WO2009059422A1

WO2009059422A1 - Rotavirus vaccine

Info

Publication number: WO2009059422A1
Application number: PCT/CA2008/001966
Authority: WO
Inventors: Denis Archambault; Aurelie Girard; Louis-Philippe Bergeron-Sandoval; Marie-Claude St-Louis; Fathey Sarhan
Original assignee: Transfert Plus S.E.C.
Priority date: 2007-11-09
Filing date: 2008-11-07
Publication date: 2009-05-14

Abstract

The present invention relates to the field of rotavirus and more particularly to VP5/VP8 Rotavirus polypeptides, polynucleotides encoding same, and their use for eliciting an immune response against rotavirus infections.

Description

ROTAVIRUS VACCINE

FIELD OF THE INVENTION

BRIEF DESCRIPTION OF THE PRIOR ART

Human rotavirus (HRV) is the leading cause of gastroenteritis and death among children worldwide. At particular risk are children under 5 years old in developing countries where the effectiveness of the live attenuated vaccines has been compromised by the difficulties associated with antigenic variation, availability, storage, administration and high production costs.

Rotaviruses have been recognized as one of the most important infectious agent that causes severe diarrhea in infants and young children since their discovery in 1973. It is estimated that rotavirus disease is responsible for about 600,000 deaths annually, mostly in developing countries.

Rotavirus-induced illness most commonly affects children between 6 and 24 months of age, and the peak prevalence of the disease generally occurs during the cooler months in temperate climates, and year-round in tropical areas. Rotaviruses are typically transmitted from person to person by the fecal-oral route with an incubation period ranging from about 1 to 3 days.

Unlike infection in the 6-month to 24-month age group, neonates are generally asymptomatic or have only mild disease. In contrast to the severe disease normally encountered in young children, most adult rotavirus infections are mild or asymptomatic because such episodes represent reinfection generally as a result of contact with children known to be excreting rotavirus.

Initial rotavirus vaccine studies suggested that bovine rotavirus strains offered partial protection to infants against heterotypic human rotaviruses even in the absence of detectable neutralizing antibody to circulating human strains. Subsequent efficacy trials, however, indicated that these bovine rotavirus vaccines are, at best, only marginally protective. For example, the RIT 4237 bovine strain rotavirus (serotype 6) appeared to be successful in preventing clinically significant diarrhea due to rotavirus infection in Finish infants. When later evaluated in developing countries, however, it was not as effective.

Rhesus rotavirus (RRV) vaccine strain MMU 18006 (serotype 3) has been shown to be immunogenic in several studies, but has been associated with mild side effects including low grade fever and watery stools.

Currently, except for the simple replenishment of metabolite solutes, the treatments generally used consist in the administration of either Rotarix® or Rota-Teq®. Rotarix® or Rota-Teq® are vaccines against various strains of rotaviruses. However, they are very expensive, might have harmful side effects since they are live vaccines, and their use is limited to very young children under the age of 8 months.

In 1998, Rotashield® was the first tetravalent HRV vaccine generated by combining HRV and RRV strains. This vaccine was suspended and withdrawed because of high numbers of intussusception cases (1 :10 000) among the vaccines. Since, only the vaccines Rotarix® and Rotateq® have been commercialized. Rotarix® is derived from a G1 HRV strain and confers immunity against G1 , G3 and G9 serotypes following two oral doses to infants between 2 and 6 months. As for Rotateq®, it was generated with human and bovine rotaviruses and confers immunity against G1 , G2, G3, G4 et P[8] HRV serotypes following tree doses to infants under 8 months. Despite rigorous safety tests and good protection correlates the Jennerian vaccines have intrinsic inconvenient and dangers linked to uncertain harmful side effects. In the case of current HRV vaccines, their use is limited to very young children and may interfere with other vaccines administered at this age.

Their virulent and replicative nature also includes highly probable infections and complications when administered to immunodeficient or autoimmune patients. These restrictions justify the necessity to develop new and safer rotavirus vaccines.

Consequently, it would be highly desirable to be provided with a new tool against rotavirus through vaccination that overcome some or all of the shortcomings of the prior art vaccines.

SUMMARY OF THE INVENTION

The present invention satisfies at least one of the above-mentioned needs. Indeed, the present invention offers a tool to induce or elicit an immune response against rotavirus which is advantageously useful against a rotavirus infection.

More specifically, an object of the invention concerns an isolated polypeptide comprising:

- a VP5 Rotavirus peptide lacking its associated C-terminus region; and

- a VP8 Rotavirus peptide.

Other objects of the invention concern a polynucleotide encoding the polypeptide of the invention, the use of said polypeptide and polynucleotide in immunogenic compositions for inducing a specific immune response against a rotavirus infection.

Still another object of the invention is to provide a method for inducing a specific immune response against a rotavirus infection in a subject, comprising the step of administering of a polypeptide, a polynucleotide or a composition according to the present invention, to said subject.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a nucleic acid sequence of a polynucleotide of the invention, for instance, the Vp8::Vp5ΔC-term OPT gene optimized for expression in mammalian cells.

Figure 2 shows a nucleic acid sequence of another polynucleotide of the invention, for instance, the Vp8::Vp5ΔC-term WT gene.

Figure 3 shows a nucleic acid sequence of a polynucleotide of the invention, for instance, the Vp8::Vp5ΔC-term OPT gene optimized for expression in plant cells.

Figure 4 shows an amino acid sequence of a polypeptide of the invention, for instance, the Vp8::Vp5ΔC-term OPT peptide.

Figure 5 shows an amino acid sequence of another polypeptide of the invention, for instance, the Vp8::Vp5ΔC-term WT peptide.

