CA2698413A1 - Rotavirus vaccine - Google Patents

Abstract

In the present invention, there is provided novel antigenic recombinant VP4 1-336::VP7, VP4 1-336::VP7::FIjb, VP41-336::FIjb, and/or VP7::FIjb fusion protein, a vaccine prepared from said fusion protein and methods of immunization and boosting.

Description

ARCHAMBAULT, Denis et al., April t 2010 ROTAVIRUS VACCINE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is the first application filed for the present invention.
TECHNICAL FIELD

[0002] The present invention relates to novel rotavirus-derived fusion proteins-based rotavirus vaccines which stimulate neutralizing antibody to multiple serotypes of human rotavirus, and methods of treating humans against rotavirus illness caused by human rotaviruses of different serotypes and expanding the titers of pre-existing neutralizing antibodies and memory of the cells producing the antibodies which are formed following primary sensitization with the novel rotavirus-derived fusion proteins.

Background of the invention [0003] 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.

[0004] In developing countries, the impact of diarrhea) disease is staggering.
For Asia, Africa and Latin America, it has been estimated that there are between 3-5 billion cases of diarrhea each year and of those cases, about 5-10 million result in death.

[0005] Rotaviruses have been recognized as one of the most important causes of severe diarrhea in infants and young children since their discovery in 1973. It is estimated that rotavirus disease is responsible for about 600-700 thousands deaths annually.

[0006] 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 of from about 1 to about 3 days.

[0007] 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 ARCHAMBAULT, Denis et a1., April ,,2010 in young children, most adult rotavirus infections are mild or asymptomatic because such episodes represent re-infection generally as a result of contact with children known to be excreting rotavirus.

[0008] 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.

[0009] Rhesus rotavirus (RRV) vaccine strain MMU18006 (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.

[0010] Currently, except for the simple replenishment of metabolite solutes, the vaccines generally used consists in the administration of either Rotarix or Rota-Teq .
Rotarix is a vaccine against one strain [G1, P1 (8)] of rotaviruses. However, it is very expensive, may have harmful side effects and its use are limited to very young children under the age of 8 months. As for Rota-Teq , it is an oral vaccine against Type G1, G2, G3, G4 & P1 (8) (Human) rotavirus.
Again here, this vaccine is very expensive may have a harmful side effect and its use is limited to very young children under the age of 8 months.

[0011] In 1998, Rotashield was the first tetravalent HRV vaccine generated by combining HRV
and RRV strains. This vaccine was suspended and withdrawn 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.

[0012] 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 2C ) ARCHAMBAULT, Denis et al., April x,2010 administered at this age.

[0013] 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 synthetic particulate vaccines.

[0014] Consequently, It would be highly desirable to be provided with a new treatment against rotavirus through vaccination that overcome some or all of the shortcomings of the prior art vaccines.

BRIEF SUMMARY OF THE INVENTION

[0015] One aspect of the present invention is to provide a new vaccine and treatment to alleviate certain of the above-mentioned problems and shortcomings in the present state of the rotavirus art. Therefore, including a molecule with adjuvant properties in fusion with the rotaviral proteins is part of the present invention. In that regard flagellin, the structural component of flagellar filament in various locomotive bacteria including that of Salmonella tiphymurium (FIjb), is the ligand of Toll-like receptor 5 (TLR5) of host cells, including immune cells.
Flagellin therefore has a strong adjuvant activity to induce protective immunity.

[0016] In accordance with the present invention there are provided antigenic recombinant VP41_336 ::VP7, VP41_336::VP7::Fljb, VP41_336::FIjb, VP7::FIjb fusion proteins as set forth in SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4, respectively, for use in production of neutralizing antibodies against HRV (Fig. 1).

[0017] The is also provided in accordance with the present invention, a vaccine composition for providing immunological protection against rotavirus illness in humans, said vaccine comprising the fusion proteins VP41_336 ::VP7, VP41.336::VP7::Fljb, VP41_336::FIjb, VP7::FIjb for eliciting antibody against HRV, in admixture with a suitable pharmaceutical carrier. The vaccine composition may for example but without limitation be formulated for oral administration.

[0018] Still in accordance with the present invention, there is provided a vaccine composition for prevention and treatment of rotavirus-related disease comprising the VP41_336::VP7, VP41_ 336::VP7::Fljb, VP41_336::Fljb, and/or VP7::Fljb fusion proteins as set forth in SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively as an effective component.
The vaccine composition may for example be adapted for administration by a route but without limitation 3 0) ARCHAMBAULT, Denis et al., April 4 2010 selected from the group consisting of intramuscular administration, intranasal administration, oral administration, transdermal administration, and transmucosal (rectal) administration.

[0019] Still in accordance with the present invention, there is provided a method for delivering from adenovectors or any other viral vector types the said antigenic VP41.33fi::VP7, VP4,_336::VP7::FIjb, VP41-336::Fljb, and/or VP7::Fljb fusion proteins recombinant described for eliciting production of neutralizing antibodies against a rotavirus once in contact with the immune system of a patient, said method comprising the steps of:

a) Inserting into a viral viral, for instance, an adenovirus vector such as non replicative or replicative virus with, for instance, an inducible cumate-based promotor (Bourbeau D.
et al. 2007, Cancer Research 67:3387-3395) nucleic acid fragment encoding VP41_ 336::VP7, VP41.336::VP7::FIjb, VP41.336::Fljb, or VP7::Fljb fusion proteins as set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 for expression upon replication of the adenovirus; and an expression plasmid such as pcDNA3.0 (Invitrogen).

b) Inoculating a mammal with the viral vector for, instance the adenovirus or the pcDNA3.0 plasmid obtained in step a) such that neutralizing antibodies against a rotavirus are being produced by the immune system of the mammal in contact with the VP41-336::VP7, VP41-336::VP7::Fljb, VP41_336::Fljb, and/or VP7::Fljb fusion proteins produced, by the adenovirus.

[0020] The medicament may be used for boosting an immune response to a previously administered rotavirus vaccine.

[0021] Still, also in accordance with the present invention, there is provided for the use of any plant parts or plant crude extract to induce immune response against a rotavirus, wherein said plant parts or plant crude extract contain the antigenic recombinant VP41_336::VP7, VP41_ 336::VP7::Fljb, VP41.336::FIjb, or VP7::FIjb fusion proteins as described herein.

[0022] Still, also in accordance with the present invention, there is provided for the use of any recombinant fusion proteins produced ex vivo in bacteria, yeast or any other cell types to be used as sub-unit vaccines induce immune response against a rotavirus, wherein said plant parts or plant crude extract contain the antigenic recombinant VP41-336::VP7, VP41_336::VP7::Fljb, VP41_ ~ Lul ARCHAMBAULT, Denis et al., April d, 2010 336::FIjb, or VP7::FIjb fusion proteins as described herein.
DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0023] Fig. I illustrates the amino acid sequences of the VP41_336::VP7, VP41_336::VP7::Fljb, VP41_336::Fljb, or VP7::Fljb fusion proteins (Wa rotavirus strain), designated as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

[0024] Fig. 2 illustrates the nucleic acid sequences (codon usage optimized for expression in mammalian cells) designated SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID
NO:8, encoding the VP41_336::VP7, VP41_336::VP7::Fljb, VP41_336::Fljb, or VP7::Fljb fusion proteins.

[0025] Fig. 3 panel A shows the expression of VP41.336 protein in 293APS
infected with recombinant Adenovirus (AdVs) by indirect immunofluorecence assay, using a rabbit antiserum as primary antibodies (1:1000) and an Alexa-fluor-green-488 coupled anti-rabbit as secondary antibodies (1:1000). Cells were infected and fixed after 48h. Cells infected with AdV(GFP) were used as negative control. As GFP is expressed in the cytoplasmic compartment, it goes out of the cell after fixing in cold methanol. No residual fluorescence interferes with the secondary antibody green fluorescence.

[0026] Fig. 3 panel B shows the expression of VP7 protein in 293APS infected with recombinant Adenovirus (AdVs) by indirect immunofluorecence assay, using a rabbit antiserum as primary antibodies (1:1000) and an Alexa-fluor-green-488 coupled anti-rabbit (1:1000) as secondary antibodies. Cells were infected and fixed after 48h. Cells infected with AdV(GFP) were used as negative control. As GFP is expressed in the cytoplasmic compartment, it goes out of the cell after fixing in cold methanol. No residual fluorescence interferes with the secondary antibody green fluorescence.

[0027] Fig. 3 panel C shows the expression of FIjB opt protein in 293APS
infected with recombinant Adenovirus (AdVs) by indirect immunofluorecence assay, using a rabbit antiserum (1:1000) as primary antibodies and a Cy3 coupled anti-rabbit as secondary antibodies (1:1000). Cells were infected and fixed after 48h. Cells infected with AdV(GFP) were used as negative control. As GFP is expressed in the cytoplasmic compartment, it goes out of the cell after fixing in cold methanol. No residual fluorescence interferes with the secondary antibody red fluorescence.

