ROTAVIRUS-LIKE PARTICLE PRODUCTION IN PLANTS

FIELD OF INVENTION

This invention relates to producing rotavirus-like particles in plants.

BACKGROUND OF THE INVENTION

Rotavirus infection is a global problem mainly affecting children under the age of five. It results in severe gastroenteritis and in worst cases death.

Rotaviruses are members of the Reoviridae family of viruses (genus Rotavirus) that affect the gastrointestinal system and respiratory tract. The name is derived from the wheel like appearance of virions when viewed by negative contrast electron microscopy. The rotavirus is usually globular shape and is named after the outer and inner shells or double-shelled capsid structure of the same. The outer capsid is about 70 nm, and inner capsid is about 55 nm in diameter, respectively. The double-shelled capsid of the rotavirus surrounds the core including the inner protein shell and genome. The genome of the rotavirus consists of double stranded RNA segments encoding at least 11 rotavirus proteins—either structural viral proteins (VP) or nonstructural proteins (NSP; Desselberger, Virus Res 190: 75-96 (2014)).

The dsRNA codes for six structural proteins (VP) and six non-structural proteins (NSP). The structural proteins comprise VP1, VP2, VP3, VP4, VP6 and VP7. Three concentric layers are formed by the assembly of VP2, VP6 and VP7 respectively, with VP4 forming “spikes” on the surface of the virus structure. VP4 is cleaved by trypsin to VP8* and VP5*. VP8* and VP5* are proteolytic products of VP4.

VP2 is a 102 kDa protein and is the most abundant protein of the viral core. It forms the inner-most structural protein layer and provides a scaffold for the correct assembly of the components and transcription enzymes of the viral core (Lawton, 2000). VP1, the largest viral protein at 125 kDa, acts as an RNA-dependent polymerase for rotavirus, creating a core replication intermediate, and associates with VP2 at its icosahedral vertices (Varani and Allain, 2002; Vende et al., 2002). VP3, a 98 kDa protein, is also directly associated with the viral genome, acting as an mRNA capping enzyme that adds a 5′ cap structure to viral mRNAs. Together, VP1 and VP3 form a complex that is attached to the outer 5-fold vertices of the VP2 capsid layer (Angel, 2007). VP6 is a 42 kDa protein which forms the middle shell of the viral core, is the major capsid protein and accounts for more than 50% of the total protein mass of the virion (González et al., 2004; Estes, 1996). It is required for gene transcription and may have a role in encapsulation of the rotavirus RNA by anchoring VP1 to VP2 in the core, as seen in 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 commonly affecting humans (Palombo, 1999). VP6 in rotavirus group A has at least four subgroups (SG), which depend on the presence or absence of SG specific epitopes: SG I, SG II, SG (I+II) and SG non-(I+II). Groups B and C lack a common group A antigen but are also known to infect humans, while group D only affects animals e.g. chickens and cows (Thongprachum, 2010).

The two outer capsid proteins VP7, a 37 kDa glycoprotein (G) and the 87 kDa protease sensitive VP4 (P), define the virus' serotypes. These two proteins induce neutralizing antibody responses and are thus used to classify rotavirus serotypes into a dual nomenclature system, depending on the G-P antigen combination (e.g. G1 P[8] or G2 P[4]) (Sanchez-Padilla et al., 2009, Rahman et al., J Clin Microbiol 41: 2088-2095 (2003)). The VP4 protein dimerizes to form 60 spikes on the outer shell of the virus, which are directly involved in the initial stages of host cell entry. The spike protein contains a cleavage site at amino acid (aa) position 248. Upon infection, it is cleaved by the protease trypsin to produce VP5 (529 aa, 60 kDa) and VP8 (246 aa, 28 kDa) (Denisova et al., 1999). This process enhances virus infectivity (cell attachment and invasion of host cell) and stabilizes the spike structure (Glass, 2006). The VP7 glycoprotein forms the third or outside layer of the virus. At present, 27 G and 35 P genotypes are known (Greenberg and Estes, 2009). VP4 and VP7 are the major antigens involved in virus neutralization and are important targets for vaccine development (Dennehy, 2007).

The non-structural proteins (NSPs) are synthesized in infected cells and function in various parts of the replication cycle or interact with some of the host proteins to influence pathogenesis or the immune response to infection (Greenberg and Estes, 2009). The rotavirus nonstructural protein, NSP4, has been shown to have multiple functions including the release of calcium from the endoplasmic reticulum (ER; Tian et al, 1995); the disruption of the ER membranes and may play an important role in the removal of the transient envelope from budding particles during viral morphogenesis (see FIG. 1); affecting membrane trafficking from the ER to the Golgi complex with its ability to bind to micro tubules (Xu et al 2000); and function as an intracellular receptor to aid in the budding of subviral particles into the ER (Tian et al 1996).

In infected mammalian cells, rotaviruses undergo a unique mode of morphogenesis to form the complete triple-layered VP2/6/4/7 viral particles (Lopez et al., 2005). The triple-layer capsid is a very stable complex which enables faecal-oral transmission and delivery of the virus into the small intestine where it infects non-dividing differentiated enterocytes near the tips of the villi (Greenberg and Estes, 2009). Firstly, the intact virus attaches to sialic acid-independent receptors via 60 VP4 dimer spikes on the surface of the virus (Lundgren and Svensson, 2001). The 60 VP4 dimer spikes on the surface of the virus allow the virus to attach to these cell receptors. VP4 is susceptible to proteolytic cleavage by trypsin which results in a conformational change that exposes additional attachment sites on the surface of the glycoprotein for interaction with a series of co-receptors.

The multi-step attachment and entry process is, however, not clearly understood but the virus is delivered across the host's plasma membrane. The VP7 outer capsid shell which is also involved in the entry process, is removed in the process and double-layered particles (DLP) are delivered into the cell cytoplasm in vesicles (FIG. 1; prior art). The DLP escapes from the vesicle and goes into non-membrane bound cytoplasmic inclusions. Early transcription of the genome by VP1 begins in particles so that dsRNA is never exposed to the cytoplasm. RNA replication and core formation takes place in these non-membrane-bound cytoplasmic inclusions. The nascent (+) RNAs are then transported into the cytoplasm and serve as templates 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 and VP6 are produced and assemble in the cytosol in virosomes and subsequently bud into the RER compartments, receiving a transient membrane envelope in the process (Lopez et al., 2005; Tian et al., 1996). In the RER, the transient envelopes of the viral particles are removed and replaced by VP4 and VP7 protein monomers, with critical involvement of rotaviral glycoprotein NSP4 (Tian et al., 1996; Lopez et al., 2005; Gonzalez et al., 2000). NSP4 functions as an intracellular receptor in the ER membrane and binds newly made subviral particles and probably also the spike protein VP4 (Tian et al., 1996). NSP4 is also toxic to humans and is the causative agent of the diarrhea. The complete, mature particles are subsequently transferred from the RER through the Golgi apparatus to the plasma membrane for secretion (Lopez et al., 2005).

A variety of different approaches have been taken to generate a rotavirus vaccine suitable to protect human populations from the various serotypes of rotavirus. These approaches include various Jennerian approaches, use of live attenuated viruses, use of virus-like particles, nucleic acid vaccines and viral sub-units as immunogens. At present there are two oral vaccines available on the market, however, these have low efficacy in due to strain variation.

U.S. Pat. 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 a variety of rotavirus vaccines and/or methods of preparing these vaccines, where the whole viral particles is used to create each of the rotavirus vaccines.

Production of rotavirus-like particles is a challenging task, as both the synthesis and assembly of one or more recombinant proteins are required. Rotavirus comprises a capsid formed by 1860 monomers of four different proteins. For RLP production the simultaneous expression and assembly of two to three recombinant proteins may be required. For example, an inner layer comprising 120 molecules of VP2, 780 molecules of VP6 (middle layer) and an outer layer of 780 molecules of the glycoprotein VP7 and 60 VP4 dimers, to form a double or triple-layered particle (Libersou et al. J. of Virology, March 2008).

Crawford et al. (J Virol. 1994 September; 68(9): 5945-5952) describe the expression of VP2, VP4, VP6, and VP7 in a baculovirus expression system. Co-expression of different combinations of the rotavirus major structural proteins resulted in the formation of stable virus-like particles (VLPs). The co-expression of VP2 and VP6 alone or with VP4 resulted in the production of VP2/6 or VP2/4/6 VLPs, which were similar to double-layered rotavirus particles. Co-expression of VP2, VP6, and VP7, with or without VP4, produced triple-layered VP2/6/7 or VP2/4/6/7 VLPs, which were similar to native infectious rotavirus particles. The VLPs maintained the structural and functional characteristics of native particles, as determined by electron microscopic examination of the particles, the presence of non-neutralizing and neutralizing epitopes on VP4 and VP7, and hemagglutination activity of the VP2/4/6/7 VLPs.

Vaccine candidates generated from rotavirus-like particles of different protein compositions have shown potential as subunit vaccines. O'Neal et al. (J. Virology, 1997, 71(11):8707-8717) show that VLPs containing VP 2 and VP6, or VP2, VP6, and VP7, and administered to mice with and without the addition of cholera toxin induced protective immunity in immunized mice. Core-like particles (CLP) and VLPs have also been used to immunize cows with VLPs more effective than CLPs in inducing passive immunity Fernandez, et al., (Vaccine, 1998, 16(5):507-516).

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

Saldana et al. (Viral Immunol. 19: 42-53 (2006)) expressed VP2 and VP6 in the cytoplasm of tomato plants. Electron microscopy studies showed that a small proportion of the proteins had assembled into 2/6 VLPs. A protective immune response was detected in mice and this may have to some extent been contributed by the non-assembled VPs. Individual proteins have been shown to elicit immune responses in mice, as in the case of VP8 and VP6 (Rodriguez-Diaz et al. Biotechnol Lett. 2011, 33(6):1169-75, Zhou et al., Vaccine 28: 6021-6027 (2010)).

Matsumura et al., (Archives of Virology 147: 1263-1270 (2002)) report bovine rotavirus A VP6 expression in transgenic potato plants. The VP6 was expressed, purified and immunogenic studies performed Immune-response in adult mice showed presence of VP6 antibodies in the sera. However, no evidence of assembled VP6 proteins was provided. It may have been that monomers or trimers of VP6 were responsible for eliciting the immune response. O'Brien et al. (2000, Virol. 270: 10444-10453) show VP6 assembly in Nicotiana benthamiana using a potato virus X (PVX) vector. Assembly of VP6 protein into icosahedral VLPs was only observed when the VP6 was fused to the PVX protein rods. Following cleavage the VP6 assembled into the icosahedral VLPs.

Codon-optimized human rotavirus VP6 has been successfully expressed in Chenopodium amaranticolor using a Beet black scorch virus (BBSV) mediated expression system. The protein was engineered as a replacement to the coat protein of BBSV. Oral immunization of female BALB/c mice with the plant based VP6 protein induced high titers of anti-VP6 mucosal IgA and serum IgG (Zhou et al., Vaccine 28: 6021-6027 (2010)). However, there was no teaching that the VP6 proteins assembled into VLPs or particles.

Rotavirus VP7 has been expressed in potato plants and was shown to produce a neutralizing immune response in mice (Yu and Langridge, 2001 Nature Biotechnol 19: 548-552). In transgenic potato plants, the VP7 gene was stable over 50 generations, with the VP7 protein from the 50th generation induced both protective and neutralizing antibodies in adult mice (Li et al., 2006, Virol 356:171-178).

Yang et al. (Yang Y M, Li X, Yang H, et al. Science China Life Science 54: 82-89 (2011)) co-expressed three rotavirus capsid proteins VP2, VP6 and VP7 of group A RV (P[8]G1) in tobacco plants and expression levels of these proteins, as well as formation of rotavirus-like particles and immunogenicity were studied. VLPs were purified from transgenic tobacco plants and analyzed by electron microscopy and Western blot. These results indicate that the plant derived VP2, VP6 and VP7 protein self-assembled into 2/6 or 2/6/7 rotavirus like particle with a diameter of 60-80 nm.

WO 2013/166609 described the production of rotavirus-like particle (RLPs) in plants, by co-expressing rotavirus structural proteins VP2, VP4, VP6 and VP7 in plants and purifying the resulting RLPs in the presence of calcium.

Rotavirus NSP4 has been expressed and purified from insect cells (Tian et al. 1996, Arch Virol. 1996; Rodriguez-Diaz et al. Protein Expr. Purif. 2003) and in E. coli (Sharif et al. Medical Journal of the Islamic Republic of Iran 2003). NSP4 has also been expressed as a fusion protein with the cholera toxin B (CTB) subunit in potato (Arakawa et al., Plant Cell Report 20 : 343-348 (2001)).

SUMMARY OF THE INVENTION

The present invention relates to producing rotavirus-like particles in plants.

It is an object of the invention to produce rotavirus-like particles in plants.

Several methods to produce a rotavirus like particle (RLP) in a plant, portion of a plant or plant cell are described.

For example, a method (A) for producing a rotavirus like particle (RLP) in a host or host cell may comprise:

a) providing a host or host cell comprising one or more nucleic acid comprising a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein and a third nucleotide sequence encoding a third rotavirus protein, the first, second and third nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
the first nucleotide sequence encoding rotavirus protein NSP4, the second nucleotide sequence encoding rotavirus protein VP6, and the third nucleotide sequence encoding one of rotavirus protein VP7 or VP4;
b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid, so that each of NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the RLP.

In the method (A) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein, the first, second, third, and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first nucleotide sequence encoding rotavirus protein NSP4, the second nucleotide sequence encoding rotavirus protein VP6, and the third and fourth nucleotide sequence encoding rotavirus protein VP2, VP4 or VP7 and wherein each of NSP4, VP6 and two of VP2, VP4 and VP7 are expressed from the one or more nucleic acid.

A method (B) to produce a rotavirus like particle (RLP) in a host or host cell is further described, the method may comprise:

a) providing a host or host cell comprising one or more nucleic acid comprising
a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein and a third nucleotide sequence encoding a third rotavirus protein, the first, second and third nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
the first, second and third nucleotide sequence encoding one of rotavirus protein NSP4, VP6 and one of rotavirus protein VP7 or VP4;
b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid, so that each of NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the RLP.

In the method (B) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein, the first, second, third, and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first, second, third and fourth nucleotide sequence encoding one of rotavirus protein NSP4, VP6 and two of rotavirus protein VP2, VP7 or VP4 and wherein each of NSP4, VP6 and two of VP2, VP7 or VP4 are expressed from the one or more nucleic acid.

In the method (A) and (B) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein and a fifth nucleotide sequence encoding a fifth rotavirus protein, the first, second, third, fourth and fifth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first, second, third, fourth and fifth nucleotide sequence encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the one or more nucleic acid.

In the method (A) or (B) as described above, if a host or host cell is provided where the one or more nucleic acid comprising a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein, a third nucleotide sequence encoding a third rotavirus protein, fourth nucleotide sequence encoding a fourth rotavirus protein and a fifth nucleotide sequence encoding a fifth rotavirus protein, the first, second, third, fourth and fifth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell, then the one or more nucleic acid may comprise the first, second, third, fourth and fifth nucleotide sequence encoding the first, second, third, fourth and fifth rotavirus protein, or the one or more nucleic acid may comprise for example two nucleic acids, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, and a second nucleic acid comprising the second, third, fourth and fifth nucleotide sequence encoding the second, third, fourth and fifth rotavirus protein, or the one or more nucleic acid may comprise for example two nucleic acids, a first nucleic acid comprising the first and second nucleotide sequence encoding the first and second rotavirus protein, and a second nucleic acid comprising the third, fourth and fifth nucleotide sequence encoding the third, fourth and fifth rotavirus protein or the one or more nucleic acid may comprise for example three nucleic acids, a first nucleic acid comprising the first and second nucleotide sequence encoding the first and second rotavirus protein, a second nucleic acid comprising the third and fourth nucleotide sequence encoding the third and fourth rotavirus protein, and a third nucleic acid comprising the fifth nucleotide sequence encoding the fifth rotavirus protein or the one or more nucleic acid may comprise for example three nucleic acids, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, and a third nucleic acid comprising the third, fourth and fifth nucleotide sequence encoding the third, fourth and fifth rotavirus protein or the one or more nucleic acid may comprise for example four nucleic acids, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, a third nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein, and a fourth nucleic acid comprising the fourth and fifth nucleotide sequence encoding the fourth and fifth rotavirus protein or the one or more nucleic acid may comprise for example five nucleic acids, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, a third nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein, a fourth nucleic acid comprising the fourth nucleotide sequence encoding the fourth rotavirus protein, and a fifth nucleic acid comprising the fifth nucleotide sequence encoding the fifth rotavirus protein.

The methods (A) or (B) as described above may further comprise the steps of:

- c) harvesting the host or host cell, and
- d) purifying the RLPs from the host or host cell, wherein the RLPs range in size from 70-100 nm.

The one or more nucleotide sequence of the method (A) or (B) as described above may be operatively linked to one or more expression enhancer. Furthermore, the expression enhancer may be selected from the group consisting of CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+.

Also described herein is a method (C) of producing a rotavirus like particle (RLP) in host or host cell comprising:

a) introducing into the host or host cell one or more nucleic acid comprising
a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein and a third nucleotide sequence encoding a third rotavirus protein, the first, second and third nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
the first nucleotide sequence encoding rotavirus protein NSP4, the second nucleotide sequence encoding rotavirus protein VP6, and the third nucleotide sequence encoding one of rotavirus protein VP7 or VP4;
b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid so that each of NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the RLP.

In the method (C) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein, the first, second, third, and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first nucleotide sequence encoding rotavirus protein NSP4, the second nucleotide sequence encoding rotavirus protein VP6, and the third and fourth nucleotide sequence encoding rotavirus protein VP2, VP4 or VP7 and wherein each of NSP4, VP6 and two of VP2, VP4 and VP7 are expressed from the one or more nucleic acid.

Also described herein is a method (D) of producing a rotavirus like particle (RLP) in host or host cell comprising:

a) introducing into the host or host cell one or more nucleic acid comprising
a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein and a third nucleotide sequence encoding a third rotavirus protein, the first, second and third nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
the first, second and third nucleotide sequence encoding one of rotavirus protein NSP4, VP6 and one of rotavirus protein VP7 or VP4;
b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid so that each of NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the RLP.

In the method (D) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein, the first, second, third, and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first, second, third and fourth nucleotide sequence encoding one of rotavirus protein NSP4, VP6 and two of rotavirus protein VP2, VP7 or VP4 and wherein each of NSP4, VP6 and two of VP2, VP7 or VP4 are expressed from the one or more nucleic acid.

In the method (C) and (D) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein and a fifth nucleotide sequence encoding a fifth rotavirus protein, the first, second, third, fourth and fifth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first, second, third, fourth and fifth nucleotide sequence encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the one or more nucleic acid.

The methods (C) or (D) as described above may further comprise the steps of:

c) harvesting the host or host cell, and
d) purifying the RLPs from the host or host cell, wherein the RLPs range in size from 70-100 nm.

In the method (C) or (D) as described above, if a host or host cell is provided where the one or more nucleic acid comprising a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein, a third nucleotide sequence encoding a third rotavirus protein, fourth nucleotide sequence encoding a fourth rotavirus protein and a fifth nucleotide sequence encoding a fifth rotavirus protein, the first, second, third, fourth and fifth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell, then in the step of introducing (step a), the one or more nucleic acid may comprise two nucleic acids, with a first nucleic acid comprising encoding the NSP4, and a second nucleic acid encoding VP2, VP4, VP6 and VP7, the ratio of an amount of the first nucleic acid relative to the second nucleic acid that is introduced into the plant, portion of a plant or plant cell is between 1:0.8 and 1:2. The ratio may also be 1:1. Alternatively, the one or more nucleic acid may comprise two nucleic acids, with a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein and the second nucleotide sequence encoding the second rotavirus protein, and a second nucleic acid comprising the third to fifth nucleotide sequence encoding the third to fifth rotavirus protein.

In the method (C) or (D) as described above, in the step of introducing (step a), the one or more nucleic acid may comprise three nucleic acids, with a first nucleic acid comprising the first and the second nucleotide sequence encoding the first and the second rotavirus protein, a second nucleic acid comprising the third and fourth nucleotide sequence encoding the third and the fourth rotavirus protein, and a third nucleic acid comprising the fifth nucleotide sequence encoding the fifth rotavirus protein, and wherein the ratio of an amount of the first nucleic acid relative to the second nucleic acid and to the third nucleic that is introduced into the plant, the portion of the plant, or the plant cell, is 1:1:1.

Alternatively, in the method (C) or (D) as described above, in the step of introducing (step a), the one or more nucleic acid may comprise three nucleic acids, with the first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, the second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, and the third nucleic acid comprising the third to fifth nucleotide sequence encoding the third to fifth rotavirus protein, and wherein the ratio of an amount of the first nucleic acid relative to the second nucleic acid and to the third nucleic that is introduced into the plant, the portion of the plant, or the plant cell, is 1:1:1.

Furthermore, in method (C) or (D) described above, in the step of introducing (step a), the one or more nucleic acid may comprise four nucleic acids, a first nucleic acid comprising the first nucleic acid encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, a third nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein, and a fourth nucleic acid comprising the fourth and the fifth nucleotide sequence encoding the fourth and fifth rotavirus protein, wherein the ratio of an amount of the first nucleic acid relative to the second nucleic acid, to the third nucleic and to the fourth nucleic acid that is introduced into the plant, the portion of the plant, or the plant cell, is 1:1:1:1.

In the method (C) or (D) as described above, in the step of introducing (step a), the one or more nucleic acid may comprise five nucleic acids, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, a third nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein, a fourth nucleic acid comprising the fourth nucleotide sequence encoding the fourth rotavirus protein, and a fifth nucleic acid comprising the fifth nucleotide sequence encoding the fifth rotavirus protein, and the ratio of an amount of the first nucleic acid relative to the second nucleic acid, to the third nucleic acid, to the fourth nucleic acid and to the fifth nucleic acid that is introduced into the plant, the portion of the plant, or the plant cell, is 1:1:1:1:1.

The one or more nucleotide sequence of the method (C) or (D) as described above may be operatively linked to one or more expression enhancer. Furthermore, the expression enhancer may be selected from the group consisting of CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+.

A method (E) of increasing incorporation of VP4, VP7, or both VP4 and VP7 in a rotavirus like particle (RLP) is also described, the method comprising:

- a) providing a host or host cell comprising one or more nucleic acid comprising
- a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein and a third nucleotide sequence encoding a third rotavirus protein, the first, second and third nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
- the first nucleotide sequence encoding rotavirus protein NSP4, the second nucleotide sequence encoding rotavirus protein VP6, and the third nucleotide sequence encoding one of rotavirus protein VP7 or VP4;
- b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid, so that each of NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when compared to the level of VP4 or VP7 produced by a second host or host cell that expresses the one or more nucleic acid that does not comprise NSP4, under the same conditions.

In the method (E) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein, the first, second, third, and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first nucleotide sequence encoding rotavirus protein NSP4, the second nucleotide sequence encoding rotavirus protein VP6, and the third and fourth nucleotide sequence encoding rotavirus protein VP2, VP4 or VP7 and wherein each of NSP4, VP6 and two of VP2, VP4 and VP7 are expressed from the one or more nucleic acid.