Figure 6 is the amino acid sequence alignment between AAA66953, AAA47290, Vp8::Vp5ΔC-term OPT and Vp8::Vp5ΔC-term WT using BioEdit v7.0.9 software. AAA66953 and AAA47290 are GeneBank accession numbers for the Wa strain human rotavirus VP4 sequence which contains the Vp8::Vp5ΔC-term sequence (amino acids 1 to 330 of VP4). AAA66953 VP4 nucleic acid sequence was chosen as template to optimize the Vp8::Vp5ΔC-term OPT gene. Identical amino acids are represented by a dot. An identity level of 99.09% was obtained between AAA66953 and Vp8::Vp5ΔC- term WT amino acid sequences. (AAA66953 is 100% identical to Vp8::Vp5ΔC-term OPT). Figure 7 shows the expression by Western immunoblotting using a rabbit antiserum, of Vp8::Vp5ΔC-term WT protein in bacteria (Panel A), Vp8::Vp5ΔC-term OPT in mammalian cells transduced with recombinant Adenovirus Ad (Vp8::Vp5ΔC-term OPT) (Panel B) and in plant cells agroinfiltrated with recombinant Agrobacterium tumefaciens (Panel C). Lane 1 : non-induced E. coli protein extract as negative control; lane 2: 3h IPTG-induced E. coli protein extract; lane 3: AdV(G FP)-infected 293APS cell (48h post infection) protein extract used as a negative control; lane 4: AdV(Vp8::Vp5ΔC-term OPT)-infected 293APS cell (48h post infection) protein extract; lane 5: non transformed Nicotiana benthamiana leaf cell protein extract; lane 6: transformed Nicotiana benthamiana leaf cell protein extract. Lower panels: Ponceau staining was used as load control. The molecular weight markers are indicated on the left side of the Figure.

Figure 8 shows the expression of Vp8::Vp5ΔC-term OPT protein in 293APS infected with recombinant AdV(Vp8::Vp5ΔC-term OPT) at an MOI of 1 , or transfected with 0.5 μg of pcDNA3.0 (Vp8::Vp5ΔC-term OPT) using an indirect immunofluorescence assay. Cells were fixed at 48h post infection or transfection. Cells infected with AdV(GFP) or transfected with pcDNA3.0 (GFP) were used as negative controls. As GFP is expressed in the cytoplasmic compartment of the cell, it leaks out of the cell upon fixation in cold methanol and hence no residual fluorescence could interfere with the secondary antibody green fluorescence.

Figure 9 shows the presence of specific anti-(Vp8::Vp5ΔC-term WT) IgG in the serum of one rabbit collected at day 60 after the primary immunization. The immunized rabbit was inoculated three times with recombinant Vp8::Vp5ΔC-term WT protein. Pre-immune serum serves as negative control. The Vp8::Vp5ΔC-term WT-specific antibodies were detected by Western blot (Panel A); lane 1 : 1 μg of recombinant Vp8::Vp5ΔC-term WT protein incubated with pre-immune rabbit serum (1 :5000 dilution); lane 2: 1μg of recombinant VP8-5ΔC-Term WT protein incubated with the rabbit serum (1 :5000 dilution) collected at day 60 after the primary immunization. The Vp8::Vp5ΔC-term WT- specific antibodies were also detected using an indirect immunofluorescence assay (Panel B). Rotavirus-infected MA104 cells were fixed 24h post infection and incubated with the rabbit serum (1:5000 dilution). Vp8::Vp5ΔC-term WT-specific IgG antibodies were detected by Alexa-fluor-green-coupled anti-rabbit IgGs (1 :1000) which were used as secondary antibodies. The serum of the immunized rabbit was shown to contain rotavirus-specific neutralizing antibodies as determined by an in vitro virus neutralization test. An antibody neutralizing titer of 128 was obtained (Panel C).

Figure 10A represents an indirect immunofluorescence assay showing the presence of specific anti-(Vp8::Vp5ΔC-term WT) IgG antibodies in the serum of mice immunized at days 0 and 14. Group A: pool of five mice were inoculated with 10⁸ TCID50 particles of AdV (Vp8::Vp5ΔC-term OPT): and group F: pool of four mice were inoculated with 100 μl of PBS (negative control). Serum samples were collected on day 35 (IF titre= 1024) following the primary immunization or inoculation. Rotavirus-infected MA104 cells were fixed 24h post infection and incubated with the mouse pooled serum (1 :1000). The Vp8::Vp5ΔC-term WT-specific antibodies were used as primary antibodies whereas anti-Alexa-fluor-green-coupled anti-mouse IgG (1 :1000) were used as secondary antibodies.

Figure 10B shows the presence of specific anti-Vp8::Vp5ΔC-term WT IgGs in the serum of individual mouse immunized at days 0 and 14 with 10⁸ TCID50 particles of AdV (Vp8::Vp5ΔC-term OPT) (Group A), as determined by Western blot. All sera from immunized mice were analysed at day 35 after primary immunization. One μg of recombinant Vp8::Vp5ΔC-term WT was deposited in each lane and incubated with the following sera. Lane 1 : negative serum from mice of group F (pool of four mice inoculated at days 0 and 14 with 100 μl of PBS) collected at day 35 after the primary inoculation; lane 2: mouse number 1 ; lane 3: mouse number 2; lane 4: mouse number 3; lane 5: mouse number 4; Lane 6: mouse number 5. Figure 11 shows the kinetics of anti-(Vp8::Vp5ΔC-term OPT) IgG antibody production in groups of mice A to F, as determined by an indirect immunofluorescence assay (IFA). Sera were collected on day 0, 14, 35, 56, and 66 and were pooled for each group for analysis. IFA titers are expressed as the reciprocal of the highest serum dilution giving positive fluorescent signal.

Figure 12 shows significant (P = 0.013) splenocyte lymphoproliferation from a group of five mice (Group A) immunized with 10⁸ TCID50 of adenovirus vector (Vp8::Vp5ΔC-term OPT) on day 0 and 14, when compared to the response of a negative control group of mice (Group F) inoculated with PBS. The cells were stimulated with 10 μg/ml of recombinant Vp8::Vp5ΔC-term WT protein. Results are expressed as stimulation index which is the ratio of optical density at 490 nm (OD₄₉₀) of cells stimulated with the antigen (Vp8::Vp5ΔC-term WT protein) to the OD₄goθf cells without antigen. The cross-bar represents the mean value of the stimulation index. The groups were compared using a Student T-test and the significance level was set at P < 0.05.

Figure 13 shows the Agro-infiltration of 3 weeks-old Nicotiana benthamiana plants at 0 and 10 days post infiltration. Control: non-infiltrated plants, Vp8::Vp5: Agrobacterium tumefaciens strain AGL1 carrying the chimeric Vp8::Vp5Δc-term OPT sequence in pGreenllO229, GFP: plants transformed with a GFP-expressing vector.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has developed a new tool for inducing an immune response against a rotavirus infection. Such a tool finds a particular advantageous application in the field of rotavirus vaccines. In this connection, the present invention relates to a polypeptide comprising a portion of the VP5 rotavirus peptide fused to a VP8 rotavirus peptide, polynucleotides encoding same and their use in compositions and methods for eliciting a specific anti-rotavirus immune response against Rotavirus- associated diseases or infections.