[0028] Fig. 4 shows the expression of VP41_336 protein and/or FIjB protein by Western immunoblotting using a specific mouse monoclonal to the VP8 fraction (amino acids 1 to 240 of (sl ARCHAMBAULT, Denis et al., April 4, 2010 VP4) (1:10,000) (Panel A and B) and a rabbit hyperimmune antiserum specific to FIjB (1:50,000) (Panel C), of various recombinant rotaviral protein in 293APS mammalian cells transduced during 48h with recombinant AdVs. AdV(GFP)-infected 293APS cell protein extract used as a negative control. The molecular weight markers are indicated on the left side of the Figure.

[0029] Fig. 5 shows the experimental design of the mouse immunizations. Eight six-week female BALB/c mice per group were immunised intra-muscularly with 108 TCID50 recombinant AdV vectors on Day 0. On day 14 and day 35, they received 100 pg of recombinant plasmid expressing the same rotaviral protein than delivered with AdV vectors. Blood (100 pI per mouse) was collected on day 0, 14 and 35 and Sera of each group were pooled. Mice were sacrificed on day 40, blood and spleen were collected to characterize humoral and cellular immune responses.

Group 1 (Eight mice) received 108TCID50 of adenovirus vector (GFP) on day 0.
They received 100 pg of plasmid pCDNA3.0- GFP on day 14 and 35.
Group 2 (Eight mice) received 108TCID50 of adenovirus vector (VP4) on day 0.
They received 100 pg of plasmid pCDNA3.0- VP4 on day 14 and 35.
Group 3 (Eight mice) received 108TCID50 of adenovirus vector (VP41.336) on day 0. They received 100 pg of plasmid pCDNA3.0- VP8 on day 14 and 35.
Group 4 (Eight mice) received 108TCID50 of adenovirus vector (VP7) on day 0.
They received 100 pg of plasmid pCDNA3.0- VP7 on day 14 and 35.
Group 5 (Eight mice) received 108TCID50 of adenovirus vector (VP41-336::FIjB) on day 0. They received 100 pg of plasmid pCDNA3.0- VP8::FIjB on day 14 and 35.
Group 6 (Eight mice) received 108TCID50 of adenovirus vector (VP7::FIjB) on day 0. They received 100 pg of plasmid pCDNA3.0- VP7::FIjB on day 14 and 35.
Group 7 (Eight mice) received 108TCID50 of adenovirus vector (VP41-336::VP7::FIjB) on day 0. They received 100 pg of plasmid pCDNA3.0- VP8::VP7 on day 14 and 35.
Group 8 (Eight mice) received 108TCID50 of adenovirus vector (VP41.336::VP7::
FIjB) on day 0.
They received 100 pg of plasmid pCDNA3.0- VP8::VP7 on day 14 and 35.

[0030] Fig. 6 shows splenocyte lymphoproliferation from eight groups of eight mice immunized with 108 TCID50 of AdV vectors on day 0 and on day 14 and 35 with recombinant plasmid. Briefly, spleen cells were suspended in RPMI cell culture medium supplemented with penicillin (100 U/ml), streptomycin (100 ug/ml), FBS 10%, 0.05 mM (3-mercaptoethanol and 10 mM HEPES, seeded in quadruplicates in a 96-well plate at a final concentration of 4.0 x 105 cells per well. The cells were stimulated with 5 pg/ml of recombinant VP41.336 protein, or VP7 protein, or FIjB, or with CsCI
prurified rotavirus (MOI=10). The cells were incubated at 37 C for a total of 72h. After 48 h, 0,5 pCi of radioactive H3-thymidine was added to each well. After 24h, the cells were harvested and 6 ~(o) ARCHAMBAULT, Denis et al., April 1, 2010 collected on a silica membrane for measurement (CPM count) of radioactive H3-thymidine incorporation in cell DNA. Results are expressed as stimulation index which is the ratio of CPM
count of cells stimulated with an antigen to CPM count of cells without antigen. The bar represents the mean value of the stimulation index with standard error.

[0031] Fig. 7 shows the kinetics of anti-rotavirus IgG antibody production in groups of mice, as determined by an indirect immunofluorescence assay (IFA). Sera were collected on day 0, 14, 35, and 40 and were pooled for each group for analysis. IFA titers are expressed as the log2 of the reciprocal of the highest serum dilution giving positive fluorescent signal.
Ma104 cells were plated into each well of a 96-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 1h at 37 C in PBS-5% bovine serum albumin. The test was performed using mouse sera and Cy3 coupled anti-mouse IgG (1:1000) secondary antibodies, each incubated for 2h at 37 C. After the antibody treatments, the cells were then visualized under fluorescence microscopy.

[0032] Fig. 8 shows kinetics of neutralizing rotavirus antibody production in groups of mice, as determined by focus fluorescent unit assay (FFU). Sera were collected on day 0, 14, 35, and 40 and were pooled for each group for analysis. FFU titers are expressed as the log2 of the reciprocal of the highest serum dilution giving a 50% reduction in focus fluorescent units compared to negative control. Sera were inactivated at 56 C 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 pg/ ml of porcine pancreatic trypsin, followed by an incubation at 37 C for 1 h.
The serum-virus mixture [25 pl per well containing 100 tissue culture infective dose (TCID)50] was then used to inoculate duplicate cultures of MA104 cells in 96-well tissue culture plates. After an in18 h. MA104 cells were fixed in cold methanol for 30 min, blocked for 1h at 37 C in PBS-5%
bovine serum albumin. The test was performed using rabbit antisera specific to VP41-336 and V137 and alexa-green-488 coupled anti-mouse IgG (1:1000) secondary antibodies, each incubated for 2h at 37 C. After the antibody treatments, the cells were then visualized under fluorescence microscopy. Fluorescent cells were defined as focus fluorescent unit.

[0033] Fig. 9 shows a synthesis of Figures 6, 7 and 8. Kinetics of IFA
specific rotavirus IgG titers, kinetics of rotavirus neutralizing antibody titers and stimulation index are presenting for each group of mice immunized according to figure 5.

[0034] Fig. 10 shows the experimental design of the mouse immunizations. Eight six-week female BALB/c mice per group were immunised intra-rectally with 108 TCID50 recombinant AdV vectors on Day 0 and day 21 s. Blood (100 pl per mouse) was collected on day 0, 21, 28, 35 and 42. Sera of CrA

ARCHAMBAULT, Denis et al., April ~ 2010 each group were pooled. Mice were sacrificed on day 42, blood and spleen were collected to characterize humoral and cellular immune responses.
Group 1 (Eight mice) received 108TCID50 of adenovirus vector (GFP) on day 0 and 14.
Group 2 (Eight mice) received 108TCID50 of adenovirus vector (VP41.336) on day 0 andl4.
Group 3 (Eight mice) received 108TCID50 of adenovirus vector (VP41.336::FIjB) on day 0 and 14.
[0035] Fig. 11 shows the kinetics of anti-rotavirus IgG antibody production in groups of mice, as determined by an indirect immunofluorescence assay (IFA). Panel A: Sera were collected on day 0, 21, 28, 35 and 42 and were pooled for each group for analysis by IFA. Panel B:
sera of individual mouse were tested on day 42. IFA titers are expressed as the log2 of the reciprocal of the highest serum dilution giving positive fluorescent signal. Mal04 cells were plated into each well of a 96-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 37 C in PBS-5%
bovine serum albumin. The test was performed using mouse sera and Cy3 coupled anti-mouse IgG
(1:1000) secondary antibodies, each incubated for 2h at 37 C. After the antibody treatments, the cells were then visualized under fluorescence microscopy.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0036] In accordance with the present invention, the rotavirus-derived fusion proteins of the present invention is believed to have the unique ability to stimulate neutralizing antibody production, which neutralizing antibodies intercepting pathogens, neutralizing human rotaviruses, so that effective protection against subsequent rotavirus illness is provided.

[0037] Thus, also in accordance with the present invention, there is provided new adenovector-based vaccines expressing the VP41_336::VP7, VP41_336::VP7::Fljb, VP41-336::FIjb, or VP7::Fljb fusion proteins which when administered will prime and stimulate neutralizing rotavirus-specific production.

[0038] The present invention also contemplates the discovery of a novel method for boosting pre-existing neutralizing antibody titers in humans following primary vaccination.
This is accomplished in. accordance with the instant invention by revaccinating (boosting) humans with the adenovector or plasmid-derived vaccines described herein, or with any other vectors including plants. In one aspect of the present invention, there is provided a combinatorial vaccination program whereby the first (or prime) vaccine consists of an inoculation of recombinant adenovectors (or any other appropriate vectors) by any administration route that transfect the target transgene in the host epithelial cells to induce the production of the fusion protein of interest to stimulate the innate and adaptive immunity of the same host followed by a boost vaccine with the same fusion proteins either ARCHAMBAULT, Denis et al., April `, 2010 inoculated as sub-unit recombinant proteins expressed in vitro prior to inoculation or delivered in the vaccinated host by any vector type (including, although not exclusive, viral, plasmid or plants)..

[0039] In forming the novel vaccines of the instant invention, depending upon the type of vaccine formed, e.g., oral, nasal, rectal, parenteral or any other route, the pharmaceutical vehicle may be sterile water, normal saline, or the like, in combination with a suitable adjuvant.
The vaccines may also be incorporated in any type of delivery systems including, although not exclusive, nanoparticles, suppository or any other types of physical delivery systems.