A method (F) of increasing incorporation of VP4, VP7, or both VP4 and VP7 in a rotavirus like particle (RLP) is also described, the method comprising:

- a) providing a host or host cell comprising one or more nucleic acid comprising
a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein and a third nucleotide sequence encoding a third rotavirus protein, the first, second and third nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
the first, second and third nucleotide sequence encoding one of rotavirus protein NSP4, VP6 and one of rotavirus protein VP7 or VP4;
- b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid, so that each of NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when compared to the level of VP4 or VP7 produced by a second host or host cell that expresses the one or more nucleic acid that does not comprise NSP4, under the same conditions.

In the method (F) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein, the first, second, third, and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first, second, third and fourth nucleotide sequence encoding one of rotavirus protein NSP4, VP6 and two of rotavirus protein VP2, VP7 or VP4 and wherein each of NSP4, VP6 and two of VP2, VP7 or VP4 are expressed from the one or more nucleic acid.

In the method (E) and (F) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein and a fifth nucleotide sequence encoding a fifth rotavirus protein, the first, second, third, fourth and fifth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first, second, third, fourth and fifth nucleotide sequence encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the one or more nucleic acid.

The one or more nucleotide sequence of the method (E) or (F) as described above may be operatively linked to one or more expression enhancer. Furthermore, the expression enhancer may be selected from the group consisting of CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+.

A method (G) of increasing incorporation of VP4, VP7, or both VP4 and VP7 in a rotavirus like particle (RLP) is also described, the method comprising:

- a) introducing into a host or host cell one or more nucleic acid comprising
- a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein and a third nucleotide sequence encoding a third rotavirus protein, the first, second and third nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
- the first nucleotide sequence encoding rotavirus protein NSP4, the second nucleotide sequence encoding rotavirus protein VP6, and the third nucleotide sequence encoding one of rotavirus protein VP7 or VP4;
- b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid, so that each of NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when compared to the level of VP4 or VP7 produced by a second host or host cell that expresses the one or more nucleic acid that does not comprise NSP4, under the same conditions.

In the method (G) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein, the first, second, third, and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first nucleotide sequence encoding rotavirus protein NSP4, the second nucleotide sequence encoding rotavirus protein VP6, and the third and fourth nucleotide sequence encoding rotavirus protein VP2, VP4 or VP7 and wherein each of NSP4, VP6 and two of VP2, VP4 and VP7 are expressed from the one or more nucleic acid.

A method (H) of increasing incorporation of VP4, VP7, or both VP4 and VP7 in a rotavirus like particle (RLP) is also described, the method comprising:

- a) introducing in a host or host cell comprising one or more nucleic acid comprising
a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein and a third nucleotide sequence encoding a third rotavirus protein, the first, second and third nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
the first, second and third nucleotide sequence encoding one of rotavirus protein NSP4, VP6 and one of rotavirus protein VP7 or VP4;
- b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid, so that each of NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when compared to the level of VP4 or VP7 produced by a second host or host cell that expresses the one or more nucleic acid that does not comprise NSP4, under the same conditions.

In the method (H) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein, the first, second, third, and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first, second, third and fourth nucleotide sequence encoding one of rotavirus protein NSP4, VP6 and two of rotavirus protein VP2, VP7 or VP4 and wherein each of NSP4, VP6 and two of VP2, VP7 or VP4 are expressed from the one or more nucleic acid.

In the method (G) and (H) as described above the one or more nucleic acid may further comprises a fourth nucleotide sequence encoding a fourth rotavirus protein and a fifth nucleotide sequence encoding a fifth rotavirus protein, the first, second, third, fourth and fifth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and

the first, second, third, fourth and fifth nucleotide sequence encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the one or more nucleic acid.

the first, second, third, fourth and fifth nucleotide sequence encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the one or more nucleic acid.

In the method (G) or (H) as described above, in the step of introducing (step a), the one or more nucleic acid may comprise two nucleic acids, with a first nucleic acid comprising encoding the NSP4, and a second nucleic acid encoding VP2, VP4, VP6 and VP7, the ratio of an amount of the first nucleic acid relative to the second nucleic acid that is introduced into the plant, portion of a plant or plant cell is between 1:0.8 and 1:2. The ratio may also be 1:1. Alternatively, the one or more nucleic acid may comprise two nucleic acids, with a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein and the second nucleotide sequence encoding the second rotavirus protein, and a second nucleic acid comprising the third to fifth nucleotide sequence encoding the third to fifth rotavirus protein.

In the method (G) or (H) as described above, in the step of introducing (step a), the one or more nucleic acid may comprise three nucleic acids, with a first nucleic acid comprising the first and the second nucleotide sequence encoding the first and the second rotavirus protein, a second nucleic acid comprising the third and fourth nucleotide sequence encoding the third and the fourth rotavirus protein, and a third nucleic acid comprising the fifth nucleotide sequence encoding the fifth rotavirus protein, and wherein the ratio of an amount of the first nucleic acid relative to the second nucleic acid and to the third nucleic that is introduced into the plant, the portion of the plant, or the plant cell, is 1:1:1.

Alternatively, in the method (G) or (H) as described above, in the step of introducing (step a), the one or more nucleic acid may comprise three nucleic acids, with the first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, the second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, and the third nucleic acid comprising the third to fifth nucleotide sequence encoding the third to fifth rotavirus protein, and wherein the ratio of an amount of the first nucleic acid relative to the second nucleic acid and to the third nucleic that is introduced into the plant, the portion of the plant, or the plant cell, is 1:1:1.

Furthermore, in the method (G) or (H) described above, in the step of introducing (step a), the one or more nucleic acid may comprise four nucleic acids, a first nucleic acid comprising the first nucleic acid encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, a third nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein, and a fourth nucleic acid comprising the fourth and the fifth nucleotide sequence encoding the fourth and fifth rotavirus protein, wherein the ratio of an amount of the first nucleic acid relative to the second nucleic acid, to the third nucleic and to the fourth nucleic acid that is introduced into the plant, the portion of the plant, or the plant cell, is 1:1:1:1.

In the method in the method (G) or (H) as described above, in the step of introducing (step a), the one or more nucleic acid may comprise five nucleic acids, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, a third nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein, a fourth nucleic acid comprising the fourth nucleotide sequence encoding the fourth rotavirus protein, and a fifth nucleic acid comprising the fifth nucleotide sequence encoding the fifth rotavirus protein, and the ratio of an amount of the first nucleic acid relative to the second nucleic acid, to the third nucleic acid, to the fourth nucleic acid and to the fifth nucleic acid that is introduced into the plant, the portion of the plant, or the plant cell, is 1:1:1:1:1.

The one or more nucleotide sequence of the method (G) or (H) as described above may be operatively linked to one or more expression enhancer. Furthermore, the expression enhancer may be selected from the group consisting of CPMV HT, CPM 160, CPMV 160+ and CPMV HT+.

The methods (G) or (H) as described above may further comprise the steps of:

c) harvesting the host or host cell, and
d) purifying the RLPs from the host or host cell, wherein the RLPs range in size from 70-100 nm.

In the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above, the one or more nucleic acid may comprise two nucleic acids, for example, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, and a second nucleic acid comprising the second to fifth nucleotide sequence encoding the second to fifth rotavirus protein. Alternatively, the one or more nucleic acid may comprise two nucleic acids, with a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein and the second nucleotide sequence encoding the second rotavirus protein, and a second nucleic acid comprising the third to fifth nucleotide sequence encoding the third to fifth rotavirus protein.

In the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above, the one or more nucleic acid may also comprise three nucleic acids, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, the second nucleotide sequence encoding the second rotavirus protein, a second nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein and fourth nucleotide sequence encoding the fourth rotavirus protein, and a third nucleic acid comprising the fifth nucleotide sequence encoding the fifth rotavirus protein. Alternatively, the one or more nucleic acid may comprise three nucleic acids, with a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, and a third nucleic acid comprising the third to fifth nucleotide sequence encoding the third to fifth rotavirus protein.

In the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above the one or more nucleic acid comprises four nucleic acids, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, a third nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein, and a fourth nucleic acid comprising the fourth and fifth nucleotide sequence encoding the fourth and fifth rotavirus protein.

Furthermore, in the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above the one or more nucleic acid may comprise five nucleic acids, with a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein, a third nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein, a fourth nucleic acid comprising the fourth nucleotide sequence encoding the fourth rotavirus protein, and a fifth nucleic acid comprising the fifth nucleotide sequence encoding the fifth rotavirus protein.

The one or more nucleotide sequence of the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above may be operatively linked to one or more expression enhancer. Furthermore, the expression enhancer may be selected from the group consisting of CPMV HT, CPM 160, CPMV 160+ and CPMV HT+.

In the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above the host or host cell may comprise insect cells, mammalian cells, plant, portion of a plant or plant cells. The plant may be Nicotiana benthamiana.

Also described herein is an RLP produced by the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) described above, wherein the RLP is a triple layered RLP comprising rotavirus protein, the rotavirus protein consists of VP2, VP4, VP6 and VP7. The RLP may not comprise NSP4.

A composition comprising an effective dose of the RLP for inducing an immune response in a subject, and a pharmaceutically acceptable carrier, and a method of inducing immunity to a rotavirus infection in a subject, that comprises administering the composition, are also described. In the method of inducing immunity, the composition may be administered to a subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

Also described herein is plant matter comprising an RLP produced by the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above.

In the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above, the one or more nucleic acid may comprise one nucleic acid comprising the first, second and third nucleotide sequence encoding the first, second and third, rotavirus protein. Furthermore, in the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above the one or more nucleic acid may comprise two nucleic acids, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, and a second nucleic acid comprising the second and third nucleotide sequence encoding the second and third rotavirus protein. Furthermore, in the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above the one or more nucleic acid may comprise two nucleic acids, a first nucleic acid comprising the first and second nucleotide sequence encoding the first and second rotavirus protein, and a second nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein. Alternatively, in the method (A), the method (B), the method (C), the method (D), the method (E), the method (F), the method (G) or the method (H) as described above the one or more nucleic acid may comprise three nucleic acids, a first nucleic acid comprising the first nucleotide sequence encoding the first rotavirus protein, a second nucleic acid comprising the second nucleotide sequence encoding the second rotavirus protein and a third nucleic acid comprising the third nucleotide sequence encoding the third rotavirus protein.

As described herein, by co-expressing NSP4 along with VP6 and VP4 or VP7, in a host or host cell, for example a plant, portion of the plant, or a plant cell, RLPs comprising increased levels of VP4, VP7, or both VP4 and VP7 are observed, when compared to the level of VP4 and VP7 in RLPs produced by a second host or host cell for example a plant, portion of a second plant, or second plant cell, that expresses the one or more nucleic acid that encodes VP6 and VP4 or VP7, and does not encode NSP4, the second host or second host cell for example a second plant, the second portion of plant, or the second plant cell, incubated or grown, under the same conditions as the host or host cell for example a plant, portion of the plant, or plant cell.

This summary of the invention does not necessarily describe all features of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

FIG. 1 shows rotavirus cell entry and replication. When rotavirus enters a cell, VP4 and VP7 are lost, forming a double layered particle (DLP). Transcription of the dsRNA commences resulting in translation of VP2, VP4, VP6 and VP7. Progeny cores with replicase activity are produced in virus factories (also called viroplasms). Late transcription occurs in these progeny cores. At the periphery of virus factories, these core are coated with VP6, forming immature DLPs that bud across the membrane of the endoplasmic reticulum, acquiring a transient lipid membrane which is modified with the ER resident viral glycoproteins NSP4 and VP7; these enveloped particles also contain VP4. As the particles move towards the interior of the ER cisternae, the transient lipid membrane and the nonstructural protein NSP4 are lost, while the virus surface proteins VP4 and VP7 rearrange to form the outermost virus protein layer, yielding mature infectious triple-layered particles (see Swiss Institute of Bioinformatics (ViralZone): viralzone.expasy.org/viralzone/all_by_species/107.html)

FIG. 2 shows rotavirus-like particle purification by ultracentrifugation on iodixanol density gradient. FIG. 2A presents the percentage of iodixanol and volume for each layer of the gradient used for the purification of rotavirus-like particles. After centrifugation, the gradient was fractionated into 1 ml fractions starting from the bottom of the tube. The approximate localization of fractions 1 to 13 are indicated by arrows. FIG. 2B shown a Coomassie-stained SDS-PAGE analysis of the protein content of fractions 1 to 10 from an iodixanol density gradient separation applied to a crude protein extract from leaves expressing rotavirus VP2, VP4, VP6 and VP7 antigens.

FIG. 3 shows rotavirus protein expression. FIG. 3A shows a Coomassie-stained SDS-PAGE analysis of fractions 2 and 3 from an iodixanol density gradient separation applied to crude protein extracts from leaves expressing VP2, VP4, VP6, VP7 in the presence or absence of NSP4. Rotavirus structural proteins VP2, VP6, VP7, VP4 were expressed, left panel, using individual constructs for each structural antigen (“single gene constructs”), with NSP4 on a separate construct; middle panel, two constructs, each having the genes of two structural antigens (“dual gene constructs”), with NSP4 on a separate construct; or right panel, a single construct for the co-expression of the four structural antigens (Quadruple gene constructs), with a separate construct for the expression of NSP4. Position of the rotavirus VP2 and VP6 antigen are shown by arrows. FIG. 3B shows a Western blot analysis of fraction F2 from the same treatments as in FIG. 3A using an anti-rotavirus VP4 or VP7 antibody as specified.

FIG. 4 shows rotavirus protein expression in the presence of an expression enhancer. FIG. 4A shows a Coomassie-stained SDS-PAGE analysis of fractions F2 and F3 from an iodixanol density gradient applied to crude protein extracts from leaves co-expressing VP2, VP4, VP6, VP7 and NSP4. Rotavirus structural proteins VP2, VP6, VP7, VP4 were expressed from single gene constructs, and one construct expressing NSP4 (left panel), or from two dual gene constructs and one construct expressing NSP4 (middle and right panel)). Each construct comprised an expression enhancer, either CPMV HT (left and middle panels) or CMPV 160 (right panel), except for NSP4 which always comprised the CPMV-HT enhancer. The ratios indicate the proportion of the Agrobacterium strains in the bacterial suspension used for transformation: left panel—five single gene constructs (VP2, VP6, VP4, VP7 and NSP4; ratio of 1:1:1:1:1); middle and right panels—dual gene constructs encoding structural proteins (VP6/2 and VP7/4) and the construct encoding non-structural protein (NSP4; ratio of 1:1:1). FIG. 4B shows a Western blot analysis of F2 from the same treatments as in FIG. 4A using an anti-rotavirus VP4 or VP7 antibody as specified. The ratios indicate the proportion of the Agrobacterium strains in the bacterial suspension used for transformation: left panel—five single gene constructs (VP2, VP6, VP4, VP7 and NSP4; ratio of 1:1:1:1:1); middle and right panels—dual gene constructs encoding structural proteins (VP6/2 and VP7/4) and the construct encoding non-structural protein (NSP4; ratio of 1:1:1).

FIG. 5 shows rotavirus protein expression. Upper panel shows a Coomassie-stained SDS-PAGE analysis of fractions F2 and F3 an iodixanol density gradient applied to crude protein extracts from leaves co-expressing VP2, VP4, VP6, VP7 and NSP4, and the lower panel shows a Western blot analysis of the corresponding F2 fraction from the upper panel. Rotavirus structural proteins VP2, VP6, VP7 and VP4 were expressed within a quadruple gene construct, and the non-structural protein NSP4 was co-expressed from a distinct single gene construct (lanes 1 and 2), or structural proteins VP2, VP6, VP7, VP4 and the non-structural protein NSP4 were expressed within a quintuple gene construct (lane 3). The ratio of agroinfiltration of the constructs is indicated. An OD of 0.4 of Agrobacterium strains in the bacterial suspension is indicated as 1. An OD of 0.6 of Agrobacterium strains in the bacterial suspension is indicated as 1.5.

FIG. 6 shows a general schematic of an example of several enhancer sequences that may be used in the constructs of the present invention. FIG. 6A and FIG. 6B show a general schematic of the CPMV HT and CPMV HT+ enhancer sequences fused to a nucleotide sequence of interest (for example encoding a rotavirus structural protein VP2, VP4, VP6, VP7, or a non-structural protein NSP4). Not all of the elements shown in FIGS. 5A or 5B may be required within the enhancer sequence. Additional elements may be included at the 3′ end of the nucleotide sequence of interest including a sequence encoding a comovirus 3′ untranslated region (CPMV 3′ UTR), or a plastocyanin 3′ UTR (3′UTR). FIGS. 6C and 6D show a general schematic of the enhancer sequence of CPMVX, and CPMVX+ (comprising CPMVX, and a stuffer fragment, which in this non-limiting example, comprises a multiple cloning site and a plant kozak sequence), as described herein. CPMVX and CPMVX+ are each shown as operatively linked to plant regulatory region at their 5′ends, and at their 3′ ends, in series, a nucleotide sequence of interest (including an ATG initiation site and STOP site), a 3′UTR, and a terminator sequence. An example of construct CPMVX as described herein, is CPMV160. An example of construct CPMVX+ as described herein, is CPMV160+.

FIG. 7 shows sequence components used to prepare construct number 1710 (2X35S/CPMV-HT/RVA(WA) VP2(opt)/NOS. FIG. 7A shows the nucleotide sequence of IF-WA_VP2(opt).s1+3c (SEQ ID NO: 19). FIG. 7B shows the nucleotide sequence of IF-WA_VP2(opt).s1−4r (SEQ ID NO: 20). FIG. 7C shows the optimized coding sequence of Rotavirus A VP2 from strain WA (SEQ ID NO: 21). FIG. 7D shows the schematic representation of construct 1191. FIG. 7E shows the nucleotide sequence of construct 1191 (SEQ ID NO: 22). FIG. 7F shows the nucleotide sequence of expression cassette number 1710 (SEQ ID NO: 23). FIG. 7G shows the amino acid sequence of VP2 from Rotavirus A WA strain (SEQ ID NO: 24). FIG. 7H shows the schematic representation of construct number 1710.

FIG. 8 shows sequence components used to prepare construct number 1713 (2X35S/CPMV-HT/RVA(WA) VP6(opt)/NOS). FIG. 8A shows the nucleotide sequence of IF-WA_VP6(opt).s1+3c (SEQ ID NO: 25). FIG. 8B shows the nucleotide sequence of IF-WA_VP6(opt).s1−4r (SEQ ID NO: 26). FIG. 8C shows the optimized coding sequence of Rotavirus A VP6 from strain WA (SEQ ID NO: 217. FIG. 8D shows the nucleotide sequence of expression cassette number 1713 (SEQ ID NO: 28). FIG. 8E shows the amino acid sequence of VP6 from Rotavirus A WA strain (SEQ ID NO: 29). FIG. 8F shows the schematic representation of construct number 1713.

FIG. 9 shows sequence components used to prepare construct number 1730 (2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS). FIG. 9A shows the nucleotide sequence of IF-Rtx_VP4(opt).s1+3c (SEQ ID NO: 30). FIG. 9B shows the nucleotide sequence of IF-Rtx_VP4(opt).s1−4r (SEQ ID NO: 31). FIG. 9C shows the optimized coding sequence of Rotavirus A VP4 from strain RVA/Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A[8] (SEQ ID NO:32). FIG. 9D shows the nucleotide sequence of expression cassette number 1730 (SEQ ID NO: 33). FIG. 9E shows the amino acid sequence of VP4 from Rotavirus A Rotarix strain (SEQ ID NO: 34). FIG. 9F shows the schematic representation of construct number 1730.

FIG. 10 shows sequence components used to prepare construct number 1734 (2X35S/CPMV-HT/RVA(Rtx) VP7(Opt)/NOS). FIG. 10A shows the nucleotide sequence of IF-TrSP+Rtx_VP7(opt).s1+3c (SEQ ID NO: 35). FIG. 10B shows the nucleotide sequence of IF-Rtx_VP7(opt).s1−4r (SEQ ID NO: 36). FIG. 10C shows the optimized coding sequence of Rotavirus A VP7 from strain RVA/Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A[8] (SEQ ID NO: 37). FIG. 10D shows the nucleotide sequence of expression cassette number 1734 (SEQ ID NO: 38). FIG. 10E shows the amino acid sequence of TrSp-VP7 from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain (SEQ ID NO: 39). FIG. 10F shows the schematic representation of construct number 1734.

FIG. 11 shows sequence components used to prepare construct number 1706 (2X35S/CPMV-HT/RVA(WA) NSP4/NOS). FIG. 11A shows the nucleotide sequence of IF-WA_NSP4.s1+3c (SEQ ID NO: 40). FIG. 11B shows the nucleotide sequence of IF-WA_NSP4.s1−4r (SEQ ID NO: 41). FIG. 11C shows the coding sequence of Rotavirus A VP6 from strain WA (SEQ ID NO: 42). FIG. 11D shows the nucleotide sequence of expression cassette number 1706 (SEQ ID NO: 43). FIG. 11E shows the amino acid sequence of NSP4 from Rotavirus A WA strain (SEQ ID NO: 44). FIG. 11F shows the schematic representation of construct number 1706.

FIG. 12 shows sequence components used to prepare construct number 1108 (2X35S/CPMV-160/RVA(WA) VP2(opt)/NOS). FIG. 12A shows the nucleotide sequence of IF(C160)-WA_VP2(opt).c (SEQ ID NO: 45). FIG. 12B shows a schematic representation of construct 1190. FIG. 12C shows the nucleotide sequence of construct 1190 (SEQ ID NO: 46). FIG. 12D shows the nucleotide sequence of expression cassette number 1108 (SEQ ID NO: 47). FIG. 12E shows a schematic representation of construct number 1108.

FIG. 13 shows sequence components used to prepare construct number 1128 (2X35S/CPMV-160/RVA(WA) VP6(opt)/NOS). FIG. 13A shows the nucleotide sequence of IF(C160)-WA_VP6(opt).c (SEQ ID NO: 48). FIG. 13B shows the nucleotide sequence of expression cassette number 1128 (SEQ ID NO: 49). FIG. 13C shows a schematic representation of construct number 1128.

FIG. 14 shows sequence components used to prepare construct number 1178 (2X35S/CPMV-160/RVA(Rtx) VP4(opt)/NOS). FIG. 14A shows the nucleotide sequence of IF(C160)-Rtx_VP4(opt).c (SEQ ID NO: 50). FIG. 14B shows the nucleotide sequence of expression cassette number 1178 (SEQ ID NO: 51). FIG. 14C shows the schematic representation of construct number 1178.

FIG. 15 shows sequence components used to prepare construct number 1199 (2X35S/CPMV-160/TrSp-RVA(Rtx) VP7(Opt)/NOS). FIG. 15A shows the nucleotide sequence of IF(C160)-TrSP+Rtx_VP7(opt).c (SEQ ID NO: 52). FIG. 15B shows the nucleotide sequence of Expression cassette number 1199 (SEQ ID NO: 53). FIG. 15C shows the schematic representation of construct number 1199.

FIG. 16 shows the schematic representation of construct number 1708 (double gene construct for the expression of VP6 and VP2 under CPMV-HT expression cassette).

FIG. 17 shows the schematic representation of construct number 1719 (double gene construct for the expression of VP7 and VP4 under CPMV-HT expression cassette).