A rotavirus-associated disease may be, for instance, diarrhea in young children, gastroenteritis and dehydration It is also worth to note that a non-exhaustive list of rotavirus-associated clinical symptoms consistent with extraintestinal rotavirus infection which the polypeptides of the invention may also be useful for, include those, such as pneumonia, exanthema, disseminated intravascular coagulation, hemophagocytic lymphohistiocytosis, coagulation, hemophagocytic lymphohistiocytosis, neurological complications such as encephalitis or encephalopathy.cerebellitis, convulsions, or seizures (Blutt and Connor, 2007).

Definitions

The term "isolated" is meant to describe a nucleic acid construct or a polypeptide that is in an environment different from that in which the nucleic acid construct or the polypeptide naturally occurs.

The term "subject" refers to any subject susceptible to be infected by a Rotavirus strain. For instance, such a subject may be, but not limited to, mice, rabbit, bovine, horse and human. More specifically, the subject consists of a human.

The term "Rotavirus strain" refers to a strain of any Groups (as classified on the basis of VP6 sequences), or any serotypes/genotypes [classified on the basis of VP7 (G) and VP4 (P) sequences (for instance, the Wa strain belongs to Group A and to the G1 P1 [8] genotype).

The term "treating" refers to a process by which the symptoms of an infection or a disease associated with a rotavirus strain are alleviated or completely eliminated. As used herein, the term "preventing" refers to a process by which symptoms of an infection or a disease associated with a rotavirus strain are obstructed or delayed. The expression "immune response" refers to an in vivo or in vitro reaction in response to a challenge by an immunogen. An immune response is generally expressed by an antibody production (e.g., neutralizing antibodies) and/or a cell- mediated immunity.

The expression "an acceptable carrier" means a vehicle for containing the compounds obtained by the method of the invention that can be administered to a subject host without adverse effects. Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.

Amino acid or nucleotide sequence "identity" is determined from an optimal global alignment between the two sequences being compared. An optimal global alignment is achieved using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. MoI. Biol. 48:443-453) or the BIOEDIT v7.0.3 software. "Identity" means that an amino acid or nucleotide at a particular position in a first polypeptide or polynucleotide is identical to a corresponding amino acid or nucleotide in a second polypeptide or polynucleotide that is in an optimal global alignment with the first polypeptide or polynucleotide. By the statement "sequence A is n% identical to sequence B", it is meant that n% of the positions of an optimal global alignment between sequences A and B consists of identical amino acid residues or nucleotides.

1. Polynucleotides and polypeptides of the invention

In a first embodiment, the present invention concerns an isolated polypeptide which comprises a VP5 Rotavirus peptide lacking its associated C-terminus region and a VP8 rotavirus peptide. As one skilled in the art may appreciate, the term "C-terminus region" in the context of the present invention when referring to the VP5 protein, preferably consists of the region spanning from about amino acid residue 331 to 775 or at an equivalent position, corresponding to position 331 to 775 of the amino acid sequence set forth in SEQ ID NO 1 or 2.

As it may be appreciated, the VP5 Rotavirus peptide lacking its associated C- terminus region may, for instance, comprise an amino acid sequence substantially identical to the amino acid sequence ranging from amino acid residue 275 to 330 or at an equivalent position, corresponding to position 275 to 330 of the amino acid sequence set forth in SEQ ID NO 1 or 2. More particularly, the VP5 Rotavirus peptide lacking its associated C-terminus region may alternatively be encoded by a nucleic acid sequence substantially identical to the nucleic acid sequence ranging from nucleotide 823 to 990, or at an equivalent position, corresponding to position 823 to 990 of the nucleotide sequence set forth in SEQ ID NO 1.

As mentioned above, the polypeptide of the invention also comprises a VP8 peptide. In this regard, the VP8 Rotavirus peptide may, for instance, comprise an amino acid sequence substantially identical to the amino acid sequence ranging from amino acid residue 1 to 274, or at an equivalent position, corresponding to position 1 to 274 of the amino acid sequence set forth in SEQ ID NO 1 or 2. Alternatively, the VP8 Rotavirus peptide may be encoded by a nucleic acid sequence substantially identical to the nucleic acid sequence ranging from nucleotide 1 to 822, or at an equivalent position, corresponding to position 1 to 822 of the nucleotide sequence set forth in SEQ ID NO 1.

As used herein, the term "equivalent position" denotes a position which, on the basis of an alignment, for instance, of the amino acid sequence of the parent VP5 protein in question with the "reference" VP5 amino acid sequence in question (for example the sequence shown in SEQ ID NO 1 or 2) so as to achieve juxtapositioning of amino acid residues/regions which are common to both, corresponds most closely to a particular position in the reference sequence in question.

By "substantially identical" when referring to an amino acid sequence, it will be understood that the polypeptide of the present invention preferably has an amino acid sequence having at least 75% identity, or even preferably 85% identity, or even more preferably 95% identity to part or all of the sequence shown in SEQ ID NOS 3 to 6, 8 and 9.

In this connection, the polypeptide of the invention may have, for instance, at least 95 % amino acid sequence identity to SEQ ID NOS 3 to 6, 8 or 9, or even more preferably consists of the amino acid sequence of SEQ ID NOS 3 to 6, 8 or 9.

In an alternate preferred embodiment, the polypeptide contemplated by the present invention may be encoded by a nucleic acid sequence having at least 85 % nucleic acid sequence identity to SEQ ID NOS 3, 5 and 8 or even more preferably, being encoded by a nucleic acid sequence consisting of nucleic acid sequence SEQ ID NOS 3, 5 or 8.

The present invention also concerns an isolated polynucleotide encoding the above mentioned polypeptide of the invention. Such a polynucleotide comprises a VP5 nucleic acid molecule encoding a VP5 Rotavirus peptide lacking its associated C- terminus region, and a VP8 nucleic acid molecule encoding a VP8 rotavirus peptide.

As previously mentioned, the VP5 nucleic acid molecule contemplated by the present invention may, for instance, comprise a nucleic acid sequence substantially identical to the nucleic acid sequence ranging from nucleotide 823 to 990 of SEQ ID NO 1. As also mentioned, the VP8 nucleic acid molecule may comprise a nucleic acid sequence substantially identical to the nucleic acid sequence ranging from nucleotide 1 to 822 of SEQ ID NO 1. By "substantially identical" when referring to a nucleic acid sequence, it will be understood that the polynucleotide of the invention preferably has a nucleic acid sequence which is at least 65 % identical, more particularly 85 % identical and even more particularly 95 % identical to part or all of the sequence shown in SEQ ID NOS 3, 5 and 8

In this connection, the polynucleotide according to the present invention may have, for instance, at least 85 % nucleic acid sequence identity to SEQ ID NO 3, 5 or 8, or even more preferably, such polynucleotide consists of the nucleic acid sequence of SEQ ID NO 3, 5 or 8.