[0040] In vivo. HRV infection is restricted to the mature tip cells of the small intestine. The infectivity of HRV is dependent on the trypsin-like treatment of the viral particle and results in the specific cleavage of outer capsid protein VP4 into polypeptides VP8 and VP5. In the gastrointestinal tract, the conformation of the highly flexible VP4 spike protein is altered by proteolytic cleavage and high pH. This allows early virus-cell interactions, cell binding, entry, hemagglutination, and neutralization.

[0041] Sequence analysis of VP4 in neutralization escape mutants suggests that the strain-specific neutralization epitopes are localized in the VP8 subunit. In contrast, the cross-reactive neutralization domains appear to be localized in the VP5 subunit. The rotavirus VP7 protein also contains important neutralizing epitopes.

[0042] To develop vaccines against HRV, the induction of a mucosal immune response is essential to block the virus in the gut prior to epithelial cell infection.
Following oral immunization through an appropriate route, multiple immune effector mechanisms will contribute to the protection at mucosal surfaces. The administered, immunogens must trigger the priming and proliferation of specific mucosal plasma cells that produce neutralizing antibody to intercept pathogens, and thus prevent mucosal invasion. The success of this approach may require the use of a strong mucosal adjuvant and an efficient vaccine inoculation vector, such as an adenovector. As an adjuvant, the Fljb described above, was selected. Fljb is an agonist of Toll-like receptor 5 which is expressed at the surface of the gut epithelial cells and immune cells for instance macrophages, dendritic cells and B cells.

[0043] Vector technology has the potential to overcome many of the problems associated with the Jennerian approach (the administration of live attenuated vaccine) and to induce protection against pathogens that invade mucosal surfaces, such as human rotavirus (HRV).
With this hypothesis in mind, the inventors selected the HRV immunogens VP41_336 , and VP7 proteins to ARCHAMBAULT, Denis et al., April 2010 be expressed alone or, in the present invention, as fusion proteins with or without Fljb (VP4,_ 336==VP7, VP41_336::VP7::Fljb, VP4,_336::Fljb, or VP7::Fljb) in any types of cells (prokayotic and eukaryotic). This is due to their efficacy to elicit neutralizing and protective antibodies blocking the HRV viruses in the gut prior to epithelial cell infection. To ensure optimal expression in plants, the DNA sequence was optimized for codon usage and synthesized by the GeneArt company (Regensburg, Germany) as per the inventor's specifications. The gene was then cloned in the appropriate adenovector (in which there is a CMV5 cumate-inducible promoter) or pcDNA3.0 vector (under the control of a CMV5 promoter) according to standard cloning and production procedures.

[0044] To the inventor's knowledge, this is the first report that shows the expression of these rotaviral fusion proteins in mammalian cells, and in plants.

[0045] The same strategy was used to construct a vector for transfection in plant cells. The rotaviral sequences of interest were also optimized for plant cell expression (GeneArt company), and similar expression results were also obtained.

[0046] The purpose of developing optimized VP41.336::VP7, VP41.336::VP7::Fljb, VP41.336::Fljb, or VP7::Fljb fusion proteins was to incorporate the encoding sequences into an eukaryotic vector, such as an adenovirus, such as AdV type 5, which is an inducible replicative system. However, with this system, the adenovirus cannot be disseminated in nature due to a defect in the packaging, such that the immunized host does not produce the virus.

[0047] Primary vaccination may be administered at 6 months to 2 years or more after birth. Of course, it should be appreciated that the longer vaccination is delayed, the more vulnerable infants and young children become to severe rotavirus disease. It is believed that the vaccines of the instant invention have the ability to induce neutralizing antibody to human rotavirus, which should be sufficient to confer protection against severe disease caused by infecting human rotavirus strains of all serotypes subsequent to vaccination.

[0048] Thus, it is believed that the instant invention, in addition to allow for a prime vaccination, now makes it possible to consistently boost titers and expand the memory of the cells that express the neutralizing antibody developed after primary immunization by revaccination. In other words, the titers and memory of the cells that produce the pre-existing neutralizing antibody induced by primary human rotavirus vaccination can be boosted or expanded to afford protection against subsequent illnesses caused by human rotavirus by practicing the methods 'of the instant cj ARCHAMBAULT, Denis et al., April J, 2010 invention.

[0049] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims. For example, one skilled in the art having the sequence in hand will be able to use many other vectors in addition to those described in the present application.

Fig. 1: Amino acid sequences of the rotavirus (Wa strain) fusion proteins SEQ ID NO:1 : VP4,_336::VP7 MASLIYRQLL TNSYSVDLHD EIEQIGSEKT QNVTINPSPF AQTRYAPVNW GHGEINDSTT VEPMLDGPYQ
....I ...l ....1....I ....I ...I ....I ...I .... I.... I ....I ...I ....I ...1 PTTFTPPNDY WILINSNTNG VVYESTNNSD FWTAVVAIEP HVNPVDRQYT IFGESKQFNV SNDSNKWKFL

....I ...l ....1....I ....I ...I ....I ...I ....I ...I ....I ...I ....I ...1 EMFRSSSQNE FYNRRTLTSD TRFVGILKYG GRVWTFHGET PRATTDSSST ANLNNISITI HSEFYIIPRS

....1 ...l 111.1 ...I ....I ...I ....I....1 ....I ...I ....I ....I ....I ...1 QESKCNEYIN NGLPPIQNTR NVVPLPLSSR SIQYKRAQVN EDIIVSKTSL WKEMQYNRDI IIRFKFGNSI
1....I ....I ....I ....l.... I ....I ...I ....I ...I .... I.... I ....I....1 VKMGGLGYKW SEISYKAANY QYNYLRDGEQ VTAHTTCSVN GVNNFSYNGG GSATMYGIEY TTILIFLISI

....I ...l ....I ...I .... I... 1 ... I....1 ....I .... I .... I.... I ....I
...1 ILLNYILKSV TRIMDYIIYR FLLITVALFA LTRAQNYGLN LPITGSMDAV YTNSTQEEVF LTSTLCLYYP

TEASTQINDG DWKDSLSQMF LTKGWPTGSV YFKEYSNIVD FSVDPQLYCD YNLVLMKYDQ SLKLDMSELA
....1 ...1 ....1....I .... I.... I ....1 ...I ....I ...I ...01....I ....I....1 DLILNEWLCN PMDVTLYYYQ QSGESNKWIS MGSSCTVKVC PLNTQTLGIG CQTTNVDSFE MIAENEKLAI

VDVVDGINHK INLTTTTCTI RNCKKLGPRE NVAVIQVGGS NVLDITADPT TNPQTERMMR VNWKKWWQVF
.... I.... I ....I ...I ....1....I ....I ..

YTIVDYINQI VQVMSKRSRS LNSAAFYYRV **

SEQ ID NO:2 : VP41_336::VP7::Fljb ....1 ...1 ....1....I ....I ...I ....I ...I .... I.... I ....I ...I .... I....
I

MASLIYRQLL TNSYSVDLHD EIEQIGSEKT QNVTINPSPF AQTRYAPVNW GHGEINDSTT VEPMLDGPYQ

PTTFTPPNDY WILINSNTNG VVYESTNNSD FWTAVVAIEP HVNPVDRQYT IFGESKQFNV SNDSNKWKFL

EMFRSSSQNE FYNRRTLTSD TRFVGILKYG GRVWTFHGET PRATTDSSST ANLNNISITI HSEFYIIPRS

QESKCNEYIN NGLPPIQNTR NVVPLPLSSR SIQYKRAQVN EDIIVSKTSL WKEMQYNRDI IIRFKFGNSI

....I ...l ....1....I .... I.... I ....I ...I ....I ...I .... I.... I ....I
...1 VKMGGLGYKW SEISYKAANY QYNYLRDGEQ VTAHTTCSVN GVNNFSYNGG GSATMYGIEY TTILIFLISI
....1 ...1 ....1....I .... I.... I ....I ...I ....I ...I .... I.... I
....I....1 ILLNYILKSV TRIMDYIIYR FLLITVALFA LTRAQNYGLN LPITGSMDAV YTNSTQEEVF LTSTLCLYYP
....I ...l 11.11 ...I .... I.... I ... I.... I ....I ...I .... I.... I
....I....1 TEASTQINDG DWKDSLSQMF LTKGWPTGSV YFKEYSNIVD FSVDPQLYCD YNLVLMKYDQ SLKLDMSELA

DLILNEWLCN PMDVTLYYYQ QSGESNKWIS MGSSCTVKVC PLNTQTLGIG CQTTNVDSFE MIAENEKLAI

....I ...I ....1....l ....1 ...1 ....I .... I .... I.... I .... I.... I ....I
...1 VDWDGINHK INLTTTTCTI RNCKKLGPRE NVAVIQVGGS NVLDITADPT TNPQTERMMR VNWKKWWQVF
....I .. I ....1....1 ....1 ...1 ...1 ...I .... I.... I .... I.... I ... .. 1 YTIVDYINQI VQVMSKRSRS LNSAAFYYRV RSATMAQVIN TNSLSLLTQN NLNKSQSALG TAIERLSSGL
....1 ...i .. I....I .... I.... I ....I ...I ....I ...I .... I.... I ....I
...1 7.10 720 730 740 750 760 770 RINSAKDDAA GQAIANRFTA NIKGLTQASR NANDGISIAQ TTEGALNEIN NNLQRVRELA VQSANSTNSQ
....I ...l ...I.... I .... I.... I ....I ...I .... I.... I .... I.... I
....I....1 SDLDSIQAEI TQRLNEIDRV SGQTQFNGVK VLAQDNTLTI QVGANDGETI DIDLKQINSQ TLGLDSLNVQ