FIG. 18 shows the schematic representation of construct number 2400 (double gene construct for the expression of VP6 and VP2 under CPMV-160 expression cassette).

FIG. 19 shows the schematic representation of construct number 2408 (double gene construct for the expression of VP7 and VP4 under CPMV-160 expression cassette).

FIG. 20 shows the schematic representation of construct number 1769 (quadruple gene construct for the expression of VP7, VP4, VP6 and VP2 under CPMV-HT expression cassette).

FIG. 21 shows the schematic representation of construct number 2441 (quintuple gene construct for the expression of VP4, VP7, NSP4, VP6 and VP2 under CPMV-HT expression cassette).

DETAILED DESCRIPTION

The following description is of a preferred embodiment.

The present invention relates to virus-like particles (VLPs) comprising one or more rotavirus structural protein (i.e. a rotavirus like particle, rotavirus VLP or RLP), and methods of producing rotavirus-like particle (RLPs) in any host, particularly in plants, a portion of a plant, or a plant cell. Other hosts might comprise, for example, insect cells and mammalian cells. The rotavirus like particle (RLP) may comprise one or more rotavirus structural protein. The RLP may triple layered. The RLP may be produced by co-expressing rotavirus structural and nonstructural proteins in plant, however, the RLP does not comprise any rotavirus nonstructural proteins.

The host or host cell may be from any source including plants, fungi, bacteria, insect and animals. In a preferred embodiment the host or host cell is a plant or plant cell.

The present invention in part provides further a method of producing a rotavirus-like particle (RLP) in a host, such as a plant, a portion of a plant, or a plant cell. The method may comprise introducing one or more nucleic acid comprising a regulatory region active in the host, such as a plant, a portion of a plant, or a plant cell, the regulatory region operatively linked to a nucleotide sequence encoding one or more rotavirus structural protein and one or more rotavirus nonstructural protein into the host, such as into a plant, portion of the plant, or plant cell. Followed by incubating the host, such as a plant, portion of the plant, or plant cell under conditions that permit the expression of the nucleic acids, thereby producing the RLP comprising one or more rotavirus structural protein. The one or more rotavirus structural protein may be rotavirus protein VP2, VP4, VP6 or VP7. The rotavirus nonstructural protein may be NSP4. The RLP may be triple layered. The RLP may comprise rotavirus structural protein VP2, VP4, VP6 and VP7, and does not comprise the nonstructural protein NSP4.

The present invention in part provides further a method of producing a rotavirus like particle (RLP) in a host or host cell, the method may comprise:

providing a host or host cell comprising one or more nucleic acid comprising a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein and a third nucleotide sequence encoding a third rotavirus protein, the first, second and third nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
the first, second and third nucleotide sequence encoding NSP4 and one or two of rotavirus protein VP2 or VP6 and one or two of rotavirus protein VP7 or VP4;
incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid, so that NSP4 and either VP2, VP4 and VP7, or VP2, VP6 and VP7, or VP2, VP6 and VP4, or VP6, VP4 and VP7, are expressed, thereby producing the RLP.

Furthermore, the one or more nucleic acid may comprise a fourth nucleotide sequence encoding a fourth rotavirus protein. The first, second, third, and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and the first nucleotide sequence, the second nucleotide sequence, the third and fourth nucleotide sequence encoding rotavirus protein VP2, or VP6, VP4 or VP7 and NSP4, wherein NSP4 and wherein either VP2 or VP6 and VP4 or VP7 are expressed from the one or more nucleic acid.

Furthermore, the present invention in part provides a method of producing a rotavirus-like particle (RLP) vaccine candidate in a host, such as a plant, a portion of the plant, or a plant cell. The method may comprise expressing in a host, such as in a plant or portion of a plant, one or more nucleic acid (R₁-R₅) comprising one or more regulatory region active in the host, such as in the plant, portion of a plant, or plant cell, the regulatory region operatively linked to nucleotide sequences R₁-R₅, wherein nucleotide sequence R₁encodes rotavirus protein X₁, nucleotide sequence R₂encodes rotavirus protein X₂, nucleotide sequence R₃encodes rotavirus protein X₃, nucleotide sequence R₄encodes rotavirus protein X₄and nucleotide sequence R₅encodes rotavirus protein X₅and each of X₁-X₅are selected from the group of rotavirus protein VP2, VP4, VP6, VP7 and NSP4, so that each of VP2, VP4, VP6, VP7 and NSP4 are expressed in the host, such as in the plant, portion of the plant, or plant cell (see Table 1). The RLP may comprise rotavirus structural protein VP2, VP4, VP6 and VP7. The RLP does not comprise nonstructural protein NSP4.

It has been found that by introducing and co-expressing rotavirus structural protein and a rotavirus non-structural protein in the host, such as a plant or portion of the plant that the yield of the RLP produced may be modulated. In particular, it has been found that by co-expressing rotavirus structural proteins along with a rotavirus non-structural protein NSP4 in the host, such as a plant, portion of the plant, or plant cell, that the incorporation of structural protein VP4 ,VP7 or both VP4 and VP7 into the RLP may be increased, when compared to the level of VP4 and VP7 produced by a second host, such as a second plant, portion of a second plant, or second plant cell that expresses the same rotavirus structural proteins but that does not express the rotavirus non-structural protein, under the same conditions.

For example a method of increasing incorporation of VP4, VP7, or both VP4 and VP7 in a rotavirus like particle (RLP) is provided. The method comprises:

- a) providing a host or host cell comprising one or more nucleic acid comprising
- a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein, a third nucleotide sequence encoding a third rotavirus protein and a fourth nucleotide sequence encoding a fourth rotavirus protein; the first, second, third and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
- the first, second, third and fourth nucleotide sequence encoding one of rotavirus protein VP7, VP4, NSP4 and VP2 or VP6;
- b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid, so that each of the VP7, VP4, NSP4 and VP2 or VP6 and are expressed, thereby producing the RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when compared to the level of VP4 or VP7 produced by a second host or host cell that expresses the one or more nucleic acid that does not comprise NSP4, under the same conditions.

Furthermore, an alternative method of increasing production of VP4 ,VP7 or both VP4 and VP7 in a rotavirus like particle (RLP) may comprises:

- a) introducing into a plant, portion of a plant or plant cell one or more nucleic acid comprising one or more regulatory operatively linked to a first, second, third, fourth and fifth nucleotide sequence, the one or more regulatory region active in the plant, portion of the plant or the plant cell, the first nucleotide sequence encoding a first rotavirus protein, the second nucleotide sequence encoding a second rotavirus protein, the third nucleotide sequence encoding a third rotavirus protein, the fourth nucleotide sequence encoding a fourth rotavirus protein and the fifth nucleotide sequence encoding a fifth rotavirus protein, each of the first, second, third, fourth or fifth nucleotide sequence encoding one of VP2, VP4, VP6, VP7 or NSP4, and
- b) incubating the plant, portion of a plant or plant cell under conditions that permit the transient expression of the one or more nucleic acid so that each of VP2, VP4, VP6, VP7 and NSP4 are transiently expressed, thereby producing the RLP with enhanced levels of VP4 and VP7 when compared to the level of VP4 and VP7 produced by a second plant, portion of a second plant, or second plant cell that expresses the one or more nucleic acid that does not comprise NSP4, under the same conditions.

If desired, the method may further comprises the steps of:

- c) harvesting the plant, portion of a plant or plant cell, and
- d) purifying the RLPs from the plant, portion of a plant or plant cell, wherein the RLPs range in size from 70-100 nm.

An alternate method of increasing production of VP4 ,VP7 or both VP4 and VP7 in a rotavirus like particle (RLP) is also provided, the method comprising:

- a) providing a plant, portion of a plant or plant cell comprising one or more nucleic acid comprising one or more regulatory region operatively linked to a first, second, third, fourth and fifth nucleotide sequence, the one or more regulatory region active in the plant, portion of the plant or the plant cell, the first nucleotide sequence encoding a first rotavirus protein, the second nucleotide sequence encoding a second rotavirus protein, the third nucleotide sequence encoding a third rotavirus protein, the fourth nucleotide sequence encoding a fourth rotavirus protein and the fifth nucleotide sequence encoding a fifth rotavirus protein, each of the first, second, third, fourth and fifth nucleotide sequence encoding one of VP2, VP4, VP6, VP7 or NSP4, and
- b) incubating the plant, portion of a plant or plant cell under conditions that permit the expression of the one or more nucleic acid, so that each of the VP2, VP4, VP6, VP7 and NSP4 are expressed, thereby producing the RLP with enhanced levels of VP4 and VP7 when compared to the level of VP4 and VP7 produced by a second plant, portion of a second plant, or second plant cell that expresses the one or more nucleic acid that does not comprise NSP4, under the same conditions.

It has been further found that by modulating the ratio between constructs comprising nucleic acids encoding rotavirus structural proteins and the non-structural protein NSP4 during transient expression in the host, such as a plant, portion of the plant, or plant cell, the yield of RLP production and the incorporation of structural proteins VP4 and VP7 into the RLP may be improved.

Without wishing to be bound by theory, the co-expression of a rotavirus non-structural protein for example NSP4 together with one or more rotavirus structural protein for example VP2, VP4, VP6 and/or VP7 may lead to an increase in incorporation of VP4 and/or VP7 into RLPs. This increase of incorporation of VP4 and/or VP7 into RLPs may occur via an increase of expression or production of the rotavirus proteins and/or because of an increase of the efficacy of the assembly of RLPs and/or an increase of recruiting of the rotavirus proteins at the RLP assembly site.

As shown in FIGS. 3A and 3B, the co-expression of rotavirus non-structural protein NSP4 together with rotavirus structural protein VP2, VP6, VP4 and VP7 in plants lead to an increase in incorporation of VP4 and VP7 into RLPs, when compared to the expression of structural protein VP2, VP6, VP4 and VP7 without the presence of NSP4 (see FIG. 3B).

The ratios of the constructs encoding the various structural and non-structural proteins that are transiently expressed in the host, such as a plant, portion of the plant, or plant cell, may be altered by providing constructs comprising nucleic acid sequences encoding rotavirus structural proteins and the non-structural protein NSP4 on two, three, four or five constructs and varying the amount of each construct during the step of introducing the construct in the host (using Agrobacterium to the plant, plant portion or plant cell). For example, five separate constructs, each encoding one structural protein and the non-structural protein, may be co-introduced at various ratios resulting co-expression of the nucleic acids at various ratios within a plant, plant portion, or plant cell. Alternatively, the nucleic acid sequences may be provided in various combinations on two, three or four constructs and the constructs co-introduced in the plant, portion of the plant, or plant cell, at various ratios, as described below.

Additionally, the nucleic acid sequences may be provided on the same construct.

“Rotavirus protein” may refer to rotavirus structural protein or rotavirus nonstructural proteins. A “rotavirus structural protein” may refer to all or a portion of a rotavirus structural protein isolated from rotavirus, present in any naturally occurring, or a variant of any naturally occurring, rotavirus strain or isolate. Thus, the term rotavirus structural protein includes a naturally occurring rotavirus structural protein, or a variant of a rotavirus structural protein that may be produced by mutation during the virus life-cycle or produced in response to selective pressure (e.g., drug therapy, expansion of host cell tropism, or infectivity, etc.). As one of skill in the art appreciates, such rotavirus structural proteins and variants thereof may be also produced using recombinant techniques. Rotavirus structural proteins may include capsid proteins such for example VP2 and VP6, surface proteins, for example VP4, or a combination of capsid and surface proteins. The structural protein may further include for example VP7.

By rotavirus “non structural protein”, “nonstructural protein”, “non-structural protein”, “NSP” or “nonstructural rotavirus protein” it is meant a protein that is encoded by the rotavirus genome, but not packaged into the viral particle. Non-limiting examples of rotavirus nonstructural proteins are rotavirus NSP4.

By “co-expressed” it is meant that two, or more than two, nucleotide sequences are expressed at about the same time within the plant, within the same tissue of the plant and within the same cells in the plant. The nucleotide sequences need not be expressed at exactly the same time. Rather, the two or more nucleotide sequences are expressed in a manner such that the encoded products have a chance to interact within a desired cellular compartment. For example, the non-structural protein may be preferably expressed either before or during the period when the structural proteins are expressed. The two or more than two nucleotide sequences can be co-expressed using a transient expression system, where the two or more sequences are introduced within the plant at about the same time, under conditions that the two or more sequences are expressed. The two or more than two sequences may be present on different constructs, and co-expression requires introduction of each of the constructs into the plant, portion of plant or plant cell, or the two or more than two sequences may be present on one construct and the construct introduced into the plant, portion of plant or plant cell.

The term “virus-like particle” (VLP), or “virus-like particles” or “VLPs” refers to structures that self-assemble and comprise one or more structural proteins such as for example rotavirus structural protein, for example but not limited to VP2, VP4, VP6, VP7, or a combination of VP2, VP4, VP6, VP7, structural protein. VLPs comprising rotavirus structural protein maybe also be referred to “rotavirus VLP”, “rotavirus-like particle (RVLP)”, “rotavirus-like particle (RLP)”, “rotavirus-like particle”, “RVLP” or “RLP”. VLPs or RLPs are generally morphologically and antigenically similar to virions produced in an infection, but lack genetic information sufficient to replicate and thus are non-infectious. VLPs may be produced in suitable eukaryotic host cells including plant host cells. Following extraction from the host cell and upon isolation and further purification under suitable conditions, VLPs may be recovered as intact structures. The RLP may be a single, double, or triple-layered RLP. Triple-layered RLPs may be obtained by the simultaneous expression of three or more rotavirus structural proteins, and as described herein, co-expression with one or more non-structural protein. For example, the co-expression of structural proteins VP2, VP6, VP7, VP4 and nonstructural protein NSP4 results in producing triple-layered RLPs.

Co-expression of VP4, along with VP2, VP6, VP7, and one or more non-structural protein as required, results in a particle with spikes that resembles native rotavirus. VP4 may be processed or cleaved to produce VP5 and VP8. This processing may take place within the host using endogenous proteases, or by co-expressing a suitable protease, for example, trypsin, a trypsin-like protease, a serine protease, a chymotrypsin-like protease, subtilisin. Alternatively, VP4 may be processed to produce VP5 and VP8 by adding a suitable protease, for example, trypsin, a trypsin-like protease, a serine protease, a chymotrypsin-like protease, subtilisin during any step of the RLP extraction procedure, or after RLP purification.

Each of the rotavirus structural proteins has different characteristics and size, and is required in different amounts for assembly into RLP. The term “rotavirus VLP”, “rotavirus virus-like particle (RVLP)”, “rotavirus virus-like particle (RLP)”, “rotavirus virus-like particle”, “RVLP” or “RLP” refers to a virus-like particle (VLP) comprising one or more rotavirus structural proteins. Example of rotavirus structural proteins may include, but are not limited to VP2, VP4 (or VP5 and VP8) VP6 and VP7 structural protein. The RLP may not comprise rotavirus nonstructural proteins.

The present invention provides for a method of producing RLPs in a plant, wherein one or more nucleic acid (N₁-N₅) comprising one or more regulatory region active in the plant are operatively linked to nucleotide sequences R₁-R₅, wherein nucleotide sequence R₁encodes rotavirus protein X₁, nucleotide sequence R₂encodes rotavirus protein X₂, nucleotide sequence R₃encodes rotavirus protein X₃, nucleotide sequence R₄encodes rotavirus protein X₄and nucleotide sequence R₅encodes rotavirus protein X₅and wherein X₁-X₅are selected from the group of rotavirus protein VP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are each selected once (see Table 1). The final set, or combination, of nucleic acids used to transform the host results in the expression of each rotavirus protein within the host resulting in expression of VP2, VP4, VP6, VP7 and NSP4 and formation of an RLP.

For example, with reference to Table 1, 2 nucleic acids (N₁and N₂), may be used to transform a host, (see example #2.1). In this case N₁comprises the R₁nucleotide sequence and R₁may encode one of VP2, VP4, VP6, VP7, or NSP4. The nucleic acid N₂comprises four sequences R₂to R₅, each of R₂to R₅encoding one of VP2, VP4, VP6, VP7, or NSP4, but not the protein encoded by R₁, so that each of the VP2, VP4, VP6, VP7 and NSP4 are expressed within the host, thereby producing the RLP. As a non-limiting example, N₁may comprise R₁which may encode VP2, and N₂may comprise R₂to R₅which may encode VP4, VP6, VP7 and NSP4 respectively.

Table 1, provides an overview of combinations, which is not to be considered limiting, of nucleic acids (N), and nucleotide sequences (R) that may be expressed or co-expressed within a host to produce an RLP comprising VP2, VP4, VP6, and VP7.

1. One Construct

1.1 Quintuple Gene Construct

As described herein, a method of producing RLPs in a plant is provided, wherein a nucleic acid (N₁; a first nucleic acid) comprising a first, second, third, fourth and fifth nucleotide sequences (R₁, R₂, R₃, R₄, R₅) encoding a first, second, third, fourth and fifth rotavirus protein (X₁, X₂, X₃, X₄, X₅) is expressed in a plant or portion of a plant (See Table 1, Combination #1.1).

Accordingly, nucleic acid N₁comprises nucleotide sequences R₁, R₂, R₃, R₄and R₅, wherein R₁encodes rotavirus protein X₁, where X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₂-R₅encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁and R₃-R₅encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁R₂, R₄, and R₅encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₃and R₅encode a rotavirus protein that is not X₄, where X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₄encode a rotavirus protein that is not X₅, with the result that a nucleotide sequence encoding for each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is comprised on nucleic acid N₁, thereby allowing for the expression of each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 in the transformed host.

The nucleic acid may comprise a nucleotide sequence R₁, wherein R₁may encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and nucleotide sequences R₂-R₅, wherein R₂-R₅encode a rotavirus protein selected from VP2, VP4, VP6, VP7 or NSP4, and wherein the rotavirus protein is not encoded by R₁. For example which is not to be considered limiting, nucleotide sequence R₁may encode rotavirus protein VP2 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP4, VP6, VP7 and NSP4, but R₂-R₅may not encode VP2. In another example which is not to be considered limiting, nucleotide sequence R₁may encode rotavirus protein VP4 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP2, VP6, VP7 and NSP4, but R₂-R₅may not encode VP4. In yet another non-limiting example, nucleotide sequence R₁may encode rotavirus protein VP6 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP7 and NSP4, but R₂-R₅may not encode VP6. In yet another example which is not to be considered limiting, nucleotide sequence R₁may encode rotavirus protein VP7 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and NSP4, but R₂-R₅may not encode VP7. In yet another example which is not to be considered limiting, nucleotide sequence R₁may encode rotavirus protein NSP4 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and VP7, but R₂-R₅may not encode NSP4.

For example, which is not to be considered limiting, the nucleotide sequence (N₁) comprises a first nucleotide sequences (R₁) encoding a first rotavirus protein, for example rotavirus protein VP7, a second nucleotide sequences (R₂) encoding a second rotavirus protein, for example rotavirus protein VP4, a third nucleotide sequences (R₃) encoding a third rotavirus protein, for example rotavirus protein NSP4, a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein, for example rotavirus protein VP6 and a fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein, for example rotavirus protein VP2.

In a further non-limiting example the nucleotide sequence (N₁) comprises a first nucleotide sequences (R₁) encoding a first rotavirus protein, for example rotavirus protein VP4, a second nucleotide sequences (R₂) encoding a second rotavirus protein, for example rotavirus protein VP7, a third nucleotide sequences (R₃) encoding a third rotavirus protein, for example rotavirus protein NSP4, a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein, for example rotavirus protein VP6 and a fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein, for example rotavirus protein VP2.

In a further non-limiting example the nucleotide sequence (N₁) comprises a first nucleotide sequences (R₁) encoding a first rotavirus protein, for example rotavirus protein VP4, a second nucleotide sequences (R₂) encoding a second rotavirus protein, for example rotavirus protein VP7, a third nucleotide sequences (R₃) encoding a third rotavirus protein, for example rotavirus protein VP6, a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein, for example rotavirus protein VP2 and a fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein, for example rotavirus protein NSP4.

In another non-limiting example the nucleotide sequence (N₁) comprises a first nucleotide sequences (R₁) encoding a first rotavirus protein, for example rotavirus protein VP7, a second nucleotide sequences (R₂) encoding a second rotavirus protein, for example rotavirus protein VP4, a third nucleotide sequences (R₃) encoding a third rotavirus protein, for example rotavirus protein VP6, a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein, for example rotavirus protein VP2 and a fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein, for example rotavirus protein NSP4. (see FIG. 5) A plant may be transformed with a single nucleic acid (N₁) comprising a first, second, third, fourth and fifth nucleotide sequences (R₁, R₂, R₃, R₄, R₅) encoding a first, second, third, fourth and fifth rotavirus protein, so that each of the first, second, third, fourth and fifth protein are expressed in the plant. The rotavirus proteins are selected from the group of rotavirus protein VP2, VP4, VP6, VP7 and NSP4, so that each of VP2, VP4, VP6, VP7 and NSP4 are expressed in the plant. The single nucleic acid may be introduced in the plant in a transient manner, or in a stable manner.

The VP4 may be processed or cleaved to produce VP5 and VP8 within the host by co-expressing a nucleic acid encoding a suitable protease, for example, trypsin, a trypsin-like protease, a serine protease, a chymotrypsin-like protease, subtilisin. Alternatively, VP4 may be processed during any step of RLP extraction, or after RLP purification by adding a sutible protease, for example, trypsin, a trypsin-like protease, a serine protease, a chymotrypsin-like protease, subtilisin.

1.2. Quadruple Gene Construct

As described herein, a method of producing RLPs in a plant is provided, wherein a nucleic acid (N₁; a first nucleic acid) comprising a first, second, third and fourth nucleotide sequences (R₁, R₂, R₃, R₄) encoding a first, second, third and fourth rotavirus protein (X₁, X₂, X₃, X₄) is expressed in a plant or portion of a plant (See Table 1, Combination #1.2).

Accordingly, nucleic acid N₁may comprises nucleotide sequences (R₁, R₂, R₃, R₄), wherein R₁encodes rotavirus protein X₁, where X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that a nucleotide sequence encoding for each rotavirus protein VP2, VP4, VP6 and NSP4 is comprised on nucleic acid N₁, thereby allowing for the expression of each rotavirus protein VP2, VP4, VP6 and NSP4 in the transformed host.

For example, which is not to be considered limiting, the nucleotide sequence (N₁) comprises a first nucleotide sequences (R₁) encoding a first rotavirus protein, for example rotavirus protein VP6, a second nucleotide sequences (R₂) encoding a second rotavirus protein, for example rotavirus protein VP4, a third nucleotide sequences (R₃) encoding a third rotavirus protein, for example rotavirus proteinVP2, and a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein, for example rotavirus protein NSP4.

Further accordingly, nucleic acid N₁may comprises nucleotide sequences (R₁, R₂, R₃, R₄), wherein R₁encodes rotavirus protein X₁, where X₁may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that a nucleotide sequence encoding for each rotavirus protein VP2, VP7, VP6 and NSP4 is comprised on nucleic acid N₁, thereby allowing for the expression of each rotavirus protein VP2, VP7, VP6 and NSP4 in the transformed host.