In another embodiment, the invention is further directed to a vector (e.g., cloning or expression vector) comprising a polynucleotide as defined above.

As used herein, the term "vector" refers to a polynucleotide construct designed for transduction/transfection of one or more cell types. Vectors may be, for example, "cloning vectors" which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors" which are designed for expression of a nucleotide sequence in a host cell, or a "viral vector" which is designed to result in the production of a recombinant virus or virus-like particle, or "shuttle vectors", which comprise the attributes of more than one type of vector.

A number of vectors suitable for stable transfection of cells and bacteria are available to the public (e.g., plasmids, adenoviruses, baculoviruses, yeast baculoviruses, plant viruses, adeno-associated viruses, retroviruses, Herpes Simplex Viruses, Alphaviruses, Lentiviruses), as are methods for constructing such cell lines. It will be understood that the present invention encompasses any type of vector comprising any of the polynucleotide molecule of the invention.

In a related aspect, the present invention provides a non-human host cell comprising a vector as defined above. The term "host cell" refers to a cell that has a new combination of nucleic acid segments that are not covalently linked to each other in nature. A new combination of nucleic acid segments can be introduced into the non- human host cell using a wide array of nucleic acid manipulation techniques available to those skilled in the art. The non-human host cell of the invention can be a eukaryotic cell (e.g., plant cell) or a prokaryotic cell (e.g., bacterial cell). The host cell can harbor a vector that is extragenomic. An extragenomic nucleic acid vector does not insert into the cell's genome. A host cell can further harbor a vector or a portion thereof that is intragenomic. The term "intragenomic" defines a nucleic acid construct incorporated within the host cell's genome.

2. Compositions and methods of use

The polypeptides and polynucleotides encoding same of the invention may be used in many ways in the induction of a specific immune response, or in the treatment and/or prevention of a Rofav/ras-associated disease or infection.

For instance, and according to an aspect of the invention, the polypeptides of the invention may be used as immunogens for the production, for instance, of specific antibodies for the treatment and/or prevention of a Rotavirus infection. Suitable antibodies may be determined using appropriate screening methods, for example by measuring the ability of a particular antibody to neutralize a Rotavirus infection in a test model. Examples of animal models include but not limited to mouse, rat and pig.

According to another aspect, the polynucleotides encoding the polypeptides of the invention may be used in a so-called DNA immunization method. That is, they can be incorporated into a vector which is replicable and expressible upon injection thereby producing the antigenic polypeptide of the invention in vivo. For example polynucleotides may be incorporated into a plasmid vector under the control of the CMV promoter or the CaMV35S promoter which are functional in eukaryotic cells (mammalian and plant cells respectively). The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method or system such as direct injection of plasmid DNA into muscles, injection of plasmid DNA with or without adjuvants, targeting cells by delivery of DNA complexed with specific carriers, injection of plasmid complexed or encapsulated in various forms of liposomes, administration of DNA with different methods of bombardment, and administration of DNA with lived vectors.

In this connection, another embodiment of the present invention relates to a composition for inducing a specific immune response against a Rotavirus infection. The composition of the present invention advantageously comprises an acceptable carrier and a polypeptide or a non-human host cell of the invention. Alternatively, the composition of the invention can comprise a polynucleotide or a vector of the invention.

Yet, another embodiment of the present invention is to provide a method for inducing a specific immune response against a rotavirus infection in a subject. Yet, a further embodiment of the present invention is to provide a method for treating and/or preventing a rotavirus infection. The methods of the invention comprise the step of administering to the subject a polypeptide, or a polynucleotide, or a composition according to the invention.

It will be understood that the methods of the invention may, for instance, further comprise a step of administering at least one booster of the polypeptide, or a polynucleotide, or a composition according to the invention, to maintain a hyperimmune state in the subject.

The amount of the components or the elements of the composition of the invention is preferably a therapeutically effective amount. A therapeutically effective amount of the contemplated component is the amount necessary to allow the same to perform their immunological role without causing overly negative effects in the subject to which the composition is administered. The exact amount of the components to be used and the composition to be administered will vary according to factors such as the type of condition being treated, the type and age of the subject to be treated, the mode of administration, as well as the other ingredients in the composition.

The composition of the invention may be given to the subject through various routes of administration. For instance, the composition may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparations may also be sterile injectable solutions or suspensions in nontoxic parenterally-acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection, by infusion or per os. Suitable dosages will vary, depending upon factors such as the amount of each of the components in the composition, the desired effect (short or long term), the route of administration, the age and the weight of the animal to be treated. Any other methods well known in the art may be used for administering the composition of the invention.

One will appreciate that the booster may be given or administered to the subject by a different route than the one used during the initial administration of the polypeptide, or a polynucleotide, or a composition of the invention to the subject.

The present invention will be more readily understood by referring to the following example. This example is illustrative of the wide range of applicability of the present invention and is not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.

EXAMPLES Material and methods

Cell lines and viruses

MA104 cells (gift from D. Yoo, University of Guelph, Ontario, Canada) and 293APS cells (gift from B. Massie, NRC-Biotechnology Research Institute, Quebec, Canada) were maintained in Dulbecco minimal essential medium (DMEM) with 5% (v/v) fetal bovine serum (FBS).

For cell infection, confluent 2-day old culture of MA104 were inoculated with the rotavirus Wa strain (classified in G1 P1A[8] serogroup, gift from D. Yoo, University of Guelph, Ontario, Canada) that was incubated for 30 minutes at 37⁰C in DMEM supplemented with 10 ug/ml porcine pancreatic trypsin (Sigma-Aldrich).The infected cells were then further incubated in FBS-free DMEM containing 5μg/ml porcine pancreatic trypsin until 90% of the cells exhibited a cytopathic effect. After three cycles of cell freezing and thawing, the cell culture supernatant was collected by centrifugation for 15 minutes at 300Og at 4⁰C. The viral titers were determined and calculated as the median tissue culture infective dose (TCID50) per ml (Archambault et al., 1988). 293PS cells were used to generate and amplify recombinant adenoviruses expressing the protein of interest. They were also used to titrate the adenovirus stock.