KAYDVKDTAV TTKAYANNGT TLDVSGLDDA AIKAATGGTN GTASVTGGAV KFDADNNKYF VTIGGFTGAD

AAKNGDYEVN VATDGTVTLA AGATKTTMPA GATTKTEVQE LKDTPAVVSA DAKNALIAGG VDATDANGAE

....I ...I ....1....1 ....I....l ....I .... 1 .Ø1 ...I ....I ....I ....I
...1 LVKMSYTDKN GKTIEGGYAL KAGDKYYAAD YDEATGAIKA KTTSYTAADG TTKTAANQLG GVDGKTEVVT
....I ...1 ....I ...I .... I.... I ....I ...I .... I.... I .... I.... I ....I
....I

IDGKTYNASK AAGHDFKAQP ELAEAAAKTT ENPLQKIDAA LAQVDALRSD LGAVQNRFNS AITNLGNTVN

NLSEARSRIE DSDYATEVSN MSRAQILQQA GTSVLAQANQ VPQNVLSLLR **.........

SEQ ID NO:3 : VP41_336::Fljb ....1 ...1 ... 1....I ....I ...I ....I ...I ....I ...I ....I ...I .... I.... I

MASLIYRQLL TNSYSVDLHD EIEQIGSEKT QNVTINPSPF AQTRYAPVNW GHGEINDSTT VEPMLDGPYQ
....1 ...I .... I.... I ....I ...I ....I ...I ....1....I ....I ...I ....I ...1 PTTFTPPNDY WILINSNTNG VVYESTNNSD FWTAVVAIEP HVNPVDRQYT IFGESKQFNV SNDSNKWKFL

....1 ...1 ....1....I .... I.... I ....I ...I ....I ...I ....I ...I ....1 ...1 EMFRSSSQNE FYNRRTLTSD TRFVGILKYG GRVWTFHGET PRATTDSSST ANLNNISITI HSEFYIIPRS

....1 .. 1 11.11 ...I ....1....l ....I .. I .... I.... I ....I ...I ....I ...I

QESKCNEYIN NGLPPIQNTR NVVPLPLSSR SIQYKRAQVN EDIIVSKTSL WKEMQYNRDI IIRFKFGNSI
....I ...l ...I ...I .... I.... I ....I ...I ....I ...I .... I.... I ....I
...1 VKMGGLGYKW SEISYKAANY QYNYLRDGEQ VTAHTTCSVN GVNNFSYNGG GSATMAQVIN TNSLSLLTQN

... ...I .... I.... I ....I ...I ....I ...I ....I ...I ....I ...I ....I ...1 NLNKSQSALG TAIERLSSGL RINSAKDDAA GQAIANRFTA NIKGLTQASR NANDGISIAQ TTEGALNEIN
.... I.... I .... I.... I ....I ...I ....I ...I .... I.... I ....I ...I
....I....1 NNLQRVRELA VQSANSTNSQ SDLDSIQAEI TQRLNEIDRV SGQTQFNGVK VLAQDNTLTI QVGANDGETI

DIDLKQINSQ TLGLDSLNVQ KAYDVKDTAV TTKAYANNGT TLDVSGLDDA AIKAATGGTN GTASVTGGAV
.... I.... I ....I ...I ....I ...I ... ...I ....I ...I .... I.... I ....I ...1 KFDADNNKYF VTIGGFTGAD AAKNGDYEVN VATDGTVTLA AGATKTTMPA GATTKTEVQE LKDTPAVVSA

.... I.... I .... I.... I ....I ...I ....I ...I ....I ...I ....I ...I ....I
...1 DAKNALIAGG VDATDANGAE LVKMSYTDKN GKTIEGGYAL KAGDKYYAAD YDEATGAIKA KTTSYTAADG
... ...I ....I ...I .... I.... I ....I ...I .I ...I .... I.... I ....I ...1 TTKTAANQLG GVDGKTEVVT IDGKTYNASK AAGHDFKAQP ELAEAAAKTT ENPLQKIDAA LAQVDALRSD
.... I.... I .... I.... I ....I ...I ....I ...I .... I.... I ....I ...I ....I
...1 LGAVQNRFNS AITNLGNTVN NLSEARSRIE DSDYATEVSN MSRAQILQQA GTSVLAQANQ VPQNVLSLLR

SEQ ID NO:4 : VP7::Fljb MYGIEYTTIL IFLISIILLN YILKSVTRIM DYIIYRFLLI TVALFALTRA QNYGLNLPIT GSMDAVYTNS
....1 ...1 ....1 ...I ....I ...I ....I ...I ....I ...I ....I ...I .I ...1 TQEEVFLTST LCLYYPTEAS TQINDGDWKD SLSQMFLTKG WPTGSVYFKE YSNIVDFSVD PQLYCDYNLV

LMKYDQSLKL DMSELADLIL NEWLCNPMDV TLYYYQQSGE SNKWISMGSS CTVKVCPLNT QTLGIGCQTT

NVDSFEMIAE NEKLAIVDVV DGINHKINLT TTTCTIRNCK KLGPRENVAV IQVGGSNVLD ITADPTTNPQ

TERMMRVNWK KWWQVFYTIV DYINQIVQVM SKRSRSLNSA AFYYRVRSAT MAQVINTNSL SLLTQNNLNK

SQSALGTAIE RLSSGLRINS AKDDAAGQAI ANRFTANIKG LTQASRNAND GISIAQTTEG ALNEINNNLQ

....1....1 1.1.1 ...I .... I.... I ... ...I ....I ...I .... I.... I ....I ...1 RVRELAVQSA NSTNSQSDLD SIQAEITQRL NEIDRVSGQT QFNGVKVLAQ DNTLTIQVGA NDGETIDIDL

....1....i ....I ...I .... I.... I ....I ...I ....I ...I ....I....1 .Ø1 ...1 KQINSQTLGL DSLNVQKAYD VKDTAVTTKA YANNGTTLDV SGLDDAAIKA ATGGTNGTAS VTGGAVKFDA

DNNKYFVTIG GFTGADAAKN GDYEVNVATD GTVTLAAGAT KTTMPAGATT KTEVQELKDT PAVVSADAKN

ALIAGGVDAT DANGAELVKM SYTDKNGKTI EGGYALKAGD KYYAADYDEA TGAIKAKTTS YTAADGTTKT

.... I.... I ....I ...I .I ... I ....I ...I ....I ...I .... I.... I ....I....1 AANQLGGVDG KTEVVTIDGK TYNASKAAGH DFKAQPELAE AAAKTTENPL QKIDAALAQV DALRSDLGAV
....1 ...1 1.111 ...I .... I.... I ....I ...I ....I ...I .... I.... I
....I....1 QNRFNSAITN LGNTVNNLSE ARSRIEDSDY ATEVSNMSRA QILQQAGTSV LAQANQVPQN VLSLLR**

Fig. 2: Nucleic acid sequences (codon usage optimized for expression in mammalian cells) encoding the rotavirus (Wa strain) fusion proteins SEQ ID NO:5 : VP41_336::VP7 ....I ...I ... ...I ....I .. ... I ... ... I.... I ... ..

.AGATCTGCCA CCATGGCCAG CCTGATCTAC CGGCAGCTGC TGACCAACAG CTACAGCGTG
... I.... ....I ...I .... I.... I ....I ...I ....I .. I ....I....1 GACCTGCACG ACGAGATCGA GCAGATCGGC AGCGAGAAAA CCCAGAACGT GACCATCAAC
....I ...I ... ...I ....I ...I ... I.... ... I.... I ... I....

CCCAGCCCCT TCGCCCAGAC CAGATACGCC CCCGTGAACT GGGGCCACGG CGAGATCAAC
... I.... I ... ...I ... ...I ... I.... ... I ....I ... I....

GACAGCACCA CCGTGGAGCC CATGCTGGAC GGCCCCTACC AGCCCACCAC CTTCACCCCC
... I ... ....I .. ... I.... I .... I.... I .... I.... I ....I....1 CCCAACGACT ACTGGATTCT GATCAACAGC AACACCAACG GCGTGGTGTA CGAGAGCACC

.... . .. . .... . .. . ....I . .. . .... . .. . .... . ...I ..:. I....

AACAACAGCG ATTTCTGGAC CGCCGTGGTG GCCATCGAGC CCCACGTGAA CCCCGTGGAC

CGGCAGTACA CCATCTTCGG CGAGAGCAAG CAGTTCAACG TGAGCAACGA CTCCAACAAG

TGGAAGTTCC TGGAAATGTT CAGAAGCAGC AGCCAGAACG AGTTCTACAA CCGGCGGACC
.... I.... ... ...I .... I.... I ....I ...I ....I .. I ... ..

CTGACCAGCG ACACCCGGTT CGTGGGCATC CTGAAGTACG GCGGCAGAGT GTGGACCTTT
... ...I ....I ...I .... I.... ... I.... ....I ...I ... ..