For example, which is not to be considered limiting, the nucleotide sequence (N₁) comprises a first nucleotide sequences (R₁) encoding a first rotavirus protein, for example rotavirus protein VP6, a second nucleotide sequences (R₂) encoding a second rotavirus protein, for example rotavirus protein VP7, a third nucleotide sequences (R₃) encoding a third rotavirus protein, for example rotavirus proteinVP2, and a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein, for example rotavirus protein NSP4.

Further accordingly, nucleic acid N₁may comprises nucleotide sequences (R₁, R₂, R₃, R₄), wherein R₁encodes rotavirus protein X₁, where X₁may be any rotavirus protein selected from the group of VP7, VP4, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP7, VP4, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP7, VP4, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP7, VP4, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that a nucleotide sequence encoding for each rotavirus protein VP7, VP4, VP6 and NSP4 is comprised on nucleic acid N₁, thereby allowing for the expression of each rotavirus protein VP7, VP4, VP6 and NSP4 in the transformed host.

For example, which is not to be considered limiting, the nucleotide sequence (N₁) comprises a first nucleotide sequences (R₁) encoding a first rotavirus protein, for example rotavirus protein VP6, a second nucleotide sequences (R₂) encoding a second rotavirus protein, for example rotavirus protein VP4, a third nucleotide sequences (R₃) encoding a third rotavirus protein, for example rotavirus proteinVP7, and a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein, for example rotavirus protein NSP4.

1.3 Triple Gene Construct

As described herein, a method of producing RLPs in a plant is provided, wherein a nucleic acid (N₁; a first nucleic acid) comprising a first, second and third nucleotide sequences (R₁, R₂, R₃) encoding a first, second and third rotavirus protein (X₁, X₂, X₃) is expressed in a plant or portion of a plant (See Table 1, Combination #1.3).

Accordingly, nucleic acid N₁may comprises nucleotide sequences (R₁, R₂, R₃), wherein R₁encodes rotavirus protein X₁, where X₁may be any rotavirus protein selected from the group of VP4, VP6 and NSP4, and wherein R₂-R₃encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP4, VP6 and NSP4, and wherein R₁and R₃encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP4, VP6 and NSP4, and wherein R₁R₂encode a rotavirus protein that is not X₃, with the result that a nucleotide sequence encoding for each rotavirus protein VP4, VP6 and NSP4 is comprised on nucleic acid N₁, thereby allowing for the expression of each rotavirus protein VP4, VP6 and NSP4 in the transformed host.

Further accordingly, nucleic acid N₁may comprises nucleotide sequences (R₁, R₂, R₃), wherein R₁encodes rotavirus protein X₁, where X₁may be any rotavirus protein selected from the group of VP7, VP6 and NSP4, and wherein R₂-R₃encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP7, VP6 and NSP4, and wherein R₁and R₃encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP7, VP6 and NSP4, and wherein R₁R₂encode a rotavirus protein that is not X₃, with the result that a nucleotide sequence encoding for each rotavirus protein VP7, VP6 and NSP4 is comprised on nucleic acid N₁, thereby allowing for the expression of each rotavirus protein VP7, VP6 and NSP4 in the transformed host.

2. Two Constructs

2.1. Quadruple Gene Construct+Single Gene Construct

The present invention also provides for a method of producing RLPs in a plant, wherein a first nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein (X₁), is co-expressed with a second nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein (X₂-X₅) (see Table 1, Combination #2.1), so that the first, second, third, fourth and fifth nucleotide sequence (R₁-R₅) are co-expressed in the plant.

In this non-limiting example, N₁comprises nucleotide sequence (R₁) and N₂comprises nucleotide sequences (R₂, R₃, R₄, R₅), wherein each rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4 is encoded in the combination of both constructs N₁and N₂, and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₂-R₅encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁and R₃-R₅encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁R₂, R₄, and R₅encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₃and R₅encode a rotavirus protein that is not X₄, where X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₄encode a rotavirus protein that is not X₅, with the result that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the host.

For example, which is not to be considered limiting, nucleotide sequence R₁may encode rotavirus protein VP2 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP4, VP6, VP7 and NSP4, but R₂-R₅may not encode VP2. In another non-limiting example nucleotide sequence R₁may encode rotavirus protein VP4 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP2, VP6, VP7 and NSP4, but R₂-R₅may not encode VP4. In yet another non-limiting example nucleotide sequence R₁may encode rotavirus protein VP6 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP7 and NSP4, but R₂-R₅may not encode VP6. In yet another example which is not to be considered limiting, nucleotide sequence R₁may encode rotavirus protein VP7 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and NSP4, but R₂-R₅may not encode VP7. In yet another non-limiting example nucleotide sequence R₁may encode rotavirus protein NSP4 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and VP7, but R₂-R₅may not encode NSP4.

The first nucleic acid (N₁) and second nucleic acid (N₂) may be introduced into the plant in the same step, or may be introduced to the plant sequentially.

For example, which is not to be considered limiting, a first nucleotide sequence (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein, for example NSP4, is co-expressed with a second nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example VP7, VP4, VP6 and VP2, respectively (see FIG. 5).

In another non-limiting example, a first nucleotide sequence (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein, for example NSP4, is co-expressed with a second nucleic acid (N₂) comprising four sequential nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example VP4, VP7, VP6 and VP2, respectively. In yet another non-limiting example, a first nucleotide sequence (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein, for example NSP4, is co-expressed with a second nucleic acid (N₂) comprising four sequential nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example VP7, VP4, VP2 and VP6, respectively. In yet another non-limiting example, a first nucleotide sequence (N₁) comprising a nuc_leotide sequence (R₁) encoding a first rotavirus protein, for example NSP4, is co-expressed with a second nucleic acid (N₂) comprising four sequential nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example VP4, VP7, VP2 and VP6, respectively. In yet another non-limiting example, a first nucleotide sequence (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein, for example NSP4, is co-expressed with a second nucleic acid (N₂) comprising four sequential nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example VP6, VP2, VP4 and VP7, respectively. In yet another non-limiting example, a first nucleotide sequence (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein, for example NSP4, is co-expressed with a second nucleic acid (N₂) comprising four sequential nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example VP6, VP2, VP7 and VP4, respectively. In yet another non-limiting example, a first nucleotide sequence (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein, for example NSP4, is co-expressed with a second nucleic acid (N₂) comprising four sequential nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example VP2, VP4, VP6 and VP7, respectively. In yet another non-limiting example, a first nucleotide sequence (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein, for example NSP4, is co-expressed with a second nucleic acid (N₂) comprising four sequential nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example VP7, VP6, VP4 and VP2, respectively. In yet another non-limiting example, a first nucleotide sequence (N₁) comprising a nuc_leotide sequence (R₁) encoding a first rotavirus protein, for example VP7, is co-expressed with a second nucleic acid (N₂) comprising four sequential nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example NSP4, VP2, VP6 and VP4, respectively. In yet another non-limiting example, a first nucleotide sequence (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein, for example VP4, is co-expressed with a second nucleic acid (N₂) comprising four sequential nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example NSP4, VP2, VP6 and VP7, respectively.

A plant that expresses a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁), may be transformed with a second nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein (X₂-X₅), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. Furthermore, a plant that expresses a first nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅) encoding a rotavirus protein X₂-X₅may be transformed with a second nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding a rotavirus protein (X₁), so that the first and second nucleotide sequences R₁-R₅are co-expressed in the plant. The rotavirus protein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and rotavirus proteins X₂-X₅may be any rotavirus protein selected from the group of VP2, VP₄, VP6, VP7 and NSP4, but not X₁, so that each rotavirus protein VP2, VP4, VP₆, VP7 and NSP4 is expressed. Furthermore, a plant may be simultaneously co-transformed with a first nucleic acid (N₂) comprising four nucleotide sequences (R₂-_R5) encoding a rotavirus protein X₂-X₅, and with a second nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding a rotavirus protein (X₁), so that the first and second nucleotide sequences R₁-R₅are co-expressed in the plant. The rotavirus protein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and rotavirus proteins X₂-X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, but not the protein selected for X₁, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed. The first nucleic acid (N₁) and second nucleic acid (N₂) may be introduced in the plant in a transient manner, or in a stable manner.

For example, which is not to be considered limiting, a plant that expresses a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) for example NSP4, may be transformed with a second nucleic acid encoding (N₂) comprising four nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein (X₂-X₅), for example VP7, VP4, VP6 and VP2, so that NSP4, VP7, VP4, VP6 and VP2 are co-expressed in the plant.

A first plant expressing a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁), may be crossed with a second plant expressing the second nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein (X₂-X₅) to produce a progeny plant (third plant) that co-expresses the first, second, third, fourth and fifth rotavirus protein (X₁-X₅). Furthermore, a first plant expressing a first nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅) encoding a rotavirus protein X₂-X₅may be crossed with a second plant expressing a second nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding a rotavirus protein (X₁), so that nucleotide sequences R₁-R₅are co-expressed in the progeny plant. The rotavirus protein may be any rotavirus protein selected from the group of rotavirus protein VP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the progeny plant.

The VP4 may be processed or cleaved to produce VP5 and VP8 within the host by co-expressing a nucleic acid encoding a suitable protease, for example, trypsin, a trypsin-like protease, a serine protease, a chymotrypsin-like protease, subtilisin. Alternatively, VP4 may be processed during any step of RLP extraction, or after RLP purification by adding a suitable protease, for example, trypsin, a trypsin-like protease, a serine protease, a chymotrypsin-like protease, subtilisin.

2.2 Triple Gene Construct+Dual Gene Construct

The present invention also provides for a method of producing RLPs in a plant, wherein a first nucleic acid (N₁) comprising two nucleotide sequences (R₁and R₂) encoding a first rotavirus protein (X₁) and second rotavirus protein (X₂) respectively, is co-expressed with a second nucleic acid (N₂) comprising three nucleotide sequences (R₃-R₅) encoding a third, fourth and fifth rotavirus proteins (X₃-X₅) (see Table 1, Combination #2.2), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant.

In a non-limiting example, N₁comprises nucleotide sequences (R₁, R₂) and N₂comprises nucleotide sequences (R₃, R₄, R₅), wherein each rotavirus protein selected from the group of VP2, VP4, VP6, V_P7and NSP4 is encoded and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₂-R₅encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁and R₃-R₅encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁R₂, R₄, and R₅encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₃and R₅encode a rotavirus protein that is not X₄, where X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₄encode a rotavirus protein that is not X₅, with the result that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the host.

Therefore, the first nucleic acid (N₁) may comprise a nucleotide sequence R₁, wherein R₁may encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and a second nucleotide sequence R₂which may encode any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, that is not encoded by R₁. The second nucleic acid (N₂) may comprise nucleotide sequences R₃-R₅, wherein R₃-R₅encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4, but not rotavirus protein that are encoded by R₁or R₂. For example, which is not to be considered limiting, nucleotide sequence R₁may encode rotavirus protein VP2 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP4, VP6, VP7 and NSP4, but R₂-R₅may not encode VP2. In another non-limiting example nucleotide sequence R₁may encode rotavirus protein VP4 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP6, VP7 and NSP4. In yet another example nucleotide sequence R₁may encode rotavirus protein VP6 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP7 and NSP4. In yet another non-limiting example nucleotide sequence R₁may encode rotavirus protein VP7 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and NSP4. In yet another non-limiting example nucleotide sequence R₁may encode rotavirus protein NSP4 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and VP7.

A plant that expresses a first nucleic acid (N₁) comprising a first and second nucleotide sequence (R₁+R₂) encoding a first and second rotavirus protein (X₁+X₂), may be transformed with a second nucleic acid (N₂) comprising three nucleotide sequences (R₃-R₅) encoding a third, fourth and fifth rotavirus protein (X₃-X₅), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. The first nucleic acid (N₁) and second nucleic acid (N₂) may be introduced in the plant in a transient manner, or in a stable manner.

Furthermore, a plant that expresses a first nucleic acid (N₂) comprising three nucleotide sequences (R₃-R₅) encoding a first, second and third rotavirus protein (X₃-X₅) may be transformed with a second nucleic acid (N₁) comprising a fourth nucleotide sequence (R₁) encoding a fourth rotavirus protein (X₁) and a fifth rotavirus protein (X₂) so that the first and second nucleic acids R₁-R₅are co-expressed in the plant. The rotavirus protein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and rotavirus proteins X₂-X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, but not X₁, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed. For example, which is not to be considered limiting, a plant that expresses a first nucleic acid (N₂) comprising three nucleotide sequences (R₃-R₅) encoding a first, second and third rotavirus proteins (X₃-X₅), for example VP7, VP4 and VP6 may be transformed with a second nucleic acid encoding (N₁) comprising a fourth and a fifth nucleotide sequences (R₁-R₂) encoding a fourth and a fifth rotavirus protein (X₁-X₂) for example VP2 and NSP4, so that NSP4, VP7, VP4, VP6 and VP2 are co-expressed in the plant. Furthermore, a plant may be simultaneously co-transformed with a first nucleic acid (N₁) comprising a first and second nucleotide sequence (R₁+R₂) encoding a first and second rotavirus protein (X₁+X₂), and a second nucleic acid (N₂) comprising three nucleotide sequences (R₃-R₅) encoding a third, fourth and fifth rotavirus protein (X₃-X₅), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. The first nucleic acid (N₁) and second nucleic acid (N₂) may be introduced in the plant in a transient manner, or in a stable manner.

A first plant expressing a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) and a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂), may be crossed with a second plant expressing the second nucleic acid (N₂) comprising three nucleotide sequences (R₃-R₅) encoding a third, fourth and fifth rotavirus protein (X₃-X₅) to produce a progeny plant (third plant) that co-expresses the first, second, third, fourth and fifth rotavirus protein (X₁-X₅). Furthermore, a first plant expressing a first nucleic acid (N₂) comprising three nucleotide sequences (R₃-R₅) encoding a rotavirus protein X₃-X₅may be crossed with a second plant expressing a second nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding a rotavirus protein (X₁) and a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂), so that nucleotide sequences R₁-R₅are co-expressed in the progeny plant. The rotavirus protein may be any rotavirus protein selected from the group of rotavirus protein VP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the progeny plant.

2.3. Triple Gene Construct+Single Gene Construct

The present invention also provides for a method of producing RLPs in a plant, wherein a first nucleic acid (N₁) comprising one nucleotide sequence (R₁) encoding a first rotavirus protein (X₁), is co-expressed with a second nucleic acid (N₂) comprising three nucleotide sequences (R₂-R₄) encoding a second, third and fourth rotavirus proteins (X₂-X₄), so that the first, second, third and fourth nucleotide sequence are co-expressed in the plant (See Table 1, Combination #2.3).

In a non-limiting example, N₁comprises nucleotide sequence (R₁) and N₂comprises nucleotide sequences (R₂, R₃, R₄), wherein each rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4 is encoded and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP2, VP4, VP6 and NSP4 is expressed in the host.

In a further non-limiting example, N₁comprises nucleotide sequence (R₁) and N₂comprises nucleotide sequences (R₂, R₃, R₄), wherein each rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4 is encoded and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP2, VP7, VP6 and NSP4 is expressed in the host.

In yet a further non-limiting example, N₁comprises nucleotide sequence (R₁) and N₂comprises nucleotide sequences (R₂, R₃, R₄), wherein each rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4 is encoded and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP4, VP7, VP6 and NSP4 is expressed in the host.

2.4 Two Double Gene Constructs

The present invention also provides for a method of producing RLPs in a plant, wherein a first nucleic acid (N₁) comprising nucleotide sequences (R₁-R₂) encoding a first and a second rotavirus proteins (X₁-X₂), is co-expressed with a second nucleic acid (N₂) comprising two nucleotide sequences (R₃-R₄) encoding a third and fourth rotavirus proteins (X₃-X₄), so that the first, second, third and fourth nucleotide sequences are co-expressed in the plant (See Table 1, Combination #2.4).

In a non-limiting example, N₁comprises nucleotide sequences (R₁, R₂) and N₂comprises nucleotide sequences (R₃, R₄), wherein each rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4 is encoded and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP2, VP4, VP6 and NSP4 is expressed in the host.

In a further non-limiting example, N₁comprises nucleotide sequences (R₁, R₂) and N₂comprises nucleotide sequences (R₃, R₄), wherein each rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4 is encoded and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP2, VP7, VP6 and NSP4 is expressed in the host.

In yet a further non-limiting example, N₁comprises nucleotide sequences (R₁, R₂) and N₂comprises nucleotide sequences (R₃, R₄), wherein each rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4 is encoded and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP4, VP7, VP6 and NSP4 is expressed in the host.

2.5 Double Gene Construct+Single Gene Construct

The present invention also provides for a method of producing RLPs in a plant, wherein a first nucleic acid (N₁) comprising nucleotide sequences (R₁-R₂) encoding a first and a second rotavirus proteins (X₁-X₂), is co-expressed with a second nucleic acid (N₂) comprising a nucleotide sequence (R₃) encoding a third rotavirus proteins (X₃), so that the first, second and third nucleotide sequences are co-expressed in the plant (See Table 1, Combination #2.5).

In a non-limiting example, N₁comprises nucleotide sequences (R₁, R₂) and N₂comprises nucleotide sequence (R₃), wherein each rotavirus protein selected from the group of VP4, VP6 and NSP4 is encoded and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP4, VP6 and NSP4, and wherein R₂-R₃encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP4, VP6 and NSP4, and wherein R₁and R₃encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP4, VP6 and NSP4, and wherein R₁and R₂encode a rotavirus protein that is not X₃, with the result that each rotavirus protein VP4, VP6 and NSP4 is expressed in the host.

In a further non-limiting example, N₁comprises nucleotide sequences (R₁, R₂) and N₂comprises nucleotide sequence (R₃), wherein each rotavirus protein selected from the group of VP7, VP6 and NSP4 is encoded and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP7, VP6 and NSP4, and wherein R₂-R₃encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP7, VP6 and NSP4, and wherein R₁and R₃encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP7, VP6 and NSP4, and wherein R₁and R₂encode a rotavirus protein that is not X₃, with the result that each rotavirus protein VP7, VP6 and NSP4 is expressed in the host.

3. Three Constructs

3.1 Two Dual Gene Constructs+One Single Gene Construct

The present invention also provides for a method of producing RLPs in a plant, wherein a first nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) and a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂), is co-expressed with a second nucleic acid (N₂) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃) and a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄) and a third nucleic acid (N₃) comprising a fifth nucleotide sequence (R₅) encoding a fifth rotavirus protein (X₅) (see Table 1, Combination #3.1) so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant.

In this non-limiting example, N₁comprises (R₁, R₂), N₂comprises (R₃, R₄) and N₃comprises (R₅), wherein each rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₂-R₅encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁and R₃-R₅encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁R₂, R₄, and R₅encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₃and R₅encode a rotavirus protein that is not X₄, where X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₄encode a rotavirus protein that is not X₅, with the result that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the host.

For example, the first nucleic acid (N₁) may comprise a nucleotide sequence R₁, wherein R₁may encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and a second nucleotide sequence R₂which may encode any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4 that is not encoded by R₁. The second nucleotide sequence may comprise nucleotide sequences R₃and R₄, wherein R₃encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4, but not a rotavirus protein that are encoded by R₁or R₂, and R₄encodes rotavirus protein VP2, VP4, VP6, VP7 or NSP4, but not a rotavirus protein that are encoded by R₁, R₂or R₃. The third nucleotide sequence may comprise nucleotide sequences R₅, wherein R₅encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4, but not a rotavirus protein that are encoded by R₁, R₂, R₂or R₄, so that each of VP2, VP4, VP6, VP7 or NSP4 are expressed in a host.

For example which is not to be considered limiting, nucleotide sequence R₁may encode rotavirus protein VP2 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP4, VP6, VP7 and NSP4, but R₂-R₅may not encode VP2. In another example nucleotide sequence R₁may encode rotavirus protein VP4 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP6, VP7 and NSP4. In yet another example nucleotide sequence R₁may encode rotavirus protein VP6 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP7 and NSP4. In yet another non-limiting example nucleotide sequence R₁may encode rotavirus protein VP7 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and NSP4. In yet another non-limiting example nucleotide sequence R₁may encode rotavirus protein NSP4 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and VP7.

For example, which is not to be considered limiting, a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein, for example VP6 and a second nucleotide sequence (R₂) encoding a second rotavirus protein, for example VP2, is co-expressed with a second nucleic acid (N₂) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein for example VP7 and a fourth nucleotide sequence (R₄) encoding a fourth rotavirus protein, for example VP4 and a third nucleic acid (N₃) comprising a fifth nucleotide sequence (R₅) encoding a fifth rotavirus protein for example NSP4. (see FIGS. 2 and 3).

A plant that expresses a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) and a second rotavirus protein (X₂), may be transformed with a second nucleic acid (N₂) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃) and a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄). The plant may be further transformed with a third nucleic acid (R₃) comprising a fifth nucleotide sequence (R₅) encoding a fifth rotavirus protein (X₅), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. Rotavirus protein X₁-X₅may be selected from the group of VP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the plant. Furthermore, a plant may be simultaneously co-transformed with a first nucleic acid (N₁) comprising a first and second nucleotide sequence (R₁+R₂) encoding a first and second rotavirus protein (X₁+X₂), a second nucleic acid (N₂) comprising a third and a fourth nucleotide sequences (R₃+R₄) encoding a third and a fourth rotavirus protein (X₃+X₄), and a third nucleic acid (N₃) comprising a fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein (X₅), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. The first nucleic acid (N₁), second nucleic acid (N₂) and third nucleic acid (N₃) may be introduced in the plant in a transient manner, or in a stable manner.

3.2 Two Single Gene Constructs+One Triple Gene Construct

An alternated method of producing RLPs in a plant is also provided, wherein a first nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) is co-expressed with a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂) and a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃), a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄) and a fifth nucleotide sequence (R₅) encoding a fifth rotavirus protein (X₅) (see Table 1, Combination #3.2) so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant.

In an alternate example, N₁comprises (R₁), N₂comprises (R₂) and N₃comprises (R_3,R₄, R₅), wherein each rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₂-R₅encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁and R₃-R₅encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁R₂, R₄, and R₅encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₃and R₅encode a rotavirus protein that is not X₄, where X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₄encode a rotavirus protein that is not X₅, with the result that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the host.

For example which is not to be considered limiting, the first nucleic acid (N₁) may comprise a nucleotide sequence R₁, wherein R₁may encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and a second nucleotide sequence R₂which may encode any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4 that is not encoded by R₁. The second nucleotide sequence may comprise nucleotide sequences R₃and R₄, wherein R₃encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4, but not a rotavirus protein that are encoded by R₁or R₂and R₄encodes rotavirus protein VP2, VP4, VP6, VP7 or NSP4, but not a rotavirus protein that are encoded by R₁, R₂or R₃. The third nucleotide sequence may comprise nucleotide sequences R₅, wherein R₅encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4, but not a rotavirus protein that are encoded by R₁, R_2,R₃or R_4,and wherein VP2, VP4, VP6, VP7 or NSP4 are encoded once. For example nucleotide sequence R₁may encode rotavirus protein VP2 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP4, VP6, VP7 and NSP4, but R₂-R₅may not encode VP2. In another example nucleotide sequence R₁may encode rotavirus protein VP4 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP6, VP7 and NSP4. In yet another example nucleotide sequence R₁may encode rotavirus protein VP6 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP7 and NSP4. In yet another example nucleotide sequence R₁may encode rotavirus protein VP7 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and NSP4. In yet another example nucleotide sequence R₁may encode rotavirus protein NSP4 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and VP7.