Sequences

To ensure optimal expression of the Vp8::Vp5ΔC-term protein in mammalian cells and plant cells, the DNA sequences based on the rotavirus Wa sequence (GeneBank accession number AAA66953) were optimized for codon usage and produced synthetically (GeneArt, Regensburg, Germany). The expressed protein was designated Vp8::Vp5ΔC-term OPT.

The recombinant Vp8::Vp5ΔC-term protein was also produced in bacteria. The wild-type (WT) sequence was obtained through a standard RT-PCR procedure using RNA from MA104 cells infected with the rotavirus Wa strain (see above), and sequenced to ascertain its viral origin (see below). The recombinant protein was designated Vp8::Vp5ΔC-term WT.

Viral RNA isolation

The rotavirus genomic RNA was extracted from infected cell supernatant using TRIzol®

Reagent (Invitrogen) according to the manufacturer's procedure.

Reverse transcription-PCR amplification, cloning, and sequencing The rotavirus genomic RNA was reverse transcribed to complementary DNA (cDNA) by using random hexadeoxyribonucleotides (pd(N)6; Amersham), as previously described (Kang et a/., 2001). The cDNA was then amplified using the WT BgIW sense (S) primer: 5' GCGGAGATCTGCCACCATGGCTTCACTCATTTATAG 3' and the WT BgIW antisense (AS) primer : 5'

GGTATTAGATCTTCATCACCCTCCATTATAGCTAAAATTGTTCACTCCA 3' through 30 successive cycles of denaturation at 94°C for 1.5 min, primer annealing at 52°C for 1.5 min, and DNA chain extension at 72°C for 2.5 min. The amplified cDNA products were cloned into pBluescript/KS+ (pBS) vector (Stratagene, La JoIIa, CA). The sequence of the inserts was confirmed through sequencing (Genome Quebec, Montreal, CA). Nucleic and the predicted corresponding amino acid sequences were aligned for comparison with the GeneBank sequence using BioEdit V7.0.9 software.

Expression of Vp8::Vp5ΔC-term WT in E. coli and production of Vp8::Vp5ΔC-term WT- specific antiserum in rabbit. Vp8::Vp5ΔC-term WT was further subcloned into pTrcHisB (Invitrogen) prokaryotic expression vector thereby allowing the sequence to be in frame with a 6 histidine-tag at the Nhb-terminal.

DH5α E. coli heat shock transformed with pTrcHisB encoding Vp8::Vp5ΔC-term WT gene were cultured and induced with 1mM IPTG for recombinant protein expression. Recombinant protein was only present in the insoluble fraction and was purified on a Ni- NTA-Hisbind^® resin (Novagen, Madisson, USA) under denaturing conditions according to the manufacturer's manual. The recombinant Vp8::Vp5ΔC-term WT protein was then dialyzed against a phosphate buffer saline (PBS) solution (pH 7.3). The purity of the protein was assessed by Western blot using an anti-His antibody (Qiagen). The concentration of the protein was determined using the Lowry method with a Bio-rad protein assay^® kit (Bio-rad). Rotavirus Vp8::Vp5ΔC-term WT polyclonal antibodies were raised by immunizing a New Zealand white rabbit according to standard procedures. The animal was inoculated subcutaneously 3 times with 250 μg of the recombinant protein mixed with an equal volume of Titermax gold adjuvant (Sigma-Aldrich), on day 0, 21 and 42. Blood samples were collected on day 0, 31 and 52. Final bleeding was performed on day 60.

Construction of recombinant plasmids for expression in eukarvotic cells The synthetic Vp8::Vp5ΔC-term OPT gene was amplified by PCR using the OPT Hind\\\ BgIW sense (S) primer: 5' CAAATAAGCTTAGATCTGCCACCATGGCCAGCCT 3' and the OPT Bgl\\/Xho\ antisense (AS) primer : 5'AACTCGAGGGGAGATCTTCAT- CAGCCGCCGTTGTAGCTGAAGTTG 3' primers allowing the addition of Hind\\\ and Xho\ restriction sites at the 5^Λand 3' terminals respectively. It was then cloned in pBS and subcloned into pCDNA3.0 (Invitrogen). Recombinant plasmid (pCDNA3.0- Vp8::Vp5ΔC-term OPT) was produced at a large scale using the Mega Qiaprep^®Spin endotoxin free kit (Qiagen).

Construction of recombinant adenoviruses

The procedures used were as per the manual of the AdEasy system (Qbiogene, Carlsbad, CA). The Vp8::Vp5ΔC-term OPT gene was first subcloned in the shuttle vector pAdenoVator-CMV5(CuO)-IRES-E1A (Bourbeau et al., 2007) at the BgIW restriction site and was verified by sequencing. A recombinant AdV with the gfp (Green Fluorescent Protein) gene as transgene was constructed as a positive control for AdV synthesis in the 293APS complementing cell line. Transgene expression is silenced in the presence of the CymR repressor and induced with cumate (50 μg/ml). The genome of the recombinant adenovirus (Ad5(ΔPS/ΔE1 B/ΔE3)transgene-IRES-E1A) was generated using the AdEasy system (Qbiogene, Carlsbad, CA) with the AdΔPS backbone and following the manufacturer's procedure. The E1A expression allowed viral DNA replication in host cells. However these adenovectors are non-disseminating because of the lack of the viral protease PS. The 293APS complementing cell line which expresses the viral protease PS (Bourbeau et al., 2007J was then used to produce viral particles that were used in the immunization experiments in mice.

Expression of Vp8::Vp5ΔC-term OPT in mammalian cells by Western immunoblotting The 293APS cells were plated into each well of a six-well plate at a density of 2.5 x 10⁵ per well, and transduced the next day with the adenovirus vector. Protein lysates were harvested when 70% of the cells exhibited a cytopathic effect and then diluted in Laemmli buffer. Proteins were separated onto a 12% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. Blots were blocked with PBS-Tween 20 (0.05 % v/v) containing 5% (w/v) of skim milk (Carnation); the blots were then incubated with either the rabbit polyclonal anti- Vp8::Vp5ΔC-term WT, or the monoclonal anti-VP8 antibody (gift from Harry Greenberg, Standford University, USA) for 1 h at 37⁰C, washed with PBS-Tween 20, and incubated with a secondary antibody coupled to horseradish peroxidase (Millipore, ViIIe St Laurent, CA). Signal detection was done using enhanced chemiluminescence (Perkin Elmer, Woodbridge, CA).