CACGGCGAGA CCCCCAGGGC CACCACCGAC AGCAGCAGCA CCGCCAACCT GAACAACATC

AGCATCACCA TCCACAGCGA GTTTTACATC ATCCCCCGGT CCCAGAAAAG CAAATGCAAC

GAGTACATCA ACAACGGCCT GCCCCCCATC CAGAACACCC GGAACGTGGT GCCCCTGCCC
... I.... I ....I ...I ....I .. .... I.... I ... ...I ... I....

CTGAGCAGCA GAAGCATCCA GTACAAGAGA GCCCAGGTCA ACGAGGACAT CATCGTGAGC
.... I.... I ....I .. ....I .. I ....I ...I ... I.... I ... I....

GGCAACAGCA TCGTGAAGAT GGGCGGCCTG GGCTACAAGT GGAGCGAGAT CAGCTACAAG
... I.... ....I .. ....I .. ... I.... ... I.... I ... I....

GCCGCCAACT ACCAGTACAA CTACCTGCGG GACGGCGAGC AGGTGACCGC CCACACCACC
... I.... ... .. ....I .. ... I.... ... I.... I ... I....

TGCAGCGTGA ACGGCGTGAA CAACTTCAGC TACAACGGCG GCGGATCTGC CACCATGTAC

GGCATCGAGT ACACCACCAT CCTGATCTTT CTGATCAGCA TCATCCTGCT GAACTACATC

CTGAAGAGCG TGACCCGGAT CATGGACTAC ATCATCTACC GGTTTCTGCT GATCACCGTG
.... I.... .... I.... ....I .. ... I.... ... I.... I ... ..

GCCCTGTTCG CCCTGACCAG AGCCCAGAAC TACGGCCTGA ACCTGCCCAT CACCGGCAGC
... ...I ... I.... I ....I .. ... I.... ... .. ....I ...1 ATGGACGCCG TGTACACCAA CAGCACCCAG GAAGAAGTCT TTCTGACCAG CACCCTGTGC

CTGTACTACC CCACCGAGGC CAGCACCCAG ATCAACGACG GCGACTGGAA GGACAGCCTG
... .. .... I.... I ... I.... I ....I ...I .... I.... I ....I....1 AGCCAGATGT TCCTGACCAA GGGCTGGCCC ACCGGCAGCG TGTACTTCAA AGAATACAGC
... .. .... I.... I ... I.... I ....I ...I ....1 ... ....I ...1 AACATCGTCG ACTTCAGCGT GGACCCCCAG CTGTACTGCG ACTACAACCT GGTGCTGATG

AAGTACGACC AGAGCCTGAA GCTGGACATG AGCGAGCTGG CCGACCTGAT CCTGAACGAG
... ...I ....I ...I .... I.... I ... I.... ... .. ... ...1 TGGCTGTGCA ACCCCATGGA CGTGACCCTG TACTACTACC AGCAGAGCGG CGAGAGCAAC
... ...1 .... I.... I ... I.... I ....I ...I .... I.... .... I....

AAGTGGATCA GCATGGGCAG CAGCTGCACC GTGAAAGTGT GCCCCCTGAA CACCCAGACC
... .. ... I.... ... I ....I .... I.... ... ...I ... ...1 CTGGGCATCG GCTGCCAGAC CACCAACGTG GACAGCTTCG AGATGATCGC CGAGAACGAG
... I.... ... I.... I ....I ...I ... I.... .... I.... I ....I....1 AAGCTGGCCA TCGTGGACGT GGTGGACGGC ATCAACCACA AGATCAACCT GACCACCACC

ACCTGCACCA TCCGGAACTG CAAGAAGCTC GGCCCTCGGG AGAACGTGGC CGTGATCCAG
... I.... I ... ...I .... I.... I ... I.... ....I .. ....I ..

GTGGGCGGCA GCAACGTGCT GGACATCACC GCCGACCCCA CCACCAACCC CCAGACCGAG
... .. ... ...I ... .. ....I ...I ....I .. ... I....

CGGATGATGC GGGTGAACTG GAAGAAATGG TGGCAGGTGT TCTACACCAT CGTGGATTAC

.... I.... .... I.... I .... I.... ... .. ... I.... I .... I.... I

ATCAACCAGA TCGTGCAGGT GATGAGCAAG CGGAGCAGAA GCCTGAACAG CGCCGCCTTC

TACTACAGAG TGTGATGAAG ATCTGGGCCC GAGCTC
SEQ ID NO:6 : VP4,.336::VP7::Fljb AGATCTGCCA CCATGGCCAG CCTGATCTAC CGGCAGCTGC TGACCAACAG CTACAGCGTG

GACCTGCACG ACGAGATCGA GCAGATCGGC AGCGAGAAAA CCCAGAACGT GACCATCAAC

CCCAGCCCCT TCGCCCAGAC CAGATACGCC CCCGTGAACT GGGGCCACGG CGAGATCAAC

. . . . . . . . . . . . . .

GACAGCACCA CCGTGGAGCC CATGCTGGAC GGCCCCTACC AGCCCACCAC CTTCACCCCC

CCCAACGACT ACTGGATTCT GATCAACAGC AACACCAACG GCGTGGTGTA CGAGAGCACC
... I.... ....I ...I .... I.... ... ...I .... I.... .... I....

AACACCAGCG ATTTCTGGAC CGCCGTGGTG GCCATCGAGC CCCACGTGAA CCCCGTGGAC
... I.... ... .. I ....I ... .. .... I.... .... I....

CGGCAGTACA CCATCTTCGG CGAGAGCAAG CAGTTCAACG TGAGCAACGA CTCCAACAAG

TGGAAGTTCC TGGAAATGTT CAGAAGCAGC AGCCAGAACG AGTTCTACAA CCGGCGGACC

CTGACCAGCG ACACCCGGTT CGTGGGCATC CTGAAGTACG GCGGCAGAGT GTGGACCTTT

CACGGCGAGA CCCCCAGGGC CACCACCGAC AGCAGCAGCA CCGCCAACCT GAACAACATC
.... I.... ... .. ... I.... .... I.... I ... I.... I .... I....

AGCATCACCA TCCACAGCGA GTTTTACATC ATCCCCCGGT CCCAGGAAAG CAAATGCAAC

GAGTACATCA ACAACGGCCT GCCCCCCATC CAGAACACCC GGAACGTGGT GCCCCTGCCC
....I ...I .... I.... I .... I.... I ....I .. ... .. ....I ...1 CTGAGCAGCA GAAGCATCCA GTACAAGAGA GCCCAGGTCA ACGAGGACAT CATCGTGAGC
... .. ....I .. ....I .. .... I.... ... I.... I ... I....

AAGACCAGCC TGTGGAAAGA GATGCAGTAC AACCGGGACA TCATCATCCG GTTCAAGTTC
... I.... I ... I.... ... I.... I ....I ...I ....I ... ....I....1 GGCAACAGCA TCGTGAAGAT GGGCGGCCTG GGCTACAAGT GGAGCGAGAT CAGCTACAAG

... .. ....I .. ....I .... ... I.... ... ...I ... I....

GCCGCCAACT ACCAGTACAA CTACCTGCGG GACGGCGAGC AGGTGACCGC CCACACCACC
... ...I .... I.... I .... I.... I ....I ...I ....I .. ....I ...1 TGCAGCGTGA ACGGCGTGAA CAACTTCAGC TACAACGGCG GCGGATCTGC CACCATGTAC
... ...I .... I.... I ....I .. ... I.... ... ...I ... I....

GGCATCGAGT ACACCACCAT CCTGATCTTT CTGATCAGCA TCATCCTGCT GAACTACATC
... .. ... .. ... I.... .... I.... I .... I.... I ....I ...1 CTGAAGAGCG TGACCCGGAT CATGGACTAC ATCATCTACC GGTTTCTGCT GATCACCGTG

GCCCTGTTCG CCCTGACCAG AGCCCAGAAC TACGGCCTGA ACCTGCCCAT CACCGGCAGC

... I.... ....I .. ... .. .... I.... ... I.... I ... I....

ATGGACGCCG TGTACACCAA CAGCACCCAG GAAGAAGTCT TTCTGACCAG CACCCTGTGC
... ...I ....I ...I ....I .. ... ...I .... I.... ....I....1 CTGTACTACC CCACCGAGGC CAGCACCCAG ATCAACGACG GCGACTGGAA GGACAGCCTG
....I ...I ... I.... ... I.... ... I.... ....I .. ....I ...1 AGCCAGATGT TCCTGACCAA GGGCTGGCCC ACCGGCAGCG TGTACTTCAA AGAATACAGC

AACATCGTCG ACTTCAGCGT GGACCCCCAG CTGTACTGCG ACTACAACCT GGTGCTGATG

AAGTACGACC AGAGCCTGAA GCTGGACATG AGCGAGCTGG CCGACCTGAT CCTGAACGAG
... I.... I ... I.... ... .. ... I.... ... ...11 ... 1...