A plant that expresses a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) may be transformed with a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂). The plant may be further transformed with a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃), a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄) and a fifth nucleotide sequence (R₅) encoding a fifth rotavirus protein (X₅), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. Rotavirus protein X₁-X₅may be selected from the group of VP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the plant. Furthermore, a plant may be simultaneously co-transformed with a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁), a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂), and a third nucleic acid (N₃) comprising a third, a fourth and a five nucleotide sequences (R₃-R₅) encoding a third, a fourth and a fifth rotavirus protein (X₃-X₅), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. The first nucleic acid (N₁), second nucleic acid (N₂) and third nucleic acid (N₃) may be introduced in the plant in a transient manner, or in a stable manner.

For example, a first plant expressing a first nucleic acid (N₁) comprising a first, second and third nucleotide sequence (R₁, R₂, and R₃) encoding a first, second and third rotavirus protein (X₁, X₂, and X₃) may be crossed with a second plant expressing a second nucleic acid (N₂) comprising a fourth nucleotide sequence (R₄) encoding a fourth rotavirus protein (X₄) to produce a progeny plant (third plant). The third plant may be crossed with a fourth plant expressing a third nucleic acid (N₃) comprising a fifth nucleotide sequence (R₅) encoding a fifth rotavirus protein (X₅) to produces a progeny plant that co-expresses the first, second, third, fourth and fifth rotavirus protein (X₁-X₅). Furthermore, a first plant expressing a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) may be crossed with a second plant expressing a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a rotavirus protein (X₂) to produce a progeny plan (third plant). The third plant may be crossed with a fourth plant expressing a third nucleic acid (N₃) comprising a third, fourth and fifth nucleotide sequence (R₃, R₄and R₅) encoding a third, fourth and fifth rotavirus protein (X₃, X₄, and X₅) to produce a progeny plant that co-expresses the first, second, third, fourth and fifth rotavirus protein (X₁-X₅). Rotavirus protein X₁-X₅may be selected from the group of VP2, VP4, VP6, VP7 and NSP4, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the plant.

3.3 Two Single Gene Constructs+One Dual Gene Construct

An alternated method of producing RLPs in a plant is also provided, wherein a first nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) is co-expressed with a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂) and a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃), and a fourth nucleotide sequence (R₄) encoding a fourth rotavirus protein (X₄), so that the first, second, third and fourth nucleotide sequence are co-expressed in the plant (See Table 1, Combination #3.3).

As a non-limiting example, N₁comprises (R₁), N₂comprises (R₂) and N₃comprises (R₃, R₄), wherein each rotavirus protein selected from the group of VP2, VP4, VP6, and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6, and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6, and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6, and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP2, VP4, VP6, and NSP4 is expressed in the host.

As another non-limiting example, N₁comprises (R₁), N₂comprises (R₂) and N₃comprises (R₃, R₄), wherein each rotavirus protein selected from the group of VP2, VP7, VP6, and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP7, VP6, and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP7, VP6, and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP7, VP6, and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP7, VP6, and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP2, VP6, VP7 and NSP4 is expressed in the host.

As a further non-limiting example, N₁comprises (R₁), N₂comprises (R₂) and N₃comprises (R_3,R₄), wherein each rotavirus protein selected from the group of VP4, VP7, VP6, and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP4, VP7, VP6, and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP4, VP7, VP6, and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP4, VP7, VP6, and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP4, VP7, VP6, and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP4, VP7, VP6, and NSP4 is expressed in the host.

3.4 Three Single Gene Constructs

An alternated method of producing RLPs in a plant is also provided, wherein a first nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) is co-expressed with a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂) and a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃), so that the first, second and third nucleotide sequence are co-expressed in the plant (See Table 1, Combination #3.4).

As a non-limiting example, N₁comprises (R₁), N₂comprises (R₂) and N₃comprises (R₃), wherein each rotavirus protein selected from the group of VP4, VP6, and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP4, VP6, and NSP4, and wherein R₂-R₃encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP4, VP6, and NSP4, and wherein R₁and R₃encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP4, VP6, and NSP4, and wherein R₁and R₂encode a rotavirus protein that is not X₃, with the result that each rotavirus protein VP4, VP6, and NSP4 is expressed in the host.

As a further non-limiting example, N₁comprises (R₁), N₂comprises (R₂) and N₃comprises (R₃), wherein each rotavirus protein selected from the group of VP7, VP6, and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP7, VP6, and NSP4, and wherein R₂-R₃encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP7, VP6, and NSP4, and wherein R₁and R₃encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP7, VP6, and NSP4, and wherein R₁and R₂encode a rotavirus protein that is not X₃, with the result that each rotavirus protein VP7, VP6, and NSP4 is expressed in the host.

4. Four Constructs
4.1. Three Single Gene Constructs+One Dual Gene Construct

Also provided herein is a method of producing RLPs in a plant, wherein a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) is co-expressed with a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂), a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃), and fourth nucleic acid (N₄) comprising a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄) and a fifth nucleotide sequence (R₅) encoding a fifth rotavirus protein (X₅) (see Table 1, Combination #4.1) so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant.

In this example, N₁comprises (R₁), N₂comprises (R₂), N₃comprises (R₃), and N₄comprises (R₄, and R₅), wherein each rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4 is encoded, and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₂-R₅encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁and R₃-R₅encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁R₂, R₄, and R₅encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₃and R₅encode a rotavirus protein that is not X₄, where X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₄encode a rotavirus protein that is not X₅, with the result that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the host.

The four nucleic acids may be introduced into a plant in any order. For example, which is not be considered limiting, a plant that expresses a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) may be transformed with a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂). The plant may be further transformed with a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃). The plant then may be further be transformed with a fourth nucleic acid (N₄) comprising a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄) and a fifth nucleotide sequence (R₅) encoding a fifth rotavirus protein (X₅), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. Furthermore, a plant may be simultaneously co-transformed with a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁), a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂), a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃), and a fourth nucleic acid (N₄) comprising a fourth and a fifth nucleotide sequences (R₄-R₅) encoding a fourth and a fifth rotavirus protein (X₄-X₅) so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. The first nucleic acid (N₁), second nucleic acid (N₂), third nucleic acid (N₃) and fourth nucleic acid (N₄) may be introduced in the plant in a transient manner, or in a stable manner.

Furthermore, a plant that expresses a first nucleic acid (N₄) comprising a first nucleotide sequence (R₄) encoding a first rotavirus protein (X₄) and a second nucleotide sequence (R₅) encoding a second rotavirus protein (X₅) may be transformed with a second nucleic acid (N₁) comprising a third nucleotide sequence (R₁) encoding a third rotavirus protein (X₁). The plant may be further transformed with a third nucleic acid (N₂) comprising a third nucleotide sequence (R₂) encoding a fourth rotavirus protein (X₃). The plant then may be further be transformed with a fourth nucleic acid (N₃) comprising a fifth nucleotide sequences (R₃) encoding a fifth rotavirus protein and a fifth nucleotide sequence (R₃) encoding a fifth rotavirus protein (X₃), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. Rotavirus protein X₁-X₅may be selected from the group of VP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the plant. The first nucleic acid (N₄), second nucleic acid (N₁), third nucleic acid (N₂) and fourth nucleic acid (N₃) may be introduced in the plant in a transient manner, or in a stable manner.

The third plant may be crossed with a fourth plant expressing a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃) to produces a progeny plant (fifth plant). The fifth plant may be crossed with a sixth plant expressing a fourth nucleic acid (N₄) comprising a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄) and a fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein (X₅) to produce a progeny plant that co-expresses the first, second, third, fourth and fifth rotavirus protein (X₁-X₅).

Furthermore, a first plant expressing a first nucleic acid (N₄) comprising a first nucleotide sequence (R₄) encoding a first rotavirus protein (X₄) and a second nucleotide sequence (R₅) encoding a second rotavirus protein (X₅) may be crossed with a second plant expressing a second nucleic acid (N₂) comprising a third nucleotide sequence (R₁) encoding a third rotavirus protein (X₁) to produce a progeny plan (third plant). The third plant may be crossed with a fourth plant expressing a third nucleic acid (N₂) comprising a fourth nucleotide sequence (R₂) encoding a fourth rotavirus protein (X₂) to produce a progeny plant (fifth plant). The fifth plant may be crossed with a sixth plant expressing a fifth nucleotide sequence (R₃) encoding a fifth rotavirus protein (X₃) to produce a progeny plant that co-expresses the first, second, third, fourth and fifth rotavirus protein (X₁-X₅). Rotavirus protein X₁-X₅may be selected from the group of VP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the plant.

4.2 Four Single Gene Constructs

Also provided herein is a method of producing RLPs in a plant, wherein a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) is co-expressed with a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂), a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃), and a fourth nucleic acid (N₄) comprising a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant (See Table 1, Combination #4.2).

In a non-limiting example, N₁comprises (R₁), N₂comprises (R₂), N₃comprises (R₃), and N₄comprises (R₄), wherein each rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4 is encoded, and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP2, VP4, VP6 and NSP4 is expressed in the host.

In another non-limiting example, N₁comprises (R₁), N₂comprises (R₂), N₃comprises (R₃), and N₄comprises (R₄), wherein each rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4 is encoded, and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP2, VP7, VP6 and NSP4 is expressed in the host.

In a further non-limiting example, N₁comprises (R₁), N₂comprises (R₂), N₃comprises (R₃), and N₄comprises (R₄), wherein each rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4 is encoded, and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₁and R₃-R₄encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₁R₂, and R₄encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, and wherein R₁-R₃encode a rotavirus protein that is not X₄, with the result that each rotavirus protein VP4, VP7, VP6 and NSP4 is expressed in the host.

5. Five Constructs
5. Five Single Gene Constructs

The present invention also provides for a method of producing RLPs in a plant, wherein a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) is co-expressed with a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂) and a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃) and fourth nucleic acid (N₄) comprising a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄) and a fifth nucleic acid (R₅) comprising a fifth nucleotide sequence (R₅) encoding a fifth rotavirus protein (X₅) (see Table 1, Combination #5) so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant.

In this non-limiting example, N₁comprises (R₁), N₂comprises (R₂), N₃comprises (R₃), N₄comprises (R₄) and N₅comprises (R₅), wherein each rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₂-R₅encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁and R₃-R₅encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁R₂, R₄, and R₅encode a rotavirus protein that is not X₃, where X₄may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₃and R₅encode a rotavirus protein that is not X₄, where X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₄encode a rotavirus protein that is not X₅, with the result that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the host.

For example, which is not to be considered limiting, nucleotide sequence R₁may encode rotavirus protein VP2 and nucleotide sequence R₂-R₅may encode in any order rotavirus protein VP4, VP6, VP7 and NSP4, but R₂-R₅may not encode VP2. In another non-limiting example nucleotide sequence R₁may encode rotavirus protein VP4 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP6, VP7 and NSP4. In yet another non-limiting example nucleotide sequence R₁may encode rotavirus protein VP6 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP7 and NSP4. In yet another example which is not to be considered limiting, nucleotide sequence R₁may encode rotavirus protein VP7 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and NSP4. In yet another non-limiting example nucleotide sequence R₁may encode rotavirus protein NSP4 and nucleotide sequences R₂-R₅may encode in any order rotavirus protein VP2, VP4, VP6 and VP7.

For example, which is not to be considered limiting, a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein, for example VP2 is co-expressed with a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein, for example VP6, a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein for example VP4, a fourth nucleic acid (N₄) comprising a fourth nucleotide sequence (R₄) encoding a fourth rotavirus protein, for example VP7 and a fifth nucleic acid (N5) comprising a fifth nucleotide sequence (R₅) encoding a fifth rotavirus protein for example NSP4 (see FIG. 3).

The five nucleic acids may be introduced into a plant in any order. For example, which is not to be considered limiting, a plant that expresses a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁) may be transformed with a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂). The plant may be further transformed with a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃). The plant then may be further transformed with a fourth nucleic acid (N₄) comprising a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄). The plant then may be further transformed with a fifth nucleic acid (N₅) comprising a fourth nucleotide sequences (R₅) encoding a fourth rotavirus protein (X₅), so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. Rotavirus protein X₁-X₅may be selected from the group of VP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the plant. Furthermore, a plant may be co-transformed simultaneously with a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁), a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂), a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃), a fourth nucleic acid (N₄) comprising a fourth nucleotide sequences (R₄) encoding a fourth and a fifth rotavirus protein (X₄), and a fifth nucleic acid (N₅) comprising a fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein (X₅) so that the first, second, third, fourth and fifth nucleotide sequence are co-expressed in the plant. The first nucleic acid (N₁), second nucleic acid (N₂), third nucleic acid (N₃), fourth nucleic acid (N₄) and fifth nucleic acid (N₅) may be introduced in the plant in a transient manner, or in a stable manner.

A first plant expressing a first nucleic acid (N₁) comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein (X₁), may be crossed with a second plant expressing a second nucleic acid (N₂) comprising a second nucleotide sequence (R₂) encoding a second rotavirus protein (X₂) to produce a progeny plant (third plant). The third plant may be crossed with a fourth plant expressing a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein (X₃) to produces a progeny plant (fifth plant). The fifth plant may be crossed with a sixth plant expressing a fourth nucleic acid (N₄) comprising a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄) to produce a progeny plant (seventh plant). The seventh plant may be crossed with an eight plant expressing a fifth nucleic acid (N₅) comprising a fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein (X₅) to produce a progeny plant that co-expresses the first, second, third, fourth and fifth rotavirus protein (X₁-X₅). Rotavirus protein X₁-X₅may be selected from the group of VP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in the plant.

Ratio of Nucleic Acids (N) used to Transform a Host

As may be seen in FIG. 5, the level of RLP accumulation in the plant, portion of the plant or plant cell, may be influenced by the ratio of the nucleic acids encoding rotavirus structural and nonstructural proteins that are expressed in a plant. For example, which is not to be considered limiting, the ratio of nucleic acids (N) that are introduced into a plant may be modified by providing different amounts of Agrobacterium, that are used to infiltrate the plant, portion of the plant or plant cell, where each Agrobacterium comprises a construct comprising a nucleic acid (N) as set out in Table 1 (and accompanying text) above. For example which is not to be considered limiting, the ratio of the structural protein-containing to nonstructural protein-containing Agrobacterium may range for example from about 0.8:1 to about 2.5:1.5 (structural protein:nonstructural protein), or any amount therebetween, for example from about 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:0.5, 2:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.5:1.1, 2.5:1.2, 2.5:1.3, 2.5:1.4, 2.5:1.5 (structural protein:nonstructural protein), or any amount therebetween. For example, as described below, the ratio of structural rotavirus protein to nonstructural protein may be varied for example by introducing different ratios of Agrobacterium containing the one or more nucleic acid (N) comprising one or more nucleotide sequence (R) encoding the rotavirus structural and non-structural proteins (X) into a host, for example a plant, portion of the plant or plant cell. For example the absorbance or optical density (OD) of Agrobacterium strains in the bacterial suspension may be used as measure to establish a ratio between Agrobacterium strains that containing the structural protein to Agrobacterium strains that contain the nonstructural protein. For example, which is not considered limiting, the OD may range for example from about 0.2:0.4 to about 1:0.6 (Agrobacterium strains containing structural protein:Agrobacterium strains containing nonstructural protein in the bacterial suspension) or any amount therebetween, for example from about 0.2:0.4, 0.3:0.4, 0.4:0.4, 0.5:0.4, 0.6:0.4, 0.7:0.4, 0.8:0.4, 0.9:0.4, 1:0.4, 0.2:0.5, 0.3:0.5, 0.4:0.5, 0.5:0.5, 0.6:0.5, 0.7:0.5, 0.8:0.5, 0.9:0.5, 1:05, 0.2:0.6, 0.3:0.6, 0.4:0.6, 0.5:0.6, 0.6:0.6, 0.7:0.6, 0.8:0.6, 0.9:0.6, 1:06, 0.3:0.4, 0.3:0.5, 0.3:0.6, 0.4:0.4, 0.4:0.5, 0.4:0.6, 0.5:0.4, 0.5:0.5, 0.5:0.6, 0.6:0.4, 0.6:0.5, 0.6:0.6, 0.7:0.4, 0.7:0.5, 0.7:0.6, 0.8:0.4, 0.8:0.5, 0.8:0.6, 0.9:0.4, 0.9:0.5, 0.9:0.6, 1:0.4, 1:0.5, 1:0.6 (Agrobacterium strains containing structural protein:Agrobacterium strains containing nonstructural protein in the bacterial suspension) or any amount therebetween. For example, which is not considered limiting, an OD of 0.4 of Agrobacterium strains in bacterial suspension may be designated as a reference of 1. Therefore a ratio of 1.5:1 of structural to non structural protein may be achieved by using an OD of 0.6 of Agrobacterium strain containing structural protein to an OD of 0.4 of Agrobacterium strain containing nonstructural protein in bacterial suspension.

The ratio of rotavirus structural protein to nonstructural protein may be varied for example by introducing different ratios of Agrobacterium containing the one or more nucleic acid (N) comprising one or more nucleotide sequence (R) encoding the rotavirus structural and non-structural proteins (X) into the plant, portion of the plant or plant cell. Alternatively, if the rotavirus structural proteins and nonstructural proteins are present on the same construct, and therefore are introduced into the plant, plant portion or plant cell, using one Agrobacterium, they may be differentially expressed within the plant, portion of the plant or plant cell using suitable promoters so that the desired ratio of rotavirus structural proteins and nonstructural proteins is obtained.

Therefore as described herein, a method is provided for increasing RLP production yield, increasing VP4 and VP7 yield, or increasing both RLPS and VP4 and VP7 yield, by modulating the ratio between the one or more nucleic acid (N) comprising one or more nucleotide sequence (R) encoding the rotavirus structural proteins (X) and the one or more nucleic acid (N) comprising one or more nucleotide sequence (R) encoding the rotavirus nonstructural proteins (X).

For example, the percentage of the Agrobacterium containing rotavirus nonstructural protein may be between 20% to 60% or any amount therebetween, of total amount of Agrobacterium use to infiltrate the plant, plant portion or plant cell. For example the percent ratio of Agrobacterium containing rotavirus nonstructural protein may be 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or any amount therebetween of the total Agrobacterium use to infiltrate the plant, plant portion or plant cell. Similarly, the percentage of Agrobacterium containing structural protein within the total amount of Agrobacterium infiltrated may be 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41% or 40% or any amount therebetween, of the total Agrobacterium use to infiltrate the plant, plant portion or plant cell.

For example, the percentage ratio of Agrobacterium containing rotavirus structural protein to Agrobacterium containing nonstructural protein may be 70%:30%, 60%:40%, 50%:50%, 40%:60% or any percentage ratio amount therebetween. For example, the percentage ratio between Agrobacterium containing structural protein and Agrobacterium containing nonstructural protein may be 50%:50%, 51%:49%, 52%:48%, 53%:47%, 54%:46%, 55%:45%, 56%:44%, 57%:43%, 58%:42%, 59%: 41%, 60%:40%, or any percentage ratio in between.

As described below, the ratio of rotavirus structural protein to rotavirus nonstructural protein may further be varied for example by differentially expressing the rotavirus structural protein and the rotavirus nonstructural protein. Expression may be varied by modulating for example replication, transcription, translation, or a combination thereof, of the rotavirus structural protein , the rotavirus nonstructural protein, or both the rotavirus structural protein and the rotavirus nonstructural protein. For example different regulatory elements, including promoters, amplification elements, enhancers or a combination thereof, may be used in addition to varying the ratio of the rotavirus structural protein-containing Agrobacterium to rotavirus nonstructural protein-containing Agrobacterium infiltrated as described above. A first set or combination of regulatory elements may be used to regulate the replication, transcription or a combination thereof, of the one or more nucleic acid comprising one or more nucleotide sequence encoding rotavirus structural protein and a second set or combination of regulatory elements may be used to regulate the replication, transcription or a combination thereof, of the one or more nucleotide sequence encoding rotavirus nonstructural protein. The first set or combination of regulatory elements is different from the second set or combination of regulatory elements and permits differential expression of the one or more nucleic acid comprising one or more nucleotide sequence encoding rotavirus structural protein and the one or more nucleic acid comprising one or more nucleotide sequence encoding rotavirus nonstructural protein to permit modulating the ratio of rotavirus structural protein:rotavirus nonstructural protein in vivo.

For example, which is not to be considered limiting, one set or combination of regulatory elements, for example the first set, may include an enhancer element for example elements obtained from CPMV, such as CPMV HT, or CPMV 160 (see FIG. 6). CMPV HT is described in U.S. 61/971,274 (which is incorporated herein by reference) and CPMV 160 is described in U.S. 61/925,852 (which is incorporated herein by reference). The enhancer element, for example those obtained from CPMV, for example CPMV HT or CPMV 160 (see FIG. 6; U.S. 61/971,274, and U.S. 61/925,852, respectively) may be absent in the other set or combination of regulatory elements, for example the second set. Alternatively, the second set may include an enhancer element (for example elements obtained from CPMV, (for example CPMV HT or CPMV 160), while the amplification element (for example elements obtained from CPMV, (for example CPMV HT or CPMV 160) may be absent in the first set or combination of regulatory elements. In a similar manner, the strength of a promoters may differ between the first and second set or combination of regulatory elements, or one of the promoters may be inducible, and the other constitutive, so that differential expression between the rotavirus structural protein relative to the rotavirus nonstructural protein is achieved in vivo.

For example, the ratio of rotavirus structural protein to nonstructural protein may be varied for example by introducing different ratios of Agrobacterium containing a first nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding first rotavirus protein for example rotavirus nonstructural protein NSP4 to Agrobacterium containing a second nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein, for example in any order rotavirus structural proteins VP2, VP4, VP6 and VP7. For example the ratio of the Agrobacterium containing a first nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding rotavirus nonstructural protein NSP4 to the Agrobacterium containing a second nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅) encoding rotavirus structural proteins VP2, VP4, VP6 and VP7 may be 0.8:1 and 1:2 (Agrobacterium containing N₁to N₂) or any amount there between for example 1:1.5 (Agrobacterium containing N₁to N₂).

Furthermore, the ratio of rotavirus structural protein to nonstructural protein may be varied by differentially expressing within the plant, portion of the plant or plant cell the rotavirus structural protein to nonstructural protein using enhancer elements. For example, the ratio of rotavirus structural protein to nonstructural protein may be varied for example by co-expressing within the plant, portion of the plant or plant cell a first nucleic acid (N₁) comprising a nucleotide sequence (R₁) encoding a first rotavirus protein for example a nonstructural protein NSP4 with a second nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein for example in any order rotavirus structural proteins VP2, VP4, VP6 and VP7, wherein the second, third, fourth and fifth nucleotide sequence are operatively linked to an enhancer sequence for example CPMV HT, CPM 160, CPMV 160+ and CPMV HT+ (described in U.S. 61/971,274, and U.S. 61/925,852, respectively which are incorporated herein by reference), as described below. In another example, the ratio of rotavirus structural protein to nonstructural protein may be varied for example by co-expressing within the plant, portion of the plant or plant cell a first nucleic acid (N₁) comprising first nucleotide sequence (R₁) encoding a first rotavirus protein for example structural protein VP6 or VP7 and second nucleotide sequence (R₂) encoding a second rotavirus protein for example structural protein VP2 or VP4, second nucleic acid (N₂) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein for example structural protein VP7 or VP6 and a fourth nucleotide sequence (R₄) encoding a fourth rotavirus protein for example structural protein VP4 or VP2 and a third nucleic acid (N₃) comprising fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein for example nonstructural protein NSP4, wherein the first, second, third and fourth nucleotide sequence are operatively linked to an enhancer sequence for example CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+, as described below.