Expression of Vp8::Vp5ΔC-term OPT in mammalians cells by indirect immunofluorescence assay (IFA)

The test was performed using anti-Vp8::Vp5ΔC-term WT antibodies as primary antibodies (1 :1000 dilution) and Alexa fluor green coupled anti-mouse secondary antibodies (1 :1000 dilution), incubated each for 2h at 37⁰C. Confluent 293APS plated (3 x 10⁵ per well) into wells of a 24-well plate were infected with the adenovirus vector [multiplicity of infection (MOI) of 1] expressing the protein of interest, or transfected (0.5 μg per well) with the pCDNA3.0- Vp8::Vp5ΔC-term OPT for 48 hours. Mock-infected cells, cells infected with an adenovirus vector expressing the Green Fluorescence Protein (GFP), or cells transfected with a pCDNA3.0 expressing GFP served as negative controls. Cells were fixed in cold methanol for 30 min, and blocked for 1 h at 37⁰C in PBS-5% bovine serum albumin before adding the antibodies. After the antibody treatments, the cells were visualized under fluorescence microscopy.

Mice and immunizations with the adenovector

Six-week-old female BALB/c mice were purchased from Charles River (St-Constant, Quebec, CA). The mice were kept under standard laboratory conditions according to the rules and regulations of the Canadian Council for Animal Care. The mice were immunised intramuscularly with 100 μl of either 10⁸ TCID50 of adenovirus vector, 10⁷ TCID50 of adenovirus vector (diluted in PBS, pH 7.3), or 100ug of recombinant plasmid (diluted in PBS, pH 7.3) on days 0, 14, 35. The mice were sacrificed on day 66 (or otherwise specified). Blood samples were collected on day 0, 14, 35, 56, and 66.

Group A (ten mice) received 10⁸ TCID50 of adenovirus vector (Vp8::Vp5ΔC-term OPT) on day 0, 14 and 35. Five mice were sacrificed on day 35.

Group B (five mice) received 10⁸ TCID50 of adenovirus vector (Vp8::Vp5ΔC-term OPT) on day 0. They received 100 μg of plasmid pCDNA3.0- Vp8::Vp5ΔC-term OPT on day 14 and 35.

Group C (five mice) received 10⁷ TCID50 of adenovirus vector (Vp8::Vp5ΔC-term OPT) on day 0, 14 and 35.

Group D (five mice) received 100 μg of plasmid pCDNA3.0-Vp8::Vp5ΔC-term OPT on day 0, 14 and 35. Group E (three mice) received 10⁸ TCID50 of adenovirus vector expressing the GFP (negative control) on day 0, 14 and 35.

Group F (four mice) received three injections of PBS pH 7.3 (negative control).

Lvmphoproliferation assay

The lymphoproliferation assay was performed based on a previously published method with some modifications (Archambault et al, 1988).) Briefly, spleen cells were suspended in RPMI cell culture medium supplemented with penicillin (100 U/ml), streptomycin (100 ug/ml), FBS 10% and 0.05 mM β-mercaptoethanol, seeded in quadruplicates in a 96-well plate at a final concentration of 5.0 * 10⁵ cells per well, and stimulated with 10 ug/ml of purified Vp8::Vp5ΔC-term WT protein. The cells were incubated at 37⁰C. After 72 h, 40 μl of MTS/PMS were added in each well. Absorbance was measured at 490 nm 3h following the MTS/PMS treatment.

The lymphoproliferation response was expressed as a stimulation index which is the ratio of optical density (OD) of cells stimulated with the antigen (Vp8::Vp5ΔC-term WT protein) to the OD of cells without antigen.

Detection of serum IqG in mice by Western immunoblottinq and indirect immunofluorescence assay (IFA)

Western immunoblotting: 1 μg of purified Vp8::Vp5ΔC-term WT protein (from bacteria, mammalian and plant cells) diluted in Laemmli buffer was loading on 15% polyacrylamide-SDS gel and transferred onto nitrocellulose membranes. Blots were incubated with mouse sera, washed, and incubated with secondary antibody coupled to horseradish peroxidase (Millipore, ville St Laurent, CA). Signal detection was done using enhanced chemiluminescence (Perkin Elmer, Woodbridge, CA). IFA: Ma104 cells were plated into each well of a six-well plate at a density of 1.0 x 10⁴ per well, infected the next day with the Wa rotavirus at a multiplicity of infection (MOI) of 1. Cells were fixed in cold methanol for 30 min, blocked for 1 h at 37⁰C in PBS-5% bovine serum albumin. The test was performed using mouse sera and Alexa fluor green coupled anti-mouse secondary antibodies (Sigma), each incubated for 2h at 37⁰C. After the antibody treatments, the cells were then visualized under fluorescence microscopy.

Seroneutralization test

The serum neutralization test was performed by a viral cytopathic effect inhibition method (Archambault et al, 1988). Sera were inactivated at 56⁰C for 30 min before testing. Serial two-fold dilutions (starting at 1 :8) of each serum were mixed with an equal volume of the Wa strain rotavirus in DMEM supplemented with 5 μg/ ml of porcine pancreatic trypsin, followed by an incubation at 37⁰C for 1 h. The serum-virus mixture [25 μl per well containing 100 tissue culture infective dose (TCID)₅₀] was then used to inoculate quadruplicate cultures of MA104 cells in 96-well tissue culture plates. After an incubation of 1 h at 37°C, 200 μl of fresh medium was added to each well. The plates were then incubated for 72 h. Neutralization titers were expressed as the reciprocal of the highest serum dilution giving 100% inhibition of the cytopathic effet.

Agro-infiltration

Two binary constructs based on pGreenllO229 or pCambia1380 were generated for transient expression assays. The Vp8::Vp5Δc-term insert sequence was optimized for codon usage into plants (Geneart) and amplified with specific primers 5'-

GAGACCATGGCTTCTCTTATCTACCGTCAG-3' and δ'-GAGATCTAGAGGGGAGCTCCTAAA-

GCTCATCCTTTTCACTACCACCGTTG-3' for blunt sub-cloning into pBSK+. Endonuclease restriction allowed ligation of the insert into the expression cassette of pTex3Δnls. Finally, the complete Vp8::Vp5Δc-term OPT cassette under the control of the CaMV 35S promoter was cloned into the expression vectors. Effector constructs were confirmed by sequencing. The plasmids were independently transformed into Agrobacterium tumefaciens strain AGL1. The transformed agrobacteria were used individually to infiltrate intact leaves of Nicotiana benthamiana according to the method described by Kane et al. (2007). To enhance transient expression of the transgenes, agrobacteria carrying the p19 suppressor of PTGS were included together with the binary vectors carrying the Vp8::Vp5Δc-term OPT gene constructs.