TGGCTGTGCA ACCCCATGGA CGTGACCCTG TACTACTACC AGCAGAGCGG CGAGAGCAAC
.... I.... I .... I.... I .... I.... I ....I ...I .... I.... ....1....1 AAGTGGATCA GCATGGGCAG CAGCTGCACC GTGAAAGTGT GCCCCCTGAA CACCCAGACC
... .. ... ...I ....I ...I ... .. ... .. ....I ...1 CTGGGCATCG GCTGCCAGAC CACCAACGTG GACAGCTTCG AGATGATCGC CGAGAACGAG
....I .. ....I .. ....I .. ... I.... ... ...I ... I....

AAGCTGGCCA TCGTGGACGT GGTGGACGGC ATCAACCACA AGATCAACCT GACCACCACC
... .. .... I.... I .... I.... I ....I ...I .... I.... I ....1....1 ACCTGCACCA TCCGGAACTG CAAGAAGCTC GGCCCTCGGG AGAACGTGGC CGTGATCCAG

.... . .. . .... . .. . ....I . .. . .... I.... .... . .. . .... I....

GTGGGCGGCA GCAACGTGCT GGACATCACC GCCGACCCCA CCACCAACCC CCAGACCGAG

... .. ... I.... ... I.... ... I.... ... ...I ... I....

CGGATGATGC GGGTGAACTG GAAGAAATGG TGGCAGGTGT TCTACACCAT CGTGGATTAC
... I.... I ... I.... ... I.... I .... I.... I ... I.... I ... I....

ATCAACCAGA TCGTGCAGGT GATGAGCAAG CGGAGCAGAA GCCTGAACAG CGCCGCCTTC
... .. ... I.... I ... I.... I ....I ...I .... I.... I .... I....

TACTACAGAG TGAGATCTGC CACCATGGCC CAGGTGATCA ACACCAACAG CCTGAGCCTG
....I .. ... I .. ....1 .. ... I.... ....I .. .... I....
2050 2060 2070, 2080 2090 2100 CTGACCCAGA ACAACCTGAA CAAGAGCCAG AGCGCCCTGG GCACCGCCAT CGAGAGACTG

AGCAGCGGCC TGCGGATCAA CTCTGCTAAG GACGATGCTG CCGGACAGGC CATCGCCAAC

== I=. .. I== == I = == I ...I .... I = .. I==

CGGTTCACCG CCAACATCAA GGGCCTGACA CAGGCCAGCC GGAACGCCAA CGACGGCATC
... I .. ... I.... ... I.... ... I.... ... I.... I ... I.... 1 AGCATCGCCC AGACCACCGA GGGAGCACTG AACGAGATCA ACAACAACCT GCAGAGAGTG

....I ...I ... I.... I ... I.... I ... I .. I ... I.... I ... I.... 1 CGGGAGCTGG CCGTGCAGAG CGCCAACAGC ACCAACTCCC AGAGCGACCT GGACAGCATC

CAGGCCGAGA TCACCCAGCG GCTGAATGAG ATCGACAGAG TGAGCGGCCA GACCCAGTTC
... .. ....I .. .. .. I .... {.... I .... I.... .... I....

AACGGCGTGA AGGTGCTGGC CCAGGACAAC ACCCTGACCA TCCAGGTCGG CGCCAACGAT

... I.... ... I.... ... I.... I .... I.... I .... I.... ... I....

GGCGAGACCA TCGACATCGA CCTGAAGCAG ATCAACAGCC AGACCCTGGG CCTGGACAGC

CTGAACGTGC AGAAAGCCTA CGACGTGAAG GACACCGCCG TGACCACCAA GGCCTACGCC
....I ...I .... I.... ... I.... I ....I ...I ....I .. ....I ...1 AACAATGGCA CCACCCTGGA CGTGAGCGGC CTGGACGACG CCGCCATCAA GGCCGCCACC

GGCGGCACCA ATGGCACCGC CAGCGTGACC GGCGGAGCCG TGAAGTTCGA CGCCGACAAC
2710 2720 2730 2740 '2750 2760 AACAAGTACT TCGTGACCAT CGGCGGCTTC ACAGGCGCCG ATGCTGCTAA GAACGGCGAC
... ...I ... ...I .... I.... I ... .. ....I .. ....I ..

TACGAGGTGA ACGTGGCCAC CGATGGCAGC GTGACCCTGG CTGCTGGCGC CACCAAGACC
... ...I .... I.... I ... I.... I ....I ...I .... I.... ....I ...1 ACCATGCCTG CTGGGGCCAC CACCAAGACA GAGGTGCAGG AACTGAAGGA CACACCCGCC
... .. ... ...I .... I.... I ....I ...I ....I .. ....I .. .

GTGGTGTCCG CCGACGCCAA GAATGCCCTG ATTGCCGGCG GAGTGGACGC CACCGACGCC

AACGGCGCCG AGCTGGTGAA GATGAGCTAC ACCGACAAGA ACGGCAAGAC AATCGAGGGC

GGCTACGCCC TGAAGGCCGG CGACAAGTAC TACGCCGCCG ACTACGACGA GGCCACAGGC

.... I.... I ... I.... I ... I.... I .... I.... ... I.... I ... I.... I

GCCATCAAAG CCAAGACCAC AAGCTACACA GCCGCCGACG GCACCACAAA GACCGCCGCC
... ...I ... .. I ... I.... I .... I.... ... I.... I ... I.... 1 AACCAGCTGG GCGGCGTGGA CGGCAAGACC GAGGTGGTGA CCATCGATGG CAAGACCTAC

AACGCCAGCA AAGCCGCCGG ACACGACTTC AAGGCCCAGC CCGAGCTGGC CGAGGCTGCC

GCCAAGACAA CCGAGAACCC CCTGCAGAAG ATCGACGCCG CCCTGGCCCA GGTGGACGCC
...:I .. .... I .. ....I .. .... I.... I .... I.... I ....I....1 CTGCGGAGCG ATCTGGGCGC CGTGCAGAAC CGGTTCAACT CCGCCATCAC CAACCTGGGC
... I.... I ... .. ... I.... I .... I.... I ....I ....I ....I....1 AACACCGTGA ACAACCTGTC CGAGGCCAGA AGCCGGATCG AGGACAGCGA CTACGCCACC
... ...I .... I.... I .... I.... I ... .. ....I .. ....I ...1 GAGGTGTCCA ACATGAGCAG GGCCCAGATC CTGCAGCAGG CCGGCACCAG CGTCCTGGCC
.... I.... ... ...I ....I .. ... .. ... .. ....I .. 1 CAGGCCAATC AGGTGCCCCA GAACGTGCTG TCCCTGCTGC GGTGATGAGG ATCCGGGCTC

GAGGGGAGAT CTGAGCTC

SEQ ID NO:7 : VP41.336::Fljb ... .. ... ...I .... I.... ... I.... ....I .. .... I.... I

AGATCTGCCA CCATGGCCAG CCTGATCTAC CGGCAGCTGC TGACCAACAG CTACAGCGTG

GACCTGCACG ACGAGATCGA GCAGATCGGC AGCGAGAAAA CCCAGAACGT GACCATCAAC
.... I.... I ... .. ....I .. ... I ... ... .. ... ...1 CCCAGCCCCT TCGCCCAGAC CAGATACGCC CCCGTGAACT GGGGCCACGG CGAGATCAAC

GACAGCACCA CCGTGGAGCC CATGCTGGAC GGCCCCTACC AGCCCACCAC CTTCACCCCC
.... I.... ....I ...I ....I .. ... I.... ... .. ... .. 1 CCCAACGACT ACTGGATTCT GATCAACAGC AACACCAACG GCGTGGTGTA CGAGAGCACC

AACAACAGCG ATTTCTGGAC CGCCGTGGTG GCCATCGAGC CCCACGTGAA CCCCGTGGAC

CGGCAGTACA CCATCTTCGG CGAGAGCAAG CAGTTCAACG TGAGCAACGA CTCCAACAAG

TGGAAGTTCC TGGAAATGTT CAGAAGCAGC AGCCAGAACG AGTTCTACAA CCGGCGGACC

CTGACCAGCG ACACCCGGTT CGTGGGCATC CTGAAGTACG GCGGCAGAGT GTGGACCTTT
.. ... .. ... I.... I .... I.... I ....I .. ....I....1 CACGGCGAGA CCCCCAGGGC CACCACCGAC AGCAGCAGCA CCGCCAACCT GAACAACATC
....I .. ... I.... ... I ....I .... I.... I .... I.... ... I....

AGCATCACCA TCCACAGCGA GTTTTACATC ATCCCCCGGT CCCAGGAAAG CAAATGCAAC
... ...I ... I.... ... I.... I .... I.... I .... I.... ....I....1 GAGTACATCA ACAACGGCCT GCCCCCCATC CAGAACACCC GGAACGTGGT GCCCCTGCCC
.... I.... I .... I.... I .... I.... I ....I ...I .... I.... .... I....

CTGAGCAGCA GAAGCATCCA GTACAAGAGA GCCCAGGTCA ACGAGGACAT CATCGTGAGC
... I.... ... .. ... I.... I .... I.... I .... I.... ... I....

AAGACCAGCC TGTGGAAAGA GATGCAGTAC AACCGGGACA TCATCATCCG GTTCAAGTTC
... I.... ... I.... ... I.... .... I.... I ... I.... I ... I ...

