant, or plant cell by a method comprising:

3. The RLP of claim 2, wherein in step a) a fourth nucleic acid comprising a fourth regulatory region active in said plant and operatively linked to a fourth nucleotide sequence encoding a fourth rotavirus structural protein is introduced into said plant, part of a plant or plant cell, and said fourth nucleic acid is expressed when said plant, part of a plant or plant cell is grown in step b).

4. The RLP of claim 2, wherein said first rotavirus structural protein is VP2, said second rotavirus structural protein is VP6, said third rotavirus structural protein is VP4, and said RLP is a double-layer RLP.

5. The RLP of claim 2, wherein said first rotavirus structural protein is VP2, said second rotavirus structural protein is VP6, said third rotavirus structural protein is VP7, and said RLP is a triple-layered RLP.

6. A composition comprising an effective amount of the RLP of any one of claims 2 to 5 and a pharmaceutically acceptable carrier.

7. A method of producing a composition comprising an effective dose of a Rotavirus Like Particle (RLP), the method comprising:

b) growing said plant, plant part or plant cell under conditions that allow transient expression of said first, second and third nucleic acids, thereby producing said RLP,

c) mixing said RLP with a pharmaceutically acceptable carrier.

8. A method of producing a Rotavirus Like Particle (RLP) in a plant, part of a plant or plant cell, the method comprising:

9. A method of producing a composition comprising an effective dose of a Rotavirus Like Particle (RLP), the method comprising:

b) growing said plant, plant part, or plant cell under conditions that allow transient expression of said first, second, and third nucleic acids, thereby producing said RLP;

c) mixing said RLP with a pharmaceutically acceptable carrier.