To verify the presence of the Vp8::Vp5Δc-term OPT sequence in plant, nucleic acid analyses were performed. Genomic DNA and total RNA were isolated from agro- infiltrated leaves (Nicotiana. benthamiana) using TRIzol reagents according to the manufacturer's instructions (Invitrogen). For RT-PCR, total RNA was subjected to reverse transcription using SuperScriptJI reverse transcriptase (Invitrogen) according to the manufacturer's recommendations. Primers 5'-

GGCTGCTAACTACCAGTACAACTACCTTAGGG-3 and 5'- GCATGCCTGCAGGTCACTGGATT-3' were designed to specifically amplify the Vp8::Vp5Δc-term OPT sequence. PCR amplification products were analyzed by electrophoresis on 0.8% agarose/ethidium bromide gels.

The expression of the Vp8::Vp5ΔC-term OPT polypeptide of the invention in Nicotiana benthamian was analysed by Western blotting as performed above. Optimal extraction of the expressed protein from transient plant tissues was achieved using TCA-acetone precipitation followed by resolubilization in 0.1 M sodium phosphate buffer containing 8M urea. Blots were incubated with rabbit antisera, washed, and incubated with secondary antibody coupled to horseradish peroxidase (Millipore, ViIIe St Laurent, CA). Signal detection was done using enhanced chemiluminescence (Perkin Elmer, Woodbridge, CA).

Example 1 : Expression of Vp8::Vp5ΔC-term WT and Vp8::Vp5ΔC-term OPT in E. coli

As the sequence encoding the Vp8::Vp5ΔC-term OPT protein was optimized for expression in mammalian cells based on GenBank sequence AAA66953, no expression, as expected, from this optimized gene was detected in prokaryotic cells. Hence the wild-type gene was used for production of the protein of interest in bacterial cells. The sequence encoding the Vp8::Vp5ΔC-term WT protein was, therefore, obtained by RT-PCR from MA104 cells infected with the Wa rotavirus strain, and then, successfully inserted into plasmid pTrcHisB. Recombinant Vp8::Vp5ΔC-term WT protein was produced in E. coli strain DH5α after 3h of IPTG induction, and was purified on Ni- NTA-Hisbind^® resin (Novagen). The purified protein was then used to immunize a laboratory rabbit. The rabbit antiserum was confirmed to contain protein-specific antibodies by Western blot (Fig. 7, panel A).

Example 2: Amino acid sequence comparison between Vp8::Vp5ΔC-term OPT and Vp8::Vp5ΔC-term WT

Amino acid alignments (Fig. 6) were performed between the amino acid sequences of Vp8::Vp5ΔC-term OPT (Fig. 4) (whose sequence is identical to that of the AAA66953 GeneBank sequence) and Vp8::Vp5ΔC-term WT (Fig. 5). The percentage of identity between the sequences was equal to 99.09 %.

Example 3 : Expression of Vp8::Vp5ΔC-term OPT in mammalian and plant cells

Western blot analysis performed using Vp8::Vp5ΔC-term WT-specific rabbit antiserum showed that the Vp8::Vp5ΔC-term OPT protein was successfully expressed in both mammalian cells transduced with recombinant Adenovirus Ad(Vp8::Vp5ΔC-term OPT) (Fig. 7, panel B) and plant cells agroinfiltrated with recombinant Agrobacterium tumefaciens (Fig. 7, panel C) (as indicated by the band at 32 KDa).

The expression of the protein was also confirmed by indirect immunofluorescence when expressed in mammalian 293APS cells infected with recombinant AdV(Vp8::Vp5ΔC- term OPT) or transfected with pcDNA3.0 (Vp8::Vp5ΔC-term OPT) (Fig. 8). Example 4: Stimulation of systemic humoral immune response in the rabbit

Specific anti-(Vp8::Vp5ΔC-term WT) IgGs were detected by Western blot (Fig. 9, panel A) in the serum (collected on day 60 after the primary inoculation) of a rabbit inoculated three times with recombinant Vp8::Vp5ΔC-term WT protein expressed in E. coli. Pre- immune serum was used as negative control.

Recombinant Vp8::Vp5ΔC-term WT-specific antibodies were also detected in the same rabbit serum (on day 60) by indirect immunofluorescence (Fig. 9, panel B) using rotavirus-infected MA104 cells as substrate antigen, and Alexa-fluor-green coupled anti- rabbit IgG as secondary antibodies.

The rabbit serum was shown to exert a rotaviral neutralizing function with a virus neutralization titer of 128 (Fig. 9, panel C).

Example 5: Stimulation of systemic humoral immune response in mice inoculated with eukaryotic vectors

Several groups of mice were inoculated with the recombinant adenovirus vector and/or the plasmid pCDNA3.0 expressing the Vp8::Vp5ΔC-term OPT protein using different regimens of immunizations. The serum of mice were collected and analyzed over time for the presence of specific antibodies by an indirect immunofluorescence assay. Inoculation of mice with the recombinant Adenovirus vector (groups A and C of mice inoculated with 10⁸ TCID50 or 10⁷ TCID50 of the adenovector, respectively) resulted in the appearance of detectable antibodies from day 14 after the primary inoculation (Fig. 11). An increase in antibody titers in both groups of mice was observed at day 35 e.g. 21 days after the secondary inoculation of the Adenovirus vector. The highest response was observed in mice inoculated with the highest dose of the adenovector (group A). Similarly, a positive antibody response, albeit to a lower level, was obtained in mice inoculated with plasmid pCDNA3.0 (group D). However, the best results in antibody response were observed in mice primed with the Adenovirus vector and boosted with the plasmid pCDNA3.0 (Group B). No antibody response was observed in control groups of mice who received an adenovector expressing the GFP protein (group E) or PBS (group F). Finally, it was shown as per Western blot that the sera of all individual mice from group A readily reacted with the recombinant Vp8::Vp5ΔC-term protein giving the expected positive band at 32 kDa (Fig. 10, panel B).

Example 6: Stimulation of mouse splenocvtes in vitro

Significant in vitro splenocyte specific lymproliferation from five mice of group A (inoculated with 10⁸ TCI D50 of the adenovirus vector (VP8-5ΔC-term OPT) on day 0 and 14) sacrificed at day 35 after the primary immunization was obtained after cell stimulation with 10 μg/ml of recombinant VP8-5ΔC-Term WT protein (P = 0.013 when compared to the values obtained from the control group of mice (group F) inoculated with PBS (Fig. 12).