GGCAACAGCA TCGTGAAGAT GGGCGGCCTG GGCTACAAGT GGAGCGAGAT CAGCTACAAG
....I ...I ... I.... ... I ... .... I.... I ... I.... I ... I ...

GCCGCCAACT ACCAGTACAA CTACCTGCGG GACGGCGAGC AGGTGACCGC CCACACCACC

TGCAGCGTGA ACGGCGTGAA CAACTTCAGC TACAACGGCG GCGGATCTGC CACCATGGCC
... I.... I ... .. ...I .. ... I.... ... I ....I ... I....

CAGGTGATCA ACACCAACAG CCTGAGCCTG CTGACCCAGA ACAACCTGAA CAAGAGCCAG
.... . .. . .... . ...I ....I . .. . .... . .. . ....I . .. . I ....I . .. .

AGCGCCCTGG GCACCGCCAT CGAGAGACTG AGCAGCGGCC TGCGGATCAA CTCTGCTAAG
... .. ... I.... I ... ...I ....I ...I .... I.... I ....I....1 GACGATGCTG CCGGACAGGC CATCGCCAAC CGGTTCACCG CCAACATCAA GGGCCTGACA

CAGGCCAGCC GGAACGCCAA CGACGGCATC AGCATCGCCC AGACCACCGA GGGAGCACTG

I.... ... I.... I ... I.... I .... I.... ... ...I ... I.... 1 AACGAGATCA ACAACAACCT GCAGAGAGTG CGGGAGCTGG CCGTGCAGAG CGCCAACAGC
... I.... ... I.... I ....I .. ... I.... ... I.... I ....I....1 ACCAACTCCC AGAGCGACCT GGACAGCATC CAGGCCGAGA TCACCCAGCG GCTGAATGAG

ATCGACAGAG TGAGCGGCCA GACCCAGTTC AACGGCGTGA AGGTGCTGGC CCAGGACAAC
... ...I .... I.... ... .. ....I ... I.... I ... I.... 1 ACCCTGACCA TCCAGGTCGG CGCCAACGAT GGCGAGACCA TCGACATCGA CCTGAAGCAG
... I.... ....I .. ... I.... .... I.... I ....I .. ... I....1 ATCAACAGCC AGACCCTGGG CCTGGACAGC CTGAACGTGC AGAAAGCCTA CGACGTGAAG
.... I.... I ... I.... ... I ....I ....I ...I ....I .. ....I....1 GACACCGCCG TGACCACCAA GGCCTACGCC AACAATGGCA CCACCCTGGA CGTGAGCGGC

CTGGACGACG CCGCCATCAA GGCCGCCACC GGCGGCACCA ATGGCACCGC CAGCGTGACC
....I ...I ... I.... I ... I.... I ....I ...I ....I .. ....I ...1 GGCGGAGCCG TGAAGTTCGA CGCCGACAAC AACAAGTACT TCGTGACCAT CGGCGGCTTC

ACAGGCGCCG ATGCTGCTAA GAACGGCGAC TACGAGGTGA ACGTGGCCAC CGATGGCACC
....I ...I .... I.... .... I.... I ... .. ... ...I ... .. 1 GTGACCCTGG CTGCTGGCGC CACCAAGACC ACCATGCCTG CTGGGGCCAC CACCAAGACA

GAGGTGCAGG AACTGAAGGA CACACCCGCC GTGGTGTCCG CCGACGCCAA GAATGCCCTG
... ...I ... I.... I ....I .. ... I ... ... ...I ... I....1 ATTGCCGGCG GAGTGGACGC CACCGACGCC AACGGCGCCG AGCTGGTGAA GATGAGCTAC

... .. ....I ...I ....I .. ... I.... ... I.... I ....I....1 1990 2000 2010 . 2020 2030 2040 ACCGACAAGA ACGGCAAGAC AATCGAGGGC GGCTACGCCC TGAAGGCCGG CGACAAGTAC

... I.... I ... .. ....I .. ... I.... ... I.... I ....I....1 TACGCCGCCG ACTACGACGA GGCCACAGGC GCCATCAAAG CCAAGACCAC AAGCTACACA
... .. ... .. ....I .. ....I ... ... I.... I ... I.... 1 GCCGCCGACG GCACCACAAA GACCGCCGCC AACCAGCTGG GCGGCGTGGA CGGCAAGACC
... .. ... I.... ... I.... .... I.... ... I.... I ... .. 1 GAGGTGGTGA CCATCGATGG CAAGACCTAC AACGCCAGCA AAGCCGCCGG ACACGACTTC
....I ...I ... I.... ... I.... ... I.... ... ...I ....I ...1 AAGGCCCAGC CCGAGCTGGC CGAGGCTGCC GCCAAGACAA CCGAGAACCC CCTGCAGAAG

ATCGACGCCG CCCTGGCCCA GGTGGACGCC CTGCGGAGCG ATCTGGGCGC CGTGCAGAAC

CGGTTCAACT CCGCCATCAC CAACCTGGGC AACACCGTGA ACAACCTGTC CGAGGCCAGA
... ...I ... ...I .... I.... I ....I ...I ....I .. ... I ...

AGCCGGATCG AGGACAGCGA CTACGCCACC GAGGTGTCCA ACATGAGCAG GGCCCAGATC
... I.... .... I.... I ... I.... I ....I ...I ....I .. ... I....

CTGCAGCAGG CCGGCACCAG CGTCCTGGCC CAGGCCAATC AGGTGCCCCA GAACGTGCTG
....I ...I I.... I ... I.... I .... I.... I .... I...

TCCCTGCTGC GGTGATGAGG ATCCGGGCTC GAGGGGAGAT CTGAGCTC
SEQ ID NO:8 : VP7::Fljb GGATCTGCCA CCATGTACGG CATCGAGTAC ACCACCATCC TGATCTTTCT GATCAGCATC

ATCCTGCTGA ACTACATCCT GAAGAGCGTG ACCCGGATCA TGGACTACAT CATCTACCGG
... .. ....I .. ....I .. ... I.... ... I.... I ... ..

TTTCTGCTGA TCACCGTGGC CCTGTTCGCC CTGACCAGAG CCCAGAACTA CGGCCTGAAC
....I ...I .... I.... I .... I.... ... .. ....I .. ....I ..

CTGCCCATCA CCGGCAGCAT GGACGCCGTG TACACCAACA GCACCCAGGA AGAAGTCTTT

CTGACCAGCA CCCTGTGCCT GTACTACCCC ACCGAGGCCA GCACCCAGAT CAACGACGGC
... I.... .... I.... ....I .. ... I ... ....I .. I ... ..

GACTGGAAGG ACAGCCTGAG CCAGATGTTC CTGACCAAGG GCTGGCCCAC CGGCAGCGTG
... I.... I ....I .. ....I .. ... ...I .... I.... I .... I.... 1 TACTTCAAAG AATACAGCAA CATCGTCGAC TTCAGCGTGG ACCCCCAGCT GTACTGCGAC
....I .. I I.... I ... I.... I .... I.... I ... ...I ... I.... 1 TACAACCTGG TGCTGATGAA GTACGACCAG AGCCTGAAGC TGGACATGAG CGAGCTGGCC
....I ...I ... I.... I ... I.... I ... I.... .... I.... I ....I....1 490 . 500 510 520 530 540 GACCTGATCC TGAACGAGTG GCTGTGCAAC CCCATGGACG TGACCCTGTA CTACTACCAG

CAGAGCGGCG AGAGCAACAA GTGGATCAGC ATGGGCAGCA GCTGCACCGT GAAAGTGTGC
.... I.... I ... ...I ... I.... ....I ...I ....I .. ... ...1 CCCCTGAACA CCCAGACCCT GGGCATCGGC TGCCAGACCA CCAACGTGGA CAGCTTCGAG
... ...I ... I ... I ... I.... I ....I ...I ....I .. ....I....1 ATGATCGCCG AGAACGAGAA GCTGGCCATC GTGGACGTGG TGGACGGCAT CAACCACAAG
... .. ... I.... I ... I.... I ....I ...I ....I .. ... I....1 ATCAACCTGA CCACCACCAC CTGCACCATC CGGAACTGCA AGAAGCTCGG CCCTCGGGAG
... .. ... I.... I ... I.... I ....I ...I ....I .. ....I....1 AACGTGGCCG TGATCCAGGT GGGCGGCAGC AACGTGCTGG ACATCACCGC CGACCCCACC
... .. ....I ...I .... I.... I ... .. ... ....I ..

ACCAACCCCC AGACCGAGCG GATGATGCGG GTGAACTGGA AGAAATGGTG GCAGGTGTTC
....I .. ....I ...I ....I .. ... .. ... .. ... I....

TACACCATCG TGGATTACAT CAACCAGATC GTGCAGGTGA TGAGCAAGCG GAGCAGAAGC

CTGAACAGCG CCGCCTTCTA CTACAGAGTG AGATCTGCCA CCATGGCCCA GGTGATCAAC
....I .. .... I.... I .... I.... ... .. ....I .. ....I ...1 ACCAACAGCC TGAGCCTGCT GACCCAGAAC AACCTGAACA AGAGCCAGAG CGCCCTGGGC
... ...I ... I.... I ....I ....I ... I.... ... .. ... I....