In another example, the ratio of rotavirus structural protein to nonstructural protein may be varied for example by co-expressing within the plant, portion of the plant or plant cell one or more nucleic acids comprising a first nucleotide sequence (R₁) encoding a first rotavirus protein for example structural protein VP6 or VP7 and second nucleotide sequence (R₂) encoding a second rotavirus protein for example structural protein VP2 or VP4, a third nucleotide sequence (R₃) encoding a third rotavirus protein for example structural protein VP7 or VP6 and a fourth nucleotide sequence (R₄) encoding fourth rotavirus protein for example structural protein VP4 or VP2, and a fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein for example nonstructural protein NSP4, wherein the first, second, third and fourth nucleotide sequence are operatively linked to an enhancer sequence for example CPM 160, CPMV 160+ and CPMV HT+, as described below and the fifth nucleotide sequence is operatively linked to CPMV HT as described below.

In another example, the ratio of rotavirus structural protein to nonstructural protein may be varied for example by co-expressing within the plant, portion of the plant or plant cell a first nucleic acid (N₁) comprising first nucleotide sequence (R₁) encoding a first rotavirus protein for example structural protein VP6 or VP7, a second nucleic acid (N₂) comprising second nucleotide sequence (R₂) encoding a second rotavirus protein for example structural protein VP2 or VP4, a third nucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding a third rotavirus protein for example structural protein VP7 or VP6, a fourth nucleic acid (N₄) comprising a fourth nucleotide sequence (R₄) encoding a fourth rotavirus protein for example structural protein VP4 or VP2 and a fifth nucleic acid (N₅) comprising fifth nucleotide sequences (R₅) encoding a fifth rotavirus protein for example nonstructural protein NSP4, wherein the first, second, third and fourth nucleotide sequence are operatively linked to an enhancer sequence for example CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+, as described below.

Amplification Elements and Enhancer Elements/Regulatory Elements

The rotavirus protein or polypeptide may be expressed in an expression system comprising a viral based, DNA or RNA, expression system, for example but not limited to, a comovirus-based expression cassette and geminivirus-based amplification element.

Enhancer elements may be used to achieve high level of transient expression of rotavirus structural and nonstructural proteins. Enhancer elements may be based on RNA plant viruses, including comoviruses, such as Cowpea mosaic virus (CPMV; see, for example, WO2007/135480; WO2009/087391; US 2010/0287670, Sainsbury F. et al., 2008, Plant Physiology; 148: 121-1218; Sainsbury F. et al., 2008, Plant Biotechnology Journal; 6: 82-92; Sainsbury F. et al., 2009, Plant Biotechnology Journal; 7: 682-693; Sainsbury F. et al. 2009, Methods in Molecular Biology, Recombinant Proteins From Plants, vol. 483: 25-39).

CPMV 160 (CPMVX) and CPMV 160+ (CPMVX+)

In an embodiment the enhancer Elements are “CPMVX” (also referred as “CPMV 160”) and/or “CPMVX+” (also referred to as “CPMV 160+”) and are described in U.S. 61/925,852 (which is incorporated herein by reference).

Expression enhancer “CPMVX” comprises a comovirus cowpea mosaic virus (CPMV) 5′ untranslated region (UTR). The 5′UTR from nucleotides 1-160 of the CPMV RNA-2 sequence (SEQ ID NO: 1), starts at the transcription start site to the first in frame initiation start codon (at position 161), which serve as the initiation site for the production of the longer of two carboxy coterminal proteins encoded by a wild-type comovirus genome segment. Furthermore a ‘third’ initiation site at (or corresponding to) position 115 in the CPMV RNA-2 genomic sequence may also be mutated, deleted or otherwise altered. It has been shown that removal of AUG 115 in addition to the removal of AUG 161 enhances expression when combined with an incomplete M protein (Sainsbury and Lomonossoff, 2008, Plant Physiology; 148: 1212-1218; WO 2009/087391; which are incorporated herein by reference).

CPMVX comprises X nucleotides of SEQ ID NO:1, where X=160, 155, 150, or 114 of SEQ ID NO:1, or a sequence that comprises between 80% to 100% sequence similarity with CPMVX, where X=160, 155, 150, or 114 of SEQ ID NO:93. This expression enhancer is generally referred to as CPMVX (see FIG. 6c).

The expression enhancer CPMVX, where X=160, consists of nucleotides 1-160 of SEQ ID NO: 1:

(SEQ ID NO: 1)

1
tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc

61
ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc

121
gatcttcaac gttgtcagat cgtgcttcgg caccagtaca

The CPMVX enhancer sequence may further be fused to a stuffer sequence, wherein the CMPVX comprises X nucleotides of SEQ ID NO:1, where X=160, 155, 150, or 114 of SEQ ID NO:1, or a sequence that comprises between 80 to 100% sequence similarity with CPMVX, where X=160, 155, 150, or 114 of SEQ ID NO:1, and the stuffer sequence comprises from 1-100 nucleotides fused to the 3′ end of the CMPVX sequence. For example, the stuffer sequence may comprise from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides, or any number of nucleotides therebetween.

If the CMPVX sequence comprises a stuffer fragment, then this expression enhancer may be referred to as CPMVX+ (see FIG. 6d), where X=160, 155, 150, 114 of SEQ ID NO:1, it may also be referred to as CMPVX comprising a stuffer sequence, or it may be referred to as CPMV160+; CPMV155+; CPMV150+; CPMV114+, when X-160, 155, 150, or 114, respectively. Constructs comprising CPMVX that do not comprise a stuffer sequence may be termed CPMVX+, where X=160, 155, 150, 114 of SEQ ID NO:1, and where the stuffer sequence is of 0 nucleotides in length.

The stuffer sequence may be modified by truncation, deletion, or replacement of the native CMPV 5′UTR sequence that is located 3′ to nucleotide 160. The modified stuffer sequence may be removed, replaced, truncated or shortened when compared to the initial or unmodified (i.e. native) stuffer sequence associated with the 5′UTR (as described in Sainsbury F., and Lomonossoff G. P., 2008, Plant Physiol. 148: pp. 1212-1218). The stuffer sequence may comprise a one or more restriction sites (polylinker, multiple cloning site, one or more cloning sites), one or more plant kozak sequences, one or more linker sequences, one or more recombination sites, or a combination thereof. For example, which is not to be considered limiting, a stuffer sequence may comprise in series, a multiple cloning site of a desired length fused to a plant kozak sequence. The stuffer sequence does not comprise a nucleotide sequence from the native 5′UTR sequence that is positioned 3′ to nucleotide 160 of the native CPMV 5′UTR, for example nucleotides 161 to 512 as shown in FIG. 1 of Sainsbury F., and Lomonossoff G. P. (2008, Plant Physiol. 148: pp. 1212-1218; which is incorporated herein by reference), or nucleotides 161-509 of prior art CPMV HT sequence. That is, the incomplete M protein present in the prior art CPMV HT sequence (FIG. 1; of Sainsbury F., and Lomonossoff G. P., 2008) is removed from the 5′UTR in the present invention.

Plant Kozak consensus sequences are known in the art (see for example Rangan et al. Mol. Biotechnol., 2008, July 39(3), pp. 207-213). Both naturally occurring and synthetic Kozak sequences may be used in the expression enhancer or may be fused to the nucleotide sequence of interest as described herein.

The plant kozak sequence may be any known plant kozak sequences (see for example L. Rangan et. al. Mol. Biotechnol. 2008), including, but not limited to the following plant consensus sequences:

(SEQ ID NO: 2; plant kingdom)

caA(A/C)a

(SEQ ID NO: 3; dicots)

aaA(A/C)a

(SEQ ID NO: 4; arabidopsis)

aa(A/G) (A/C)a

The plant kozak sequence may also be selected from the group of:

(SEQ ID NO: 5)

AGAAA

(SEQ ID NO: 6)

AGACA

(SEQ ID NO: 7)

AGGAA

(SEQ ID NO: 8)

AAAAA

(SEQ ID NO: 9)

AAACA

(SEQ ID NO: 10)

AAGCA

(SEQ ID NO: 11)

AAGAA

(SEQ ID NO: 12)

AAAGAA

(SEQ ID NO: 13)

AAAGAA

(SEQ ID NO: 14; Consensus sequence)

(A/-)A(A/G) (A/G) (A/C)A.

The expression enhancer CPMVX, or CPMVX+, may be operatively linked at the 5′ end of the enhancer sequence with a regulatory region that is active in a plant, and operatively linked to a nucleotide sequence of interest at the 3′ end of the expression enhancer (FIG. 6c), in order to drive expression of the nucleotide sequence of interest within a plant host.

CPMV HT+, CPMV HT+[WT115], CPMV HT+[511]

In another embodiment the enhancer elements is “CPMV HT+” which is described in U.S. 61/971,274 (which is incorporated herein by reference). Expression enhancer “CPMV HT+” (see FIG. 6b) comprises a comovirus 5′ untranslated region (UTR) and a modified, lengthened, or truncated stuffer sequence.

A plant expression system comprising a first nucleic acid sequence comprising a regulatory region, operatively linked with one or more than one expression enhancer as described herein (e.g. CPMV HT+, CPMV HT+[WT115], CPMV HT+[511]), and a nucleotide sequence encoding a rotavirus structural or nonstructural proteins is also provided. Furthermore, a nucleic acid comprising a promoter (regulatory region) sequence, an expression enhancer (e.g. CPMV HT+ or CPMV HT+[WT115]) comprising a comovirus 5′UTR and a stuffer sequence with a plant kozak sequence fused to one or more nucleic acid sequences encoding a rotavirus structural or nonstructural proteins are described. The nucleic acid may further comprise a sequence comprising a comovirus 3′ untranslated region (UTR), for example, a plastocyanin 3′ UTR, or other 3′UTR active in a plant, and a terminator sequence, for example a NOS terminator, operatively linked to the 3′ end of the nucleotide sequence encoding a rotavirus structural or nonstructural proteins (referred to as nucleotide of interest in FIG. 6a), so that the nucleotide sequence encoding the rotavirus structural or nonstructural proteins is inserted upstream from the comovirus 3′ untranslated region (UTR), plastocyanin 3′ UTR, or other 3′UTR sequence.

SEQ ID NO:15 comprises a “CPMV HT” expression enhancer as known in the prior art (e.g. FIG. 1 of Sainsbury and Lomonossoff 2008, Plant Physiol. 148: pp. 1212-1218; which is incorporated herein by reference). CPMV HT includes the 5′UTR sequence from nucleotides 1-160 of SEQ ID NO:15 with modified nucleotides at position 115 (cgt), and an incomplete M protein with a modified nucleotide at position 162 (acg), and lacks a plant kozak sequence (5′UTR: nucleotides 1-160; incomplete M protein underlined, nucleotides 161-509). SEQ ID NO:15 also includes a multiple cloning site (italics, nucleotides 510-528) which is not present in the prior art CPMV HT sequence:

SEQ ID NO: 15

1
tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc

61
ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc

121
gatcttcaac gttgtcagat cgtgcttcgg caccagtaca custom-character

ttttctt tcactgaagc

181

gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc

241

ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc

301

atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt

361

gacgaggtat tgttgcctgt acttctttct tcttcttctt gctgattggt tctataagaa

421

atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt

481

taagcttctg tatattctqc ccaaatttg
t cgggccc

CPMV HT+ with a plant kozak consensus sequence is provided in SEQ ID NO:16 (nucleotide 1-160, 5′UTR, including modified ATG at positions 115 (GTG) lower case bold and italics; stuffer fragment comprising: an incomplete M protein underlined, nucleotides 161-509, with modified nucleotide at 162 (ACG); a multiple cloning site, italics, nucleotides 510-528; and a consensus plant kozak sequence, caps and bold, nucleotides 529-534).

(SEQ ID NO: 16)

1
tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc

61
ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc

121
gatcttcaac gttgtcagat cgtgcttcgg caccagtaca custom-character

SEQ ID NO:17 (“CPMV HT+511”) comprises a segment of the native sequence of the CPMV RNA 2 genome from nucleotides 1-154. The 5′UTR sequence from nucleotides 1-511 of SEQ ID NO:17 comprises modified “atg” sequences at positions 115 (“g” in place of “a”; italics bold) and 162 (“c” in place of “t”; italics bold), and an incomplete M protein (underlined) from nucleotides 161-511. CPMV HT+511 comprises a native M protein kozak consensus sequence (nucleotides 508-511; bold):

SEQ ID NO: 17

1
tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc

61
ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgc custom-character

agc

121
gatcttcaac gttgtcagat cgtgcttcgg caccagtaca custom-character

Another non-limiting example of a CPMV HT+ enhancer sequence is provided by the sequence of SEQ ID NO:18 (CPMV HT+[WT115]). Expression cassettes or vectors comprising CPMV HT+ and including a plant regulatory region in operative association with the expression enhancer sequence of SEQ ID NO: 18, and the transcriptional start site (ATG) at the 3′ end fused to a nucleotide sequence encoding rotavirus structural or nonstructural protein are also part o the present invention.

SEQ ID NO: 18 (CPMV HT+[WT115]) nucleotide 1-160, 5′UTR, with an ATG at position 115-117, lower case bold; stuffer fragment comprising: an incomplete M protein underlined, nucleotides 161-509; with a modified ATG at position 161-153 lower case bold, and underlined, a multiple cloning site, italics, nucleotides 510-528; and a plant kozak sequence, caps and bold, nucleotides 529-534).

(SEQ ID NO: 18)

1
tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc

61
ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgc custom-character

agc

121
gatcttcaac gttgtcagat cgtgcttcgg caccagtaca custom-character

The plant kozak sequence of SEQ ID NO:18 may be any plant kozak sequence, including but not limited, to one of the sequences of SEQ ID NO's: 2-14.

A plant expression system comprising a first nucleic acid sequence comprising a regulatory region, operatively linked with one or more than one expression enhancer as described herein (e.g. CPMV HT+, CPMV HT+[WT115], CPMV HT+[511]), and a nucleotide sequence encoding a rotavirus structural or nonstructural protein is also provided. Furthermore, a nucleic acid comprising a promoter (regulatory region) sequence, an expression enhancer (e.g. CPMV HT+ or CPMV HT+[WT115]) comprising a comovirus 5′UTR and a stuffer sequence with a plant kozak sequence fused to one or more nucleic acid sequences encoding rotavirus structural or nonstructural protein are described. The nucleic acid may further comprise a sequence comprising a comovirus 3′ untranslated region (UTR), for example, a plastocyanin 3′ UTR, or other 3′UTR active in a plant, and a terminator sequence, for example a NOS terminator, operatively linked to the 3′end of the nucleotide sequence encoding rotavirus structural or nonstructural protein (referred to as nucleotide of interest in FIG. 6a), so that the nucleotide sequence encoding rotavirus structural or nonstructural protein is inserted upstream from the comovirus 3′ untranslated region (UTR), plastocyanin 3′ UTR, or other 3′UTR sequence.

The occurrence of RLPs produced using the methods described herein may be detected using any suitable method for example density gradient centrifugation or size exclusion chromatography. RLPs may be assessed for structure and size, for example by electron microscopy, size exclusion chromatography, or other techniques that would be evident to one of skill in the art.

For size exclusion chromatography, total soluble proteins may be extracted from plant tissue by homogenizing (Polytron) sample of frozen-crushed plant material in extraction buffer, and insoluble material removed by centrifugation. Precipitation with ice cold acetone or PEG may also be of benefit. The soluble protein is quantified, and the extract passed through a Sephacryl™ column, for example a Sephacryl™ S500 column. Blue Dextran 2000 may be used as a calibration standard. Following chromatography, fractions may be further analyzed by immunoblot to determine the protein complement of the fraction.

The separated fraction may be for example a supernatant (if centrifuged, sedimented, or precipitated), or a filtrate (if filtered), and is enriched for proteins, or suprastructure proteins, and include higher molecular weight, particles such as single-layered (sl), double-layered (dl) or triple-layered (tl) RLPs.

The separated fraction may be further processed to isolate, purify, concentrate or a combination thereof, the proteins, suprastructure proteins or higher-order particles by, for example, additional centrifugation steps, precipitation, chromatographic steps (e.g. size exclusion, ion exchange, affinity chromatography), tangential flow filtration, or a combination thereof. The presence of purified proteins, suprastructure proteins or higher-order particles such as RLPs, may be confirmed by, for example, native or SDS-PAGE, Western analysis using an appropriate detection antibody, capillary electrophoresis, electron microscopy, or any other method as would be evident to one of skill in the art.

The RLP's produced according to the present invention may be purified, partially purified from a plant, portion of a plant or plant matter, or may be administered as an oral vaccine, using methods as know to one of skill in the art.

RLP purification may involve gradient centrifugation, for example sucrose, iodixanol, OptiPrep™ or cesium chloride (CsCl) density gradients may be used to purify or partially purify the RLPs from transformed plant biomass. As shown for example in FIG. 4, an iodixanol step gradient or iodixanol continuous gradient might be used to purify the RLP and/or expressed rotavirus structural proteins.

Calcium (Ca2+) concentration has been shown to be important for the triple-layer particle (TLP) to double layer particle (DLP) transformation and is strain dependent (see for example Martin et al. Journal of Virology, January 2002, which is incorporated herein by reference). Complete loss of the outer-capsid proteins from TLPs (TLP decapsidation) takes place in the nanomolar range of [Ca2+]. Therefore the extraction and/or purification of RLP may be performed in the presence of calcium, and the step of gradient centrifugation may be performed in the presence of calcium, for example in the present of CaCl2. The concentration of CaCl2 maybe between for example, 1 mM and 1000 mM, or any amount there between, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 50, 600, 650, 700, 750, 800, 850, 900, 950 mM or any amount therebetween.

The plants, or plant fragments may be minimally processed. By the term “minimal processing” it is meant plant matter, for example, a plant or portion thereof comprising a protein of interest and/or the RLP which is partially purified to yield a plant extract, homogenate, fraction of plant homogenate or the like (i.e. minimally processed). Partial purification may comprise, but is not limited to disrupting plant cellular structures thereby creating a composition comprising soluble plant components, and insoluble plant components which may be separated for example, but not limited to, by centrifugation, filtration or a combination thereof. In this regard, proteins secreted within the extracellular space of leaf or other tissues could be readily obtained using vacuum or centrifugal extraction, or tissues could be extracted under pressure by passage through rollers or grinding or the like to squeeze or liberate the protein free from within the extracellular space. Minimal processing could also involve preparation of crude extracts of soluble proteins, since these preparations would have negligible contamination from secondary plant products. Further, minimal processing may involve aqueous extraction of soluble protein from leaves, followed by precipitation with any suitable salt. Other methods may include large scale maceration and juice extraction in order to permit the direct use of the extract. The RLPs may be purified or extracted using any suitable method for example mechanical or biochemical extraction.

The one or more rotavirus structural protein may be synthesized at an amount up to 2 g per kilogram of plant fresh weight. For example, the amount of synthesized structural protein maybe between 1 and 2 g per kilogram of fresh weight, or any amount there between, such as 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 g per kilogram of fresh weight or any amount therebetween. For example, the structural protein may be synthesized at an amount up to 1.54 g per kilogram of plant fresh weight.

The size (i.e. the diameter) of the above-defined RLPs, maybe measures for example by dynamic light scattering (DLS) or electron microscope (EM) techniques, is usually between 50 to 110 nm, or any size therebetween. For example, the size of the intact RLP structure may range from about 70 nm to about 110 nm, or any size therebetween, such as 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm or any size therebetween.

Nucleotide Sequences

The present invention further provides a nucleic acid comprising a nucleotide sequence encoding one or more rotavirus structural protein operatively linked to a regulatory region active in a plant. The nucleotide sequence may be optimized for example for human codon usage or plant codon usage. Furthermore one or more rotavirus structural protein may be operatively linked to one or more than one amplification elements. In addition one or more rotavirus structural protein may be operatively linked to one or more than one compartment targeting sequence. The one or more rotavirus structural protein encoded by the nucleotide sequence may be for example VP2, VP4, VP6 or VP7. Furthermore the one or more rotavirus structural protein encoded by the nucleotide sequence may be for example from any rotavirus group A to G, but more preferably from rotavirus group A. Furthermore, the one or more rotavirus structural protein encoded by the nucleotide sequence maybe from any rotavirus strain having a genotype of any combinations of G- and P-types from G1 to G27 and from P1 to P34, and 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 A WA strain, rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain or rotavirus SA11 strain.

A nucleic acid sequence referred to in the present invention, may be “substantially homologous”, “substantially similar” or “substantially identical” to a sequence, or a compliment of the sequence if the nucleic acid sequence hybridize to one or more than one nucleotide sequence or a compliment of the nucleic acid sequence as defined herein under stringent hybridization conditions. Sequences are “substantially homologous” “substantially similar” “substantially identical” when at least about 70%, or between 70 to 100%, or any amount therebetween, for example 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100%, or any amount therebetween, of the nucleotides match over a defined length of the nucleotide sequence providing that such homologous sequences exhibit one or more than one of the properties of the sequence, or the encoded product as described herein.

For example the present invention provides an isolated polynucleotide comprising a nucleotide sequence which encodes one or more rotavirus protein, for example a structural or nonstructural rotavirus protein, that is 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 identical to sequences as defines for example in SEQ ID NOs: 21, 27, 32, 37 or 42. The polynucleotide may be human codon optimized by any of the methods known in the art. The nucleotide sequence may enclode for example a rotavirus protein that is 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 identical the amino acid sequence of SEQ ID NOs: 24, 29, 34, 39 or 44.

Furthermore, the present invention provides RLPS that comprise rotavirus structural proteins that are for example encoded by nucleic acids that are 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 identical to sequences as defines for example in SEQ ID NOs: 21, 27, 32, 37 or 42.

Such a sequence similarity or identity may be determined using a nucleotide sequence comparison program, such as that provided within DNASIS (using, for example but not limited to, the following parameters: GAP penalty 5, # of top diagonals 5, fixed GAP penalty 10, k tuple 2, floating gap 10, and window size 5). However, other methods of alignment of sequences for comparison are well-known in the art for example the algorithms of Smith & Waterman (1981, Adv. Appl. Math. 2:482), Needleman & Wunsch (J. Mol. Biol. 48:443, 1970), Pearson & Lipman (1988, Proc. Nat'l. Acad. Sci. USA 85:2444), and by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and BLAST, available through the NIH.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 supplement), or using Southern or Northern hybridization under stringent conditions (see Maniatis et al., in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982). Preferably, sequences that are substantially homologous exhibit at least about 80% and most preferably at least about 90% sequence similarity over a defined length of the molecule.