Example 7: Transient expression by agro-infiltration of Nicotiana benthamiana

Plants at various developmental stages and various post-infection conditions were tested to determine the optimal conditions for maximal protein accumulation. Optimal expression was acquired 10 days post-infiltration as determined for example by the GFP signal (Fig. 13). As also shown in Fig. 13, the expressed VP8::VP5Δc-term OPT polypeptide of the invention is not toxic to the plant. Similar results were obtained with the pCambia1380 vector (not shown).

As shown in Fig. 7, panel C, the immunoblot analysis confirmed that VP8::VP5Δc-term OPT polypeptide of the invention was successfully expressed in plants (using in this example the pGreen II0229 vector). The chimeric protein shows a molecular weight of ~32 kDa identical to that of the wild type protein produced in bacteria (Fig. 7, panel A) and to that of the VP8::VP5Δc-term OPT polypeptide expressed in mammalian cells (Fig. 7, panel B).

REFERENCES

Archambault, D., G. Morin, Y. Elazhary, R. S. Roy, and J. H. Joncas. 1988. Immune response of pregnant heifers and cows to bovine rotavirus inoculation and passive protection to rotavirus infection in newborn calves fed colostral antibodies or colostral lymphocytes. American Journal of Veterinary Research 49:1084-1091.

Blutt, S. E. and M. E. Conner. 2007. Rotavirus: to the gut and beyond! Current Opinion in Gastroenterology 2007, 23:39-43

Bourbeau D., C. J. Lau, J. Jaime, Z. Koty, S. P. Zehntner, G. Lavoie, A. M. Mes- Masson, J. Nalbantoglu, and B. Massie. 2007. Improvement of antitumor activity by gene amplification with a replicating but nondisseminating adenovirus. Cancer Research 67:3387-3395.

Kane N.A., Z. Agharbaoui, A. O. Diallo, H. Adam, Y. Tominaga, F. Ouellet, and F. Sarhan. 2007. TaVRT2 represses transcription of the wheat vernalization gene TaVRNL Plant Journal 51 :670-680.

Kang G., T. Raman T, J. Green, C.I. Gallimore, and D. W. Brown. 2001. Distribution of rotavirus G and P types in north and south Indian children with acute diarrhoea in 1998- 99. Trans R Soc Trop Med Hyg. 95(5):491-2.

Claims

1. An isolated polypeptide comprising:

- a VP5 Rotavirus peptide lacking its associated C-terminus region; and

- a VP8 Rotavirus peptide.

2. The polypeptide according to claim 1 , wherein the VP5 Rotavirus peptide lacking its associated C-terminus region comprises an amino acid sequence substantially identical to the amino acid sequence ranging from amino acid 275 to 330 of SEQ ID NO 1 or 2.

3. The polypeptide according to claim 1 , wherein the VP5 Rotavirus peptide lacking its associated C-terminus region is encoded by a nucleic acid sequence substantially identical to the nucleic acid sequence ranging from nucleotide 823 to 990 of SEQ ID NO

1.

4. The polypeptide according to claim 1 , wherein the VP8 Rotavirus peptide comprises an amino acid sequence substantially identical to the amino acid sequence ranging from amino acid 1 to 274 of SEQ ID NO 1 or 2.

5. The polypeptide according to claim 1 , wherein the VP8 Rotavirus peptide is encoded by a nucleic acid sequence substantially identical to the nucleic acid sequence ranging from nucleotide 1 to 822 of SEQ ID NO 1.

6. The polypeptide according to claim 1 , having at least 95 % amino acid sequence identity to SEQ ID NO 3 to 6, 8 or 9.

7. The polypeptide according to claim 1 , consisting of the amino acid sequence of SEQ ID NO 3 to 6, 8 or 9.

8. The polypeptide according to claim 1 , being encoded by a nucleic acid sequence having at least 85 % nucleic acid sequence identity to SEQ ID NO 3, 5 or 8.

9. The polypeptide according to claim 1 , being encoded by a nucleic acid sequence consisting of nucleic acid sequence SEQ ID NO 3, 5 or 8.

10. An isolated polynucleotide comprising :

- a VP5 nucleic acid molecule encoding a VP5 Rotavirus peptide lacking its associated C-terminus region; and

- a VP8 nucleic acid molecule encoding a VP8 Rotavirus peptide.

11. The polynucleotide according to claim 10, wherein the VP5 nucleic acid molecule comprises a nucleic acid sequence substantially identical to the nucleic acid sequence ranging from nucleotide 823 to 990 of SEQ ID NO 3, 5 or 8.

12. The polynucleotide according to claim 10, wherein the VP8 nucleic acid molecule comprises a nucleic acid sequence substantially identical to the nucleic acid sequence ranging from nucleotide 1 to 822 of SEQ ID NO 1.

13. The polynucleotide according to claim 10, having at least 85% nucleic acid sequence identity to SEQ ID NO 3, 5 or 8.

14. The polynucleotide according to claim 10, consisting of the nucleic acid sequence of SEQ ID NO 3, 5 or 8.

15. An expression or cloning vector comprising a polynucleotide as defined in any one of claims 10 to 14.

16. A non-human host cell comprising a vector as defined in claim 15.

17. An immunogenic composition for inducing a specific immune response against a Rotavirus infection, comprising a polypeptide according to any one of claim 1 to 9, a polynucleotide according to any one of claims 10 to 14, a vector as defined in claim 15 or a non-human host cell as defined in claim 16, and an acceptable carrier or vehicle.

18. Use of a polypeptide of any one of claims 1 to 9, or of a polynucleotide according to any one of claims 10 to 14, or of a composition according to claim 15, for inducing a specific immune response against a rotavirus infection.

19. Use of a polypeptide of any one of claims 1 to 9, or of a polynucleotide according to any one of claims 10 to 14, or of a composition according to claim 15, for the treatment and/or prevention of a rotavirus infection.

20. A method for inducing a specific immune response against a rotavirus infection in a subject, comprising the step of administering of a polypeptide of any one of claims 1 to 9, or of a polynucleotide according to any one of claims 10 to 14, or of a composition according to claim 15, to said subject.

21. A method for treating and/or preventing a rotavirus infection, comprising the step of administering of a polypeptide of any one of claims 1 to 9, or of a polynucleotide according to any one of claims 10 to 14, or of a composition according to claim 15, to a subject in need thereof.