ACCGCCATCG AGAGACTGAG CAGCGGCCTG CGGATCAACT CTGCTAAGGA CGATGCTGCC

.... I.... I ... I.... I .... I.... I ... .. ... .. ... ..

GGACAGGCCA TCGCCAACCG GTTCACCGCC AACATCAAGG GCCTGACACA GGCCAGCCGG
....I ...I ... I.... I .... I.... I ... I ... ... ...I ... ..

AACGCCAACG ACGGCATCAG CATCGCCCAG ACCACCGAGG GAGCACTGAA CGAGATCAAC
.... I.... ... I.... I .... I.... I ... ...I ....I .. I ....I ...1 AACAACCTGC AGAGAGTGCG GGAGCTGGCC GTGCAGAGCG CCAACAGCAC CAACTCCCAG

AGCGACCTGG ACAGCATCCA GGCCGAGATC ACCCAGCGGC TGAATGAGAT CGACAGAGTG
... .. I ... I.... I ... I.... I .... I.... I ... I.... I .... I.... 1 AGCGGCCAGA CCCAGTTCAA CGGCGTGAAG GTGCTGGCCC AGGACAACAC CCTGACCATC

CAGGTCGGCG CCAACGATGG CGAGACCATC GACATCGACC TGAAGCAGAT CAACAGCCAG
... I.... .... I.... ... I.... .... I.... I .... I.... ... ..

ACCCTGGGCC TGGACAGCCT GAACGTGCAG AAAGCCTACG ACGTGAAGGA CACCGCCGTG
... ...I .... I.... I ... .. ... I.... .... I.... I ... ..

ACCACCAAGG CCTACGCCAA CAATGGCACC ACCCTGGACG TGAGCGGCCT GGACGACGCC

GCCATCAAGG CCGCCACCGG CGGCACCAAT GGCACCGCCA GCGTGACCGG CGGAGCCGTG
....I .. ....I ...I .... I.... I ... .. ... .. ....I ...1 AAGTTCGACG CCGACAACAA CAAGTACTTC GTGACCATCG GCGGCTTCAC AGGCGCCGAT
... .. ....I ...I ....I .. ... .. ... .. ....I ...1 GCTGCTAAGA ACGGCGACTA CGAGGTGAAC GTGGCCACCG ATGGCACCGT GACCCTGGCT
....I .. .... I.... I .... I.... I ... .. ....I .. ....I ...1 GCTGGCGCCA CCAAGACCAC CATGCCTGCT GGGGCCACCA CCAAGACAGA GGTGCAGGAA
... ...I ... I ... ... I ... .... I.... I .... I.... I .... I ...

CTGAAGGACA CACCCGCCGT GGTGTCCGCC GACGCCAAGA ATGCCCTGAT TGCCGGCGGA
... .. ... .. ....I .. ... I.... ... I.... I ... ..
1930 19.40 1950 1960 1970 1980 GTGGACGCCA CCGACGCCAA CGGCGCCGAG CTGGTGAAGA TGAGCTACAC CGACAAGAAC
... ...I ... ...I ....I .. ... I.... ... .. I ... I....

GGCAAGACAA TCGAGGGCGG CTACGCCCTG AAGGCCGGCG ACAAGTACTA CGCCGCCGAC

TACGACGAGG CCACAGGCGC CATCAAAGCC AAGACCACAA GCTACACAGC CGCCGACGGC
.... I.... I ... .. I ... I.... I .... I.... I ... I.... I .... I....

ACCACAAAGA CCGCCGCCAA CCAGCTGGGC GGCGTGGACG GCAAGACCGA GGTGGTGACC

ATCGATGGCA AGACCTACAA CGCCAGCAAA GCCGCCGGAC ACGACTTCAA GGCCCAGCCC
.... I.... I .... I.... I ... I ....I ....I ... I ... I.... I ... I ... 1 GAGCTGGCCG AGGCTGCCGC CAAGACAACC GAGAACCCCC TGCAGAAGAT CGACGCCGCC

.... . .. . I . . . . .. . I .... I.... I .... I.... I ....I . .. . I .... .
...1 CTGGCCCAGG TGGACGCCCT GCGGAGCGAT CTGGGCGCCG TGCAGAACCG GTTCAACTCC

GCCATCACCA ACCTGGGCAA CACCGTGAAC AACCTGTCCG AGGCCAGAAG CCGGATCGAG

GACAGCGACT ACGCCACCGA GGTGTCCAAC ATGAGCAGGG CCCAGATCCT GCAGCAGGCC
....I ...I ....I .. I ... .. I .... I.... I .... I.... I ....I ... 1 GGCACCAGCG TCCTGGCCCA GGCCAATCAG GTGCCCCAGA ACGTGCTGTC CCTGCTGCGG

TGATGAGGAT CCGGGCTCGA GGGGAGATCT GAGCTC

Claims (14)

1. Antigenic recombinant VP4 1-336::VP7, VP4 1-336::VP7::FIjb, VP4 1-336::FIjb, or VP7::FIjb fusion proteins as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID
NO:4, respectively, for use in production of neutralizing antibodies against a rotavirus (Fig. 1)..

2. Antigenic recombinant fusion proteins as set forth in claim 1, fused to another rotavirus protein and/or adjuvant, for use in the production of vaccine against a rotavirus.

3. A vaccine composition for providing immunological protection against rotavirus illness in humans, said vaccine comprising the fusion proteins VP4 1-336::VP7, VP4 1-336::VP7::FIjb, VP4 1-336::FIjb, and/or VP7::FIjb for eliciting antibody production against HRV, in admixture with a suitable pharmaceutical carrier.

4. The vaccine composition of claim 3, wherein said vaccine is formulated for any type of administration including oral, transmucosal (rectal) and parenteral administration.

5. A vaccine composition for prevention and treatment of rotavirus-related disease comprising the recombinant fusion proteins VP4 1-336::VP7, VP4 1-336::VP7::FIjb, VP4 1-336::FIjb, and/or VP7::FIjb fusion proteins as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, and SEQ ID NO:4, respectively, as effective components.

6. The vaccine composition of Claim 5, wherein said composition is adapted for administration by a route selected from the group consisting of intramuscular administration, intranasal administration, oral administration, transdermal administration, and trans-mucosal (rectal) administration.

7. Methods for producing the antigenic recombinant VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-336::Fljb, and/or VP7::Fljb fusion proteins of claim 1 in any type of cells (prokaryotic and eukaryotic cells), for eliciting production of neutralizing antibodies against a rotavirus once in contact with the immune system of a patient.

8. Methods for producing the antigenic recombinant VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-336::Fljb, and/or VP7::Fljb fusion proteins of claim 1 in bacterial, insect or yeast cells, or any other type of cells, for production ex vivo of said recombinant fusion proteins, which may be used for immunization with any types of vehicle material or delivery systems, eliciting the production of neutralizing antibodies against a rotavirus once in contact with the immune system of a patient.

9. A method for producing the antigenic recombinant VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-336::Fljb, and/or VP7::Fljb fusion proteins of claim 1 in a mammalian cell using plasmid and/or any type of viral vectors including adenovectors (replicative or non replicative vectors) and baculovirus systems, for eliciting production of neutralizing antibodies against a rotavirus once in contact with the immune system of a patient, said method comprising the steps of:

a) introducing a nucleic acid fragment encoding VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-336::Fljb, or VP7::Fljb fusion proteins as set forth in SEQ ID NO:5, SEQ ID
NO:6, SEQ
ID NO:7, and SEQ ID NO:8, respectively, for expression under control of a promoter into a mammalian cell for expression of said VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-336::Fljb, and/or VP7::Fljb fusion proteins.

b) inoculating a mammal with the said vectors obtained in step a) such that neutralizing antibodies against a rotavirus are being produced by the immune system of the mammal in contact with the VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-336::Fljb, or VP7::Fljb fusion proteins.

10. A method for producing the antigenic VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-331,::Fljb, and/or VP7::Fljb fusion proteins of claim 1 in a plant cell, for eliciting production of antibodies against rotavirus once in contact with the immune system of a patient, said method comprising the steps of:

a) introducing a nucleic acid fragment encoding VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-336::Fljb, and/or VP7::Fljb fusion proteins as set forth in SEQ ID NO:5, SEQ
ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively for expression under control of a promoter into a plant cell for expression of said VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-336::Fljb, or VP7::Fljb fusion proteins, upon culture of said plant cell; and b) recovering said VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-336::Fljb, and/or VP7::Fljb fusion protein from the plant cell.

11. The method of claim 10, wherein the vector further comprises the tobacco etch virus leader sequence and the endoplasmic reticulum retention signal SEKDEL.

12. The use of antigenic recombinant VP4 1-336::VP7, VP4 1-336::VP7::Fljb, VP4 1-336::Fljb, and/or VP7::Fljb fusion proteins as defined in claim 1 or 2, or of a vaccine composition as defined in anyone of claims 3 to 6 for the manufacture of a medicament for generating a rotaviral disease-reducing immunogenic response.

13. The use of Claim 12, wherein the vaccine composition is adapted for administration by a route selected from the group consisting of intramuscular administration, intranasal administration, oral administration, transdermal administration, and transmucosal (rectal) administration.

14. The use of claim 8 or 9 or 10 in the preparation of a medicament for boosting an immune response to a previously administered rotavirus vaccine.