An example of one such stringent hybridization conditions may be overnight (from about 16-20 hours) hybridization in 4×SSC at 65° C., followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes. Alternatively an exemplary stringent hybridization condition could be overnight (16-20 hours) in 50% formamide, 4×SSC at 42° C., followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes, or overnight (16-20 hours), or hybridization in Church aqueous phosphate buffer (7% SDS; 0.5M NaPO4 buffer pH 7.2; 10 mM EDTA) at 65° C., with 2 washes either at 50° C. in 0.1×SSC, 0.1% SDS for 20 or 30 minutes each, or 2 washes at 65° C. in 2×SSC, 0.1% SDS for 20 or 30 minutes each for unique sequence regions.

A nucleic acid encoding a rotavirus structural polypeptide may be described as a “rotavirus nucleic acid”, a “rotavirus nucleotide sequence”, a “rotavirus nucleic acid”, or a “rotavirus nucleotide sequence”. For example, which is not to be considered limiting, a virus-like particle comprising one or more rotavirus structural protein or rotavirus structural polypeptide, may be described as a “rotavirus VLP”, “RVLP” or “RLP”.

Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. The process of optimizing the nucleotide sequence coding for a heterologously expressed protein can be an important step for improving expression yields. The optimization requirements may include steps to improve the ability of the host to produce the foreign protein.

“Codon optimization” is defined as modifying a nucleic acid sequence for enhanced expression in cells 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 frequently or most frequently used in the genes of another organism or species. Various species exhibit particular bias for certain codons of a particular amino acid.

The present invention includes synthetic polynucleotide sequences that have been codon optimized for example the sequences have been optimized for human codon usage or plant codon usage. The codon optimized polynucleotide sequences may then be expressed in plants. More specifically the 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 the sequences optimized for human codon increases the guanine-cytosine content (GC content) of the sequence and improves expression yields in plants.

There are different codon-optimisation techniques known in the art for improving, the translational kinetics of translationally inefficient protein coding regions. These techniques mainly rely on identifying the codon usage for a certain host organism. If a certain gene or sequence should be expressed in this organism, the coding sequence of such genes and sequences will then be modified such that one will replace codons of the sequence of interest by more frequently used codons of the host organism.

Amino Acid Sequences

Non-limiting examples of rotavirus structural protein are rotavirus protein VP2, VP4, VP6 and VP7, and a fragment of VP2, VP4, VP6 and VP7. Non-limiting examples of VP2, VP4, VP6 and VP7, or fragments of VP2, VP4, VP6 and VP7 protein that may be used according to the present invention include those VP2, VP4 VP6 and VP7 protein from rotavirus strain G9 P[6], rotavirus A WA strain, rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain and rotavirus SA11 strain. For example, but not limited to Rotarix-A41CB052A: VP4 (accession #JN849113), VP7: (accession #JN849114), rotavirus A WA strain: VP2 (accession #X14942), VP4: (accession #L34161), VP6 (accession #K02086), VP7: (accession #GU723327), NSP4 (accession #K02032), rotavirus SA11 strain: VP2 (accession #NC_011506), VP4 (accession #NC_011510), VP6 (accession #NC_011509), VP7 (accession #NC_011503) and NSP4 (accession #NC_011504).

An example of a VP2 structural protein, which is not to be considered limiting, is set forth in the amino acid sequence of SEQ ID NO: 24. Furthermore, the VP2 structural protein may comprise the sequence set forth in SEQ ID NO: 24, or a sequence having at least about 90-100% sequence similarity thereto, including any percent similarity within these ranges, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto. In addition, a VP2 structural protein may be encoded by a nucleotide sequence as set forth in SEQ ID NO:21 or 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 a VP4 structural protein, which is not to be considered limiting, is set forth in the amino acid sequence of SEQ ID NO: 34. Furthermore, the VP4 structural protein may comprise the sequence set forth in SEQ ID NO: 34, or a sequence having at least about 90-100% sequence similarity thereto, including any percent similarity within these ranges, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto. In addition, a VP4 structural protein may be encoded by a nucleotide sequence as set forth in SEQ ID NO: 32 or 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 a VP6 structural protein, which is not to be considered limiting, is set forth in the amino acid sequence of SEQ ID NO: 29. Furthermore, the VP6 structural protein may comprise the sequence set forth in SEQ ID NO: 29, or a sequence having at least about 90-100% sequence similarity thereto, including any percent similarity within these ranges, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto. In addition, a VP6 structural protein may be encoded by a nucleotide sequence as set forth in SEQ ID NO:27 or 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 a VP7 structural protein, which is not to be considered limiting, is set forth in the amino acid sequence of SEQ ID NO: 39. Furthermore, the VP7 structural protein may comprise the sequence set forth in SEQ ID NO: 39, or a sequence having at least about 90-100% similarity thereto, including any percent similarity within these ranges, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto. In addition, a VP7 structural protein may be encoded by a nucleotide sequence as set forth in SEQ ID NO:37 or 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 a NSP4 structural protein, which is not to be considered limiting, is set forth in the amino acid sequence of SEQ ID NO: 44. Furthermore, the NSP4 nonstructural protein may comprise the sequence set forth in SEQ ID NO: 44, or a sequence having at least about 90-100% similarity thereto, including any percent similarity within these ranges, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto. In addition, a NSP4 nonstructural protein may be encoded by a nucleotide sequence as set forth in SEQ ID NO: 42 or 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 may be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0 algorithm. Techniques for computing 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 ALTSCHUL et al. (1990, J Mol. Biol. 215: 403-410) and ALTSCHUL et al. (1997, Nucleic Acids Res. 25: 3389-3402).

Without wishing to be bound by theory, the protein concentration and ratio of the different rotavirus structural proteins may be important for the assembly efficiency of RLPs. Therefore multiplicity and time of infection, may be important to manipulate protein concentration and the overall assembly efficiency of RLPs in plants.

The construct of the present invention may be transiently expressed in a plants or portion of a plant. A transient expression system relying on the epichromosomal expression of recombinant Agrobacterium tumefaciens in a plant, portion of a plant or plant cell may be used to express the rotavirus structural protein, targeted to various cell compartments or sub-compartments. A transient expression system allows for a high production speed. Furthermore, large amounts of protein can be attained within a few days after infiltration of recombinant Agrobacterium in plants (Rybicki, 2010; Fischer et al., 1999). It is also possible to express long gene sequences and have more than one gene simultaneously expressed in the same cell, allowing for efficient assembly of multimeric proteins (Lombardi et al., 2009).

The nucleotide sequences encoding for the rotavirus structural proteins and nonstructural proteins may be transferred into the plant host using 1, 2, 3, 4 or 5 transformed Agrobacterium tumefaciens strains (as described in Table 1 and accompanying text

During transient expression post-transcriptional gene silencing may limit the expression of the heterologous proteins in plants. The co-expression of a suppressor of silencing, for example, but not limited to Nss from Tomato spotted wilt virus may be used to counteract the specific degradation of transgene mRNAs (Brigneti et al., 1998). Alternate suppressors of silencing are well known in the art and may be used as described herein (Chiba et al., 2006, Virology 346:7-14; which is incorporated herein by reference), for example but not limited to HcPro, TEV-p1/HC-Pro (Tobacco etch virus-p1/HC-Pro), BYV-p21, p19 of Tomato bushy stunt virus (TBSV p19), capsid protein of Tomato crinkle 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), p16 of Blueberry scorch virus, (BScV-p16), p23 of Citrus tristexa virus (CTV-p23), p24 of Grapevine leafroll-associated virus-2, (GLRaV-2 p24), p10 of Grapevine virus A, (GVA-p10), p14 of Grapevine virus B (GVB-p14), p10 of Heracleum latent virus (HLV-p10), or p16 of Garlic common latent virus (GCLV-p16). Therefore, a suppressor of silencing, for example HcPro, TEV-p1/HC-Pro, BYV-p21, TBSV p19, TCV-CP, CMV-2b, PVX-p25, PVM-p11, PVS-p11, BScV-p16, CTV-p23, GLRaV-2 p24, GBV-p14, HLV-p10, GCLV-p16or GVA-p10, may be co-expressed along with one or more rotavirus structural or non structural protein for example VP2, VP4, VP6, VP7 and NSP4 or a combination thereof, to further ensure high levels of protein production within a plant or portion of a plant.

The present invention also provides a methods as described above, wherein an additional (second, third, fourth, fifth or sixth) nucleotide sequence is expressed within the plant, the additional (second, third, fourth, fifth or sixth) nucleotide sequence encoding a suppressor of silencing is operatively linked with an additional (second, third, fourth, fifth or sixth) regulatory region that is active in the plant. 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-2 p24, GBV-p14, HLV-p10, GCLV-p16 or GVA-p10.

As described below, transient expression methods may be used to express the constructs of the present invention (see Liu and Lomonossoff, 2002, Journal of Virological Methods, 105:343-348; which is incorporated herein by reference). Alternatively, a vacuum-based transient expression method, as described by Kapila et al., 1997, which is incorporated herein by reference) may be used. These methods may include, for example, but are not limited to, a method of Agro-inoculation or Agro-infiltration, syringe infiltration, however, other transient methods may also be used as noted above. With Agro-inoculation, Agro-infiltration, or syringe infiltration, a mixture of Agrobacteria comprising the desired nucleic acid enter the intercellular spaces of a tissue, for example the leaves, aerial portion of the plant (including stem, leaves and flower), other portion of the plant (stem, root, flower), or the whole plant. After crossing the epidermis the Agrobacteria infect and transfer t-DNA copies into the cells. The t-DNA is episomally transcribed and the mRNA translated, leading to the production of the protein of interest in infected cells, however, the passage of t-DNA inside the nucleus is transient.

To aid in identification of transformed plant cells, the constructs of this invention may be further manipulated to include plant selectable markers. Useful selectable markers include enzymes that provide for resistance to chemicals such as an antibiotic for example, gentamycin, hygromycin, kanamycin, or herbicides such as phosphinothrycin, glyphosate, chlorosulfuron, and the like. Similarly, enzymes providing for production of a compound identifiable by colour change such as GUS (beta-glucuronidase), or luminescence, such as luciferase or GFP, may be used.

Also considered part of this invention are transgenic plants, plant cells or seeds containing the constructs as described herein. Methods of regenerating whole plants from plant cells are also known in the art. In general, transformed plant cells are cultured in an appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be used to establish repetitive generations, either from seeds or using vegetative propagation techniques. Transgenic plants can also be generated without using tissue cultures.

The use of the terms “regulatory region”, “regulatory element” or “promoter” in the present application is meant to reflect a portion of nucleic acid typically, but not always, upstream of the protein coding region of a gene, which may be comprised of either DNA or RNA, or both DNA and RNA. When a regulatory region is active, and in operative association, or operatively linked, with a gene of interest, this may result in expression of the gene of interest. A regulatory element may be capable of mediating organ specificity, or controlling developmental or temporal gene activation. A “regulatory region” may includes promoter elements, core promoter elements exhibiting a basal promoter activity, elements that are inducible in response to an external stimulus, elements that mediate promoter activity such as negative regulatory elements or transcriptional enhancers. “Regulatory region”, as used herein, may also includes elements that are active following transcription, for example, regulatory elements that modulate 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 proximal to the coding region.

In the context of this disclosure, the term “regulatory element” or “regulatory region” typically refers to a sequence of DNA, usually, but not always, upstream (5′) to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at a particular site. However, it is to be understood that other nucleotide sequences, located within introns, or 3′ of the sequence may also contribute to the regulation of expression of a coding region of interest. An example of a regulatory element that provides for the recognition for RNA polymerase or other transcriptional factors 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 comprised of adenosine and thymidine nucleotide base pairs usually situated approximately 25 base pairs upstream of a transcriptional start site. A promoter element comprises a basal promoter element, responsible for the initiation of transcription, 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. A regulatory region that is developmentally regulated, or controls the differential expression of a gene under its control, is activated within certain organs or tissues of an organ at specific times during the development of that organ or tissue. However, some regulatory regions that are developmentally regulated may preferentially be active within certain organs or tissues at specific developmental stages, they may also be active in a developmentally regulated manner, or at a basal level in other organs or tissues within the plant as well. Examples of tissue-specific regulatory regions, for example see-specific a regulatory region, include the napin promoter, and the cruciferin promoter (Rask et al., 1998, J. Plant Physiol. 152: 595-599; Bilodeau et al., 1994, Plant Cell 14: 125-130). An example of a leaf-specific promoter includes the plastocyanin promoter (see U.S. Pat. No. 7,125,978, which is incorporated herein by reference).

An inducible regulatory region is one that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA sequences or genes will not be transcribed. Typically the protein factor that binds specifically to an inducible regulatory region to activate transcription may be present in an inactive form, which is then directly or indirectly converted to the active form by the inducer. However, the protein factor may also be absent. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound or a physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus. A plant cell containing an inducible regulatory region may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or similar methods. Inducible regulatory elements may be derived from either plant or non-plant genes (e.g. Gatz, C. and Lenk, L R. P., 1998, Trends Plant Sci. 3, 352-358; which is incorporated by reference). Examples, of potential inducible promoters include, but not limited to, tetracycline-inducible promoter (Gatz, C.,1997, Ann. Rev. Plant Physiol. Plant Mol. Biol. 48,89-108; which is incorporated by reference), steroid inducible promoter (Aoyama. T. and Chua, N. H.,1997, Plant 1. 2, 397-404; which is incorporated by reference) and ethanol-inducible promoter (Salter, M. G., et al, 1998, Plant Journal 16, 127-132; Caddick, M. X., et al, 1998, Nature Biotech. 16, 177-180, which are incorporated by reference) cytokinin inducible IB6 and CKI 1 genes (Brandstatter, I. and Kieber, 1.1., 1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996, Science 274,982-985; which are incorporated by reference) and the auxin inducible element, DR5 (Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971; which is incorporated by reference).

A constitutive regulatory region directs the expression of a gene throughout the various parts of a plant and continuously throughout plant development. Examples of known constitutive regulatory elements include promoters associated with the CaMV 35S transcript (Odell et al., 1985, Nature, 313: 810-812), the 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. Pat. No. 5,428,147, which is incorporated herein by reference), and triosephosphate isomerase 1 (Xu et. al., 1994, Plant Physiol. 106: 459-467) genes, the maize ubiquitin 1 gene (Cornejo et al, 1993, Plant Mol. Biol. 29: 637-646), the Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al, 1995, Plant Mol. Biol. 29: 637-646), and the tobacco translational initiation factor 4A gene (Mandel et al, 1995, Plant Mol. Biol. 29: 995-1004).

The term “constitutive” as used herein does not necessarily indicate that a gene under control of the constitutive regulatory region is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types even though variation in abundance is often observed. Constitutive regulatory elements may be coupled with other sequences to further enhance the transcription and/or translation of the nucleotide sequence to which they are operatively linked. For example, the CPMV-HT system is derived from the untranslated regions of the Cowpea mosaic virus (CPMV) and demonstrates enhanced translation of the associated coding sequence. By “native” it is meant that the nucleic acid or amino acid sequence is naturally occurring, or “wild type”. By “operatively linked” it is meant that the particular sequences, for example a regulatory element and a coding region of interest, interact either directly or indirectly to carry out an intended function, such as mediation or modulation of gene expression. The interaction of operatively linked sequences may, for example, be mediated by proteins that interact with the operatively linked sequences.

The RLP produced within a plant may produce a rotavirus VP7 structural protein comprising plant-specific N-glycans. Therefore, this invention also provides for a RLP comprising VP7 having plant specific N-glycans.

Furthermore, modification of N-glycan in plants is known (see for example U.S. 60/944,344; which is incorporated herein by reference) and VP7 having modified N-glycans may be produced. VP7 comprising a modified glycosylation pattern, for example with reduced fucosylated, xylosylated, or both, fucosylated and xylosylated, N-glycans may be obtained, or VP7 having a modified glycosylation pattern may be obtained, wherein the protein lacks fucosylation, xylosylation, or both, and comprises increased galactosylation. Furthermore, modulation of post-translational modifications, for example, the addition of terminal galactose may result in a reduction of fucosylation and xylosylation of the expressed VP7 when compared to a wild-type plant expressing VP7.

For example, which is not to be considered limiting, the synthesis of VP7 having a modified glycosylation pattern may be achieved by co-expressing VP7 along with a nucleotide sequence encoding beta-1.4 galactosyltransferase (GalT), for example, but not limited to mammalian GalT, or human GalT however GalT from another sources may also be used. The catalytic domain of GalT may also be fused to a CTS domain (i.e. the cytoplasmic tail, transmembrane domain, stem region) of N-acetylglucosaminyl transferase (GNT1), to produce a GNT1-GalT hybrid enzyme, and the hybrid enzyme may be co-expressed with VP7. The VP7 may also be co-expressed along with a nucleotide sequence encoding N-acetylglucosaminyl transferase III (GnT-III), for example but not limited to mammalian GnT-III or human GnT-III, GnT-III from other sources may also be used. Additionally, a GNT1-GnT-III hybrid enzyme, comprising the CTS of GNT1 fused to GnT-III may also be used.

Therefore the present invention also provides RLPs comprising VP7 having modified N-glycans.

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

Table 2 lists sequences provided in various embodiments of the invention.

TABLE 2

SEQ ID

NO
Description
Page/FIG.

1
expression enhancer CPMVX

2
plant kingdom kozak consensus sequence

3
Dicots kozak consensus sequence

4

Arabidopsis kozak consensus sequence

5-13
plant kozak sequences

14
Kozak consensus sequence

15
CPMV HT

16
CPMV HT+

17
CPMV HT+ 511

18
CPMV HT+ [WT115]

19
IF-WA_VP2(opt).s1 + 3c
FIG. 7A

20
IF-WA_VP2(opt).s1 − 4r
FIG. 7B

21
Optimized coding sequence of Rotavirus A VP2
FIG. 7C

from strain WA

22
Construct 1191
FIG. 7E

23
Expression cassette number 1710
FIG. 7F

24
Amino acid sequence of VP2 from Rotavirus A WA
FIG. 7G

strain

25
IF-WA_VP6(opt).s1 + 3c
FIG. 8A

26
IF-WA_VP6(opt).s1 − 4r
FIG. 8B

27
Optimized coding sequence of Rotavirus A VP6
FIG. 8C

from strain WA

28
Expression cassette number 1713
FIG. 8D

29
Amino acid sequence of VP6 from Rotavirus A WA
FIG. 8E

strain

30
IF-Rtx_VP4(opt).s1 + 3c
FIG. 9A

31
IF-Rtx_VP4(opt).s1 − 4r
FIG. 9B

32
Optimized coding sequence of Rotavirus A VP4
FIG. 9C

from strain RVA/Vaccine/USA/Rotarix-

A41CB052A/1988/G1P1A[8]

33
Expression cassette number 1730
FIG. 9D

34
Amino acid sequence of VP4 from Rotavirus A
FIG. 9E

Rotarix strain

35
IF-TrSP + Rtx_VP7(opt).s1 + 3c
FIG. 10A

36
IF-Rtx_VP7(opt).s1 − 4r
FIG. 10B

37
Optimized coding sequence of Rotavirus A VP7
FIG. 10C

from strain RVA/Vaccine/USA/Rotarix-

A41CB052A/1988/G1P1A[8]

38
Expression cassette number 1734
FIG. 10D

39
Amino acid sequence of TrSp-VP7 from Rotavirus
FIG. 10E

A vaccine USA/Rotarix-

A41CB052A/1988/G1P1A[8] strain

40
IF-WA_NSP4.s1 + 3c
FIG. 11A

41
IF-WA_NSP4.s1 − 4r
FIG. 11B

42
Coding sequence of Rotavirus A NSV4 from strain
FIG. 11C

WA

43
Expression cassette number 1706
FIG. 11D

44
Amino acid sequence of NSP4 from Rotavirus A
FIG. 11E

WA strain

45
IF(C160)-WA_VP2(opt).c
FIG. 12A

46
Construct 1190
FIG. 12C

47
Expression cassette number 1108
FIG. 12D

48
IF(C160)-WA_VP6(opt).c
FIG. 13A

49
Expression cassette number 1128
FIG. 13B

50
IF(C160)-Rtx_VP4(opt).c
FIG. 14A

51
Expression cassette number 1178
FIG. 14B

52
IF(C160)-TrSP + Rtx_VP7(opt).c
FIG. 15A

53
Expression cassette number 1199
FIG. 15B

The present invention will be further illustrated in the following examples.

EXAMPLES
Example 1
Materials and Methods

TABLE 3

Constructs

De-

De-

Constr. #
scription
FIG.
Constr.#
scription
FIG.

1108
160-VP2
3A/3B
1706
CPMV-HT
3A/3B

NSP4
4A/4B

1128
160-VP6
3A/3B
1708
CPMV-HT
3A/3B

VP6/2
4A/4B

1178
160-VP4
3A/3B
2408
160-VP7/4
3A/3B

4A/4B

1199
160-VP7
3A/3B
1769
CPMV-HT
3A/3B/5

VP7/4/6/2

1710
CPMV-HT
4A/4B
2441
CPMV-HT
5

VP2

VP4/7/

NSP4/6/2

1713
CPMV-HT
4A/4B
2400
160-VP6/2
4A/4B

VP6

1730
CPMV-HT
4A/4B
1719
CPMV-HT
4A/4B

VP4

VP7/4

1734
CPMV-HT
4A/4B

VP7

1. 2X35S/CPMV-HT/RVA(WA) VP2(opt)/NOS (Construct Number 1710)

An optimized sequence encoding VP2 from Rotavirus A WA strain was cloned into 2X35S-CPMV-HT-NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the VP2 coding sequence was amplified using primers IF-WA_VP2(opt).s1+3c (FIG. 7a, SEQ ID NO: 19) and IF-WA_VP2(opt).s1−4r (FIG. 7B, SEQ ID NO: 20), using optimized VP2 gene sequence (FIG. 7C, SEQ ID NO :21) as template. For sequence optimization, VP2 protein sequence (Genbank accession number CAA33074) was backtranslated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned in 2X35S/CPMV-HT/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct number 1191 (FIG. 7D) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 1191 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV-HT-based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in FIG. 7E (SEQ ID NO: 22). The resulting construct was given number 1710 (FIG. 7F, SEQ ID NO: 23). The amino acid sequence of VP2 from Rotavirus A strain WA is presented in FIG. 7G (SEQ ID NO: 24). A representation of plasmid 1710 is presented in FIG. 7H.

2. 2X35S/CPMV-HT/RVA(WA) VP6(opt)/NOS (Construct Number 1713)

An optimized sequence encoding VP6 from Rotavirus A WA strain was cloned into 2X35S-CPMV-HT-NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the VP6 coding sequence was amplified using primers IF-WA_VP6(opt).s1+3c (FIG. 8A, SEQ ID NO: 25) and IF-WA_VP6(opt).s1−4r (FIG. 8B, SEQ ID NO: 26), using optimized VP6 gene sequence (FIG. 8C, SEQ ID NO:27) as template. For sequence optimization, VP6 protein sequence (Genbank accession number AAA47311) was backtranslated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned in 2X35S/CPMV-HT/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct number 1191 (FIG. 7D) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 1191 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV-HT-based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in FIG. 7E (SEQ ID NO: 22). The resulting construct was given number 1713 (FIG. 8D, SEQ ID NO: 28). The amino acid sequence of VP6 from Rotavirus A strain WA is presented in FIG. 8E (SEQ ID NO: 29). A representation of plasmid 1713 is presented in FIG. 8F.

3. 2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS (Construct Number 1730)

An 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 Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the VP4 coding sequence was amplified using primers IF-Rtx_VP4(opt).s1+3c (FIG. 9A, SEQ ID NO: 30) and IF-Rtx_VP4(opt).s1−4r (FIG. 9B, SEQ ID NO: 31), using optimized VP4 gene sequence (FIG. 9C, SEQ ID NO: 32) as template. For sequence optimization, VP4 protein sequence (Genbank accession number AEX30660) was backtranslated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned in 2X35S/CPMV-HT/NOS expression cassette using In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct number 1191 (FIG. 7D) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 1191 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV-HT-based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in FIG. 7E (SEQ ID NO: 22). The resulting construct was given number 1730 (FIG. 9D, SEQ ID NO: 33). The amino acid sequence of VP4 from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] is presented in FIG. 9E (SEQ ID NO: 34). A representation of plasmid 1730 is presented in FIG. 9F.

4. 2X35S/CPMV-HT/TrSp-RVA(Rtx) VP7(opt)/NOS (Construct Number 1734)

An optimized sequence encoding VP7 with a truncated version of the native signal peptide from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain was cloned into 2X35S-CPMV-HT-NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the VP7 coding sequence was amplified using primers IF-TrSP+Rtx_VP7(opt).s1+3c (FIG. 10A, SEQ ID NO: 35) and IF-Rtx_VP7(opt).s1−4r (FIG. 10B, SEQ ID NO: 36), using optimized VP7 gene sequence (corresponding to nt 88-891 from FIG. 10C, SEQ ID NO: 37) as template. For sequence optimization, VP7 protein sequence (Genbank accession number AEX30682) was backtranslated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned in 2X35S/CPMV-HT/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct number 1191 (FIG. 7D) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 1191 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV-HT-based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in FIG. 7E (SEQ ID NO: 22). The resulting construct was given number 1734 (FIG. 10D, SEQ ID NO: 38). The amino acid sequence of VP7 with truncated signal peptide from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain is presented in FIG. 10E (SEQ ID NO: 39). A representation of plasmid 1734 is presented in FIG. 10F.

5. 2X35S/CPMV-HT/RVA(WA) NSP4/NOS (Construct Number 1706)

A sequence encoding NSP4 from Rotavirus A WA strain was cloned into 2X35S-CPMV-HT-NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the NSP4 coding sequence was amplified using primers IF-WA_NSP4.s1+3c (FIG. 11A, SEQ ID NO: 40) and IF-WA_NSP4.s1−4r (FIG. 11B, SEQ ID NO: 41), using synthesized NSP4 gene (corresponding to nt 42-569 from GenBank accession number K02032) (FIG. 11C, SEQ ID NO: 42) as template. The PCR product was cloned in 2X35S/CPMV-HT/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct number 1191 (FIG. 7D) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 1191 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV-HT-based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in FIG. 7E (SEQ ID NO: 22). The resulting construct was given number 1706 (FIG. 11D, SEQ ID NO: 43). The amino acid sequence of NSP4 from Rotavirus A strain WA is presented in FIG. 11E (SEQ ID NO: 44). A representation of plasmid 1706 is presented in FIG. 11F.

6. 2X35S/CPMV-160/RVA(WA) VP2(opt)/NOS (Construct Number 1108)

An optimized sequence encoding VP2 from Rotavirus A WA strain was cloned into 2X35S/CPMV-160/NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the VP2 coding sequence was amplified using primers IF(C160)-WA_VP2(opt).c (FIG. 12A, SEQ ID NO: 45) and IF-WA_VP2(opt).s1−4r (FIG. 7B, SEQ ID NO: 20), using optimized VP2 gene sequence (FIG. 7C, SEQ ID NO: 21) as template. For sequence optimization, VP2 protein sequence (Genbank accession number CAA33074) was backtranslated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned in 2X35S/CPMV-160/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct number 1190 (FIG. 12B) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 1190 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV-160-based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in FIG. 12C (SEQ ID NO: 46). The resulting construct was given number 1108 (FIG. 12D, SEQ ID NO: 47). The amino acid sequence of VP2 from Rotavirus A strain WA is presented in FIG. 7G (SEQ ID NO: 24). A representation of plasmid 1108 is presented in FIG. 12E.

7.—2X35S/CPMV-160/RVA(WA) VP6(opt)/NOS (Construct Number 1128)—

An optimized sequence encoding VP6 from Rotavirus A WA strain was cloned into 2X35S/CPMV-160/NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the VP6 coding sequence was amplified using primers IF(C160)-WA_VP6(opt).c (FIG. 13A, SEQ ID NO: 48) and IF-WA_VP6(opt).s1−4r (FIG. 8B, SEQ ID NO: 26), using optimized VP6 gene sequence (FIG. 8C, SEQ ID NO: 27) as template. For sequence optimization, VP6 protein sequence (Genbank accession number AAA47311) was backtranslated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned in 2X35S/CPMV-160/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct number 1190 (FIG. 11B, SEQ ID NO:40) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 1190 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV-160-based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in FIG. 11C (SEQ ID NO: 41). The resulting construct was given number 1128 (FIG. 13B, SEQ ID NO: 49). The amino acid sequence of VP6 from Rotavirus A strain WA is presented in FIG. 8E (SEQ ID NO: 28). A representation of plasmid 1128 is presented in FIG. 13C.

8. X35S/CPMV-160/RVA(Rtx) VP4(opt)/NOS (Construct Number 1178)

An optimized sequence encoding VP4 from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain was cloned into 2X35S/CPMV-160/NOS in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the VP4 coding sequence was amplified using primers IF(C160)-Rtx_VP4(opt).c (FIG. 14A, SEQ ID NO: 50) and IF-Rtx_VP4(opt).s1−4r (FIG. 9B, SEQ ID NO: 30), using optimized VP4 gene sequence (FIG. 9C, SEQ ID NO: 31) as template. For sequence optimization, VP4 protein sequence (Genbank accession number AEX30660) was backtranslated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned in 2X35S/CPMV-160/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct number 1190 (FIG. 11B, SEQ ID NO: 40) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 1190 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV-160-based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in FIG. 11C (SEQ ID NO: 41). The resulting construct was given number 1178 (FIG. H2, SEQ ID NO: H2). The amino acid sequence of VP4 from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] is presented in FIG. 9E (SEQ ID NO: 33). A representation of plasmid 1178 is presented in FIG. 14C.

9. 2X35S/CPMV-160/TrSp-RVA(Rtx) VP7(opt)/NOS (Construct Number 1199)

An optimized sequence encoding VP7 with a truncated version of the native signal peptide from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain was cloned into 2X35S/CPMV-160/NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the VP7 coding sequence was amplified using primers IF(C160)-TrSP+Rtx_VP7(opt).c (FIG. 15A, SEQ ID NO: 52) and IF-Rtx_VP7(opt).s1−4r (FIG. 10B, SEQ ID NO: 35), using optimized VP7 gene sequence (corresponding to nt 88-891 from FIG. 10C, SEQ ID NO: 36) as template. For sequence optimization, VP7 protein sequence (Genbank accession number AEX30682) was backtranslated and optimized for human codon usage, GC content and mRNA structure. The PCR product was cloned in 2X35S/CPMV-160/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct number 1190 (FIG. 11B, SEQ ID NO: 40) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 1190 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV-160-based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in FIG. 11C (SEQ ID NO: 41). The resulting construct was given number 1199 (FIG. 15B, SEQ ID NO: 53). The amino acid sequence of VP7 with truncated signal peptide from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain is presented in FIG. 10E (SEQ ID NO: 38). A representation of plasmid 1199 is presented in FIG. 15C.

10. Double Gene Construct for the Expression of VP6 and VP2 Under CPMV-HT Expression Cassette (Construct Number 1708)

A single vector for the co-expression of VP6 from Rotavirus A WA strain and VP2 from Rotavirus A WA strain under the control of CPMV-HT expression system was assembled using the following restriction enzyme/ligase-based method. Donor plasmid DNA (construct number 1710; 2X35S/CPMV-HT/RVA(WA) VP2(opt)/NOS) (FIG. 7F, SEQ ID NO: 23) was digested with AvrII (located before the 2X35S promoter) and AscI (located after the NOS terminator) restriction enzymes and the fragment corresponding to 2X35S/CPMV-HT/RVA(WA) VP2(opt)/NOS expression cassette was gel-purified. This fragment was then inserted into the acceptor construct number 1713 (2X35S/CPMV-HT/RVA(WA) VP6(opt)/NOS) (FIG. 8D, SEQ ID NO: 28) linearized using XbaI and AscI restriction enzymes (both sites are located after the NOS terminator of VP6 expression cassette). The resulting construct was given number 1708. A representation of plasmid 1708 is presented in FIG. 16.

11. Double Gene Construct for the Expression of VP7 and VP4 Under CPMV-HT Expression Cassette (Construct Number 1719)

A single vector for the co-expression of VP7 with a truncated version of the native signal peptide from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain and VP4 from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain under the control of CPMV-HT expression system was assembled using the following restriction enzyme/ligase-based method. Donor plasmid DNA (construct number 1730; 2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS) (FIG. 9D, SEQ ID NO: 32) was digested with AvrII (located before the 2X35S promoter) and AscI (located after the NOS terminator) restriction enzymes and the fragment corresponding to 2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS expression cassette was gel-purified. This fragment was then inserted into the acceptor construct number 1734 (2X35S/CPMV-HT/TrSp-RVA(Rtx) VP7(opt)/NOS) (FIG. 10D, SEQ ID NO: 37) linearized using XbaI and AscI restriction enzymes (both sites are located after the NOS terminator of VP7 expression cassette). The resulting construct was given number 1719. A representation of plasmid 1719 is presented in FIG. 17.

12. Double Gene Construct for the Expression of VP6 and VP2 Under CPMV-160 Expression Cassette (Construct Number 2400)

A single vector for the co-expression of VP6 from Rotavirus A WA strain and VP2 from Rotavirus A WA strain under the control of CPMV-160 expression system was assembled using the following restriction enzyme/ligase-based method. Donor plasmid DNA (construct number 1108; 2X35S/CPMV-160/RVA(WA) VP2(opt)/NOS) (FIG. 12D, SEQ ID NO: 47) was digested with AvrII (located before the 2X35S promoter) and AscI (located after the NOS terminator) restriction enzymes and the fragment corresponding to 2X35S/CPMV-160/RVA(WA) VP2(opt)/NOS expression cassette was gel-purified. This fragment was then inserted into the acceptor construct number 1128 (2X35S/CPMV-160/RVA(WA) VP6(opt)/NOS) (FIG. 13B, SEQ ID NO: 49) linearized using XbaI and AscI restriction enzymes (both sites are located after the NOS terminator of VP6 expression cassette). The resulting construct was given number 2400. A representation of plasmid 2400 is presented in FIG. 18.

13. Double Gene Construct for the Expression of VP7 and VP4 Under CPMV-160 Expression Cassette (Construct Number 2408)

A single vector for the co-expression of VP7 with a truncated version of the native signal peptide from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain and VP4 from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain under the control of CPMV-160 expression system was assembled using the following restriction enzyme/ligase-based method. Donor plasmid DNA (construct number 1178; 2X35S/CPMV-160/RVA(Rtx) VP4(opt)/NOS) (FIG. 14B, SEQ ID NO: 51) was digested with AvrII (located before the 2X35S promoter) and AscI (located after the NOS terminator) restriction enzymes and the fragment corresponding to 2X35S/CPMV-160/RVA(Rtx) VP4(opt)/NOS expression cassette was gel-purified. This fragment was then inserted into the acceptor construct number 1199 (2X35S/CPMV-160/TrSp-RVA(Rtx) VP7(opt)/NOS) (FIG. 15B, SEQ ID NO: 53) linearized using XbaI and AscI restriction enzymes (both sites are located after the NOS terminator of VP7 expression cassette). The resulting construct was given number 2408. A representation of plasmid 2408 is presented in FIG. 19.

14. Quadruple Gene Construct for the Expression of VP7, VP4, VP6 and VP2 Under CPMV-HT Expression Cassette (Construct Number 1769)

A single vector for the co-expression of VP7 with a truncated version of the native signal peptide from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain, VP4 from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain, VP6 from Rotavirus A WA strain and VP2 from Rotavirus A WA strain under the control of CPMV-HT expression system was assembled using the following restriction enzyme/ligase-based method. Donor plasmid DNA (construct number 1730; 2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS) (FIG. 9D, SEQ ID NO: 32) was digested with AvrII (located before the 2X35S promoter) and AscI (located after the NOS terminator) restriction enzymes and the fragment corresponding to 2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS expression cassette was gel-purified. This fragment was then inserted into the acceptor construct number 1734 (2X35S/CPMV-HT/TrSp-RVA(Rtx) VP7(opt)/NOS) (FIG. 10D, SEQ ID NO: 37) linearized using XbaI and AscI restriction enzymes (both sites are located after the NOS terminator of VP7 expression cassette). Ligation of cohesive ends produced by AvrII and XbaI destroyed the original restriction sites producing a temporary acceptor vector with the same unique XbaI and AscI restriction enzyme sites at the end of the NOS terminator of the second expression cassettes (from left to right T-DNA). VP6 (construct number 1713; FIG. 8D, SEQ ID NO: 28) and VP2 (construct number 1710; FIG. 7F, SEQ ID NO: 23) expressed under CPMV-HT expression system were then inserted sequentially in the resulting temporary acceptor vector using the same digestion strategy to give the final VP7/VP4/VP6/VP2 construct. The resulting construct was given number 1769. A representation of plasmid 1769 is presented in FIG. 20.

15. Quintuple Gene Construct for the Expression of VP4, VP7, NSP4, VP6 and VP2 Under CPMV-HT Expression Cassette (Construct Number 2441)

A single vector for the co-expression of VP4 from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain, VP7 with a truncated version of the native signal peptide from Rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain, NSP4 from Rotavirus A WA strain, VP6 from Rotavirus A WA strain and VP2 from Rotavirus A WA strain under the control of CPMV-HT expression system was assembled using the following restriction enzyme/ligase-based method. Donor plasmid DNA (construct number 1734; 2X35S/CPMV-HT/TrSp-RVA(Rtx) VP7(opt)/NOS) (FIG. 10D, SEQ ID NO: 37) was digested with AvrII (located before the 2X35S promoter) and AscI (located after the NOS terminator) restriction enzymes and the fragment corresponding to 2X35S/CPMV-HT/TrSp-RVA(Rtx) VP7(opt)/NOS expression cassette was gel-purified. This fragment was then inserted into the acceptor construct number 1730 (2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS) (FIG. 9D, SEQ ID NO: 32) linearized using XbaI and AscI restriction enzymes (both sites are located after the NOS terminator of VP4 expression cassette). Ligation of cohesive ends produced by AvrII and XbaI destroyed the original restriction sites producing a temporary acceptor vector with the same unique XbaI and AscI restriction enzyme sites at the end of the NOS terminator of the second expression cassettes (from left to right T-DNA). NSP4 (construct number 1706; FIG. 11D, SEQ ID NO: 42), VP6 (construct number 1713; FIG. 8D, SEQ ID NO: 28) and VP2 (construct number 1710; FIG. 7F, SEQ ID NO: 23) expressed under CPMV-HT expression system were then inserted sequentially in the resulting temporary acceptor vector using the same digestion strategy to give the final VP4/VP7/NSP4/VP6/VP2 construct. The resulting construct was given number 2441. A representation of plasmid 2441 is presented in FIG. 21.

Example 2
Co-Expression of NSP4 Increases VP4 and VP4 Incorporation into RLPs

The rotavirus VP2, VP4, VP6 and VP7 structural antigens were transiently co-expressed in Nicotiana benthamiana plants in the presence or absence of a NSP4 expression construct using agroinfiltration as described in example 1. Crude protein extracts from RLP producing plants contain large amounts of host protein as shown by the banding pattern in Coomassie-stained SDS-PAGE (FIG. 2B, load). Rotavirus-like particles can be separated from plant proteins by ultracentrifugation on a iodixanol density gradient. After centrifugation, analysis of the fractions from iodixanol density gradient showed that the RLPs migrated to the 35% iodixanol fraction (F2 and F3 in FIG. 2B) while the majority of the host proteins remained in the 25-30% iodixanol fractions (F4-F10 in FIG. 2B). RLPs from plants co-expressing rotavirus structural antigens were purified on iodixanol density gradients and the analysis of the RLP containing fractions (F2 and F3) showed that RLPs can be produced efficiently, irrespectively of the number of gene per construct as shown in FIG. 3A with single, dual and quadruple gene constructs. The results obtained also showed that the co-expression of NSP4 reduced RLP expression (compare fractions under −NSP4 and +NSP4 in FIG. 3A). Note that equal volumes of each fraction were loaded on the gel to compare RLP content per volume.

RLP-containing fraction 2 from the same experiments were analyzed by western blot to evaluate the impact of NSP4 co-expression on VP4 and VP7 incorporation. For that comparison, equal amounts of RLPS were loaded on the gel. The western blot results obtained showed stronger signals for VP4 and VP7 on the RLPs produced in the presence of NSP4 (FIG. 3B, compare lanes under −NSP4 and +NSP4). These results clearly indicate that the co-expression of NSP4 increased VP4 and VP7 incorporation on the surface of the RLPs.

The genes encoding the four rotavirus antigens and the non-structural protein NSP4 were cloned into CPMV-HT and CPMV160 for comparison of expression. Co-expression studies followed by extraction and purification by ultracentrifugation in iodixanol density gradient showed that both expression efficiently produced RLPs, as demonstrated by the amount of VP6 in fractions 2 and 3 of the gradient (FIG. 4A), and the amount of VP4 and VP7 in fraction 2 from the same treatments (FIG. 4B). This study also showed that, when using the CPMV-HT system for expression of the rotavirus proteins, single gene constructs produced as much RLPs as dual gene constructs (FIG. 4A, left panel vs middle panel) and resulted in similar coverage with the surface antigens, VP4 and VP7 (FIG. 4B, left panel vs middle panel).

A quintuple gene construct (comprising 5 genes on the same plasmid) has been evaluated for the co-expression of the four structural antigens with NSP4. As shown in FIG. 5, the use of quintuple gene construct resulted in similar RLP production level as with the use of a quadruple gene construct with the NSP4 gene on a separate plasmid (FIG. 5, top panel), as well as comparable levels of VP4 and VP7 incorporation (FIG. 5, lower panel).

Agrobacterium Transformation

All plasmids were used to transform Agrobacterium tumefaciens (AGL1; ATCC, Manassas, Va. 20108, USA) by electroporation (Mattanovich et al., 1989, Nucleic Acid Res. 17:6747) alternatively, heat shock using CaCl2-prepared competent cells (XU et al., 2008, Plant Methods 4) may be used. The integrity of the plasmids in the A. tumefaciens strains created was confirmed by restriction mapping.

Preparation of Plant Biomass, Inoculum, Agroinfiltration, and Harvesting

Nicotiana benthamiana plants were grown from seeds in flats filled with a commercial peat moss substrate. The plants were allowed to grow in the greenhouse under a 16/8 photoperiod and a temperature regime of 25° C. day/20° C. night. Three weeks after seeding, individual plantlets were picked out, transplanted in pots and left to grow in the greenhouse for three additional weeks under the same environmental conditions.

Agrobacteria transfected with each construct were grown in a LB medium from vegetal origin and supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 50 μg/ml kanamycin pH5.6 until they reached an OD600 between 0.6 and 2.5. Agrobacterium suspensions were mixed to reach appropriate ratio for each construct and brought to 2.5× OD600 with infiltration medium (10 mM MgCl2 and 10 mM MES pH 5.6). A. tumefaciens suspensions were stored overnight at 4° C. On the day of infiltration, culture batches were diluted with infiltration medium and allowed to warm before use. Whole plants of N. benthamiana were placed upside down in the bacterial suspension in an air-tight stainless steel tank under a vacuum of 20-40 Torr for 2-min. Following infiltration, plants were returned to the greenhouse for a 9 day incubation period until harvest. Harvested biomass was kept frozen (−80° C.) until use for purification of particles.

Extraction and Screening by Ultracentrifugation of Rotavirus-Like Particles

Proteins were extracted from frozen biomass by mechanical extraction in a blender with 2 volumes of extraction buffer (TNC: 10 mM Tris pH 7.4, 140 mM NaCl, 10 mM CaCl2). The slurry was filtered through a large pore nylon filter to remove large debris and centrifuged 5000 g for 5 min at 4° C. The supernatant was collected and centrifuged again at 5000 g for 30 min (4° C.) to remove additional debris. The supernatant is then loaded on a discontinuous iodixanol density gradient.

Analytical density gradient centrifugation was performed as follows. 38 ml tubes containing discontinuous iodixanol density gradient in TNC buffer (1.2 ml at 45%, 2 ml at 35%, 5 ml at 30% and 5 ml at 25% of iodixanol) were prepared and overlaid with 25 ml of the extracts containing the rotavirus-like particles. The gradients were centrifuged at 120 000 g for 4 hours (4° C.). After centrifugation, 1 ml fractions were collected from the bottom to the top and fractions 2 and 3 (corresponding to 35% iodixanol) were analysed by SDS-PAGE combined to protein staining or Western blot.

SDS-PAGE and Immunoblotting

Protein concentrations were determined by the BCA protein assay (Pierce Biochemicals, Rockport, Ill.). Proteins were separated by SDS-PAGE under reducing conditions using Criterion™ TGX Stain-Free™ precast gels (Bio-Rad Laboratories, Hercules, Calif.) and proteins were visualized with Gel Doc™ EZ imaging system (Bio-Rad Laboratories, Hercules, Calif.).

For immunoblotting, electrophoresed proteins were electrotransferred onto polyvinylene difluoride (PVDF) membranes (Roche Diagnostics Corporation, Indianapolis, Ind.). Prior to immunoblotting, the membranes were blocked with 5% skim milk and 0.1% Tween-20 in Tris-buffered saline (TBS-T) for 16-18 h at 4° C.

Immunoblotting was performed by incubation with a suitable antibody (Table 4) in 2% skim milk in TBS-Tween 20 0.1%. Secondary antibodies used for chemiluminescence detection were as indicated in Table 4, diluted as indicated in 2% skim milk in TBS-Tween 20 0.1% Immunoreactive complexes were detected by chemiluminescence using luminol as the substrate (Roche Diagnostics Corporation, Indianapolis, Ind.).

TABLE 4

Electrophoresis conditions, antibodies, and dilutions

for immunoblotting of rotavirus antigens.

Rota-
Electro-

virus
phoresis

anti-
condi-

Secondary

gen
tion
Primary antibody
Dilution
antibody
Dilution

VP4
Re-
Rabbit serum from
1:30 000
Goat anti-
1:10 000

ducing
immunized Rabbit

rabbit (JIR

with recombinant

111-035-

VP4 (in house)

144)

VP7
Re-
Rabbit serum from
1:50 000
Goat anti-
1:10 000

ducing
immunized Rabbit

rabbit (JIR

with recombinant

111-035-

VP7 (in house)

144)

All citations are hereby incorporated by reference.

The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

ROTAVIRUS-LIKE PARTICLE PRODUCTION IN PLANTS

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